European Journal of Pharmaceutics and Biopharmaceutics (v.63, #2)

Editorial by Robert Gurny (iii).

A new drug nanocarrier consisting of chitosan and hydoxypropylcyclodextrin by Francesca Maestrelli; Marcos Garcia-Fuentes; Paola Mura; Maria José Alonso (79-86).
The objective of the present work was to develop a new drug nanocarrier consisting of nanoparticles made of chitosan and cyclodextrins. The rationale behind the design of this new nanosystem was to simultaneously implement the cyclodextrin drug complexation power and the inherent properties of chitosan nanoparticles, in a unique delivery system. The complexation with the cyclodextrin permits the solubilization as well as the protection for sensitive drugs, whereas the entrapment in the chitosan network is expected to facilitate their absorption. Chitosan nanoparticles including hydroxypropylcyclodextrins could be prepared by the ionic crosslinking of chitosan with sodium tripolyphosphate in the presence of cyclodextrins. Two hydrophobic drugs, triclosan and furosemide, were selected as models for complexation with the cyclodextrin and further entrapment in the chitosan nanocarrier. The resulting nanosystems were thoroughly characterized for their size and zeta potential and also for their ability to associate and deliver the complexed drugs. The results showed that the size of the nanoparticles was slightly affected by the incorporation of cyclodextrins, whereas the ζ potential did not suffer a significant modification. Moreover, the complexation of the drugs with the cyclodextrin facilitated their entrapment into the nanoparticles, increasing up to 4 and 10 times (for triclosan and furosemide, respectively) the final drug loading of the nanoparticles. These results led to the conclusion that the drug–cyclodextrin complex was efficiently retained in the nanoparticulate structure. Finally, the in vitro release profile observed for these nanoparticles was characterized by an initial fast release followed by a delayed release phase. In conclusion, this new nanosystem offers an interesting potential for the transmucosal delivery of hydrophobic compounds.
Keywords: Nanoparticle drug carriers; Chitosan; Cyclodextrins; Class IV drugs;

Freeze-drying was recently applied to improve the long-term storage stability of nanoparticles. Nanocapsules have a thin polymeric envelope that may not withstand the stresses of such process. So, cryoprotectants and lyoprotectants are usually added to the formulation to protect these vectors during freezing and desiccation steps. The aim of this paper was to investigate the importance of the vitrification of cryoprotectants on the stabilization of nanocapsules during freezing, desiccation, and storage steps. Furthermore, the effect of stabilizer crystallization on the conservation of nanocapsules properties was studied. Finally, the effect of temperature storage and relative humidity on the stability of nanocapsules was tested through an accelerated stability study. Results indicate that nanocapsules stabilization during the different steps of freeze-drying requires their dispersion within a vitrified matrix of amorphous excipient to protect them against the stress of freezing and dehydration. The crystallization of this stabilizer during the freezing, the desiccation or the storage steps can destabilize these fragile particles. Electron spectroscopy for chemical analysis revealed the adsorption of nanocapsules at the interface ice/liquid during the freezing step. Such adsorption must be avoided in the case of freeze-drying of immuno-nanoparticles to preserve the native structure of proteins attached to their surface.
Keywords: Freeze-drying; Nanocapsules; Vitrification; Crystallization; Cryoprotectant; Electron spectroscopy for chemical analysis;

