European Journal of Pharmaceutics and Biopharmaceutics (v.71, #2)

APV Diary (S1-S2).

Lipid nanoparticles for parenteral delivery of actives by Medha D. Joshi; Rainer H. Müller (161-172).
The present review compiles the applications of lipid nanoparticles mainly solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and lipid drug conjugates (LDC) in parenteral delivery of pharmaceutical actives. The attempts to incorporate anticancer agents, imaging agents, antiparasitics, antiarthritics, genes for transfection, agents for liver, cardiovascular and central nervous system targeting have been summarized. The utility of lipid nanoparticles as adjuvant has been discussed separately. A special focus of this review is on toxicity caused by these kinds of lipid nanoparticles with a glance on the fate of lipid nanoparticles after their parenteral delivery in vivo viz the protein adsorption patterns.
Keywords: Lipid nanoparticles; Solid lipid nanoparticles (SLN); Nanostructured lipid carriers (NLC); Lipid drug conjugates (LDC); Parenteral delivery;

Follicular transport route – Research progress and future perspectives by Fanny Knorr; Jürgen Lademann; Alexa Patzelt; Wolfram Sterry; Ulrike Blume-Peytavi; Annika Vogt (173-180).
The important role of hair follicles as penetration pathways and reservoir structures for topically applied compounds has been validated in numerous animal models as well as in humans. Follicular penetration rates are modulated by regional variations in size and proportions and the functional status. Advances have especially been made in the targeting of hair follicle-associated cell populations including antigen-presenting cells and stem cells. Improved investigative methods based on differential stripping, spectrophotometry and confocal laser scanning microscopy have led to the determination of the penetration profiles and kinetics for a multiplicity of drugs and drug delivery systems. The observation that particulate delivery systems aggregate and remain in hair follicle openings and their penetration along the follicular duct occurs in a size-dependent manner, which has led to advanced concepts of targeted drug delivery of bioactive compounds in the field of solid particles, as well as semi-solid particles, such as liposomes. This review summarizes the recent progress in this field, and underlines the necessity for pilot studies in human volunteers to further the development of clinical applications for follicular targeting.
Keywords: Hair follicles; Skin; Penetration; Stem cells; Sebaceous glands; Nanoparticles; Microparticles; Liposomes; Transcutaneous vaccination;

Short- and long-term stability study of lyophilized solid lipid nanoparticles for gene therapy by A. del Pozo-Rodríguez; M.A. Solinís; A.R. Gascón; J.L. Pedraz (181-189).
Most studies in gene therapy are focused on developing more efficient non-viral vectors, ignoring their stability, even though physically and chemically stable vectors are necessary to achieve large easily shipped and stored batches. In the present work, the effect of lyophilization on the morphological characteristics and transfection capacity of solid lipid nanoparticles (LyoSLN) and SLN-DNA vectors (Lyo(SLN-DNA)) has been evaluated. The lyophilized preparations were stored under three different sets of temperature and humidity ICH conditions: 25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH. After lyophilization we found an increase in particle size which did not imply a reduction of “in vitro” transfection capacity. Stability studies of formulations lyophilized with trehalose showed that SLNs were physically stable during 9 months at 25 °C/60%RH and 6 months at 30 °C/65%RH. This stability was lost when harder conditions were employed (40 °C/75%RH). LyoSLNs maintained or increased the transfection efficacy (from 19% to approximately 40% EGFP positive cells) over time only at 25 °C/60%RH and 30 °C/65%RH. Lyo(SLN-DNA) resulted in almost no transfection under all conditions. LyoSLNs showed less DNA condensation capacity, whereas in Lyo(SLN-DNA) the plasmid became strongly bound, hampering the transfection. Furthermore, the storage of lyophilized lipoplexes stabilized with the disaccharide trehalose did not affect cell viability.
Keywords: Gene therapy; Non-viral vectors; Solid lipid nanoparticles; Lyophilization; Stability study;

In vivo investigation of tolerance and antitumor activity of cisplatin-loaded PLGA-mPEG nanoparticles by George Mattheolabakis; Era Taoufik; Sylva Haralambous; Michael L. Roberts; Konstantinos Avgoustakis (190-195).
The tolerance of BALB/c mice to different doses of blank and cisplatin-loaded PLGA-mPEG nanoparticles and the in vivo anticancer activity of these nanoparticles on SCID mice xenografted with colorectal adenocarcinoma HT 29 cells were investigated. Nanoparticles with an average size of 150–160 nm and approximately 2% w/w cisplatin content were prepared by a modified emulsification and solvent evaporation method. Normal BALB/c mice tolerated three weekly intravenous injections of a relatively high dose of blank PLGA-mPEG nanoparticles (500 mg/kg, equivalent to about 10 mg nanoparticles/mouse) and three weekly intravenous injections of a high dose of nanoparticle-entrapped cisplatin (10 mg/kg). Also, histopathology examination indicated that there were no differences in the kidneys or spleens from animals treated with cisplatin-loaded nanoparticles or blank nanoparticles compared to the untreated control group. A moderate granulation of protoplasm of hepatic cells was observed in the livers from mice treated with cisplatin-loaded nanoparticles and blank nanoparticles, however, both the hepatic lobe and the portal hepatis maintained their normal architecture. The cisplatin-loaded PLGA-mPEG nanoparticles appeared to be effective at delaying tumor growth in HT 29 tumor-bearing SCID mice. The group of mice treated with cisplatin-loaded nanoparticles exhibited higher survival rate compared to the free cisplatin group. The results justify further evaluation of the in vivo antitumor efficacy of the PLGA-mPEG/cisplatin nanoparticles.
Keywords: Poly(lactide-co-glycolide)-methoxy-poly(ethylene glycol) nanoparticles; PLGA-mPEG; Cisplatin; Cytotoxicity; HT 29 cells; Anticancer activity;

