BMP-silk composite matrices heal critically sized femoral defects.

Clinical drawbacks of bone grafting prompt the search for alternative bone augmentation technologies such as use of growth and differentiation factors, gene therapy, and cell therapy. Osteopromotive matrices are frequently employed for the local delivery and controlled release of these augmentation agents. Some matrices also provide an osteoconductive scaffold to support new bone growth. In this study, silkworm-derived silk fibroin was evaluated as an osteoconductive matrix for healing critical sized mid-femoral segmental defects in nude rats. Four treatment groups were assessed over eight weeks: silk scaffolds (SS) with recombinant human BMP-2 (rhBMP-2) and human mesenchymal stem cells (HMSC) that had been pre-differentiated along an osteoblastic lineage ex vivo (Group I; pdHMSC/rhBMP-2/SS); SS with rhBMP-2 and undifferentiated HMSCs (Group II; udHMSC/rhBMP-2/SS); SS and rhBMP-2 alone (Group III; rhBMP-2/SS); and empty defects (Group IV). Bi-weekly radiographs revealed a progressive and similar increase in Group I-III mean defect mineralization through post-operative week (POW) 8. Radiographs, dual energy x-ray absorptiometry, and micro-computed tomography confirmed that Groups I-III exhibited similar substantial and significantly (p<0.05) greater defect mineralization at POW 8 than the unfilled Group IV defects which remained void of bone. No significant differences in Groups I-III defect healing at POW 8 were apparent using these same assays or mechanical testing. Histology at POW 8 revealed moderately good bridging of the parent diaphyseal cortices with woven and lamellar bone bridging islands of silk matrix in Groups I and III. Group II defects possessed comparatively less new bone which was most abundant adjacent to the parent bone margins. Elsewhere the silk matrix was more often enveloped by poorly differentiated loose fibrous connective tissue. Group IV defects showed minimal new bone formation. None of the treatment groups attained the mean mineralization or the mean biomechanical strength of identical defects implanted with SS and pdHMSCs alone in a previous study. However, addition of rhBMP-2 to SS prompted more bone than was previously generated using udHMSC/SS or SS alone. These data imply the clinical potential of silk scaffolds and rhBMP-2 as composite osteopromotive implants when used alone or with select stem cell populations. Additional studies in larger species are now warranted.

Silk based biomaterials to heal critical sized femur defects.

Bone auto- and allografts have inherent drawbacks, therefore the treatment of non-unions and critical size defects in load bearing long bones would benefit from the use of osteopromotive biodegradable, biocompatible and mechanically durable matrices to enhance migration or delivery of cell populations and/or morphogens/cytokines. Silk fibroin biomaterial scaffolds were evaluated as osteopromotive matrices in critical sized mid-femoral segmental defects in nude rats. Four treatment groups were assessed over 8 weeks in vivo: silk scaffolds (SS) with human mesenchymal stem cells (hMSCs) that had previously been differentiated along an osteoblastic lineage in vitro (group I; pdHMSC/SS); SS with undifferentiated hMSCs (group II; udHMSC/SS); SS alone (group III; SS); and empty defects (group IV). When hMSCs were cultured in vitro in osteogenic medium for 5 weeks, bone formation was characterized with bimodal peak activities for alkaline phosphatase at 2 and 4 weeks. Calcium deposition started after 1 week and progressively increased to peak at 4 weeks, reaching cumulative levels of deposited calcium at 16 mug per mg scaffold wet weight. In vivo osteogenesis was characterized by almost bridged defects with newly formed bone after 8 weeks in group I. Significantly (P < 0.01) greater bone volumes were observed with the pdHMSC/SS (group I) implants than with groups II, III or IV. These three groups failed to induce substantial new bone formation and resulted in the ingrowth of cells with fibroblast-like morphology into the defect zone. The implantation of pdHMSC/SS resulted in significantly (P < 0.05) greater maximal load and torque when compared to the other treatment regimens. The pdHMSC/SS implants demonstrated osteogenic ability in vitro and capacity to thrive towards the healing of critical size femoral segmental defects in vivo. Thus, these new constructs provide an alternative protein-based biomaterial for load bearing applications.

Silk fibroin as an organic polymer for controlled drug delivery.

