International Journal of Pharmaceutics (v.496, #2)

Bioactivity of noble metal nanoparticles decorated with biopolymers and their application in drug delivery by Mahendra Rai; Avinash P. Ingle; Indarchand Gupta; Adriano Brandelli (159-172).
Antibacterial activity of AgNPs Antiviral activity of AgNPsDisplay OmittedThe unique properties of nanomaterials can be applied to solve different problems including new ways of drug delivery. Noble metal nanoparticles are most promising because they have been used for medicinal purposes since ancient time. It is evident from the past studies that the metallic nanoparticles are much more effective against various microorganisms when compared to their conventional counterparts. However, decoration of such nanoparticles with biomaterials add more advantages to their antimicrobial activity. Decoration of metal nanoparticles with biopolymers is a quite new area of research. Studies performed hitherto shown that nanoparticles of noble metals like silver, gold and platinum demonstrated better antibacterial, antifungal and antiviral activities when conjugated with biopolymers. The development of such technology has potential to develop materials that are more effective in the field of health science. Considering the importance and uniqueness of this concept, the present review aims to discuss the use of biopolymer-decorated metal nanoparticles for combating various diseases caused by microbial pathogens. Moreover, the nanotoxicity aspect has also been discussed.
Keywords: Biopolymers; Silver nanoparticles; Gold nanoparticles; Platinum nanoparticles; Drug delivery; Toxicity;

Double emulsion solvent evaporation techniques used for drug encapsulation by Muhammad Iqbal; Nadiah Zafar; Hatem Fessi; Abdelhamid Elaissari (173-190).
Encapsulation of hydrophilic and lipophilic molecules via double emulsion techniques.Display OmittedDouble emulsions are complex systems, also called “emulsions of emulsions”, in which the droplets of the dispersed phase contain one or more types of smaller dispersed droplets themselves. Double emulsions have the potential for encapsulation of both hydrophobic as well as hydrophilic drugs, cosmetics, foods and other high value products. Techniques based on double emulsions are commonly used for the encapsulation of hydrophilic molecules, which suffer from low encapsulation efficiency because of rapid drug partitioning into the external aqueous phase when using single emulsions. The main issue when using double emulsions is their production in a well-controlled manner, with homogeneous droplet size by optimizing different process variables. In this review special attention has been paid to the application of double emulsion techniques for the encapsulation of various hydrophilic and hydrophobic anticancer drugs, anti-inflammatory drugs, antibiotic drugs, proteins and amino acids and their applications in theranostics. Moreover, the optimized ratio of the different phases and other process parameters of double emulsions are discussed. Finally, the results published regarding various types of solvents, stabilizers and polymers used for the encapsulation of several active substances via double emulsion processes are reported.
Keywords: Double emulsion; Solvent evaporation; Encapsulation; Theranostics; Control release; Drug delivery;

Recently superparamagnetic iron oxide nanoparticles (SPIONs) have been extensively used in cancer therapy and diagnosis (theranostics) via magnetic targeting, magnetic resonance imaging, etc. due to their remarkable magnetic properties, chemical stability, and biocompatibility. However, the magnetic properties of SPIONs are influenced by various physicochemical and synthesis parameters. So, this review mainly focuses on the influence of spin canting effects, introduced by the variations in size, shape, and organic/inorganic surface coatings, on the magnetic properties of SPIONs. This review also describes the several predominant chemical synthesis procedures and role of the synthesis parameters for monitoring the size, shape, crystallinity and composition of the SPIONs. Moreover, this review discusses about the latest developments of the inorganic materials and organic polymers for encapsulation of the SPIONs. Finally, the most recent advancements of the SPIONs and their nanopackages in combination with other imaging/therapeutic agents have been comprehensively discussed for their effective usage as in vitro and in vivo theranostic agents in cancer treatments.
Keywords: Magnetic nanoparticles; Superparamagnetic iron oxide; Synthesis and surface engineering; Spin canting; Cancer theranostics; Magnetic resonance imaging; Hyperthermia;

Polyethylene glycols in oral and parenteral formulations—A critical review by Rampurna P. Gullapalli; Carolyn L. Mazzitelli (219-239).
Display OmittedPolyethylene glycols (PEGs) are frequently employed as vehicles in oral and parenteral dosage forms. PEGs have low toxicity, are miscible with aqueous fluids in all proportions, and dissolve many poorly aqueous soluble compounds. Compounds with poor aqueous solubility and resulting poor bioavailability and considerable individual variability in the absorption were shown to provide exceptionally high bioavailability and reduced inter-subject variability in plasma concentrations when dosed as solutions or suspensions in PEGs. The advantages offered by PEGs, however, are not without potential challenges that must also be considered and which are the focus of this review. First, PEGs often may have high solubilizing power for some poorly aqueous soluble compounds, the high affinity of these vehicles for water can potentially lead to precipitation of the dissolved compounds when the formulations encounter an aqueous environment in vitro or in vivo, resulting in reduced bioavailability of the compounds. Second, PEGs, due to the presence of hydroxyl groups in their structures, are reactive with compounds dissolved within, resulting in the formation of degradation products. Third, PEGs, due to the presence of recurring ether groups in their polymer chains, are also inherently susceptible to autooxidative reactions, resulting in the formation of highly reactive products, which degrade several compounds formulated with PEGs. The objective is to review the applications and limitations of PEGs in pharmaceutical dosage forms and discuss solutions to mitigate challenges that may potentially arise from their use.
Keywords: Polyethylene glycols; Solubilization; Bioavailability; Precipitation; Autooxidation; Degradation;

Display OmittedThromboembolic complications are the most common preventable cause of mortality and morbidity in trauma patients. Thrombosis is also the common cause of ischemic heart disease (acute coronary syndrome), stroke, and venous thromboembolism. Heparin, as a potent anticoagulant, has been used in clinical practice for more than five decades and remains the major medicine for the prevention and treatment of venous thromboembolism. However it binds to the endothelium and has a high affinity for plasma proteins resulting in a short half-life and unpredictable bioavailability. Transdermal drug delivery can address the problems of short half-life and unpredictable bioavailability. Other advantages of transdermal drug delivery include convenience, improved patient compliance, prompt termination of dosing and avoidance of the first-pass effect. This review focuses on different approaches used for transdermal delivery of heparin.
Keywords: Transdermal; Heparin; Drug delivery; Sonophoresis; Electroporation; Iontophoresis;

Particle contamination of parenteralia and in-line filtration of proteinaceous drugs by Benjamin Patrick Werner; Gerhard Winter (250-267).
Display OmittedProtein drug products play an important role in the treatment of severe diseases. However, due to the instability of these complex molecules, protein aggregates can form which can compromise drug safety and efficacy including immunogenic reactions. In-line filtration during the administration of these drugs can serve as a final safeguarding step to protect patients from risks associated with proteinaceous particles. A unique analysis of more than 300 marketed protein drug products revealed that already around 16% of all these products are filtered during preparation or administration. Further, the research revealed that no standardized filtration practice exists. Broad variances regarding filter membrane or pore size are found and sometimes no specifications are mentioned at all. The benefits as well as possible negative impacts of filtration like filter shedding, extractables or drug adsorption are critically assessed. Several proposals to improve the current filtration practice and to expand the number of in-line filtered protein drug products are made. The suggestions include the demand for the specific usage of one filter membrane type, the establishment of a filtration routine for unfiltered protein drugs and a statistical analysis between filtered and non-filtered products with similar formulations to identify possible differences in the immunogenicity rate.
Keywords: Particulate matter; Particle; Protein; Biopharmaceutical; In-line filtration; Immunogenicity;

Display OmittedTargeting of drug delivery systems (DDSs) to specific intracellular organelles (i.e., subcellular targeting) has been investigated in numerous publications, but targeting efficiency of these systems is seldom reported. We searched scientific publications in the subcellular DDS targeting field and analyzed targeting efficiency and major formulation parameters that affect it. We identified 77 scientific publications that matched the search criteria. In the majority of these studies nanoparticle-based DDSs were applied, while liposomes, quantum dots and conjugates were used less frequently. The nucleus was the most common intracellular target, followed by mitochondrion, endoplasmic reticulum and Golgi apparatus. In 65% of the publications, DDSs surface was decorated with specific targeting residues, but the efficiency of this surface decoration was not analyzed in predominant majority of the studies. Moreover, only 23% of the analyzed publications contained quantitative data on DDSs subcellular targeting efficiency, while the majority of publications reported qualitative results only. From the analysis of publications in the subcellular targeting field, it appears that insufficient efforts are devoted to quantitative analysis of the major formulation parameters and of the DDSs’ intracellular fate. Based on these findings, we provide recommendations for future studies in the field of organelle-specific drug delivery and targeting.
Keywords: Targeted drug delivery; Subcellular targeting; Drug delivery systems; Decoration with targeting residues; Targeting efficiency;

Display OmittedIn drug formulations containing polymer excipients, the effects of the polymer on the dissolved free drug concentration and resulting dissolution or release can be important, especially for poorly soluble drugs. In this study, an in vitro method based on pulsatile microdialysis (PMD) was developed to quantitatively determine dissolved free concentrations of drugs in the presence of polymers in aqueous media in situ (e.g., in place within the system being characterized). Formulations were made by dissolving various ratios of the drug griseofulvin and polymer PVP K30 in water and allowing the mix to equilibrate. A PMD probe was immersed in each mixture and the dissolved free drug concentrations were determined in the PMD samples. The experimental procedure and the equations used for data analysis are presented. To assess the consistency of data, a binding model was fit to the data obtained using PMD by calculating the dissolved free drug fraction fD for each drug–polymer ratio in solution, and obtaining the product of the binding stoichiometry and binding constant (νK per mole of polymer) from the slope of a plot of (1−fD )/fD vs. the molar polymer concentration. For comparison, equilibrium binding experiments were also performed at 23 C, and the determined value of νK was similar to the value found using PMD. Experiments were performed at three temperatures, and a plot of ln (νK) vs. 1/T was linear and a binding enthalpy of −110.9 ± 4.4 J/mol of monomer was calculated from its slope. It was concluded that PMD can be used to determine the dissolved free drug concentrations in situ, which allows characterization of the drug–polymer interaction, even for low drug concentrations. This information may be important in modeling the dissolution or release of drugs from formulations containing polymers.
Keywords: Pulsatile microdialysis; Drug–polymer interaction; Dissolved free drug; Dissolved free fraction; Binding enthalpy; In situ;

Display OmittedAn increased number of amorphous formulations of poorly water soluble drugs are being introduced into the market due to their higher transient solubility and thus faster absorption and higher bioavailability. While most amorphous drug products contain a single drug substance, there is a growing trend towards co-formulating compounds in the same dosage form to improve patient compliance. The purpose of the present work was to evaluate the dissolution behavior and maximum achievable solution concentrations of amorphous solid dispersions of co-formulated ritonavir and lopinavir, and to compare the results with individual amorphous solid dispersion formulations. Dispersions of ritonavir and lopinavir were prepared in polyvinylpyrrolidone (PVP) or hydroxypropylmethylcellulose acetate succinate (HPMCAS) at a 20% (w/w) total drug loading, both alone and in combination, at three different lopinavir:ritonavir weight ratios. Amorphous films containing both drugs, but no polymer, were also prepared. The dissolution behavior of the dispersions and the amorphous films in non-sink conditions was evaluated, using ultracentrifugation to separate any colloidal material from molecularly dissolved drug. Nanoparticle tracking analysis was used to characterize colloidal material formed during the dissolution process. Results from the dissolution study revealed that, although supersaturated solutions resulted following dissolution, the maximum achievable concentration of each drug, when present in combination, was dramatically lower than when the individual dispersions were dissolved. The maximum achievable solution concentration for systems containing both drugs was found to decrease as the mole fraction of the drug in the amorphous phase decreased. The type of polymer used to formulate the dispersion also appeared to influence the dissolution behavior whereby the HPMCAS dispersions dissolved rapidly, resulting in the generation of a nanodroplets, while the PVP dispersions did not produce as many colloidal species. These results highlight the need to consider potential decreases in achievable supersaturation for formulations containing more than one amorphous compound.
Keywords: Combination products; Amorphous; Solubility; Dissolution;

Drug release from slabs and the effects of surface roughness by George Kalosakas; Dimitra Martini (291-298).
Display OmittedWe discuss diffusion-controlled drug release from slabs or thin films. Analytical and numerical results are presented for slabs with flat surfaces, having a uniform thickness. Then, considering slabs with rough surfaces, the influence of a non-uniform slab thickness on release kinetics is numerically investigated. The numerical release profiles are obtained using Monte Carlo simulations. Release kinetics is quantified through the stretched exponential (or Weibull) function and the resulting dependence of the two parameters of this function on the thickness of the slab, for flat surfaces, and the amplitude of surface fluctuations (or the degree of thickness variability) in case of roughness. We find that a higher surface roughness leads to a faster drug release.
Keywords: Controlled drug release; Slabs/thin films; Surface roughness; Diffusion; Monte Carlo simulations; Stretched exponential release curves;

An evaluation of the adhesion of solid oral dosage form coatings to the oesophagus by John D. Smart; Sian Dunkley; John Tsibouklis; Simon Young (299-303).
An evaluation of the adhesion of solid oral dosage form coatings to the oesophagus.Display OmittedThere is a requirement for the development of oral dosage forms that are adhesive and allow extended oesophageal residence time for localised therapies, or are non-adhesive for ease of swallowing. This study provides an initial assessment of the in vitro oesophageal retention characteristics of several widely utilised pharmaceutical coating materials. To this end, a previously described apparatus has been used to measure the force required to pull a coated disc-shaped model tablet across a section of excised oesophageal tissue. Of the materials tested, the well-studied mucoadhesive polymer sodium alginate was found to be associated with significant oesophageal adhesion properties that was capable of ‘self-repairing’. Hydroxypropylmethylcellulose exhibited less pronounced bioadhesive behaviour and blending this with plasticiser or with low molecular weight polymers and surfactants did not significantly affect this. Low molecular weight water soluble polymers, were found to behave similarly to the uncoated glass control disc. Polysorbates exhibited bioadhesion behaviour that was majorly influenced by the nature of the surfactant. The insoluble polymer ethylcellulose, and the relatively lipophilic surfactant sorbitan monooleate were seen to move more readily than the uncoated disc, suggesting that these may have a role as ‘easy-to-swallow’ coatings.
Keywords: Oesophageal adhesion; Easy-to-swallow; Mucoadhesion; Non-adhesive coatings;

An in-vitro exploration of permeation enhancement by novel polysulfonate thiomers by Arshad Mahmood; Sonja Bonengel; Flavia Laffleur; Muhammad Ijaz; Gintare Leonaviciute; Andreas Bernkop-Schnürch (304-313).
Display OmittedThe study was aimed to synthesize preactivated polysulfonate thiomers using poly(4-styrenesulfonic acid-co-maleic acid) (PSSA-MA) and to evaluate their permeation enhancing properties. PSSA-MA-cysteamine (PC) conjugates with 2-mercaptonicotinic acid (2MNA) having different degree of preactivation (PC1608-2MNA, PC2300-2MNA, PC3100-2MNA) were synthesized from the subsequent PSSA-MA-cysteamine thiomers (PC1608, PC2300 and PC3100). The permeation-enhancing features were evaluated by in-vitro models using low-molecular size marker sodium fluorescein (Na-Flu) and with high-molecular size marker fluorescein isothiocyanate-dextran (FD4). Associating the influence of degree of preactivation on permeation enhancement, following rank order PC3100-2MNA > PC3100 > PC2300-2MNA > PC2300 > PC1608-2MNA > PC1608 > PSSA-MA > control was observed on Caco-2 cell monolayers and with little change in sequence on freshly excised rat intestine. The apparent permeability (P app) was improved 3.16-fold for Na-Flu and 3.51-fold for FD4 on Caco-2 cell monolayers. Similarly, 4.17- and 3.60-fold improved P app values were observed on freshly excised rat intestine for Na-Flu and FD4, respectively. More pronounced permeation effects on rat intestine compared to Caco-2 cell monolayer by the thiomer/preactivated conjugates indicated their mucus-interpenetration capability in addition to mucoadhesion. Thus relatively low molecular weight (LMW) preactivated polysulfonate thiomers could to a higher extent enhance permeation on membranes covered by mucus layer compared to high molecular weight thiomers/preactivated conjugates that usually exhibit higher permeation enhancing effects on Caco-2 cell monolayer.
Keywords: Poly(4-styrenesulfonic acid-co-maleic acid); Thiomer; Preactivated conjugate; Permeation enhancing; Mucus interpenetration;

Display OmittedOral thin film (OTF) is a preparation of postage stamp size, with advantages of flexible, tasty and without water for oral administration. A commercial product (Zentrip®) was developed for people who suffered from motion sickness. In order to improve the mechanical strength of Zentrip®, OTF containing meclizine hydrochloride (MH) was designed and prepared using the solvent casting method. The characteristics of the prepared OTF were evaluated using micrometer, auto stripping tester, DSC, X-ray diffraction. ATR-FTIR was employed to investigate the interaction between drug and polymer. The thickness of MH OTF obtained was 0.116 ± 0.004 mm, the tensile strength was 17.37 ± 1.54 N mm−2 and the drug dissolution at 5 min was more than 80% both in distilled water and 0.1 mol/L HCL. DSC and XRD showed MH was amorphous in the polymer. ATR-FTIR indicated the MH molecules inserted into the network structure of polymer, which resulted in an inhibition of drug recrystallization. The C max of Zentrip® and MH OTF were 1.46 ± 0.44 μg/mL and 1.91 ± 0.51 μg/mL, and the AUC were 10.38 ± 2.93 μg h/mL and 13.74 ± 3.23 μg h/mL, respectively. Compared with Zentrip®, MH OTF successfully overcome the weakness of mechanical strength, possessed faster dissolution profile and showed bioequivalence in pharmacokinetics, deserving to a further development.
Keywords: Oral thin film; Meclizine hydrochloride; Tensile strength; Dissolution rate; Drug-polymer interaction; Bioequivalence;

