International Journal of Pharmaceutics (v.358, #1-2)

High protein concentration solutions are becoming increasingly important in the pharmaceutical industry. The solution behavior of proteins at high concentrations can markedly differ from that predicted based on dilute solution analysis due to thermodynamic non-ideality in these solutions. The non-ideality observed in these systems is related to the protein–protein interactions (PPI). Different types of forces play a key role in determining the overall nature and extent of these PPI and their relative contributions are affected by solute and solvent properties. However, individual contributions of these forces to the solution properties of concentrated protein solutions are not fully understood. The role of PPI, driven by these intermolecular forces, in governing solution rheology and physical stability of high protein concentration solutions is discussed from the point of view of pharmaceutical product development. Investigation of protein self-association and aggregation in concentrated protein solutions is crucial for ensuring the safety and efficacy of the final product for the duration of the desired product shelf life. Understanding rheology of high concentration protein solutions is critical for addressing issues during product manufacture and administration of final formulation to the patient. To this end, analysis of solution viscoelastic character can also provide an insight into the nature of PPI affecting solution rheology.
Keywords: High protein concentration solutions; Protein–protein interactions; Virial coefficient; Rheology; Viscosity; Viscoelasticity; Self-association; Aggregation;

Dry coating, a novel coating technology for solid pharmaceutical dosage forms by Yanfeng Luo; Jesse Zhu; Yingliang Ma; Hui Zhang (16-22).
Dry coating is a coating technology for solid pharmaceutical dosage forms derived from powder coating of metals. In this technology, powdered coating materials are directly coated onto solid dosage forms without using any solvent, and then heated and cured to form a coat. As a result, this technology can overcome such disadvantages caused by solvents in conventional liquid coating as serious air pollution, high time- and energy-consumption and expensive operation cost encountered by liquid coating. Several dry coating technologies, including plasticizer-dry-coating, electrostatic-dry-coating, heat-dry-coating and plasticizer-electrostatic-heat-dry-coating have been developed and extensively reported. This mini-review summarized the fundamental principles and coating processes of various dry coating technologies, and thoroughly analyzed their advantages and disadvantages as well as commercialization potentials.
Keywords: Dry coating; Powder coating; Solid pharmaceutical dosage form; Plasticizer; Electrostatic coating;

Lyotropic liquid crystal preconcentrates for the treatment of periodontal disease by A. Fehér; E. Urbán; I. Erős; P. Szabó-Révész; E. Csányi (23-26).
The aim of our study was to develop water-free lyotropic liquid crystalline preconcentrates, which consist of oils and surfactants with good physiological tolerance and spontaneously form lyotropic liquid crystalline phase in aqueous environment. In this way these preconcentrates having low viscosity can be injected into the periodontal pocket, where they are transformed into highly viscous liquid crystalline phase, so that the preparation is prevented from flowing out of the pocket due to its great viscosity, while drug release is controlled by the liquid crystalline texture. In order to follow the structure alteration upon water absorption polarization microscopical and rheological examinations were performed. The water absorption mechanism of the samples was examined by the Enslin-method. Metronidazole-benzoate was used as active agent the release of which was characterized via in vitro investigations performed by means of modified Kirby–Bauer disk diffusion method. On the grounds of the results it can be stated that the 4:1 mixture of the investigated surfactants (Cremophor EL, Cremophor RH40) and oil (Miglyol 810) formed lyotopic liquid crystalline phases upon water addition. Polarization microscopic examinations showed that samples with 10–40% water content possessed anisotropic properties. On the basis of water absorption, rheological and drug release studies it can be concluded that the amount of absorbed water and stiffness of lyotropic structure influenced by the chemical entity of the surfactant exerted major effect on the drug release.
Keywords: Periodontal disease; Lyotropic liquid crystals; Kirby–Bauer disk diffusion method; Drug release;

Synthesis of lidocaine-loaded PLGA microparticles by flow focusing by M.A. Holgado; J.L. Arias; M.J. Cózar; J. Alvarez-Fuentes; A.M. Gañán-Calvo; M. Fernández-Arévalo (27-35).
In the present work, two methods for the preparation of lidocaine-loaded PLGA microparticles are compared. The differences between the polymeric particles obtained by solvent evaporation (SEVM) or flow focusing (FF) were studied by means of scanning electron microscopy and surface thermodynamics determinations. A detailed investigation of the capabilities of the polymer particles to load this drug is described. The physical state of the drug in the polymeric particles and the existence of interactions between both entities were studied by differential scanning calorimetry. The main factors determining the lidocaine incorporation and the release kinetics were the synthesis procedure followed, the amount of drug dissolved in the organic phase during the synthesis routine, the type of polymer (molecular weight and end chemical groups) and the size and the hydrophobic/hydrophilic properties of the particles. The FF technology allowed higher drug incorporations and slower release kinetics. The release studies showed a biphasic profile probably due to diffusion-cum-degradation mediated processes.
Keywords: Drug delivery systems; Flow focusing; Lidocaine; Poly(lactic-co-glycolic) acid; Solvent evaporation; Surface thermodynamics;

Copolymers of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with N,N-dimethylacrylamide (DMAA) formed insoluble interpolymer complexes with mucin from porcine stomach at pH 9.0. The complex formation based on boronate–sugar interactions took place between the similarly charged macromolecules and resulted in coacervate particles formation, which depended both on pH and ionic strength of the solution. The coacervation rate displayed a maximum at the intermediate DMAA-NAAPBA copolymer: mucin weight ratio, that is a pattern typical of interpolymer complex formation. The effective hydrodynamic particle diameter of the coacervates monotonously grew from 155 ± 20 nm up to 730 ± 120 nm in 2 days in 0.1 M sodium bicarbonate buffer solution, pH 9.0. Electrophoretic mobility of the resultant nanoparticles was intermediate between those of individual polymers, whereas the particles zeta-potential was −7.5 ± 0.4 mV in the above buffer solution. Pre-treatment of the inner mucosal epithelium of excised male pig urethras with 5% (w/v) solutions of acrylamide-NAAPBA or DMAA-NAAPBA copolymers at pH 8.8 allowed for tight occlusion of the lumen by poly(vinyl alcohol) – borax gel injected via a two-way catheter. Leakage of 0.15 M NaCl solution through the thus occluded organs could be prevented, while the leakage through the organs occluded by the gel without the pre-treatment was unavoidable. The gel plug could be quickly dissolved on demand after injection of 5% (w/v) aqueous fructose solution into the lumen. The described technique may be useful for temporal occlusion of mucosal lumens in living organisms. In contrast to the conventional mucoadhesive polymers like polyacrylic acid or chitosan, the boronate-containing copolymers display their mucoadhesivity at weakly alkaline pH of 8–9 and physiological ionic strength.
Keywords: Boronic acid; Controlled release; Drug delivery; Mucoadhesive; Smart material;