Single dose and multiple dose studies of itraconazole nanoparticles by Jason M. Vaughn; Jason T. McConville; David Burgess; Jay I. Peters; Keith P. Johnston; Robert L. Talbert; Robert O. Williams (95-102).
The objective of this study was to determine and compare the lung and serum concentrations in mice following oral and pulmonary dosing of amorphous nanoparticulate itraconazole (ITZ) compositions as well as the Sporanox® oral solution (itraconazole/Janssen). Second, the steady state partitioning of ITZ in lung tissue and circulatory compartments following repeated oral and pulmonary dosing was determined. The pulmonary formulation (ITZ-pulmonary) consisted of ITZ, polysorbate 80, and poloxamer 407 in a 1:0.75:0.75 ratio and the oral formulation (ITZ-oral) consisted of ITZ, PEG 8000, poloxamer 188, and sorbitan monooleate 80 in a 1:1:2:1 ratio. Mice were dosed every 12 h by nebulization with ITZ-pulmonary, or by oral gavage with ITZ-oral or Sporanox oral solution (n  = 12 per study arm). ITZ-pulmonary achieved significantly greater (>10-fold) lung tissue concentrations compared to the Sporanox oral solution and ITZ-oral. There were no statistical differences between the two oral formulations. ITZ-pulmonary achieved significantly greater lung levels per unit serum concentration compared to the orally dosed ITZ compositions. High and sustained lung tissue concentrations were achieved via inhalation of an amorphous nanoparticulate ITZ-pulmonary composition while maintaining serum levels which are above the minimum lethal concentration (MLC) of Aspergillus fumigatus.
Keywords: Itraconazole; Nanoparticles; Spray freezing into liquid; Amorphous; Pharmacokinetics; Lung deposition;

Effect of hydrogen bonding interactions on the release mechanism of felodipine from nanodispersions with polyvinylpyrrolidone by Evangelos Karavas; Georgios Ktistis; Aristotelis Xenakis; Emmanouel Georgarakis (103-114).
Solid dispersion systems are widely investigated for the dissolution enhancement of poorly water soluble drugs. Nevertheless, very limited commercial use has been achieved due to the poor predictability of such systems caused by the lack of a basic understanding of the dissolution optimization mechanism. In the present study an investigation of the release mechanism is performed for solid dispersion systems composed by polyvinylpyrrolidone (PVP) and felodipine (FEL), based on a correlation of their hydrophilicity with the intensity of interactions. The existing interactions were evaluated by using NMR and UV spectroscopy while molecular simulation techniques were also enabled. It was found that the interactions that take place correspond to the creation of hydrogen bonds. The correlation between the intensity of interactions and the concentration of PVP in the matrix showed a sigmoid function. The interactions are impressively increased for polymer concentration exceeding 75% (w/w). This phenomenon was well explained by using the molecular simulation technique. A similar sigmoid pattern was found for the function between dissolution profiles and polymer concentration in the matrix, indicating that the intensity of interactions promotes the dissolution enhancement. Investigation of the solubility and the particle size distribution of FEL in the binary system appeared to have similar behaviour indicating that the interactions affect the release profile through these two factors. The hydrophilicity of PVP does not significantly affect this enhancement as the contact angle was found to be linear to PVP concentration. Microscopic observation of the dissolution behaviour showed that FEL remains in fine dispersion in aqueous solution, verifying the release mechanism.
Keywords: Felodipine; Solid dispersions; Nanodispersions; Interactions; Release mechanism;

Aqueous nanoscale lipid dispersions consisting of carnauba wax–decyl oleate mixtures acting as carriers or accompanying vehicles for inorganic sunscreens such as barium sulfate, strontium carbonate, and titanium dioxide were prepared by high pressure homogenization. For the manufacture of these nanosuspensions, three pigment concentrations (%wt), namely 2, 4, and 6, and two carnauba wax–decyl oleate ratios, 1:1 and 2:1, were used, being some of these combinations chosen for stability studies. Six-month physical stability tests at 4, 20, and 40 °C selecting the mean particle size and the polydispersity index of the nanosuspensions as reference parameters were performed. Centrifugation tests of the nanosuspensions assessed by transmission electron microscopy and by the determination of the content of pigments and carnauba wax in the separated fractions were done. The mean particle sizes and the polydispersity indices of the nanosuspensions were not altered after six-month storages at 20 and at 40 °C. However, the storage of those at 4 °C considerably increased the particle size and polydispersity of the systems, particularly when wax–oil ratios (2:1) were used for the entrapment of the pigments. Transmission electron micrographs of centrifuged samples denoted the presence of three major fractions showing the different types of particles integrated into the nanosuspensions. Furthermore, it was observed that not all the carnauba wax participated in the entrapment of the pigment. Regarding the amount of pigment being encapsulated or bonded by the wax–oil matrices, entrapment efficiencies higher than 85.52% were reported.
Keywords: Nanosuspensions; Inorganic sunscreens; Physical stability; Centrifugation; Entrapment efficiency; Transmission electron microscopy;

