Trophically and topographically functionalized silk fibroin nerve conduits for guided peripheral nerve regeneration.

Artificial nerve conduits (NC) can be used as an alternative to autologous nerve grafts to enhance the repair of small nerve gaps. Current NC lack adequate molecular and structural functionalities. Thus, we developed silk fibroin NC (SF NC) that were loaded with glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) and topographically functionalized with aligned and non-aligned SF nanofibers. The SF NC were produced from fully functionalized SF membranes on which initial experiments were performed. DRG (dorsal root ganglions) sensory neurons and spinal cord (SpC) motor neurons, both from chicken embryos, exhibited an augmented length and rate of axonal outgrowth parallel to the aligned nanofibers. In addition, glial cells from DRG proliferated and migrated in close association and even slightly ahead of the outgrowing axons. On the contrary, axonal and glial growth was slower and randomly oriented on non-aligned nanofibers. The DRG and SpC explants were also inserted into the lumen of the finished SF NC. The unidirectional orientation of axo-glial outgrowth from the explants evidenced the preservation of the trophic and topographical functionalities in the SF NC. Bioactive GDNF and NGF were released in vitro from SF NC over 4 weeks. Thus, the developed functionalized SF NC hold promise to enhance functional recovery of injured peripheral nerves.

Ultrasonic atomization and subsequent polymer desolvation for peptide and protein microencapsulation into biodegradable polyesters.

Peptide and protein microencapsulation into poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) microspheres continues to represent a technological challenge in terms of product sterility and up-scaling. The primary objective of this study was to examine the feasibility of a novel method for peptide and protein entrapment into PLA and PLGA microspheres, particularly suitable for up-scaling and aseptic processing. The method involves ultrasonic atomization of an organic polymer solution combined with subsequent organic solvent extraction by a hardening agent. The study evaluated the critical atomization conditions, the required molecular cohesion parameters of polymer solvents and hardening agent for particle preparation as well as the quality of entrapment and release as a function of polymer and peptide/protein type. Suitable polymer solvents and hardening agents were restricted to defined domains of fractional cohesion parameters: f(p) = 0.2-0.35 and f(h) = 0.2-0.4 for the polymer solvents, and f(p) = 0-0.1 and f(h) = 0-0.25 for the hardening agents. Microsphere size (0.1-100 micro m) was largely controlled by the viscosity of the atomized solution. Microencapsulation of the freely water-soluble bovine serum albumin and tetrapeptide thymocartin yielded modest efficiencies of 12-35%, whereas the slightly water-soluble octapeptide vapreotide pamoate was entrapped with 63-93% efficiency. Drug release was mainly governed by the polymer type, lasting over 100 days for BSA entrapped in PLA microspheres and; 20 days for vapreotide pamoate in PLGA 50 : 50 and for thymocartin in PLA. Very importantly, the novel method was readily accommodated within a laminar air-flow cabinet. Under aseptic conditions, sterile microspheres could be prepared. In conclusion, the novel method described may have potential in industrial environments.

Solvent extraction employing a static micromixer: a simple, robust and versatile technology for the microencapsulation of proteins.

The potential of a static micromixer for the production of protein-loaded biodegradable polymeric microspheres by a modified solvent extraction process was examined. The mixer consists of an array of microchannels and features a simple set-up, consumes only very small space, lacks moving parts and offers simple control of the microsphere size. Scale-up from lab bench to industrial production is easily feasible through parallel installation of a sufficient number of micromixers ('number-up'). Poly(lactic-co-glycolic acid) microspheres loaded with a model protein, bovine serum albumin (BSA), were prepared. The influence of various process and formulation parameters on the characteristics of the microspheres was examined with special focus on particle size distribution. Microspheres with monomodal size distributions having mean diameters of 5-30 micro m were produced with excellent reproducibility. Particle size distributions were largely unaffected by polymer solution concentration, polymer type and nominal BSA load, but depended on the polymer solvent. Moreover, particle mean diameters could be varied in a considerable range by modulating the flow rates of the mixed fluids. BSA encapsulation efficiencies were mostly in the region of 75-85% and product yields ranged from 90-100%. Because of its simple set-up and its suitability for continuous production, static micromixing is suggested for the automated and aseptic production of protein-loaded microspheres.