In this study, we synthesized and characterized two methoxy poly(ethylene glycol)-block-poly(caprolactone) (MePEG-b-PCL) amphiphilic diblock copolymers, both based on MePEG with a molecular weight of 5000 g/mol (114 repeat units) and PCL block lengths of either 19 or 104 repeat units. Nanoparticles were formed from these copolymers by a nanoprecipitation and dialysis technique. The MePEG114-b-PCL19 copolymer was water soluble and formed micelles that had a hydrodynamic diameter of 40 nm at all copolymer concentrations tested, and displayed a relatively low core microviscosity. The practically water insoluble MePEG114-b-PCL104 copolymer formed nanoparticles with a larger hydrodynamic diameter, which was dependent on copolymer concentration, and possessed a higher core microviscosity than the MePEG114-b-PCL19 micelles, characteristic of nanospheres. The micelles solubilized a maximum of 1.6% w/w of the hydrophobic anticancer agent, paclitaxel (PTX), and released 92% of their drug payload over 7 days, as compared to the nanospheres, which solubilized a maximum of 3% w/w of PTX and released 60% over the same period of time. Both types of nanoparticles were found to be hemocompatible, causing only minimal hemolysis and no changes in plasma coagulation times as compared to control. Upon in vitro incubation in human plasma, PTX solubilized by micelles had a plasma distribution similar to free drug. The majority of PTX was associated with the lipoprotein deficient plasma (LPDP) fraction, which primarily consists of albumin and alpha-1-acid glycoprotein. In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction.
Keywords: Micelle; Nanosphere; Nanoparticle; Paclitaxel; Amphiphilic block copolymer; Plasma distribution; Hemocompatibility;

Benefits of nanoencapsulation for the hypercin-mediated photodetection of ovarian micrometastases by Magali Zeisser-Labouèbe; Florence Delie; Robert Gurny; Norbert Lange (207-213).
The high recurrence and lethality of ovarian cancer at advanced stages is problematic, especially due to the development of numerous micrometastases scattered throughout the abdominal cavity. Fluorescence photodetection (PD) used in combination with surgical resection of malignant tissues has been suggested to improve recovery. Based on promising in vivo results for the detection of bladder cancer, hypericin (Hy), a natural photosensitizer (PS), stands as a good candidate for the photodetection of ovarian cancer. However, due to its hydrophobicity, systemic administration of Hy is problematic. Polymeric nanoparticles (NPs) help to overcome these delivery and stability problems and enable intravenous administration of Hy. In this study, Hy-loaded NPs of polylactic acid were produced with the following properties: (i) mean size of 268 nm, (ii) negative zeta potential, (iii) low residual surfactant and (iv) drug loading of 3.7 % (w/w). The potential of hypericin-loaded nanoparticles for the fluorescence photodetection of ovarian metastases in Fischer 344 rats bearing ovarian tumours was compared to free drug. The selectivity of Hy administered with both formulations was assessed first by fluorescence endoscopy, and then quantified after tissue extraction. The results showed an improved selective accumulation of Hy in ovarian micrometastases when NPs were used.
Keywords: Ovarian cancer; Fluorescence photodetection; Nanoparticle; Hypericin; In vivo; Drug delivery system;

Drug resistance limits the success of many anticancer drugs. Reduced accumulation of the drug at its intracellular site of action because of overexpression of efflux transporters such as P-glycoprotein (P-gp) is a major mechanism of drug resistance. In this study, we investigated whether photodynamic therapy (PDT) using methylene blue, also a P-gp inhibitor, can be used to enhance doxorubicin-induced cytotoxicity in drug-resistant tumor cells. Aerosol OT (AOT)-alginate nanoparticles were used as a carrier for the simultaneous cellular delivery of doxorubicin and methylene blue. Methylene blue was photoactivated using light of 665 nm wavelength. Induction of apoptosis and necrosis following treatment with combination chemotherapy and PDT was investigated in drug-resistant NCI/ADR-RES cells using flow cytometry and fluorescence microscopy. Effect of encapsulation in nanoparticles on the intracellular accumulation of doxorubicin and methylene blue was investigated qualitatively using fluorescence microscopy and was quantitated using HPLC. Encapsulation in AOT-alginate nanoparticles significantly enhanced the cytotoxicity of combination therapy in resistant tumor cells. Nanoparticle-mediated combination therapy resulted in a significant induction of both apoptosis and necrosis. Improvement in cytotoxicity could be correlated with enhanced intracellular and nuclear delivery of the two drugs. Further, nanoparticle-mediated combination therapy resulted in significantly elevated reactive oxygen species (ROS) production compared to single drug treatment. In conclusion, nanoparticle-mediated combination chemotherapy and PDT using doxorubicin and methylene blue was able to overcome resistance mechanisms and resulted in improved cytotoxicity in drug-resistant tumor cells.
Keywords: Nanoparticles; Photodynamic therapy; Nuclear delivery; Drug efflux; Reactive oxygen species; Photosensitizer; Cellular delivery; Cytotoxicity;

In this study, doxorubicin (DOX) was physically incorporated into pH-sensitive micelles made from a mixture of poly(l -histidine)-b-poly(ethylene glycol)/poly(l -lactide)-b-poly(ethylene glycol) (75/25, wt.%). The DOX-loaded mixed micelles were formulated using dialysis methods and optimal DOX incorporation was achieved at a drug/polymer feed ratio of 0.2 (wt./wt.) when a proper amount of aqueous phase (0.2, v./v.) was added into the common solvent (DMSO) solution, followed by dialysis at 4 °C. Based on the results obtained from dynamic light scattering, UV–Vis absorption, and fluorescence experiments, it was demonstrated that the encapsulated drugs were mainly located inside the hydrophobic micelle cores, well protected and inaccessible to the exterior molecules. Under in vitro conditions, although the microstructure of the micelles was altered below pH 8.0 by the encapsulated drugs, the drug-loaded micelles still exhibited a desirable ability to control the drug release in response to tumor extracellular pH.
Keywords: Polymeric mixed micelles; Poly(l -histidine); pH-sensitive; Anticancer drug delivery; Doxorubicin; Drug incorporation; In vitro kinetics;