Colloidal dispersions of the pre-equilibrated cubic phase in the monoolein/poloxamer 407/water system, which are under investigation as potential drug carriers, often contain a considerable fraction of undesired non-cubic particles, particularly when prepared with high concentrations of poloxamer. Recent investigations revealed that the non-cubic particles can be transformed into particles of cubic internal structure by heat treatment. The present study investigates the effect of drug loading on the non-cubic to cubic transformation process during autoclaving of the dispersions. The results indicate that the process can also proceed in dispersions loaded with different concentrations of ubidecarenone, tocopheryl acetate, betamethasone-17-valerate, chloramphenicol or miconazole. At low concentration, none of the drugs had pronounced influence on the autoclaved dispersions whereas with increasing drug concentration different effects were observed. Depending on the type of drug no effects (betamethasone-17-valerate), increasing particle size of the dispersions (chloramphenicol, miconazole) or phase separation upon autoclaving (high load of miconazole) was observed. Except for loading with high amounts of chloramphenicol, which led to the formation of cubic phase particles already without additional heat treatment, the properties of the thermally untreated dispersions were virtually unaffected by drug incorporation.
Keywords: Colloidal drug carriers; Dispersions of cubic phase; Autoclaving; Monoolein; Drug loading; Ultrastructural transformation;

Stabilization of all-trans retinol by loading lipophilic antioxidants in solid lipid nanoparticles by Jun-Pil Jee; Soo-Jeong Lim; Jeong-Sook Park; Chong-Kook Kim (134-139).
Loading of drugs into the solid matrix of solid lipid nanoparticles (SLNs) can be one of effective means to protect them against chemical degradation. In this study, the SLNs for all-trans retinol (AR) were formulated to improve the stability of AR, whose chemical instability has been a limiting factor in its clinical use. First of all, the physicochemical properties of AR-loaded SLNs, including mean particle diameter and zeta potential, were modulated by changing the total amount of surfactant mixture and the mixing ratio of eggPC and Tween 80 as surfactant mixture. The AR-loaded SLNs formulation was irradiated with a 60-W bulb to investigate the photostability. The extent of photodegradation was measured by high-performance liquid chromatography. The mean particle diameter and zeta potential of the smallest SLNs were 96 nm and −28 mV, respectively. The loading of AR in optimized SLNs formulations rather decelerated the degradation of AR, compared with AR solution dissolved in methanol. Our subsequent study showed that the co-loading of antioxidants greatly enhanced the stability of AR loaded in SLNs, compared with those loaded in SLNs without antioxidant. The photostability at 12 h of AR in SLNs was enhanced folds (43% approximately) higher than that in methanol solution (about 11%). Furthermore, the protecting effect of antioxidants was greatly dependent on the type of antioxidant. Taken together, AR could be effectively stabilized by being loaded in SLNs together with an antioxidant BHT–BHA.
Keywords: All-trans retinol; Solid lipid nanoparticles; Stability; Antioxidant;

Influence of liposphere preparation on butyl-methoxydibenzoylmethane photostability by Valentina Iannuccelli; Nicoletta Sala; Rosanna Tursilli; Gilberto Coppi; Santo Scalia (140-145).
The incorporation of butyl-methoxydibenzoylmethane (BMDBM), one of the most efficient and frequently used UV-A blockers, into lipospheres was examined in order to decrease the light-induced sunscreen degradation. Lipospheres, obtained by the melt technique and using tristearin as the lipid material and hydrogenated phosphatidylcholine as the emulsifier, showed proper features in terms of size (10–40 μm), BMDBM loading level (21.63% ± 0.90%, w/w) and physical state. Photolysis studies, involving irradiation of lipospheres with simulated sunlight before and after their introduction in emulsion formulations, demonstrated a relevant enhancement of the encapsulated sunscreen photostability in comparison with unencapsulated BMDBM.
Keywords: Lipospheres; Sunscreens; Butyl-methoxydibenzoylmethane; Physical state; Photostability;