The pharmaceutical utility of silk fibroin (SF) materials for drug delivery was investigated. SF films were prepared from aqueous solutions of the fibroin protein polymer and crystallinity was induced and controlled by methanol treatment. Dextrans of different molecular weights, as well as proteins, were physically entrapped into the drug delivery device during processing into films. Drug release kinetics were evaluated as a function of dextran molecular weight, and film crystallinity. Treatment with methanol resulted in an increase in beta-sheet structure, an increase in crystallinity and an increase in film surface hydrophobicity determined by FTIR, X-ray and contact angle techniques, respectively. The increase in crystallinity resulted in the sustained release of dextrans of molecular weights ranging from 4 to 40 kDa, whereas for less crystalline films sustained release was confined to the 40 kDa dextran. Protein release from the films was studied with horseradish peroxidase (HRP) and lysozyme (Lys) as model compounds. Enzyme release from the less crystalline films resulted in a biphasic release pattern, characterized by an initial release within the first 36 h, followed by a lag phase and continuous release between days 3 and 11. No initial burst was observed for films with higher crystallinity and subsequent release patterns followed linear kinetics for HRP, or no substantial release for Lys. In conclusion, SF is an interesting polymer for drug delivery of polysaccharides and bioactive proteins due to the controllable level of crystallinity and the ability to process the biomaterial in biocompatible fashion under ambient conditions to avoid damage to labile compounds to be delivered.

Effect of scaffold design on bone morphology in vitro.

Silk fibroin is an important polymer for scaffold designs, forming biocompatible and mechanically robust biomaterials for bone, cartilage, and ligament tissue engineering. In the present work, 3D biomaterial matrices were fabricated from silk fibroin with controlled pore diameter and pore interconnectivity, and utilized to engineer bone starting from human mesenchymal stem cells (hMSC). Osteogenic differentiation of hMSC seeded on these scaffolds resulted in extensive mineralization, alkaline phosphatase activity, and the formation of interconnected trabecular- or cortical-like mineralized networks as a function of the scaffold design utilized; allowing mineralized features of the tissue engineered bone to be dictated by the scaffold features used initially in the cell culture process. This approach to scaffold predictors of tissue structure expands the window of applications for silk fibroin-based biomaterials into the realm of directing the formation of complex tissue architecture. As a result of slow degradation inherent to silk fibroin, scaffolds preserved their initial morphology and provided a stable template during the mineralization phase of stem cells progressing through osteogenic differentiation and new extracellular matrix formation. The slow degradation feature also facilitated transport throughout the 3D scaffolds to foster improved homogeneity of new tissue, avoiding regions with decreased cellular density. The ability to direct bone morphology via scaffold design suggests new options in the use of biodegradable scaffolds to control in vitro engineered bone tissue outcomes.

Biophysical and biological studies of end-group-modified derivatives of Pep-1.

Pep-1 is a tryptophane-rich cell-penetrating peptide (CPP) that has been previously proposed to bind protein cargoes by hydrophobic assembly and translocate them across cellular membranes. To date, however, the molecular mechanisms responsible for cargo binding and translocation have not been clearly identified. This study was conducted to gain insight into the interaction between Pep-1 with its cargo and the biological membrane to identify the thereby involved structural elements crucial for translocation. We studied three peptides differing in their N- and C-termini: (i) Pep-1, carrying an acetylated N-terminus and a C-terminal cysteamine elongation, (ii) AcPepWAmide, with an acetylated N-terminus and an amidated C-terminus, and (iii) PepW, with two free termini. Thioredoxin (TRX) and beta-galactosidase were used as protein cargoes. To study CPP-membrane interactions, we performed biophysical as well as biological assays. To mimic biological membranes, we used phospholipid liposomes in a dye leakage assay and surfactant micelles for high-resolution NMR studies. In addition, membrane integrity, cell viability, and translocation efficiency were analyzed in HeLa cells. An alpha-helical structure was found for all peptides in the hydrophobic N-terminal region encompassing residues 4-13, whereas the hydrophilic region remained unstructured in the presence of micelles. Our results show that the investigated peptides interacted with the micelles as well as with the protein cargo via their tryptophan-rich domain. All peptides displayed an orientation parallel to the micelle surface. The C-terminal cysteamine group formed an additional membrane anchor, leading to more efficient translocation properties in cells. No membrane permeabilization was observed, and our data were largely compatible with an endocytic pathway for cellular uptake.

Structure of peptide solutions: a light scattering and numerical study.