Display OmittedGenipin, a natural and non-toxic cross linker, was used to prepare cross linked floating kappa carrageenan/sodium carboxymethyl cellulose hydrogels and the effect of genipin on hydrogels characterization was investigated. Calcium carbonates were employed as gas forming agents. Ranitidine hydrochloride was used as drug. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA) were carried out to study the changes in the characteristics of hydrogels. Furthermore, scanning electron microscope (SEM) was performed to study microstructure of hydrogels. The result showed that all formulated hydrogels had excellent floating behavior. It was discovered that the cross linking reaction showed significant effect on gel strength, porosity and swelling ratio compared to non-cross linked hydrogels. It was found that the drug release was slower and lesser after being cross linked. Microstructure study shows that cross linked hydrogels exhibited hard and rough surface. Therefore, genipin can be an interesting cross linking agent for controlled drug delivery in gastrointestinal tract.
Keywords: Floating hydrogel; Genipin cross linking; Kappa carrageenan/sodium carboxymethyl cellulose; Characterization;

Stabilization and immune response of HBsAg encapsulated within poly(lactic-co-glycolic acid) microspheres using HSA as a stabilizer by Wenjuan Xu; Jintian He; Guanghao Wu; Fangfang Xiong; Huijuan Du; Gaizhen Wang (332-341).
SEC-HPLC assay showed that co-encapsulation of HSA could significantly inhibit HBsAg aggregation and preserve the stability of HBsAg during microsphere preparation process and release period.Display OmittedThe aim of this study was to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres containing hepatitis B virus surface antigen (HBsAg) using human serum albumin (HSA) as a stabilizer. Lyophilization and emulsification of HBsAg solution with dichloromethane caused a considerable loss of HBsAg antigenicity. Thus, the effects of HSA and trehalose on HBsAg recovery during lyophilization and emulsification were investigated. Adding HSA to HBsAg solutions significantly improved antigen recovery to >90% during lyophilization and emulsification. The effects of co-encapsulated HSA on the characteristics of the PLGA microspheres and stability of HBsAg released from the microspheres were also investigated. The in vitro release test showed that HBsAg was released from the PLGA microspheres continuously over seventy days. A large amount of released HBsAg was inactive without co-encapsulation of HSA. On the contrary, with HSA co-encapsulation, the released HBsAg retained approximately 90% of its antigenicity. The single injection of the HBsAg-HSA-loaded PLGA microspheres in rats resulted in higher anti-HBsAg IgG and Th1 cytokine levels than the single injection of the HBsAg-loaded microspheres or two injections of the conventional aluminum-adjuvanted HBsAg vaccine. Based on these findings, the HBsAg-HSA-loaded PLGA microspheres could be an effective carrier for HBsAg and form a promising depot system.
Keywords: Hepatitis B virus surface antigen; Vaccine stabilization; Microsphere; Poly(d,l-lactic-co-glycolic acid); Human serum albumin; Single-shot vaccine;

Proliposome powders prepared using a slurry method for the generation of beclometasone dipropionate liposomes by Iftikhar Khan; Sakib Yousaf; Sneha Subramanian; Oshadie Korale; Mohamed Albed Alhnan; Waqar Ahmed; Kevin M.G. Taylor; Abdelbary Elhissi (342-350).
Display OmittedA novel “slurry method” was described for the preparation of proliposome powders using soya phosphatidylcholine (SPC) with cholesterol (1:1) and for incorporation of beclometasone dipropionate (BDP) at 2 mole% of the total lipid phase. Proliposomes made with a range of lipid to sucrose carrier ratios were studied in terms of surface morphology using scanning electron microscopy (SEM) and thermal properties using differential scanning calorimetry (DSC). Following hydration of proliposomes, the resultant vesicles were compared to liposomes made using the traditional proliposome method, in terms of vesicle size and drug entrapment efficiency. SEM showed that sucrose was uniformly coated with lipid regardless of lipid to carrier ratio. Liposomes generated using the slurry proliposome method tended to have smaller median size than those generated with the conventional proliposome method, being in the range of 4.72–5.20 μm and 5.89–7.72 μm respectively. Following centrifugation of liposomes using deuterium oxide (D2O) as dispersion medium, vesicles entrapping BDP were separated as a floating creamy layer, whilst the free drug was sedimented as crystals. Drug entrapment was dependent on formulation composition and preparation method. When 1:15 w/w lipid to carrier was used, liposomes generated using the slurry method had an entrapment efficiency of 47.05% compared to 18.67% for those generated using the conventional proliposome method. By contrast, liposomes made by the thin-film hydration method had an entrapment efficiency of 25.66%. DSC studies using 50 mole% BDP demonstrated that the drug was amorphous in the proliposome formulation and tended to crystallize on hydration, resulting in low drug entrapment. In conclusion, a novel approach to the preparation of proliposomes using a slurry method has been introduced, offering higher entrapment for BDP than liposomes made using the conventional proliposome method and those prepared by thin-film hydration technique.
Keywords: Characterization; Drug development; Proliposome; Liposome; Manufacture;

Ex vivo evaluation of a microneedle array device for transdermal application by Sunaina Indermun; Yahya E. Choonara; Pradeep Kumar; Lisa C. du Toit; Girish Modi; Sandy van Vuuren; Regina Luttge; Viness Pillay (351-359).
Display OmittedA new approach of transdermal drug delivery is the use of microneedles. This promising technique offers the potential to be broadly used for drug administration as it enables the dramatic increase in permeation of medicaments across the stratum corneum. The potential of microneedles has evolved to spawn a plethora of potential transdermal applications. In order to advance the microneedle capabilities and possibly revolutionize advanced drug delivery, this study introduces a novel transdermal electro-modulated hydrogel–microneedle array (EMH–MNA) device composed of a nano-porous, embeddable ceramic microneedle array as well as an optimized EMH for the electro-responsive delivery of indomethacin through the skin. The ex vivo permeation as well as drug release experiments were performed on porcine skin tissue to ascertain the electro-responsive capabilities of the device. In addition, the microbial permeation ability of the microneedles across the viable epidermis in both microneedle-punctured skin as well as hypodermic needle-punctured skin was determined. Ex vivo evaluation of the EMH–MNA device across porcine skin demonstrated that without electro-stimulation, significantly less drug release was obtained (±0.4540 mg) as compared to electro-stimulation (±2.93 mg).
Keywords: Electro-modulated hydrogel; Microneedles; Micro-organisms; Transdermal drug delivery; Indomethacin;

Hybrid aerogel preparations as drug delivery matrices for low water-solubility drugs by Peter Veres; Ana M. López-Periago; István Lázár; Javier Saurina; Concepción Domingo (360-370).
Designed hybrid aerogels (silica & gelatin) for immediate and sustained release of hydrophobic acid drugs.Display OmittedA comprehensive study of 14 hybrid aerogels of different composition with applications in drug delivery has been carried out. The overall objective was to modulate the release behavior of drug-impregnated aerogels, from an almost instantaneous release to a semi-retarded delivery prolonged during several hours, through internal surface functionalization. The designed hybrid aerogels were composed of silica and gelatin and functionalized with either phenyl, long (16) hydrocarbon chain or methyl moiety. As model systems, three class II active agents (pK a  < 5.5), ibuprofen, ketoprofen and triflusal, were chosen to impregnate the aerogels. The work relied on the use of supercritical fluid technology for both the synthesis and functionalization of the hybrid aerogels, as well as for the impregnation with an active agent using supercritical CO2 as a solvent. For the impregnated aerogels, in vitro release profiles were recorded under gastric and intestinal pH-conditions using HPLC techniques. The release behavior observed for the three studied drugs was explained considering the measured dissolution profiles of the crystalline drugs, the aerogel composition and its functionalization. Such features are considered of great interest to tailor the bioavailability of drugs with low water solubility.
Keywords: Supercritical CO2; Hybrid aerogel; Silica; Gelatin; Acid drugs;

Design and evaluation of surface and adjuvant modified PLGA microspheres for uptake by dendritic cells to improve vaccine responses by Aiala Salvador; Kerrie J. Sandgren; Frank Liang; Elizabeth A. Thompson; Richard A. Koup; José Luis Pedraz; Rosa Maria Hernandez; Karin Loré; Manoli Igartua (371-381).
Display Omitted
Keywords: PLGA; Human primary dendritic cells; Polyethyleneimine; Monophosphoryl lipid A; Polyinosinic-polycytidilic acid; α-galactosylceramide;

Display OmittedThe poor aqueous solubility of BCS Class II drugs represents a major challenge for oral dosage form development. Using celecoxib (CXB) as model drug, the current study adopted a novel solid phospholipid nanoparticle (SPLN) approach and compared the effect of two commonly used industrial manufacturing methods, spray- and freeze-drying, on the solubility and dissolution enhancement of CXB. CXB was formulated with Phospholipoid E80 (PL) and trehalose at different CXB:PL:trehalose ratios, of which 1:10:16 was the optimal formulation. Spherical amorphous SPLNs with average diameters <1 μm were produced by spray-drying; while amorphous ‘matrix’-like structures of solid PL dispersion with larger particle sizes were prepared by freeze-drying. Formulations from both methods significantly enhanced the dissolution rates, apparent solubility, and molecularly dissolved concentration of CXB in phosphate buffer (PBS, pH 6.5) and in biorelevant fasted state simulated intestinal fluid (FaSSIF, pH 6.5) (p  < 0.05). While similar dissolution rates were found, the spray-dried SPLNs had a larger enhancement in apparent solubility (29- to 132-fold) as well as molecular solubility (18-fold) of CXB at equilibrium (p  < 0.05). The strong capability of the spray-dried SPLNs to attain ‘true’ supersaturation state makes them a promising approach for bioavailability enhancement of poorly soluble drugs.
Keywords: Solid phospholipid nanoparticles; Amorphous solid dispersion; Solubility; Spray-drying; Freeze-drying; Celecoxib;

Enhanced antioxidation via encapsulation of isooctyl p-methoxycinnamate with sodium deoxycholate-mediated liposome endocytosis by Yongtai Zhang; Lina Shen; Kai Zhang; Teng Guo; Jihui Zhao; Nana Li; Nianping Feng (392-400).
Display OmittedIsooctyl p-methoxycinnamate(OMC) is a commonly used chemical ultraviolet B sunscreen that suffers rapid degradation with current delivery systems following sun exposure. In this study, deoxycholate-mediated liposome (DOC-LS) endocytosis was employed to improve the antioxidation effects of OMC following topical administration, and the in vitro cell uptake was investigated to understand the enhanced cutaneous absorption of the drug via this nanocarrier. Following topical application, structural changes in the stratum corneum were identified. With the increase of DOC content, the drug deposition in skin decreased; from this, a DOC-LS formulation was selected that showed significantly more drug delivery in skin than did the other preparations (P  < 0.05). DOC-LS decreased skin resistance, suggesting its ability to induce skin barrier disruption. In vitro HaCaT keratinocyte cell uptake of coumarin-6 incorporated in the two types of phosphatidylcholine (PC) vesicles (i.e., LS or DOC-LS) yielded similar fluorescence intensities following incubation for different periods (P  < 0.05). However, CCC-ESF-1 embryonic fibroblast cell uptake of the fluorescence revealed time-dependence, and the emitted light from DOC-LS incubated cells was stronger than that from cells incubated with LS (P  < 0.05). These findings might be associated with the endocytic pathway of HaCaT, which mainly exhibited adsorption or physical adhesion of the fluorescent vesicles, whereas CCC-ESF-1 markedly internalized the PC vesicles via the lysosomes, as shown by intracellular fluorescence co-location studies. Following loading with the same amount of OMC, the DOC-LS vesicles exhibited superior skin tissue antioxidative capacity among the preparations tested, corroborating the in vivo skin drug deposition results. Thus, our results suggest that DOC-LS is a promising system for OMC dermal delivery without promoting skin irritation, which is quite advantageous for therapeutic purposes.
Keywords: Transdermal; Transfersomes; Cell uptake; Superoxide dismutase; Methane dicarboxylic aldehyde;

Liposomally formulated phospholipid-conjugated indocyanine green for intra-operative brain tumor detection and resection by Akiko Suganami; Yasuo Iwadate; Sayaka Shibata; Masamichi Yamashita; Tsutomu Tanaka; Natsuki Shinozaki; Ichio Aoki; Naokatsu Saeki; Hiroshi Shirasawa; Yoshiharu Okamoto; Yutaka Tamura (401-406).
Display OmittedSome tumor-specific near-infrared (NIR) fluorescent dyes such as indocyanine green (ICG), IDRye800CW, and 5-aminolevulinic acid have been used clinically for detecting tumor margins or micro-cancer lesions. In this study, we evaluated the physicochemical properties of liposomally formulated phospholipid-conjugated ICG, denoted by LP-iDOPE, as a clinically translatable NIR imaging nanoparticle for brain tumors. We also confirmed its brain-tumor-specific biodistribution and its characteristics as the intra-operative NIR imaging nanoparticles for brain tumor surgery. These properties of LP-iDOPE may enable neurosurgeons to achieve more accurate identification and more complete resection of brain tumor.
Keywords: Brain tumor surgery; Indocyanine green (ICG); Liposome; Near-infrared (NIR); Intra-operative imaging;

Measuring the sticking of mefenamic acid powders on stainless steel surface by Zihua Wang; Umang V. Shah; Dolapo Olusanmi; Ajit S. Narang; Munir A. Hussain; John F. Gamble; Michael J. Tobyn; Jerry Y.Y. Heng (407-413).
Display OmittedThis study proposes an approach for quantifying the amount of pharmaceutical powder adhering (quality attribute) to the metals surfaces. The effect of surface roughness (detrimental attribute) on the amount of powder sticking to a stainless steel surface for a model pharmaceutical material is also qualitatively determined. Methodology to quantify powder adhesion to surfaces utilises a texture analyser and HPLC. The approach was validated to qualitatively investigate effect of metal surface roughness on adhesion of mefenamic acid. An increase in metal surface roughness resulted in an increase in cohesion. By increasing the average roughness from 289 nm to 407 nm, a 2.5 fold increase in amount adhering to metal was observed, highlighting the role of surface roughness on adhesion. The simplicity in experimental design with no requirement of specialised equipment and operational ease makes the approach very easy to adopt. Further, ease in interpreting results makes this methodology very attractive.
Keywords: Adhesion quantification; Sticking; Surface roughness; Optical profilometer; HPLC;

Fabrication of controlled-release budesonide tablets via desktop (FDM) 3D printing by Alvaro Goyanes; Hanah Chang; Daniel Sedough; Grace B. Hatton; Jie Wang; Asma Buanz; Simon Gaisford; Abdul W. Basit (414-420).
Display OmittedThe aim of this work was to explore the feasibility of using fused deposition modelling (FDM) 3D printing (3DP) technology with hot melt extrusion (HME) and fluid bed coating to fabricate modified-release budesonide dosage forms. Budesonide was sucessfully loaded into polyvinyl alcohol filaments using HME. The filaments were engineered into capsule-shaped tablets (caplets) containing 9 mg budesonide using a FDM 3D printer; the caplets were then overcoated with a layer of enteric polymer. The final printed formulation was tested in a dynamic dissolution bicarbonate buffer system, and two commercial budesonide products, Cortiment® (Uceris®) and Entocort®, were also investigated for comparison. Budesonide release from the Entocort® formulation was rapid in conditions of the upper small intestine while release from the Cortiment® product was more delayed and very slow. In contrast, the new 3D printed caplet formulation started to release in the mid-small intestine but release then continued in a sustained manner throughout the distal intestine and colon. This work has demonstrated the potential of combining FDM 3DP with established pharmaceutical processes, including HME and film coating, to fabricate modified release oral dosage forms.
Keywords: Three dimensional printing; Modified release; Fused filament fabrication; Budesonide; Inflammatory bowel disease; Colonic delivery;

Evaluation of the physicochemical and functional stability of diluted REMSIMA ® upon extended storage—A study compliant with NHS (UK) guidance by Benjamin L. Young; Monika Ali Khan; Terry J. Chapman; Richard Parry; Maria A. Connolly; Andrew G. Watts (421-431).
Display OmittedA newly licensed biosimilar product containing infliximab as the active pharmaceutical ingredient has recently been marketed under the brand name Remsima®. We have evaluated the stability of Remsima® diluted in sodium chloride solution and stored in polyolefin bags at 2–8 °C using a range of techniques to assess the physico-chemical and functional integrity of the drug over time. The methods and techniques employed are fully compliant with NHS (UK) guidance for evaluating the stability of biologicals, enabling the data to be used for the application of an extended shelf-life to Remsima products in the UK, when prepared under a Section 10 exemption or a Specials Licence. The results clearly demonstrate physico-chemical and functional stability of the drug over the 7 day period of the study, when prepared as described here under aseptic conditions in accordance with the Summary of manufacturers Product Characteristics.
Keywords: Remsima; Infliximab; Biosimilar; Antibody; Shelf-life; Extended storage; Compliance; Stability study;