Liquid crystalline phases of monoolein and water for topical delivery of cyclosporin A: Characterization and study of in vitro and in vivo delivery by Luciana B. Lopes; João L.C. Lopes; Dionéia C.R. Oliveira; José A. Thomazini; M. Tereza J. Garcia; Márcia C.A. Fantini; John H. Collett; M. Vitória L.B. Bentley (146-155).
Reverse cubic and hexagonal phases of monoolein have been studied as drug delivery systems. The present study was aimed at investigating whether these systems enhance the cutaneous penetration of cyclosporin A (CysA) in vitro (using porcine ear skin) and in vivo (using hairless mice). Different mesophases were obtained depending on CysA concentration. CysA at 4% allowed the formation of reverse cubic and hexagonal phases in a temperature range of 25–40 °C. At 8%, CysA induced the formation of other phases, which might be due to an interaction between the polar groups of the peptide and monoolein. In vitro, the cubic phase increased the penetration of CysA in the stratum corneum (SC) and epidermis plus dermis ([E + D]) at 12 h post-application. The reverse hexagonal phase increased CysA penetration in [E + D] at 6 h and percutaneous delivery at 7.5 h post-application. In vivo, both liquid crystalline phases increased CysA skin penetration. Topical application of these systems, though, induced skin irritation after a 3-day exposure. These results demonstrate that liquid crystalline systems of monoolein are effective in optimizing the delivery of peptides to the skin. The skin irritation observed after topical application of cubic and hexagonal phases should be minimized for their safe use as topical delivery systems.
Keywords: Cubic phase; Reverse hexagonal phase; Monoolein; Skin penetration; Cyclosporin A;

Fluorescence resonance energy transfer: Evaluation of the intracellular stability of polyplexes by Miriam Breunig; Uta Lungwitz; Renate Liebl; Achim Goepferich (156-165).
The investigation of intracellular mechanisms of non-viral nucleic acid delivery systems has provided great impetus for the improvement of their efficacy. Especially the intracellular release of the nucleic acid from the non-viral carrier system may be a relevant criterion for the high transfection efficiency of certain polymers. Therefore, we evaluated fluorescence resonance energy transfer (FRET) in combination with confocal laser scanning microscopy or flow cytometry as tool to determine the intracellular disintegration of polyplexes built with plasmid DNA and linear polyethylenimine. In microscopy, which allowed for an observation of polyplexes within single cells, sensitized emission measurement and acceptor photobleaching have been tested towards quantitative FRET analysis. In contrast, the whole cell population was analyzed by the flow cytometry-based method. We suggest that FRET is a useful tool to evaluate the intracellular disintegration of polyplexes built with various polymers.
Keywords: Non-viral gene delivery; Polyethylenimine; Fluorescence resonance energy transfer; Confocal laser scanning microscopy; Flow cytometry;

Development of a novel method for the preparation of submicron particles based on thiolated chitosan by Andreas Bernkop-Schnürch; Andreas Heinrich; Alexander Greimel (166-172).
It was the aim of this study to develop a simple method for the production of thiolated chitosan particles without being ionically crosslinked. In the first step, thiolated chitosan was ionically gelated with tripolyphosphate (TPP) and sulphate in aqueous solution forming submicron particles and microparticles, respectively. In the next step, thiol groups in and on the particles were partially oxidized forming stabilizing inter- and intramolecular disulfide bonds. As the degree of oxidation can be controlled during the production process, the share of thiol and disulfide groups can be adjusted on demand. Thereafter the polyanions were removed. Utilizing this novel preparation method stable particles of a mean size of 366 ± 30 nm and a zeta potential of around + 11.3 ± 1.3 mV can be produced using TPP as ionic crosslinker. On average 83% of all thiol groups were oxidized. In contrast, particles did not remain stable after removing sulphate as temporary auxiliary ionic crosslinker. Neither ionically nor covalently crosslinked particles were degraded by lysozyme under physiological conditions.Utilizing the novel method described here allows a simple production of thiolated chitosan particles without losing the cationic charge of chitosan.
Keywords: Chitosan; Submicron particles; Microparticles; Thiomers; Crosslinking;