New environmental sensitive system for colon-specific delivery of peptidic drugs by Barbara Luppi; Federica Bigucci; Teresa Cerchiara; Roberto Mandrioli; Anna Maria Di Pietra; Vittorio Zecchi (44-49).
Nano and micro preparative technologies for the realization of pharmaceutical carriers represent an actual strategy for reaching the therapeutic success of drugs, particularly in the case of peptidic drugs. Vancomycin is here entrapped in carriers composed by a swellable, mucoadhesive and biodegradable albumin core, coated with fatty acids able to improve a colon-specific release. Bovine serum albumin nanospheres (core) were prepared from protein solutions using a coacervation method followed by thermal cross-linking at different temperature, or from protein solutions at different pHs using a coacervation method followed by thermal cross-linking at 75 °C. Solid nanospheres were collected by freeze-drying, loaded by soaking from solutions of vancomycin and subsequently coated with myristic, palmitic or stearic acid by spray-drying technique obtaining microcapsules. Nanosphere dimensions and polydispersity, drug loading capacity, swelling ability and mucoadhesion properties were evaluated, as well as in vitro release behaviour. The results indicated that nanospheres present an adequate loading capacity, a great swelling tendency and good mucoadhesion ability. Moreover, albumin cores showed a pH-dependent release according to the structure of thermally denaturated protein in different experimental conditions, while microcapsules showed a pH-dependent release according to the different fatty acids solubility in acidic and alkaline media.
Keywords: Albumin nanospheres; Swelling; Mucoadhesion; Enzymatic destabilization; Colon-specific delivery; Peptidic drugs;

This study investigated the effect of ion-pairing of anionic polyelectrolytes: our novel poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) (PEG-OVSDM) and poly(ethylene glycol)-block-poly(l-aspartic acid) (PEG-PAA) with cationic lysozyme on retention of protein stability during emulsification. Soluble lysozyme recovery after exposure to the deleterious interface was 42–88% (when ion-paired with PEG-OVSDM, PEG-OVSDM concentration dependent) compared to only 30% for free lysozyme. PEG-OVSDM provided a higher stabilization of lysozyme than PEG-PAA (36–60%). Lysozyme when recovered in the aqueous phase and analyzed by chromatography, enzymatic assay, fluorescence, and mass spectrometry showed no significant physicochemical change when compared with a lysozyme standard. Lysozyme was incorporated into poly(lactide-co-glycolide) (PLGA) microspheres via the typical double emulsion method. Incorporation of lysozyme complexes led to a higher encapsulation efficiency and loading amount, and a lower incidence of insoluble lysozyme aggregates compared to the control microspheres containing lysozyme only. More significantly, ion-pairing was able to dramatically reduce the initial lysozyme release to 18% compared with 50% from control microspheres and provided an overall better control of protein release. PEG-PAA was less effective than PEG-OVSDM in controlling the release probably due to weaker interactions between this polyelectrolyte and lysozyme. Manipulation of such polyelectrolyte–protein complexation may play a role in protein-controlled delivery.
Keywords: PEG-b-oligo(vinyl sulfadimethoxine); Ion-pairing; Lysozyme stabilization; Aqueous/organic interface; PLGA microspheres; Release modulation;

Relationship between dissolution efficiency of Oxazepam/carrier blends and drug and carrier molecular descriptors using multivariate regression analysis by Annalisa Cutrignelli; Angela Lopedota; Adriana Trapani; Giancarlo Boghetich; Massimo Franco; Nunzio Denora; Valentino Laquintana; Giuseppe Trapani (60-68).
Quantitative structure–property relationships were developed for predicting the enhancement of dissolution rate of the model lipophilic drug Oxazepam (Oxa) from blends (BLs) with 12 structurally different carriers at three different drug/carrier weight ratios (1/5, 1/10, and 1/20). To this end, 36 BLs were prepared by the solvent-evaporation method and characterized by spectroscopic (FT-IR), thermoanalytical (DSC) and X-ray diffraction studies. The dissolution rate of the examined systems was quantified by log DE/DEOxa, where DE and DEOxa are the dissolution efficiencies of the BL and pure drug, respectively. Twenty molecular descriptors, including parameters for size, lipophilicity, cohesive energy density (CED), and hydrogen bonding capacity were calculated and together with the experimental melting point (MP), were used in multivariate analysis. Twelve pertinent variables were detected after looking at the results of principal component analysis (PCA) and cluster analysis, and reliable six-descriptor models generated by Partial Least Squares-Projection to Latent Structures (PLS) method. Satisfactory coefficient of determination values were obtained (i.e., R 2 equal to 0.794 and Q 2 equal to 0.705). The equations generated can predict with reasonable accuracy the dissolution rate increase of the model lipophilic drug/carrier BLs.
Keywords: Dissolution efficiency; Oxazepam; Blends/solid dispersions; Prediction; PLS regression;

Compressibility and compactibility of granules produced by wet and dry granulation by C. Bacher; P.M. Olsen; P. Bertelsen; J.M. Sonnergaard (69-74).
The bulk properties, compactibility and compressibility of granules produced by wet and dry granulation were compared applying a rotary tablet press, three different morphological forms of calcium carbonate and two particle sizes of sorbitol. Granules from both granulation methods possessed acceptable flow properties; however, the ground (Mikhart) and cubic (Scoralite) calcium carbonate demonstrated better die-filling abilities in the tablet press than the scalenhedral calcium carbonate (Sturcal). The wet processed granules showed in general larger compression properties. This was explained as these granules were mechanical stronger and had a higher initial porosity. In some cases, a large particle surface area of calcium carbonate and sorbitol resulted in a small, insignificant improvement of the consolidation characteristics. A correlation between the compression and compaction characteristics was demonstrated.
Keywords: Roller compaction; Granulation; Compressibility; Compactibility; Morphology; Particle size;

The purpose of the study was to examine the suitability of the Spraytec® laser diffraction technique for measuring the size distribution of aerosol particles generated from dry powder inhalators. A range of formulations with different dispersion properties were produced by spray-drying. The percentage of particles below 5.0 μm of these formulations was measured by laser diffraction (Mastersizer® 2000 and Spraytec®) and inertial impaction (MsLI and NGI) using various inhaler devices and at different flow rates between 30 and 100 l/min. Linear relationships and correlations (R 2  > 0.9) existed between the results obtained from, on one hand, the Mastersizer® 2000 and the Spraytec®, and, on the other hand, the MsLI and the Spraytec® regardless of flow rates and inhaler devices. The Spraytec® could be a reliable technique for the development, evaluation and quality control of dry powder aerosol formulations.
Keywords: Spraytec®; Dry powder aerosols; Inertial impaction; Laser diffraction; Particle size distribution;

Development and evaluation of new sustained-release floating microspheres by Ninan Ma; Lu Xu; Qifang Wang; Xiangrong Zhang; Wenji Zhang; Yang Li; Lingyu Jin; Sanming Li (82-90).
A type of multi-unit floating alginate (Alg) microspheres was prepared by the ionotropic gelation method with calcium carbonate (CaCO3) being used as gas-forming agent. Attempts were made to enhance the drug encapsulation efficiency and delay the drug release by adding chitosan (Cs) into the gelation medium and by coating with Eudragit, respectively. The gastrointestinal transit of optimized floating sustained-release microspheres was compared with that of the non-floating system manufactured from identical material using the technique of gamma-scintigraphy in healthy human volunteers. It was found that the drug encapsulation efficiency of Cs–Alg microspheres was much higher than that of the Ca–Alg microspheres, and coating the microspheres with Eudragit RS could extend the drug release significantly. Both uncoating and coating microspheres were able to continuously float over the simulated gastric fluid (SGF) for 24 h in vitro. Prolonged gastricretention time (GRT) of over 5 h was achieved in the volunteer for the optimized coating floating microspheres (FM). In contrast, non-floating system (NFM) could be emptied within 2.5 h. In the present study, a multi-unit system with excellent floating ability, optimum drug entrapment efficiency and suitable drug release pattern has been developed.
Keywords: Floating drug delivery system; Sustained-release; Alginate microsphere; Gamma-scintigraphy;