Innovative intragastric ascaridole floating tablets: Development, optimization, and in vitro–in vivo evaluation by Qiang Zhao; Baoan Gao; Lulu Ma; Jianhao Lian; Li Deng; Jianming Chen (432-439).
Display OmittedGastro-floating tablets of ascaridole, a volatile oil were developed to prolong the gastric residence time and thereby, enhance local therapeutic efficacy. The tablets were optimized and prepared by direct compression techniques using hydroxypropylmethylcellulose (HPMC K15M) and polyethylene oxide (PEO WSRN-750) as hydrophilic matrices and calcium carbonate (CaCO3) as a gas-generating agent.In vitro evaluation of the prepared tablets was performed by determining the hardness, friability, content uniformity, and weight variation. In addition, floating lag time, total floating time, and drug release behavior were evaluated. Finally, optimized tablets were subjected to stability and in vivo gamma scintigraphy studies. Results showed that the formulated tablets were white, smooth, and flat in appearance and met the Chinese Pharmacopoeia (ChP) criteria for weight variation, drug content, and friability. The tablets had satisfactory buoyancy and sustained drug release profile that followed non-Fickian kinetics. In vivo gamma scintigraphy suggests that the floating tablet did not adhere to the stomach mucous but were retained in the stomach for extended periods of 5.80 ± 0.50 h following administration, indicating that gastro retentive time of ascaridole tablets increased owing to the floating principle.
Keywords: Ascaridole; Intragastric floating tablet; Evaluation; Drug release; Gamma scintigraphy;

Neochamaejasmin B increases the bioavailability of chamaechromone coexisting in Stellera chamaejasme L. via inhibition of MRP2 and BCRP by Lanying Pan; Kui Zeng; Xiangjun Wang; Huichang Bi; Haihong Hu; Ming Huang; Yan Lou; Su Zeng (440-447).
Display OmittedChamaechromone and neochamaejasmin B (NCB) are the most abundant components in the dried roots of the toxic perennial herb Stellera chamaejasme L. and have pharmacological activities. The objective of this study was to investigate the transport mechanism of these two components in vivo and in vitro. The transport and cellular accumulation studies in Madin–Darby canine kidney (MDCK) cells overexpressing human multidrug resistance protein 2 (MRP2) or P-gp and LLC-PK1 cells overexpressing human breast cancer resistance protein (BCRP) were performed. The results showed that chamaechromone was a good substrate of MRP2 and BCRP but not a substrate of P-gp. NCB was found to be a MRP2 inhibitor in transfected cells and significantly enhanced the cellular accumulation of chamaechromone in MDCK cells overexpressing MRP2. Similar results were obtained in LLC-PK1-BCRP cells. In addition, the influence of NCB on the bioavailability of chamaechromone following their co-administration was also determined in rats. The results showed that the area under the plasma concentration–time curve and maximal plasma concentration of chamaechromone in rats were increased by 48.9% and 81.9%, respectively. The mechanism of improving the oral bioavailability of chamaechromone was attributable to the inhibition of the BCRP and MRP2-mediated efflux of chamaechromone by NCB.
Keywords: Chamaechromone; Neochamaejasmin B; MRP2; BCRP; Bioavailability; Inhibition;

Display OmittedThe tack of a pressure sensitive adhesive (PSA) is not an inherent material property and strongly depends on the measurement conditions. Following the concept of a measurement system analysis (MSA), influencing factors of the probe tack test were investigated by a design of experiments (DoE) approach. A response surface design with 38 runs was built to evaluate the influence of detachment speed, dwell time, contact force, adhesive film thickness and API content on tack, determined as the maximum of the stress strain curve (σ max). It could be shown that all investigated factors have a significant effect on the response and that the DoE approach allowed to detect two-factorial interactions between the dwell time, the contact force, the adhesive film thickness and the API content. Surprisingly, it was found that tack increases with decreasing and not with increasing adhesive film thickness.
Keywords: Pressure sensitive adhesive; Probe tack test; Rheology; Measurement system analysis; Design of experiments; Transdermal patch;

Display OmittedThe amorphous form of a drug has higher water solubility and faster dissolution rate than its crystalline form. However, the amorphous form is less thermodynamically stable and may recrystallize during manufacturing and storage. Maintaining the amorphous state of drug in a solid dosage form is extremely important to ensure product quality. The purpose of this study was to quantitatively determine the amount of amorphous indomethacin (INDO) formed in the Soluplus® solid dispersions using thermoanalytical and Fourier transform infrared (FTIR) spectral curve-fitting techniques. The INDO/Soluplus® solid dispersions with various weight ratios of both components were prepared by air-drying and heat-drying processes. A predominate IR peak at 1683 cm−1 for amorphous INDO was selected as a marker for monitoring the solid state of INDO in the INDO/Soluplus® solid dispersions. The physical stability of amorphous INDO in the INDO/Soluplus® solid dispersions prepared by both drying processes was also studied under accelerated conditions. A typical endothermic peak at 161 °C for γ-form of INDO (γ-INDO) disappeared from all the differential scanning calorimetry (DSC) curves of INDO/Soluplus® solid dispersions, suggesting the amorphization of INDO caused by Soluplus® after drying. In addition, two unique IR peaks at 1682 (1681) and 1593 (1591) cm−1 corresponded to the amorphous form of INDO were observed in the FTIR spectra of all the INDO/Soluplus® solid dispersions. The quantitative amounts of amorphous INDO formed in all the INDO/Soluplus® solid dispersions were increased with the increase of γ-INDO loaded into the INDO/Soluplus® solid dispersions by applying curve-fitting technique. However, the intermolecular hydrogen bonding interaction between Soluplus® and INDO were only observed in the samples prepared by heat-drying process, due to a marked spectral shift from 1636 to 1628 cm−1 in the INDO/Soluplus® solid dispersions. The INDO/Soluplus® solid dispersions prepared by both drying processes could keep the amorphous state of INDO in the INDO/Soluplus® solid dispersions at the accelerated storage condition.
Keywords: Indomethacin (INDO); Soluplus®; Amorphous; Solid dispersion; DSC; FTIR; Curve-fitting technique; Physical stability;

Modified local diatomite as potential functional drug carrier—A model study for diclofenac sodium by Jelena Janićijević; Danina Krajišnik; Bojan Čalija; Bojana Nedić Vasiljević; Vladimir Dobričić; Aleksandra Daković; Milan D. Antonijević; Jela Milić (466-474).
Display OmittedDiatomite makes a promising candidate for a drug carrier because of its high porosity, large surface area, modifiable surface chemistry and biocompatibility. Herein, refined diatomite from Kolubara coal basin, which complied with the pharmacopoeial requirements for heavy metals content and microbiological quality, was used as a starting material. Inorganic modification of the starting material was performed through a simple, one-step procedure. Significant increase in adsorbent loading with diclofenac sodium (DS) was achieved after the modification process (∼373 mg/g) which enabled the preparation of comprimates containing therapeutic dose of the adsorbed drug. Adsorption of DS onto modified diatomite resulted in the alteration of the drug’s XRD pattern and FTIR spectrum. In vitro drug release studies in phosphate buffer pH 7.5 demonstrated prolonged DS release over 8 h from comprimates containing DS adsorbed on modified diatomite (up to 37% after 8 h) and those containing physical mixture of the same composition (up to 45% after 8 h). The results of in vivo toxicity testing on mice pointed on potential safety of both unmodified (starting) and modified diatomite. All these findings favor the application of diatomite as a potential functional drug carrier.
Keywords: Diatoms; Porous silica; Inorganic modification; Adsorption; Drug delivery; Prolonged drug release;

Interactions of dendritic glycopolymer with erythrocytes, red blood cell ghosts and membrane enzymes by Dominika Wrobel; Anna Janaszewska; Dietmar Appelhans; Brigitte Voit; Maria Bryszewska; Jan Maly (475-488).
Display OmittedInteractions between maltose functionalized hyperbranched poly(ethylene imine)s (95% maltose decoration denoted as Mal-PEI A; 33% maltose decoration denoted as Mal-PEI B) and red blood cells (RBCs) and between red blood cell membranes were investigated. We monitored the degree of hemolysis, the change in cell shape, the influence of polymers on the fluidity of the cell membrane and some cell membrane enzymes to determine their possible cytotoxic impact on them. To observe the extent of hemolysis, the RBCs were incubated with different concentrations of Mal-PEIs. The first significant lysis of RBCs was observed after 6 h of incubation. Prolongation of the incubation time increased the number of ruptured cells. Moreover, we observed that Mal-PEI B was more hemolytic than Mal-PEI A in buffer solution. In contrast, an incubation of RBCs with Mal-PEIs in human plasma significantly decreased the hemolytic process and showed higher hemolytic property of Mal-PEI A compared to Mal-PEI B. Also several changes in the shape of the RBCs occurred after incubation with Mal-PEIs. Some of the erythrocytes shrank (echinocytes), but their morphology generally remained unchanged during the incubation. As shown by fluorescence experiments, both polymers induced the increase of fluidity of RBCs membranes. In summary, both types of hyperbranched poly(ethylene imine)s were practically non-hemolytic even at high polymer concentrations. Mal-PEI B was slightly more noxious than the Mal-PEI A in a buffer solution, while in blood plasma, the situation was opposite. Decrease of Na+/K+ ATPase and total ATPase enzymes activity was related with molecule size and number of maltose groups on the surface of molecule. The low hemolytic properties only observed at higher concentration (100 μM and 400 μM) indicated that Mal-PEIs are promising macromolecules in the area of drug delivery systems.
Keywords: Dendritic glycopolymer; Erythrocytes; Hemolysis; Nanoparticles;

Improvement of mechanical properties of pellet containing tablets by thermal treatment by Zsombor Csobán; Barnabás Kállai-Szabó; Nikolett Kállai-Szabó; István Sebe; Péter Gordon; István Antal (489-496).
Display OmittedBatches of partially spray-dried lactose tablets with three different initial tensile strength (∼20 N, ∼35 N, ∼50 N) were made. Changes along a 24 h long thermal treatment at 100 °C in tensile strength, friability, individual mass, water content, disintegration time, average free volume and wetting properties were evaluated. Caffeine containing gastroresistant pellets were gained by drug layering and filmcoating of inert microcrystalline cellulose pellet cores in fluid bed equipment. Shape, size, mechanical properties, drug content and dissolution profile of the coated pellets were determined. Batches of pellet containing tablets with three different pellet-filler ratios were compressed where partially spray-dried lactose was used as a filler-binder material.Characteristics of pellet containing tablets were evaluated before and after a 24 h long thermal treatment at 100 °C. Results shown that the poor initial mechanical properties (friability, tensile strength) were improved by thermal exposure while there were no remarkable alterations in drug release profiles.
Keywords: Tablet hardness; Thermal treatment; Reduced friability; Multiple-unit pellet system (MUPS); Spray-dried lactose; Dissolution profile;

Carboxymethyl starch mucoadhesive microspheres as gastroretentive dosage form by Marc Lemieux; Patrick Gosselin; Mircea Alexandru Mateescu (497-508).
Display OmittedCarboxymethyl starch microspheres (CMS-MS) were produced from carboxymethyl starch powder (CMS-P) with a degree of substitution (DS) from 0.1 to 1.5 in order to investigate the influence of DS on physicochemical, drug release and mucoadhesion properties as well as interactions with gastrointestinal tract (GIT) epithelial barrier models. Placebo and furosemide loaded CMS-MS were obtained by emulsion-crosslinking with sodium trimetaphosphate (STMP). DS had an impact on increasing equilibrium water uptake and modulating drug release properties of the CMS-MS according to the surrounding pH. The transepithelial electrical resistance (TEER) of NCI-N87 gastric cell monolayers was not influenced in presence of CMS-MS, whereas that of Caco-2 intestinal cell monolayers decreased with increasing DS but recovered initial values at about 15 h post-treatment. CMS-MS with increasing DS also enhanced furosemide permeability across both NCI-N87 and Caco-2 monolayers at pH gradients from 3.0 to 7.4. Mucoadhesion of CMS-MS on gastric mucosa (acidic condition) increased with the DS up to 55% for a DS of 1.0 but decreased on neutral intestinal mucosa to less than 10% with DS of 0.1. The drug release, permeability enhancement and mucoadhesive properties of the CMS-MS suggest CMS-MS with DS between 0.6 and 1.0 as suitable excipient for gastroretentive oral delivery dosage forms.
Keywords: Carboxymethyl starch microspheres; Drug delivery; Cell viability; Permeability; Mucoadhesion; Gastroretentive dosage form;

Endocytosis of fluorescent cyclodextrins by intestinal Caco-2 cells and its role in paclitaxel drug delivery by Katalin Réti-Nagy; Milo Malanga; Éva Fenyvesi; Lajos Szente; György Vámosi; Judit Váradi; Ildikó Bácskay; Pálma Fehér; Zoltán Ujhelyi; Eszter Róka; Miklós Vecsernyés; György Balogh; Gábor Vasvári; Ferenc Fenyvesi (509-517).
Display OmittedCyclodextrins are widely used excipients in pharmaceutical formulations. They are mainly utilized as solubilizers and absorption enhancers, but recent results revealed their effects on cell membranes and pharmacological barriers. In addition to the growing knowledge on their interaction with plasma membranes, it was confirmed that cyclodextrins are able to enter cells by endocytosis. The number of the tested cyclodextrins was limited, and the role of this mechanism in drug absorption and delivery is not known. Our aim was to examine the endocytosis of fluorescently labeled hydroxypropyl-β-cyclodextrin, random methyl-β-cyclodextrin and soluble β-cyclodextrin polymer, and the cellular uptake of the fluorescent paclitaxel derivative-random methyl-β-cyclodextrin complex. The studied cyclodextrin derivatives were able to enter Caco-2 intestinal cells and localized in vesicles in the cytoplasm, while their permeability was very limited through Caco-2 monolayers. We demonstrated for the first time that the fluorescent paclitaxel derivative and rhodamine-labeled random methyl-β-cyclodextrin were detected in the same intracellular vesicles after treating cells with their inclusion complex. These results indicate that the endocytosis of cyclodextrin complexes can contribute to drug absorption processes.
Keywords: Cyclodextrin; Endocytosis; Caco-2; Paclitaxel;

Optimised transdermal delivery of pravastatin by Cornel Burger; Minja Gerber; Jan L. du Preez; Jeanetta du Plessis (518-525).
Display OmittedWiechers’ programme “Formulating for Efficacy” initiated a new strategy to optimise the oil phase of topical formulations in order to achieve optimal transdermal drug delivery. This new approach uses the “Delivery Gap Theory” on any active pharmaceutical ingredients (APIs) to test if it could enhance transdermal drug delivery. The aim of the study was to formulate six different semi-solid formulations (three creams and three emulgels) with 2% pravastatin as the API in order to investigate the “Delivery Gap Principle”, by determining which formulation would deliver pravastatin best to the target-site (system circulation). The three cream- and three emulgel formulations had different polarities, i.e. a formulation with polarity equal to that of the stratum corneum (optimised), a non-polar (lipophilic)- and a polar (hydrophilic)-formulation. Franz cell diffusion studies were executed over 12 h and the optimised emulgel (2.578 μg/cm2) had the highest median amount per area obtained. Tape stripping followed the diffusion studies and in the stratum corneum–epidermis, the hydrophilic emulgel (1.448 μg/ml) contained the highest median pravastatin concentration and the epidermis–dermis the optimised emulgel (0.849 μg/ml) depicted the highest pravastatin concentration. During this study, it was observed that when both emulgel and cream formulations were compared; the emulgels enhanced the delivery of pravastatin more than the creams.
Keywords: Pravastatin; Wiechers; Transdermal delivery; Formulation; Delivery gap theory; Franz cell;

Display OmittedThe aim of this research was to prepare and characterize an alginate-based wound dressing containing vancomycin immobilized at the silica surface. The silica samples functionalized with amine, diol and carboxylic acid groups were loaded with 7.8, 5.7 and 7.1 wt.% of the antibiotic respectively. The immobilized drug was encapsulated in alginate or gelatin/alginate gels and the average concentration of vancomycin was about 10 mg per g of the dried gel. The effect of functional organic groups at the silica surface on the release rate of the drug was investigated. Only the drug immobilized at Si-amine in alginate matrix was found to demonstrate slower release from the proposed wound dressing. The in vitro release profiles for other silica carriers did not show significant differences in relation to the free loaded drug. The presence of gelatin had a favourable impact on the slowing down of the drug release from the dressing with a double barrier. All the gels studied with vancomycin immobilized at the silica surface demonstrated antimicrobial activity against various bacteria. A reduction of the drug dose to a half had no effect on changing microbiological activity of gels.
Keywords: Vancomycin; Alginate; Silica; Release profile; Biological activity; Wound dressing;

