International Journal of Pharmaceutics (v.428, #1-2)

Characterization of starch Pickering emulsions for potential applications in topical formulations by Diana Marku; Marie Wahlgren; Marilyn Rayner; Malin Sjöö; Anna Timgren (1-7).
The aim of this work has been to characterize starch based Pickering emulsions as a first step to evaluate their possible use as vehicles for topical drug delivery. A minor phase study of emulsions with high oil content has been performed. Emulsion stability against coalescence over eight weeks and after mild centrifugation treatment has been studied. The particle size, rheological properties and in vitro skin penetration of emulsions containing three different oils (Miglyol, paraffin and sheanut oil) was investigated. It was shown that it is possible to produce oil in water starched stabilised Pickering emulsions with oil content as high as 56%. Furthermore, this emulsions show good stability during storage over eight weeks and towards mild centrifugation. The particle size of the systems are only dependent on the ratio between oil and starch and for liquid oils the type of oil do not affect the particle size. The type of oil also affects the cosmetic and rheological properties of the creams but did not affect the transdermal diffusion in in vitro tests. However, it seems as if the Pickering emulsions affected the transport over the skin, as the flux was twice that of what has been previously reported for solutions.
Keywords: Quinoa starch; Pickering emulsions; Topical formulation; Emulsion stability; Particle stabilised emulsions;

In vitro and in vivo characterization of amorphous, nanocrystalline, and crystalline ziprasidone formulations by Avinash G. Thombre; Jaymin C. Shah; Kazuko Sagawa; W. Brett Caldwell (8-17).
Ziprasidone, commercially available as Geodon® capsules, is an atypical antipsychotic used in the treatment of schizophrenia and bipolar disorder. It is a BCS Class II drug that shows up to a 2-fold increase in absorption in the presence of food. Because compliance is a major issue in this patient population, we developed and characterized solubilized formulations of ziprasidone in an effort to improve absorption in the fasted state, thereby resulting in a reduced food effect. Three formulations utilizing solubilization technologies were studied: (1) an amorphous inclusion complex of ziprasidone mesylate and a cyclodextrin, (2) a nanosuspension of crystalline ziprasidone free base, and (3) jet-milled ziprasidone HCl coated crystals made by spray drying (CCSD) the drug with hypromellose acetate succinate. The formulations were characterized by in vitro methods appropriate to each particular solubilization technology. These studies confirmed that ziprasidone mesylate – cyclodextrin was an amorphous inclusion complex with enhanced dissolution rates. The ziprasidone free base crystalline nanosuspension showed a mean particle size of 274 nm and a monomodal particle size distribution. In a membrane permeation test, the CCSD showed a 1.5-fold higher initial flux compared to crystalline ziprasidone HCl. The three formulations were administered to fasted beagle dogs and their pharmacokinetics compared to Geodon® capsules administered in the fed state. The amorphous complex and the nanosuspension showed increased absorption in the fasted state, indicating that solubilized formulations of ziprasidone have the potential to reduce the food effect in humans.
Keywords: Ziprasidone; Solubilization; Cyclodextrin; Nanosuspension; Bioavailability enhancement;

Aminoalkyl methacrylate copolymers for improving the solubility of tacrolimus. I: Evaluation of solid dispersion formulations by Takatsune Yoshida; Ippei Kurimoto; Keiichi Yoshihara; Hiroyuki Umejima; Naoki Ito; Shunsuke Watanabe; Kazuhiro Sako; Akihiko Kikuchi (18-24).
The aim of this study was to investigate the effect of Eudragit E®/HCl (E-SD) on the reprecipitation of a poorly water-soluble drug, tacrolimus. To evaluate the inhibition of reprecipitation of E-SD, reprecipitation studies on tacrolimus were conducted using a dissolution test method. Solubility of tacrolimus was measured at regular intervals in a dissolution media, in which tacrolimus was dissolved in ethanol, and the test media contained additives for inhibiting precipitation. Supersaturation profiles of tacrolimus were observed, and were maintained for 24 h only with E-SD. Solid dispersion formulations of tacrolimus prepared with hydroxypropylmethylcellulose (HPMC) or E-SD in different drug/carrier ratios were also investigated. Solid dispersions prepared with E-SD showed higher solubility of tacrolimus compared with that of HPMC. In the E-SD formulation, the drug solubility influences to drug/carrier ratio. The formulation of drug/E-SD (1/5) showed the highest drug solubility. Thus, it may be inferred that a definite drug/carrier ratio exists to increase drug solubility. Further, by mixing E-SD the solid dispersion prepared with HPMC showed enhanced drug solubility.
Keywords: Tacrolimus; Solid dispersion; Solubilization; Eudragit® E; Supersaturation; Reprecipitation inhibitor;