Novel method for stratum corneum pore size determination using positron annihilation lifetime spectroscopy by Yoshiaki Itoh; Akira Shimazu; Yasuyuki Sadzuka; Takashi Sonobe; Shigeru Itai (91-95).
Positron annihilation lifetime spectroscopy (PALS) is a powerful tool for the investigation of microstructure. Three main classes of materials, metals, semiconductors and polymers, have been studied by using this technique. But, relatively few investigations have been performed in the biological sciences. PALS provides important information on pore properties and free volume at the molecular level. Our PALS study showed that Yucatan miniature pig stratum corneum separated with heat and trypsin digestion had a longer positron annihilation lifetime than cyclodextrins. This indicates that the stratum corneum has larger pores and/or free volume than cyclodextrins, whose pores have a diameter of 0.5–0.8 nm and a torus height of 0.79 nm. Positron annihilation spectroscopy may be developed as a new technique for the detection of nano-pore properties and free volume in the biological sciences.
Keywords: Stratum corneum; Positron annihilation lifetime spectroscopy (PALS); Ortho-positronium (o-Ps); Positron; 22Na;

Evaluation of gatifloxacin penetration into skeletal muscle and lung by microdialysis in rats by Leandro Tasso; Clarissa C. Bettoni; Laura K. Oliveira; Teresa Dalla Costa (96-101).
This study aimed to investigate gatifloxacin distribution into skeletal muscle and lung interstitial fluid by microdialysis and to correlate free tissue and free plasma levels of the drug. Microdialysis recoveries were determined in vitro by extraction efficiency and retrodialysis at 80, 160 and 400 ng/ml resulting in 33.5 ± 1.3%, 33.1 ± 1.2%, 31.8 ± 2.7% and 31.4 ± 2.6%, 33.1 ± 2.2%, 30.6 ± 3.3%, respectively. In vivo recovery by retrodialysis in Wistar rats’ skeletal muscle and lung were 29.1 ± 1.0% and 30.7 ± 1.4%, respectively. The recovery was constant and independent on the method or media used. Gatifloxacin tissue penetration was investigated after intravenous dosing of 6 mg/kg to Wistar rats. Free skeletal muscle, lung and plasma profiles were virtually super imposable resulting in similar area under the curve (AUC0–9) of 3888 ± 734 ng h/ml, 4138 ± 1071 ng h/ml and 3805 ± 577 ng h/ml, respectively (α  = 0.05). The tissue distribution factors were 1.02 and 1.08 for muscle and lung relative to plasma. In conclusion, free plasma levels are a good surrogate for gatifloxacin active levels at the infection site.
Keywords: Gatifloxacin; Skeletal muscle penetration; Lung penetration; Microdialysis; Wistar rats;

Physicochemical effects of terpenes on organogel for transdermal drug delivery by Perry Fung Chye Lim; Xiang Yang Liu; Lifeng Kang; Paul Chi Lui Ho; Sui Yung Chan (102-107).
It is accepted that terpenes are effective penetration enhancers to promote the passage of drugs or chemicals through the human skin barrier. However the physical and chemical changes of a pharmaceutical vehicle induced by the incorporation of terpenes have not been explored. Thus, this study examines the effects of three terpenes (linalool, cineole, limonene) on the rheology and chemical stability of an organogel composed of dibutyllauroylglutamide (GP1) and propylene glycol (PG). At a given GP1 concentration, oxygen-containing linalool and cineole decreased gel moduli (elastic and viscous) and brittleness, and the reverse was obtained for hydrocarbon limonene. Probably, linalool and cineole interfered with hydrogen bonding between GP1 molecules while limonene could have initiated a phase separation-mediated gelation, changing the gel morphology. Microcalorimetry detected minute heat endotherms for gels (with and without terpenes) subjected to accelerated heat testing. These heat changes could arise from a small degree of structural disruption of the gel network. Heat endotherms normalized with respect to GP1 content were used to assess gel chemical stability. Although the terpenes altered rheology, they did not significantly affect the chemical stability of the gels. This is the first in the literature that reports the effect of penetration enhancers, such as terpenes, on the physical, rheological and chemical characteristics of a model pharmaceutical formulation for topical and transdermal drug delivery.
Keywords: Terpenes; Transdermal; Organogel; Rheology; Microcalorimetry;

Stability of lyophilized human growth hormone by Maya S. Salnikova; C. Russell Middaugh; J. Howard Rytting (108-113).
To evaluate relationships between the extent of protein–excipient interactions, structural relaxation of an amorphous matrix, and the physico-chemical stability of a protein, human growth hormone (hGH) was lyophilized with sucrose and trehalose in a 1:2 weight ratio. The protein–excipient interactions were analyzed immediately after lyophilization with isoperibol solution calorimetry (ISC), water sorption analysis (WSA), differential scanning calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR). The physical and chemical stability of hGH during storage at 50 °C was monitored by reverse phase (RP)–HPLC, SEC–HPLC and UV absorption spectroscopy. The hGH formulation containing sucrose demonstrated greater protein–excipient interactions and faster initial relaxation times compared to the trehalose formulation. Although both formulations had similar chemical stability (rate of deamidation), physical stabilities (e.g. degree of aggregation) were different. The hGH/sucrose formulation manifested a higher rate and lower extent of insoluble aggregate formation. The decreased amount of aggregation in the sucrose formulation could be correlated with a greater extent of protein–excipient interactions and the presence of a more homogeneous mixture. In contrast, the higher rate of aggregation in the sucrose formulation could be directly correlated with the higher molecular mobility of the matrix.
Keywords: Lyophilization; Protein; Growth hormone; Structural relaxation; Tg; Isoperibol solution calorimetry; Water sorption analysis; Differential scanning calorimetry; Fourier Transform Infrared Spectroscopy;

Paclitaxel tumor biodistribution and efficacy after intratumoral injection of a biodegradable extended release implant by Ariella Shikanov; Sergey Shikanov; Boris Vaisman; Jacob Golenser; Abraham J. Domb (114-120).
The aim of this study was to investigate the effectiveness of paclitaxel controlled release from intratumorally injected polymer.The effectiveness of paclitaxel–polymer formulation injected intratumorally was tested in mouse bladder tumor model. To determine paclitaxel biodistribution in tumor at predetermined time periods the tumor was excised, frozen and sectioned, and the paclitaxel concentrations were determined in the tumor tissue and in plasma by HPLC. Histopathological evaluation of the necrosis and inflammation was performed on tumor sections.In the paclitaxel/polymer group mice were injected intratumorally with 0.2 ml of the 10% (w/w) paclitaxel formulation, the tumor disappeared completely 5 days after injection, and mice survived till the end of the study (50 days post-tumor cells inoculation). In biodistribution studies, the highest paclitaxel concentration in the tumor tissue was 40 μg/mg 1 day after the intratumoral injection and decreased gradually during 10 days to 5 μg/mg that is still high enough to induce cytotoxic effect, and the necrotic effect of paclitaxel on the tumors was confirmed by histopathology.Treatment with local injection of polymer–paclitaxel formulation inhibited the growth of solid tumors. Distribution studies of paclitaxel after intratumoral injection showed high and effective drug concentrations in tumor.
Keywords: Injectable biodegradable polymer; Paclitaxel; Poly(ester anhydride); Anti-tumor efficacy; Controlled release; Tumor distribution;