Induction of a cytotoxic T-cell response to HIV-1 proteins with short synthetic peptides and human compatible adjuvants.

The goal of this study was the induction of a strong CTL response against multiple CTL epitopes present in HIV proteins using short synthetic peptides. Four HLA-A2.1 restricted peptides (RT 476-484, p17 77-85, gp41 814-823, RT 956-964) that showed stable binding to the HLA-A2.1 molecule in an in vitro binding assay were able to elicit a strong specific immune response in HLA-A2.1 transgenic mice when injected with IFA or Montanide. The use of biodegradable microspheres (MS) as adjuvant was also successfully tested for all peptides. When the peptides were injected as a mixture the response was weaker as compared to individual injections of the peptides indicating the occurrence of immunodominance (ID). We are currently investigating whether ID can be overcome by a combined injection of peptide loaded MS with different release patterns. Taken together, it seems feasible to induce a specific CTL response in HLA-A2.1 transgenic mice against several HIV proteins using short synthetic peptides and human compatible adjuvants.

Release kinetics and immunogenicity of parvovirus microencapsulated in PLA/PLGA microspheres.

The aim of this work was to examine the immunogenicity of microencapsulated inactivated duck parvovirus in Muscovy duck (Cairina moschata) and goose. Inactivated duck parvovirus suspension was microencapsulated into 14-17 kDa poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA50:50H) by coacervation. The in vitro antigen release from individual and mixed PLA and PLGA50:50H microspheres (MS) was biphasic with an initial lag-phase of approx. 10 days followed by a relatively constant release over additional 12 days. By varying the composition of PLA+PLGA50:50H MS mixtures from 3+1 to 1+3, the release kinetics could be altered and controlled efficiently. The antigen-loaded MS were injected subcutaneously into ducks. The immune response, expressed as virus neutralisation (VN) titres, after single administration of MS was modest, i.e. below 200 over the 6 weeks tested, unless the animals were pre-immunised 3 weeks before injecting the MS. The weak immune response was attributed to the low dose injected and inappropriate antigen release kinetics. With pre-immunised animals, however, the results were encouraging and showed that the encapsulated parvovirus was immunogenic.

Stabilizing insulin-like growth factor-I in poly(D,L-lactide-co-glycolide) microspheres.

This study aimed at developing a controlled drug delivery system for recombinant human insulin-like growth factor-I (IGF-I) for localized delivery in bone healing. IGF-I was microencapsulated into an end-group uncapped 14 kDa poly(D,L-lactide-co-glycolide) 50:50 (PLGA 50:50) by solvent extraction from a W(1)/O/W(2) dispersion. Prior to encapsulation, IGF-I was exposed to ultrasonication in a water/dichloromethane dispersion, and its stability tested in the presence and absence of various excipients in the W(1) phase. HPLC and RIA were used for the assessment of IGF-I stability. Microencapsulated IGF-I was tested again for its structural intactness and also for in vitro release from various formulations containing appropriate co-encapsulated excipients. A specific fat cell assay was used to determine the biological activity of released IGF-I. Moderate ultrasonic treatment of aqueous IGF-I/dichloromethane mixtures caused approx. 50% IGF-I degradation. However, IGF-I was fully protected when bovine serum albumin, succinylated gelatin or poly(ethyleneglycol) were added to the aqueous IGF-I. Co-encapsulation of these excipients protected efficiently the protein upon microencapsulation. IGF-I release from microsphere formulations was sustained for up to 13 days featuring a moderately pulsatile pattern, depending on the microsphere composition. Typically, the amounts of IGF-I released within the first 24 h (burst) and during the second release pulse were in the order of 20 and 40%, respectively, of the total dose. The biological activity of released IGF-I was confirmed at selected time-points by the fat cell assay. In conclusion, the developed microspheres proved to be suitable to release biologically intact IGF-I over up to 13 days, a time-period considered to be relevant to promote bone fracture healing.