Regulation of human monocarboxylate transporter 4 in skeletal muscle cells: The role of protein kinase C (PKC) by Katsuya Narumi; Masaki Kobayashi; Sho Otake; Ayako Furugen; Natsuko Takahashi; Jiro Ogura; Shirou Itagaki; Takeshi Hirano; Hiroaki Yamaguchi; Ken Iseki (25-32).
In the present study, to clarify the role of protein kinase C (PKC) in the regulation of monocarboxylate transporter 4 (MCT4) expression, we examined the regulation mechanism of MCT4 expression in human rhabdomyosarcoma (RD) cells, an in vitro skeletal muscle model. Exposure of RD cells to PMA, a PKC activator, for 24 h resulted in a two-fold increase in the amount of lactic acid in the growth medium. In parallel to an increase in lactic acid release from RD cells, the level of MCT4 mRNA and protein were also significantly increased in RD cells. A PKC inhibitory study indicated that PMA-induced stimulation of MCT4 expression can be mediated through a novel PKC isoform, especially PKCδ. Moreover, rottlerin, a selective PKCδ inhibitor, decreased PMA-induced MCT4 promoter activity. Deletion and mutational analysis suggested that the potential hypoxia-response elements (HREs) played a major role in the observed modulation of MCT4 expression by PMA. Furthermore, we found that small interfering RNA (siRNA)-mediated knockdown of hypoxia-inducible factor 1α (HIF-1α) significantly inhibited PMA-induced MCT4 promoter activity. Our results show that the effects of PMA on MCT4 expression are mediated through an indirect pathway partially involving PKCδ and HIF-1α transcription factor.
Keywords: Monocarboxylate transporter 4; Skeletal muscle; Protein kinase C; Hypoxia-inducible factor 1α;

RETRACTED: Suppression of efflux transporters in the intestines of endotoxin-treated rats by Mikio Tomita; Yusuke Takizawa; Atsushi Kanbayashi; Hiroyuki Murata; Ayako Tanaka; Mariko Nakaike; Megumi Hatanaka; Tomomi Kai; Masahiro Hayashi (33-38).
This article has been retracted at the request of Publisher Drug Metabolism and Disposition.The integrity of the research findings in the retracted article is undisputed.The Publisher apologizes for any inconvenience caused.

A comparative study of roll compaction of free-flowing and cohesive pharmaceutical powders by Shen Yu; Bindhu Gururajan; Gavin Reynolds; Ron Roberts; Michael J. Adams; Chuan-Yu Wu (39-47).
Roll compaction is widely adopted as a dry granulation method in the pharmaceutical industry. The roll compaction behaviour of feed powders is primarily governed by two parameters: the maximum pressure and the nip angle. Although the maximum pressure can be measured directly using pressure sensors fitted in the rolls, it is not a trivial task to determine the nip angle, which is a measure of the size of the compaction zone and hence the degree of compression. Thus a robust approach based upon the calculation of the pressure gradient, which can be obtained directly from experiments using an instrumented roll compactor, was developed. It has been shown that the resulting nip angles are comparable to those obtained using the methods reported in literature. Nevertheless, the proposed approach has distinctive advantages including (1) it is based on the intrinsic features of slip and no-slip interactions between the powder and roll surface and (2) it is not necessary to carry out wall friction measurements that involve plates that may not be representative of the roll compactor in terms of the surface topography and surface energy. The method was evaluated by investigating the effect of roll speed for two pharmaceutical excipients with distinctive material properties: microcrystalline cellulose (MCC) and di-calcium phosphate dihydrate (DCPD). It was found that the maximum pressure and nip angle for DCPD, which is a cohesive powder, decrease sharply with increasing roll speed whereas they are essentially independent of roll speed for MCC, which is an easy flowing powder. The roll compaction behaviour of MCC–DCPD mixtures with various compositions was also investigated in order to evaluate the effect of flowability. It was found that the nip angle and maximum pressure generally increased with improved flowability of the feed powders.
Keywords: Roll compaction; Granulation; Flowability; Compressibility; Maximum pressure; Nip angle;