Display OmittedThe foreign body reaction is the major cause of the dysfunction and relatively short lifetime associated with implanted glucose biosensors. An effective strategy to maintain sensor functionality is to apply biocompatible coatings that elute drug to counter the negative tissue reactions. This has been achieved using dexamethasone releasing poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a polyvinyl alcohol (PVA) hydrogel coating. Accordingly, the biosensor lifetime relies on the duration and dose of drug release from the coating. To achieve long-term drug release mixed populations of microspheres have been used. In the current study, microspheres were prepared by blending low (25 KDa) and high (113 KDa) molecular weight PLGA at different mass ratios to overcome problems associated with mixing multiple populations of microspheres. “Real-time” in vitro studies demonstrated dexamethasone release for approximately 5 months. An accelerated method with discriminatory ability was developed to shorten drug release to less than 2 weeks. An in vivo pharmacodynamics study demonstrated efficacy against the foreign body reaction for 4.5 months. Such composite coatings composed of PLGA microspheres prepared using polymer blends could potentially be used to ensure long-term performance of glucose sensors.
Keywords: Polymer blends; PLGA; Microspheres; Foreign body reaction; Glucose biosensors; Long-term drug release; Accelerated release testing;

Additive manufacturing of scaffolds with dexamethasone controlled release for enhanced bone regeneration by Pedro F. Costa; Ana M. Puga; Luis Díaz-Gomez; Angel Concheiro; Dirk H. Busch; Carmen Alvarez-Lorenzo (541-550).
Display OmittedThe adoption of additive manufacturing in tissue engineering and regenerative medicine (TERM) strategies greatly relies on the development of novel 3D printable materials with advanced properties. In this work we have developed a material for bone TERM applications with tunable bioerosion rate and dexamethasone release profile which can be further employed in fused deposition modelling (the most common and accessible 3D printing technology in the market). The developed material consisted of a blend of poly-ϵ-caprolactone (PCL) and poloxamine (Tetronic®) and was processed into a ready-to-use filament form by means of a simplified melt-based methodology, therefore eliminating the utilization of solvents. 3D scaffolds composed of various blend formulations were additively manufactured and analyzed revealing blend ratio-specific degradation rates and dexamethasone release profiles. Furthermore, in vitro culture studies revealed a similar blend ratio-specific trend concerning the osteoinductive activity of the fabricated scaffolds when these were seeded and cultured with human mesenchymal stem cells. The developed material enables to specifically address different regenerative requirements found in various tissue defects. The versatility of such strategy is further increased by the ability of additive manufacturing to accurately fabricate implants matching any given defect geometry.
Keywords: Additive manufacturing; Tissue engineering; Regenerative medicine; Bone regeneration; Controlled drug release;

New polymorphs of 9-nitro-camptothecin prepared using a supercritical anti-solvent process by Yinxia Huang; Hongdi Wang; Guijin Liu; Yanbin Jiang (551-560).
Display OmittedRecrystallization and micronization of 9-nitro-camptothecin (9-NC) has been investigated using the supercritical anti-solvent (SAS) technology in this study. Five operating factors, i.e., the type of organic solvent, the concentration of 9-NC in the solution, the flow rate of 9-NC solution, the precipitation pressure and the temperature, were optimized using a selected OA16 (45) orthogonal array design and a series of characterizations were performed for all samples. The results showed that the processed 9-NC particles exhibited smaller particle size and narrower particle size distribution as compared with 9-NC raw material (Form I), and the optimum micronization conditions for preparing 9-NC with minimum particle size were determined by variance analysis, where the solvent plays the most important role in the formation and transformation of polymorphs. Three new polymorphic forms (Form II, III and IV) of 9-NC, which present different physicochemical properties, were generated after the SAS process. The predicted structures of the 9-NC crystals, which were consistent with the experiments, were performed from their experimental XRD data by the direct space approach using the Reflex module of Materials Studio. Meanwhile, the optimal sample (Form III) was proved to have higher cytotoxicity against the cancer cells, which suggested the therapeutic efficacy of 9-NC is polymorph-dependent.
Keywords: 9-Nitro-camptothecin; Polymorph; Micronization; Supercritical anti-solvent process; solvent; Physicochemical properties; Cytotoxicity;

Functional stability of 3D8 scFv, a nucleic acid-hydrolyzing single chain antibody, under different biochemical and physical conditions by Joungmin Lee; Hyunjoon Park; Minjae Kim; Youngsil Seo; Yeonjin Lee; Sung June Byun; Sukchan Lee; Myung-Hee Kwon (561-570).
Display Omitted3D8 single-chain Fv (scFv) is a catalytic nucleic acid antibody with anti-viral activity against a broad spectrum of viruses. Here we investigated the functional stability of 3D8 scFv to provide a basis for engineering a 3D8 scFv derivative and for developing stable formulations with improved stability and potential use as an anti-viral agent. The stability of 3D8 scFv was assessed by measuring its DNA-hydrolyzing activity under different biochemical and physical conditions using a fluorescence resonance energy transfer (FRET)-based method. In addition, the anti-influenza (H9N2) effect of 3D8 scFv was evaluated in A549 cells. 3D8 scFv was stable at 50 °C for 6 h at pH 7.2, for 3 days at pH 4–10 at 37 °C and 30 days at pH 4–8 at 37 °C. The stability was not affected by a reducing condition, freeze–thawing for up to 30 cycles, or lyophilization. Evaluation of the anti-virus effect showed that cells treated with 32–128 units of 3D8 scFv showed a 50% decrease in influenza replication compared to untreated cells. Based on its enzymatic stability in various biochemical and physical environments, 3D8 scFv holds good potential for development as an anti-viral therapeutic.
Keywords: Antibody stability; Anti-nucleic acid antibody; Catalytic antibody; DNA hydrolysis; Anti- influenza activity;

Twin screw wet granulation: Effect of process and formulation variables on powder caking during production by Mohammed F. Saleh; Ranjit M. Dhenge; James J. Cartwright; Michael J. Hounslow; Agba D. Salman (571-582).
Display OmittedThis work focuses on monitoring the behaviour and the mass of the built up/caking of powder during wet granulation using Twin Screw Granulator (TSG). The variables changed during this work are; powder (α-lactose monohydrate and microcrystalline cellulose (MCC)), the screw configuration (conveying and kneading elements) and the weight percentage of hydroxypropyl-methyl cellulose (HPMC) dissolved in the granulation liquid (i.e. changing liquid viscosity). Additionally, the effect of these variables on the size distribution, of the granules produced, was determined. The experiments were conducted using an acrylic transparent barrel. A stainless steel barrel was then used to conduct the two extreme granulation liquid viscosities with two different screw configurations, using lactose only. This was done to compare the findings to those obtained from the transparent barrel for validation purpose.These variables showed to affect the behaviour and the mass of the powder caking as well as the size distribution of granules. Overall, the use of kneading element resulted in uniform behaviour in caking with higher mass. Furthermore, increasing the amount of HPMC resulted in a reduction of the mass of powder caking for lactose, while showing inconsistent trend for MCC. Furthermore, lactose showed to have a greater tendency to cake in comparison to MCC.The results, for lactose, obtained from the stainless steel barrel compared well with their corresponding conditions from the transparent barrel, as the screw configuration and HPMC mass varied.
Keywords: Twin screw; Screw configuration; HPMC; Viscosity; Online caking monitoring; Continuous granulation;

Development and preclinical pharmacokinetics of a novel subcutaneous thermoresponsive system for prolonged delivery of heparin by Maja Radivojša Matanović; Pegi Ahlin Grabnar; Dario Voinovich; Samuel Golob; Mojca Božič Mijovski; Iztok Grabnar (583-592).
Display OmittedHeparin is still widely used for treatment and prevention of thromboembolic diseases. Due to specific physicochemical properties, it requires frequent parenteral injections. In this study we present the development and in vitro evaluation of an advanced delivery system for prolonged subcutaneous release of heparin. The delivery system consisted of an in situ forming thermoresponsive poloxamer-based platform combined with pH-responsive polyelectrolyte heparin/chitosan nanocomplexes. Thermoresponsive hydrogels were tested for gelation temperature, gel dissolution and in vitro heparin release, whereas polyelectrolyte nanocomplexes were physico-chemically characterized, as well as tested for in vitro cytotoxicity and in vitro heparin release. Hydrogel combined of two poloxamers demonstrated the highest gelation temperature (28.6 °C), while the addition of hydroxypropyl methylcellulose prolonged gel dissolution. On the other hand, nanocomplexes’ dispersions, prepared at 1:1 heparin/chitosan mass ratio and in the concentration range 0.375–1.875 mg/mL, demonstrated mean diameter <400 nm and zeta potential >34 mV. Pharmacokinetics of selected formulations (thermoresponsive hydrogel, nanocomplexes and a dual system consisting of nanocomplexes incorporated into thermoresponsive hydrogel) were studied in rats. Heparin plasma concentration-time profiles revealed a double-peak phenomenon, probably due to heparin diffusion inside the polymer matrix and gel dissolution. Pharmacokinetic parameters were determined by a non-linear mixed effects modeling approach. It was demonstrated that thermoresponsive hydrogel with heparin/chitosan nanocomplexes enabled the lowest absorption rate of heparin into systemic circulation and provided heparin concentration above the prophylaxis threshold for 5 days. In situ gelling thermoresponsive matrix combined with chitosan nanocomplexes present a promising delivery system for heparin, requiring less frequent administration during long-term treatment.
Keywords: Thermoresponsive hydrogels; Polyelectrolyte complexes; Heparin; Subcutaneous administration; Pharmacokinetic model;

Display OmittedThe aim of the present study was to evaluate the gel-forming polysaccharide psyllium in the preparation of mucoadhesive patches for the controlled release of chlorhexidine (CHX) to treat pathologies in the oral cavity, using the casting-solvent evaporation technique. A number of different film-forming semi-synthetic polymers, such as sodium carboxymethyl cellulose (SCMC) and hydroxypropylmethyl cellulose (HPMC) were evaluated for comparison. The patch formulations were characterized in terms of drug content, morphology surface, swelling and mucoadhesive properties, microbiology inhibition assay and in vitro release tests. Three ex-vivo testswere carried out using porcine mucosa: an alternative dissolution test using artificial saliva that allows contemporary measurement of dissolution and mucoadhesion, a permeation test through the mucosa and the measurement of mucoadhesion using a Nouy tensile tester, as the maximum force required for the separation of the patch from the mucosa surface. The patches were also examined for determination of the minimum inhibitory concentration in cultures of Escherichia coli and Staphylococcus aureus. All the patches incorporating psyllium were found suitable in terms of external morphology, mucoadhesion and controlled release of the drug: in the presence of psyllium the drug displays prolonged zero-order release related to slower swelling rate of the system.
Keywords: Buccal patch; Chlorhexidine base; Psyllium; Swelling; Mucoadhesion; Controlled release; Antibacterial activity;

Display OmittedThe present study evaluates the feasibility of particulate carriers of a biodegradable polymer polyethylene sebacate (PES) as an alternative to Freund’s adjuvant in the design of a peptide vaccine formulation. The vaccine formulation comprised of PES and the antigen KLH conjugated 80 kDa HSA peptide-1 dissolved in N-methyl-2-pyrrolidone (NMP)/NMP-water as solvent. The antigen revealed good stability and the formulations were readily syringeable. Intradermal injection of the formulations resulted in the formation of PES particulates in situ at the site of injection. The NMP formulations revealed larger particulates which elicited no immunogenic response when injected in rabbits. On the other hand the NMP-water formulation revealed formation of microparticles which were significantly smaller in size, in combination with a small fraction of nanoparticles. It elicited an antibody titer up to 1:3200 in rabbits following intradermal injection. Western blot confirmed generation of antibodies specific to the peptide. Contraceptive efficacy was confirmed by loss of sperm motility and head-to-head agglutination of sperms in the treatment group. Unlike the severe reactions observed with administration of Freund’s adjuvant, only mild hypersensitivity reaction was observed with the PES formulations. The mild reaction coupled with the contraceptive efficacy observed suggested PES particulates as a viable alternative to Freund’s adjuvant.
Keywords: Contraceptive peptide vaccine; 80 kDa HSA peptide-1; Polyethylene sebacate; Nanoparticles and microparticles; Freund adjuvant;

Effect of the surface free energy of materials on the lamination tendency of bilayer tablets by Kitti Papós; Péter Kása; Ilija Ilič; Sandra Blatnik-Urek; Géza Regdon; Stane Srčič; Klára Pintye-Hódi; Tamás Sovány (609-613).
Display OmittedDosage forms with fixed dose combinations of drugs is a frequent and advantageous mode of administration, but their production involves a number of technological problems. Numerous interactions in a homogeneous vehicle may be avoided through the use of layered tablets. The mechanical properties of these dosage forms depend on numerous process parameters and material characteristics. The aim of the present study was a detailed investigation of the relationships between the surface characteristics and deformation properties of tableting materials and the tendency of bilayer tablets to undergo lamination.Bilayer tablets were compressed from unlubricated materials with different plastic–elastic properties and surface free energies according to a mixed 2 and 3-level half-replicated factorial design. The results revealed that the surface characteristics play the main role in the lamination of layered tablets and the effect of the plastic–elastic behavior cannot be interpreted without a knowledge of these properties.
Keywords: Bilayer tablets; Lamination tendency; Surface free energy; Plasto-elastic behaviour;

Display OmittedViral vectors are common tools in gene therapy to deliver foreign therapeutic sequences in a specific target population via their natural cellular entry mechanisms. Incorporating such vectors in implantable systems may provide strong alternatives to conventional gene transfer procedures. The goal of the present study was to generate different hydrogel structures based on alginate (AlgPH155) and poloxamer PF127 as new systems to encapsulate and release recombinant adeno-associated viral (rAAV) vectors. Inclusion of rAAV in such polymeric capsules revealed an influence of the hydrogel composition and crosslinking temperature upon the vector release profiles, with alginate (AlgPH155) structures showing the fastest release profiles early on while over time vector release was more effective from AlgPH155 + PF127 [H] capsules crosslinked at a high temperature (50 °C). Systems prepared at room temperature (AlgPH155 + PF127 [C]) allowed instead to achieve a more controlled release profile. When tested for their ability to target human mesenchymal stem cells, the different systems led to high transduction efficiencies over time and to gene expression levels in the range of those achieved upon direct vector application, especially when using AlgPH155 + PF127 [H]. No detrimental effects were reported on either cell viability or on the potential for chondrogenic differentiation. Inclusion of PF127 in the capsules was also capable of delaying undesirable hypertrophic cell differentiation. These findings are of promising value for the further development of viral vector controlled release strategies.
Keywords: rAAV gene transfer; Alginate/poloxamer systems; Human MSCs; Differentiation potential; Release profile; Hydrogel structure;

Display OmittedMelt granulation in fluid bed processors is an emerging technique, but literature data regarding the modeling of this granulation method are lacking. In the present study different techniques (response surface analysis, multilayer perceptron neural network, and partial least squares method) were applied for modeling of spray-on fluidized bed melt granulation. Experiments were organized in line with central composite design. The effect of binder content and spray air pressure on granule properties was evaluated. The results obtained indicate that binder content can be identified as a critical factor controlling the granule size and size distribution. It was found that agglomeration mechanism involved, i.e., granule shape, can be greatly influenced by binder properties. The spray air pressure was identified as critical process parameter affecting granule flowability. The results presented indicate that application of in silico tools enables enhanced understanding and better control of novel pharmaceutical processes, such as melt granulation in fluidized bed. The artificial neural networks and partial least squares method were found to be superior to response surface methodology in prediction of granule properties. According to the results obtained, application of more advanced empirical modeling techniques complementary to design of experiments can be a suitable approach in defining the design space and optimization of spray-on fluidized bed melt granulation.
Keywords: Spray-on melt granulation; Fluid bed; Experimental design; Artificial neural networks; Response surface methodology; Partial least squares;

Amperometric monitoring of quercetin permeation through skin membranes by Jadwiga Rembiesa; Hala Gari; Johan Engblom; Tautgirdas Ruzgas (636-643).
Display OmittedTransdermal delivery of quercetin (QR, 3,3′,4′,5,7-pentahydroxyflavone), a natural flavonoid with a considerable antioxidant capacity, is important for medical treatment of, e.g., skin disorders. QR permeability through skin is low, which, at the same time, makes the monitoring of percutaneous QR penetration difficult. The objective of this study was to assess an electrochemical method for monitoring QR penetration through skin membranes. An electrode was covered with the membrane, exposed to QR solution, and electrode current was measured. The registered current was due to electro-oxidation of QR penetrating the membrane. Exploiting strict current-QR flux relationships diffusion coefficient, D, of QR in skin and dialysis membranes was calculated. The D values were strongly dependent on the theoretical model and parameters assumed in the processing of the amperometric data. The highest values of D were in the range of 1.6–6.1 × 10−7  cm2/s. This was reached only for skin membranes pretreated with buffer-ethanol mixture for more than 24 h. QR solutions containing penetration enhancers, ethanol and l-menthol, definitely increased D values. The results demonstrate that electrochemical setup gives a possibility to assess penetration characteristics as well as enables monitoring of penetration dynamics, which is more difficult by traditional methods using Franz cells.
Keywords: Quercetin; Flavonoids; Electrochemistry; Skin permeation; Penetration enhancers; Diffusion coefficient;