The aim of this study was to enhance the loading of dalargin (enkephalin derivatives) a hydrophilic drug and loperamide HCl (non-opiate antidiarrheal agent) a lipophilic drug candidates within PEGylated nanoparticles. A novel nanoencapsulation method based on the concept of s/o/w and ion pairing followed by solvent diffusion was adopted. The copolymers with three different mPEG densities (5%, 12% and 17%) were employed separately in combination with two different grades of dextran sulphate (DS) 5000 and 500,000 MW in the preparations. Nanoparticles prepared from copolymers with increasing mPEG densities, showed an insignificant (p  > 0.05) increasing trend of drug loading, this was however significantly increased when DS5000 was included in the preparations. The particle size remains unchanged after dalargin loading, with no significant (p  > 0.05) alteration in the neutral zeta potential compared to that of the preparations without DS5000. Considering that a dalargin ion pair could also have a neutral charge, it was not advisable to conclude its incorporation, as the size remain unchanged, which would otherwise increase if an ion pair was incorporated within the core of nanoparticles. Therefore, it was expected that a dalargin ion pair might be located outside the core as a separate particulate entity or reside in the hydrophilic shell of the nanoparticles. A loperamide HCl ion pair showed significant (p  < 0.05) increase in size when incorporated; at the same time it provided a neutral zeta potential despite adding negatively charged DS5000 in the preparation, hence it seemed encapsulated. Inclusion of DS500,000 in the preparation further increased the drug loading of dalargin and loperamide HCl. However, a significant (p  < 0.05) negative zeta potential was noted in both cases which suggested that excess charge was still available on the surface of nanoparticles which could trap further amounts of drug on the surface rather than inside the core of nanoparticles. During in vitro evaluation of drug loaded nanoparticles, dalargin released as quickly as free drug, when loperamide HCl showed almost burst free sustained release profile with respect to the release of their free drug solutions, suggested that ion pairing approach was more pronounced for loperamide HCl formulation.
Keywords: s/o/w; Ion pairing; Microencapsulation; PEGylation; Nanoparticles;

Nanoparticles for skin penetration enhancement – A comparison of a dendritic core-multishell-nanotransporter and solid lipid nanoparticles by Sarah Küchler; Michal R. Radowski; Tobias Blaschke; Margitta Dathe; Johanna Plendl; Rainer Haag; Monika Schäfer-Korting; Klaus D. Kramer (243-250).
Nanosized particles are of growing interest for topical treatment of skin diseases to increase skin penetration of drugs and to reduce side effects. Effects of the particle structure and size were studied loading nile red to dendritic core-multishell (CMS) nanotransporters (20–30 nm) and solid lipid nanoparticles (SLNs, 150–170 nm).Interaction properties of CMS nanotransporters with the dye molecules – attachment to the carrier surface or incorporation in the carrier matrix – were studied by UV/Vis and parelectric spectroscopy. Pig skin penetration was studied ex vivo using a cream for reference. Interactions of SLN and skin were followed by scanning electron microscopy, internalisation of the particles by viable keratinocytes by laser scanning microscopy.Incorporating nile red into a stable dendritic nanoparticle matrix, dye amounts increased eightfold in the stratum corneum and 13-fold in the epidermis compared to the cream. Despite SLN degradation at the stratum corneum surface, SLN enhanced skin penetration less efficiently (3.8- and 6.3-fold). Viable human keratinocytes showed an internalisation of both nanocarriers.In conclusion, CMS nanotransporters can favour the penetration of a model dye into the skin even more than SLN which may reflect size effects.
Keywords: Dendritic core-multishell nanotransporters; Solid lipid nanoparticles; Skin penetration; Cellular uptake; Nile red;

Human serum albumin (HSA) nanoparticles were manufactured by desolvation. Transferrin or transferrin receptor monoclonal antibodies (OX26 or R17217) were covalently coupled to the HSA nanoparticles using the NHS-PEG-MAL-5000 crosslinker. Loperamide was used as a model drug since it normally does not cross the blood–brain barrier (BBB) and was bound to the nanoparticles by adsorption. Loperamide-loaded HSA nanoparticles with covalently bound transferrin or the OX26 or R17217antibodies induced significant anti-nociceptive effects in the tail-flick test in ICR (CD-1) mice after intravenous injection, demonstrating that transferrin or these antibodies covalently coupled to HSA nanoparticles are able to transport loperamide and possibly other drugs across the BBB. Control loperamide-loaded HSA nanoparticles with IgG2a antibodies yielded only marginal effects.
Keywords: Drug targeting; Human serum albumin (HSA); Nanoparticle size; Transferrin; Transferrin receptor; OX26 mAb; Blood–brain barrier; Loperamide;