Carboxymethyl starch: Chitosan monolithic matrices containing diamine oxidase and catalase for intestinal delivery by Carmen Calinescu; Bruno Mondovi; Rodolfo Federico; Pompilia Ispas-Szabo; Mircea Alexandru Mateescu (48-56).
The capacity of carboxymethyl starch (CMS):Chitosan monolithic tablets to protect diamine oxidase and/or catalase therapeutic enzymes against simulated gastric fluid (SGF) and to control their delivery in simulated intestinal fluid (SIF) was investigated. Enzyme formulations loaded with grass pea seedlings diamine oxidase (PSDAO) vegetal extract, catalase, or PSDAO associated to catalase, were obtained by direct compression. The CMS:Chitosan (1:1) matrix afforded a good gastric protection to PSDAO and to catalase, when each enzyme was formulated separately. Variable amounts of DAO were delivered in the SIF containing pancreatin, with maximal release reached at about 8 h, a time convenient for tablets to attain the colon. Up to 50% of the initial enzymatic activity of catalase formulated with CMS:Chitosan was found after 8 h in SIF. For the CMS:Chitosan tablets of bi-enzymatic formulations containing PSDAO:Catalase, the releases of DAO and of catalase were synchronized. The hydrogen peroxide (product of DAO activity) was decomposed by the catalase liberated in the same SIF environment. The proposed formulations could allow novel therapeutic approaches for the treatment of inflammatory bowel diseases, intestinal cancers or pseudo-allergic reactions.
Keywords: Diamine oxidase vegetal extract; Catalase; Carboxymethyl high amylose starch; Chitosan; Oral tablet administration; Inflammatory bowel diseases;

Optimization of matrix tablets controlled drug release using Elman dynamic neural networks and decision trees by Jelena Petrović; Svetlana Ibrić; Gabriele Betz; Zorica Đurić (57-67).
The main objective of the study was to develop artificial intelligence methods for optimization of drug release from matrix tablets regardless of the matrix type. Static and dynamic artificial neural networks of the same topology were developed to model dissolution profiles of different matrix tablets types (hydrophilic/lipid) using formulation composition, compression force used for tableting and tablets porosity and tensile strength as input data. Potential application of decision trees in discovering knowledge from experimental data was also investigated.Polyethylene oxide polymer and glyceryl palmitostearate were used as matrix forming materials for hydrophilic and lipid matrix tablets, respectively whereas selected model drugs were diclofenac sodium and caffeine. Matrix tablets were prepared by direct compression method and tested for in vitro dissolution profiles. Optimization of static and dynamic neural networks used for modeling of drug release was performed using Monte Carlo simulations or genetic algorithms optimizer. Decision trees were constructed following discretization of data.Calculated difference (f 1) and similarity (f 2) factors for predicted and experimentally obtained dissolution profiles of test matrix tablets formulations indicate that Elman dynamic neural networks as well as decision trees are capable of accurate predictions of both hydrophilic and lipid matrix tablets dissolution profiles. Elman neural networks were compared to most frequently used static network, Multi-layered perceptron, and superiority of Elman networks have been demonstrated. Developed methods allow simple, yet very precise way of drug release predictions for both hydrophilic and lipid matrix tablets having controlled drug release.
Keywords: Matrix tablets; Neural networks; Decision trees; Drug release modeling; Controlled release;

A novel method to enhance the efficiency of drug transdermal iontophoresis delivery by using complexes of drug and ion-exchange fibers by Che Xin; Wang Li-hong; Yuan Yue; Gao Ya-nan; Wang Qi-fang; Yang Yang; Li San-ming (68-75).
The main reason for generally low efficiency of the transdermal iontophoretic drug delivery is that the fraction of the total current contributed by the drug ions is very small. The objective of this study was to find a method to increase the fraction of the total current contributed by the drug ions so as to enhance the drug's iontophoretic delivery. Iontophoretic transport of diclofenac solution and diclofenac assisted by ion exchange materials, including ion-exchange resin, ion-exchange membrane and ion-exchange fiber, across the rat skin were investigated. Both in vitro and in vivo iontophoretic transport experiments showed the amount of diclofenac permeated across rat skin from the diclofenac-fibers was highest among those from the diclofenac simple solutions and ion exchange materials complexes. The results of this study suggested that there is an enhancement of drug across rat skin by ion-exchange fibers in ion-exchange fibers assisted iontophoresis. The present study has demonstrated the potential of a new approach using ion-exchange fibers to enhance transdermal iontophoretic transport of an ionizable drug.
Keywords: Iontophoresis; Transdermal; Ion-exchange fiber; Transport enhancement; Diclofenac;

Dual function of tributyrin emulsion: Solubilization and enhancement of anticancer effect of celecoxib by Sung Nam Kang; Soon-Seok Hong; Mi-Kyung Lee; Soo-Jeong Lim (76-81).
Tributyrin, a triglyceride analogue of butyrate, can act as a prodrug of an anticancer agent butyrate after being cleaved by intracellular enzymes. We recently demonstrated that the emulsion containing tributyrin as an inner oil phase possesses a potent anticancer activity. Herein we sought to develop tributyrin emulsion as a carrier of celecoxib, a poorly-water soluble drug with anticancer activity. Combined treatment of human HCT116 colon cancer cells with free celecoxib plus tributyrin emulsion inhibited the cellular proliferation more effectively than that of each drug alone, suggesting the possibility of tributyrin emulsion as a potential celecoxib carrier. The mean droplet size of emulsions tended to increase as the tributyrin content in emulsion increases and the concentration of celecoxib loaded in emulsions was affected by tributyrin content and the initial amount of celecoxib, but not by the total amount of surfactant mixture. The concentration of celecoxib required to inhibit the growth of HCT116 and B16-F10 cancer cells by 50% was 2.6- and 3.1-fold lowered by loading celecoxib in tributyrin emulsions, compared with free celecoxib. These data suggest that the anticancer activity of celecoxib was enhanced by loading in tributyrin emulsions, probably due to the solubilization capacity and anticancer activity of tributyrin emulsion.
Keywords: Tributyrin; Emulsion; Celecoxib; Cancer; Formulation;