Display OmittedCisplatin (CDDP) is widely used anticancer drug for various solid tumors including lung cancer. However, its indiscriminate distribution causes serious adverse effects and limits its therapeutic effect. In this study, by using hyaluronic acid (HA) we synthesized a complex of CDDP (HA-CDDP), by utilizing ionic interaction between Pt2+ of CDDP with carboxyl group of HA. The mean HA-CDDP particle size was 208.5 nm in PBS according to dynamic light scattering which was also confirmed by TEM, which could exert tumor-targeting property by enhanced permeability and retention (EPR) effect. The CDDP loading in this preparation was 13% (w/w), and release rate of free CDDP from the HA-CDDP complex at physiological pH (7.4) was ∼20%/day. However, in acidic pH the release was much faster, i.e., ∼95% of CDDP was released in 72 h at pH 5.5. Moreover, HA-CDDP showed a 2.5-fold higher tumor accumulation than free CDDP whereas no increase of distribution was found in most normal tissues. In addition, because HA receptor CD44 is overexpressed in many tumor cells, we also observed CD44-based endocytosis of HA-CDDP in mouse lung carcinoma LCC cells. These findings together suggest that HA-CDDP may show tumor-selective cytotoxicity by taking advantage of EPR effect, weak acidic environment of tumor tissues (e.g., pH 6 ∼ 7), as well as CD44-based intracellular uptake. As expected, HA-CDDP exhibited much improved therapeutic effect than free CDDP in mouse LCC tumor model, whereas no apparent side effect was found. These findings may shed some light on the potential utility of HA for development of tumor-targeted polymeric CDDP drugs, which need further investigations.
Keywords: EPR effect; Tumor targeting; Antitumor therapy; Hyaluronic acid; Cisplatin;

Display OmittedThe manufacture of porous polycaprolactone (PCL) scaffolds containing three different drugs, namely 5-fluorouracil, nicotinamide and triflusal, was investigated in this work with the aim of obtaining bioactive systems with controlled drug delivery capabilities. The scaffolds were prepared by means of a supercritical CO2 (scCO2) foaming technique by optimizing the drug loading process. This was achieved by dissolving the drugs in organic solvents miscible with scCO2 and by mixing these drug/solvent solutions with PCL powder. The as prepared mixtures were further compressed to eliminate air bubbles and finally processed by the scCO2 foaming technique. ScCO2 saturation and foaming conditions were optimized to create the porosity within the samples and to allow for the concomitant removal of the organic solvents. Physical and chemical properties of porous scaffolds, as well as drug content and delivery profiles, were studied by HPLC. The results of this study demonstrated that the composition of the starting PCL/drug/solvent mixtures affected polymer crystallization, scaffold morphology and pore structure features. Furthermore, it was found that drug loading efficiency depended on both initial solution composition and drug solubility in scCO2. Nevertheless, in the case of highly scCO2-soluble drugs, such as triflusal, loading efficiency was improved by adding a proper amount of free drug inside of the pressure vessel. The drug delivery study indicated that release profiles depended mainly upon scaffolds composition and pore structure features.
Keywords: Drug release; Polycaprolactone; Pore structure; Scaffolds; scCO2 foaming;

Impact of the nature and concentration of plasticizers on the ability of PVC to sorb drug by H. Al Salloum; J. Saunier; C. Aymes-Chodur; H. Barakat; N. Yagoubi (664-675).
Display OmittedThe sorption of a drug by an infusion set may dramatically reduce the drug delivery efficiency. In this paper, we investigated how the drug sorption, in static conditions, is affected by the plasticizer’s nature and ratio in the case of plasticized PVC, one of the most common material for infusion set tubing. Within the study, the drug concentration in diazepam solutions was studied after contact with PVC films containing different amounts of DEHP, DEHT, TOTM and DINCH® plasticizers. Moreover the partition coefficients between material and water were calculated. The drug sorption levels were equivalent for the different plasticizers and there was a plasticizer ratio for which the drug uptake was enhanced. As a consequence, the amount of sorbed drug might not be only linked to the amount of plasticizer in the film and to the solubility of the drug in the plasticizer alone: it must probably depend on specific interactions between plasticizer and PVC.
Keywords: Plasticizer; DEHP; PVC; Drug sorption; Partition coefficients; Diazepam;

Factors influencing native and acylated peptide release from biodegradable MPs in gel combination.Display OmittedPolymeric microparticles (MPs)-in-gel formulations for extended delivery of octreotide were developed. We investigated influence of polymer composition on acylation of octreotide and kinetics of release during in vitro release from biodegradable polymeric formulations. Polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA) and polyethylene glycol (PEG) based triblock (TB ≈ PCL10k-PEG2k-PCL10k) and pentablock (PBA ≈ PLA3k-PCL7k-PEG2k-PCL7k-PLA3k and PBB ≈ PGA3k-PCL7k-PEG2k-PCL7k-PGA3k) polymers were investigated. Octreotide was encapsulated in MPs using methanol–oil/water emulsion solvent evaporation method. The particles were characterized for size, morphology, encapsulation efficiency, drug loading and in vitro release. Release samples were subjected to HPLC analysis for quantitation and HPLC-MS analysis for identification of native and chemically modified octreotide adducts. Entrapment efficiency of methanol-oil/water method with TB, PBA and PBB polymers were 45%, 60%, and 82%, respectively. A significant fraction of released octreotide was acylated from lactide and glycolide based PBA (53%) and PBB (92%) polymers. Substantial amount of peptide was not released from PBB polymers after 330 days of incubation. Complete release of octreotide was achieved from TB polymer over a period of 3 months with minimal acylation of peptide (13%). PCL based polymers resulted in minimal acylation of peptide and hence may be suitable for extended peptide and protein delivery. Conversely, polymers having PLA and PGA blocks may not be appropriate for peptide delivery due to acylation and incomplete release.
Keywords: Polycaprolactone; Polyglycolic acid; Polylactic acid; Hydrogel; Peptide; Microparticles; Drug delivery; Sustained delivery; Peptide chemical stability;

Starch-free grewia gum matrices: Compaction, swelling, erosion and drug release behaviour by E.I. Nep; K. Asare-Addo; M.U. Ghori; B.R. Conway; A.M. Smith (689-698).
Display OmittedPolysaccharides are suitable for application as hydrophilic matrices because of their ability to hydrate and swell upon contact with fluids, forming a gel layer which controls drug release. When extracted from plants, polysaccharides often contain significant quantities of starch that impacts upon their functional properties. This study aimed to evaluate differences in swelling, erosion and drug release from matrix tablets prepared from grewia gum (GG) and starch-free grewia gum (GDS) extracted from the stems of Grewia mollis. HPMC was used as a control polymer with theophylline as a model drug. Swelling, erosion, and in-vitro release were performed in deionized water, pH 1.2 and pH 6.8 media. The Vergnaud and Krosmeyer-Peppas model were used for swelling and drug release kinetics, respectively. However, linear regression technique was used to determine the erosion rate. GDS compacts were significantly harder than the native GG and HPMC compacts. GDS matrices exhibited the fastest erosion and drug release in deionised water and phosphate buffer compared with the GG and HPMC. At pH 1.2, GDS exhibited greater swelling than erosion, and drug release was similar to GG and HPMC. This highlights the potential of GDS as a matrix for controlled release similar to HPMC and GG at pH 1.2 but with a more rapid release at pH 6.8. GDS may have wider application in reinforcing compacts with relatively low mechanical strength.
Keywords: HPMC K4M; Grewia gum; Starch-free grewia gum; Matrix tablets; Theophylline;

GX1-mediated anionic liposomes carrying adenoviral vectors for enhanced inhibition of gastric cancer vascular endothelial cells by Dan Xiong; Zhongbing Liu; Tierong Bian; Juan Li; Wenjun Huang; Pei Jing; Li Liu; Yunlong Wang; Zhirong Zhong (699-708).
Display OmittedGastric cancer is a highly lethal malignancy and its 5-year survival rate remains depressed in spite of multiple treatment options. Targeting drug delivery to tumor vasculature may be a promising strategy for gastric cancer therapy, for it can block the nutrition source of tumor and inhibit the metastasis and invasion in a certain extent. In present study, we have prepared the drug-targeting delivery system of peptide GX1-mediated anionic liposomes carrying adenoviral vectors (GX1-Ad5-AL), in which the tumor suppressor gene of PTEN was integrated into DNA of Ad5 and the GX1 peptide could play targeting role to vascular of gastric cancer. The inhibition ability of GX1-Ad5-AL to human gastric cancer cell lines (SGC-7901) and human umbilical vein endothelial cells (HUVEC) was evaluated by MTT assay. Further, the cell migration assay was carried out in transwell inserts and the cells uptaking of GX1-Ad5-AL was detected by confocal laser scanning microscopy. The experimental results indicated that the average cell proliferation inhibition rates resulted from the drug delivery system of GX1-Ad5-AL in SGC-7901 and HUVEC were 68.36% and 64.13%, respectively which were higher than that resulted from GX1 or Ad5-AL. Meanwhile, results of cell migration experiment demonstrated that GX1-Ad5-AL could significantly suppress the migration of gastric cancer cell of SGC-7901. Moreover, both the imaging from confocal laser scanning microscopy and the quantitative analysis of fluorescence intensity showed that, GX1-Ad5-AL was more easily uptaken by SGC-7901 cells, as compared to Ad5-AL. Therefore, the formulation of GX1-Ad5-AL was effective for enhancing the inhibition effect and suppressing the migration of gastric cancer vascular endothelial cells.
Keywords: Peptide GX1; Anionic liposomes; Adenoviral vectors; Gastric cancer vascular endothelial cells; Cell migration; Cell proliferation inhibition;

Encapsulation of volatiles by homogenized partially-cross linked alginates by Pavan K. Inguva; Shing Ming Ooi; Parind M. Desai; Paul W.S. Heng (709-716).
Display OmittedCross-linked calcium alginate gels are too viscous to be efficaciously incorporated into spray dried formulations. Thus, viscosity reduction is essential to ensure the processability of calcium alginate gels to be sprayed. Viscosity reduction by high pressure homogenization can open new formulation possibilities. Presently, testing of microcapsule integrity is also limited because either single particle tests neglect collective particle behaviours in bulk or bulk testing methods are often associated with single compressions which may not fully characterize individual particle strengths. The aim of this study was sub-divided into three objectives. First objective was to evaluate the impact of high pressure homogenization on gel viscosity. Second objective was to explore the use of the homogenized gels with modified starch for microencapsulation by spray drying. The final objective was to develop a stamping system as microcapsule strength tester that can assess microcapsules in bulk and evaluate the impact of multiple compressions. Collectively, this study would lead towards developing a pressure-activated patch of microcapsules with encapsulated volatiles and the method to assess the patch efficacy. The alginate gels largely experienced an exponential decay in viscosity when homogenized. Furthermore, the homogenized gels were successfully incorporated in spray drying formulations for microencapsulation. The custom-designed microcapsule strength tester was successfully used and shown to possess the required sensitivity to discern batches of microcapsules containing volatiles to have different release profiles. Addition of homogenized gels strengthened the microcapsules only at high wall to core ratios with low mass-load alginate gels. High mass-load gels weaken the microcapsules, exhibiting a higher release at low stamping pressures and wrinkling on the microcapsules surface.
Keywords: Cross-linked alginates; High pressure homogenization; Viscosity; Spray drying; Microencapsulation; Volatiles release; Pressure-activated patch;

A linear free energy analysis of PAMPA models for biological systems by Jia He; Michael H. Abraham; William E. Acree; Yuan H. Zhao (717-722).
PAMPA processes • do not correlate with biological processes ○ except for intestinal absorption □.Display OmittedEquation coefficients in the Abraham linear free energy relationship have been obtained for a number of parallel artificial membrane permeability assays, PAMPA, and have been compared to equation coefficients for several biological processes and a large number of water-solvent partitions. From a principal component analysis of the equation coefficients we show that one particular PAMPA (Wohnsland and Faller, 2001) is a good model for human intestinal absorption, but we could find no PAMPA that was a good model for rat brain permeation, mouse brain permeation, blood–brain distribution, skin permeation or water–skin partition, although models that include PAMPA and other descriptors may be useful. The water–octanol system is a poor model for all the biological process we have studied, but water-nonpolar solvent partitions are good models for permeation through egg lecithin membranes.
Keywords: PAMPA; Linear free energy relationship; Abraham equation; Water-solvent partitions; Principal component analysis;

Comparative studies on the properties of glycyrrhetinic acid-loaded PLGA microparticles prepared by emulsion and template methods by Hong Wang; Guangxing Zhang; Hong Sui; Yanhua Liu; Kinam Park; Wenping Wang (723-731).
Display OmittedThe O/W emulsion method has been widely used for the production of poly (lactide-co-glycolide) (PLGA) microparticles. Recently, a template method has been used to make homogeneous microparticles with predefined size and shape, and shown to be useful in encapsulating different types of active compounds. However, differences between the template method and emulsion method have not been examined. In the current study, PLGA microparticles were prepared by the two methods using glycyrrhetinic acid (GA) as a model drug. The properties of obtained microparticles were characterized and compared on drug distribution, in vitro release, and degradation. An encapsulation efficiency of over 70% and a mean particle size of about 40 μm were found for both methods. DSC thermograms and XRPD diffractograms indicated that GA was highly dispersed or in the amorphous state in the matrix of microparticles. The emulsion method produced microparticles of a broad size distribution with a core–shell type structure and many drug-rich domains inside each microparticle. Its drug release and matrix degradation was slow before Day 50 and then accelerated. In contrast, the template method formed microparticles with narrow size distribution and drug distribution without apparent drug-rich domains. The template microparticles with a loading efficiency of 85% exhibited a zero-order release profile for 3 months after the initial burst release of 26.7%, and a steady surface erosion process as well. The same microparticles made by two different methods showed two distinguished drug release profiles. The two different methods can be supplementary with each other in optimization of drug formulation for achieving predetermined drug release patterns.
Keywords: PLGA microparticles; Hydrogel template method; O/W emulsion method; Glycyrrhetinic acid;

Nano-curcumin prepared via supercritical: Improved anti-bacterial, anti-oxidant and anti-cancer efficacy by Maobin Xie; Dejun Fan; Zheng Zhao; Zhi Li; Gang Li; Yufeng Chen; Xiaowen He; Aizheng Chen; Jiashen Li; Xiaofen Lin; Min Zhi; Yi Li; Ping Lan (732-740).
Display OmittedCurcumin (CM) possesses multiple biological activities. However, poor water solubility and low bioavailability limit its application in biomedical fields. CM nanoparticles (NPs) (230–240 nm) were prepared by solution-enhanced dispersion via supercritical CO2 (SEDS) (22–22.5 MPa pressure, 31–32.5 °C temperature) and its biological functions were evaluated in this study. The Minimum inhibitory concentration of CM NPs against S. aureus (∼250 μg/mL) was lower than CM-DMSO (∼500 μg/mL). Meanwhile, CM NPs showed effective anti-oxidant ability at a concentration raging from 125 to 2000 μg/mL. CM NPs showed time-dependent intracellular internalization ability, resulting in an enhanced anti-cancer effect on colorectal cancer cells (HCT116), and the mechanism could be explained by cell cycle arrest in G2/M phase associated with inducing apoptotic cells. Moreover, CM NPs exhibited reduced cytotoxicity on normal cells (NCM460) compared to CM-DMSO and 5-Fu. In conclusion, CM NPs prepared via SEDS showed potentials in biomedical applications.
Keywords: Supercritical CO2; Curcumin; Anti-bacterial; Anti-oxidant; Anti-cancer;

Molecular interaction between glimepiride and Soluplus®-PEG 4000 hybrid based solid dispersions: Characterisation and anti-diabetic studies by Joy Nneji Reginald-Opara; Anthony Attama; Kenneth Ofokansi; Chukwuebuka Umeyor; Frankline Kenechukwu (741-750).
Display OmittedThe objective of this study was to evaluate a novel blend of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol 6000 grafted copolymer (Soluplus®) and polyethylene glycol (PEG) 4000 for solubility enhancement, physicochemical stability and anti-diabetic efficacy of the produced solid dispersions containing glimepiride, a biopharmaceutics classification system (BCS) class II sulphonylurea. Different batches of glimepiride solid dispersions (SD) were prepared by the solvent evaporation method using the individual polymers and blends of the polymers at different ratios. The Soluplus®-PEG 4000 (sol-PEG) hybrid polymer based glimepiride solid dispersions were characterized by differential scanning calorimetry (DSC), fourier transform infrared (FTIR) spectroscopy, micromeritics and dissolution studies. In vivo anti-diabetic activity was determined by measuring the changes in blood glucose concentrations in albino rats. The solid dispersions showed good flow properties and excellent practical yield. Drug content and release from the different formulations increased when Soluplus® was used as the main matrix polymer. The kinetics of drug release from all the solid dispersions followed first order. Solid state characterization confirmed the formation of amorphous glimepiride solid dispersions in the Sol-PEG hybrid polymer and no strong drug–polymer interaction was observed. The blood glucose reduction in albino rats by the Sol-PEG-Glim SDs was significantly (p  < 0.05) higher and more sustained when compared with the plain drug sample and commercially available product. Optimized SD batches (SP1 and SP3) showed a reduction in blood glucose level from 100% to 9.81% and 8.97%, respectively, at T max of 3 h. The Sol-PEG-Glim SD was found to be stable over a period of 6 months (at 40 °C, 70% RH) with no significant changes in the drug content. Thus, the Sol-PEG polymeric hybrids represent a promising tool for enhanced delivery of glimepiride.
Keywords: Solid dispersions; Anti-diabetic; Hybrid polymers; Bioavailability; Soluplus®; Dissolution studies; Glimepiride;

