International Journal of Pharmaceutics (v.436, #1-2)

Engineering drug ultrafine particles of beclomethasone dipropionate for dry powder inhalation by Li-Min Xu; Qian-Xia Zhang; Yue Zhou; Hong Zhao; Jie-Xin Wang; Jian-Feng Chen (1-9).
The engineering ultrafine drug particles of beclomethasone dipropionate for dry powder inhalation administration were prepared by combining microfluidic antisolvent precipitation (a), high-pressure homogenization (b) and spray drying (c).Beclomethasone dipropionate (BDP), which is a member in the inhaled glucocorticosteroid class, is commonly used in the treatment of asthma by pulmonary delivery. The purpose of this study is to prepare ultrafine BDP particles for dry powder inhalation (DPI) administration by combining microfluidic antisolvent precipitation without surfactant, high-pressure homogenization (HPH) and spray drying. T-junction microchannel was adopted for the preparation of needle-like BDP particles. The needle-like particles could be easily broken down into smaller particles during HPH, which were assembled into uniform low-density spherical BDP aggregates by spray drying. The effects of the operation parameters, such as the flow rates of BDP methanol solution and antisolvent, the overall flow rate, the BDP concentration, and the change of the injection phase on BDP particle size were explored. The results indicated that the BDP particle size greatly decreased with the reduction of BDP solution flow rate and the increase of antisolvent flow rate. However, the BDP particle size firstly decreased and then increased with the increase of the overall flow rate and the increase of BDP concentration. Also, BDP solution as the injection phase could form the smaller BDP particles. 10 HPH cycles are enough to forming short rod-like particles. After spray drying, the BDP spherical aggregates with a 2–3 μm size could be achieved. They have an excellent aerosol performance, 2.8 and 1.4 times as many as raw BDP and vacuum-dried BDP particles, respectively.
Keywords: T-junction microchannel; Beclomethasone dipropionate; Homogenization; Spray drying; Dry powder inhalation;

Quaternized chitosan-coated nanofibrous materials containing gossypol: Preparation by electrospinning, characterization and antiproliferative activity towards HeLa cells by Milena Ignatova; Nevena Manolova; Reneta Toshkova; Iliya Rashkov; Elena Gardeva; Liliya Yossifova; Marin Alexandrov (10-24).
Nanofibrous polylactide-based materials loaded with a natural polyphenolic compound gossypol (GOS) with antitumor properties were prepared by electrospinning. The nanofibrous materials were coated with a thin film of crosslinked quaternized chitosan (QCh). GOS incorporated in the nanofibrous mats was in the amorphous state. GOS release was diffusion-controlled and its in vitro release profiles depended on the mat composition. The nanofibrous materials exhibited high cytotoxicity towards HeLa tumor cells. Interestingly, it was particularly pronounced in the case of fibrous materials, which contain both QCh and GOS. The observed strong antiproliferative effect of the nanofibrous mats was mainly due to induction of cell apoptosis.
Keywords: Electrospinning; Gossypol; Quaternized chitosan; Polylactide; Antitumor activity; Apoptosis;

To establish the biodistribution profile of the PLGA nanoparticles with dual surface modifications of PEG and folic acid (FA) in mice xenografted with MDA-MB-231 human breast cancer cells with high expression of folate receptor (FR); and to illustrate that the modified nanoparticles can target the loaded indocyanine green (ICG) to the tumor with high FR expression.ICG-loaded nanoparticles were prepared with PLGA (non-modified nanoparticles, NM-NP) or mPEG-PLGA and FA-PLGA (dual modified nanoparticles, DM-NP). Biodistribution of the ICG-loaded nanoparticles (1.25 mg/kg) after i.v. injection was investigated on athymic mice transplanted with MDA-MB-231 tumor.ICG concentration in plasma from the DM-NP group was significantly (p  < 0.05) higher than the NM-NP group from 90 min to the end of the study (12 h). After 4 h, the drug concentration in the tumor tissue from the DM-NP started to be significantly (p  < 0.05) higher than the NM-NP until 12 h. Compared to the NM-NP, the DM-NP increased the AUC0–12 h in plasma by 245% and the AUC0–12 h in tumor by 194%, while decreased the AUC0–12 h in liver by 13%.The accumulation of DM-NP into the tumor was significantly higher than NM-NP due to the long circulation and FR-mediated uptake.
Keywords: Indocyanine green (ICG); Folic acid; Folate-receptor (FR) targeted; PEG; Biodistribution; Breast cancer;

Investigation of formulation factors affecting in vitro and in vivo characteristics of a galantamine transdermal system by Chun-Woong Park; Dao-Danh Son; Ju-Young Kim; Tack-Oon Oh; Jung-Myung Ha; Yun-Seok Rhee; Eun-Seok Park (32-40).
Because of low treatment compliance with the Alzheimer disease patients, there have been clinical needs for the alternative administration route to effective and well-tolerated approaches of galantamine (). In this study, drug-in-adhesive transdermal patches with galantamine were prepared and evaluated in vitro and in vivo. The in vitro permeation studies indicated that DT-2510 was the most suitable pressure-sensitive-adhesive and oleic acid was the most promising enhancer for galantamine drug-in-adhesive patch. The optimized galantamine drug-in-adhesive patch could be physicochemically stable for 28 days at 40 °C/75% RH. The in vivo studies of the optimized galantamine drug-in-adhesive patch showed high absolute bioavailability of around 80% and sustained effect on the drug plasma levels for 24 h. The in vitro and in vivo studies of galantamine drug-in-adhesive patches with different pressure-sensitive-adhesive functional groups showed a strong correlation between the skin permeation rate and the area under the curve. The results suggest that the transdermal application of galantamine drug-in-adhesive patches might be the alternative dosage form to have good efficacy and tolerability for the treatment of Alzheimer disease.
Keywords: Galantamine; Transdermal patch; Skin permeation rate; Bioavailability; In vitro and in vivo characteristics;

Mechanochemically induced disordered structures of vincamine: The different mediation of two cross-linked polymers by Dritan Hasa; Beatrice Perissutti; Michele Remo Chierotti; Roberto Gobetto; Iztok Grabnar; Alois Bonifacio; Stefano Dall’Acqua; Sergio Invernizzi; Dario Voinovich (41-57).
The aims of this research were to prepare highly bioavailable binary cogrounds (vincamine–AcDiSol® or PVP-Cl) by means of a mechanochemical process and to study the mediation of each polymer in the induction of physical transformations of the drug.From a set of fifteen cogrounds for each crosslinked polymer, two samples were selected in each group on the basis of the AUC of in vitro dissolution profiles with the help of a statistical comparison. The chosen samples were analysed by means of TEM, XRPD, Raman-spectroscopy/imaging, SSNMR, also including the study of 1H spin–lattice relaxation times. The research encompassed in vivo oral absorption studies in rats, pharmacokinetic analysis and physical stability studies during 1 year. An intimate drug–polymer mixing was found in the coground samples with domain average dimensions smaller than 100 Å; this reflected in a remarkable enhancement of the in vitro and in vivo bioavailability. Different disordered states were detected in the coground samples as a function of cogrinding time and the type and amount of polymer used.Though both crosslinked polymers produced a remarkable enhancement of the oral bioavailability, coground systems based on AcDiSol® are preferable in terms of pharmacokinetic performance and physical stability.
Keywords: Vincamine; Mechanochemical activation; Cross-linked polymers; Physico-chemical characterisation; Bioavailability; Physical stability;

Characterization and evaluation of solid self-microemulsifying drug delivery systems with porous carriers as systems for improved carbamazepine release by Mladen Milović; Jelena Djuriš; Ljiljana Djekić; Dragana Vasiljević; Svetlana Ibrić (58-65).
The purpose of this study was to investigate solid self-microemulsifying drug delivery system (SSMEDDS), as potential delivery system for poorly water soluble drug carbamazepine (CBZ). Self-microemulsifying drug delivery system (SMEDDS) was formulated using the surfactant polyoxyethylene 20 sorbitan monooleate [Polysorbate 80] (S), the cosurfactant PEG-40 hydrogenated castor oil [Cremophor® RH40] (C) and the oil caprylic/capric triglycerides [Mygliol® 812] (O). Four different adsorbents with high specific surface area were used: Neusilin® UFL2, Neusilin® FL2 (magnesium aluminometasilicate), Sylysia® 320 and Sylysia® 350 (porous silica). Microemulsion area at the surfactant to cosurfactant ratio (K m) 1:1 was evaluated and for further investigation SMEDDS with SC/O ratio 8:2 was selected. Solubilization capacity of selected SMEDDS for CBZ was 33.771 ± 0.041 mg/ml. Rheological measurements of unloaded and CBZ-loaded SMEDDS at water content varied from 10 to 60% (w/w) were conducted. It has been found that CBZ has great influence on rheological behaviour of investigated system upon water dilution. Photon correlation spectroscopy has shown the ability of CBZ-loaded SMEDDS to produce microemulsion droplet size. SSMEDDS improved release rate of CBZ, but the type of adsorbent significantly affects release rate of CBZ. For SSMEDDS with different magnesium aluminometasilicate adsorbents, release rate of CBZ decreased with increasing specific surface area due to entrapment of liquid SMEDDS inside the pores and its gradual exposure to dissolution medium. With porous silica adsorbents no difference in release rate was found in comparison to physical mixtures. In physical mixtures at 12.5% (w/w) CBZ content, presence of amorphous CBZ led to high dissolution rate.
Keywords: Solid self-microemulsifying drug delivery systems; Magnesium aluminometasilicate; Porous silica; Carbamazepine; Dissolution rate improvement;

Double crosslinked interpenetrated network in nanoparticle form for drug targeting—Preparation, characterization and biodistribution studies by Anca N. Jătariu (Cadinoiu); Mihaela N. Holban; Cătălina A. Peptu; Anca Sava; Marcel Costuleanu; Marcel Popa (66-74).
The development of polymer nanosystems able to target and control/sustain the drug delivery is still considered an important desideratum in pharmaceutical research. The present study reports the preparation of nanoparticles based on chitosan and gelatin, using a reverse emulsion-double crosslinking (ionic followed by covalent one) technique. The nanoparticles structural and morphological characteristics (diameter and size distribution), their swelling capacity in aqueous media of different pH (4 and 7.4) and their ability to include and release poorly water-soluble drugs were seen to be influenced by the composition of the polymer mixture and by the surfactants concentration. Also, nanoparticles biodistribution after intraperitoneal or intravenous administration was evaluated by polymer marking with fluorescein. Particles ability to penetrate different organs (liver, heart, lungs, and less brain, gums, testicles) was increased when injected intravenously.
Keywords: Chitosan; Gelatin; Nanoparticles; Double crosslinking; Biodistribution; Chloramphenicol;

Exploring the assembly process and properties of novel crosslinker-free hyaluronate-based polyelectrolyte complex nanocarriers by Anita Umerska; Krzysztof J. Paluch; Iwona Inkielewicz-Stępniak; Maria Jose Santos-Martinez; Owen I. Corrigan; Carlos Medina; Lidia Tajber (75-87).
The aim of this work was to study the formulation of pharmaceutically relevant polyelectrolyte complex nanoparticles (NPs) composed of hyaluronic acid (HA) and chitosan (CS) containing no crosslinkers. The influence of polymer mixing ratio, concentration and molecular weight as well as the type of counterion in chitosan salt on properties of the resulting NPs was examined. Formulations and their components were studied by laser light scattering, viscosity, infrared spectroscopy and microscopy. Physical stability, isoelectric points and cytotoxicity of selected NPs were determined.By appropriate modification of HA molecular weight, stable and non-sedimenting NPs were successfully formed. Sonication was found to be an effective method to reduce the molecular weight of HA from 2882 ± 25 to 176 ± 4 kDa with no chemical changes in the HA structure observed. High molecular weight CS formed micron-sized entities at all compositions investigated. Positively and negatively charged NPs were obtained depending on the mixing ratio of the polymers, with CS glutamate NPs yielding more negatively charged particles compared to CS chloride NPs. The smallest NPs (149 ± 11 nm) were formed using HA with molecular weight of 176 kDa. Cytotoxicity of NPs was dependent on environmental pH but HA was found to exert cytoprotective effects on Caco-2 cells
Keywords: Hyaluronic acid; Chitosan; Nanoparticle; pH titration; Stability; Cytotoxicity;

Time-engineeringed biphasic drug release by electrospun nanofiber meshes by Li-Ya Huang; Christopher Branford-White; Xia-Xia Shen; Deng-Guang Yu; Li-Min Zhu (88-96).
A drug-loaded nanofiber mesh which could achieve time-engineeringed biphasic release was fabricated through sequential electrospinning. The drug to polymer ratio of each single mesh was allocated and designed before the tri-layered meshes were created. The resultant meshes had the following construction: (i) the first drug-loaded mesh (top side), (ii) the second drug-loaded mesh (second side), and (iii) the third drug-loaded mesh (bottom side). The drug release speed and duration were controlled by designing morphological features of the electrospun meshes such as the fiber diameter and mesh thickness. An in vitro release experiment revealed that the tri-layered construction with distinct morphological features of each component mesh can provide biphasic drug release. The time-engineeringed dual release system using the multilayered electrospun nanofiber meshes was proved to be a useful formulation when achieving controlled drug release at different times.
Keywords: Biphasic release; Tri-layered meshes; Nanotechnology; Electrospinning;

Biophysical and biological investigation of DNA nano-complexes with a non-toxic, biodegradable amine-modified hyperbranched polyester by Regina Reul; Juliane Nguyen; Adam Biela; Elena Marxer; Udo Bakowsky; Gerhard Klebe; Thomas Kissel (97-105).
Designed for gene therapy of chronic diseases, HBP–DEAPA 60 is a non-toxic biodegradable amine modified hyperbranched polyester. This candidate was chosen from a series of hyperbranched polymers for further characterization as it showed the best transfection efficiency and fastest degradation rate. HBP–DEAPA 60/DNA complexes were investigated with regard to stability, uptake and formation to gain a better insight into HBP–DEAPA 60/DNA complex properties. We investigated HBP–DEAPA 60/DNA complex uptake into A 549 cells by FACS and CLSM. Their stability was investigated by a heparin displacement assay as well as by DNAse I assay. Morphology was shown by AFM. HBP–DEAPA 60/DNA complex formation was further characterized in terms of thermodynamic parameters. We studied the conformation of DNA in nano-complexes via circular dichroism (CD) spectroscopy for different NP ratios. Thermodynamic studies showed that binding enthalpies were endothermic; the nano-complex formation was entropically driven. Although PEI/DNA and HBP–DEAPA 60/DNA complexes showed similar behavior with regard to uptake, heparin stability, DNA helicality and their entropically driven complex formation they differ in their binding constant K a and in their ability to protect the DNA from DNAse. Concerning K a and DNAse stability, HBP–DEAPA/DNA complexes should be further optimized. This shows that different characterization studies are necessary to fully characterize polyplex stability and properties.
Keywords: Hyperbranched polyester; Dendritic polymers; Circular Dichroism; Isothermal calorimetry; Uptake studies;

Aerosolized liposomal amphotericin B: Prediction of lung deposition, in vitro uptake and cytotoxicity by Mélanie Fauvel; Cécile Farrugia; Nicolas Tsapis; Claire Gueutin; Odile Cabaret; Christian Bories; Stéphane Bretagne; Gillian Barratt (106-110).
To predict the efficacy and toxicity of pulmonary administration of liposomal amphotericin B (L-AMB) for the treatment or the prevention of pulmonary invasive aspergillosis, a multistage liquid impinger was used to estimate the concentrations of drug that could be attained in different lung compartments after nebulization. The highest concentration of amphotericin B was found in the alveolar compartment, where it was calculated that the concentration in the lung surfactant could reach 54 μM or more when 21.6 μmoles of drug as liposomes was nebulized. The uptake and toxicity of L-AMB were studied in vitro using the A549 human lung epithelial cell line. Uptake was time and concentration-dependent and reached intracellular concentrations exceeding the minimal inhibitory concentrations for most Aspergillus species. The toxicity of L-AMB toward these cells, estimated by the MTT reduction assay, was reduced compared with the conventional form, deoxycholate amphotericin B (D-AMB), with an IC50 value of about 120 μM after 24 h of exposure for D-AMB, but only a 13% reduction in viability for 200 μM L-AMB at 24 h. These results indicate that aerosol therapy with nebulized L-AMB could be efficient but that doses need to be carefully controlled to avoid toxicity.
Keywords: Liposomal amphotericin B; A549; Nebulization; MTT;

In vitro controlled release of antihypertensive drugs intercalated into unmodified SBA-15 and MgO modified SBA-15 matrices by Iuliana Florentina Alexa; Maria Ignat; Roxana Florentina Popovici; Daniel Timpu; Eveline Popovici (111-119).
The use of nanotechnology in medicine and more specifically in drug delivery systems is set to spread rapidly. In order to broaden the range of matrices and implicitly to develop the class of drug delivery systems based on diffusion mechanism, in this study the starting materials, SBA-15 powder matrices, were engineered by MgO modification for antihypertensive drugs intercalation. Captopril and aliskiren were used as drug models. All powders, unmodified and MgO-modified silica matrices, and their corresponding drug-loaded samples were characterized by X-ray diffraction, N2 adsorption and desorption, FTIR spectroscopy and scanning electron microscopy. The studied drug carriers were tested in the controlled drug release process and the influence of the silica pore morphology and geometry on drug release profiles was extensively studied. In order to analyze the data obtained from the in vitro release studies and to evaluate the kinetic release mechanism, the Korsmeyer and Peppas equation was used. The obtained drug delivery system based on MgO–SBA-15 matrix exhibits exciting structural features and is therefore promising for its use as antihypertensive drug delivery system, having potential therapeutic benefits resulting in safe and effective management of captopril and aliskiren adsorption and in vitro release.
Keywords: Mesoporous silica; Captopril; Aliskiren; Controlled release; Drug delivery;

Thiomers: Influence of molar mass on in situ gelling properties by Fabian Hintzen; Flavia Laffleur; Federica Sarti; Gul Shahnaz; Andreas Bernkop-Schnürch (120-126).
The aim of this study was to investigate the influence of molar mass of thiolated polymers (thiomers) on their in situ gelling properties. Chitosan–thioglycolic acid (chitosan–TGA) and pectin–cysteine (pectin–Cys) of increasing molar mass were chosen to produce in situ gels in combination with carbamide peroxide. Low molar mass chitosan (∼2 kDa) was prepared by oxidative degradation with NaNO2, whereas pectin was depolymerized by heat treatment. Thiomers, displaying 1271–1616 μmol (chitosan–TGA) and 305–403 μmol (pectin–Cys) free thiol groups per gram polymer, were synthesized via amide bond formation mediated by a carbodiimide. The results showed that a reduction of molar mass combined with increased concentrations of both cationic chitosan–TGA and anionic pectin–Cys leads to higher final viscosities and to a higher relative increase in viscosity within 60 min and 180 min, respectively. Using this method, the dynamic viscosity of a very low molar mass chitosan–TGA (∼2 kDa) could be increased 100,000-fold within 60 min and 390,000-fold within 180 min. In view of these in situ gelling properties carbohydrate thiomers might be useful for various pharmaceutical applications such as vehicle for drug delivery or as wound dressing material.
Keywords: Molar mass; Thiomers; Depolymerization; In situ gelling; Crosslinking;