From wax matrix dosage forms, drug and water-soluble polymer are released into the external solvent over time. As a consequence, the pore volume inside the wax matrix particles is increased and the diffusion coefficient of the drug is altered. In the present study, we attempted to derive a novel empirical mathematical model, namely, a time-dependent diffusivity (TDD) model, that assumes the change in the drug's diffusion coefficient can be used to predict the drug release from spherical wax matrix particles. Wax matrix particles were prepared by using acetaminophen (APAP), a model drug; glyceryl monostearate (GM), a wax base; and aminoalkyl methacrylate copolymer E (AMCE), a functional polymer that dissolves below pH 5.0 and swells over pH 5.0. A three-factor, three-level (33) Box–Behnken design was used to evaluate the effects of several of the variables in the model formulation, and the release of APAP from wax matrix particles was evaluated by the paddle method at pH 4.0 and pH 6.5. When comparing the goodness of fit to the experimental data between the proposed TDD model and the conventional pure diffusion model, a better correspondence was observed for the TDD model in all cases. Multiple regression analysis revealed that an increase in AMCE loading enhanced the diffusion coefficient with time, and that this increase also had a significant effect on drug release behavior. Furthermore, from the results of the multiple regression analysis, a formulation with desired drug release behavior was found to satisfy the criteria of the bitter taste masking of APAP without lowering the bioavailability. That is to say, the amount of APAP released remains below 15% for 10 min at pH 6.5 and exceeds 90% within 30 min at pH 4.0. The predicted formulation was 15% APAP loading, 8.25% AMCE loading, and 400 μm mean particle diameter. When wax matrix dosage forms were prepared accordingly, the predicted drug release behavior agreed well with experimental values at each pH level. Therefore, the proposed model is feasible as a useful tool for predicting drug release behavior, as well as for designing the formulation of wax matrix dosage forms.
Keywords: Wax matrix; Mathematical model; Time-dependent diffusivity; AMCE;

Small- and wide-angle X-ray scattering (SWAXS) for quantification of aspirin content in a binary powder mixture by A. Hodzic; M. Llusa; S.D. Fraser; O. Scheibelhofer; D.M. Koller; F. Reiter; P. Laggner; J.G. Khinast (91-95).
This article presents a novel application of small and wide angle X-ray scattering (SWAXS) in the assessment of aspirin and lactose content in a binary pharmaceutical powder formulation. It is shown that the content correlates with the intensity of the SAXS signal and the intensity of polymorph fingerprints in the WAXS spectra that are collected from the same samples. Because the polymorph WAXS fingerprints and the SAXS signal are two independent characteristics of the same sample, simultaneous SWAXS analysis provides the basis for a dual independent assessment of the same contents.
Keywords: API content; Polymorphism; SWAXS;

Measuring the retention, or residence time, of dosage forms to biological tissue is commonly a qualitative measurement, where no real values to describe the retention can be recorded. The result of this is an assessment that is dependent upon a user's interpretation of visual observation. This research paper outlines the development of a methodology to quantitatively measure, both by image analysis and by spectrophotometric techniques, the retention of material to biological tissues, using the retention of polymer solutions to ocular tissue as an example. Both methods have been shown to be repeatable, with the spectrophotometric measurement generating data reliably and quickly for further analysis.
Keywords: Bioadhesion; Mucoadhesion; Image analysis; UV/vis spectroscopy; In vitro models; Residence time; Drug delivery;