Microencapsulated enterotoxigenic Escherichia coli and detached fimbriae for peroral vaccination of pigs.

The feasibility of peroral immunisation with microencapsulated Escherichia coli and detached fimbriae to prevent enterotoxigenic E. coli infections in pigs was examined. For this E. coli and fimbriae were microencapsulated into poly(lactide-co-glycolide) microspheres by spray-drying. Various microsphere formulations designed to deliver priming and booster doses were fed to new-born and weaned pigs. The pigs were challenged 19 days after the booster dose by peroral administration of an infective dose of the homologous E. coli. Serum IgA antibody titres and excretion of challenge E. coli, as indicators for colonisation, were determined. The data showed that no significant serum antibodies were induced, and E. coli colonisation was not reduced by the peroral administration of the various antigen-loaded microspheres. These results are in contradiction to some of the previously published experiments typically in rats or rabbits, where model antigens or unpractical immunisation procedures have frequently been used.

Technological considerations related to the up-scaling of protein microencapsulation by spray-drying.

Research and development of therapeutics and vaccines based on biodegradable polymers are intensive and one of the most promising fields in controlled drug delivery. However, new applications necessitate successful technology transfer and industrial scale-ups. In an endeavour to produce clinical samples of a single-administration tetanus vaccine based on poly(lactide-co-glycolide) microspheres, we report on technological parameters that are of importance in the up-scaling of the spray-drying process. The results show that an up-scaling of the encapsulation of protein vaccines or drug by spray-drying is feasible, but that additives, the type of polymer solvent, the polymer concentration, the w/o ratio and the product collection method influence process and product quality.

Control of gentamicin release from a calcium phosphate cement by admixed poly(acrylic acid).

The aim of this work was to develop a calcium phosphate cement (CPC) providing controlled release of the antibiotic gentamicin sulfate (GS) over at least 1 week. The CPC was made of beta-tricalcium phosphate [beta-TCP; beta-Ca(3)(PO(4))(2)], monocalcium phosphate monohydrate [MCPM; Ca(H(2)PO(4))(2). H(2)O] and water. Release of GS was controlled by admixture of poly(acrylic acid) (PAA). The effects on the GS release kinetics of the molecular weight of PAA, of the amount of admixed PAA, and of the pH of the release medium were investigated. A typical cement sample weighed 3.6 g and contained 100 mg of GS and between 0 and 150 mg of PAA. In the following, PAA content is expressed as the weight ratio, lambda, with respect to GS. At a low PAA content in the CPC (lambda < 0.7), GS was released over 1-2 days according to a square-root-of-time kinetics, but not all GS was released. The unreleased GS fraction increased from 0 to 58% with an increase of PAA content (up to lambda = 0.7). At high PAA content (lambda > 0.7), GS was released over a period of up to 8 days according to a combination of a square-root-of-time and a zero-order kinetics. The total GS fraction released increased again from 58 to 100% with an increase of the amount of PAA (up to lambda = 1.5). These observations were explained by molecular interaction between PAA and GS resulting in gel formation. The maximum fraction of GS released from the cement was indeed a function of the solubility of the PAA-GS (coacervate) complex in the release medium. Thus, GS release was controlled by two mechanisms: (1) diffusion of free GS molecules through the porous cement (square-root-of-time kinetics); and (2) dissociation of GS from the PAA-GS complex (zero-order kinetics). The first mechanism was predominant at low lambda, whereas the second mechanism became important at high lambda and later release times. As the solubility of the PAA-GS complex decreased with an increase in PAA molecular weight, the higher molecular weight PAA yielded more prolonged release periods of up to 8 days. Interestingly, the use of 450 kDa PAA at lambda = 1.00 provided an almost constant release profile over a period of 7 days. Gel formation between PAA and GS was explained in terms of hydrogen bonding of PAA carboxyl groups with GS amino groups. The molar ratio between carboxyl groups and amino groups in the gel was estimated to be approximately 1.9. In conclusion, admixture of PAA into calcium phosphate cement appeared to be a very elegant tool to control the release of the antibiotic over a period of 7 to 8 days.