Chitosan/cyclodextrin nanoparticles can efficiently transfect the airway epithelium in vitro by Desirée Teijeiro-Osorio; Carmen Remuñán-López; María José Alonso (257-263).
The main goal of the present study was to investigate the potential of a new generation of hybrid polysaccharide nanocarriers, composed of chitosan (CS) and anionic cyclodextrins (CDs), for gene delivery to the airway epithelium. More specifically, these nanocarriers were investigated with regard to their ability to enter epithelial cells and promote gene expression in the Calu-3 cell culture model.In the search for the most suitable nanocarrier composition for gene delivery, the effect of CS molecular weight (Mw) on the nanocarriers characteristics and their ability to transfect cells was investigated. Thus, hybrid CS/CD nanoparticles were prepared with two different CS Mw, medium (110 kDa) and low (10 kDa), and loaded with pSEAP (plasmid DNA model that encodes the expression of secreted alkaline phosphatase). The resulting nanoparticles presented an adequate size range (100–200 nm, depending on CS Mw), a positive surface charge (+22 to +35 mV) and very high DNA association efficiency values (>90%). Cellular uptake studies showed that the nanoparticles were effectively internalized by the cells, providing a good indication of their potential as gene carriers. The transfection efficiency of the different formulations, measured by the concentration of secreted gene product (SEAP), indicated that all the nanoparticles were able to elicit a significantly higher response than the naked DNA (control), the transfection efficiency being more important for low MwCS nanoparticles than for those composed of medium MwCS. Overall, this report is the first evidence of the potential of a new generation of safe polysaccharide nanocarriers for gene delivery to the airway epithelium.
Keywords: Calu-3 cells; Chitosan; Cyclodextrin; Nanoparticles; Nanocarriers; Gene/DNA delivery;

The objective of this study was to load preformed highly porous microparticles with drug. The microparticles were prepared by a modified multiple emulsion (w/o/w) solvent evaporation method with the addition of pore formers (NaCl into the internal aqueous phase or of glycerol monooleate to the poly(lactide-co-glycolide) (PLGA) polymer phase). The drug-free solidified microparticles were then washed with either water (for NaCl) or hexane (for glycerol monooleate) to extract the pore formers. The drug was then loaded into the preformed porous microparticles by incubation in aqueous drug solutions followed by air- or freeze-drying. The drug was strongly bound to the polymeric surface with air-dried microparticles. A biphasic drug release with an initial rapid release phase (burst effect) was followed by a slower release up to several weeks. The initial burst was dependent on the drug loading and could be significantly reduced by wet (non-aqueous) temperature curing.
Keywords: Biodegradable drug delivery systems; Initial burst; Poly(lactide-co-glycolide); Porous microparticles; Solvent evaporation method;

Intraorally fast-dissolving particles of a poorly soluble drug: Preparation and in vitro characterization by Riikka Laitinen; Eero Suihko; Kaisa Toukola; Mikko Björkqvist; Joakim Riikonen; Vesa Pekka Lehto; Kristiina Järvinen; Jarkko Ketolainen (271-281).
In this study, the dissolution rate of a poorly soluble drug, perphenazine (PPZ) was improved by a solid dispersion technique to permit its usage in intraoral formulations. Dissolution of PPZ (4 mg) in a small liquid volume (3 ml, pH 6.8) within one minute was set as the objective. PVP K30 and PEG 8000 were selected for carriers according to the solubility parameter approach and their 5/1, 1/5 and 1/20 mixtures with PPZ (PPZ/polymer w/w) were prepared by freeze-drying from 0.1 N HCl solutions. The dissolution rate of PPZ was improved with all drug/polymer mixture ratios compared to crystalline or micronized PPZ. A major dissolution rate improvement was seen with 1/5 PPZ/PEG formulation, i.e. PPZ was dissolved completely within one minute. SAXS, DSC and XRPD measurements indicated that solid solutions of amorphous PPZ in amorphous PVP or in partly amorphous PEG were formed. DSC and FTIR studies suggested that PPZ dihydrochloride salt was formed and hydrogen bonding was occurred between PPZ and the polymers. It was concluded that molecular mixing together with salt formation promoted the dissolution of PPZ, especially in the case of the 1/5 PPZ/PEG dispersion, making it a promising candidate for use in intraoral formulations.
Keywords: Solid dispersion; Solid solution; Dissolution enhancement; PVP; PEG; Amorphous;

Nano-salbutamol dry powder inhalation: A new approach for treating broncho-constrictive conditions by Bhavna; Farhan Jalees Ahmad; Gaurav Mittal; Gaurav K. Jain; Geena Malhotra; Roop K. Khar; Aseem Bhatnagar (282-291).
Nanoparticle DPI is known to have deeper lung penetration but its clinical utility as a potentially better treatment option needs to be evaluated in the light of higher expected mucociliary movement of the nanoparticles compared to micronized DPI. The objective of this study was to make nano-salbutamol sulphate (SBS) DPI, radiolabel it with Tc-99m using a novel surface labeling methodology, characterize the formulation and assess its in vitro and in vivo deposition in healthy human volunteers to estimate its bioavailability in the target area. Nano-SBS with a mean particle of 60.71 ± 35.99 nm was produced using liquid anti-solvent precipitation method. The drug particles were spherical, pure and crystalline. Anderson cascade impaction showed that blend formulations of Nano-SBS exhibited significantly higher respirable fraction of 45.2% compared to the known behavior of micronized salbutamol sulphate blends. Though the particle size tended to increase due to solid phase interaction after blending with lactose, there was definitive correlation between the radiolabeled and non-radiolabeled forms. In 10 healthy volunteers, lower oropharyngeal depositions (25.3 ± 4.5%) were observed with nano-SBS formulation compared to micronized SBS formulation (58.4 ± 6.1%). Furthermore, Nano-SBS formulations showed nearly 2.3-fold increase in total lung deposition compared to micronized SBS. The in vivo deposition data and the ratio of peripheral to central lung deposition (P/C) of 1.12 ± 0.4 indicate that Nano-SBS is evenly distributed within different lung regions. As demonstrated for SBS, nano-sizing may enhance regional deposition and thus provide an attractive particle engineering option for the development of blend formulations for inhalation delivery.
Keywords: Nano-sizing; Anti solvent precipitation; Spray drying; Salbutamol sulphate; Lung deposition; Inhalation;