Influence of in line monitored fluid bed granulation process parameters on the stability of Ethinylestradiol by Katrin Roßteuscher-Carl; Sabine Fricke; Michael C. Hacker; Michaela Schulz-Siegmund (751-758).
Display OmittedEthinylestradiol (EE) as a highly active and low dosed compound is prone to oxidative degradation. The stability of the drug substance is therefore a critical parameter that has to be considered during drug formulation. Beside the stability of the drug substance, granule particle size and moisture are critical quality attributes (CQA) of the fluid bed granulation process which influence the tableting ability of the resulting granules. Both CQA should therefore be monitored during the production process by process analytic technology (PAT) according to ICH Q8.This work focusses on the effects of drying conditions on the stability of EE in a fluid-bed granulation process. We quantified EE degradation products 6-alpha-hydroxy-EE, 6-beta-hydroxy-EE, 9(11)-dehydro-EE and 6-oxo-EE during long time storage and accelerated conditions. PAT-tools that monitor granule particle size (Spatial filtering technology) and granule moisture (Microwave resonance technology) were applied and compared with off-line methods. We found a relevant influence of residual granule moisture and thermic stress applied during granulation on the storage stability of EE, whereas no degradation was found immediately after processing. Hence we conclude that drying parameters have a relevant influence on long term EE stability.
Keywords: Fluid bed granulation; Ethinylestradiol; Process analytical technologies; Spatial filtering technology; Microwave resonance technology;

Formulation and in-vitro evaluation of directly compressed controlled release matrices of Losartan Potassium using Ethocel Grade 100 as rate retarding agent by Kamran Ahmad Khan; Gul Majid Khan; Muhammad Zeeshan Danish; Akhlaq; Haroon Khan; Fazal Rehman; Saifullah Mehsud (759-765).
Display OmittedCurrent study was aimed to develop 200 mg controlled release matrix tablets of Losartan Potassium using Ethocel 100 Premium and Ethocel 100 FP Premium as rate controlling polymer. In-vitro studies were performed according to USP Method-I in phosphate buffer (PH 6.8) using pharma test dissolution apparatus. The temperature of the dissolution medium was kept constant at 37 ± 0.5 °C at 100 rpm. Flow properties, physical quality control tests, effect of polymer size and drug-to-polymers ratios were studied using different kinetics models such as 1st-order, zero-order, Hixon Crowell model, Highuchi model and Power law. Difference factor f 1 and similarity factor f 2 were applied for dissolution profiles against Cardaktin® tablets used as a reference formulation. The matrices with polymer ethocel 100 FP Premiums have prolonged the drug release rate as compared to polymer ethocel 100 Premiums. The n values matrices with polymer ethocel grade 100 ranged from 0.603 to 0.857 indicating that the drug release occurred by anomalous non fickian diffusion kinetics while then value of reference Cardaktin® tablet was measured as 0.125 indicating that these tablets do not follow power law. The dissolution profiles of test formulations were different than that of reference Cardaktin®. This suggests the polymer Ethocel grade 100 can be proficiently incorporated in fabrication and development of once a day controlled release matrix tablets.
Keywords: Losartan Potassium; Controlled release; Ethocel grade 100; Matrix tablets;

Display OmittedThe limited solubility of BCS class II drugs diminishes their dissolution and thus reduces their bioavailability. Our aim in this study was to develop and optimize a spray dried emulsion containing indomethacin as a model for Class II drugs, Labrasol®/Transuctol® mixture as the oily phase, and maltodextrin as a solid carrier. The optimization was carried out using a 23 full factorial design based on two independent variables, the percentage of carrier and concentration of Poloxamer® 188. The effect of the studied parameters on the spray dried yield, loading efficiency and in vitro release were thoroughly investigated. Furthermore, physicochemical characterization of the optimized formulation was performed. In vivo bioavailability, ulcerogenic capability and histopathological features were assessed. The results obtained pointed out that poloxamer 188 concentration in the formulation was the predominant factor affecting the dissolution release, whereas the drug loading was driven by the carrier concentration added. Moreover, the yield demonstrated a drawback by increasing both independent variables studied. The optimized formulation presented a complete release within two minutes thus suggesting an immediate release pattern as well, the formulation revealed to be uniform spherical particles with an average size of 7.5 μm entrapping the drug in its molecular state as demonstrated by the DSC and FTIR studies. The in vivo evaluation, demonstrated a 10-fold enhancement in bioavailability of the optimized formulation, with absence of ulcerogenic side effect compared to the marketed product. The results provided an evidence for the significance of spray dried emulsion as a leading strategy for improving the solubility and enhancing the bioavailability of class II drugs.
Keywords: Dry emulsion; Factorial design; Spray drying; Class II; Bioavailability; Dissolution;

Optical diagnostics study of air flow and powder fluidisation in Nexthaler®—Part I: Studies with lactose placebo formulation by I. Pasquali; C. Merusi; G. Brambilla; E.J. Long; G.K. Hargrave; H.K. Versteeg (780-791).
Display OmittedEffective drug delivery to the lungs by a DPI device requires the air-stream through the device to have sufficient power to aerosolise the powder. Furthermore, sufficient turbulence must be induced, along with particle-wall and particle–particle collisions, in order to de-aggregate small drug particles from large carrier particles. As a result, the emitted and the fine particle doses produced by many commercially available DPI devices tend to be strongly affected by the natural inter-patient variability of the inhaled air flow. The Nexthaler® is a multi-dose breath-actuated dry-powder inhaler with minimum drug delivery-flow rate dependency and incorporating a dose protector. The actuation mechanism of the dose-protector ensures that the dose is only exposed to the inhaled air flow if the flow has sufficient power to cause complete aerosolisation. For this study, a proprietary lactose placebo powder blend was filled into “transparent” Nexthaler® to allow application of high-speed imaging and particle image velocimetry (PIV) techniques to successfully interrogate and reveal details of the powder entrainment and emission processes coupled with characterisation of the flow environment in the vicinity of the mouthpiece exit.The study showed that fluidisation of the bulk of the powder occurs very quickly (∼20 ms) after withdrawal of the dose protector followed by powder emission from the device within ∼50 ms thereafter. The bulk of the metered placebo dose was emitted within 100–200 ms. The visualisation study also revealed that a very small fraction of powder fines is emitted whilst the dose protector still covers the dosing cup as the flow rate through the device accelerates. The PIV results show that the flow exiting the device is highly turbulent with a rotating flow structure, which forces the particles to follow internal paths having a high probability of wall impacts, suggesting that the flow environment inside the Nexthaler® DPI will be very beneficial for carrier-drug de-aggregation.
Keywords: Powder fluidization; Dry powder inhaler; Breath-actuated; Optical diagnostics; High-speed imaging; Particle image velocimetry; Powder emission;

In-line near infrared spectroscopy during freeze-drying as a tool to measure efficiency of hydrogen bond formation between protein and sugar, predictive of protein storage stability by Maarten A. Mensink; Pieter-Jan Van Bockstal; Sigrid Pieters; Laurens De Meyer; Henderik W. Frijlink; Kees van der Voort Maarschalk; Wouter L.J. Hinrichs; Thomas De Beer (792-800).
Display OmittedSugars are often used as stabilizers of protein formulations during freeze-drying. However, not all sugars are equally suitable for this purpose. Using in-line near-infrared spectroscopy during freeze-drying, it is shown here that hydrogen bond formation during freeze-drying, under secondary drying conditions in particular, can be related to the preservation of the functionality and structure of proteins during storage. The disaccharide trehalose was best capable of forming hydrogen bonds with the model protein, lactate dehydrogenase, thereby stabilizing it, followed by the molecularly flexible oligosaccharide inulin 4 kDa. The molecularly rigid oligo- and polysaccharides dextran 5 kDa and 70 kDa, respectively, formed the least amount of hydrogen bonds and provided least stabilization of the protein. It is concluded that smaller and molecularly more flexible sugars are less affected by steric hindrance, allowing them to form more hydrogen bonds with the protein, thereby stabilizing it better.
Keywords: Near-infrared (NIR) spectroscopy; Water-replacement; Vitrification; Molecular flexibility; Solid-state stability; Fourier transform infrared (FTIR) spectroscopy;

Melatonin/HPβCD complex: Microwave synthesis, integration with chitosan scaffolds and inhibitory effects on MG-63CELLS by Bedriye Topal; Damla Çetin Altındal; Menemşe Gümüşderelioğlu (801-811).
Display OmittedIn this study, melatonin/2-hydroxypropyl-β-cyclodextrin (HPβCD) inclusion complex was prepared by using microwave irradiation in a very short time. Phase-solubility studies indicated a linear increase in the solubility of melatonin with HPβCD demonstrating Higuchi’s AL-type phase solubility profiles and the formation of 1:1 stoichiometric inclusion complexes. Also, the ability of inclusion complex formation was promoted at microwave conditions compared to room conditions. The structure of inclusion complex was determined by FTIR, DSC, XRD, 1H NMR and 13C NMR studies. Both of pure melatonin and melatonin/HPβCD inclusion complex were loaded into the chitosan scaffolds that were prepared by freeze-drying method and the effect of released melatonin on human osteosarcoma cells (MG-63) was investigated in vitro. Approximately 9 mM melatonin concentration caused time dependent cell death by reducing the proportion of the cells in the G2/M phase rather than S phase. In conclusion, melatonin/HPβCD inclusion complex loaded chitosan scaffolds could be considered as an alternative system for the human osteosarcoma therapy.
Keywords: 2-Hydroxypropyl-β-cyclodextrin; Melatonin; Microwave; Inclusion complex;

Display OmittedPraziquantel is the drug of choice to treat several parasitic infections including the neglected tropical disease schistosomiasis. Due to its low aqueous solubility, cyclodextrins have been tested as potential host candidates to prepare praziquantel inclusion complexes with improved solubility. For the first time, the interactions of praziquantel with β-cyclodextrin and β-cyclodextrin derivatives (methyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin) were investigated using high resolution solid-state NMR spectroscopy. The results of this work confirmed that solid-state NMR experiments provided structural characterization, demonstrating the formation of inclusion complexes most probably with PZQ adopting an anti conformation, also the most likely in amorphous raw PZQ. Further information on the interaction of praziquantel with methyl-β-cyclodextrin was obtained from proton rotating-frame relaxation time measurements, sensitive to kilohertz-regime motions but modulated by spin-diffusion. Evidences were presented in all cases for praziquantel complexation through the aromatic ring. In addition, 1:2 drug:carrier molar ratio appears to be the most probable and therefore suitable stoichiometry to improve pharmaceutical formulations of this antischistosomal drug.
Keywords: Schistosomiasis disease; Praziquantel; β-cyclodextrin derivatives; Inclusion complex; Solid-state NMR;

Macromolecular crowding of molecular imprinting: A facile pathway to produce drug delivery devices for zero-order sustained release by Lei Tang; Chun-Yan Zhao; Xian-Hua Wang; Rong-Shan Li; Jin-Rong Yang; Yan-Ping Huang; Zhao-Sheng Liu (822-833).
Display OmittedThis paper reported the facile fabrication of drug delivery devices for zero-order sustained release by molecular crowding strategy of molecularly imprinting technology. Crowding-assisted molecularly imprinting polymers (MIPs) matrices were prepared by free-radical precipitation polymerization using aminoglutethimide (AG) as a model drug. The crowding effect was achieved by adding polystyrene as a macromolecular co-solute in pre-polymerization mixture. The MIP prepared under the non-MMC condition and the two corresponding non-imprinted particles were tested as controlled vehicles. The release profiles presented zero-order behaviors from two crowding-assisted polymers, the duration of approximately 18 h for the crowding-assisted MIP and 10 h for the crowding-assisted NIP, respectively while AG were all very rapid released from the other two controlled particles (85% occurring in the first hour). The BET surface area and pore volume of the crowding-assisted MIP were about ten times than those of the controlled MIP. The value of imprinting factor is 6.02 for the crowding-assisted MIP and 1.19 for the controlled MIP evaluated by the equilibrium adsorption experiment. Furthermore, the values of effective diffusivity (D eff) obtained from crowding-assisted MIP (10−17  cm2/s) was about two orders of magnitude smaller than those from the controlled MIP, although the values of free drug diffusivity (D) were all found in the order of 10−13  cm2/s. Compared with the commercial AG tablet, the MMC-assisted MIP gave a markedly high relative bioavailability of 266.3%, whereas the MMC-assisted NIP gave only 57.7%. The results indicated that the MMC condition can modulate the polymer networks approaciate to zero-order release of the drug and maintain the molecular memory pockets, even if under the poor polymerization conditions of MIPs preparation.
Keywords: Molecularly imprinted polymer; Molecular crowding; Zero-order release; Burst effect; Aminoglutethimide;

Absorption of ipratropium and l-carnitine into the pulmonary circulation of the ex-vivo rat lung is driven by passive processes rather than active uptake by OCT/OCTN transporters by Ghaith Al-Jayyoussi; Daniel F. Price; Katharina Kreitmeyr; John P. Keogh; Mathew W. Smith; Mark Gumbleton; Christopher J. Morris (834-841).
Display OmittedThe organic cation transporters OCT and OCTN have been reported to play a significant role in the cellular uptake of substrates within in vitro lung cells. However, no studies to date have investigated the effect of these transporters upon transepithelial absorption of substrates into the pulmonary circulation. We investigated the contribution of OCT and OCTN transporters to total pulmonary absorption of l-carnitine and the anti-muscarinic drug, ipratropium, across an intact isolated perfused rat lung (IPRL). The results obtained from the IPRL were contrasted with active transport in vitro using three human pulmonary cell lines and primary rat alveolar epithelial cells. Ex-vivo studies showed that OCT/OCTN transporters do not play a role in the overall pulmonary absorption of l-carnitine or ipratropium, as evidenced by the effect of chemical inhibition of these transporters upon pulmonary absorption. In contrast, in vitro studies showed that OCT/OCTN transporters play a significant role in cellular accumulation of substrates with preferential uptake of ipratropium by OCTs, and of l-carnitine uptake by OCTNs. The results show that in vitro uptake studies cannot be predictive of airway to blood absorption in vivo. Nevertheless, localised submucosal pulmonary concentrations of inhaled drugs and their pulmonary pharmacodynamic profiles may be influenced by OCT/OCTN transport activity.
Keywords: Organic cation transporters, OCT1, OCT2, OCT3, OCTN; SLC22; Isolated perfused rat lung (IPRL); Transporters; Lung; Pulmonary absorption;

Feasability of a new process to produce fast disintegrating pellets as novel multiparticulate dosage form for pediatric use by Thanh Huong Hoang Thi; Siham Lhafidi; Simone Pinto Carneiro; Marie-Pierre Flament (842-849).
Display OmittedNovel orally disintegrating system based on multiparticulate form was developed, offering an alternative to encounter major issues in the design of dosage form for pediatric patients, i.e., the difficulty in swallowing large solid dosage form (tablet or capsule), and the requirement to cover a broad range of doses for different age groups. Microcrystalline cellulose-based pellets containing acetaminophen were prepared via extrusion/spheronization followed by freeze-drying. The in vitro disintegration behavior of these pellets was quantitatively measured with a texture analyzer. Mercury intrusion and gas adsorption techniques, scanning electron microscopy of pellet surface and cross-section were performed in order to characterize their internal porous structure. Pellets characteristics such as size distribution, sphericity, friability and drug release were also determined. The developing process was able to produce pellets containing high drug loading (25, 50 and up to 75%, w/w) with good sphericity (aspect ratio ∼1) and low friability. The pellets exhibited an instantaneous disintegration upon contact with water, which was indicated by two parameters: the disintegration onset was approximating to 0, and the disintegration time less than 5 s. The fast disintegration behavior is correlated with the pellet internal structure characterized by a capillary network with pore diameter varying from 0.1 to 10 μm. Such a structure not only ensured a rapid disintegration but it also offers to freeze-dried pellets adequate mechanical properties in comparison with conventional freeze-dried forms. Due to pellet disintegration, fast dissolution of acetaminophen was achieved, i.e., more than 90% of drug released within 15 min. This novel multiparticulate system offers novel age-appropriate dosage form for pediatric population owing to their facility of administration (fast disintegration) and dosing flexibility (divided and reduced-size solid form).
Keywords: Pediatric dosage form; Fast disintegration; Extrusion/spheronization; Freeze-drying;

Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization by Pedro Fonte; Francisca Araújo; Vítor Seabra; Salette Reis; Marco van de Weert; Bruno Sarmento (850-862).
Display OmittedThe purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol at 10% (w/v). Insulin-loaded PLGA nanoparticles with co-encapsulated lyoprotectants achieved a mean particle size of 386–466 nm, and a zeta potential ranging between −34 and −38 mV, dependent on the lyoprotectant used. Formulations had association efficiencies and loading capacities of 85–91% and 10–12%, respectively. The lyophilization process increased the colloidal stability of nanoparticles, and maintained their spherical shape and smooth surface, particularly in presence of lyoprotectants. XRPD revealed that the lyophilizates of nanoparticles with co-encapsulated lyoprotectants were amorphous, whereas formulations with externally added lyoprotectants, except trehalose, showed crystallinity. FTIR assessment showed that co-encapsulating lyoprotectants better preserved insulin structure upon lyophilization with a spectral area overlap of 82–87%, compared to only 72% in lyoprotectant absence. These results were confirmed by circular dichroism spectroscopy. Surprisingly, the simultaneous co-encapsulation and addition of lyoprotectants was detrimental to protein stabilization. The insulin in vitro release studies demonstrated that formulations with co-encapsulated trehalose, glucose, sucrose, fructose and sorbitol achieved 83%, 69%, 70%, 77% and 74%, respectively after 48 h. In contrast, formulations added with those lyoprotectants prior lyophilization showed a lower release rate not higher than 60% after 48 h. This work gives rise to a different promising strategy of co-encapsulating lyoprotectants and therapeutic proteins, to better stabilize protein structure upon lyophilization.
Keywords: PLGA nanoparticles; Insulin; Co-encapsulation; Lyophilization; Lyoprotectants; Protein stability;