Enhanced brain accumulation of pazopanib by modulating P-gp and Bcrp1 mediated efflux with canertinib or erlotinib by Mukul Minocha; Varun Khurana; Bin Qin; Dhananjay Pal; Ashim K. Mitra (127-134).
Primary objective of this investigation was to delineate the differential impact of efflux transporters P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) on brain disposition and plasma pharmacokinetics of pazopanib. In addition, this research investigated whether inhibition of these efflux transporters with clinically relevant efflux modulators canertinib or erlotinib could be a viable strategy for improving pazopanib brain delivery. In vitro assays with MDCKII cell monolayers suggested that pazopanib is a high affinity substrate for Bcrp1 and a moderate substrate for P-gp. Co-incubation with specific transport inhibitors restored cell accumulation and completely abolished the directionality of pazopanib flux. Brain and plasma pharmacokinetic studies were conducted in FVB wild type mice in the absence and presence of specific transport inhibitors. Drug levels in plasma and brain were determined using a validated high performance liquid chromatography method using vandetanib as an internal standard. In vivo studies indicated that specific inhibition of either P-gp (by zosuquidar or LY335979) or Bcrp1 (by Ko143) alone did not significantly alter pazopanib brain accumulation. However, dual P-gp/Bcrp1 inhibition by elacridar (GF120918), significantly enhanced pazopanib brain penetration by ∼5-fold without altering its plasma concentrations. Thus, even though Bcrp1 showed higher affinity towards pazopanib in vitro, in vivo at the mouse BBB both P-gp and Bcrp1 act in concert to limit brain accumulation of pazopanib. Furthermore, erlotinib and canertinib as clinically relevant efflux modulators efficiently abrogated directionality in pazopanib efflux in vitro and their co-administration resulted in 2–2.5-fold increase in pazopanib brain accumulation in vivo. Further pre-clinical and clinical investigations are warranted as erlotinib or canertinib may have a synergistic pharmacological effect in addition to their primary role of pazopanib efflux modulation as a combination regimen for the treatment of recurrent brain tumors.
Keywords: Pazopanib; Pharmacokinetics; Brain; P-glycoprotein (P-gp); Breast cancer resistance protein (Bcrp1); Erlotinib; Canertinib;

Squalenoyl prodrug of paclitaxel: Synthesis and evaluation of its incorporation in phospholipid bilayers by Maria Grazia Sarpietro; Sara Ottimo; Donatella Paolino; Annalisa Ferrero; Franco Dosio; Francesco Castelli (135-140).
1,1′,2-Trisnorsqualenoic acid was conjugated to paclitaxel to obtain the squalenoyl–paclitaxel prodrug with the aim to improve the incorporation in phospholipid bilayers. Differential scanning calorimetry technique was employed to compare the interaction of squalenoyl–paclitaxel prodrug and free paclitaxel with phospholipid bilayers. The possibility of using lipid vesicles as carrier for the prodrug was also evaluated. An increased encapsulation into phospholipid bilayers of squalenoyl–paclitaxel with respect to the free drug was observed. The ability of lipid vesicles to retain the loaded prodrug was also observed which make this system to be considered as carrier for the prodrug.
Keywords: Paclitaxel; Prodrug; Phospholipid bilayers; DSC; Squalenoyl–paclitaxel;

An innovative bi-layered wound dressing made of silk and gelatin for accelerated wound healing by Sorada Kanokpanont; Siriporn Damrongsakkul; Juthamas Ratanavaraporn; Pornanong Aramwit (141-153).
In this study, the novel silk fibroin-based bi-layered wound dressing was developed. Wax-coated silk fibroin woven fabric was introduced as a non-adhesive layer while the sponge made of sericin and glutaraldehyde-crosslinked silk fibroin/gelatin was fabricated as a bioactive layer. Wax-coated silk fibroin fabrics showed improved mechanical properties compared with the non-coated fabrics, but less adhesive than the commercial wound dressing mesh. This confirmed by results of peel test on both the partial- and full-thickness wounds. The sericin-silk fibroin/gelatin spongy bioactive layers showed homogeneous porous structure and controllable biodegradation depending on the degree of crosslinking. The bi-layered wound dressings supported the attachment and proliferation of L929 mouse fibroblasts, particularly for the silk fibroin/gelatin ratio of 20/80 and 0.02% GA crosslinked. Furthermore, we proved that the bi-layered wound dressings promoted wound healing in full-thickness wounds, comparing with the clinically used wound dressing. The wounds treated with the bi-layered wound dressings showed the greater extent of wound size reduction, epithelialization, and collagen formation. The superior properties of the silk fibroin-based bi-layered wound dressings compared with those of the clinically used wound dressings were less adhesive and had improved biological functions to promote cell activities and wound healing. This novel bi-layered wound dressing should be a good candidate for the healing of full-thickness wounds.
Keywords: Silk fibroin; Gelatin; Silk sericin; Bi-layered wound dressing; Wound healing;

The ability of non-steroidal anti-inflammatory drugs (NSAIDs) to injure the small intestine has been well established in humans and animals. Muscovite is one kind of natural clay consisting of an insoluble double silicate of aluminum and magnesium. It has been developed and marketed in China for the treatment of gastric diseases. The present study was designed to examine the effects of intragastric treatment of muscovite on the intestinal damage induced by administration of diclofenac in rat.Male SD rats were treated with muscovite for 9 days, with concomitant treatment with anti-inflammatory doses of diclofenac on the final 5 days. The anatomical lesion, villous height, the thickness and the section area of small intestine were quantitatively analyzed. The change of ultrastructural organization was observed. Endotoxin level in blood was measured by photometry. Epidermal growth factor was observed by immunohistochemistry.Muscovite decreased the macroscopic and histologic damage induced by diclofenac in the rat small intestine. In the muscovite group, villous height (139.8 ± 13.2 μm) was higher than which of the model group (86.6 ± 17.1 μm) (P  < 0.05). The index of the thickness and the section area was higher than model group. LPS level in the portal blood of muscovite (0.84 ± 1.17 EU/ml) was lower than model group (4.52 ± 0.98 EU/ml) (P  < 0.05). The EFG of muscovite group was higher significantly compared with the model group (P  < 0.05).Muscovite can protect the small intestine from the damage induced by diclofenac in the conscious rat. Muscovite can repair NSAID-induced intestinal damage at least in part because of significant lesion in mechanical barrier function and reduction in epidermal growth factor.
Keywords: Muscovite; Non-steroidal anti-inflammatory drugs; Intestine; Protect;

Solubility enhancement of desloratadine by solid dispersion in poloxamers by Nemanja Kolašinac; Kyriakos Kachrimanis; Irena Homšek; Branka Grujić; Zorica Đurić; Svetlana Ibrić (161-170).
The present study investigates the possibility of using poloxamers as solubility and dissolution rate enhancing agents of the poorly water soluble drug substance desloratadine that can be used for the preparation of immediate release tablet formulation. Two commercially available poloxamer grades (poloxamer P 188 and poloxamer P 407) were selected, and solid dispersions (SDs) containing different weight ratio of poloxamers and desloratadine were prepared by a low temperature melting method. All SDs were subjected to basic physicochemical characterization by thermal and vibrational spectroscopy methods in order to evaluate the efficiency of poloxamers as solubility enhancers. Immediate release tablets were prepared by direct compression of powdered solid dispersions according to a General Factorial Design, in order to evaluate the statistical significance of two formulation (X 1 – type of poloxamer in SD and X 2 – poloxamer ratio in SD) and one process variable (X 3 – compression force) on the drug dissolution rate. It was found that desloratadine in SDs existed in the amorphous state, and that can be largely responsible for the enhanced intrinsic solubility, which was more pronounced in SDs containing poloxamer 188. Statistical analysis of the factorial design revealed that both investigated formulation variables exert a significant effect on the drug dissolution rate. Increased poloxamer ratio in SDs resulted in increased drug dissolution rate, with poloxamer 188 contributing to a faster dissolution rate than poloxamer 407, in accordance with the results of intrinsic dissolution tests. Moreover, there is a significant interaction between poloxamer ratio in SD and compression force. Higher poloxamer ratio in SDs and higher compression force results in a significant decrease of the drug dissolution rate, which can be attributed to the lower porosity of the tablets and more pronounced bonding between poloxamer particles.
Keywords: Solid dispersion; Desloratadine; Poloxamer; Amorphous state; Drug release;

Influence of compaction properties and interfacial topography on the performance of bilayer tablets by Niranjan Kottala; Admassu Abebe; Omar Sprockel; Ilgaz Akseli; Faranak Nikfar; Alberto M. Cuitiño (171-178).
Bilayer tablets are generating great interest recently as they can achieve controlled delivery of different drugs with pre-defined release profiles. However, the production of such tablets has been facing great challenges as the layered tablets are prone to delaminate or fracture in the individual layers due to insufficient bonding strength of layers and adhesion at the interfaces. This paper will provide an insight into the role of interfacial topography on the performance of the bilayer tablets. In this study, two widely used pharmaceutical excipients: microcrystalline cellulose and lactose were investigated. Bilayer tablets were manufactured with a range of first and second layer compression forces. A crack of known dimensions was introduced at the interface to investigate the crack propagation mechanisms upon axially loading the bilayer tablet, and to determine the stress intensity factor (K I) of the interface (will be discussed in a separate paper). The results indicated that a strong dependency of the strength of bilayer tablets and mode of crack propagation on the material and compaction properties. The results showed that the strength of bilayer tablets increased with the increase of interfacial roughness, and the first layer and second layer forces determined the magnitude of interfacial roughness for both plastic and brittle materials. Further, the results also indicated that layer sequence and compaction forces played a key role in influencing the strength of the bilayer tablets. For the same (first and second layer) force combination, interfacial strength is higher for the tablets made of brittle material in the first layer. It was observed that interfacial strength decreased with the increase of lubricant concentration. The studies showed that the effect of lubricant (i.e. reduction in compact strength with the increase of lubricant concentration) on the strength of compacts is higher for tablets made of plastic material as compared to the tablets made of brittle material.
Keywords: Bilayer tablets; Delamination; Interfacial topography; Axial testing; Stylus profilometry; (Surface) roughness;

Drug delivery by topical route for the treatment of onychomycosis, a nail fungal infection, is challenging due to the unique barrier properties of the nail plate which imparts high resistance to the passage of antifungal drugs. Permeation enhancers are used in transungual formulations to improve the drug flux across the nail plate. Selection of the effective permeation enhancer among the available large pool of permeation enhancers is a difficult task. Screening the large number of permeation enhancers using conventional Franz diffusion cells is laborious and expensive. The objective of present study was to evolve a simple, accurate and rapid method for screening of transungual drug permeation enhancers based on the principle of hydration of nail plate. The permeation enhancer which affects the structural or physicochemical properties of nail plate would also affect their hydration capacity. Two screening procedures namely primary and secondary screenings were evolved wherein hydration and uptake of ciclopirox olamine by nail plates were measured.Hydration enhancement factor, HEF24 and drug uptake enhancement factor, UEF24 were determined for screening of 23 typical permeation enhancers. The Pearson's correlation coefficient between HEF24 and UEF24 was determined. A good agreement between the HEF24 and UEF24 data proved the validity of the proposed nail plate hydration model as a screening technique for permeation enhancers.
Keywords: Onychomycosis; Transungual; Ciclopirox olamine; Permeation enhancer;

Design, synthesis and evaluation of N-acetyl glucosamine (NAG)–PEG–doxorubicin targeted conjugates for anticancer delivery by Smita K. Pawar; Archana J. Badhwar; Firuza Kharas; Jayant J. Khandare; Pradeep R. Vavia (183-193).
Efficacy of anticancer drug is limited by the severe adverse effects induced by drug; therefore the crux is in designing delivery systems targeted only to cancer cells. Toward this objectives, we propose, synthesis of poly(ethylene glycol) (PEG)–doxorubicin (DOX) prodrug conjugates consisting N-acetyl glucosamine (NAG) as a targeting moiety. Multicomponent system proposed here is characterized by 1H NMR, UV spectroscopy, and HPLC. The multicomponent system is evaluated for in vitro cellular kinetics and anticancer activity using MCF-7 and MDA-MB-231 cells. Molecular modeling study demonstrated sterically stabilized conformations of polymeric conjugates. Interestingly, PEG–DOX conjugate with NAG ligand showed significantly higher cytotoxicity compared to drug conjugate with DOX. In addition, the polymer drug conjugate with NAG and DOX showed enhanced internalization and retention effect in cancer cells, compared to free DOX. Thus, with enhanced internalization and targeting ability of PEG conjugate of NAG–DOX has implication in targeted anticancer therapy.
Keywords: Doxorubicin; N-Acetyl glucosamine; Prodrug; Glutathione; Cellular internalization;

Design, optimization and in vitro evaluation of reverse micelle-loaded lipid nanocarriers containing erlotinib hydrochloride by Sandy Vrignaud; José Hureaux; Séverine Wack; Jean-Pierre Benoit; Patrick Saulnier (194-200).
Erlotinib hydrochloride (ERLO) belongs to the tyrosine kinase inhibitor family and is used for the treatment of pancreatic cancers. In the present study, ERLO was entrapped in lipid nanocarriers by means of reverse micellar incorporation. This study aims to optimize the formulation of ERLO-loaded nanoparticles. Surfactants forming reverse micelles in Labrafac® were filled with ERLO under various conditions. Both the initial amount of drug incubated with reverse micelles and the surfactant composing the reverse micelles are crucial parameters for reverse micelle capacity to load ERLO. The optimal loading system for reverse micelles was obtained with a mix of sorbitan trioleate (Span® 85) and Labrafac® oil at a 1:1 (w/w) ratio. Reverse micelle composition influenced the nanocarrier's hydrodynamic diameter, polydispersity index, and zeta potential. In lipid nanoparticles formulated by using the phase inversion temperature (PIT) method, ERLO entrapment efficiency was around 56%. In vitro, the efficacy of ERLO-loaded nanocarriers on BxPC-3 pancreatic adenocarcinoma cells was comparable to free ERLO, and led to a cell death rate of around 40%.
Keywords: Lipid nanoparticles; Tyrosine kinase inhibitor; PIT method; BxPC-3 cells; Sorbitan esters; Polyoxyethylene sorbitan fatty acid esters;

Improved antibacterial activity of cephalosporins loaded in magnetic chitosan microspheres by Carmen Mariana Chifiriuc; Alexandru Mihai Grumezescu; Crina Saviuc; Cristina Croitoru; Dan Eduard Mihaiescu; Veronica Lazar (201-205).
Putative mechanism for the improved delivery of the antibiotic in active forms from the chitosan matrix: the positively charged polymeric vehicles intimately interact with the negatively charged bacterial surface, delivering the antibiotic with a higher yield (a) than that reached by the antibiotic alone, dispersed in solution (b).During the present study, we have evaluated magnetic chitosan as a potential drug delivery device, by specifically determining if chitosan could elute antibiotics in an active form that would be efficacious in inhibiting Staphylococcus aureus and Escherichia coli growth. We have demonstrated that the incorporation of cephalosporins of second, third and fourth generation into magnetic chitosan microspheres can possibly lead to an improved delivery of antibiotics in active forms, probably due to the inherent properties of chitosan.
Keywords: Magnetic chitosan beads; Cephalosporins; Antibacterial activity; Chitosan microspheres;

Diffusion and permeation profiles of urea (left), caffeine (middle) and diclofenac sodium (right) across SC lipid model membranes and human SC.The impact of the lipophilic penetration enhancer, oleic acid (OA), on the barrier properties of stratum corneum (SC) lipid model membranes was investigated based on diffusion and permeation studies of model drugs covering a broad range of lipophilicities. Diffusion and permeation experiments of urea, caffeine and diclofenac sodium were conducted using Franz-type diffusion cells. HPLC and capillary electrophoresis techniques were employed to analyze the amount of permeated drug. An incorporation of OA to the SC lipid model membranes did not change the relation between the diffusion and permeation behavior of model drugs presented previously for SC lipid model membranes without OA. The fastest rate of diffusion through SC lipid model membranes occurred in the case of the most hydrophilic drug, urea. In the case of permeation studies of caffeine and diclofenac sodium across SC lipid model systems, the permeability parameters were either equal or slightly larger in favor of the most lipophilic drug, diclofenac sodium.OA had a pronounced impact on the barrier properties of SC lipid model membranes. It caused the impairment of the barrier function of the SC lipid model membrane with Cer [AP] (phytosphingosine-based ceramide), however, surprisingly improved the barrier properties of the SC lipid model system with Cer [EOS] (sphingosine-based acylceramide).
Keywords: Stratum corneum; SC lipid model membrane; Diffusion; Permeation; Penetration enhancer; Oleic acid;

This review aims to awake an interest in the determination of the tensile strength of tablets of various shapes using a variety of direct and indirect test methods. The United States Pharmacopoeia monograph 1217 () has provided a very good approach to the experimental determination of and standards for the mechanical strength of tablets. Building on this monograph, it is hoped that the detailed account of the various methods provided in this review will encourage industrial and academic scientists involved in the development and manufacture of tablet formulations to take a step forward and determine the tensile strength of tablets, even if these are not simply flat disc-shaped or rectangular. To date there are a considerable number of valid test configurations and stress equations available, catering for many of the various shapes of tablets on the market. The determination of the tensile strength of tablets should hence replace the sole determination of a breaking force, because tensile strength values are more comparable and suggestions for minimum and/or maximum values are available. The review also identifies the gaps that require urgent filling. There is also a need for further analysis using, for example, Finite Element Method, to provide correct stress solutions for tablets of differing shapes, but this also requires practical experiments to find the best loading conditions, and theoretical stress solutions should be verified with practical experiments.
Keywords: Direct tensile testing; Equi-biaxial tensile strength; Fracture envelope; Indirect tensile testing; Mechanical strength; Tablets; Tensile strength; United States Pharmacopoeia USP35/NF30;