Trifluralin liposomal formulations active against Leishmania donovani infections by Manuela Carvalheiro; João Jorge; Carla Eleutério; Ana F. Pinhal; Ana C. Sousa; José G. Morais; M. Eugénia M. Cruz (292-296).
The purpose of this study was to increase the therapeutic index of the antiparasitic drug, trifluralin (TFL), to allow its parenteral administration without the need of toxic solvents. This was achieved by incorporating TFL in liposomes with high loading capacity. These formulations were stable in freeze-dried form during at least one year and in frozen form during at least three months. Therapeutic activity, assessed on a visceral model of infection, showed that TFL liposomes reduced the number of parasites by up to one third or one half as compared to negative control and to free TFL, respectively.
Keywords: Trifluralin; Dinitroanilines; Liposomes; Leishmaniasis; Therapeutic activity;

Gelation of microemulsions and release behavior of sodium salicylate from gelled microemulsions by Guilong Feng; Yun Xiong; Hong Wang; Yajiang Yang (297-302).
A novel gelled microemulsion was prepared in the presence of the low molecular weight gelator N-stearine-N′-stearyl-l-phenylalanine at a very low concentration. It is completely different from the conventional microemulsion-based gels (MBGs) usually formed by polymeric gelling agents, such as gelatin, agar and κ-carrageenan. The microemulsion consists of i-propyl myristate, Tween 80, propylene glycol and water. The gelled microemulsions showed good thermo-reversibility. The gel-to-sol transition temperature (T GS) of gelled microemulsion depends upon the concentration of gelator and the composition of the microemulsions. The gelation mechanism was investigated by polarized optical microscopy (POM) and FT-IR. POM images show elongated and strand-like crystallites formed by the aggregation of the gelator, ultimately resulting in the gelation of the microemulsion. FT-IR analysis indicates that intermolecular hydrogen bonds are responsible for the formation of gelator aggregates. Water-soluble sodium salicylate was used as a model drug for the investigation of the release from the gelled microemulsions. The release profiles exhibited a controlled release and followed the first-order release kinetics. The release rates decreased with an increase of the gelator and isopropyl myristate contents. These results reveal potential applications of gelled microemulsion in drug delivery systems.
Keywords: Gelled microemulsion; Gelator; Sodium salicylate; Release behavior;

The purpose of this study was to investigate the cytokine production and liver injury induced by lipoplexes prepared with DOTMA/cholesterol and DOTAP/cholesterol liposomes with various mixing ratios in mice. Lipoplexes were prepared with pCMV-Luc and DOTMA/cholesterol or DOTAP/cholesterol liposomes. After intravenous administration into the mice, organ luciferase activity and serum TNFα and ALT were measured to evaluate the transfection efficacy, cytokine production and liver injury. After intravenous administration of these lipoplexes, basically the serum TNFα and ALT levels were in agreement with the transfection efficacy of the lipoplexes. The cytokine production and liver injury were markedly suppressed by reducing the pDNA dose, and achieved normal levels at a pDNA dose of 0.47 mg/kg. As far as the effects of the charge ratio at this low pDNA dose are concerned, the charge ratios of the lipoplexes affected the transfection efficacy, but not the cytokine production and liver injury. After intravenous administration of either DOTAP/cholesterol or DOTMA/cholesterol liposomes, serum TNFα and ALT levels were normal, suggesting that liver injury as well as cytokine production was caused by lipoplexes, but not by cationic liposomes. This information will be valuable for the future optimization of the preparation conditions of lipoplexes for use in clinical gene therapy.
Keywords: Plasmid DNA; Cationic lipids; Lipoplexes; Cytokine; Liver injury;

Oral microemulsions of paclitaxel: In situ and pharmacokinetic studies by Adwoa O. Nornoo; HaiAn Zheng; Luciana B. Lopes; Boris Johnson-Restrepo; Kurunthachalam Kannan; Rachel Reed (310-317).
The overall goal of this study was to develop cremophor-free oral microemulsions of paclitaxel (PAC) to enhance its permeability and oral absorption. The mechanism of this enhancement, as well as characteristics of the microemulsions relevant to the increase in permeability and absorption of the low solubility, low permeability PAC was investigated. Phase diagrams were used to determine the macroscopic phase behavior of the microemulsions and to compare the efficiency of different surfactant-oil mixtures to incorporate water. The microemulsion region on the phase diagrams utilizing surfactant-myvacet oil combinations was in decreasing order: lecithin: butanol: myvacet oil (LBM, 48.5%) > centromix CPS: 1-butanol: myvacet oil (CPS, 45.15%) > capmul MCM: polysorbate 80: myvacet oil (CPM, 27.6%) > capryol 90: polysorbate 80: myvacet oil (CP-P80, 23.9%) > capmul: myvacet oil (CM, 20%). Oil-in-water (o/w) microemulsions had larger droplet sizes (687–1010 nm) than the water-in-oil (w/o) microemulsions (272–363 nm) when measured using a Zetasizer nano series particle size analyzer. Utilizing nuclear magnetic resonance spectroscopy (NMR), the self-diffusion coefficient (D) of PAC in CM, LBM and CPM containing 10% of deuterium oxide (D2O) was 2.24 × 10−11, 1.97 × 10−11 and 0.51 × 10−11  m2/s, respectively. These values indicate the faster molecular mobility of PAC in the two w/o microemulsions (CM and LBM) than the o/w microemulsion – CPM. The in situ permeability of PAC through male CD-IGS rat intestine was 3- and 11-fold higher from LBM and CM, respectively, than that from the control clinical formulation, Taxol® (CE, cremophor: ethanol) in a single pass perfusion study. PAC permeability was significantly increased in the presence of the pgp/CYP3A4 inhibitor cyclosporine A (CsA). This enhancement may be attributed to the pgp inhibitory effect of the surfactants, oil and/or the membrane perturbation effect of the surfactants. The oral disposition of PAC in CM, LBM and CPM compared to CE was studied in male CD-IGS rats after a single oral dose (20 mg/kg). The area-under-the-curve of PAC in CM was significantly larger than LBM, CPM and CE. Oral microemulsions of PAC were developed that increased both the permeability and AUC of PAC as compared to CE.
Keywords: Microemulsion; Paclitaxel; Permeability; In situ; Oral; Pharmacokinetics;