Development and in vivo evaluation of an implantable nano-enabled multipolymeric scaffold for the management of AIDS dementia complex (ADC) by Sheri-lee Harilall; Yahya E. Choonara; Lomas Kumar Tomar; Charu Tyagi; Pradeep Kumar; Lisa C. du Toit; Girish Modi; Dinesh Naidoo; Sunny E. Iyuke; Michael P. Danckwerts; Viness Pillay (863-877).
Display OmittedThis study reports the use of biocompatible and biodegradable polymers for the formulation and design of an implantable multipolymeric drug delivery device (MDDD) for the management of AIDS dementia complex (ADC), a debilitating condition affecting the cognitive, motor and behavioral systems in HIV+ individuals. A 3-factor Box-Behnken statistical design was employed for the optimization of nanoparticle and multipolymeric scaffold formulations. Fifteen formulations were generated using the Box-Behnken template, which were assessed for physicochemical and physicomechanical characterization. The optimised nanoparticle formulation yielded nanoparticles measuring 68.04 nm in size and zeta potential (ZP) of −13.4 mV was calculated for the colloidal system. In an attempt to further retard drug release and to formulate a device for implantation in the frontal lobe of the brain, nanoparticles were dispersed within a multipolymeric matrix. Matrix erosion was calculated at 28% for multipolymeric scaffold and a matrix resilience of 4.451% was observed 30 days post exposure to PBS, indicating slow degradation of the MDDD. In vivo studies showed 12.793 ng/mL and 35.225 ng/mL AZT level in plasma and CSF. In view of the physicomechanical properties, in vitro and in vivo drug release kinetics of MDDD makes it a potential candidate for the management of the ADC.
Keywords: Nanoparticles; Polymeric scaffold; AIDS dementia complex management; Implantable device;

Characteristics of silver nanoparticles in vehicles for biological applications by Kristina Kejlová; Věra Kašpárková; Daniel Krsek; Dagmar Jírová; Hana Kolářová; Markéta Dvořáková; Kateřina Tománková; Veronika Mikulcová (878-885).
Display OmittedSilver nanoparticles (AgNPs) have been used for decades as anti-bacterial agents in various industrial fields such as cosmetics, health industry, food storage, textile coatings and environmental applications, although their toxicity is not fully recognized yet. Antimicrobial and catalytic activity of AgNPs depends on their size as well as structure, shape, size distribution, and physico-chemical environment. The unique properties of AgNPs require novel or modified toxicological methods for evaluation of their toxic potential combined with robust analytical methods for characterization of nanoparticles applied in relevant vehicles, e.g., culture medium with/without serum and phosphate buffered saline.
Keywords: Silver nanoparticles; Particle size and distribution; Dynamic light scattering; Transmission electron microscopy; Phosphate buffered saline; Culture medium;

Inhalable liposomal dry powder of gemcitabine-HCl: Formulation, in vitro characterization and in vivo studies by Manit Gandhi; Tosha Pandya; Ravi Gandhi; Sagar Patel; Rajashree Mashru; Ambikanandan Misra; Hemal Tandel (886-895).
Display OmittedPulmonary drug delivery system facilitates local instillation of anticancer drugs to lungs which has proven to be pioneering approach for treatment of lung cancer. This approach led the groundwork for delivering liposomal formulation directly to lungs. Gemcitabine-HCl is currently considered as most effective drug for management of lung cancer. However, its application is limited owing to its metabolism by enzymes present in plasma resulting in reduced efficacy and higher toxicity. In present study, lyophilisation technique was used to convert liposomes into dry powder inhaler, which was formulated using emulsification solvent evaporation technique. The physicochemical properties including size, morphology, entrapment efficiency, loading efficiency etc. of formulated liposomes were evaluated. The prepared liposomal DPI (LDPI) formulations were then examined for solid state characteristics and aerosol performance using cascade impactor. From all the formulations prepared, the LDPI formulated using trehalose as cryoprotectant presented required properties along with desirable deposition pattern. Finally, the optimized formulation was selected for in vitro cell line studies; in vivo studies and stability study. This formulated inhalable particles offers a promising approach for the management of lung cancer through regional chemotherapy.
Keywords: Gemcitabine-HCl; Regional chemotherapy; Lung cancer; Liposomes; Dry powder inhaler; Pulmonary pharmacokinetics;

β-CD-dextran polymer for efficient sequestration of cholesterol from phospholipid bilayers: Mechanistic and safe-toxicity investigations by Dominik Stelzl; Thorbjørn Terndrup Nielsen; Terkel Hansen; Massimiliano di Cagno (896-902).
Display OmittedThe aim of this work was to investigate the suitability of β-cyclodextrin-dextran (BCD-dextran) polymer as cholesterol sequestering agent in vitro. For this purpose, BCD-dextran-cholesterol complexation was studied by phase solubility studies as well as with a specifically designed in vitro model based on giant unilamellar vesicles (GUVs) to evaluate the ability of this polymer to sequestrate cholesterol from phospholipid bilayers. Cholesterol-sequestering ability of BCD-dextran was also investigated on different cell lines relevant for the hematopoietic system and results were correlated to cells toxicity. BCD-dextran polymer was capable of extracting significant amount of cholesterol from phospholipid bilayers and to a higher extent in comparison to available β-cyclodextrins (BCDs). The ability of BCD-dextran in sequestering cholesterol resulted also very high on cell lines relevant for the hematopoietic system. Moreover, BCD-dextran resulted less toxic on cell cultures due to higher selectivity in sequestering cholesterol in comparison to MBCD (that sequestrated also significant amounts of cholesteryl esters). In conclusion, BCD-dextran resulted an extremely efficient cholesterol-sequestering agent and BCD-dextran resulted more selective to cholesterol extraction in comparison to other BCDs (therefore of lower cytotoxicity). This phenomenon might play a key role to develop an efficient treatment for hypercholesterolemia based on cholesterol segregation.
Keywords: β-cyclodextrins; β-cyclodextrin-dextran polymers; Cholesterol depletion; Safe-toxicity studies; Giant unilamellar vesicles;

The targeted delivery of doxorubicin with transferrin-conjugated block copolypeptide vesicles by Brian S. Lee; Allison T. Yip; Alison V. Thach; April R. Rodriguez; Timothy J. Deming; Daniel T. Kamei (903-911).
Display OmittedWe previously investigated the intracellular trafficking properties of our novel poly(l-glutamate)60-b-poly(l-leucine)20 (E60L20) vesicles (EL vesicles) conjugated to transferrin (Tf). In this study, we expand upon our previous work by investigating the drug encapsulation, release, and efficacy properties of our novel EL vesicles for the first time. After polyethylene glycol (PEG) was conjugated to the vesicles for steric stability, doxorubicin (DOX) was successfully encapsulated in the vesicles using a modified pH-ammonium sulfate gradient method. Tf was subsequently conjugated to the vesicles to provide active targeting to cancer cells and a mode of internalization into the cells. These Tf-conjugated, DOX-loaded, PEGylated EL (Tf-DPEL) vesicles exhibited colloidal stability and were within the allowable size range for passive and active targeting. A mathematical model was then derived to predict drug release from the Tf-DPEL vesicles by considering diffusive and convective mass transfer of DOX. Our mathematical model reasonably predicted our experimentally measured release profile with no fitted parameters, suggesting that the model could be used in the future to manipulate drug carrier properties to alter drug release profiles. Finally, an in vitro cytotoxicity assay was used to demonstrate that the Tf-DPEL vesicles exhibited enhanced drug carrier efficacy in comparison to its non-targeted counterpart.
Keywords: Vesicle; Block copolypeptide; Drug delivery; Drug release; Doxorubicin; Mathematical modeling;

Chitosan grafted low molecular weight polylactic acid for protein encapsulation and burst effect reduction by Antonio Di Martino; Pavel Kucharczyk; Jiri Zednik; Vladimir Sedlarik (912-921).
Display OmittedChitosan and chitosan-grafted polylactic acid as a matrix for BSA encapsulation in a nanoparticle structure were prepared through a polyelectrolyte complexation method with dextran sulfate. Polylactic acid was synthetized via a polycondensation reaction using the non-metal-based initiator methanesulfonic acid and grafted to the chitosan backbone by a coupling reaction, with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as the condensing agent. The effect of concentration of the polymer matrix utilized herein on particle diameter, ζ-potential, encapsulation efficiency, and the release kinetic of the model protein bovine serum albumin at differing pH levels was investigated. The influence of pH and ionic strength on the behavior of the nanoparticles prepared was also researched. Results showed that grafting polylactic acid to chitosan chains reduced the initial burst effect in the kinetics of BSA release from the structure of the nanoparticles. Furthermore, a rise in encapsulation efficiency of the bovine serum albumin and diminishment in nanoparticle diameter were observed due to chitosan modification. The results suggest that both polymers actually show appreciable encapsulation efficiency; and release rate of BSA. CS-g-PLA is more suitable than unmodified CS as a carrier for controlled protein delivery.
Keywords: Nanoparticles; Chitosan; Polylactic acid; Bovine serum albumin; Controlled drug delivery;

Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties by C. Gamazo; H. Bussmann; S. Giemsa; A.I. Camacho; Daisy Unsihuay; N. Martín-Arbella; J.M. Irache (922-930).
Display OmittedUnderstanding how nanoparticles are formed and how those processes ultimately determine the nanoparticles’ properties and their impact on their capture by immune cells is key in vaccination studies. Accordingly, we wanted to evaluate how the previously described poly (anhydride)-based nanoparticles of the copolymer of methyl vinyl ether and maleic anhydride (NP) interact with macrophages, and how this process depends on the physicochemical properties derived from the method of preparation. First, we studied the influence of the desolvation and drying processes used to obtain the nanoparticles. NP prepared by the desolvation of the polymers in acetone with a mixture of ethanol and water yielded higher mean diameters than those obtained in the presence of water (250 nm vs. 180 nm). In addition, nanoparticles dried by lyophilization presented higher negative zeta potentials than those dried by spray-drying (−47 mV vs. −35 mV). Second, the influence of the NP formulation on the phagocytosis by J774 murine macrophage-like cell line was investigated. The data indicated that NPs prepared in the presence of water were at least three-times more efficiently internalized by cells than NPs prepared with the mixture of ethanol and water. Besides, lyophilized nanoparticles appeared to be more efficiently taken up by J744 cells than those dried by spray-drying. To further understand the specific mechanisms involved in the cellular internalization of NPs, different pharmacological inhibitors were used to interfere with specific uptake pathways. Results suggest that the NP formulations, particularly, nanoparticles prepared by the addition of ethanol:water, are internalized by the clathrin-mediated endocytosis, rather than caveolae-mediated mechanisms, supporting their previously described vaccine adjuvant properties.
Keywords: Nanoparticle; Macrophage; Antigen presenting cell; Cellular uptake; Clathrin;

Biocompatible microemulsions of a model NSAID for skin delivery: A decisive role of surfactants in skin penetration/irritation profiles and pharmacokinetic performance by Marija N. Todosijević; Miroslav M. Savić; Bojan B. Batinić; Bojan D. Marković; Mirjana Gašperlin; Danijela V. Ranđelović; Milica Ž. Lukić; Snežana D. Savić (931-941).
Display OmittedTo elaborate the decisive role of surfactants in promotion of aceclofenac’ skin absorption, potentially avoiding irritation, we developed non-ionic microemulsions varying natural or synthetic surfactants: sucrose esters (laurate or myristate) vs. polysorbate 80. A comprehensive physicochemical characterization indicated no significant influence of the solubilized nonsteroidal anti-inflammatory drug on the bicontinuous structure of blank formulations. To evaluate skin tolerability of isopropyl alcohol, a sucrose ester-based microemulsion containing transcutol P as a cosurfactant was also developed. The measured skin parameters strongly depended on the (co)surfactant type, showing higher compatibility of the microemulsions containing sucrose ester and isopropyl alcohol. In vitro release results, in vivo tape stripping and pharmacokinetics in rats confirmed superiority of the sucrose ester- over polysorbate-based microemulsions (total amounts of aceclofenac penetrated 60.81 ± 5.97 and 60.86 ± 3.67 vs. 27.00 ± 5.09 μg/cm2, and its maximum plasma concentrations 275.57 ± 109.49 and 281.31 ± 76.76 vs. 150.23 ± 69.74 ng/ml for sucrose laurate- and myristate- vs. polysorbate 80-based microemulsions, respectively). Hence, sugar-based excipients increased delivery of aceclofenac through stratum corneum by increasing its fluidity, showing overall more satisfying safety profiles. In conclusion, sucrose ester-based microemulsions proved to be promising carriers for dermal/transdermal aceclofenac delivery.
Keywords: Bicontinuous microemulsion; Sucrose ester; Aceclofenac; Skin irritation potential; Tape stripping; Pharmacokinetics;

The study on cholangiocellular cell lines was conducted with mTHPP-loaded nanoparticles with different polymers as matrices. Nanoparticles were characterized and the effect of the carrier system was examined in vitro. Comparison of free substance, embedded substance and different materials revealed severe differences for cellular uptake and in vitro activity after activation by illumination.Display OmittedThe photodynamic therapy with porphyrin derivatives is an established approach to targeted tumor therapy, but is still afflicted with disadvantages of the physicochemical characteristics of the photosensitizer. To overcome drug-related restrictions in photodynamic therapy, three 5,10,15,20-tetrakis(m-hydroxyphenyl) porphyrin (mTHPP)-loaded nanoparticulate formulations based on poly(dl-lactide-co-glycolide) (PLGA), poly(d,l-lactide) (PLA), and Eudragit® E were prepared in a consistent diameter range and compared with free mTHPP in vitro. Formulation behavior was investigated in two different cholangiocellular cell lines, EGI-1 and TFK-1.High cytotoxicity was shown for all photosensitizer loaded nanoparticle (NP) formulations and free mTHPP, with EC50 values ranging from 0.2 to 1.3 μM. PLA based NP were not as effective in all performed tests as other formulations. Nanoparticulate embedded mTHPP remained photodynamically active and resulted in caspase-3 activation even at low concentrations of 250 nM. PLGA based NP exhibited highest caspase-3 activation.For all formulations an effective intracellular accumulation of mTHPP was observed, whereby for mTHPP-Eudragit® E-NP a 200-fold drug accumulation was shown.Polymer based nanoparticles were shown to be an effective and highly active transport vehicle for the photosensitizer mTHPP in vitro. Problems like low solubility of free drug can be circumvented by successful embedding into nanoparticulate carrier systems, maintaining therapeutic effects of the photosensitizer.
Keywords: Drug delivery system; Photodynamic therapy; Cholangiocarcinoma; Nanoparticle; Cytotoxicity; Polymer influence;

Characterization of smart auto-degradative hydrogel matrix containing alginate lyase to enhance levofloxacin delivery against bacterial biofilms by German A. Islan; Cecilia Dini; Laura C. Bartel; Alejandro D. Bolzán; Guillermo R. Castro (953-964).
Display OmittedThe aim of the present work is the characterization of smart auto-degradable microspheres composed of calcium alginate/high methoxylated pectin containing an alginate lyase (AL) from Sphingobacterium multivorum and levofloxacin. Microspheres were prepared by ionotropic gelation containing AL in its inactive form at pH 4.0. Incubation of microspheres in Tris–HCl and PBS buffers at pH 7.40 allowed to establish the effect of ion-chelating phosphate on matrix erodability and suggested an intrinsically activation of AL by turning the pH close to neutrality. Scanning electron and optical microscopies revealed the presence of holes and surface changes in AL containing microspheres. Furthermore, texturometric parameters, DSC profiles and swelling properties were showing strong changes in microspheres properties. Encapsulation of levofloxacin into microspheres containing AL showed 70% efficiency and 35% enhancement of antimicrobial activity against Pseudomonas aeruginosa biofilm. Levofloxacin release from microspheres was not changed at acidic pH, but was modified at neutral pH in presence of AL. Advantageously, only gel matrix debris were detectable after overnight incubation, indicating an autodegradative gel process activated by the pH. Absence of matrix cytotoxicity and a reduction of the levofloxacin toxicity after encapsulation were observed in mammalian CHO-K1 cell cultures. These properties make the system a potent and versatile tool for antibiotic oral delivery targeted to intestine, enhancing the drug bioavailability to eradicate bacterial biofilm and avoiding possible intestinal obstructions.
Keywords: Alginate; Pectin; Levofloxacin; Alginate lyase; Controlled release; Autodegradable biogels;