Using a drug to structure its release matrix and release profile by Michael A. Brook; Alison C. Holloway; Kenneth K. Ng; Michael Hrynyk; Carly Moore; Ryan Lall (121-127).
Silicone elastomers have proven to be useful implantable release matrices for hydrophobic drugs. However, their utility for the release of hydrophilic materials is less well developed and, even with the addition of polar excipients such as poly(ethylene oxide) (PEO), burst release profiles are often observed—achieving longer term release is more challenging. We report that linoleic acid, initially used to solubilize polar, cationic nicotine in silicone precursors, additionally acted to change the internal morphology of resulting silicone + PEO elastomers. The unexpected consequence of this change was a change in the distribution of hydrophilic domains of PEO/drug within the silicone and the ability to control the rate of release of the drug in vitro. The relationship between excipients, silicone morphology, and release profile is examined.
Keywords: Nicotine delivery; Implantable silicone elastomer; Controlled morphology; In vitro sustained release; Surface active drug complexes;

Improved delivery of cromolyn from oral proliposomal beads by Deepali D. Deshmukh; William R. Ravis; Guru V. Betageri (128-136).
Proliposomal bead formulations for improved oral delivery of cromolyn (BCS Class III compound) were formulated. Phospholipid (distearylphosphatidylcholine)–cholesterol-surfactant (Tween 80/sodium cholate) systems were spray-coated on beads containing cromolyn sodium and the dosage forms were characterized for vesicle formation and encapsulation efficiency. Delivery of cromolyn sodium from this novel dosage form was evaluated across the Caco-2 and everted rat intestinal sac model. Spontaneous formation of vesicles upon dilution of beads was observed. Enhancement in cromolyn transport was higher with phospholipids-surfactant proliposomal formulations compared to surfactant-free lipid formulations or pure surfactant solutions, most significant enhancement being with formulations with low surfactant concentration. No evidence of cellular damage to Caco-2 monolayers (e.g. significant decrease in the TEER) or change in transport and tissue accumulation of a marker molecule in the intestinal tissue model was observed. This indicated enhancement of transport via transcellular routes and not due to the modulation of the tight junctions or cell disruption. Results suggest that phospholipids-surfactant proliposomal beads offer a good potential for improved oral delivery of cromolyn.
Keywords: Proliposomal beads; Cromolyn; Transport; Caco-2; Intestinal sac;

The objective of this study was to investigate the effects of experimental conditions for measuring the water vapor transmission rate (WVTR) of high-density polyethylene (HDPE) bottles using a steady-state sorption method. Bottles were filled with desiccant, closed with caps and heat induction sealed, and then stored in stability chambers at controlled temperature and relative humidity. Weight gain of the bottles was determined every 1 or 2 weeks until a linear weight gain profile was obtained. WVTR of the bottles was determined from the slope of the linear portion of the weight gain versus time profile. The effects of desiccants and temperature/humidity were studied. Results show that, with a sufficient amount of anhydrous calcium chloride in bottles, a negligibly low and sufficiently constant headspace humidity is maintained, and a steady-state permeation rate is achieved. For all 8 sizes of bottles used in this study, steady-state was achieved in 1 or 2 weeks after the experiment was started. This method provided reproducible WVTR data for HDPE bottles. Apparent moisture permeability of all 8 sizes of bottles was (2.3 ± 0.3) × 10−7, (2.6 ± 0.2) × 10−7, and (3.4 ± 0.2) × 10−7  cm2/s at 25 °C, 30 °C, 40 °C, respectively. Moisture permeability determined from the current study was similar to data reported in the literature, indicating that the steady-state weight gain method can be used to obtain reliable WVTR of containers for pharmaceutical products.
Keywords: HDPE bottles; WVTR; Steady-state; Permeation;

The aim of the present study was to use the in vitro human skin sandwich system in order to quantify the influence of formulation variables on intrafollicular hydrocortisone permeation. The investigated variables were the pH and the viscosity of the topical formulation as well as the presence of chemical enhancers (carvone, menthone, oleic acid and sodium lauryl sulphate). Furthermore, skin sandwich hydration was also varied in order to determine if the method itself can be run using only partially hydrated skin tissues. It was determined that the follicular contribution to hydrocortisone flux decreased marginally with increasing alkalinity in the pH range 3–8.8. Intrafollicular penetration was markedly reduced when HPMC gels were used instead of an aqueous solution. Pretreating the skin with chemical enhancers also reduced the follicular contribution to flux, probably due to permeabilisation of the continuous stratum corneum. Furthermore, it was not possible to satisfactorily modify the skin sandwich method so that it could be deployed using less hydrated skin.
Keywords: Skin sandwich; Hair follicles; Transdermal; Hydrocortisone; Transfollicular; Skin;

Effect of HPβCD on solubility and transdermal delivery of capsaicin through rat skin by Peng Zi; Xinghao Yang; Huifen Kuang; Yanshuang Yang; Lili Yu (151-158).
We evaluated the ability of hydroxypropyl-β-cyclodextrin (HPβCD) to influence the percutaneous absorption of capsaicin (CP) through isolated rat skin. Phase solubility analysis and phase distribution studies suggested the potential of HPβCD as a solubilizer and permeation enhancer for CP. In vitro permeation studies showed the trend that, the penetration flux (J s) of CP increased with the increasing concentration of HPβCD from 0 to 2.20% (w/v), and then decreased dramatically when the concentration of HPβCD kept on increasing up to 15% (w/v). 2.20% (w/v) of HPβCD provided both just adequate solubilization and preferred J s for the permeation of CP (0.075%, w/v). Similar change patterns of the permeation parameters were also observed in the hydrogels, but the J s of CP was reduced significantly along with the increasing concentration of Carbopol U21. Histological analysis showed an invasive action of HPβCD on the stratum corneum (SC) of rat skin, which could only reduce the lag time (T L) but could not increase the J s of CP. On the other hand, the complexation of HPβCD with CP could attenuate this invasive action. It is inferred that excess of HPβCD could not only disturb the percutaneous absorption of CP but also disrupt the structure of SC.
Keywords: Capsaicin; Hydroxypropyl-β-cyclodextrin; Hydrogel; In vitro percutaneous studies; Histological analysis;

A comparative study of a range of polymeric microspheres as potential carriers for the inhalation of proteins by Neeraj Sivadas; Desmond O’Rourke; Aoife Tobin; Vivienne Buckley; Zeibun Ramtoola; John G. Kelly; Anthony J. Hickey; Sally-Ann Cryan (159-167).
The aim of this study was to compare protein-loaded inhalable microparticles manufactured using a range of biocompatible polymers including hydroxypropyl cellulose (HPC), chitosan, hyaluronic acid, alginate, gelatin, ovalbumin and poly(lactide-co-glycolide) (PLGA). Spray-drying was used to prepare microparticles containing bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC). Particles of respirable size and high protein loading were obtained. No evidence of BSA degradation was seen from PAGE analysis. The microparticles were mixed with mannitol as a carrier and powder aerosolization was assessed with a multi-dose dry powder inhaler (DPI) using a multi-stage cascade impactor. The mass median aerodynamic diameter (MMAD) ranged between 2.9 and 4.7 μm. Potential polymer toxicity in the lungs was compared by impinging the particles on Calu-3 monolayers and assessing the cytotoxicity, induction of cytokine release, changes in transepithelial permeability and electrical resistance. No toxic effects were observed with most of the polymers though some evidence of compromised cell monolayer integrity was seen for PLGA and ovalbumin. PLGA and gelatin microparticles caused a significant increase in IL-8 release. Of the polymers studied, PLGA showed the greatest toxicity. Certain polymers showed particular promise for specific protein delivery needs in the lungs, such as HPC to improve flow properties, sodium hyaluronate for controlled release, and chitosan and ovalbumin for systemic delivery.
Keywords: Pulmonary drug delivery; Protein; Microparticles; Calu-3; Toxicity;

Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake by Shuanghui Luo; Zhiying Wang; Viral Kansara; Dhananjay Pal; Ashim. K. Mitra (168-176).
The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [14C]ascorbic acid ([14C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di-substituted benzenes. Transepithelial transport of [14C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase-mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy.Uptake of AA was found to be saturable with a K m of 83.2 μM and V max of 94.2 pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy-dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca2+/CaM mediated pathways. [14C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group, i.e., halogen- and nitro-substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A–B) as well as basal to apical side (B–A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile-substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs.
Keywords: Ascorbic acid (AA); SVCT; Uptake and transport; Substrate specificity; Regulation; MDCK-MDR1;

A doxorubicin (DOX)-carrier micellar system consisting of poly(histidine)(5K)-b-poly(ethylene glycol)(2K) and poly(l-lactic acid)(3K)-b-PEG(2K)-folate has been developed targeting the early endosomal pH and it have been convincingly proved that intracellular high dose strategy using such micelles is effective in overcoming multidrug resistance (MDR) of cancer cells. Due to the low DOX concentrations in the micelle solution obtained by dialysis and the lack of long-term stability of the micelles, stable and lyophilized micelle formulations were the subject of investigation reported here by using excipients of sucrose, PEG or Pluronic. The reconstituted micelle solutions were examined by particle size, pH sensitivity, and cytotoxicity for MDR cells and the results were compared with the non-lyophilized micelles. Among tested excipients, Pluronic F127 (33 wt%) added to the polymer/drug solution prior to dialysis resulted in a reconstituted product stable for a week and presented equivalent benefits as the fresh micelle formulation. The blank micelles did not present any apparent systemic toxicity in mice up to 2400 mg/kg i.v. injection (800 mg/(kg day) for 3 days). The brief toxicity of reconstituted DOX loaded micelles was examined by the maximum tolerated dose (MTD), which was approximately 7.5-fold higher than free DOX and guaranteeing further animal toxicity and efficacy study.
Keywords: Poly(l-histidine)-b-PEG; pH-sensitive micelle; Multidrug resistance; Reconstitution; Pluronic F127; Maximum tolerated dose;

Formulation of monolayered films with drug and polymers of opposing solubilities by V.A. Perumal; D. Lutchman; I. Mackraj; T. Govender (184-191).
The aim of this study was to prepare and characterise monolayered multipolymeric films (MMFs) comprising of a hydrophilic drug (Propranolol HCl) (PHCl) and polymers of opposing solubilities. Films were prepared by emulsification and casted by a new approach using a silicone-molded tray with individual wells. MMFs comprising of PHCl with Eudragit® 100 (EUD100) and Chitosan (CHT), i.e. films with drug and polymers of opposing solubilities were successfully prepared (PHCl:EUD100:CHT; 1:10:0.5) and demonstrated uniform and reproducible drug content (100.71 ± 2.66%), thickness (0.442 ± 0.030 mm), mucoadhesivity (401.40 ± 30.73 mN) and a controlled drug release profile. Drug release followed Higuchi's square-root model. Maximum swelling of the films occurred after 1 h and 28.26% of the films eroded during the 8-h test period. Mechanical testing revealed that the MMFs displayed a greater abrasion resistance, were more elastic and also required more energy to break, rendering them tougher and more suitable for buccal delivery than the monopolymeric PHCl:EUD100 film. The inclusion of CHT to the film led to a more porous surface morphology. The surface pH of the films remained constant at neutral pH. This study confirmed the potential of the above MMFs as a promising candidate for buccal delivery of PHCl.
Keywords: Films; Buccal; Drug release; Physicochemical/mechanical characterisation;

Thermally Stimulated Depolarisation Currents (TSDC) measurements on d(−)-salicin have been carried out in the temperature region from −165 °C up to 150 °C. The slow molecular mobility was characterised in the crystal and in the glassy state. The value of the steepness index or fragility (T g—normalized temperature dependence of the relaxation time) was obtained by Differential Scanning Calorimetry (DSC) from the analysis of the scanning rate dependency of T g. The existence of an unknown polymorph of salicin is also reported.
Keywords: Molecular mobility; Amorphous materials; Glass transition; Fragility; β-Relaxation; Polymorphism;

The objective of the present investigation was to clarify the mechanism by which Labrafac Lipophile WL 1349 (WL 1349) enhanced the oral bioavailability (BA) of hydroxysafflor yellow A (HSYA), the representative low permeable hydrophilic (biopharmaceutic classification system (BCS) Class III) drug. HSYA–phospholipid complex was prepared, and dissolved into WL 1349 with a certain surfactant to form a stable oil solution. Oral administration of HSYA aqueous solution at a dosage of 4.5 mg/kg resulted a low plasma HSYA concentration with C max and AUC0–8 h values of 0.105 μg/ml and 10.29 μg min/ml, respectively. HSYA–phospholipid complex oil solution with the same administration and dosage increased the plasma HSYA concentration significantly with C max and AUC0–8 h values of 2.063 μg/ml and 381.145 μg min/ml, respectively. The results showed that WL 1349 could improve oral absorption of HSYA remarkably. Bioavailability investigations were performed to show WL 1349 dosage independent from HSYA absorption within the dosage from 1 ml/kg to 9 ml/kg. The test of bile duct ligation in rats showed that the oil solution containing WL 1349 did not result in detectable plasma HSYA concentration, but HSYA aqueous solution had the same AUC0–8 h as the bile duct was not ligated. The in vitro lipolysis experiments of WL 1349 showed that WL 1349 was emulsified by deoxycholate, and then was hydrolyzed to fatty acids and monoglycerides by pancreatic lipase rapidly. The lipolysis products of WL 1349, caprylic acid, capric acid and caprylic and capric acid monoglycerides all improved the BA of HSYA in vivo. The results above indicated the emulsifying by bile, and hydrolysis to fatty acids and monoglycerides by pancreatic lipase was one of the enhancing mechanisms of HSYA–phospholipid complex oil solution absorption.
Keywords: Hydroxysafflor yellow A; Labrafac Lipophile WL 1349; Bioavailability; Absorption enhancing mechanism;

The aim of this paper was to study the swelling and diffusion behaviors of calcium polysaccharide gel (CaPG) films prepared by an interfacial complexation technique, a new gel formation method that allowed calcium ions to diffuse from a source to form gel films with polysaccharide (i.e., alginate or pectin). The dynamic swelling behavior of CaPG films showed that swelling was a function of time. Most CaPG films showed a maximum amount of water absorption during the first few hours. The films swelled less in water and acidic media but extensively swelled in 0.1 M NaCl. The rehydration of the dry films in the acidic media or the 0.1 M NaCl solution also lead to the extraction of most of the calcium ions from the CaPG within 4 h or less. Partitioning and diffusion of a model drug, theophylline (TPL), were measured through CaPG films equilibrated in different media. The partition and diffusion coefficients of TPL through CaPG films were found to vary, depending upon polysaccharide type, concentration and equilibration medium. The results suggest that both partition and pore mechanisms operated concurrently in the transport of TPL through CaPG films equilibrated in different media.
Keywords: Polysaccharide film; Alginate; Pectin; Swelling; Diffusion coefficient;