The transepithelial transport rates of compounds after deposition as aerosolised particles onto respiratory cell layers and allowing dissolution in the cell surface secretions has not been reported in a comprehensive manner to date. Here, the twin-stage impinger (TSI) was used to deposit potentially respirable particles (aerodynamically <6.4 μm) of varying molecular weight dextrans labelled with fluorescein isothiocyanate (FITC-dex) onto Calu-3 cells, a model of the bronchial epithelium. The TSI functioned as a particle size segregator, with >96% of the deposited particles being geometrically <6.4 μm (as measured by microscopy) and the particles being deposited discretely with a uniform distribution. Cell layers tolerated particle deposition at an air flow of 60 L/min. A small reduction in transepithelial electrical resistance (TER) of <10% occurred initially, but the original TER was recovered within 10 min and there was no significant effect on apparent permeability (P app) of FITC-dex 4 over 4 h. Interleukin 8 (IL-8) secretion in the apical and basolateral directions over 24 h was not increased by exposure to the TSI and particle deposition. The rate of FITC-dex 4 (4 kDa) transport across the cell layer after deposition and dissolution of the particles in the cell surface secretions was ∼20-fold higher (P  < 0.05) than if applied as a solution. The volume of cell surface secretions was estimated by tracer dilution (3.44 ± 1.90 μl, mean ± SEM) and this value was used to calculate the P app of compound once deposited as a particle. The Papp value was found to be similar to that obtained when the compound was applied in solution (P  < 0.05). Thus, the increased transport rate was attributable to the differences in donor chamber solute concentration rather than any change in the permeability of the cell layer itself. Following particle deposition, transport of FITC-dex with molecular weights between 4 and 70 kDa correlated well (r 2  = 0.918) with reported in vivo canine pulmonary clearance after intratracheal instillation of dextrans of similar molecular weight. The use of the TSI and the Calu-3 cell line for the assessment of compound dissolution and transport rates after particle deposition may allow more realistic analyses to be made with respect to the in vivo situation.
Keywords: Twin-stage impinger; Calu-3; Deposition; Particle;

The objective of this study was to identify the important factors for the drug permeability and mucoadhesion of casted free pectin/chitosan combination films. The factors varied were: the type of pectin (low and high methoxyl pectin) and the ratio pectin:chitosan (25:75, 50:50 and 75:25). The model drug used for measuring drug permeability was paracetamol. A texture analyzer was used for measuring mucoadhesion by using two different setups: (1) in vitro tensile tests measuring the detachment force of films versus a mucin dispersion and (2) ex vivo shear tests measuring the friction forces between pre-hydrated films and fresh porcine small intestine, with the system immersed in phosphate buffer, pH 6.8.The type of pectin used in the combination films did not have a significant effect on the drug permeability. The ex vivo mucoadhesion test revealed significant differences between low and high methoxyl pectin only for the 50:50 pectin:chitosan films. For that type of film, the peak and friction forces were highest for high methoxyl pectin. Both the mucoadhesion and drug permeability generally increased with decreasing amounts of pectin relative to chitosan in the films.
Keywords: Mucoadhesion; Pectin; Degree of methoxylation; Chitosan; Film casting; Texture analyzer;

A Caco-2 cell based screening method for compounds interacting with MRP2 efflux protein by Sanna Siissalo; Jenni Hannukainen; Johanna Kolehmainen; Jouni Hirvonen; Ann Marie Kaukonen (332-338).
The aim of this work was to develop a screening method for MRP2 efflux substrates using the well-characterized, human-based intestinal Caco-2 cell model as a platform. MRP2 has a significant role in drug absorption and disposition and is known to co-operate with phase II metabolic enzymes. Caco-2 cells grown in a 96-well plate were loaded with non-fluorescent CDCFDA (diacetate ester of 5(6)-carboxy-2′,7′-dichlorofluorescein), which is hydrolyzed to fluorescent CDCF by intracellular esterases. De-esterification in Caco-2 was comparable to that in porcine liver esterases. CDCFDA enters the cells passively, while CDCF is effluxed out of the cells by the apically localized MRP2 and/or basolateral MRPs. The method was optimized with regard to several factors. In the concluding protocol, Caco-2 cells are grown on clear 96-well plates for 8 days. The loading conditions were optimized to 10 min incubation with 5 μM CDCFDA. The highest responses were obtained for samples taken at t  = 30 min. The samples were analyzed in black 96-well plates with a fluorescence plate reader. The Caco-2 based method utilizing the probe pair CDCFDA/CDCF provides a fast screening tool for MRP2 substrates and/or inhibitors, along with compounds having metabolites formed in Caco-2 that interact with MRP2.
Keywords: Caco-2 cell line; Screening method; MRP2 efflux protein; Intracellular de-esterification; CDCF; 5(6)-Carboxy-2′,7′-dichlorofluorescein;