We investigated the interactions between protein molecules in solution, in particular for low salt concentrations and thus strong electrostatic interactions where a treatment based on the second virial coefficient is not sufficient. Static and dynamic light scattering experiments on solutions containing the peptide human calcitonin (hCT) were combined with calculations based on the Ornstein-Zernike equation with the hypernetted chain (HNC) closure and computer simulations within the primitive electrolyte model. The simulation illustrates the distribution of proteins in solution and the formation of (transient) protein aggregates. It furthermore allows us to predict the physical stability of hCT solutions in dependence of ionic strength, pH and hCT concentration.

Cationic stearylamine-containing biodegradable microparticles for DNA delivery.

Technical aspects for preparing a new type of cationic stearylamine (SA)-containing microparticle as a potential drug delivery system for negatively charged therapeutics were investigated. Cationic biodegradable microparticles based on poly(lactide) and poly(lactide-co-glycolide) were prepared upon incorporation of SA either by solvent evaporation or by spray-drying. Water-insoluble SA offers the advantage over other water-soluble cationic compounds that it can be dissolved directly in the organic solution together with the polymers. This facilitated the subsequent preparation of the microparticle formulations. Particle size was controlled by the respective process parameters, resulting in either large polymer aggregates within the range 50-100 micro m or small spherical microparticles within the range 1-10 micro m. The incorporation of SA into the formulations also improved particle characteristics in terms of re-dispersibility, reduced sticking, and particle size uniformity. Both circular plasmid DNA (5 kbp) and linear salmon DNA (0.5 kbp) were efficiently adsorbed to the cationic SA microparticle surfaces. Preliminary tests on the release of DNA from spray-dried SA microparticles showed an immediate burst release, which was followed by a delayed second release phase for more than 4 weeks. The cationic SA microparticles might provide a potential drug-delivery system to improve the efficacy for protein and DNA-type therapeutics.

Uptake and metabolism of a dual fluorochrome Tat-nanoparticle in HeLa cells.

The ability to use magnetic nanoparticles for cell tracking, or for the delivery of nanoparticle-based therapeutic agents, requires a detailed understanding of probe metabolism and transport. Here we report on the development and metabolism of a dual fluorochrome version of our tat-CLIO nanoparticle termed Tat(FITC)-Cy3.5-CLIO. The nanoparticle features an FITC label on the tat peptide and a Cy3.5 dye directly attached to the cross-linked coating of dextran. This nanoparticle was rapidly internalized by HeLa cells, labeling 100% of cells in 45 min, with the amount of label per cell increasing linearly with time up to 3 h. Cells loaded with nanoparticles for 1 h retained 40-60% of their FITC and Cy3.5 labels over a period of 72 h in label-free media. Over a period of 144 h, or approximately 3.5 cell divisions, the T2 spin-spin relaxation time of cells was not significantly changed, indicating retention of the iron oxide among the dividing cell population. Using confocal microscopy and unfixed cells, both dyes were nuclear and perinuclear (broadly cytoplasmic) after Tat(FITC)-Cy3.5-CLIO labeling. Implications of the rapid labeling and slow excretion of the Tat(FITC)-Cy3.5-CLIO nanoparticle are discussed for cell tracking and drug delivery applications.

Presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters.

Psoriasis is a chronic inflammatory skin disease. Its treatment is based on the inhibition of proliferation of epidermal cells and interference in the inflammatory process. A new systemic antipsoriasis drug, which consists of dimethylfumarate and ethylhydrogenfumarate in the form of their calcium, magnesium and zinc salts has been introduced in Europe with successful results. In the present study, a homologous series of mono- and diesters of fumaric acid has been studied with respect to the sites and kinetics of presystemic ester degradation using pancreas extract, intestinal perfusate, intestinal homogenate and liver S9 fraction. In addition, intestinal permeability has been determined using isolated intestinal mucosa as well as Caco-2 cell monolayers, in order to obtain estimates of the fraction of the dose absorbed for these compounds. Relationships between the physicochemical properties of the fumaric acid esters and their biological responses were investigated. The uncharged diester dimethylfumarate displayed a high presystemic metabolic lability in all metabolism models. It also showed the highest permeability in the Caco-2 cell model. However, in permeation experiments with intestinal mucosa in Ussing-type chambers, no undegraded DMF was found on the receiver side, indicating complete metabolism in the intestinal tissue. The intestinal permeability of the monoesters methyl hydrogen fumarate, ethyl hydrogen fumarate, n-propylhydrogen fumarate and n-pentyl hydrogen fumarate increased with an increase in their lipophilicity, however, their presystemic metabolism rates likewise increased with increasing ester chain length. It is concluded that for fumarates, an increase in intestinal permeability of the more lipophilic derivatives is counterbalanced by an increase in first-pass extraction.