Characterization of surfactants in an oil-in-water emulsion-based vaccine adjuvant using MS and HPLC–MS: Structural analysis and quantification by Jean-François Cotte; Sylvain Sonnery; Fabien Martial; Jean Dubayle; François Dalençon; Jean Haensler; Olivier Adam (233-239).
Mass spectrometry and high performance liquid chromatography coupled to mass spectrometry have been used to better characterize two surfactants, a polyethoxylated alcohol and a sorbitan oleate.Mass spectrometry (MS) and high performance liquid chromatography coupled to mass spectrometry (HPLC–MS) techniques were developed to characterize two surfactants, cetheareth-12 and sorbitan oleate, used to manufacture AF03, an emulsified oil-in-water (O/W) adjuvant. MS was first used to characterize the chemical structure and determine the composition of the two surfactants. The two surfactants appeared as complex products, in particular with respect to the nature of the fatty alcohols and the distribution of the number of ethylene oxides in cetheareth-12, and with respect to the different sorbitan-bound fatty acids (oleic, linoleic and palmitic acids) in sorbitan oleate. Subsequently, once the ions of interest were determined and selected, HPLC–MS was developed and optimized to quantify and to “quality control” the two surfactants as raw materials and as ingredients in the final O/W emulsion bulk and filled products.
Keywords: Sorbitan oleate; Cetheareth-12; AF03; Mass spectrometry; HPLC; Quantification;

Preparation, characterization and in vitro release study of a glutathione-dependent polymeric prodrug Cis-3-(9H-purin-6-ylthio)-acrylic acid-graft-carboxymethyl chitosan by Xiao-Yu Gong; Yi-Hua Yin; Zhi-Jun Huang; Bo Lu; Pei-Hu Xu; Hua Zheng; Fu-Liang Xiong; Hai-Xing Xu; Xiong Xiong; Xing-Bao Gu (240-247).
Self-assembly of PTA-g-CMCS and the release of 6-MP from the polymeric prodrug.In this work, an amphiphilic polymeric prodrug Cis-3-(9H-purin-6-ylthio)-acrylic acid-graft-carboxymethyl chitosan (PTA-g-CMCS) was designed and synthesized. In aqueous solution, this grafted polymer can self-assemble into spherical micelles with a size ranging from 104 to 285 nm and zeta potential ranging from −12.3 to −20.1 mV. For the release study, less than 24% of 6-Mercaptopurine (6-MP) was released from PTA-g-CMCS1 in the media containing 2 and 100 μM glutathione (GSH), whereas 37%, 54% and 75% of 6-MP was released from the media with GSH of 1, 2 and 10 mM, respectively. Besides, pH and drug content of the polymeric prodrug only presented slight influence on the 6-MP release. MTT assay demonstrated that this system had higher inhibition ratio on HL-60 cells (human promyelocytic leukemia cells) in the presence of GSH and lower cytotoxicity on mouse fibroblast cell line (L929). Therefore, this nano-sized system is glutathione-dependent, and it can be employed as a potential carrier for the controlled release of 6-MP.
Keywords: Carboxymethyl chitosan; 6-Mercaptopurine; GSH-dependent release; Polymeric prodrug;

An arginine derivative contained nanostructure lipid carriers with pH-sensitive membranolytic capability for lysosomolytic anti-cancer drug delivery by Sai Li; Zhigui Su; Minjie Sun; Yanyu Xiao; Feng Cao; Aiwen Huang; Hongying Li; Qineng Ping; Can Zhang (248-257).
Lysosomal escape capability of AL-containing pH-sensitive membranolytic BSA-AL-NLCs, as well as the schematic illustration of hypothetic lysosomolysis mechanism.By inserting l-arginine lauril ester (AL) into nanostructure lipid carriers (NLCs) and then coating with bovine serum albumin (BSA), pH-sensitive membranolytic and lysosomolytic nanocarriers (BSA-AL-NLCs) were developed. Hemolysis assay demonstrated the pH-sensitive biomembrane disruptional capability of AL and BSA-AL-NLCs. BSA-AL-NLCs did not disrupt biomembrane at pH 7.4 even at high concentration, exhibited ideal feasibility as lysosomolytic drug delivery nanoparticles without cytotoxicity. Confocal Laser Scanning Microscope (CLSM) images confirmed the lysosomolytic capability of BSA-AL-NLCs after internalized into MCF-7 (human breast cancer cell) via endosome–lysosome path in vitro. Paclitaxel (PTX) loaded BSA-AL-NLCs displayed pH-dependent release in vitro. In MCF-7 viability test with MTT assays, both the blank NLCs did not exhibit cellular toxicity. Of particular interest, the in vitro cell experiments demonstrated that the anti-tumor effect of PTX-loaded BSA-AL-NLCs was preferable to BSA-NLCs, even comparable with PTX solution, which indicated that AL served to facilitate lysosomal escape of BSA-AL-NLCs so as to improve the anti-cancer effect. Biodistribution and anti-cancer activity in vivo confirmed the improved tumor targeting and anti-cancer efficacy of BSA-AL-NLCs. The study suggested that the simple and small molecule of AL may render more nanocarriers lysosomolytic capability with lower cytotoxicity, as well as improved therapeutic index of loaded active agents.
Keywords: l-Arginine lauril ester; pH-sensitive; Lysosomolytic; Nanostructure lipid carriers; Bovine serum albumin; Paclitaxel;

Stabilized micelles as delivery vehicles for paclitaxel by Krassimira Yoncheva; Patricia Calleja; Maite Agüeros; Petar Petrov; Ivanka Miladinova; Christo Tsvetanov; Juan M. Irache (258-264).
Schematic presentation describing the improved pharmacokinetic parameters achieved by administration of paclitaxel-loaded stabilized Pluronic micelles (PTX-PM). For comparison, the same parameters calculated after administration of paclitaxel in the form of commercial solution (Taxol®).Paclitaxel is an antineoplastic drug used against a variety of tumors, but its low aqueous solubility and active removal caused by P-glycoprotein in the intestinal cells hinder its oral administration. In our study, new type of stabilized Pluronic micelles were developed and evaluated as carriers for paclitaxel delivery via oral or intravenous route. The pre-stabilized micelles were loaded with paclitaxel by simple solvent/evaporation technique achieving high encapsulation efficiency of approximately 70%. Gastrointestinal transit of the developed micelles was evaluated by oral administration of rhodamine-labeled micelles in rats. Our results showed prolonged gastrointestinal residence of the marker encapsulated into micelles, compared to a solution containing free marker. Further, the oral administration of micelles in mice showed high area under curve of micellar paclitaxel (similar to the area of i.v. Taxol®), longer mean residence time (9-times longer than i.v. Taxol®) and high distribution volume (2-fold higher than i.v. Taxol®) indicating an efficient oral absorption of paclitaxel delivered by micelles. Intravenous administration of micelles also showed a significant improvement of pharmacokinetic parameters of micellar paclitaxel vs. Taxol®, in particular higher area under curve (1.2-fold), 5-times longer mean residence time and lower clearance, indicating longer systemic circulation of the micelles.
Keywords: Stabilized micelles; Paclitaxel; Pluronic; Gastrointestinal transit; Oral absorption;

A comparison of the physicochemical properties and a sensory test of Acyclovir creams by Yutaka Inoue; Kayoko Furuya; Miruto Matumoto; Isamu Murata; Masayuki Kimura; Ikuo Kanamoto (265-271).
In external medicine, types and ratios of additives are not necessarily the same for well-known brand-name drugs and generic drugs. This study sought to compare the physicochemical properties and sensory test results of a brand-name Acyclovir (ACV) cream and two generic ACV creams. Near-infrared (NIR) spectroscopy revealed changes in absorption spectra attributed to differences in the oil and water content of the 3 creams. In addition, ACV-B and ACV-C had similar NIR absorption spectra. Microscopic examination revealed crystallization in each of the creams and droplets in ACV-C. Powder X-ray diffraction measurement revealed diffraction peaks due to ACV for ACV-A and ACV-B. Assessment of viscoelasticity indicated that stress of subjection to 35 °C caused no changes in the viscoelasticity of ACV-B and ACV-C in comparison to stress of subjection to 25 °C but it did cause the viscoelasticity of ACV-A to decrease. ACV-A had a greater tolerance to stress and a higher viscosity, tan  δ, and yield value than the other 2 creams. Results of a sensory test revealed significant differences in adhesiveness, spreadability, and feel for ACV-A in comparison to ACV-B and ACV-C. Thus, differences in the viscosity and elasticity of the creams due to differences in types and ratios of additives were noted. These differences are surmised to be differences in physical properties. In addition, results suggested that viscoelasticity and spreadability in the sensory test reflected differences in physical properties.
Keywords: Acyclovir; Generic drug; Viscosity; Elasticity; Sensory test;

Preparation and characterization of 5-fluorouracil-loaded PLLA–PEG/PEG nanoparticles by a novel supercritical CO2 technique by Cheng Zhang; Guodong Li; Yihan Wang; Fuying Cui; Jien Zhang; Qingshan Huang (272-281).
In this work, 5-fluorouracil-loaded- poly(l-lactic)-polyethylene glycol/polyethylene glycol (5-FU-loaded-PLLA–PEG/PEG) nanoparticles were prepared using a novel reverse emulsion-solution enhanced dispersion by supercritical fluids (reverse emulsion-SEDS) technique in an effort to obtain an efficient drug delivery system. In the experiment, 5-FU and PEG were dissolved in water PLLA–PEG was dissolved in organic solution, the aqueous solution was added dropwise to the organic solution under magnetic stirring, a reverse emulsion was immediately formed. The reverse emulsion was dried by a SEDS process so that 5-FU-loaded-PLLA–PEG/PEG nanoparticles (5-FU-NPs) were obtained. The particle size, size distribution, surface morphology, and physical and chemical properties of the 5-FU-NPs were investigated by laser diffraction particle size analysis, scanning electron microscopy (SEM), Fourier transform infrared spectrometry (FTIR) and thermogravimetric analysis (TGA). The drug encapsulation efficiency (EE), drug loading (DL), in vitro release profile and pharmacokinetics of 5-FU-NPs in rat plasma were investigated by high-performance liquid chromatography (HPLC). The in vivo tumor inhibition effect, increase in lifespan and hepatotoxicity of placebo-NPs, 5-FU and 5-FU-NPs were determined using H22 tumor-bearing ICR mice. These results collectively suggest that 5-FU-NPs prepared using SEDS have potential anti-tumor applications as a controlled drug release dosage form without harmful drug toxicity.
Keywords: Supercritical CO2; 5-Fluorouracil; Poly(l-lactic)-polyethylene glycol; Reverse emulsion-SEDS; Nanoparticles; Drug delivery system;

Long-term stability of diluted solutions of the monoclonal antibody rituximab by Muriel Paul; Victoire Vieillard; Emmanuel Jaccoulet; Alain Astier (282-290).
As it is an important challenge for pharmacists to access the stability of monoclonal antibodies because of the widespread of centralized preparation units, we conducted a study to evaluate the physicochemical and biological stability of diluted rituximab at 1 mg/mL over six months at 4 °C. We also conducted the study at 40 °C to demonstrate that all methods employed were stability indicating. Various protein characterization methods were used to determine changes in physicochemical properties of rituximab, including size-exclusion chromatography, dynamic light scattering, turbidimetry, cation-exchange chromatography, second-derivative ultraviolet and infrared spectroscopy, and peptide mapping. Cell culture was used to assess biological stability. We demonstrated that diluted rituximab stored at 4 °C in polyolefine bags remained stable for at least six months. No physical or chemical instability was observed, and the biological activity was fully maintained. Size exclusion chromatography did not show polymerization or fragmentation. No difference was noticed in the hydrodynamic diameter of RTX. No additional peak or decrease in the areas under curve was found by cation exchange chromatography. The thermal aggregation curves and their derived thermodynamic parameters were unchanged. The primary, secondary and tertiary structures of the protein were not modified. Finally, the direct cytotoxic effect of rituximab was fully maintained.
Keywords: Rituximab; Physicochemical stability; Biological stability; Diluted solution; Monoclonal antibody;

Preparation and quality assessment of itraconazole transfersomes by Wen-sheng Zheng; Xia-qin Fang; Lu-lu Wang; Yu-jia Zhang (291-298).
Drug-loading transfersomes were prepared through screening and optimizing formulation with itraconazole at a mean entrapment efficiency of 80%.Drug-loading transfersomes were prepared with itraconazole, a lipophilic drug, as a model drug to investigate the key factor affecting transfersomes quality and to evaluate their qualities. Drug-loading transfersomes were prepared using film dispersion method. The quality of transfersomes was evaluated by HPLC, transmission electron microscope, particle size analyzer and in vitro release. Itraconazole transfersomes was transparent solution in ivory white color with a mean entrapment efficiency of about 80%. The shape of hollow vesicles was spheroidal with the diameter of approximately 100 nm, and the zeta potential of 45 mV, which had a good transdermal effect. It can be concluded via single-factor investigation that the quality of transfersomes is significantly affected by solvent, salt ion concentration and homogenization pressure and so on. The preparation method obtained through screening and optimizing formulation and technology is feasible and the quality can be controlled.
Keywords: Transfersomes; Itraconazole; Quality assessment; In vitro release;

Drug delivery strategies for the treatment of malignant gliomas by Daniela Allhenn; Maryam Alsadat Shetab Boushehri; Alf Lamprecht (299-310).
As primary brain tumors, malignant gliomas are known to be one of the most insidious types of brain cancer afflicting the humans. The current standard strategy for the treatment of malignant gliomas includes the surgical resection of the tumor when possible, followed by a combination of radiotherapy and/or a certain chemotherapeutic protocol. However, due to the short mean survival, frequent recurrences, and poor prognosis associated with the tumors, new therapeutic strategies are investigated consecutively. These novel drug delivery approaches can be subdivided as systemic and local drug administration. This review focuses on localized drug delivery strategies for the treatment of malignant gliomas, including the injections, infusions, trans-nasal delivery systems, convection enhanced delivery (CED) systems, and various types of polymeric implants. Furthermore, systemic strategies to increase the drug penetration into the brain, such as temporary disruption of the blood brain barrier (BBB), chemical modification of the available therapeutic substances, and utilization of endogenous transport systems will be briefly discussed.
Keywords: Gliomas; Localized drug delivery; Systemic drug delivery; Polymeric implants; Endogenous transport systems;

Biopharmaceutics classification and intestinal absorption study of apigenin by Jianjun Zhang; Dapeng Liu; Yanting Huang; Yuan Gao; Shuai Qian (311-317).
The aim of the study was to characterize the biopharmaceutics classification system (BCS) category of apigenin (AP) using intrinsic dissolution rate (IDR) and rat intestinal permeability, and to investigate the intestinal absorption mechanism of AP in rats. In the present investigation, equilibrium solubility and intrinsic dissolution rate (IDR) of AP were estimated in phosphate buffers. Effective intestinal permeability (P eff ) of AP was determined using single-pass intestinal perfusion (SPIP) technique in four segments (duodenum, jejunum, ileum and colon) of rat intestine at three concentrations (10, 50 and 100 μg/ml). The aqueous solubility of AP in tested phosphate buffers was very poor with maximum solubility of 2.16 μg/ml at pH 7.5. The IDR of AP was very low with a value of 0.006 mg/min/cm2. The minimum and maximum P eff s determined by SPIP were 0.198 × 10−4 and 0.713 × 10−4  cm/s at jejunum and duodenum site, respectively. In addition, the concentration-dependent permeability behavior was observed in the duodenum and jejunum, which suggested that AP was transported by both passive and active carrier-mediated saturable mechanism in these two intestinal segments. However, the observed concentration-independent permeability behavior in ileum and colon indicated primarily passive transport mechanism of absorption of AP in the last two intestinal segments. AP was classified as class II drug of the BCS due to its low solubility and high intestinal permeability. AP could be well absorbed in the whole intestine with the main absorption site at duodenum. The absorption of AP in four intestinal segments exhibited different transport mechanisms.
Keywords: Apigenin; Biopharmaceutics classification system; Intrinsic dissolution rate; Permeability; Single-pass intestinal perfusion;

Efficacy and toxicity of a tropically stable lipid-based formulation of amphotericin B (iCo-010) in a rat model of invasive candidiasis by Fady Ibrahim; Pavel Gershkovich; Olena Sivak; Ellen K. Wasan; Karen Bartlett; Kishor M. Wasan (318-323).
The objective of this work was to assess the antifungal activity of a tropically stable formulation of amphotericin B (AmB) (iCo-010) over short period of treatment in a rat model of invasive candidiasis. The rats were infected with Candida albicans (ATCC 18804); 48 h later, the animals were assigned either to a control group, AmBisome® group (5 mg/kg QD), or iCo-010 groups (0.5, 1, 2.5, 5 and 10 mg/kg TID). The animals were treated for two days and then sacrificed 18 h following the completion of the treatment. The blood, liver, lungs, kidneys and spleen were harvested to assess the colony forming units in the samples. There was no significant difference in the reduction of the fungal burden in the organs between the AmBisome® and iCo-010 groups except in the spleen and liver. There was a linear correlation between the antifungal activity in renal tissues and the administered doses of iCo-010. The plasma creatinine levels were not significantly different among the control and all the treatment groups. Oral iCo-010 has high efficacy against invasive candidiasis in renal and pulmonary tissues. Longer treatment period than the two-days regimen should be considered for higher therapeutic efficacy of iCo-010 in all the tissues.
Keywords: Amphotericin B; Lipid-based formulations; Invasive candidiasis; Efficacy; Rats; Candida albicans;

Molecular expression and functional activity of sodium dependent multivitamin transporter in human prostate cancer cells by Mitesh Patel; Ramya Krishna Vadlapatla; Sujay Shah; Ashim K. Mitra (324-331).
Nutrient transporters expressed on cell membrane have been targeted for enhancing bioavailability of poorly permeable drugs. Sodium dependent multivitamin transporter (SMVT) is once such carrier system, utilized for improving drug targeting to specific tissues. Therefore, the main objective of this study is to characterize SMVT in human derived prostate cancer cells (PC-3). Reverse transcription polymerase chain reaction (RT-PCR) analysis has provided product band at 774 bp, specific to SMVT. The mechanism and intracellular regulation of [3H]-biotin is also studied. [3H]-biotin uptake is found to be time and concentration dependent with K m and V max values of 19 ± 2 μM and 23 ± 1 pmol/min/mg protein, respectively. The uptake process is saturable in micromolar concentration range but linear in nanomolar concentration range. [3H]-biotin uptake shows significant sodium, temperature, pH and energy dependency. The process is strongly inhibited by unlabeled biotin and structural analogs such as desthiobiotin, pantothenate, lipoate and valeric acid. Intracellular regulatory pathways such as Ca2+/calmodulin and PKC pathway but not PTK pathway appears to play an important role in modulating [3H]-biotin uptake. This study for the first time confirms the molecular expression of SMVT and demonstrates that SMVT, responsible for biotin uptake is functionally active in PC-3 cells.
Keywords: Biotin; SMVT; Cellular uptake mechanism; Prostate cancer; Drug delivery;