Electronic structure and UV spectrum of fenofibrate in solutions by Yuan Le; Jian-Feng Chen; Min Pu (214-218).
The structure and UV spectra of fenofibrate have been evaluated in gas phase and in solutions using time dependent density functional theory (TDDFT) method at the B3LYP/6-31G(d), B3LYP/6-311G(d,p) and B3LYP/6-311++G(d,p) levels. The solvent effects have been taken into account based on the polarizable continuum model (PCM). The computed results appear that the introduction of dielectric medium has slight effect on the molecular geometry of fenofibrate. There is one allowed excited state presenting the strongest oscillator strength in the UV region, which is associated with the HOMO → LUMO and HOMO-1 → LUMO transition both in gas phase and in solutions. The prediction of the λ max in THF, ethanol and DMSO is 285 nm, 286 nm and 287 nm, respectively, which are in a good agreement with experimental data of 284 nm, 285 nm and 288 nm.The results demonstrate that TDDFT-PCM is a useful tool for study of the electronic absorption in solutions.
Keywords: Fenofibrate; Solvent effects; Electronic structure; Excited states; UV absorption;

Cyclosporine A (CsA), Rapamycin (RAPA), Tacrolimus (FK-506) and Mycophenolate mofetil (MMF) are immunosuppressants that are widely used in solid organ transplant patients. However, some of these drugs have been reported to cause dyslipidemia in patients. Our aim was to determine the effects of these drugs on in vitro cholesteryl ester transfer protein (CETP), hepatic lipase (HL) and lipoprotein lipase (LPL) activity within human plasma.We measured CETP activity in human normolipidemic plasma with and without drug treatment, by measuring the incorporation of labeled cholesteryl ester into lipoproteins. To further confirm the result, we also measured recombinant CETP (rCETP) activity with and without drug treatment. We measured HL and LPL activity in post-heparin normal human plasma in the presence and absence of the drugs by measuring the release of fatty acids from radiolabeled triolein.We found an increase in CETP activity in human normolipidemic plasma and rCETP treated with CsA and RAPA. By contrast, CETP activity was not altered significantly in the presence of FK-506 and MMF. LPL activity in post-heparin normal human plasma was suppressed following the co-incubation with CsA, RAPA, FK-506 or MMF whereas HL activity remained unaffected.The increase in CETP activity and suppression in LPL activity following CsA and RAPA treatment observed in the present study may be associated with elevated LDL cholesterol levels and hypertriglyceridemia seen in patients administered these drugs.
Keywords: Immunosuppressant; Dyslipidemia; Cholesteryl ester transfer protein; Hepatic lipase; Lipoprotein lipase;

Curcuma drugs and curcumin regulate the expression and function of P-gp in Caco-2 cells in completely opposite ways by Xiao-Long Hou; Kyoko Takahashi; Ken Tanaka; Katsuhiko Tougou; Feng Qiu; Katsuko Komatsu; Koichi Takahashi; Junichi Azuma (224-229).
Curcumin is a phenolic compound isolated from rhizomes of C. longa, C. aromatica and other Curcumas except C. zedoaria. Recently, both curcumin and Curcumas have become prevalent as supplement. P-gp has been reported as an important determinant for drug absorption in small intestine. In this study, Caco-2 cell monolayers were treated with methanol extracts of Curcumas (0.1 mg/ml) or curcumin (30 μM) for 72 h to investigate the relationship between the potential affects of Curcumas and curcumin on P-gp. [3H]-digoxin and rhodamine 123 were used to evaluate P-gp activity. All Curcumas significantly increased the activity of P-gp by up-regulating the expressions of P-gp protein and MDR1 mRNA levels. Interestingly, contrary to Curcumas, curcumin treatment inhibited the activity of P-gp with a decrease in P-gp protein and MDR1 mRNA expression levels. Curcumas might alter the pharmacokinetics of co-administrated drugs by up-regulating the function and expression levels of intestinal P-gp. However, curcumin has no relationship with the inductive effect of Curcumas since curcumin showed an opposite effects. Caution should be exercised when Curcumas or curcumin are to be consumed with drugs that are P-gp substrates because Curcumas and curcumin might regulate the function of P-gp in completely opposite ways.
Keywords: Curcuma; Curcumin; P-gp;

Site-directed PEGylation as successful approach to improve the enzyme replacement in the case of prolidase by C. Colonna; B. Conti; P. Perugini; F. Pavanetto; T. Modena; R. Dorati; P. Iadarola; I. Genta (230-237).
The first aim of this work was to perform site-directed PEGylation of the enzyme prolidase at sulphydril groups by methoxy-polyethylene glycol-maleimide (Mal-PEG, Mw 5000 Da) in order to obtain a safe conjugation product more stable than the native enzyme. Prolidase is a cytosolic aminoacyl-l-proline hydrolase whose deficiency causes the onset of rare autosomal recessive disorder called prolidase deficiency (PD). The second purpose of this work was to investigate whether biodegradable chitosan nanoparticles loaded with PEGylated prolidase could be effective in releasing active enzyme inside fibroblasts as a possible therapeutic approach for PD. The SDS-PAGE analysis and the ESI-MS spectra confirmed the presence of the PEGylated prolidase: in particular the main conjugation product (m/z  = about 65,000 Da) corresponded to the enzyme with two residues of Mal-PEG. In this study it was demonstrated the lack of toxicity (MTT assay) and the prolonged activity (40.6 ± 2.6% after 48 h of incubation at 37 °C) of the PEGylated enzyme. The PEGylated prolidase loaded chitosan nanoparticles had spherical shape, narrow size distribution (271.6 ± 45.5 nm), a positive zeta-potential (15.93 ± 0.26 mV) with a good preparation yield (54.6 ± 3.6%) and protein encapsulation efficiency (44.8 ± 4.6%). The ex vivo evaluation of prolidase activity on PD fibroblasts individuated a good level of prolidase activity replaced (about 72% after only 2 days of incubation) up to 10 days with improved morphological cell features.
Keywords: Site-directed PEGylation; Enzyme stability; Chitosan nanoparticles; Enzyme replacement therapy; Prolidase deficiency;

Experimental observations of dry powder inhaler dose fluidisation by Rob Tuley; John Shrimpton; Matthew D. Jones; Rob Price; Mark Palmer; Dave Prime (238-247).
Dry powder inhalers (DPIs) are widely used to deliver respiratory medication as a fine powder. This study investigates the physical mechanism of DPI operation, assessing the effects of geometry, inhalation and powder type on dose fluidisation. Patient inhalation through an idealised DPI was simulated as a linearly increasing pressure drop across three powder dose reservoir geometries permitting an analysis of shear and normal forces on dose evacuation. Pressure drop gradients of 3.3, 10 and 30 kPa s−1were applied to four powder types (glass, aluminium, and lactose 6 and 16% fines) and high speed video of each powder dose fluidisation was recorded and quantitatively analysed. Two distinct mechanisms are identified, labelled ‘fracture’ and ‘erosion’. ‘Fracture’ mode occurs when the initial evacuation occurs in several large agglomerates whilst ‘erosion’ mode occurs gradually, with successive layers being evacuated by the high speed gas flow at the bed/gas interface. The mechanism depends on the powder type, and is independent of the reservoir geometries or pressure drop gradients tested. Both lactose powders exhibit fracture characteristics, while aluminium and glass powders fluidise as an erosion. Further analysis of the four powder types by an annular shear cell showed that the fluidisation mechanism cannot be predicted using bulk powder properties.
Keywords: Dry powder inhaler; Fluidisation; Lactose; Powder flow; Inhalation; Shear cello;