A novel technique for the production of nano- and micro-particulate formulations of poorly water-soluble drugs has been developed. This technique involves the use of static mixer elements to provide fast precipitation by continuous turbulent mixing of two liquid flows, an aqueous phase and an organic phase, respectively. The objective of this study was to develop the mixer technique by investigating the influence of the element number on the particle size of the resulting dispersions. Four model active pharmaceutical ingredients (APIs) with a variety of polymers, lipids or surfactants underwent intensive mixing and the final suspensions showed a narrow size distribution. Parameters such as the flow rate and the temperature of the precipitated organic–aqueous phases were also significant in the reduction of particle size. Further development of the mixing technique led to reproducible and stable formulations with minimal excipient amounts. These formulations were spray- or freeze-dried to improve stability.
Keywords: Mixer technique; Poorly water soluble drugs; Precipitation; Microparticles; Nanoparticles;

In order to achieve implants which provide sustained release of gentamicin, microparticles based on a 50/50 Resomer® 503/Resomer® 502H blend were combined with collagen in order to achieve their fixation and to utilize the favorable effect of collagen on wound healing. Ethylene oxide treatment as well as β- and γ-irradiation were tested for sterilization of the collagen/PLGA-microparticle composite. All methods resulted in a decrease of molecular weight and glass transition temperature of polymer raw material and microparticles. In addition, ethylene oxide treatment yielded aggregation of microparticles leading to a substantial increase in the initially liberated gentamicin dose. Furthermore, chemical changes of gentamicin after ethylene oxide sterilization could be identified using NMR spectroscopy. Despite a decrease in the molecular weight and glass transition temperature after irradiation, neither morphological changes of the composites nor changes regarding the gentamicin release profile from β- and γ-sterilized material were observed. Free radicals, which could only be detected in gentamicin drug substance and at marginal level in gentamicin-loaded MPs, disappeared within 4 weeks. Additional microbiological testing verified the microbiological activity of gentamicin liberated from β-sterilized composites. Storage of β-sterilized composite at 4 °C/35% r.h. for 3 months did not influence morphology, molecular weight, glass transition temperature, and release profiles of microparticles and composites. However, at 25 °C/60% r.h. and 40 °C/75% r.h. a marked decrease in molecular weight and glass transition temperature resulted. This effect was due to a higher humidity, water uptake into polymers, and subsequent hydrolysis of polymers and microparticles, which was more pronounced for RG 502H because of its hydrophilicity. Upon storage at 25 °C/60% r.h. and 40 °C/75% r.h. particles collapsed resulting in an increased gentamicin liberation. Thus, all sterilization techniques have their pros and cons, but based on drug release profile and chemical changes of gentamicin irradiation treatment appears to be more suitable for collagen/gentamicin-loaded PLGA microparticle composites.
Keywords: β-Irradiation; Collagen; ESR; Ethylene oxide; γ-Irradiation; Gentamicin; Microparticles; NMR; PLGA; Sterilization;

In this study, we have optimized different formulations of DNA encapsulated into PLGA microspheres by correlating the protocol of preparation and the molecular weight and composition of the polymer, with the main characteristics of these systems in order to design an efficient non-viral gene delivery vector. For that, we prepared poly(d,l-lactic-co-glycolic acid) (PLGA) microparticles with an optimized water–oil–water double emulsion process, by using several types of polymers (RG502, RG503, RG504, RG502H and RG752), and characterized in terms of size, zeta potential, encapsulation efficiency (EE%), morphology, DNA conformation, release kinetics, plasmid integrity and erosion. The size of the particles ranged between 0.7 and 5.7 μm depending on the protocol of formulation and the molecular mass of the polymer used. The microspheres prepared by using in their formulation polymers of high molecular weight (RG503 and RG504) were bigger in size than in the case of using a lower molecular weight polymer (RG502). The EE (%) of plasmid DNA increased with increasing the molecular mass of the polymer and by using the most hydrophilic polymer RG502H, which contains terminal acidic groups in its structure. The plasmid could be encapsulated without compromising its structural and functional integrity. Also a protective effect of PLGA on endonuclease digestion is observed. Plasmid DNA release from microspheres composed of low molecular weight or hydrophilic polymers, like RG502H, was faster than from particles containing high molecular weight or hydrophobic polymers. These PLGA microspheres could be an alternative to the viral vectors used in gene therapy, given that may be used to deliver genes and other bioactive molecules, either very rapidly or in a controlled manner.
Keywords: Poly(d,l-lactic-co-glycolic acid) (PLGA); Microparticles; Gene delivery; Biodegradable polymers; Plasmid DNA; Microencapsulation;