In vitro evaluation of natural and methylated cyclodextrins as buccal permeation enhancing system for omeprazole delivery by Ana Figueiras; Juliane Hombach; Francisco Veiga; Andreas Bernkop-Schnürch (339-345).
In this work the enhancing effect of cyclodextrins on the buccal permeation of a hydrophobic model drug, omeprazole was studied. First, the influence of the complexation with cyclodextrins in the absence and in the presence of an alkali agent, l-arginine, on the drug stability was checked at neutral conditions since omeprazole alone is only stable in basic conditions. In vitro transbuccal permeation of omeprazole non-complexed and complexed with β- and methyl-β-cyclodextrin and in presence of l-arginine was examined using freshly obtained porcine buccal mucosa. Tissue viability after incubation with sample solutions was assessed using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) biochemical assay and histological evaluation. The toxicity of the sample solutions on buccal mucosa was evaluated by measuring lactate dehydrogenase activity. The present results show that complexation with cyclodextrins increases drug stability at neutral conditions; furthermore, l-arginine contributed to higher drug stability. Permeation studies indicate an increase on drug permeation in complexed form of 1.1- and 1.7-fold for β-cyclodextrin and methyl-β-cyclodextrin, respectively. The presence of l-arginine increases drug permeation 1.4-fold in omeprazole complexed with β-cyclodextrin and 2.4-fold in the inclusion complex formed with methyl-β-cyclodextrin. The cell viability of the buccal mucosa after a 3 h incubation period, with all sample solutions, remained around 70% and lactate dehydrogenase assay showed that studied cyclodextrins, even in the presence of an alkali agent are not cytotoxic for porcine buccal mucosa. Histological evaluation of the tissue demonstrated that the buccal epithelium remains viable after 3 h of incubation with sample solutions.
Keywords: Buccal mucosa; Cyclodextrins; l-Arginine; Omeprazole; Permeation; Stability;

In order to better understand the mechanism of destabilization of liposomes used as drug carriers for oral administration by bile salts, the insertion and partition of sodium taurocholate (TC) into small unilamellar vesicles (SUV) and multilayers (ML) of dipalmitoylphosphatidylcholine (DPPC) were examined by continuous turbidity analysis and DSC. Optical density was recorded during the progressive solubilisation of DPPC SUV and ML into DPPC/TC mixed micelles by varying the rate of TC addition and the temperature. The results show that the insertion and diffusion of TC in the DPPC membrane is a slow process influenced by the polymorphism of the lipid, independently of its organisation. This dynamic study mimics physiological phenomena of the digestion of liposomes. In the gastrointestinal tract, DPPC SUV would be more resistant to TC than egg phosphatidylcholine (EPC) SUV [K. Andrieux, L. Forte, S. Lesieur, M. Paternostre, M. Ollivon, C. Grabielle-Madelmont, Insertion and partition of sodium taurocholate into egg phosphatidylcholine vesicles, Pharm. Res. 21 (2004) 1505–1516] because of the lower insertion of TC into DPPC bilayer at 37 °C at low TC concentration in the medium (fasted conditions). At high TC concentration (postprandially or after lipid absorption), the use of DPPC to prepare liposomes will delay or reduce the liberation of a drug encapsulated into liposomes in the gastrointestinal tract. As a conclusion, the addition of DPPC appears an attractive strategy to formulate orally administered liposomes.
Keywords: Liposome; Phospholipid; Polymorphism; Bile salt; Turbidity; DSC; Solubilisation mechanism;

Use of spray-dried chitosan acetate and ethylcellulose as compression coats for colonic drug delivery: Effect of swelling on triggering in vitro drug release by J. Nunthanid; M. Luangtana-anan; P. Sriamornsak; S. Limmatvapirat; K. Huanbutta; S. Puttipipatkhachorn (356-361).
Spray-dried chitosan acetate (CSA) and ethylcellulose (EC) were used as new compression coats for 5-aminosalicylic acid tablets. Constrained axial or radial swelling of pure CSA and EC/CSA tablets in 0.1 N HCl (stage I), Tris–HCl, pH 6.8 (stage II), and acetate buffer, pH 5.0 (stage III), was investigated. Factors affecting in vitro drug release, i.e., % weight ratios of coating polymers, dip speeds of dissolution apparatus or pH of medium or colonic enzyme (β-glucosidase) in stage III, and use of a super disintegrant in core tablets, were evaluated. Swollen CSA gel dissolved at lower pH and became less soluble at higher pH. The mechanism of swelling was Fickian diffusion fitting well into both Higuchi’s and Korsmeyer–Peppas models. EC:CSA, at 87.5:12.5% weight ratio, provided lag time rendering the tablets to reach stage III (simulated colonic fluid of patients), and the drug was released over 90% within 12 h. The system was a dual time- and pH-control due to the insolubility of EC suppressing water diffusion and the swelling of CSA in the stages I and II. The erosion of CSA gel in the stage III induced the disintegration of the coat resulting in rapid drug release. The lower dip speed and higher pH medium delayed the drug release, while a super disintegrant in the cores enhanced the drug release and no enzyme effect was observed.
Keywords: Chitosan acetate; Compression-coated tablets; Swelling; Ethylcellulose; Colonic drug delivery;

The objective was to compare the in vivo distribution profiles of betamethasone 17-valerate (BMV) across the stratum corneum (SC) following (a) delivery from gelled and un-gelled formulations, and (b) two different skin cleaning procedures at the end of the application period. BMV was dissolved in gelled and un-gelled vehicles comprising either medium chain triglycerides (MCT) or a brand microemulsion (ME). The BMV concentration was adjusted to 80% of saturation and applied to the forearms of healthy volunteers. After 2 h, the treated skin site was cleaned either with a dry paper towel or with an isopropyl alcohol swab, and the SC was then progressively removed by repeated adhesive tape-stripping. BMV distribution profiles across the SC showed reasonable reproducibility, and that delivery from the ME was significantly superior to that from MCT. Gelled vehicles were less efficiently removed from the skin surface by dry wiping than un-gelled formulations. Removing excess formulation more aggressively with isopropyl alcohol resulted in a lower apparent uptake of drug into the SC. Excess gelled formulation may be trapped in the skin ‘furrows’, and requires an efficient skin cleaning procedure to ensure its complete removal.
Keywords: Topical bioavailability; Corticosteroids; Dermatopharmacokinetics; Tape-stripping; Stratum corneum; Vehicle effects;