Degradation mechanism and stability of 5-aminolevulinic acid.

The physiological substance and precursor of the heme synthesis 5-aminolevulinic acid (ALA) is a promising prodrug for photodiagnosis and photodynamic therapy of epithelial tumors, particularly in urological and gynecological tissues. For the clinical use of this substance, a chemically stable and sterile drug formulation is required. In the present study, degradation mechanism of ALA in aqueous solution and possibilities to improve its stability were examined. A capillary electrophoretic method was developed that was suitable for the quantification of ALA and of two degradation products. The intermediate degradation product was 2, 5-dicarboxyethyl-3,6-dihydropyrazine, which was further oxidized to 2,5-dicarboxyethylpyrazine. The structures of the degradation products were proven by (1)H and (13)C nuclear magnetic resonance spectroscopy. ALA degradation was very efficiently inhibited by adjusting the pH of the aqueous solution to a value <5 and by purging with nitrogen. Additives such as antioxidants did not improve the ALA stability. These results demonstrated that low pH ALA aqueous solution may be one possible dosage form to be considered for market introduction.

Validation of a capillary electrophoresis method for determination of 5-aminolevulinic acid and degradation products.

A capillary electrophoresis method was developed for simultaneous quantification of 5-aminolevulinic acid (ALA) and its degradation products 2,5-dicarboxyethyl-3,6-dihydropyrazine and 2,5-dicarboxyethylpyrazine in aqueous solution within a total analysis time of 9 min. The optimized method was validated with respect to specificity, precision, linearity, limits of detection and quantitation, and robustness. The degradation products were quantified with respect to the ALA peak. A related micellar electrokinetic chromatography method, involving the addition of sodium dodecylsulfate to the running buffer solution, was applied for direct injection of an oil-in-water emulsion containing ALA, i.e. without sample pretreatment.

Revisiting PLA/PLGA microspheres: an analysis of their potential in parenteral vaccination.

Poly(lactide) and poly(lactide-co-glycolide) microspheres have been studied for controlled antigen delivery and immune response enhancement for more than a decade. Early developments of such vaccines were basically technology-driven, stemming from the well-established biocompatibility of these polymers in concert with their innate properties to tailor rates of bioerosion and release. More recently, other features have become equally or even more appealing, such as the adjuvancy of such microspheres and their ability to elicit cellular effector responses, so-called cytotoxic T-cell responses, in addition to antibody responses observed already in the very early studies. In this review, we intended to revisit microsphere-based vaccines designed for the parenteral route and attempted to outline major developmental issues, as well as to analyze immunological fundamentals and data associated with antigen delivery by microspheres.

In vitro and in vivo evaluation of a somatostatin analogue released from PLGA microspheres.

The purpose of this study was to design poly(lactide-co-glycolide) (PLGA) microspheres for the continuous delivery of the somatostatin analogue, vapreotide, over 2-4 weeks. The microspheres were produced by spray-drying and the desired characteristics, i.e. high encapsulation efficiency and controlled release over 2-4 weeks, achieved through optimizing the type of polymer, processing solvent, and co-encapsulated additive. The in vitro release was tested in fetal bovine serum preserved with 0.02% of thiomersal. Furthermore, formulations were injected intramuscularly into rats to obtain pharmacokinetic profiles. Encapsulation efficiency was between 34 and 91%, depending on the particular formulation. The initial peptide release (within 6 h) was lowest, i.e. <20%, when acetic acid was used as processing solvent and highest, i.e. 57%, with dichloromethane. The various co-encapsulated additives generally lowered the encapsulation efficiency by 15-30%. The best formulation in terms of low burst and effective drug serum levels (>1 ng/ml) over 21-28 days in rats was the one made with end-group uncapped PLGA 50:50, the solvent acetic acid and the additive polyethyleneglycol. In conclusion, the optimization of formulation parameters allowed us to produce vapreotide-loaded PLGA microspheres of suitable characteristics for therapeutic use.