Controlled release of lipase from Candida rugosa loaded into hydrogels of N-isopropylacrylamide and itaconic acid by Nikola Milašinović; Zorica Knežević-Jugović; Nedeljko Milosavljević; Jovanka Filipović; Melina Kalagasidis Krušić (332-340).
The series of poly(N-isopropylacrylamide-co-itaconic acid) hydrogels, with lipase from Candida rugosa as a model protein, were synthesized by free radical copolymerization. The composition of hydrogels was varied by monomers ratio, crosslinking agent concentration and amounts of lipase, which was loaded by in situ polymerization. All samples were characterized regarding morphology. The investigation of hydrogel swelling properties revealed their pH and temperature sensitive character. Protein loading efficiency, release profiles and the specific activity yield of the released lipase were also investigated as a function of hydrogel composition, protein content and pH, at the physiological temperature of 37 °C. Copolymers of N-isopropylacrylamide and itaconic acid presented high lipase loading efficiency. Another very important feature of these copolymers was that the protein release kinetic strongly depended on the pH value of the medium. The diffusion exponents values around 1 denoted that these hydrogel compositions could be adjusted to follow near zero-order kinetics. Namely, hydrogel formulations released low amounts of lipase at pH 2.20, but much higher released protein quantities were observed at pH 6.80 enabling these copolymers to be attractive candidates as site specific protein oral drug delivery systems.
Keywords: Hydrogels; N-isopropylacrylamide; Itaconic acid; Candida rugosa lipase; Controlled release;

Thiolated Eudragit nanoparticles for oral insulin delivery: Preparation, characterization and in vivo evaluation by Yan Zhang; Xiaorong Wu; Lingkuo Meng; Yu Zhang; Ruiting Ai; Na Qi; Haibing He; Hui Xu; Xing Tang (341-350).
In the present study thiolated Eudragit L100 (Eul) based polymeric nanoparticles (NPs) were employed to develop an oral insulin delivery system. Sulfydryl modification was achieved by grafting cysteine to the carboxylic acid group of Eudragit L100, which displayed maximum conjugate level of 390.3 ± 13.4 μmol thiol groups per gram. Eudragit L100-cysteine (Eul-cys) and Eul nanoparticles were prepared by the precipitation method, in which reversible swelling of pH-sensitive material was used for insulin loading and release. Nanoparticles were characterized in terms of their particle size, morphology, loading efficiency (LE%) and in vitro insulin release behavior. The NPs had an average size of 324.2 ± 39.0 nm and 308.8 ± 35.7 nm, maximal LE% of 92.2 ± 1.7% and 96.4 ± 0.5% for Eul-cys and Eul, respectively. The release profile of NPs in vitro showed pH-dependent behavior. Circular dichroism (CD) spectroscopy analysis proved that the secondary structure of the insulin released from NPs was unchanged compared with native insulin. The mucoadhesion study in vitro showed that Eul-cys NPs produced a 3-fold and 2.8-fold increase in rat jejunum and ileum compared with unmodified polymer NPs, respectively, which was due to the immobilization of thiol groups on Eudragit L100. Oral administration of insulin-loaded Eul-cys NPs produced a higher and prolonged hypoglycemic action, and the corresponding relative bioavailability of insulin was found to be 7.33 ± 0.33%, an increase of 2.8-fold compared with Eul NPs (2.65 ± 0.63%). This delivery system is a promising novel tool to improve the absorption of protein and peptide drugs in the intestinal tract.
Keywords: Thiomer; Eudragit L100; Insulin nanoparticles; Mucoadhesion; pH-responsive; Oral administration;

Three different kinds of silica (S2, S1 and SBA-15) with different particle sizes (130, 430 nm and 1–2 μm) and different pore characteristics (i.e. pore size and shape) were developed as oral vaccine immunological adjuvants and the relationship between the silica architecture and immunological properties was investigated. The silica particles were characterized using SEM, TEM and nitrogen adsorption. Model antigen bovine serum albumin (BSA) was successfully entrapped into the silica pores to produce a sustained release vaccine delivery system. Compared with the responsiveness induced by parenteral administration of BSA emulsified in Freund's complete adjuvant (FCA), oral immunization with the silica/BSA formulation produced a stimulated humoral and mucosal (sIgA) response. The IgG and IgA titers induced by loading BSA was as follows: S1 > S2 > SBA-15. The highest IgG and IgA titers of S1 were attributed to its large honeycombed pores and the optimal particle diameter of 430 nm. The corresponding IgG1 and IgG2a titers were also investigated to confirm that BSA loaded in nanoparticles by oral immunization can induce both T-helper 1- and T-helper 2- (Th1 or Th2) mediated responses. We believe that the results of our research will open up new avenues for the formulation of oral vaccines.
Keywords: Porous silica nanoparticles; Oral vaccine adjuvant; Silica architecture;

Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: Alternatives to synthetic surfactants in the pharmaceutical field? by Eléonore Bouyer; Ghozlene Mekhloufi; Véronique Rosilio; Jean-Louis Grossiord; Florence Agnely (359-378).
Emulsions are widely used in pharmaceutics for the encapsulation, solubilization, entrapment, and controlled delivery of active ingredients. In order to answer the increasing demand for clean label excipients, natural polymers can replace the potentially irritative synthetic surfactants used in emulsion formulation. Indeed, biopolymers are currently used in the food industry to stabilize emulsions, and they appear as promising candidates in the pharmaceutical field too. All proteins and some polysaccharides are able to adsorb at a globule surface, thus decreasing the interfacial tension and enhancing the interfacial elasticity. However, most polysaccharides stabilize emulsions simply by increasing the viscosity of the continuous phase. Proteins and polysaccharides may also be associated either through covalent bonding or electrostatic interactions. The combination of the properties of these biopolymers under appropriate conditions leads to increased emulsion stability. Alternative layers of oppositely charged biopolymers can also be formed around the globules to obtain multi-layered “membranes”. These layers can provide electrostatic and steric stabilization thus improving thermal stability and resistance to external treatment. The novel biopolymer-stabilized emulsions have a great potential in the pharmaceutical field for encapsulation, controlled digestion, and targeted release although several challenging issues such as storage and bacteriological concerns still need to be addressed.
Keywords: Emulsion; Stabilization; Biopolymer; Drug delivery systems; Layer-by-layer;

Galactosylated chitosan nanoparticles for hepatocyte-targeted delivery of oridonin by Dandan Zheng; Cunxian Duan; Dianrui Zhang; Lejiao Jia; Guangpu Liu; Yue Liu; Feihu Wang; Caiyun Li; Hejian Guo; Qiang Zhang (379-386).
In this study, oridonin-loaded nanoparticles coated with galactosylated chitosan (ORI-GC-NP) were prepared for tumor targeting and their characteristics were evaluated for the morphologies, particle size and zeta potential. Oridonin-loaded nanoparticles (ORI-NP) without galactosylated chitosan were prepared as a control. The entrapment efficiency of ORI-GC-NP and ORI-NP were 72.15% and 85.31%, respectively. The in vitro drug release behavior from nanoparticles displayed biphasic drug release pattern with initial burst release and consequently sustained release. Next, the pharmacokinetics and tissue distribution of ORI-GC-NP, ORI-NP and ORI solution were carried out. Pharmacokinetic analysis showed that ORI-GC-NP and ORI-NP could prolong the drug plasma levels compared with ORI solution. Meanwhile, the distribution of ORI-GC-NP to liver was higher than that of ORI-NP and free drug. In conclusion, ORI-GC-NP, as a promising intravenous drug delivery system for ORI, could be developed as an alternative to the conventional ORI preparations.
Keywords: Galactosylated chitosan; Nanoparticles; Oridonin; Tumor targeting;

Two-layered dissolving microneedles formulated with intermediate-acting insulin by Yukako Ito; Muneyuki Hirono; Keizo Fukushima; Nobuyuki Sugioka; Kanji Takada (387-393).
Two-layered dissolving microneedles (DMs) containing intermediate-acting insulin, protamine sulfate insulin (PSI), were prepared. Then a pharmacodynamic study was performed to evaluate the prolonged hypoglycemic effects in rats. The DMs were approximately 497 ± 5 μm long, with 303 ± 3 μm diameter at their base. The length of the insulin loaded space was 182 ± 4 μm. PSI contents in DMs were 0.51 ± 0.02 IU. A three-month stability study showed that 99.9 ± 1.4% of PSI was recovered at 4 °C. As the temperature increased to 40 °C, recovery decreased to 97.5 ± 2.0%. PSI was released within 5 min from DMs. Hypoglycemic effects of PSI DMs were evaluated in rats where subcutaneous injection preparations were used as references. Total area above the plasma glucose level (% of the pre-dose level) vs. time curve as an index of hypoglycemic effect was 144.0 ± 16.0% h and 243.3 ± 8.5% h for PSI DMs at 1.46 and 3.28 IU/kg. The relative pharmacologic availability of PSI from DMs were 100.2 ± 9.8% and 91.4 ± 4.1%. No significant difference of hypoglycemic curves was found between DMs and injection solutions, which suggests the usefulness of two-layered DMs of PSI for the displacement therapy of sc injection preparation.
Keywords: Insulin analog; Dissolving microneedles; Protamine sulfate insulin; Two-layered; Transdermal delivery; Relative pharmacological availability; Rats;

(a) PLGA particles formed by homogenization without CO2. (b) PLGA particles formed by homogenization in the presence of CO2 acting as plasticizer. (c) Evolution of the particle size during high pressure homogenization. Display OmittedThe study explores the enabling role of near-critical CO2 as a reversible plasticizer in the high pressure homogenization of polymer particles, aiming at their comminution as well as at the formation of drug–polymer composites. First, the effect of near-critical CO2 on the homogenization of aqueous suspensions of poly lactic-co-glycolic acid (PLGA) was investigated. Applying a pressure drop of 900 bar and up to 150 passes across the homogenizer, it was found that particles processed in the presence of CO2 were generally of microspherical morphology and at all times significantly smaller than those obtained in the absence of a plasticizer. The smallest particles, exhibiting a median x 50 of 1.3 μm, were obtained by adding a small quantity of ethyl acetate, which exerts on PLGA an additional plasticizing effect during the homogenization step. Further, the study concerns the possibility of forming drug–polymer composites through simultaneous high pressure homogenization of the two relevant solids, and particularly the effect of near-critical CO2 on this process. Therefore, PLGA was homogenized together with crystalline S-ketoprofen (S-KET), a non-steroidal anti-inflammatory drug, at a drug to polymer ratio of 1:10, a pressure drop of 900 bar and up to 150 passes across the homogenizer. When the process was carried out in the presence of CO2, an impregnation efficiency of 91% has been reached, corresponding to 8.3 wt.% of S-KET in PLGA; moreover, composite particles were of microspherical morphology and significantly smaller than those obtained in the absence of CO2. The formation of drug–polymer composites through simultaneous homogenization of the two materials is thus greatly enhanced by the presence of CO2, which increases the efficiency for both homogenization and impregnation.
Keywords: Homogenization; Polymer; PLGA; Microspheres; Carbon dioxide; Supercritical; Plasticization;

A new cocrystal and salts of itraconazole: Comparison of solid-state properties, stability and dissolution behavior by Anna Shevchenko; Luis M. Bimbo; Inna Miroshnyk; Jorma Haarala; Kristýna Jelínková; Kaisa Syrjänen; Bert van Veen; Juha Kiesvaara; Hélder A. Santos; Jouko Yliruusi (403-409).
Cocrystallization and salt formation have been shown to entail substantial promise in tailoring the physicochemical properties of drug compounds, in particular, their dissolution and hygroscopicity. In this work, we report on the preparation and comparative evaluation of a new cocrystal of itraconazole and malonic acid and two new hydrochloric salts (dihydrochloride and trihydrochloride) of itraconazole. The intrinsic dissolution rate, hygroscopicity, and thermodynamic stability were determined for the obtained solid-state forms and compared to itraconazole–succinic acid (2:1) cocrystal. The results show that the solid-state forms with higher intrinsic dissolution rate are less stable. Both itraconazole salts exhibited the highest dissolution rate, but also demonstrated high hygroscopicity at relative humidity above 70%. The new cocrystal, in contrast, were found to increase the dissolution rate of the parent drug by about 5-fold without compromising the hygroscopicity and the stability. This study demonstrates that, for dissolution rate enhancement of poorly water-soluble weak bases, cocrystallization is a more suitable approach than hydrochloric salt formation.
Keywords: Cocrystal; Salt; Dissolution; Preformulation; Hygroscopicity; Thermodynamic stability; Phase transition;

Aggregation behavior and in vitro biocompatibility study of octopus-shaped macromolecules based on tert-butylcalix[4]arenes by Denitsa Momekova; Desislava Budurova; Elena Drakalska; Stoycho Shenkov; Georgi Momekov; Barbara Trzebicka; Nikolay Lambov; Emil Tashev; Stanislav Rangelov (410-417).
A series of products based on tert-butylcalix[4]arene have been synthesized by anionic polymerization of ethylene oxide. The resulting products are amphiphilic octopus-shaped macromolecules, consisting of a hydrophobic calix[4]arene core and four arms of hydrophilic poly(ethylene oxide) chains. In aqueous solutions the polyoxyethylated tert-butylcalix[4]arenes were found to self-associate above certain CMC determined by dye solubilization technique. The light scattering study reveals that the polyoxyethylated tert-butylcalix[4]arenes form aggregates of narrow size distribution and hydrodynamic diameters ranging from about 155 to 245 nm and aggregation numbers from tens to hundreds macromolecules per particle depending on the degree of polymerization of the PEO chains. An in vitro biocompatibility study showed that the tested compounds are practically devoid of intrinsic cytotoxic and hemolytic effects and moreover they failed to modulate the mitogen-induced interleukin-2 release from the human T-lymphocyte cell line Jurkat E6-1. Taken together the excellent in vitro biocompatibility profile and the favorable physicochemical characteristics of the tested polyoxyethylated calix[4]arenes give us reason to consider them as promising for further evaluation as drug delivery platforms.
Keywords: Calix[4]arenes; PEO; Self-assembled structures; Biocompatibility; Cytotoxicity; Hemolysis; IL-2;

The present study investigates the ability of a polyelectrolyte complex, composed of O-carboxymethyl chitosan (O-CMC) and carbopol cross linked with calcium, as a pH-sensitive carrier for acid susceptible drugs. DSC studies were performed to confirm the formation of O-CMC–carbopol complex. Double endothermic peaks in thermogram of polyelectrolyte beads reflect the molecular changes brought in after cross-linking. FT-IR spectroscopy was used to reveal peak variation of the carboxylic groups as a function of pH 1.2 and pH 6.8. The formation of polyelectrolyte complex, on account of electrostatic interactions between the ―COO group of carbopol and the ―NH3 + group of O-CMC, was also confirmed by FT-IR studies. Swelling of the O-CMC–carbopol film showed a pH-dependent profile that was affected by calcium ion concentration. The swelling rate was more significant at intestinal pH because the ionization of carboxylic acid group on O-CMC and carbopol creates electrostatic repulsion. Release behavior of drug is relative to the viscosity of solution and the ionic interaction between O-CMC and carbopol. Mucous glycoprotein assay revealed that ionization of carboxylic group on the beads at intestinal pH formed a strong hydrogen bond with mucin, which was responsible for the prominent mucoadhesive property thus prolonging the intestinal residence time.
Keywords: O-Carboxymethyl chitosan; Carbopol; Rabeprazole sodium; Mucoadhesive; Ionic gelation method; Polyelectrolyte complex;

Similar molecular descriptors determine the in vitro drug permeability in endothelial and epithelial cells by Jenni J. Hakkarainen; Jari Pajander; Riikka Laitinen; Marjukka Suhonen; Markus M. Forsberg (426-443).
Endothelial and epithelial cells are commonly used for assessing blood–brain barrier (BBB) permeability of the drug candidates. However, the additional value provided by the endothelial cells as an in vitro BBB drug permeability model is not clear. The aim of the study was to identify the molecular descriptors that impact on drug permeability through the primary bovine brain microvessel endothelial cell model (BBMEC) and compare descriptors with those determined for epithelial cell models. In addition, we intended to clarify the reasons for previously reported similar in vitro–in vivo correlations between endothelial and epithelial cell models and to evaluate whether BBMEC can provide additional value. The permeability of model drugs through the BBMEC was determined. Principal component analysis (PCA) model was created with twenty-two model drugs and this model was used to interpret the molecular descriptors. The present study demonstrates that hydrophobic interactions, the balance between the hydrophilic and lipophilic moieties in the drug, hydrophilic interactions and hydrogen bonding interactions are the key descriptors depicting drug permeability through the BBMEC. There were no clear differences between the molecular descriptors determining the in vitro permeability of drugs in the endothelial and epithelial cells. In conclusion, the predominance of passive permeability in in vitro setups may explain the similar in vitroin vivo correlations previously obtained between endothelial and epithelial cell models. Therefore, the present results further support previous findings that epithelial cell models can be used instead of laborious primary endothelial cells as an in vitro BBB permeability model when passive transport is mainly being evaluated.
Keywords: Blood–brain barrier; BBMEC; In vitro permeability; Principal component analysis;

Transferrin-conjugated boron nitride nanotubes: Protein grafting, characterization, and interaction with human endothelial cells by Gianni Ciofani; Serena Del Turco; Giada Graziana Genchi; Delfo D’Alessandro; Giuseppina Basta; Virgilio Mattoli (444-453).
Transferrin-grafted boron nitride nanotubes (green fluorescent) are actively internalized by human umbilical vein endothelial cells (in red, f-actin staining; in blue, nucleus staining).In this paper we report on a covalent grafting of boron nitride nanotubes with human transferrin. After silanization of the nanotube wall, transferrin was linked to the nanotubes through carbamide binding. The obtained transferrin-conjugated boron nitride nanotubes (tf-BNNTs) resulted stable in aqueous environments and were characterized in terms of scanning electron microscopy, transmission electron microscopy, size distribution analysis and Z-potential measurement. Effective covalent grafting of transferrin was demonstrated by Fourier transform infrared spectroscopy and UV–Vis spectrophotometry. The obtained tf-BNNTs were thereafter tested on human umbilical vein endothelial cells (HUVECs); in particular cellular up-take was investigated by confocal, scanning and transmission electron microscopy, demonstrating the key role of transferrin during the internalization process. Here reported for the first time in the literature, the covalent BNNT functionalization with a targeting ligand represents a fundamental step towards BNNT exploitation as smart and selective nanocarriers in a number of nanomedicine applications.
Keywords: Boron nitride nanotubes; Transferrin; HUVECs; Selective up-take; Cell targeting;