Role of benzyl alcohol in the prevention of heat-induced aggregation and inactivation of hen egg white lysozyme by Monu Kumari Goyal; Ipsita Roy; Uttam Chand Banerjee; Vikas Kumar Sharma; Arvind Kumar Bansal (367-376).
The aim of the study was to investigate the stability of a model protein, lysozyme, in the presence of the commonly used preservative benzyl alcohol. Techniques including lytic assay, size exclusion chromatography, circular dichroism, differential scanning calorimetry, native polyacrylamide gel electrophoresis and dynamic light scattering were used to study the overall stability of lysozyme in the presence of benzyl alcohol. The stability of lysozyme against thermal stress was higher in the presence of benzyl alcohol. In the presence of 0.5%, 0.9% and 2% v/v benzyl alcohol, the enzyme showed 33%, 42% and 75% residual activity, respectively, when exposed to 75 °C for 2 h, as compared to the 22% activity of control sample. A gradual increase in the size of aggregates was observed for the control sample relative to the samples containing benzyl alcohol, as a result of loss of monomer concentration. The effect was found to be concentration-dependent with 2% benzyl alcohol showing maximum prevention of heat-induced unfolding and aggregation. This effect is remarkable since the thermal transition temperature of the enzyme decreases in the presence of benzyl alcohol. Benzyl alcohol favours the thermal denaturation of lysozyme but stabilizes the lysozyme against the heat-induced aggregation.
Keywords: Lysozyme; Benzyl alcohol; Multi-dose formulation; Protein stabilization; Circular dichroism;

The influence of the preparation methods on the inclusion of model drugs in a β-cyclodextrin cavity by P.J. Salústio; G. Feio; J.L. Figueirinhas; J.F. Pinto; H.M. Cabral Marques (377-386).
The work aims to prove the complexation of two model drugs (ibuprofen, IB and indomethacin, IN) by β-cyclodextrin (βCD), and the effect of water in such a process, and makes a comparison of their complexation yields. Two methods were considered: kneading of a binary mixture of the drug, βCD, and inclusion of either IB or IN in aqueous solutions of βCD. In the latter method water was removed by air stream, spray-drying and freeze-drying. To prove the formation of complexes in final products, optical microscopy, UV spectroscopy, IR spectroscopy, DSC, X-ray and NMR were considered. Each powder was added to an acidic solution (pH = 2) to quantify the concentration of the drug inside βCD cavity. Other media (pH = 5 and 7) were used to prove the existence of drug not complexed in each powder, as the drugs solubility increases with the pH. It was observed that complexation occurred in all powders, and that the fraction of drug inside the βCD did not depend neither on the method of complexation nor on the processes of drying considered.
Keywords: Ibuprofen; Indomethacin; Cyclodextrin; Freeze-drying; Spray-drying; Physical mixture; Kneading; NMR; X-ray; Optical microscopy; FT-IR spectroscopy; UV spectroscopy; DSC;

Mechanism of drug release from polymethacrylate-based extrudates and milled strands prepared by hot-melt extrusion by Jessica Albers; Rainer Alles; Karin Matthée; Klaus Knop; Julia Schulze Nahrup; Peter Kleinebudde (387-394).
The aim of the study was the formulation of solid dispersions of the poorly water-soluble drug celecoxib and a polymethacrylate carrier by hot-melt extrusion. The objectives were to elucidate the mechanism of drug release from obtained extrudates and milled strands addicted to the solid-state properties of the solid dispersions and to examine and eliminate stability problems occurring under storage, exposure of mechanical stress, and in vitro dissolution.Transparent extrudates containing up to 60% drug could be prepared with a temperature setting below the melting point of celecoxib. XRPD and DSC measurements indicated the formation of a glassy solid solution, where the drug is molecularly dispersed in the carrier. The amorphous state of the glassy solid solution could be maintained during the exposure of mechanical stress in a milling process, and was stable under storage for at least 6 months. Solid-state properties and SEM images of extrudates after dissolution indicated a carrier-controlled dissolution, whereby the drug is molecularly dispersed within the concentrated carrier layer. The glassy solid solution showed a 58-fold supersaturation in 0.1 N HCl within the first 10 min, which was followed by a recrystallization process. Recrystallization could be inhibited by an external addition of HPMC.
Keywords: Celecoxib; Polymethacrylate; Hot-melt extrusion; Solid dispersion; Dissolution; Intrinsic dissolution; Stability; Recrystallization inhibition;

On the physical interpretation of the initial bending of a Shapiro–Konopicky–Heckel compression profile by Ingvild Klevan; Josefina Nordström; Annette Bauer-Brandl; Göran Alderborn (395-401).
The relationship between the natural logarithm of the tablet porosity and the applied pressure is used to describe the compression behavior of a powder. Such a relationship, here referred to as a Shapiro–Konopicky–Heckel (SKH) profile, is usually divided into three regions, of which the first often is non-linear. The objective of this work was to address the question of the mechanisms controlling the compression and the bending of the first region of a SKH profile for dense particles. In this paper, the first region was described by the Shapiro General Compression Equation, from which a compression parameter was derived as a measure of the bending. The results indicate that for powders undergoing significant particle rearrangement at low applied pressures, the particle rearrangement is the major cause for the initial bending of the SKH profile. For powders showing limited particle rearrangement, the initial bending is mainly caused by the change in particle diameter due to particle fragmentation. It is concluded that the evaluation of the first region of a SKH profile in terms of bending may be used to assess particle fragmentation. The SKH profile could hence be a useful tool to describe powder compression behavior in terms of particle fragmentation and particle deformation from one single compression analysis.
Keywords: Powder compression; Heckel; Shapiro; Fragmentation; Powder compression classification system; Powder technology;

by Richard Süverkrüp (402).

by Katharina M. Picker-Freyer (402-403).

by Katharina M. Picker-Freyer (403).

by Hartmut Glaeser (404-405).

by Katharina M. Picker-Freyer (404).