Oral colon targeted delivery systems for treatment of inflammatory bowel diseases: Synthesis, in vitro and in vivo assessment by Amal H. El-Kamel; Alaa A.-M. Abdel-Aziz; Amal J. Fatani; Hussein I. El-Subbagh (248-255).
The aim of this study was to investigate the potential of prodrugs of some non-steroidal anti-inflammatory drugs (NSAIDs) as colon targeted delivery systems for treatment of inflammatory bowel diseases. Naproxen, sulindac and flurbiprofen (Fbp) were used. The carboxylic group of those drugs was conjugated onto the amino group of l-aspartic acid or the hydroxyl group of α- or β-cyclodextrin (CyD). Prodrugs hydrolysis in buffers of pH range 1.2–7.2 and in rat gastrointestinal tract homogenates and the effect of oral pretreatment of rats with clindamycin on the hydrolysis of the prodrugs was examined. Additionally, the effect of oral administration of Fbp-β-CyD prodrug on the experimentally induced colitis in rats was evaluated. The in vivo inflammatory response was assessed macroscopically, histologically and by measurement of reduced glutathione (GSH) levels in colon tissues. No significant hydrolysis of the proposed seven prodrugs in buffers having pH range of 1.2–7.2 was observed over 72 h. Negligible % of drug released from Fbp-α-CyD or Fbp-β-CyD prodrugs was detected in rat stomach contents, intestinal tissues and intestinal contents homogenates. On the other hand, Fbp-α-CyD and Fbp-β-CyD prodrugs released about 60% Fbp within 4 h in rat colon homogenate. Oral pretreatment of rats with clindamycin significantly reduced % Fbp released from Fbp-α-CyD or Fbp-β-CyD prodrugs. Oral administration of Fbp-β-CyD to rats after induction of colitis significantly attenuated the severity of the colonic injury and reduced the score of the macroscopic and microscopic damage. Additionally, there was a significant increase in the level of GSH. The present study provided an evidence that Fbp-β-CyD prodrug may be beneficial in treatment of inflammatory bowel disease.
Keywords: Synthesis; Colon; NSAIDs; Aspartic acid; Cyclodextrin; Prodrug; In vivo/in vitro;

Salbutamol sulphate nanoparticles have been simultaneously prepared and coated with l-leucine in the gas phase. Three different ways of coating can be separated based on the operation temperatures used in an aerosol flow reactor. Below the temperature of l-leucine sublimation, formation of the l-leucine layer on the core particle surface takes place via diffusion of l-leucine molecules on the droplet surfaces during droplet drying. At intermediate temperatures, the extent of sublimation of l-leucine depends notably on the concentration, and thus partial evaporation was expected. The l-leucine coating was solely formed via vapor deposition at high reactor temperatures when complete sublimation of l-leucine was obtained. The geometric mean diameter of the core salbutamol particles was approximately 65 nm. In general, particle size increased with the addition of l-leucine. The size distribution remained the same or broadened when the coating layer of the particles was formed via surface diffusion whereas notable narrowing of the distribution was observed when the coating was formed via vapor deposition. Upon desublimation and heterogeneous nucleation on the surfaces of smooth, spherical core particles, l-leucine formed a discontinuous coating with leafy crystals a few nanometers in size.
Keywords: Aerosol; Coating; l-Leucine; Nanoparticles; Pharmaceutical; Physical vapor deposition; Salbutamol sulphate;

Cationic lipid-coated magnetic nanoparticles associated with transferrin for gene delivery by Xiaogang Pan; Jingjiao Guan; Jung-Woo Yoo; Arthur J. Epstein; L. James Lee; Robert J. Lee (263-270).
Cationic lipid-coated magnetic nanoparticles (MPs) associated with transferrin were evaluated as gene transfer vectors in the presence of a static magnetic field. MPs were prepared by chemical precipitation and were surface-coated with cationic lipids, composed of DDAB/soy PC (60:40 mole/mole). These cationic MPs were then combined with polyethylenimine (PEI) condensed plasmid DNA, followed by transferrin. The resulting magnetic electrostatic complexes retained relatively compact particle size and showed complete DNA condensation. Their transfection activity in the presence of a static magnetic field was evaluated by luciferase and green fluorescent protein (GFP) reporter genes. The magnetic complexes exhibited up to 300-fold higher transfection activity compared to commonly used cationic liposomes or cationic polymer complexes, based on luciferase assay. The enhancement in transfection activity was maximized when the cells were exposed to the vectors for a relatively short period of time (15 min), or were treated in media containing 10% serum. Incorporation of transferrin further improved transfection efficiency of the cationic MPs. However, when cells were incubated for 4 h in serum-free media, magnetic and non-magnetic vectors showed similar transfection efficiencies. In conclusion, transferrin-associated cationic MPs are excellent gene transfer vectors that can mediate very rapid and efficient gene transfer in vitro in the presence of a magnetic field.
Keywords: Gene delivery; Magnetic nanoparticles; Transferrin; Cationic lipid; Magnetofection;

A formyl group-ended poly(dl-lactic acid) (PLA-aldehyde), synthesized in the same manner as reported previously, was utilized to produce the polymeric marker for PLA-related nanoparticles. Namely, pyrene-ended poly(dl-lactic acid) (PLA-pyrene) was prepared as a polymeric marker by the reductive amination of PLA-aldehyde and aminopyrene. Methoxypolyethylene glycol amine–poly(dl-lactic acid) block copolymer (PLA–(MeO-PEG) nanoparticles loaded with PLA-pyrene were prepared, and examined on retention of PLA-pyrene in the nanoparticles, and biodisposition in normal and sarcoma-180 solid tumor-bearing mice. PLA-pyrene was retained stably in PLA–(MeO-PEG) nanoparticles in a PBS-ethanol (7:3, v/v) mixture and a plasma-PBS (1:1, v/v) mixture, indicating that PLA-pyrene might be a useful marker of PLA–(MeO-PEG) nanoparticles themselves. After i.v. injection in normal rats, the plasma level of PLA-pyrene was very high for initial 8 h, and accumulated gradually into organs, especially spleen and liver. After i.v. injection in tumor-bearing mice, similar biodistribution profiles of PLA-pyrene were observed, and PLA-pyrene was accumulated well in tumor, suggesting that PLA–(MeO-PEG) nanoparticles should be delivered efficiently to solid tumors. It is suggested that PLA-pyrene might be a useful probe of the nanoparticles themselves. In addition, it was demonstrated that PLA–(MeO-PEG) nanoparticles should be a useful drug carrier for passive tumor targeting.
Keywords: PLA–(MeO-PEG) nanoparticle; PLA-pyrene; Biodisposition; Normal mice; Tumor-bearing mice;