Thermoresponsive hydrogels with low toxicity from mixtures of ethyl(hydroxyethyl) cellulose and arginine-based surfactants by Maria Teresa Calejo; Anna-Lena Kjøniksen; Aurora Pinazo; Lourdes Pérez; Ana Maria S. Cardoso; Maria C. Pedroso de Lima; Amália S. Jurado; Sverre Arne Sande; Bo Nyström (454-462).
Ethyl(hydroxyethyl) cellulose (EHEC) is known to form hydrogels in water at elevated temperatures in the presence of an ionic surfactant. In this paper, the potential use of arginine-based surfactants is explored considering the production of a low toxicity thermoresponsive hydrogel for pharmaceutical and biomedical applications. The interactions between EHEC and the monomeric surfactant Nα-lauroyl-l-arginine methyl ester (LAM) and two gemini surfactants N α,N ω -bis(N α-acylarginine) α,ω-dialkyl amides were evaluated by Rheo-Small Angle Light Scattering measurements. The complex viscosity of the systems was dependent on surfactant concentration and temperature. Under specific conditions, soft gels of homogeneous structure were produced. The cloud point (CP) of the EHEC–LAM system varied significantly with surfactant concentration, while only moderate CP changes were found in the presence of the gemini surfactants. Finally, the effect of the surfactants on the viability of a human cell line was evaluated. Despite the lower toxicity of LAM, the superior gel forming efficiency of the gemini surfactants at lower concentrations revealed their advantageous suitability as components of a biocompatible thermoresponsive gel system.
Keywords: EHEC; Thermoresponsive gel system; Rheo-SALS; Amino acid-based surfactants; Biocompatibility;

Development of a new LDL-based transport system for hydrophobic/amphiphilic drug delivery to cancer cells by Veronika Huntosova; Diana Buzova; Dana Petrovajova; Peter Kasak; Zuzana Nadova; Daniel Jancura; Franck Sureau; Pavol Miskovsky (463-471).
Low-density lipoproteins (LDL), a natural in vivo carrier of cholesterol in the vascular system, play a key role in the delivery of hydrophobic/amphiphilic photosensitizers to tumor cells in photodynamic therapy of cancer. To make this delivery system even more efficient, we have constructed a nano-delivery system by coating of LDL surface by dextran. Fluorescence spectroscopy, confocal fluorescence imaging, stopped-flow experiments and flow-cytometry were used to characterize redistribution of hypericin (Hyp), a natural occurring potent photosensitizer, loaded in LDL/dextran complex to free LDL molecules as well as to monitor cellular uptake of Hyp by U87-MG cells. It is shown that the redistribution process of Hyp between LDL molecules is significantly suppressed by dextran coating of LDL surface. The modification of LDL molecules by dextran does not inhibit their recognition by cellular LDL receptors and U-87 MG cellular uptake of Hyp loaded in LDL/dextran complex appears to be similar to that one observed for Hyp transported by unmodified LDL particles. Thus, it is proposed that dextran modified LDL molecules could be used as a basis for construction of a drug transport system for targeted delivery of hydrophobic/amphiphilic drugs to cancer cells expressing high level of LDL receptors.
Keywords: Drug delivery; Low-density lipoproteins; Dextran; Hypericin; Fluorescence; Photodynamic therapy;

Single-pass intestinal perfusion to establish the intestinal permeability of model drugs in mouse by Elvira Escribano; Xavier García Sala; Jorge Salamanca; Claudia Roig Navarro; Josep Queralt Regué (472-477).
The aim of the present work was to study the intestinal permeabilities (P eff) of five model drugs: furosemide, piroxicam, naproxen, ranitidine and amoxicillin in the in situ intestinal perfusion technique in mice and compare them with corresponding rat and human in vivo P eff values.The main experimental conditions were: mice CD1 30–35 g, test drug concentrations in perfusion experiments (the highest dose strength dissolved in 250 mL of PBS pH 6.2) and flow rate of 0.2 mL/min. The test compounds were assayed following a validated HPLC method. The effective permeability coefficients at steady-state were calculated after correcting the outlet concentration following the gravimetric correction method proposed by . The permeability coefficient values ranged from 0.1751 ± 0.0756 × 10−4  cm/s for ranitidine to 17.19 ± 4.16 × 10−4  cm/s for naproxen.The mouse method correctly assigned the BCS permeability classification of a given drug and a correlation between mouse permeability data and the fraction of an oral dose absorbed in humans was achieved (FA = 1 − exp(−34,745·P eff(mouse)); R  = 0.9631).Based on the results obtained, we conclude that mouse can be considered a valuable tool in the evaluation of intestinal permeability in order to predict the extent of human gastrointestinal absorption following oral administration of a drug.
Keywords: Mouse intestine; Perfusion; Permeability; Model drugs; Biopharmaceutics classification system;

In vivo evaluation of the highly soluble oral β-cyclodextrin–Sertraline supramolecular complexes by Joel J. Passos; Frederico B. De Sousa; Iram M. Mundim; Ricardo R. Bonfim; Robson Melo; Alice F. Viana; Eveline D. Stolz; Milene Borsoi; Stela M.K. Rates; Rubén D. Sinisterra (478-485).
The aim of the present work was to evaluate the antidepressant like-effect and plasma concentration of Sertraline (SRT) using an inclusion complex (IC) with β-cyclodextrin (βCD) in mice. This supramolecular system was prepared using two different molar ratios at 1:1 and 1:2 SRT:βCD and both were characterized to assess the drug inclusion into the host cavity. Based on the X-ray powder diffraction, Fourier transform infrared spectroscopy and thermal analysis the interaction between host and guest molecules could be suggested. This result indicates that the freeze drying process was efficient to prepare the ICs, when these are compared with the physical mixtures. By comparing the solid state results of 1:1 and 1:2 ICs no significant chemical or structural changes were identified between these systems. However, in vivo experiments indicated that the host–guest ratio was able to modify the SRT activity. Mice treated with both ICs (20 mg kg−1, p.o.) have shown lower immobility time in the tail suspension test in comparison with mice treated with free SRT (20 mg kg−1, p.o.). Mice spontaneous locomotor activity was not affected by any treatment. Higher SRT plasma concentration was determined after 30 min of treatment with 1:1 IC in comparison with free SRT, demonstrating the IC greater drug transport efficacy.
Keywords: Sertraline; β-Cyclodextrin; Inclusion complex; Freeze-dryer; In vivo experiments; Behavior test;

Roller compaction process development and scale up using Johanson model calibrated with instrumented roll data by Vishwas V. Nesarikar; Chandrakant Patel; William Early; Nipa Vatsaraj; Omar Sprockel; Robert Jerzweski (486-507).
Roller compaction is a dry granulation process used to convert powder blends into free flowing agglomerates. During scale up or transfer of roller compaction process, it is critical to maintain comparable ribbon densities at each scale in order to achieve similar tensile strengths and subsequently similar particle size distribution of milled material. Similar ribbon densities can be reached by maintaining analogous normal stress applied by the rolls on ribbon for a given gap between rolls. developed a model to predict normal stress based on material properties and roll diameter. However, the practical application of Johanson model to estimate normal stress on the ribbon is limited due to its requirement of accurate estimate of nip pressure i.e. pressure at the nip angle. Another weakness of Johanson model is the assumption of a fixed angle of wall friction that leads to use of a fixed nip angle in the model. To overcome the above mentioned limitations, we developed a novel approach using roll force equations based on a modified Johanson model in which the requirement of pressure value at nip angle was eliminated. An instrumented roll on WP120 roller compactor was used to collect normal stress data measured at three locations across the width of a roll (P1, P2, P3), as well as gap and nip angle data on ribbon for placebo and various active blends along with corresponding process parameters. The nip angles were estimated directly using experimental pressure profile data of each run.The roll force equation of Johanson model was validated using normal stress, gap, and nip angle data of the placebo runs. The calculated roll force values compared well with those determined from the roll force equation provided for the Alexanderwerk® WP120 roller compactor. Subsequently, the calculation was reversed to estimate normal stress and corresponding ribbon densities as a function of gap and RFU (roll force per unit roll width). A placebo model was developed and calibrated using a subset of placebo run data obtained on WP120. The roll force values were calculated using vendor supplied equation. The nip angle was expressed as a function of gap and RFU. The nip angle, gap and RFU were used in a new roll force equation to estimate normal stress P2 at the center of the ribbon. Using ratios P1/P2 and P3/P2 from the calibration data set, P1 and P2 were estimated. The ribbon width over which P1, P2, and P3 are effective was determined by minimizing sum square error between the model predicted vs. experimental ribbon densities of the calibration set. The model predicted ribbon densities of the placebo runs compared well with the experimental data. The placebo model also predicted with reasonable accuracy the ribbon densities of active A, B, and C blends prepared at various combinations of process parameters.The placebo model was then used to calculate scale up parameters from WP120 to WP200 roller compactor. While WP120 has a single screw speed, WP200 is equipped with a twin feed screw system. A limited number of roller compaction runs on WP200 was used as a calibration set to determine normal stress profile across ribbon width. The nip angle equation derived from instrumented roll data collected on WP120 was applied to estimate nip angles on WP200 at various processing conditions. The roll force values calculated from vendor supplied equation and the nip angle values were used in roll force equation to estimate normal stress P2 at the tip of the feed screws. Based on feed screw design, it was assumed that the normal stress at the center of the ribbon was equal to those calculated at the tip of the feed screws. The ratio of normal stress at the edge of the ribbon Pe to the normal stress P2 at the feed screw tip was optimized to minimize sum square error between model predicted vs. experimental ribbon densities of the calibration set. The model predicted ribbon densities of the batches prepared on WP200 compared well with the experimental data thus indicating success of the scale up procedure.For the demonstration purpose, the model was also calibrated using instrumented roll data of active C batches. This would be applicable when sufficient amount of API is available or placebo model cannot predict ribbon density of active batches.
Keywords: Roller compaction; Johanson model; Instrumented roll; Process design space; Ribbon normal stress; Ribbon density;

(A) Hyperglycaemia (glucose) disrupts blood–brain barrier (BBB) integrity leading to leakage of molecules (fluorescein) from blood into brain parenchyma. (B) Dendrimers (PAMAM) scavenge glucose excess and restore BBB integrity.We hypothesized that BBB is impaired in rat model of streptozotocin-induced diabetes and can be sealed by poly(amido)amine dendrimers G4.0 (PAMAM G4), which reveal anti-glycation activity.The BBB permeabilization was monitored in rats with the 60-day streptozotocin-diabetes and non-diabetic animals, using three fluorescent dyes (given intraperitoneally) differing in molecular weight: fluorescein, fluorescein isothiocyanate (FITC)–dextran and Evans blue. All animals were administered for 2 months with either PAMAM G4 dendrimer or placebo. The fluorescence intensities of the injected fluorescent markers were recorded in the homogenates of selected brain regions.The highest accumulations of the used fluorescent dyes were observed for fluorescein, predominantly in thalamus, hippocampus, frontal cortex, striatum and cerebellum. FITC-dextran leaked to much smaller extent, however, higher permeabilization for FITC–dextran was revealed in pons–medulla oblongata, frontal and parietal cortex of diabetic compared to control animals. Evans blue leaked very slowly into striatum and pons–medulla oblongata in diabetic rats.The treatment of diabetic animals with PAMAM G4 significantly reduced blood glucose concentration and hallmarks of late diabetic complications, compared to non-treated diabetic animals. PAMAM G4 significantly reduced diabetes-induced permeabilization of BBB, which remained in line with the reduced blood glucose and the amelioration of the biochemical hallmarks of severe hyperglycaemia.
Keywords: Blood–brain barrier; Experimental diabetes; Non-enzymatic glycosylation of proteins; Poly(amido)amine (PAMAM) dendrimers;

Nebulization of ultradeformable liposomes: The influence of aerosolization mechanism and formulation excipients by Abdelbary M.A. Elhissi; Joanna Giebultowicz; Anna A. Stec; Piotr Wroczynski; Waqar Ahmed; Mohamed Albed Alhnan; David Phoenix; Kevin M.G. Taylor (519-526).
Ultradeformable liposomes are stress-responsive phospholipid vesicles that have been investigated extensively in transdermal delivery. In this study, the suitability of ultradeformable liposomes for pulmonary delivery was investigated. Aerosols of ultradeformable liposomes were generated using air-jet, ultrasonic or vibrating-mesh nebulizers and their stability during aerosol generation was evaluated using salbutamol sulphate as a model hydrophilic drug. Although delivery of ultradeformable liposome aerosols in high fine particle fraction was achievable, the vesicles were very unstable to nebulization so that up to 98% drug losses were demonstrated. Conventional liposomes were relatively less unstable to nebulization. Moreover, ultradeformable liposomes tended to aggregate during nebulization whilst conventional vesicles demonstrated a “size fractionation” behaviour, with smaller liposomes delivered to the lower stage of the impinger and larger vesicles to the upper stage. A release study conducted for 2 h showed that ultradeformable liposomes retained only 30% of the originally entrapped drug, which was increased to 53% by inclusion of cholesterol within the formulations. By contrast, conventional liposomes retained 60–70% of the originally entrapped drug. The differences between ultradeformable liposomes and liposomes were attributed to the presence of ethanol or Tween 80 within the elastic vesicle formulations. Overall, this study demonstrated, contrary to our expectation, that materials included with the aim of making the liposomes more elastic and ultradeformable to enhance delivery from nebulizers were in fact responsible for vesicle instability during nebulization and high leakage rates of the drug.
Keywords: Aerosol; Pulmonary; Nebulizer; Stability; Transferosome; Ultradeformable;

Poly(acrylic acid) microspheres loaded with lidocaine: Preparation and characterization for arterial embolization by Dai-Chao Cui; Wan-Liang Lu; Er-A Sa; Meng-Jie Gu; Xiao-Jing Lu; Tian-Yuan Fan (527-535).
A new embolic agent, poly(acrylic acid) microspheres, was synthesized for embolization. Lidocaine was loaded on the microspheres to relief pain caused by embolization.A new embolic agent, poly(acrylic acid) microspheres (PMs), was synthesized and the cytocompatibility was proved by mouse L929 fibroblast cells. An analgesic drug, lidocaine, was loaded on the PMs to relief pain caused by embolization. PMs and lidocaine loaded microspheres (LMs) were characterized by investigating infrared spectrum, morphology, particle size, and equilibrium water contents (EWC). A series of tests were employed to evaluate the elasticity of PMs, LMs and Embosphere™, including once compression, twice compression, and stress relaxation test. The pressures of PMs and LMs passing through a catheter were measured on line by our new designed device. Drug release was studied with T-cell apparatus. The properties of PMs and LMs were proved to be suitable for embolization. Both PMs and LMs in this study might be potential embolic agents in the future.
Keywords: Poly(acrylic acid); Microspheres; Embolization; Lidocaine; Sustained release;

The purpose of this study was to develop a propofol microemulsion with a low concentration of free propofol in the aqueous phase. Propofol microemulsions were prepared based on single-factor experiments and orthogonal design. The optimal microemulsion was evaluated for pH, osmolarity, particle size, zeta potential, morphology, free propofol in the aqueous phase, stability, and pharmacokinetics in beagle dogs, and comparisons made with the commercial emulsion, Diprivan®. The pH and osmolarity of the microemulsion were similar to those of Diprivan®. The average particle size was 22.6 ± 0.2 nm, and TEM imaging indicated that the microemulsion particles were spherical in appearance. The concentration of free propofol in the microemulsion was 21.3% lower than that of Diprivan®. Storage stability tests suggested that the microemulsion was stable long-term under room temperature conditions. The pharmacokinetic profile for the microemulsion showed rapid distribution and elimination compared to Diprivan®. We conclude that the prepared microemulsion may be clinically useful as a potential carrier for propofol delivery.
Keywords: Propofol; Microemulsion; Emulsion; Free propofol;

Preservation of biological activity of glial cell line-derived neurotrophic factor (GDNF) after microencapsulation and sterilization by gamma irradiation by P. Checa-Casalengua; C. Jiang; I. Bravo-Osuna; B.A. Tucker; I.T. Molina-Martínez; M.J. Young; R. Herrero-Vanrell (545-554).
A main issue in controlled delivery of biotechnological products from injectable biodegradable microspheres is to preserve their integrity and functional activity after the microencapsulation process and final sterilization. The present experimental work tested different technological approaches to maintain the biological activity of an encapsulated biotechnological product within PLGA [poly (lactic-co-glycolic acid)] microspheres (MS) after their sterilization by gamma irradiation. GDNF (glial cell line-derived neurotrophic factor), useful in the treatment of several neurodegenerative diseases, was chosen as a labile model protein. In the particular case of optic nerve degeneration, GDNF has been demonstrated to improve the damaged retinal ganglion cells (RGC) survival.GDNF was encapsulated in its molecular state by the water-in-oil-in-water (W/O/W) technique or as solid according to the solid-in-oil-in-water (S/O/W) method. Based on the S/O/W technique, GDNF was included in the PLGA microspheres alone (S/O/W 1) or in combination with an antioxidant (vitamin E, Vit E) (S/O/W 2). Microspheres were sterilized by gamma-irradiation (dose of 25 kGy) at room and low (−78 °C) temperatures. Functional activity of GDNF released from the different microspheres was evaluated both before and after sterilization in their potential target cells (retinal cells).Although none of the systems proposed achieved with the goal of totally retain the structural stability of the GDNF-dimer, the protein released from the S/O/W 2 microspheres was clearly the most biologically active, showing significantly less retinal cell death than that released from either W/O/W or S/O/W 1 particles, even in low amounts of the neurotrophic factor.According to the results presented in this work, the biological activity of biotechnological products after microencapsulation and sterilization can be further preserved by the inclusion of the active molecule in its solid state in combination with antioxidants and using low temperature (−78 °C) during gamma irradiation exposure.
Keywords: Poly (lactic-co-glycolic acid); Microspheres; Polymeric drug delivery systems; Protein delivery; Gamma irradiation; Neuroprotection; GDNF (glial cell line-derived neurotrophic factor); Biological activity; Sterilization;

Novel gene transfer vectors based on artificial recombinant multi-functional oligopeptides by Kai Han; Juan Yang; Si Chen; Jing-Xiao Chen; Chen-Wei Liu; Cao Li; Han Cheng; Ren-Xi Zhuo; Xian-Zheng Zhang (555-563).
Viral vectors, except for their safety concern, have shown high efficiency in both delivery and expression of gene. Here, a series of new gene carriers, comprised of short peptide subunits with special functions to imitate viral vectors, were designed and three vectors, (C18)2KH4R8GDS, AcKH4R8GDS and (C18)2KH4R8, designated as ARM1, ARM2, ARM3, respectively, were synthesized and evaluated. The transfection efficiency in vitro was studied in terms of 293T, HepG2 and HeLa cell lines. It was found that the transfection efficiency was enhanced significantly for the vectors (ARM1 and ARM3) with double hydrophobic aliphatic tails. Interestingly, the conjugation of RGDS sequence in vectors displayed no obvious difference in cell adhesion for all of the three cell lines. Moreover, confocal laser scanning microscope results indicated that the peptide/pDNA complexes can enter the cell and nuclei successfully. On the other hand, all the vectors displayed low cytotoxicity. The artificial recombinant multi-block oligopeptides (ARMs) demonstrated here might give a promising potential of the peptide-based vectors in gene therapy.
Keywords: Peptide; Gene delivery; Transfection efficiency; Cytotoxicity;