Solid dispersions of itraconazole for inhalation with enhanced dissolution, solubility and dispersion properties by Christophe Duret; Nathalie Wauthoz; Thami Sebti; Francis Vanderbist; Karim Amighi (103-113).
The purpose of this study was to produce a dry powder for inhalation (DPI) of a poorly soluble active ingredient (itraconazole: ITZ) that would present an improved dissolution rate and enhanced solubility with good aerosolization properties. Solid dispersions of amorphous ITZ, mannitol and, when applicable, d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) were produced by spray-drying hydro-alcoholic solutions in which all agents were dissolved. These dry formulations were characterized in terms of their aerosol performances and their dissolution, solubility and physical properties. Modulate differential scanning calorimetry and X-ray powder diffraction analyses showed that ITZ recovered from the different spray-dried solutions was in an amorphous state and that mannitol was crystalline. The inlet drying temperature and, indirectly, the outlet temperature selected during the spray-drying were critical parameters. The outlet temperature should be below the ITZ glass transition temperature to avoid severe particle agglomeration. The formation of a solid dispersion between amorphous ITZ and mannitol allowed the dry powder to be produced with an improved dissolution rate, greater saturation solubility than bulk ITZ and good aerosol properties. The use of a polymeric surfactant (such as TPGS) was beneficial in terms of dissolution rate acceleration and solubility enhancement, but it also reduced aerosol performance. For example, significant dissolution rate acceleration (f 2  < 50) and greater saturation solubility were obtained when introducing 1% (w/w) TPGS (mean dissolution time dropped from 50.4 min to 36.9 min and saturation solubility increased from 20 ± 3 ng/ml to 46 ± 2 ng/ml). However, the fine particle fraction dropped from 47 ± 2% to 37.2 ± 0.4%. This study showed that mannitol solid dispersions may provide an effective formulation type for producing DPIs of poorly soluble active ingredients, as exemplified by ITZ.
Keywords: Itraconazole; Pulmonary aspergillosis; Dry powder for inhalation; Spray-drying; Amorphous; Solid dispersion;

Modification of the C16Y peptide on nanoparticles is an effective approach to target endothelial and cancer cells via the integrin receptor by Nobuhito Hamano; Yoichi Negishi; Azusa Fujisawa; Maya Manandhar; Hinako Sato; Fumihiko Katagiri; Motoyoshi Nomizu; Yukihiko Aramaki (114-117).
Liposomes have been explored as potential drug and gene-delivery particles. In recent years, tumor-targeted liposomes have been developed to improve the efficacy of antitumor treatment. The C16Y peptide is a modified C16 peptide, which is derived from the laminin γ1 chain, and binds to integrins αvβ3 and α5β1 on endothelial cells. In this study, we prepared integrin-targeted C16Y peptide-modified liposomes (C16Y-L) to enhance the intracellular uptake of drugs and genes specifically into tumor tissues. The selectivity of C16Y-L for endothelial cells and cancer cells, which strongly express integrins αvβ3 and α5β1, was assessed by flow cytometry and confocal microscopy. The cellular uptake of C16Y-L by both cell types was higher than uptake of the un-labeled and scramble peptide-modified liposomes. Next, to ascertain the involvement of receptor-mediated endocytosis in the process, these cells were treated with C16Y-L for 1 h at 37 °C or at 4 °C. We found that uptake was also dependent on the temperature. Moreover, to evaluate whether the uptake depended on an integrin–ligand interaction, we examined the inhibition of C16Y-L uptake using recombinant integrin αVβ3 and found that the cellular uptake of C16Y-L treated with αVβ3 integrin decreased. This result suggests that C16Y-L can selectively target cells that highly express integrin αVβ3. Thus, the modification of the C16Y peptide on a Drug Delivery System (DDS) carrier may be a feasible approach for drug or gene delivery into tumors.
Keywords: Liposomes, Integrin, Peptide, Tumor targeting;

In situ controlled crystallization as a tool to improve the dissolution of Glibenclamide by Amal Ali Elkordy; Ayobami Jatto; Ebtessam Essa (118-120).
For pharmaceutical purpose, micro-sized drugs are needed for many delivery systems, such as pulmonary and oral drug delivery systems. Many strategies have been employed to reduce the particle size of poorly water soluble drugs. Microcrystals could be produced by controlled association of drug in order to obtain naturally grown particles. The aim of this work was to increase the aqueous solubility and dissolution of Glibenclamide. The in situ controlled crystallization process was conducted in the presence of the non-ionic surfactants, Cremophor RH40 and Solutol HS-15 (0.75 and 1.5%, w/v), as protective stabilizing agents against agglomeration. In addition, these surfactants inhibit P-glycoprotein that reduces intestinal absorption of Glibenclamide by efflux transportation. Crystal shape was changed and particle size was reduced by about 15-folds, compared to control untreated drug. Differential Scanning Calorimetry (DSC) results indicated no interaction between the drug and the stabilizer. Microcrystals showed marked increase in the drug dissolution, Solutol HS-15 at 1.5% (w/v) concentration showing the highest dissolution efficiency. It could be concluded that in situ controlled crystallization using surfactants are promising method to improve dissolution of Glibeclamide as a model poorly water soluble drug.
Keywords: Controlled crystallization; Glibenclamide; Cremophor RH40; Solutol HS-15;