Au@SiO2 core–shell structure involved with methotrexate: Fabrication, biodegradation process and bioassay explore by Xiaolei Huo; Chaofan Dai; Deying Tian; Shuping Li; Xiaodong Li (965-975).
A new strategy is proposed to synthesize the Au@SiO2 core–shell structure (*), with methotrexate (MTX) loaded within it. Furthermore, the MTX molecules could be continuously released from such core–shell structure, along with the collapse of SiO2.Display OmittedA new strategy is proposed to synthesize a kind of Au@SiO2 core–shell structure with methotrexate (MTX) loaded within it. Firstly, MTX molecules are attracted to the surface and vicinity of Au nanoparticles (NPs). Then the enriched MTX molecules on the surface of Au NPs have a good chance to be wrapped into the core–shell structure when SiO2 is uniformly deposited on the Au core. Secondly, the effect of Au amount and MTX content on the drug-loading capacity is emphatically studied and the result shows that core–shell structure plays a vital role in drug loading. In addition, the biodegradation process is also examined in phosphate buffer solution (PBS) at 37 °C. The results show that the biodegradation of Au-MTX@SiO2 core–shell structure can be divided into two stages: the release of drug together with the fragmentation of core–shell structure and the subsequent dissolution of SiO2 layers. Lastly, in vitro bioassay tests give the evidence that obvious tumor inhibition can be achieved in presence of Au-MTX@SiO2 NPs even at low concentration and the efficacy can be greatly enhanced by the photothermal therapy on Au cores.
Keywords: Au; SiO2; Methotrexate; Core–shell structure; Drug delivery;

Effect of sterilization on the physical stability of brimonidine-loaded solid lipid nanoparticles and nanostructured lipid carriers by Noha S. El-Salamouni; Ragwa M. Farid; Amal H. El-Kamel; Safaa S. El-Gamal (976-983).
Display OmittedNanoparticulate delivery systems have recently been under consideration for topical ophthalmic drug delivery. Brimonidine base-loaded solid lipid nanoparticles and nanostructured lipid carrier formulations were prepared using glyceryl monostearate as solid lipid and were evaluated for their physical stability following sterilization by autoclaving at 121  °C for 15 min. The objective of this work was to evaluate the effect of autoclaving on the physical appearance, particle size, polydispersity index, zeta potential, entrapment efficiency and particle morphology of the prepared formulations, compared to non-autoclaved ones. Results showed that, autoclaving at 121  °C for 15 min allowed the production of physically stable formulations in nanometric range, below 500 nm suitable for ophthalmic application. Moreover, the autoclaved samples appeared to be superior to non-autoclaved ones, due to their increased zeta potential values, indicating a better physical stability. As well as, increased amount of brimonidine base entrapped in the tested formulations.
Keywords: Brimonidine; Ocular; Glaucoma; Autoclaving; Solid lipid nanoparticles; Nanostructured lipid carriers;

Green fabricated reduced graphene oxide: evaluation of its application as nano-carrier for pH-sensitive drug delivery by Naxin Ma; Baohua Zhang; Jing Liu; Pei Zhang; Zhonghao Li; Yuxia Luan (984-992).
Green fabricated reduced graphene oxide rGO is demonstrated to be a good candidate as an ideal nano-carrier for drug delivery and controlled release.Display OmittedA green and mild approach for the preparation of reduced graphene oxide (rGO) was proposed by using riboflavin-5′-phosphate sodium salt dihydrate as a reducing reagent and stabilizer without any other reagent. The fabricated nano-rGO was systematically evaluated for its application as nano-carrier for pH-sensitive drug delivery. The hemolytic toxicity test indicated the as-prepared nano-rGO had negligible hemolytic activity, which demonstrating its safety in drug delivery system. Doxorubicin hydrochloride (DOX) as a model drug was successfully attached onto the surface of nano-rGO via strong π–π stacking interaction. Compared with common carriers, the obtained DOX-loaded nano-rGO nanohybrid exhibited characteristics of high drug loading content, good stability, pH-sensitive and sustainable release of drugs. Cytotoxicity assay results suggested such nanohybrid exhibited effective cytotoxicity to MCF-7 and A549 cells by nonspecific endocytosis mechanism. Therefore, the present green fabricated rGO could be a good candidate as an ideal nano-carrier for drug delivery and controlled release.
Keywords: Green chemistry; Graphene oxide; Reduced graphene oxide; Drug carrier;

Display OmittedOral administration of insulin remains a challenge due to its poor enzymatic stability and inefficient permeation across epithelium. We herein developed a novel self-assembled polyelectrolyte complex nanoparticles by coating insulin-loaded dodecylamine-graft-γ-polyglutamic acid micelles with trimethyl chitosan (TMC). The TMC material was also conjugated with a goblet cell-targeting peptide to enhance the affinity of nanoparticles with epithelium. The developed nanoparticle possessed significantly enhanced colloid stability, drug protection ability and ameliorated drug release profile compared with graft copolymer micelles or ionic crosslinked TMC nanoparticles. For in vitro evaluation, Caco-2/HT29-MTX-E12 cell co-cultures, which composed of not only enterocyte-like cells but also mucus-secreting cells and secreted mucus layer, were applied to mimic the epithelium. Intracellular uptake and transcellular permeation of encapsulated drug were greatly enhanced for NPs as compared with free insulin or micelles. Goblet cell-targeting modification further increased the affinity of NPs with epithelium with changed cellular internalization mechanism. The influence of mucus on the cell uptake was also investigated. Ex vivo performed with rat mucosal tissue demonstrated that the nanoparticle could facilitate the permeation of encapsulated insulin across the intestinal epithelium. In vivo study preformed on diabetic rats showed that the orally administered nanoparticles elicited a prolonged hypoglycemic response with relative bioavailability of 7.05%.
Keywords: Nanoparticles; Oral delivery; Insulin; Epithelium; CSKSSDYQC;

Preparation and evaluation of amoxicillin loaded dual molecularly imprinted nanoparticles for anti-Helicobacter pylori therapy by Zhihui Wu; Jiapeng Hou; Yuyan Wang; Miaolin Chai; Yan Xiong; Weiyue Lu; Jun Pan (1006-1014).
Display OmittedThis paper reports studies on preparation and evaluation of amoxicillin loaded dual molecularly imprinted nanoparticles (Amo/Dual-MIPs) designed for anti-H. pylori therapy. Both MNQA and AmoNa were chosen as templates to prepare Dual-MIPs using inverse microemulsion polymerization method. NQA was modified with myristic acid (MNQA) to become amphiphilic and assist in leaving NQA cavities on the surface of Dual-MIPs for H. pylori adhesion. AmoNa was applied to produce imprinting sites in Dual-MIPs for rebinding AmoNa to exert its anti-H. pylori effect. Batch rebinding test demonstrated a preferential rebinding effect of NQA toward the Dual-MIPs. In vivofluorescence imaging showed the prolonged residence time of Dual-MIPs in H. pylori infected mice stomachs after intragastric administration of nanoparticles.In vivo H. pylori clearance tests indicated Amo/Dual-MIPs had a better aniti-H. pylori effect than amoxicillin powder did. In conclusion, Amo/Dual-MIPs may provide an alternative drug delivery strategy for anti-H. pylori therapy.
Keywords: Dual molecularly imprinted nanoparticles; Lpp20; Amoxicillin sodium; Template rebinding; anti-H. pylori therapy;

Disc-shaped polyoxyethylene glycol glycerides gel nanoparticles as novel protein delivery vehicles by Doroty Codoni; Jonathan Cowan; Jenna Bradley; William J. McAuley; Maria A. O'Connell; Sheng Qi (1015-1025).
Display OmittedDisc-shaped nanoparticles with high aspect ratios have been reported to show preferential cellular uptake in vitro by mammalian cells. However, engineering and producing such disc-shaped nanoparticles are often complex. This study reports for the first time the use of a single, approved pharmaceutical excipient to prepare stable disc-shaped nanoparticles with a high aspect ratio via a simple, organic solvent free process. These disc-shaped nanoparticles were formed by fragmentation of stearoyl macrogol-32 glycerides (Gelucire 50/13) hydrogels. The nanoparticles showed good physical stability as a result of their outer coating of polyethylene glycol (PEG) that is a part of Gelucire composition. Using lysozyme as a model hydrophilic protein, these nanoparticles demonstrated a good loading capacity for hydrophilic macromolecules, mainly via surface adsorption. As a result of the higher hydrophobicity of the core of the nano-discs, the loading efficiency of hydrophobic model components, such as Coumarin-6, was significantly increased in comparison to the model hydrophilic compound. These Gelucire nano-discs exhibited no cytotoxicity at the tested level of 600 μg/ml for Caco-2 cells. Rapid in vitro cellular uptake of the disc-shaped nanoparticles by Caco-2 cells was observed. This rapid internalisation was attributed to the high aspect ratio of the disc-shape nanoparticles which provides a high contact surface area between the particles and cells and may lower the strain energy required for membrane deformation during uptake. The results of this study demonstrate the promising potential of Gelucire nano-discs as effective nanocarriers for drug delivery and which can be manufactured using a simple solvent-free process.
Keywords: Drug delivery; Polyoxyethylene glycol glycerides; Disc-shape nanoparticles; Cellular uptake;

Antibody fragment-armed mesoporous silica nanoparticles for the targeted delivery of bevacizumab in ovarian cancer cells by Ying Zhang; Jing Guo; Xiao-Ling Zhang; Da-Peng Li; Ting-Ting Zhang; Fu-Feng Gao; Nai-Fu Liu; Xiu-Gui Sheng (1026-1033).
Display OmittedIn order to enhance the therapeutic efficacy and intracellular concentration of bevacizumab (BVC), we have designed a novel tumor endothelial marker 1 (TEM1)/endosialin (Ab-/scFv)-conjugated mesoporous silica nanoparticles (MSN) to target ovarian cancer cell. The Ab-/scFv-conjugated MSN were prepared by the conjugation of amine functional group of antibody of the carboxyl group of MSN. The resultant MSN was nanosized, spherical shaped, and exhibited a controlled release phenomenon in pH 7.4 conditions. Furthermore, BMSN/Ab was found to increase the cellular uptake and intracellular distribution of BVC in OVCAR-5 cancer cells. The Ab- conjugated MSN exhibited a superior anticancer effect with profound apoptosis in cancer cells in a time- and concentration dependent manner. Consistently, BMSN/Ab effectively inhibited the colony formation in transwell plate. Finally, BMSN/Ab showed a notable increase in the proportion of cells in G2/M phase of cell cycle indicating promising anticancer efficacy profile. Overall, Ab-/scFv-conjugated MSN might provide an effective strategy for the therapeutic management of ovarian cancers.
Keywords: Antibody; Bevacizumab; Mesoporous silica nanoparticles; Apoptosis; Ovarian cancer;

Skin delivery by block copolymer nanoparticles (block copolymer micelles) by Faiza Laredj-Bourezg; Marie-Alexandrine Bolzinger; Jocelyne Pelletier; Jean-Pierre Valour; Marie-Rose Rovère; Batoule Smatti; Yves Chevalier (1034-1046).
Display OmittedBlock copolymer nanoparticles often referred to as “block copolymer micelles” have been assessed as carriers for skin delivery of hydrophobic drugs. Such carriers are based on organic biocompatible and biodegradable materials loaded with hydrophobic drugs: poly(lactide)-block-poly(ethylene glycol) copolymer (PLA-b-PEG) nanoparticles that have a solid hydrophobic core made of glassy poly(d,l-lactide), and poly(caprolactone)-block-poly(ethylene glycol) copolymer (PCL-b-PEG) nanoparticles having a liquid core of polycaprolactone. In vitro skin absorption of all-trans retinol showed a large accumulation of retinol in stratum corneum from both block copolymer nanoparticles, higher by a factor 20 than Polysorbate 80 surfactant micelles and by a factor 80 than oil solution. Additionally, skin absorption from PLA-b-PEG nanoparticles was higher by one order of magnitude than PCL-b-PEG, although their sizes (65 nm) and external surface (water-swollen PEG layer) were identical as revealed by detailed structural characterizations. Fluorescence microscopy of histological skin sections provided a non-destructive picture of the storage of Nile Red inside stratum corneum, epidermis and dermis. Though particle cores had a different physical states (solid or liquid as measured by 1H NMR), the ability of nanoparticles for solubilization of the drug assessed from their Hildebrand solubility parameters appeared the parameter of best relevance regarding skin absorption.
Keywords: Skin delivery; Cutaneous absorption; Block copolymer; Nanoparticle;

Airway administration of a highly versatile peptide-based liposomal construct for local and distant antitumoral vaccination by Zahra Kakhi; Benoît Frisch; Line Bourel-Bonnet; Joseph Hemmerlé; Françoise Pons; Béatrice Heurtault (1047-1056).
Display OmittedWith the discovery of tumor-associated antigens such as ErbB2, vaccination is considered as a promising strategy to prevent the development of cancer or treat the existing disease. Among routes of immunization, the respiratory route provides the opportunity to develop non-invasive approach for vaccine delivery. In the current study, this administration route was used in order to investigate the potency of a highly versatile di-epitopic liposomal construct to exhibit local or distant antitumoral efficiency after prophylactic or therapeutic vaccination in mice. Well-characterized liposomes, containing the ErbB2 (p63–71) TCD8+ and HA (p307–319) TCD4+ peptide epitopes and the Pam2CAG adjuvant, were formulated and administered into the airway of naïve BALB/c mice. The nanoparticle vaccine candidate induced local and specific systemic immune response, as measured by immune cell infiltration and chemokine and cytokine production in BALF or lung tissue, and by spleen T-cell activation ex vivo, respectively. This potent immune response resulted in an efficient antitumor activity against both lung and solid s.c. tumors. Interestingly, the antitumor efficacy was observed after both prophylactic and therapeutic vaccinations, which are the most judicious ones to fight cancer. Our data showed an undeniable interest of liposomal peptide-based vaccines in antitumor vaccination by the respiratory route, opening new perspectives for cancer treatment.
Keywords: Liposome; Peptide-based vaccine; Cancer; Vaccination; Respiratory route; Mucosal immunization;

Display OmittedDue to the high morbidity and mortality of cancer, it has become an urgent matter to develop an effective and a safe treatment strategy. Nanoparticles (NP) based drug delivery systems have gained much attention nowadays but they faced a paradoxical issue in delivering drugs into tumors: NP with large size were characterized with weak tumor penetration, meanwhile NP with small size resulted in poor tumor retention. To solve this problem, we proposed a multistage drug delivery system which could intelligently shrink its size from large size to small size in the presence of matrix metalloproteinase-2 (MMP-2) which were highly expressed in tumor tissues, therefore the multistage system could benefit from its large size for better retention effect in tumor and then shrunk to small size to contribute to better penetration efficiency. The multistage drug delivery system, RGD–DOX–DGL–GNP, was constructed by 155.4 nm gelatin NP core (the substrate of MMP-2) and surface decorated with doxorubicin (DOX) and RGD peptide conjugated dendritic poly-l-lysine (DGL, 34.3 nm in diameter). In vitro, the size of multistage NP could effectively shrink in the presence of MMP-2. Thus, the RGD–DOX–DGL–GNP could penetrate deep into tumor spheroids. In vivo, this multistage drug delivery system showed higher tumor retention and deeper penetration than both DOX–DGL and DOX–GNP. Consequently, RGD–DOX–DGL–GNP successfully combined the advantages of dendrimers and GNP in vivo, resulting in an outstanding anti-tumor effect. In conclusion, the multistage drug delivery system could intelligently shrink from large size to small size in the tumor microenvironment and displayed better retention and penetration efficiency, making it an impressing system for cancer treatment.
Keywords: Tumor penetration and retention; Breast cancer; Multistage drug delivery system; Matrix metalloproteinase-2; Gelatin nanoparticles;

Nebulized treatment is an important delivery option for the young, elderly, and those with severe chronic respiratory disease, but there is a lack of new nebulized drug products being produced for these patients, leading to the potential for under-treatment. This communication describes a new drug development paradigm as a timely solution to this issue. Often, drug development is initiated with nebulizers in the early stages, to provide cheaper and faster drug development, and then switched to inhaler devices in later clinical trials to address the majority of patients. However, the waste of resource on parallel development of the inhaler can be large due to the high early attrition rate of new drug development. The new paradigm uses the nebulizer to continue drug development through to market, and initiates inhaler development after completion of the riskier early phase studies. New drug safety and efficacy can be assessed faster and more efficiently by using a nebulized formulation rather than developing an inhaler. The results of calculations of expected net present value showed that the new paradigm produced higher expected net present values than the conventional model over a range of economic scenarios. This new paradigm could therefore provide improved returns on investments, as well as more modern drugs in nebulized form for those patients unable to use inhalers.
Keywords: Estimated net present value; Drug development; Nebulizer; Inhaler; Life cycle management;

Display OmittedThe total uncertainty of quantitative microbiological methods, used in pharmaceutical analysis, consists of several components. The analysis of the most important sources of the quantitative microbiological methods variability demonstrated no effect of culture media and plate-count techniques in the estimation of microbial count while the highly significant effect of other factors (type of microorganism, pharmaceutical product and individual reading and interpreting errors) was established. The most appropriate method of statistical analysis of such data was ANOVA which enabled not only the effect of individual factors to be estimated but also their interactions. Considering all the elements of uncertainty and combining them mathematically the combined relative uncertainty of the test results was estimated both for method of quantitative examination of non-sterile pharmaceuticals and microbial count technique without any product. These data did not exceed 35%, appropriated for a traditional plate count methods.
Keywords: Pharmaceutical analysis; Quantitative microbiological method; Uncertainty;