Liposomal bismuth-ethanedithiol formulation enhances antimicrobial activity of tobramycin by Majed Halwani; Shanna Blomme; Zacharias E. Suntres; Misagh Alipour; Ali O. Azghani; Aseem Kumar; Abdelwahab Omri (278-284).
Pseudomonas aeruginosa and Burkholderia cenocepacia (formally, genomovar III genotype of Burkholderia cepacia complex) have emerged as serious opportunistic resistant pathogens in patients with cystic fibrosis (CF). We have developed a liposomal formulation containing bismuth-ethanedithiol (BiEDT) and tobramycin to overcome bacterial resistance. The stability of liposomal BiEDT-tobramycin (LipoBiEDT-TOB) was studied in phosphate buffered saline (PBS) and human pooled plasma at 4 and 37 °C. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) for free tobramycin and LipoBiEDT-TOB against clinical isolates of P. aeruginosa and B. cenocepacia were determined by the broth dilution method. The toxicity profile and the influence on bacterial adhesion of LipoBiEDT-TOB formulation were determined using a human lung carcinoma cell line (A549). LipoBiEDT-TOB exhibited lower MICs than the conventional antibiotic (0.25 mg/L vs. 1024 mg/L) and eradicated this highly resistant bacterial strain of P. aeruginosa (PA-48913) at very low concentrations (4 mg/L vs. 4096 mg/L). LipoBiEDT-TOB was significantly less toxic when compared to the free BiEDT, as evaluated by the MTT and LDH assay. The LipoBiEDT-TOB formulation suppressed bacterial adhesion (B. cenocepacia M13642R) to A549 cells. These data suggest that the novel LipoBiEDT-TOB drug delivery system could be utilized as a new strategy to enhance the efficacy of existing antibiotics against resistant organisms that commonly affect individuals with chronic lung infections.
Keywords: Liposomal-(bismuth ethanedithiol); MICs; MBCs; Toxicity; Bacterial adhesion; Burkholderia cenocepacia; Pseudomonas aeruginosa; Staphylococcus aureus;

Intranasal nanoemulsion based brain targeting drug delivery system of risperidone by Mukesh Kumar; Ambikanandan Misra; A.K. Babbar; A.K. Mishra; Puspa Mishra; Kamla Pathak (285-291).
The objective of investigation was to prepare nanoemulsion containing risperidone (RSP) to accomplish the delivery of drug to the brain via nose. Risperidone nanoemulsion (RNE) and mucoadhesive nanoemulsion (RMNE) were characterized for drug content, pH, percentage transmittance, globule size and zeta potential. Biodistribution of RNE, RMNE, and risperidone solution (RS) in the brain and blood of Swiss albino rats following intranasal (i.n.) and intravenous (i.v.) administration was examined using optimized technetium labeled (99mTc-labeled) RSP formulations. Gamma scintigraphy imaging of rat brain following i.v. and i.n. administrations were performed to ascertain the localization of drug in brain. The brain/blood uptake ratio of 0.617, 0.754, 0.948, and 0.054 for RS (i.n.), RNE (i.n.), RMNE (i.n.), and RNE (i.v.), respectively, at 0.5 h are indicative of direct nose to brain transport bypassing the blood–brain barrier. Higher drug transport efficiency (DTE%) and direct nose to brain drug transport (direct transport percentage, DTP%) for mucoadhesive nanoemulsions indicated more effective and best brain targeting of RSP amongst the prepared nanoemulsions. Studies conclusively demonstrated rapid and larger extent of transport of RSP by RMNE (i.n.) when compared to RS (i.n.), RNE (i.n.) and RNE (i.v.) into the rat brain.
Keywords: Risperidone; Brain targeting; Intranasal delivery; Nanoemulsion; Biodistribution; Radiolabel;

Nanocapsule@xerogel microparticles containing sodium diclofenac: A new strategy to control the release of drugs by Letícia Sias da Fonseca; Rodrigo Paulo Silveira; Alberto Marçal Deboni; Edilson Valmir Benvenutti; Tânia M.H. Costa; Sílvia S. Guterres; Adriana R. Pohlmann (292-295).
The aim of this work was to evaluate the potentiality to control the drug release of a new architecture of microparticles organized at the nanoscopic scale by assembling polymeric nanocapsules at the surface of drug-loaded xerogels. Xerogel was prepared by sol–gel method using sodium diclofenac, as hydrophilic drug model, and coated by spray-drying. After coating, the surface areas decreased from 82 to 28 m2/g, the encapsulation efficiency was 71% and SEM analysis showed irregular microparticles coated by the nanocapsules. Formulation showed satisfactory gastro-resistance presenting drug release lower than 3% (60 min) in acid medium. In water, the pure drug dissolved 92% after 5 min, uncoated drug-loaded xerogel released 60% and nanocapsule coated drug-loaded xerogel 36%. After 60 min, uncoated drug-loaded xerogel released 82% and nanocapsule coated drug-loaded xerogel 62%. In conclusion, the new system was able to control the release of the hydrophilic drug model.
Keywords: Nanoparticle-coated microparticles; Nanocapsule-coated xerogels; Sol–gel; Spray-drying; Sodium diclofenac;

A targeted delivery system for inflammatory bowel disease (IBD), Eudragit L100 (EuL)-coated chitosan (Ch)–succinyl-prednisolone (SP) conjugate microspheres (Ch–SP-MS/EuL), were designed and examined in vivo for efficacy and toxicity. Their preparation was conducted in the same manner as previously; that is, by synthesis of the conjugate by carbodiimide coupling of Ch and SP, conversion into microspheres (Ch–SP-MS), and coating of Ch–SP-MS with EuL. Experimental colitis was induced by instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) into the colon in rats. Drugs were administered once or twice a day intragastrically for three consecutive days. Visible colitis severity, colon/body weight ratio and myeloperoxidase activity were measured as inflammatory indices. Toxicity was examined from the decrease in the thymus/body weight ratio. Efficacy was dose-dependent and the greatest in the order Ch–SP-MS/EuL > Ch–SP-MS > prednisolone (PD) alone, and Ch–SP-MS/EuL showed excellent recovery of colitis states. Toxicity was the greatest in the order PD ≫ Ch–SP-MS > Ch–SP-MS/EuL. Ch–SP-MS and Ch–SP-MS/EuL reduced significantly the thymic atrophy caused by PD. It was demonstrated that Ch–SP-MS/EuL enhanced effectiveness of PD and reduced toxic side effects of PD greatly. Also, these results established the prediction by previous in vitro and in vivo studies.
Keywords: Eudragit L100-coated chitosan–succinyl-prednisolone conjugate microspheres; 2,4,6-Trinitrobenzenesulfonic acid-induced colitis; Efficacy; Toxicity;

Solubilisation of drugs in micellar solutions of diblock copolymers of ethylene oxide and styrene oxide by Michael Crothers; Nagíla M.P.S. Ricardo; Frank Heatley; S. Keith Nixon; David Attwood; Colin Booth (303-306).
The solubilisation of two poorly soluble drugs, furosemide and nabumetone, in micellar solutions of diblock copolymers of ethylene oxide and styrene oxide has been studied at 25 and 37 °C and solubilisation capacities compared with published values for griseofulvin and docetaxel. Solubilisation in the micelle core, corrected for the different proportions of poly(styrene oxide) in the copolymers, was similar for all four drugs. The highest solubilisation capacities were found for a copolymer with worm-like micelles.
Keywords: Solubilisation; Block copolymers; Micelles;

by Alexander T. Florence (307).

by Alexander T. Florence (308).

Noticeboard (309).