Lyophilised ready-to-use formulations of PEG-PCL-PEI nano-carriers for siRNA delivery by Thomas Endres; Mengyao Zheng; Moritz Beck-Broichsitter; Thomas Kissel (121-124).
The purpose of the present study was to transfer aqueous PEG-PCL-PEI nano-suspensions into dry ready-to-use formulations, suitable for delivery of siRNA. Therefore, freshly prepared nano-suspensions were lyophilised with glucose as lyoprotectant. Firstly, the required glucose concentration for sufficient stabilisation of unloaded carriers was determined via dynamic light scattering. Morphology of fresh and rehydrated carriers was visualised by cryogenic scanning electron microscopy. Subsequently, the feasibility of siRNA loading before and after lyophilisation was investigated. For both strategies complex diameter and in vitro transfection efficiency were determined and correlated to freshly prepared samples. Hydrodynamic diameter (95.2 ± 1.4 nm) and size distribution (0.132 ± 0.019) of unloaded nano-suspension were restored after rehydration by addition of ≥1.5% of glucose before lyophilisation. Moreover, after loading of rehydrated carriers with siRNA, no significant difference in complex size was observed as compared to freshly prepared ones. Stabilisation of pre-formed carrier/siRNA complexes during lyophilisation is feasible at elevated N/P (e.g. 20) and glucose concentrations above 5%. As determined via real-time-PCR, lyophilised samples were as active as freshly prepared ones regarding transfection efficiency. In conclusion, lyophilisation is an effective technique to produce physically stable PEG-PCL-PEI formulations. These general findings may be applicable to further particulate gene delivery systems to shelf ready-to-use formulations.
Keywords: Cationic triblock copolymer; Nano-carrier; Freeze-drying; siRNA; Gene delivery;

Adsorption kinetics of plasma proteins on ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles by M. Jansch; P. Stumpf; C. Graf; E. Rühl; R.H. Müller (125-133).
In this study the kinetics of plasma protein adsorption onto ultrasmall superparamagnetic iron oxide (USPIO) particles have been analyzed and compared to previously published kinetic studies on polystyrene particles (PS particles), oil-in-water nanoemulsions and solid lipid nanoparticles (SLNs). SPIO and USPIO nanoparticles are commonly used as magnetic resonance imaging (MRI) enhancers for tumor imaging as well as in drug delivery applications. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to determine the plasma protein adsorption onto the citrate/triethylene glycol-stabilized iron oxide surface. The results indicate that the existence of a Vroman effect, a displacement of previously adsorbed abundant proteins, such as albumin or fibrinogen, respectively, on USPIO particles has to be denied. Previously, identical findings have been reported for oil-in-water nanoemulsions. Furthermore, the protein adsorption kinetics differs dramatically from that of other solid drug delivery systems (PS, SLN). More relevant for the in vivo fate of long circulating particles is the protein corona after several minutes or even hours. Interestingly, the patterns received after an incubation time of 0.5 min to 240 min are found to be qualitatively and quantitatively similar. This leads to the assumption of a long-lived (“hard”) protein corona around the iron oxide nanoparticles.
Keywords: Protein adsorption; 2-D PAGE; Drug targeting; Biodistribution; USPIO; Iron oxide nanoparticles;

In the current study, poly lactic acid (PLA) modified hollow crosslinked poly(hydroxyethyl methacrylate) (PHEMA) microspheres have been prepared.In the current study, poly lactic acid (PLA) modified hollow crosslinked poly(hydroxyethyl methacrylate) (PHEMA) microspheres have been prepared, in order to obtain a stimulus-responsive, biocompatible carrier with sustained drug release properties. The synthetical process consisted of the preparation of poly(methacrylic acid)@poly(hydroxyethyl methacrylate-co-N,N′-methylene bis(acrylamide)) microspheres by a two stage distillation-precipitation polymerization technique using 2,2′-azobisisobutyronitrile as initiator. Following core removal, a PLA coating of the microspheres was formed, after ring opening polymerization of dl-lactide, attributing the initiator's role to the active hydroxyl groups of PHEMA. The anticancer drug daunorubicin (DNR) was selected for the study of loading and release behavior of the coated microspheres. The loading capacity of the PLA modified microspheres was found to be four times higher than that of the parent ones (16% compared to 4%). This coated microspherical carrier exhibited a moderate pH responsive drug release behavior due to the pH dependent water uptake of PHEMA, and PLA hydrolysis. The in vitro cytotoxicity of both the parent and the DNR-loaded or empty modified hollow microspheres has been also examined on MCF-7 breast cancer cells. The results showed that although the empty microspheres were moderately cytotoxic, the DNR-loaded microspheres had more potent anti-tumor effect than the free drug. Therefore, the prepared coated microspheres are interesting drug delivery systems.
Keywords: Daunorubicin; Drug delivery system; Hollow microspheres; pH response; PLA modification; Ring opening polymerization;