Ambiguities in the preclinical quality assessment of microparticulate vaccines.

Vaccination techniques do not always stimulate immunity because of the inappropriate mobilization of immune responses, and the frequency of vaccinations required is impractical in many developing countries. Such limitations have spurred the development of new vaccine-delivery approaches. Microparticles made of poly(lactide-co-glycolide) can induce adaptive immunity after a single administration of a vaccine. However, the preclinical assessment of such vaccines is not standardized, making it difficult to compare pharmaceutical with immunological data. The relevance of and the ambiguity in the assessment of microparticulate vaccines with respect to the current knowledge on immunity are discussed, in addition to the application of this knowledge to rational vaccine design.

Gentamicin encapsulation in PLA/PLGA microspheres in view of treating Brucella infections.

In view of treating intracellular Brucella infections, microspheres made of poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) were developed as delivery system for the cationic and highly hydrophilic antibiotic gentamicin sulphate. Drug microencapsulation by spray drying yielded microspheres with regular morphology, an average particle size of approximately 3 micrometer and encapsulation efficiencies of up to 45%. Different copolymers of similar molecular weights gave varying encapsulation efficiencies and particle size distributions. The encapsulation efficiency generally increased with polymer hydrophilicity, except for the hydrophilic copolymer PLGA50:50H carrying carboxylic end groups. Encapsulation also depended on the pH value of the aqueous drug solution to be encapsulated. Moreover, increasing nominal gentamicin sulphate loading yielded lower efficiencies. For comparison, some formulations were also prepared by a (W(1)/O)W(2)-solvent evaporation method, which yielded lower encapsulation efficiencies, in the order of 13%. Finally, drug bioactivity was found to remain intact after microencapsulation, MS storage and MS incubation in aqueous medium. The results suggest that PLA/PLGA microspheres prepared by spray drying may be an appropriate delivery system for gentamicin sulphate to be used in the treatment of intracellular Brucella infections.

Towards clinical testing of a single-administration tetanus vaccine based on PLA/PLGA microspheres.

The availability of single-administration vaccines would assist in the control of global mortality caused by infectious diseases where protection can be achieved only upon repeated immunisations with appropriate vaccines. Biodegradable microspheres of poly(lactide-co-glycolide) have been studied pre-clinically for this purpose and shown to be promising for several protein and sub-unit antigens. In view of preparing a microsphere-based tetanus vaccine for clinical trials, final candidate vaccine-formulations were pre-clinically optimised here. Specifically, the importance of particular materials and processing for the induction of neutralising antibodies in guinea pigs were examined. The most efficacious vaccines were small-sized (<5 microm), co-adjuvanted with admixed alum and fabricated from fast-degrading polymers. Interestingly, the immunogenicity was less influenced by the type of antigen-stabilising excipient, the number of microsphere populations mixed together, or the microencapsulation technology, i.e. spray-drying versus coacervation, used. On the basis of these, we plan to prepare clinical samples for safety and immunogenicity testing in man.

Immunogenicity of single-dose diphtheria vaccines based on PLA/PLGA microspheres in guinea pigs.

Biodegradable polyester microspheres (MS) have shown potential for single-dose vaccines. This study examined the immunogenicity of diphtheria toxoid (Dtxd) microencapsulated in different types of poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) MS prepared by the methods of spray-drying and coacervation. We investigated the influence of polymer type (PLGA 50:50 of low M(w); PLA of high M(w); end-group stearylated PLAs of low M(w)) and co-encapsulated excipients (BSA and/or trehalose) on Dtxd content, in vitro release and immunogenicity in guinea pigs. The co-encapsulated trehalose lowered the Dtxd entrapment efficiency in the spray-dried particles from 75 to 56%, whereas albumin alone had no effect in the spray-drying, but improved the encapsulation in the coacervation process. With the hydrophobic, end-group stearylated PLAs, Dtxd could only be encapsulated in the presence of albumin. Guinea pigs immunised with Dtxd-MS made with the relatively hydrophilic PLGA 50:50 exhibited specific and sustained antibody responses over 40 weeks, comparable to the responses to alum-adjuvanted toxoid. In contrast, undetectable or very low antibody responses were determined after immunisation with MS made with hydrophobic polymers. Surprisingly, large (15-60 microm) and small (1-5 microm) MS gave comparable primary antibody responses. In conclusion, the data presented confirm the feasibility of MS vaccines to induce strong, long-lasting protective antibody responses after a single immunisation.