Preparation, characterization, and in vivo anti-ulcer evaluation of pantoprazole-loaded microparticles by R.P. Raffin; L.M. Colomé; A.R. Pohlmann; S.S. Guterres (198-204).
Pantoprazole is an important drug in the treatment of acid-related disorders. This work concerns the preparation and characterization of gastro-resistant pantoprazole-loaded microparticles prepared using an O/O emulsification/solvent evaporation technique. The in vivo activity of the pantoprazole-loaded Eudragit® S100 microparticles was carried out in rats. Furthermore, tablets containing the microparticles were also investigated. Microparticles presented spherical and smooth morphologies (SEM) and they remained intact in the inner surface of tablets. DSC and IR analyses showed that pantoprazole was physically and molecularly dispersed in the polymer. In vivo anti-ulcer evaluation showed that the microparticles were able to protect rat stomachs against ulcer formation, while the drug aqueous solution did not present activity. Drug dissolution profiles from tablets demonstrated slower release than untabletted microparticles. Weibull equation was the best model for describing the drug release profiles from microparticles and tablets. As regards the acid protection, tablets showed a satisfactory drug protection in acid medium (61.05 ± 8.09% after 30 min).
Keywords: Microparticles; Pantoprazole; Emulsification/solvent evaporation; Polymer; Gastric resistance; Tablet; Release profile; In vivo ulcer evaluation;

Leuprolide acetate-loaded poly(lactide-co-glycolide) (PLGA RG503H) microparticles prepared by the solvent evaporation method had a tri-phasic drug release pattern over a duration of up to 2 months. An initial release was followed by a slow drug release phase and a final rapid drug release. The objective of this study was to identify parameters, which shift the release profile from the tri-phasic to a more continuous release profile. Varying formulation and processing parameters (e.g., drug loading, volume of the external aqueous phase, using low molecular weight PLGA, different microparticle drying methods) affected the initial release (burst) but did not influence the drug release thereafter. The addition of the hydrophilic polymer polyvinylpyrrolidone (PVP) led to the formation of more porous microparticles. This influenced the initial release but did not change the tri-phasic drug release pattern. The inclusion of medium chain triglycerides (MCT) successfully shifted the tri-phasic pattern to a continuous release profile. MCT accelerated the leuprolide release in the second, slow release phase and reduced it in the final rapid release phase. MCT led to the formation of microparticles with an irregular surface and a highly porous inner structure. Differential scanning calorimetry (DSC) revealed a high encapsulation efficiency of MCT (88–105%) in the microparticles and an unchanged glass transition temperature (T g) of PLGA.
Keywords: Tri-phasic release; Initial release; Leuprolide acetate; Polyvinylpyrrolidone; Medium chain triglyceride;