Liposomal diclofenac eye drop formulations targeting the retina: Formulation stability improvement using surface modification of liposomes by Takuya Fujisawa; Hiroko Miyai; Kohei Hironaka; Toshimasa Tsukamoto; Kohei Tahara; Yuichi Tozuka; Masaki Ito; Hirofumi Takeuchi (564-567).
The modifying liposome surface with polyvinyl alcohol or its derivative prevented the liposome aggregation and/or fusion during remote loading process, leading to effective retinal delivery.An efficient liposomal formulation for targeting the retina was produced as an optimal means of distributing therapeutic agents to the retina. Diclofenac was used as a model compound for liposome encapsulation, and the release rate and distribution to the retina were investigated. The calcium acetate gradient method was found to be the optimal method for encapsulating diclofenac into liposomes. Entrapment efficiency using this method was greater than 97%, whereas conventional hydration method achieved 51.3%. The resultant formulation obtained with the gradient method caused aggregation and/or fusion of liposomes. To avoid inhibition of retinal delivery due to the aggregation of the carrier, surface modification was performed simultaneously with the gradient method. The increase in particle size of the liposomal formulation clearly was inhibited for a long time in the presence of polyvinyl alcohol or its derivative. This observation may be explained by surface modification of the liposomes by physisorption or anchoring effect of polymers on the surface of the lipid bilayer. Furthermore, the sustained release profile of the diclofenac formulation was retained after modification. An in vivo animal study revealed that concentration of the accumulated diclofenac in the retina–choroid was enhanced 1.8-fold by surface-modified liposome entrapment compared to that of the unaltered diclofenac solution.
Keywords: Diclofenac; Liposome; Calcium acetate gradient method; PVA; Eye drop; Retina;

New polymer–halloysite hybrid materials—potential controlled drug release system by Adi Ghebaur; Sorina A. Garea; Horia Iovu (568-573).
New hybrid materials based on a natural host (halloysite), biodegradable polymer (poly(vinyl alcohol)) and diphenhydramine hydrochloride as drug were synthesized and tested as a potential controlled drug delivery system.The formation of these hybrid materials was proved using different characterization methods like Spectroscopic techniques (FTIR, XPS and UV–vis), thermogravimetrical analysis and scanning electron microscopy (SEM). The hybrid materials exhibit different features regarding the drug release. In vitro drug release tests showed that there are several factors which exhibit a strong influence on the drug release rate. Thus the initial drug concentration used at the hybrid materials synthesis, the presence of a polymer in the hybrid composition and the pH value of the release medium are the most important factors.
Keywords: Halloysite; Drug; Polymer; Delivery system;

Enhanced preclinical efficacy of tamoxifen developed as alginate–cysteine/disulfide bond reduced albumin nanoparticles by A. Martínez; E. Muñiz; I. Iglesias; J.M. Teijón; M.D. Blanco (574-581).
Tamoxifen (TMX) is the most common clinical choice for the treatment of advanced or metastatic estrogen-dependent breast cancer. However, research on new challenging therapies is necessary due to its undesirable side effects and the limitation of the treatment only to the oral route. In this study, the antitumor activity of TMX-loaded nanoparticles based on different mixtures of alginate–cysteine and disulfide bond reduced bovine serum albumin was tested in vivo in MCF-7 nude mice xenograft model. These systems showed an enhancement of the TMX antitumor activity, since lower tumor evolutions and lower tumor growth rates were observed in mice treated with them. Moreover, histological and immunohistochemical studies revealed that treatments with TMX-loaded nanoparticles showed the most regressive and less proliferative tumor tissues. TMX biodistribution studies determined that TMX-loaded nanoparticles caused more accumulation of the drug into the tumor site with undetectable levels of TMX in plasma, reducing the possibility of delivering TMX to other not-targeted organs and, consequently, developing possible side effects. Thus, these TMX nanoparticulate systems are expected to provide a novel approach to the treatment of breast cancer in the future.
Keywords: Tamoxifen; Breast cancer; Alginate–cysteine; Bovine serum albumin; Nanoparticles; Xenograft tumor;

Cholorpheniramine tannate complexes: Physicochemical, chemometric, and taste masking evaluation by Ziyaur Rahman; Ahmed S. Zidan; Saeed R. Khan; Indra K. Reddy; Mansoor A. Khan (582-592).
The focus of present investigation was to evaluate the tannic acid (TA) complexes of cholorpheniramine maleate (CPM) and characterize it by a variety of physicochemical, dissolution, and electronic tongue methods. The complexes were prepared in various molar ratios by solvent evaporation method. They were characterized by spectroscopic, thermal, powder X-ray, electronic tongue, solubility and dissolution methods. FTIR (infrared red) spectra showed complex formation between the TA and CPM. Complex formation has significantly lowered the drug solubility and sustained its release for more than 24 h in phosphate buffer pH 6.8. On the contrary, the release was much faster in the presence of Avicel PH 113 in the same molar ratio complex. The complex formulation has suppressed the bitter taste of CPM as indicated by Euclidean distance in electronic tongue evaluation. NIR-CI (near infrared chemical imaging) showed lower skew value that indicated the homogenous distribution of formulation components. The chemometric models were also developed using the NIR data. The model based on second derivative data was better in predicting the TA and CPM loading as indicated by higher values of R, R 2 and lower values of root mean square error and standard errors. Furthermore, it has a better accuracy and less biased in comparison to other models. In conclusion, the CPM tannate has a sustained release behavior and excipients play a major role in modifying its release. Additionally, the complexes with varying molar ratio of tannate to CPM have differential taste masking abilities than that of the pure drug.
Keywords: Cholorpheniramine maleate; Tannic acid; Complex; Solubility; Dissolution; Bitter taste; Chemometric model;

Physicochemical characterization of GBV-C E1 peptides as potential inhibitors of HIV-1 fusion peptide: Interaction with model membranes by Maria Jesús Sánchez-Martín; Antonio Cruz; M. Antònia Busquets; Isabel Haro; M. Asunción Alsina; Montserrat Pujol (593-601).
Four peptide sequences corresponding to the E1 protein of GBV-C: NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10) and QAGLAVRPGKSAAQLVGE (P18) were studied as they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP). In this work, the surface properties of the E1 peptide sequences are investigated and their physicochemical characterization is done by studying their interaction with model membranes; moreover, their mixtures with HIV-1 FP were also studied in order to observe whether they are capable to modify the HIV-1 FP interaction with model membranes as liposomes or monolayers. Physicochemical properties of peptides (pI and net charge) were predicted showing similarities between P7 and P8, and P10 and HIV-1 FP, whereas P18 appears to be very different from the rest. Circular dichroism experiments were carried out showing an increase of the percentage of α-helix of P7 and P8 when mixed with HIV-1 FP corroborating a conformational change that could be the cause of their inhibition ability. Penetration experiments show that all the peptides can spontaneously insert into phospholipid membranes. Analysis of compression isotherms indicates that the peptides interact with phospholipids and the E1 peptides modify the compression isotherms of HIV-1 FP, but there is one of the peptides that excelled as the best candidate for inhibiting the activity of HIV-1 FP, P7, and therefore, that could be potentially used in future anti-HIV-1 research.
Keywords: Hepatitis GB virus C; Synthetic peptide; Lipid monolayer; Compression isotherms; HIV-1 FP; Circular dichroism;

Chitosan-based mucoadhesive tablets for oral delivery of ibuprofen by Ioannis A. Sogias; Adrian C. Williams; Vitaliy V. Khutoryanskiy (602-610).
Chitosan and its half-acetylated derivative have been compared as excipients in mucoadhesive tablets containing ibuprofen. Initially the powder formulations containing the polymers and the drug were prepared by either co-spray drying or physical co-grinding. Polymer–drug interactions and the degree of drug crystallinity in these formulations were assessed by infrared spectroscopy and differential scanning calorimetry. Tablets were prepared and their swelling and dissolution properties were studied in media of various pHs. Mucoadhesive properties of ibuprofen-loaded and drug-free tablets were evaluated by analysing their detachment from pig gastric mucosa over a range of pHs. Greater polymer–drug interactions were seen for spray-dried particles compared to co-ground samples and drug loading into chitosan-based microparticles (41%) was greater than the corresponding half-acetylated samples (32%). Swelling and drug release was greater with the half-acetylated chitosan tablets than tablets containing the parent polymer and both tablets were mucoadhesive, the extent of which was dependent on substrate pH. The results illustrate the potential sustained drug delivery benefits of both chitosan and its half-acetylated derivative as mucoadhesive tablet excipients.
Keywords: Chitosan; Mucoadhesion; Tablets; Spray-drying; Ibuprofen; Crystallinity;

Amphotericin B (AmB) lipid nanoemulsions were prepared and characterized and their suitability for pulmonary delivery via nebulization was evaluated. AmB nanoemulsions were prepared by sonicating and vortexing the drug with two commercially available lipid nanoemulsions: the Intralipid® or Clinoleic®. Loading the nanoemulsions with the drug slightly increased the size of the lipid droplets and did not affect the zeta potential of the nanoemulsions. The loading efficiency of AmB was found to be 87.46 ± 2.21% in the Intralipid® nanoemulsions and 80.7 ± 0.70% in the Clinoleic® formulation. This respectively corresponded to 21.86 mg and 20.19 mg of AmB being successfully loaded in the nanoemulsions. On aerosolization using a Pari Sprint jet nebulizer, both nanoemulsions produced very high drug output which was approximately 90% for both formulations. Using the two-stage impinger, the Clinoleic® emulsion had higher fine particle fraction (FPF) than the Intralipid®, since the Clinoleic® displayed higher deposition of AmB in the lower impinger stage (exceeding 80%), compared to 57% for the Intralipid®. Overall, the ease of preparation of the AmB lipid nanoemulsions, along with their in vitro nebulization performance suggest that lipid nanoemulsions could be successful nanocarriers for delivery of AmB to the peripheral respiratory airways.
Keywords: Aerosol; Amphotericin B; Nanoemulsion; Nebulizer; Pulmonary;

Novel lipid based oral formulation of curcumin: Development and optimization by design of experiments approach by Yogesh B. Pawar; Hitesh Purohit; Guru Raghavendra Valicherla; Bhushan Munjal; Shantanu V. Lale; Sarsvatkumar B. Patel; Arvind Kumar Bansal (617-623).
The clinical utility of curcumin (CRM) is limited due to its poor oral bioavailability. Lipid based oral formulations (LBOFs) are emerging as useful oral drug delivery systems for ‘difficult to deliver’ molecules like CRM. In present study, we report novel Type IV LBOF for CRM using Gelucire 44/14, Labrasol, Vit. E TPGS and PEG 400 with superior CRM loading and enhanced oral bioavailability. The optimization of LBOF for CRM loading and post dilution droplet size was carried out by design of experiments (DoE) approach with Box–Behnken design. Oral bioavailability of optimized LBOF (O-LBOF) was evaluated in male Sprague-Dawley (SD) rats at a dose of 250 mg/kg. Raw CRM (control) showed C max and AUC0–∞ of 32.29 ng/ml and 38.07 ng h/ml, respectively. O-LBOF improved C max and AUC0–∞ by 11.6 and 35.8 folds respectively over control.
Keywords: Curcumin; Lipid based oral formulations; Design of experiments; Solubility; Bioavailability;

Screening paediatric rectal forms of azithromycin as an alternative to oral or injectable treatment by Tina Kauss; Karen Gaudin; Alexandra Gaubert; Boubakar Ba; Serena Tagliaferri; Fawaz Fawaz; Jean-Louis Fabre; Jean-Michel Boiron; Xavier Lafarge; Nicholas J. White; Piero L. Olliaro; Pascal Millet (624-630).
The aim of this study was to identify a candidate formulation for further development of a home or near-home administrable paediatric rectal form of a broad-spectrum antibiotic – specially intended for (emergency) use in tropical rural settings, in particular for children who cannot take medications orally and far from health facilities where injectable treatments can be given. Azithromycin, a broad-spectrum macrolide used orally or intravenously for the treatment of respiratory tract, skin and soft tissue infections, was selected because of its pharmacokinetic and therapeutic properties. Azithromycin in vitro solubility and stability in physiologically relevant conditions were studied. Various pharmaceutical forms, i.e. rectal suspension, two different rectal gels, polyethylene glycol (PEG) suppository and hard gelatin capsule (HGC) were assessed for in vitro dissolution and in vivo bioavailability in the rabbit. Azithromycin PEG suppository appears to be a promising candidate.
Keywords: Azithromycin; Antibiotic; Rectal; Formulation; Bioavailability;

Solubilisation capacity of Brij surfactants by Maria E.N.P. Ribeiro; Carolina L. de Moura; Mariano G.S. Vieira; Nilce V. Gramosa; Chiraphon Chaibundit; Marcos C. de Mattos; David Attwood; Stephen G. Yeates; S. Keith Nixon; Nágila M.P.S. Ricardo (631-635).
The aim of this study was to investigate the potential of selected Brij non-ionic surfactants for enhancing the solubility of poorly water-soluble drugs. Griseofulvin was selected as a model drug candidate enabling comparisons to be made with the solubilisation capacities of other poly(ethylene oxide)-based copolymers. UV/Vis and 1H NMR spectroscopies were used to quantify the enhancement of solubility of griseofulvin in 1 wt% aqueous micellar solutions of Brij 78 (C18H37E20), Brij 98 (C18H35E20) and Brij 700 (C18H37E100) (where E represents the OCH2CH2 unit of the poly(ethylene oxide) chain) at 25, 37 and 40 °C. Solubilisation capacities (S cp expressed as mg griseofulvin per g Brij) were similar for Brij 78 and 98 (range 6–11 mg g−1) but lower for Brij 700 (3–4 mg g−1) as would be expected for the surfactant with the higher ethylene oxide content. The drug loading capacity of micelles of Brij 78 was higher than many di- and triblock copolymers with hydrophilic E-blocks specifically designed for enhancement of drug solubility.
Keywords: Solubilisation; Micelles; Griseofulvin; Block copolymers; Brij surfactants;

Accelerated blood clearance of PEGylated liposomes containing doxorubicin upon repeated administration to dogs by Takuya Suzuki; Masako Ichihara; Kenji Hyodo; Eiichi Yamamoto; Tatsuhiro Ishida; Hiroshi Kiwada; Hiroshi Ishihara; Hiroshi Kikuchi (636-643).
The accelerated blood clearance phenomenon involving anti-PEG IgM production has been recognized as an important issue for the design and development of PEGylated liposomes. Here, we show that empty PEGylated liposomes and Doxil, PEGylated liposomes containing doxorubicin, both caused anti-PEG IgM production and thereby a rapid clearance of the second and/or third dose of Doxil in Beagle dogs in a lipid-dose, inverse-dependent manner. It appears that the pharmacokinetic profile of the second and third administration of Doxil reflected the presence of anti-PEG IgM circulating in the blood. Doxil plus an excess amount of empty PEGylated liposomes rather enhanced the production of anti-PEG IgM compared to Doxil of the same doxorubicin dose. During sequential administration, increasing the lipid dose of Doxil in each dose by the addition of empty PEGylated liposomes strongly attenuated the magnitude of the ABC phenomenon during the effectuation phase of a second and third dose of Doxil. Our results suggest that the pre-clinical study of anti-cancer drug-containing PEGylated liposomes with dogs must be carefully designed and performed with monitoring of the anti-PEG IgM and liposomal drugs circulating in the blood.
Keywords: Polyethylene glycol; PEGylated liposomes; Accelerated blood clearance phenomenon; Doxorubicin; Doxil; Dog;

The aim of this work was to incorporate a hydrophobic compound benzophenone (BP) into core–shell nanoparticles (NPs) and to study its release. The core of these NPs is based on polylactic acid (PLA) and serves as a nanocontainer to accommodate BP. The shell is constituted of a β-cyclodextrin polymer (Poly-β-CD) which ensures the control of BP release through non-covalent host–guest interactions. Studies were focused on the preparation, physico-chemical analysis of the BP-loaded NPs and on the elucidation of the release mechanisms. The main features which are the slow kinetics, the dilution-induced release and the total release of encapsulated BP are in good agreement with a mechanism mainly controlled by diffusion of BP and by its binding with the β-CDs cavities present at the surface of NPs.
Keywords: Core–shell nanoparticles; Release kinetics; Host–guest interactions;

Phase behavior of itraconazole–phenol mixtures and its pharmaceutical applications by Chun-Woong Park; Heidi M. Mansour; Tack-Oon Oh; Ju-Young Kim; Jung-Myung Ha; Beom-Jin Lee; Sang-Cheol Chi; Yun-Seok Rhee; Eun-Seok Park (652-658).
The aims of this study were to examine the phase behavior of itraconazole–phenol mixtures and assess the feasibility of topical formulations of itraconazole using eutectic mixture systems. Itraconazole–phenol eutectic mixtures were characterized using differential scanning calorimetry, Fourier transform infrared spectroscopy, 1H-nuclear magnetic resonance, and powder X-ray diffractometry. The skin permeation rates of itraconazole–phenol eutectic formulations were determined using Franz diffusion cells fitted with excised hairless mouse skins. Itraconazole can form eutectic compounds with phenol, and the hydrogen-bonding interactions between the carbonyl group in the itraconazole and hydroxyl group in phenol play a major role in itraconazole–phenol eutectic formation. Despite its high molecular weight and hydrophobicity, the drug (i.e., itraconazole) can be permeated through excised hairless mouse skins from itraconazole–phenol eutectic formulations. The findings of this study emphasize the capabilities of the topical application of itraconazole via external preparations.
Keywords: Itraconazole; Phenol; Eutectic mixture; Topical; Skin permeation;