Importance of the test medium for the release kinetics of a somatostatin analogue from poly(D,L-lactide-co-glycolide) microspheres.

The determination of in vitro release kinetics of peptides from poly(d,l-lactide-co-glycolide) (PLGA) microspheres generally requires optimization of the test conditions for a given formulation. This is particularly important when in vitro/in vivo correlation should be determined. Here, the somatostatin analogue vapreotide pamoate, an octapeptide, was microencapsulated into PLGA 50:50 by spray-drying. The solubility of this peptide and its in vitro release kinetics from the microspheres were studied in various test media. The solubility of vapreotide pamoate was approximately 20-40 microg/ml in 67 mM phosphate buffer saline (PBS) at pH 7.4, but increased to approximately 500-1000 microg/ml at a pH of 3.5. At low pH, the solubility increased with the buffer concentration (1-66 mM). Very importantly, proteins (aqueous bovine serum albumin (BSA) solution or human serum) appeared to solubilize the peptide pamoate, resulting in solubilities ranging from 900 to 6100 microg/ml. The release rate was also greatly affected by the medium composition. Typically, in PBS of pH 7.4, only 33+/-1% of the peptide were released within 4 days, whereas 53+/-2 and 61+/-0.9% were released in 1% BSA solution and serum, respectively. The type of medium was found critical for the estimation of the in vivo release. The in vivo release kinetics of vapreotide pamoate from PLGA microspheres following administration to rats were qualitatively in good agreement with those obtained in vitro using serum as release medium. Finally, sterilization by gamma-irradiation had only a minor effect on the in vivo pharmacokinetics.

Sustained release of heparin from polymeric particles for inhibition of human vascular smooth muscle cell proliferation.

Vascular smooth muscle cell (SMC) growth plays an important role in atherosclerosis, restenosis and venous bypass graft disease. With systemic drug administration no effective therapy for restenosis and venous bypass graft disease is available. This could be due to low local concentrations of the drugs at the target site. A directed delivery of drugs to tissues with a sustained release system during percutaneous transluminal coronary angioplasty (PTCA) or during bypass surgery could provide high concentrations of drugs at the target site and avoid systemic side effects. In the present study heparin was encapsulated by spray-drying into biodegradable poly(D, L-lactic-co-glycolic acid) (PLGA) to obtain a system for prolonged drug release. SMC were cultured from saphenous vein explants obtained from patients undergoing coronary bypass surgery. Cell proliferation was measured by [(3)H]thymidine incorporation. Heparin release from PLGA 50:50 microspheres in an isoosmolar PBS buffer (pH=7.4) showed a triphasic profile with an initial burst (completed after 24 h), a dormant period and a final stage with increased release rate, which lasted about 10-14 days. Cell proliferation as measured by [(3)H]thymidine incorporation was markedly stimulated by platelet-derived growth factor-BB (PDGF-BB) (5 ng/ml) or serum (5%). Proliferation of SMC was equally reduced (50%; P<0.05; n=9-11) by native heparin or heparin released from PLGA microspheres, while PLGA microspheres without heparin loading had no effect on [(3)H]thymidine incorporation in human SMC. Similar results were also obtained when SMC were stimulated with 5% serum instead of PDGF-BB (50%; P<0.05; n=6). Thus, heparin encapsulated into PLGA microspheres was released over a prolonged period of time and thereby effectively reduced human SMC proliferation stimulated either with PDGF or serum. Biodegradable PLGA microspheres may also be used to encapsulate other antiproliferative agents and provide a new approach for local drug delivery after PTCA. This may help to prevent restenosis after PTCA or to reduce graft disease after coronary bypass graft surgery.