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.

Surface modification of PLGA microspheres.

Microspheres made of poly(lactic-co-glycolic acid) (PLGA) are biocompatible and biodegradable, rendering them a promising tool in the context of drug delivery. However, nonspecific adsorption of plasma proteins on PLGA micro- and nanospheres is a main limitation of drug targeting. Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), physisorbed on flat metal oxide surfaces, has previously been shown to suppress protein adsorption drastically. The goal of our work was to characterize the efficiency of the protein repellent character of PLL-g-PEG on PLGA microspheres and to show the feasibility of introducing functional groups on the PLGA microspheres via functionalized PLL-g-PEG. To quantify the adsorbed amount of protein, a semiquantitative method that uses confocal laser scanning microscopy (CLSM) was applied. The first part of the experiment confirms the feasibility of introducing specific functional groups on PLL-g-PEG-coated PLGA microspheres. In the second part of the experiment, PLL-g-PEG-coated PLGA microspheres show a drastic decrease of adsorbed proteins by two orders of magnitude in comparison to uncoated PLGA microspheres. Low protein-binding, functionalizable microspheres provide a fundamental basis for the design of drug delivery and biosensor systems.

Encapsulation of plasmid DNA in PLGA-stearylamine microspheres: a comparison of solvent evaporation and spray-drying methods.

Stearylamine, a positively charged hydrophobic molecule, was tested as a formulation agent for the encapsulation of a model plasmid in PLGA microspheres. The primary objective was to compare the spray-drying and double emulsion solvent evaporation methods and evaluate their suitability for fabricating PLGA-stearylamine plasmid-entrapped microspheres. A luciferase reporter gene plasmid (pGL3-Con) was formulated into microspheres using a 64 kDa PLGA 50:50 polymer blended with stearylamine (SA) at a range of concentrations up to 15%m/m, by the solvent evaporation and spray-drying methods. The microspheres were characterized regarding their size distributions, zeta potentials and morphology by laser diffraction, electrophoretic mobility and scanning electron microscopy (SEM), respectively. Formulated plasmid extracts were assessed for physical damage by agarose gel electrophoresis, and the in vitro biological activity was determined by transfection of a human embryo kidney epithelial (293) cell line. Size distribution analysis showed that SA reduced the median diameters of spray-dried particles from 8.32 to 3.64 microns, with a corresponding reduction in the spread of the distribution, but solvent evaporation microspheres showed an increased median diameter on addition of SA. Concentrations of SA above 10%(m)/(m) resulted in disruption of the smooth morphology of the solvent evaporation particles. There was a SA concentration-dependent tendency in the increase of surface positive charge and resistance to serum nuclease assault and in vitro expression of luciferase protein. These results show that SA and possibly other charged hydrophobic molecules may be useful agents in the formulation of particulate DNA vaccines by both methods.

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.

Transport of alkamides from Echinacea species through Caco-2 monolayers.

To gain more insights into the human intestinal absorption of alkamides from Echinacea species, transport studies were performed with the human adenocarcinoma colonic cell line Caco-2 (ATCC) as a model to assess the epithelial transport of dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides (1/ 2). 30 minutes after apical loading of 25 microg/ml 1/ 2, about 15 % of these alkamides were detectable on the basolateral side. Close monitoring of the transport during 6 hours revealed a nearly complete transport to the basolateral side after 4 hours and no significant metabolism was observable. Transport experiments performed at 4 degrees C showed only a slight decrease in transport, which is a strong hint that dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides (1/ 2) cross biological membranes by passive diffusion. Nearly the same results were obtained after preincubation of the Caco-2 cells with lipopolysaccharides (LPS) or phorbol 12-myristate-13-acetate (PMA) to mimic an inflammatory status. These results support the assumption that the alkamides can be easily transported from the intestinum and hence may contribute to the in vivo effects of Echinacea preparations.

Transforming growth factor beta-3 crystals as reservoirs for slow release of active TGF-beta3.