Mucoadhesive chitosan based films, incorporated with insulin loaded nanoparticles (NPs) made of poly(ethylene glycol)methyl ether-block-polylactide (PEG-b-PLA) have been developed and characterised. Blank-NPs were prepared by double emulsion solvent evaporation technique with varying concentrations of the copolymer (5 and 10%, w/v). The optimised formulation was loaded with insulin (model protein) at initial loadings of 2, 5 and 10% with respect to copolymer weight. The developed NPs were analysed for size, size distribution, surface charge, morphology, encapsulation efficiency and drug release. NPs showing negative (ζ)-potential (<−6 mV) with average diameter > 300 nm and a polydispersity index (P.I.) of ≈0.2, irrespective of formulation process, were achieved. Insulin encapsulation efficiencies of 70% and 30% for NPs-Insulin-2 and NPs-Insulin-5 were obtained, respectively. The in vitro release behaviour of both formulations showed a classic biphasic sustained release of protein over 5 weeks which was influenced by pH of the release medium. Optimised chitosan films embedded with 3 mg of insulin loaded NPs were produced by solvent casting with homogeneous distribution of NPs in the mucoadhesive matrix, which displayed excellent physico-mechanical properties. The drug delivery system has been designed as a novel platform for potential buccal delivery of macromolecules.
Keywords: Buccal delivery; Chitosan films; Insulin; Nanoparticles; Poly(ethylene glycol)methyl ether-block-polylactide;

Cyclodextrin-based nanogels for pharmaceutical and biomedical applications by Maria D. Moya-Ortega; Carmen Alvarez-Lorenzo; Angel Concheiro; Thorsteinn Loftsson (152-163).
Hydrophilic nanogels combine the advantages of hydrogels with certain advantages that are inherent in their nanoscale size. Similar to macrogels, nanogels can contain and protect drugs and regulate their release by incorporating high-affinity functional groups, stimuli-responsive conformations and biodegradable bonds into the polymer network. Similar to nanoparticles, nanogels can easily be administered in liquid form for parenteral drug delivery. The nanoscale size of nanogels gives them a high specific surface area that is available for further bioconjugation of active targeting agents. Biodistribution and drug release can be modulated through size adjustments. The incorporation of hydrophilic cyclodextrin (CD) moieties into the polymeric network of the nanogels provides them with a drug loading and release mechanism that is based on the formation of inclusion complexes without decreasing the hydrophilicity of the network. The covalent attachment of CD molecules to the chemically crosslinked networks may enable the CDs to display fully their ability to form complexes, while simultaneously preventing drug release upon media dilution. The preparation, characterization and advantages for pharmaceutical and biomedical applications of CD-based nanogels are reviewed in this article.
Keywords: Nanogels; Cyclodextrins; Crosslinking; Key–lock; Polymerization;

Thiolated chitosan nanoparticles for the nasal administration of leuprolide: Bioavailability and pharmacokinetic characterization by Gul Shahnaz; Anja Vetter; Jan Barthelmes; Deni Rahmat; Flavia Laffleur; Javed Iqbal; Glen Perera; Wolfgang Schlocker; Sarah Dünnhaput; Patrick Augustijns; Andreas Bernkop-Schnürch (164-170).
Improved nasal transport of leuprolide based on thiolated chitosan–thioglycolic acid (chitosan–TGA) nanoparticles (NPs).The purpose of this study was to develop thiolated nanoparticles to enhance the bioavailability for the nasal application of leuprolide. Thiolated chitosan–thioglycolic acid (chitosan–TGA) and unmodified chitosan nanoparticles (NPs) were developed via ionic gelation with tripolyphosphate (TPP). Leuprolide was incorporated during the formulation process of NPs. The thiolated (chitosan–TGA) NPs had a mean size of 252 ± 82 nm, a zeta potential of +10.9 ± 4 mV, and payload of leuprolide was 12 ± 2.8. Sustained release of leuprolide from thiolated NPs was demonstrated over 6 h, which might be attributed to inter- and/or intramolecular disulfide formation within the NPs network. Ciliary beat frequency (CBF) study demonstrated that thiolated NPs can be considered as suitable additives for nasal drug delivery systems. Compared to leuprolide solution, unmodified NPs and thiolated NPs provoked increased leuprolide transport through porcine nasal mucosa by 2.0 and 5.2 folds, respectively. The results of a pharmacokinetic study in male Sprague–Dawley rats showed improved transport of leuprolide from thiolated NPs as compared to leuprolide solution. Thiolated NPs had a 6.9-fold increase in area under the curve, more than 4-fold increase in elimination half-life, and a ∼3.8-fold increase in maximum plasma concentration compared to nasal solution alone. The relative nasal bioavailability (versus s.c. injection) of leuprolide thiolated NPs calculated on the basis of AUC(0–6) was about 19.6% as compared to leuprolide solution 2.8%. The enhanced bioavailability of leuprolide is likely due to facilitated transport by thiolated NPs rather than improved release.
Keywords: Thiolated polymer; Nanoparticles (NPs); Bioavailability; Leuprolide; Nasal delivery;