In vitro antimicrobial activity of nanoconjugated vancomycin against drug resistant Staphylococcus aureus by Subhankari Prasad Chakraborty; Sumanta Kumar Sahu; Panchanan Pramanik; Somenath Roy (659-676).
The mounting problem of antibiotic resistance of Staphylococcus aureus has prompted renewed efforts toward the discovery of novel antimicrobial agents. The present study was aimed to evaluate the in vitro antimicrobial activity of nanoconjugated vancomycin against vancomycin sensitive and resistant S. aureus strains. Folic acid tagged chitosan nanoparticles are used as Trojan horse to deliver vancomycin into bacterial cells. In vitro antimicrobial activity of nanoconjugated vancomycin against VSSA and VRSA strains was determined by minimum inhibitory concentration, minimum bactericidal concentration, tolerance and disc agar diffusion test. Cell viability and biofilm formation was assessed as indicators of pathogenicity. To establish the possible antimicrobial mechanism of nanoconjugated vancomycin, the cell wall thickness was studied by TEM study. The result of the present study reveals that nano-sized vehicles enhance the transport of vancomycin across epithelial surfaces, and exhibits its efficient drug-action which has been understood from studies of MIC, MBC, DAD of chitosan derivative nanoparticle loaded with vancomycin. Tolerance values distinctly showed that vancomycin loaded into nano-conjugate is very effective and has strong bactericidal effect on VRSA. These findings strongly enhanced our understanding of the molecular mechanism of nanoconjugated vancomycin and provide additional rationale for application of antimicrobial therapeutic approaches for treatment of staphylococcal pathogenesis.
Keywords: Vancomycin resistant Staphylococcus aureus; Nanoconjugated vancomycin; Minimum inhibitory concentration; Cell wall thickness; Cell viability; Na+, K+-ATPase activity;

Fluxgate magnetorelaxometry: A new approach to study the release properties of hydrogel cylinders and microspheres by S. Wöhl-Bruhn; E. Heim; A. Schwoerer; A. Bertz; S. Harling; H. Menzel; M. Schilling; F. Ludwig; H. Bunjes (677-684).
Hydrogels are under investigation as long term delivery systems for biomacromolecules as active pharmaceutical ingredients. The release behavior of hydrogels can be tailored during the fabrication process. This study investigates the applicability of fluxgate magnetorelaxometry (MRX) as a tool to characterize the release properties of such long term drug delivery depots. MRX is based on the use of superparamagnetic core-shell nanoparticles as model substances. The feasibility of using superparamagnetic nanoparticles to study the degradation of and the associated release from hydrogel cylinders and hydrogel microspheres was a major point of interest. Gels prepared from two types of photo crosslinkable polymers based on modified hydroxyethylstarch, specifically hydroxyethyl starch-hydroxyethyl methacrylate (HES-HEMA) and hydroxyethyl starch-polyethylene glycol methacrylate (HES-P(EG)6MA), were analyzed. MRX analysis of the incorporated nanoparticles allowed to evaluate the influence of different crosslinking conditions during hydrogel production as well as to follow the increase in nanoparticle mobility as a result of hydrogel degradation during release studies. Conventional release studies with fluorescent markers (half-change method) were performed for comparison. MRX with superparamagnetic nanoparticles as model substances is a promising method to analyze pharmaceutically relevant processes such as the degradation of hydrogel drug carrier systems. In contrast to conventional release experiments MRX allows measurements in closed vials (reducing loss of sample and sampling errors), in opaque media and at low magnetic nanoparticle concentrations. Magnetic markers possess a better long-term stability than fluorescent ones and are thus also promising for the use in in vivo studies.
Keywords: Hydrogel cylinders; Microspheres; Magnetorelaxometry; Magnetic nanoparticles; Degradation; Release;

Preparation, structural analysis, and properties of tenoxicam cocrystals by Jagdishwar R. Patel; Robert A. Carlton; Thomas E. Needham; Clinton O. Chichester; Frederick G. Vogt (685-706).
Cocrystals of tenoxicam, a non-steroidal anti-inflammatory drug, are screened, prepared, and characterized in this study. Nine tenoxicam cocrystals were identified using solvent-drop grinding (SDG) techniques. Structural characterization was performed using powder X-ray diffraction (PXRD), differential scanning calorimetry, and multinuclear solid-state NMR (SSNMR). Thermal analysis, PXRD, and 1D SSNMR are used to detect solvates and phase mixtures encountered in SDG cocrystal screening. 2D SSNMR methods are then used to confirm cocrystal formation and determine structural aspects for selected cocrystals formed with saccharin, salicylic acid, succinic acid, and glycolic acid in comparison to Forms I and III of tenoxicam. Molecular association is demonstrated using cross-polarization heteronuclear dipolar correlation (CP-HETCOR) methods involving 1H and 13C nuclei. Short-range 1H–13C CP-HETCOR and 1H–1H double-quantum interactions between atoms of interest, including those engaged in hydrogen bonding, are used to reveal local aspects of the cocrystal structure. 15N SSNMR is used to assess ionization state and the potential for zwitterionization in the selected cocrystals. The tenoxicam saccharin cocrystal was found to be similar in structure to a previously-reported cocrystal of piroxicam and saccharin. The four selected cocrystals yielded intrinsic dissolution rates that were similar or reduced relative to tenoxicam Form III.
Keywords: Cocrystal; Physical characterization; Solid-state NMR; X-ray diffraction; Differential scanning calorimetry; Dissolution rate; Polymorphism;

Distribution of cholesterol across the lipid, aqueous and sediment phases following lipolysis in the presence of water control (n  = 6), stigmastanol powder (262.5 mg, n  = 6), stigmastanol formulation (262.5 mg, n  = 5) and DAPP (262.5 mg, n  = 7) under simulated fed state conditions. *P  < 0.05 multiple comparisons versus control group.An in vitro lipolysis model was utilized to study the effect of stigmastanol (lipophilic phytosterol) and disodium ascorbyl phytostanol phosphate (DAPP) (modified hydrophilic phytostanol) on intestinal processing of cholesterol to gain further understanding of their cholesterol lowering mechanism. Lipolysis results showed that stigmastanol, if given in powder alone, had no effect on cholesterol processing probably due to its poor solubility. Stigmastanol suspension formulation re-distributed cholesterol from aqueous phase to oil and sediment phases. The water soluble DAPP has changed cholesterol distribution even more significantly by transferring cholesterol from aqueous phase to sediment phase. Moreover, the results provided evidence that DAPP inhibited triglyceride digestion in vitro. Considering DAPP as a surfactant with the same lipophilic sterol ring as bile salt, its ability to inhibit triglyceride lipolysis may be due to its competition with bile salt for the substrate surface, thereby hindering the lipolysis of triglyceride and inhibiting cholesterol solubilization with the lipolysis products. It can be speculated that the cholesterol lowering mechanism of DAPP during intestinal digestion is related to its ability to act as a surfactant closely resembling bile salt.
Keywords: Stigmastanol; Disodium ascorbyl phytostanol phosphate; Cholesterol; Bile salt; Surfactant; Lipolysis;

The impact of training set data distributions for modelling of passive intestinal absorption by Taravat Ghafourian; Alex A. Freitas; Danielle Newby (711-720).
This study presents regression and classification models to predict human intestinal absorption of 645 drug and drug like compounds using percentage human intestinal values from the published dataset by . The problem with this dataset and other datasets in the literature is there are more highly than poorly absorbed compounds. Any models developed using these datasets will be biased towards highly absorbed compounds and not applicable for use in industry where now more compounds are likely to be poorly absorbed. The study compared two training sets, TS1, a balanced (50:50) distribution of highly and poorly absorbed compounds created by under-sampling the majority high absorption compounds, with TS2, a randomly selected training set with biased distribution towards highly absorbed compounds. The regression results indicate that the best models were those developed using the balanced dataset (TS1). Also for classification, TS1 led to the most accurate models and the highest specificity value of 0.949. In comparison, TS2 led to the highest sensitivity with a value of 0.939. Thus, under-sampling the majority class of the highly absorbed compounds leads to a balanced training set (TS1) that can achieve more applicable in silico regression and classification models for the use in the industry.
Keywords: Intestinal absorption; QSAR; Oral absorption; Training set; Regression; Classification;

Many recently developed drugs encounter delivery issues due to their high lipophilicity and poor aqueous solubility. This study reports the development of a novel hybrid nanocarrier known as polymer-oil nanostructured carrier (PONC), in which highly lipophilic drugs such as all-trans-retinoic acid (ATRA) and indomethacin pre-solubilized in oil phase were dispersed in a polymeric matrix of poly(d,l-lactic-co-glycolic acid) (PLGA). In comparison to the standard PLGA only nanoparticles, PONC substantially increased the encapsulation efficiency of ATRA and indomethacin by up to 259% and 124%, respectively. Differential scanning calorimetry analysis revealed that the inclusion of oil introduced nanostructure into the polymeric matrix of the carrier. This feature facilitated more uniform distribution of the drug molecules which subsequently led to improved drug release kinetics with significantly reduced burst release effects (p  < 0.05). PONC was lyophilizable, remained physically stable when stored at low temperatures, and demonstrated low non-specific carrier toxicity. Delivery of ATRA by PONC also significantly improved its anticancer activity over the standard PLGA only nanoparticles (p  < 0.05). Our findings have therefore validated a promising delivery system that integrates the advantages of lipid-based (e.g. efficient encapsulation of highly lipophilic drugs) and polymeric colloidal carriers (e.g. uniform size, good stability), plus potential therapeutic benefits for delivery of poorly water-soluble anticancer drugs like ATRA.
Keywords: Nanomedicine; PLGA; Controlled drug delivery; Lipophilic drug; All-trans retinoic acid;

Investigation of surfactants suitable for stabilizing of latanoprost by Akiko Ochiai; Moriyuki Ohkuma; Kazumi Danjo (732-737).
The content of latanoprost, a therapeutic drug for glaucoma, is likely to decrease in solution. In a previous study, we confirmed that this was associated with latanoprost adsorption to the container and hydrolysis of latanoprost, and established a formulation of latanoprost eye drop solution that can be stored at room temperature. In addition, we clarified that a surfactant added to the formulation stabilized latanoprost by forming complex micelle. In this study, we investigated the influence of structure of surfactants in the stabilization of latanoprost.Non-ionic surfactants involving the polyoxyethylene chain (additive mol number: 20 or more) and the straight-chain alkyl group, with an HLB of 15.0 or more, markedly inhibited a decrease in the latanoprost content and its degradation. These surfactants are soluble in the formulation of eye drop solution, leading to micelle formation even at a low concentration, and they are not influenced by the temperature. Therefore, they may exhibit marked stabilization effects. In addition, there was no influence on the stability of latanoprost when adding benzalkonium chloride, as a preservative, to a formulation involving these surfactants.
Keywords: Latanoprost; Stabilization; Surfactant; Micelle; Structure;

PLGA-microparticles with 4.7 μm in diameter were prepared by the double emulsion technique and loaded with 1.7 μg fluorescein/mg PLGA mimicking a hydrophilic API. In an effort to further elucidate the release and bioadhesive characteristics of lectin-grafted formulations in vitro, the particles were coated with wheat germ agglutinin. The microparticles exhibited thermo-responsive release since no free fluorescein was detected at 4 °C or room temperature. At body temperature, however, more than 80% of the payload was released within 48 h. The adhesion of lectin-grafted particles to Caco-2 monolayers, which were applied as a model for the human intestinal epithelium, exceeded that of plain ones 1.5-fold as also observed by fluorescence microscopy. Furthermore, the amount of model drug bound and taken up into the cells was 5.8-fold higher after incubation for 4 h at 37 °C as compared to fluorescein in solution. According to fluorescence imaging a considerable amount of the total fluorescein payload was accumulated intracellularily after incubation for 5 h at 37 °C. These findings not only confirm the utility of bioadhesives of the second generation for improved absorption of low molecular weight hydrophilic compounds but also indicate storage stability of such suspensions at 4 °C and room temperature without any premature loss of API.
Keywords: Bioadhesion; Caco-2; Microparticle; PLGA; Thermo-responsive; WGA;

In this study, we combined a high-throughput screening method, differential scanning fluorimetry (DSF), with design of experiments (DoE) methodology to evaluate the effects of several formulation components on the thermostability of granulocyte colony stimulating factor (G-CSF). First we performed a primary buffer screening where we tested thermal stability of G-CSF in different buffers, pH values and buffer concentrations. The significance of each factor and the two-way interactions between them were studied by multivariable regression analysis. pH was identified as most critical factor regarding thermal stability. The most stabilizing buffer, sodium glutamate, and sodium acetate were determined for further investigations. Second we tested the effect of 6 naturally occurring extremolytes (trehalose, sucrose, ectoine, hydroxyectoine, sorbitol, mannitol) on the thermal stability of G-CSF, using a central composite circumscribed design. At low pH (3.8) and low buffer concentration (5 mM) all extremolytes led to a significant increase in thermal stability except the addition of ectoine which resulted in a strong destabilization of G-CSF. Increasing pH and buffer concentration led to an increase in thermal stability with all investigated extremolytes. The described systematic approach allowed to create a ranking of stabilizing extremolytes at different buffer conditions.
Keywords: Design of experiments; Protein formulation; High-throughput screening; Thermal stability; Differential scanning fluorimetry; Quality by design;

Chemico-physical investigation of tenofovir loaded polymeric nanoparticles by Daniela Belletti; Giovanni Tosi; Flavio Forni; Maria Cristina Gamberini; Cecilia Baraldi; Maria Angela Vandelli; Barbara Ruozi (753-763).
Tenofovir (PMPA), an acyclic nucleoside phosphonate analog, is one of the most important drugs used for the HIV treatment. Unfortunately, several adverse reactions are related to its i.v. administration owing to the saturation of an anionic renal transporter. In order to improve the drug administration, the PMPA was embedded into a new type of nanocarriers based on poly-(d,l-lactide-co-glycolide) (PLGA) and/or chitosan (CH). The strategies for the preparation of nanoparticles (Nps) with a more efficient drug loading respect to the one reported in the literature for PMPA nanoencapsulation were investigated. CH was added in the first inner emulsion or in the external phase during the second emulsion of water/oil/water (W/O/W) Nps. The addition of CH in the first inner emulsion was the most promising technique. The Nps have a Z-average of 230 nm, a Z-potential of −3 mV and an EE% of 15 that was 2.5–3 times higher than that obtained with PLGA Nps or CH Nps. In vitro release studies showed a limited control on drug release in phosphate buffer (pH 7.4) while an initial burst effect followed by a slow drug release was observed in acidic receiving phase (pH 4.6). These results suggest the PLGA/CH Nps should be an effective and attractive anti-HIV drug carrier to study the cellular uptake and drug delivery on target cells such as macrophages.
Keywords: PMPA; Nps; PLGA; CH; Raman spectra; DSC analysis;

Transdermal delivery of 8-methoxypsoralene mediated by polyamidoamine dendrimer G2.5 and G3.5—In vitro and in vivo study by Katarzyna Borowska; Stanisław Wołowiec; Kazimierz Głowniak; Elwira Sieniawska; Sebastian Radej (764-770).
In this work, we have focused on 8-methoxypsoralene (8-MOP) complexed with G2.5 and G3.5 poly(amido amine) (PAMAM) dendrimers. The purpose of this study was to investigate the efficacy of half-generation G2.5 and G3.5 PAMAM dendrimers conjugated with 8-MOP for delivery of 8-MOP in vitro study through polivinyldifluoride membrane (PVDE) and prepared pig ear skin (PES) using Franz diffusion and in vivo study through the skin of experimental animals (hairless rat skin). The tissue concentration of 8-MOP in hairless rat skin was analyzed by high performance liquid chromatography (HPLC) after 1 and 2 h. Detailed distribution of 8-MOP in skin layers and cellular structures were analyzed using laser scanning microscopy (CLSM). In vitro and in vivo studies showed that half-generation G2.5 and G3.5 PAMAM dendrimers are able to facilitate transdermal delivery of 8-MOP. G2.5 PAMAM dendrimer appeared to be more effective 8-MOP penetration enhancer than G3.5 PAMAM dendrimer, but in vivo the differences are not statistically significant. The concept of using G2.5 and G3.5 PAMAM dendrimers as carriers seems to be a promising method for the delivery of 8-MOP for PUVA (psoralen-UV-A) therapy.
Keywords: G2.5; G3.5; PAMAM dendrimers; 8-MOP; Host–guest complexes; PUVA; Transdermal; Confocal laser scanning microscopy;

Synthesis, characterization and in vitro assessment of the magnetic chitosan–carboxymethylcellulose biocomposite interactions with the prokaryotic and eukaryotic cells by Alexandru Mihai Grumezescu; Ecaterina Andronescu; Anton Ficai; Coralia Bleotu; Dan Eduard Mihaiescu; Mariana Carmen Chifiriuc (771-777).
Preparation and characterization of CS/Fe3O4/CMC composite scaffolds including the morphology, crystallinity, and the in vitro efficacy as antibiotic delivery vehicles as well as their influence on the eukaryotic cells are reported. The results demonstrated that the magnetic polymeric composite scaffolds are exhibiting structural and functional properties that recommend them for further applications in the biomedical field. They improve the activity of currently used antibiotics belonging to penicillins, macrolides, aminoglycosides, rifampicines and quinolones classes, representing potential macromolecular carriers for these antimicrobial substances, to achieve extracellular and intracellular targets. The obtained systems are not cytotoxic and do not influence the eukaryotic HCT8 cell cycle, representing potential tools for the delivery of drugs in a safe, effective and less expensive manner.
Keywords: Prokaryotic cells; Eukaryotic cells; Magnetic chitosan; Magnetic cellulose; Magnetic composite;

Carboxylic modified spherical mesoporous silicas аs drug delivery carriers by Margarita D. Popova; Ágnes Szegedi; Iliyan N. Kolev; Judit Mihály; Borislav S. Tzankov; Georgi Tz. Momekov; Nikolai G. Lambov; Krassimira P. Yoncheva (778-785).
The present study deals with the development and functionalization of mesoporous silica nanoparticles as drug delivery platforms. Spherical MCM-41 and SBA-15 silicas with different pore sizes (2.7 nm and 5.5 nm, respectively) were post-synthesis modified applying a new, two step process. The initial step was the modification with 3-amino-propyltriethoxysilane, and the next was the reaction with succinic anhydride in toluene in order to obtain carboxylic modified mesoporous carriers. The carboxylic-functionalized mesoporous materials were characterized by XRD, nitrogen physisorption, TEM, ATR FT-IR spectroscopy. The successful carboxylic functionalization was proved by the changes of the zeta potential of the mesoporous materials before and after modification. The parent and the carboxylic-modified MCM-41 and SBA-15 materials showed high adsorption capacity (approximately 50 wt.%, except for non-functionalized MCM-41) for sulfadiazine that possesses amino functional groups. Mesoporous structure peculiarities lead to different adsorption capacities on the carriers. In vitro release studies showed slower release rate of sulfadiazine from carboxylic modified MCM-41 and SBA-15 mesoporous particles compared to the non modified ones. Both non loaded and drug-loaded silica materials demonstrated no cytotoxicity on Caco-2 cell line. The functionalized mesoporous systems are appropriate drug delivery platforms due to their biocompatibility and the possibility to modify drug release.
Keywords: Carboxylic modification; Spherical mesoporous silica; Drug release; Sulfadiazine;

pH-induced alterations in SMA conformation causes the co-polymer-based liposomes to fuse with endosomes and mediate cytosolic delivery of encapsulated drugs which ultimately leads to enhanced chemotherapy.pH-responsive polymers render liposomes pH-sensitive and facilitate the intracellular release of encapsulated payload by fusing with endovascular membranes under mildly acidic conditions found inside cellular endosomes. The present study reports the use of high-molecular weight poly(styrene-co-maleic acid) (SMA), which exhibits conformational transition from a charged extended structure to an uncharged globule below its pK 1 value, to confer pH-sensitive property to liposomes. The changes in the co-polymer chain conformation resulted in destabilization of the liposomes at mildly acidic pH due to vesicle fusion and/or channel formation within the membrane bilayer, and ultimately led to the release of the encapsulated cargo. The vesicles preserved their pH-sensitivity and stability in serum unlike other polymer-based liposomes and exhibited no hemolytic activity at physiological pH. The lysis of RBCs at endosomal pH due to SMA-based liposome-induced alterations in the bilayer organization leading to spherocyte formation indicated the potential of these vesicles to mediate cytosolic delivery of bio-active molecules through endosome destabilization. The SMA-loaded liposomes exhibiting excellent cytocompatibility, efficiently delivered chemotherapeutic agent 5-Fluorouracil (5-FU) within colon cancer cells HT-29 in comparison to neat liposomes. This caused increased cellular-availability of the drug, which resulted in enhanced apoptosis and highlighted the clinical potential of SMA-based vesicles.
Keywords: Poly(styrene-co-maleic acid); Liposomes; pH-sensitive; Cytocompatible; Apoptosis;