Transforming growth factor betas (TGF-betas) play critical roles in many diseased states and injury repair processes. Exogenous delivery of TGF-beta may thus have therapeutic applications. Here, crystals of TGF-beta3 (TGF-beta3) are being evaluated as protected reservoirs for sustained local release. A sensitive Mv1Lu cell growth inhibition assay established that in vitro, active TGF-beta3 can be delivered from physically stable crystals. Non-sink release experiments revealed that crystal solubility at pH 7.4 was higher in cell culture medium (2.7+/-0.1 microg/ml) than in saline buffers (approximately 1-1.5 microg/ml, P<0.05). Addition of serum induced a five-fold delay in equilibration of soluble-crystal TGF-beta3. Semi-sink experiments cumulated in higher TGF-beta3 release than under non-sink conditions; the observed steady states correlated with crystal solubility and the frequency of buffer exchange. Release of TGF-beta3 from crystals was also strongly dependent on solubility changes as affected by pH. At neutral pH the solubilities were the lowest, and increased with both higher and lower pH. The results indicate that TGF-beta3 crystals may have promising features for local pH-triggered sustained-release applications.

Evaluation of particle uptake in human blood monocyte-derived cells in vitro. Does phagocytosis activity of dendritic cells measure up with macrophages?

This work focuses on microparticles as potential antigen delivery systems to target professional antigen-presenting cells. Surface modified polystyrene microparticles were administered to human-derived macrophages (MPhis) and dendritic cells (DCs) in vitro to evaluate the phagocytosis activity of each cell type. To discriminate between internalised particles and those closely attached to the outside of the cells, particle internalisation was verified by confocal laser scanning microscopy. Especially positively charged particles tend to stick to the outer cell membrane and may lead to false positive results when measured by conventional microscopy. In contrast, fluorescence microscopy in combination with an extracellular fluorescence quenching agent (trypan blue) allows the unequivocal assessment of particle uptake for screening purposes. For this assay, the fluorescent label needs to be in direct contact to the quenching agent and cannot be localised inside the particle core. Different types of microparticles varying in size, surface-material and zeta potential resulted in vast differences regarding their uptake by MPhis and DCs as well as the maturation of DCs. Negatively-charged carboxylated and bovine serum albumin-coated particles were phagocytosed by MPhis to a relatively small extent. Interestingly, phagocytosis of these particles was still significantly lower in DCs while positively charged poly-L-lysine (PLL) coated particles induced high phagocytosis activity in both cell types. By comparing our results with literature data, we conclude that phagocytosis activity of DCs and MPhis largely depends on particle size and surface charge and is also influenced by the character of bulk and coating material. PLL can be directed to DCs and MPhis with comparable efficiency and, in addition, induce maturation of DCs.

Hydrophilic poly(DL-lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells.

Biodegradable poly(lactide-co-glycolide) (PLGA) microspheres have a proven track record for drug delivery and are suggested to be ideal carrier systems to target therapeutics into phagocytic cells such as macrophages (MPhis) and dendritic cells (DCs). Microspheres prepared by spray-drying from different PLGA-type polymers were evaluated regarding their effect on phagocytosis, intracellular degradation and viability of human-derived macrophages MPhis and DCs. Even the microspheres prepared from the most hydrophilic polymer RG502H, were efficiently phagocytosed by primary human MPhis and DCs. Interestingly, uptake of PLGA microspheres by DCs as potent immune modulator cells was almost as efficient as uptake by the highly phagocytic MPhis. Phagocytosed microspheres remained inside the cells until decay with none of the microsphere preparations induced significant apoptosis or necrotic cell death. Acidic pH and the phagosomal environment inside the cells enhanced microsphere decay and release of encapsulated material. Degradation of microspheres consisting of the most hydrophilic PLGA polymer RG502H occurred in a reasonable time frame of less than 2 weeks ensuring the release of encapsulated drug during the life span of the cells. To explore important technical and biological aspects of DNA microencapsulation, we have studied DNA loading and in vitro DNA release of microspheres from different PLGA type polymers. Hydrophobicity and molecular weight of the PLGA polymers had profound influence on both the encapsulation efficiency of DNA and its release kinetics in vitro: the hydrophilic polymers showed higher encapsulation efficiency and faster release of intact DNA compared to the hydrophobic ones. These results suggest that microspheres from the PLGA polymer RG502H have improved characteristics for DNA delivery to human MPhis and DCs.

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.

Diamonds in the rough: protein crystals from a formulation perspective.

The focus of the present review is to address the use of protein crystals in formulation design. Although this idea has been present for some time, i.e., insulin crystals were first reported back in 1920s, macromolecular crystallization has not received as much attention as the other methods for stabilizing protein drug candidates. The prospective potential of crystalline protein formulations in light of new advances in the field of macromolecular crystallization was reviewed, and the basic concepts and the tools now available for developing protein crystals into drug formulations are introduced. In addition, formulation challenges and regulatory demands, along with examples of current applications of protein crystals, are presented.