Design of cyclic RKKH peptide-conjugated PEG liposomes targeting the integrin α2β1 receptor by Nina Ø. Knudsen; Raymond M. Schiffelers; Lene Jorgensen; Jens Hansen; Sven Frokjaer; Camilla Foged (171-177).
A novel cyclic peptide was conjugated to the distal ends of poly(ethylene glycol) (PEG) chains on PEGylated liposomes for targeting to the human integrin α2β1 receptor.Peptide conjugation to the surface of stealth liposomes has been studied for liposomal drug targeting to cells expressing specific receptors to provide a site-specific drug delivery. This study investigated the potential of peptide-conjugated liposomes for targeting cells expressing the human integrin α2β1 receptor. A 12 amino acid head-to-tail cyclic peptide derived from the Jararhagin protein containing the Arg-Lys-Lys-His (RKKH)-specific binding site was conjugated to the distal ends of poly(ethylene glycol) (PEG) chains on PEGylated liposomes. Epithelial cells expressing the receptor showed increased cellular association and uptake of peptide-conjugated liposomes at 4 °C, compared to liposomes conjugated with a non-specific peptide. The interaction between cells and peptide-conjugated liposomes was significantly increased at 37 °C suggesting that a possible uptake mechanism might be energy-dependent endocytosis. In keratinocyte cell cultures, the ligand-conjugated liposomes loaded with the vitamin D3 analogue calcipotriol induced transcription of the gene encoding the antimicrobial peptide cathelicidin, which is activated through the vitamin D3 receptor upon binding of vitamin D3 analogues. This suggests that the liposomes are internalized and that calcipotriol is delivered intracellularly and released in an active form. In conclusion, the 12 amino acid head-to-tail cyclic RKKH peptide seems promising for targeting of liposomes to the integrin α2β1 receptor.
Keywords: Liposomes; Integrin alpha 2; Targeting; Calcipotriol; Drug delivery; Nanomedicine;

The equilibrium of the formation of polyamidoamine dendrimer (PAMAM-NH2 G4) and an oncological drug, 5-fluorouracil (FU) in water at room temperature has been examined. Using calorimetric titration, the number of active sites in the dendrimer combining the drug molecules and the equilibrium constant of the dendrimer–drug complex were estimated. The addition of the drug to the dendrimer active sites is an exothermic process. This process is accompanied by a beneficial change in entropy. The number of drug molecules combined by the polymer was confirmed by means of 1H NMR spectroscopy. 1HNMR measurements show that the dendrimer macromolecule binds the drug molecules with superficial protonated or unprotonated amine groups.
Keywords: PAMAM-NH2 G4 dendrimer; 5-Flurouracil; Isothermal titration calorimetry; 1H NMR spectroscopy; Binding parameters;

Nanocomposite formation between alpha-glucosyl stevia and surfactant improves the dissolution profile of poorly water-soluble drug by Hiromasa Uchiyama; Yuichi Tozuka; Masahiro Nishikawa; Hirofumi Takeuchi (183-186).
The formation of a hybrid-nanocomposite using α-glucosyl stevia (Stevia-G) and surfactant was explored to improve the dissolution of flurbiprofen (FP). As reported previously, the dissolution amount of FP was enhanced in the presence of Stevia-G, induced by the formation of an FP and Stevia-G-associated nanostructure. When a small amount of sodium dodecyl sulfate (SDS) was present with Stevia-G, the amount of dissolved FP was extremely enhanced. This dissolution-enhancement effect was also observed with the cationic surfactant of dodecyl trimethyl ammonium bromide, but not with the non-ionic surfactant of n-octyl-β-d-maltopyranoside. To investigate the dissolution-enhancement effect of Stevia-G/SDS mixture, the pyrene I 1/I 3 ratio was plotted versus the Stevia-G concentration. The pyrene I 1/I 3 ratio of Stevia-G/SDS mixture had a sigmoidal curve at lower Stevia-G concentrations compared to the Stevia-G solution alone. These results indicate that the Stevia-G/SDS mixture provides a hydrophobic core around pyrene molecules at lower Stevia-G concentrations, leading to nanocomposite formation between Stevia-G and SDS. The nanocomposite of Stevia-G/SDS showed no cytotoxicity to Caco-2 cells at a mixture of 0.1% SDS and 1% Stevia-G solution, whereas 0.1% SDS solution showed high toxicity. These results suggest that the nanocomposite formation of Stevia-G/SDS may be useful way to enhance the dissolution of poorly water-soluble drugs without special treatment.
Keywords: α-glucosyl stevia; Flurbiprofen; Surfactant; Dissolution enhancement; Nanocomposite;