Long-term stability, biocompatibility and oral delivery potential of risperidone-loaded solid lipid nanoparticles by A.C. Silva; A. Kumar; W. Wild; D. Ferreira; D. Santos; B. Forbes (798-805).
A solid lipid nanoparticles (SLN) formulation to improve the oral delivery of risperidone (RISP), a poorly water-soluble drug, was designed and tested. Initially, lipid–RISP solubility was screened to select the best lipid for SLN preparation. Compritol®-based formulations were chosen and their long-term stability was assessed over two years of storage (at 25 °C and 4 °C) by means of particle size, polydispersity index (PI), zeta potential (ZP) and encapsulation efficiency (EE) measurements. SLN shape was observed by transmission electron microscopy (TEM) at the beginning and end of the study. The oxidative potential (OP) of the SLN was measured and their biocompatibility with Caco-2 cells was evaluated using the (4,5-dimethylthiazol-2-yl)2,5-dyphenyl-tetrazolium bromide (MTT) assay. In vitro drug release and transport studies were performed to predict the in vivo release profile and to evaluate the drug delivery potential of the SLN formulations, respectively. The RISP-loaded SLN systems were stable and had high EE and similar shape to the placebo formulations before and after storage. Classical Fickian diffusion was identified as the release mechanism for RISP from the SLN formulation. Biocompatibility and dose-dependent RISP transport across Caco-2 cells were observed for the prepared SLN formulations. The viability of SLN as formulations for oral delivery of poorly water-soluble drugs such as RISP was illustrated.
Keywords: Risperidone; Solid lipid nanoparticles; Oral delivery; Biocompatibility; Caco-2 cells;

Formation of drug/surfactant catanionic vesicles and their application in sustained drug release by Yue Jiang; Feifei Li; Yuxia Luan; Wenting Cao; Xiaoqing Ji; Lanxia Zhao; Longlong Zhang; Zhonghao Li (806-814).
The catanionic vesicles formed by the double-chain surfactant (AOT) and the oppositely charged drug exhibit promising applications in drug delivery systems.The aggregation behavior of the cationic drug/anionic surfactant vesicles formed by tetracaine hydrochloride (TH) and double-chain surfactant, sodium bis(2-ethylhexyl)sulfosuccinate (AOT), was investigated. By controlling the molar ratio of TH to AOT, a transition from catanionic vesicles to micelles was observed. The catanionic aggregates exhibited different charge properties, structures, interaction enthalpies and drug release behaviors depending on the composition. To characterize the cationic drug/anionic surfactant system, transmission electron microscopy (TEM), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), conductivity, turbidity and zeta potential (ζ) measurements were performed. The drug release results indicate that the present drug-containing catanionic vesicles have promising applications in drug delivery systems. Furthermore, the percentage of drug distributed in the catanionic vesicles or micelles can be obtained by comparing the cumulative release of the corresponding aggregates with the pure drug solution.
Keywords: Surfactant; Drug; Catanionic vesicle; Drug release;

Preparation and characterization of teniposide PLGA nanoparticles and their uptake in human glioblastoma U87MG cells by Liqian Mo; Lianbing Hou; Dan Guo; Xiaoyan Xiao; Ping Mao; Xixiao Yang (815-824).
Many studies have demonstrated the uptake mechanisms of various nanoparticle delivery systems with different physicochemical properties in different cells. In this study, we report for the first time the preparation and characterization of teniposide (VM-26) poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) and their cellular uptake pathways in human glioblastoma U87MG cells. The nanoparticles prepared with oil-in-water (O/W) single-emulsion solvent evaporation method had a small particle size and spherical shape and provided effective protection against degradation of teniposide in PBS solution. Differential scanning calorimeter (DSC) thermograms concluded that VM-26 was dispersed as amorphous or disordered crystalline phase in the PLGA matrix. A cytotoxicity study revealed that, in a 24 h period, blank PLGA NPs had no cytotoxicity, whereas teniposide-loaded PLGA NPs (VM-26-NPs) had U87MG cytotoxicity levels similar to free teniposide. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) images showed the distribution and degradation processes of nanoparticles in cells. An endocytosis inhibition test indicated that clathrin-mediated endocytosis and macropinocytosis were the primary modes of engulfment involved in the internalization of VM-26-NPs. Our findings suggest that PLGA nanoparticles containing a sustained release formula of teniposide may multiplex the therapeutic effect and ultimately degrade in lysosomal within human glioblastoma U87MG cells.
Keywords: Teniposide; PLGA; Nanoparticle; Endocytosis; Clathrin; Macropinocytosis;

Elastin-like polypeptide (ELP) is a macromolecular carrier with thermally responsive properties that can passively accumulate in solid tumors and additionally aggregate in tumor tissue when exposed to hyperthermia. In this study, ELP was conjugated to the anticancer drug doxorubicin (DOXO) and three different cell penetrating peptides (CPP) in order to inhibit tumor growth in mice compared to free doxorubicin. Fluorescence microscopy studies in MCF-7 breast carcinoma cells demonstrated that the three different CPP–ELP–DOXO conjugates delivered doxorubicin to the cell nucleus. All CPP–ELP–DOXO conjugates showed cytotoxicity with IC50 values in the range of 12–30 μM at 42 °C, but the ELP carrier with SynB1 as the cell penetrating peptide had the lowest intrinsic cytotoxicity. Therefore, the antitumor efficacy of SynB1–ELP–DOXO was compared to doxorubicin under hyperthermic conditions. C57BL/6 female mice bearing syngeneic E0771 murine breast tumors were treated with either free doxorubicin or the SynB1–ELP–DOXO conjugate with or without focused hyperthermia on the tumor. Under hyperthermic conditions, tumor inhibition with SynB1–ELP–DOXO was 2-fold higher than under therapy with free doxorubicin at the equivalent dose, and is thus a promising lead candidate for optimizing thermally responsive drug polymer conjugates.
Keywords: Elastin-like polypeptide (ELP); Doxorubicin; 6-(Maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH, INNO-206); Acid-sensitive prodrugs; Cell penetrating peptide (CPP); Hyperthermia;

In this work a new formulation of inhalable rifampicin-loaded PLGA microspheres (RIF-MS) is proposed for the management of tuberculosis treatment. For their formulation, the non-biodegradable polyvinyl alcohol surfactant was replaced with a biodegradable and biocompatible sucrose ester, sucrose palmitate. The effects of critical process and formulation parameters have been investigated and the obtained microspheres were characterized in terms of size, morphology, encapsulation efficiencies and RIF release profile. The optimized RIF-MS showed high drug loading (34.2%, w/w), an aerodynamic diameter compliant with deep lung delivery and an in vitro gradual and almost complete drug release over a week. The drug release data fitted well to the Higuchi models suggesting a drug release governed by Fickian diffusion. The RIF-MS uptake qualitative and quantitative studies on ex vivo rat alveolar macrophages (AM) revealed an efficient internalization of RIF-MS and their location in the perinuclear area. RIF intracellular levels were 7-fold higher in AM incubated with RIF-MS than with an equivalent amount of free RIF.
Keywords: Cell uptake; Alveolar macrophage; PLGA microsphere; Rifampicin; Sucrose palmitate;

Penetratin-functionalized PEG–PLA nanoparticles for brain drug delivery by Huimin Xia; Xiaoling Gao; Guangzhi Gu; Zhongyang Liu; Quanyin Hu; Yifan Tu; Qingxiang Song; Lei Yao; Zhiqing Pang; Xinguo Jiang; Jun Chen; Hongzhuan Chen (840-850).
Nanoparticulate drug delivery system possesses distinct advantages for brain drug delivery. However, its amount that reach the brain is still not satisfied. Cell-penetrating peptides (CPPs), short peptides that facilitate cellular uptake of various molecular cargo, would be appropriate candidates for facilitating brain delivery of nanoparticles. However, such effect could be deprived by the rapid systemic clearance of CPPs-functionalized nanoparticles due to their positive surface charge. Penetratin (CPP with relatively low content of basic amino acids) was here functionalized to poly(ethylene glycol)–poly(lactic acid) nanoparticles (NP) to achieve desirable pharmacokinetic and biodistribution profiles for brain drug delivery. The obtained penetratin-NP showed a particle size of 100 nm and zeta potential of −4.42 mV. The surface conjugation of penetratin was confirmed by surface chemical compositions analysis via X-ray photo electron spectroscopy. In MDCK–MDR cell model, penetratin-NP presented enhanced cellular accumulation via both lipid raft-mediated endocytosis and direct translocation processes with the involvement of Golgi apparatus, lysosome and microtubules. In vivo pharmacokinetic and biodistribution studies showed that penetratin-NP exhibited a significantly enhanced brain uptake and reduced accumulation in the non-target tissues compared with low-molecular-weight protamine (CPP with high arginine content)-functionalized nanoparticles. These data strongly implicated that penetratin-NP might represent a promising brain-targeting drug delivery system. The findings also provided an important basis for the optimization of brain drug delivery systems via surface charge modulation.
Keywords: Blood–brain barrier (BBB); Nanoparticles; Brain targeting; Cell-penetrating peptides; Penetratin;

Antimicrobial properties of cyclodextrin–antiseptics-complexes determined by microplate laser nephelometry and ATP bioluminescence assay by Susanne Finger; Cornelia Wiegand; Hans-Jürgen Buschmann; Uta-Christina Hipler (851-856).
Antimicrobial effects of substances can be determined with different methods that measure distinct parameters. Thus, a comparison of the results obtained can be difficult. In this study, two in vitro methods were employed to determine concentration and time dependent effects of cyclodextrin (CD)-complexes with the antiseptics chlorhexidine diacetate (CHX), iodine (IOD) and polihexanide (PHMB) on Candida albicans and Malassezia pachydermatis. Using both, microplate laser nephelometry and the ATP bioluminescence assay, it could be shown that CD–antiseptics-complexes tested exhibited significant antifungal effects with the exception of γ–CD–CHX in the case of C. albicans. Microplate laser nephelometry (MLN) is an optical method and enables a quantitative determination of particle concentrations in solution. By means of this method, microbial growth under influence of potential antimicrobial substances can be monitored over a prolonged time period. In addition, the antimicrobial activity was analyzed by measurement of the microbial adenosine triphosphate (ATP) content with a bioluminescent assay. The luminescent signal is directly proportional to the amount of ATP, and thus, a linear function of the number of living microbial cells present. Both methods were compared according to the half maximal inhibitory concentration (IC50) calculated and the statistical evaluation of Pearson's correlation coefficient (r). In summary, it could be demonstrated that both methods yield similar results although they differ in the parameter.
Keywords: Antimycotic; Bioluminescence; Candida albicans; Cyclodextrin-complex; Laser nephelometry; Malassezia pachydermatis;

Effect of the stability of hydrogen-bonded ion pairs with organic amines on transdermal penetration of teriflunomide by Honglei Xi; Dongmei Cun; Zhongyan Wang; Lei Shang; Wenting Song; Liwei Mu; Liang Fang (857-861).
The aim of this work was to investigate the effect of the stability of hydrogen-bonded ion pairs with organic amines on transdermal penetration of teriflunomide (TEF). Five organic amines, diethylamine (DEtA), triethylamine (TEtA), diethanolamine (DEA), triethanolamine (TEA), and N-(2′-hydroxyethanol)-piperdine (NP), were chosen to form ion pairs with TEF separately, and the passage of each TEF ion pair through the rabbit skin was evaluated in vitro. FTIR and 1H NMR spectroscopy were performed to confirm the formation of ion pairs between TEF and organic amines in solution. The stability parameter of ion pairs in terms of ion-pair lifetimes (T life) was calculated from the NMR data. TEF could form ion pairs with these amines via hydrogen bond. The formation of ion pairs enhanced the percutaneous absorption of TEF except TEF–DEA. It was found that, for most studied organic amines, the longer the ion-pair lifetime, the higher the flux of skin permeation. The stability of TEF ion pairs was a pivotal factor influencing the skin permeation of TEF.
Keywords: Teriflunomide; Hydrogen-bonded ion pairs; FTIR; 1H NMR; Stability; Transdermal;

Prediction of the compressibility of complex mixtures of pharmaceutical powders by V. Busignies; V. Mazel; H. Diarra; P. Tchoreloff (862-868).
The model we proposed was able to predict with a good precision the final density of the compacts of three different complex mixtures at different compaction pressures.The development of predictive models for the pharmaceutical compaction process is of great interest for not only the formulation step but also in the context of the quality by design development. This paper deals with the prediction of the compressibility, i.e. the prediction of the evolution of the density and the porosity of the compact along with the compaction pressure, both “in-die” (during the compaction) and “out-of-die (after the ejection of the compact). For this purpose, four different mixtures composed of five different pharmaceutical products were studied using a rotative press simulator. The excipients and formulations were chosen to be as near as possible to real industrial formulations. Using the volume as an additive property and a reformulation of the Kawakita equation as a function of the density, it was possible to predict the density of the compact both “in-die” and “out-of-die” with a good accuracy (residuals <3.5%). In most of the cases, for the pressure levels used in the pharmaceutical industry, the absolute error on the prediction of the porosity was below 2%. This study demonstrates that this approach could be well suited to predict the compressibility of real pharmaceutical formulations in the industrial context.
Keywords: Compaction; Compressibility; Kawakita; Mixtures; Formulation; Modeling;

Application of organogels as oral controlled release formulations of hydrophilic drugs by Kazunori Iwanaga; Mineo Kawai; Makoto Miyazaki; Masawo Kakemi (869-872).
We previously demonstrated that organogels prepared from soybean oil using 12-hydroxy stearic acid as a gelator can slowly release ibuprofen, a model lipophilic drug. In this study, we investigated the applicability of organogels as controlled release formulations of hydrophilic drugs. The release rates of theophylline and ofloxacin, which are used as model hydrophilic drugs, were significantly slower than those of ibuprofen and antipyrine (model lipophilic drugs). Furthermore, no erosion was noted during drug release from organogels. Lipophilic drug molecules are released after diffusion in organogels because all molecules fully dissolve in the gel. On the other hand, hydrophilic drug molecules need to be dissolved before they diffuse in the organogel, prior to their release from the gel. Therefore, it is speculated that the release rates of hydrophilic drugs are slower than those of lipophilic drugs. To confirm the usefulness of organogels in controlled release formulations in vivo, organogels containing ibuprofen, ofloxacin, theophylline or antipyrine were intraduodenally administered to rats. All drugs used in this study were rapidly absorbed when administered in aqueous suspensions. In contrast, the drug concentrations in plasma after administration in organogels were lower; however, the lower concentrations of drugs sustained for 10 h after administration. With organogel administration, the mean residence time of drugs was longer than that with aqueous suspension administration. In conclusion, organogels are potential candidates for controlled release formulations of not only lipophilic drugs, but also hydrophilic drugs.
Keywords: Organogel; Controlled release; Hydrophilic drugs; Intestinal absorption; Drug delivery system;

A novel approach to crystallisation of nanodispersible microparticles by spray drying for improved tabletability by Krzysztof J. Paluch; Lidia Tajber; Bożena Adamczyk; Owen I. Corrigan; Anne Marie Healy (873-876).
High-dose API powders which are to be tableted by direct compression should have high compactibility and compressibility. This note reports on a novel approach to the manufacture of crystalline powders intended for direct compaction with improved compactibility and compressibility properties. The poorly compactable API, chlorothiazide, was spray dried from a water/acetone solvent mix producing additive-free nanocrystalline microparticles (NCMPs) of median particle size 3.5 μm. Tablets compacted from NCMPs had tensile strengths ranging from 0.5 to 4.6 MPa (compared to 0.6–0.9 MPa for tablets of micronised CTZ) at compression forces ranging from 6 kN to 13 kN. NCMP tablets also had high porosities (34–20%) and large specific surface areas (4.4–4.8 m2/g). The time taken for tablets made of NCMPs to erode was not statistically longer (p  > 0.05) than for tablets made of micronised CTZ. Fragmentation of NCMPs on compression was observed. The volume fraction of particles below 1 μm present in the suspension recovered after erosion of NCMP tablets was 34.8 ± 3.43%, while no nanosized particles were detected in the slurry after erosion of compacted micronised CTZ.
Keywords: Spray drying; Tabletability; Specific surface area; Nanocrystalline microparticles; Erosion; Nanosuspension;

Application of artificial neural networks (ANNs) and genetic programming (GP) for prediction of drug release from solid lipid matrices by Sinan Güres; Aleksander Mendyk; Renata Jachowicz; Przemysław Dorożyński; Peter Kleinebudde (877-879).
A model based on artificial neural networks and genetic programming was derived, which is able to predict the release profiles of solid lipid extrudates of different dimensions.The aim of the present study was to develop a semi-empirical mathematical model, which is able to predict the release profiles of solid lipid extrudates of different dimensions. The development of the model was based on the application of ANNs and GP. ANNs' abilities to deal with multidimensional data were exploited. GP programming was used to determine the constants of the model function, a modified Weibull equation. Differently dimensioned extrudates consisting of diprophylline, tristearin and polyethylene glycol were produced by the use of a twin-screw extruder and their dissolution behaviour was studied. Experimentally obtained dissolution curves were compared to the calculated release profiles, derived from the semi-empirical mathematical model.
Keywords: Solid lipid extrusion; Artificial neural networks; Genetic programming; Release profile;