In vivo induction of mucosal immune responses by intranasal administration of chitosan microspheres containing Bordetella bronchiseptica DNT by Mi Lan Kang; Sang Gyun Kang; Hu-Lin Jiang; Seung Won Shin; Deog Yong Lee; Jeong-Min Ahn; Nabin Rayamahji; In-Kyu Park; Sung Jae Shin; Chong-Su Cho; Han Sang Yoo (215-220).
In vitro immune-stimulating activities of Bordetella bronchiseptica dermonecrotoxin (BBD)-loaded in chitosan microspheres (CMs) were reported with a mouse alveolar macrophage cell line (RAW264.7). Based on the report, in vivo activity of immune-induction was investigated by intranasal administration of the BBD-loaded CMs into mice. BBD was loaded into the CMs prepared by an ionic gelation process with tripolyphosphate. Mice were immunized by direct administration of the BBD-loaded CMs into the nasal cavity. After immunization of the mice, BBD-specific immune responses (IgG and IgA titers) were measured in sera, nasal wash, and saliva by ELISA. BBD-specific IgA titers in the nasal cavity were time- and dose-dependently increased by the administration. Similar phenomena were observed in the analysis of systemic IgA and IgG in sera. However, the antibody in saliva was undetectable by ELISA. These results suggested that direct vaccination via the nasal cavity was effective for targeting nasal-associated lymphoid tissues, and that CMs were an efficient adjuvant in nasal mucosal immunity for atrophic rhinitis vaccine.
Keywords: Bordetella bronchiseptica; DNT; Chitosan microspheres; Mucosal immunity; Nasal administration;

Effect of GDNF-releasing biodegradable microspheres on the function and the survival of intrastriatal fetal ventral mesencephalic cell grafts by Anne Clavreul; Laurence Sindji; Anne Aubert-Pouëssel; Jean-Pierre Benoît; Philippe Menei; Claudia N. Montero-Menei (221-228).
The transplantation of fetal ventral mesencephalic (FVM) cell suspensions into the brain striatal system is an alternative approach for the treatment of Parkinson’s disease (PD). However, one objection to this procedure is the relatively poor survival of implanted cells. Attempts have been made to improve the survival of grafted dopaminergic neurons using glial cell line-derived neurotrophic factor (GDNF). Nevertheless, the clinical application of GDNF is limited, due to the difficulties in administering a protein to the brain tissue and due to the ubiquity of its receptor, thus leading to neurological side effects. A strategy to deliver GDNF in the brain based on the intracerebral implantation of biodegradable poly(d,l-lactic acid-co-glycolic acid) sustained release microspheres has been developed. Such microparticles can be easily implanted by sterotaxy in precise and functional areas of the brain without causing damage to the surrounding tissue. Moreover, the release profile of the GDNF-loaded microspheres showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses. The present study shows that the implantation of GDNF-loaded microspheres at a distance to the site of FVM cells in the 6-hydroxydopamine-lesioned rat model of PD improves dopaminergic graft survival and function. Furthermore, the unloaded and the GDNF-loaded microspheres, when they are mixed with FVM cells, may provide a mechanical support and a 3D environment inducing differentiation and increased function of dopaminergic neurons. Taken together, these results show that GDNF microspheres represent an efficient delivery system for cell transplantation studies.
Keywords: Parkinson’s disease; Cell therapy; GDNF; Transplantation; Microspheres; Poly(lactic acid-co-glycolic acid);

Subcutaneous delivery of insulin loaded poly(fumaric-co-sebacic anhydride) microspheres to type 1 diabetic rats by Stacia Furtado; Danielle Abramson; Liat Simhkay; Daniel Wobbekind; Edith Mathiowitz (229-236).
The method of phase inversion nanoencapsulation (PIN) and microencapsulation was used to produce biodegradable poly(fumaric-co-sebacic anhydride) (p(FASA)) microspheres that contain insulin. Microspheres were characterized by SEM and a laser light scattering technique to determine particle size distribution. Insulin stability was determined by RP and SEC HPLC. Release rate studies were conducted and microspheres were administered subcutaneously (SQ) to type 1 diabetic rats to determine the bioactivity of insulin at three different dosages. Pharmacokinetic parameters for SQ experiments were measured using the trapezoidal rule by plotting average plasma insulin level (PIL) vs. time and determining peak concentration (C P), the time of peak concentration (T P), duration of PIL curve (D), and relative bioavailability (RB). When our insulin containing formulation was analyzed by HPLC, there was no evidence of high molecular weight transformation (HMWT) or deamidated products. In addition, we effectively altered the onset, peak, and duration of insulin action after SQ injection.
Keywords: Subcutaneous delivery; Insulin; Microspheres; Poly(fumaric-co-sebacic anhydride); Diabetic rat;

by Thomas Kissel (237).

by Richard Süverkrüp (238).

by Geoffrey Lee (238-239).