Peripheral nerve regeneration with sustained release of poly(phosphoester) microencapsulated nerve growth factor within nerve guide conduits

Prolonged delivery of neurotrophic proteins to the target tissue is valuable in the treatment of various disorders of the nervous system. We have tested in this study whether sustained release of nerve growth factor (NGF) within nerve guide conduits (NGCs), a device used to repair injured nerves, would augment peripheral nerve regeneration. NGF-containing polymeric microspheres fabricated from a biodegradable poly(phosphoester) (PPE) polymer were loaded into silicone or PPE conduits to provide for prolonged, site-specific delivery of NGF. The conduits were used to bridge a 10 mm gap in a rat sciatic nerve model. Three months after implantation, morphological analysis revealed higher values of fiber diameter, fiber population and fiber density and lower G-ratio at the distal end of regenerated nerve cables collected from NGF microsphere-loaded silicone conduits, as compared with those from control conduits loaded with either saline alone, BSA microspheres, or NGF protein without microencapsulation. Beneficial effects on fiber diameter, G-ratio and fiber density were also observed in the permeable PPE NGCs. Thus, the results confirm a long-term promoting effect of exogenous NGF on morphological regeneration of peripheral nerves. The tissue-engineering approach reported in this study of incorporation of a microsphere protein release system into NGCs holds potential for improved functional recovery in patients whose injured nerves are reconstructed by entubulation.     

Multifunctional alginate microspheres for biosensing, drug delivery and magnetic resonance imaging

This research aims to develop and investigate a multifunctional implantable system capable of biosensing, drug delivery and magnetic resonance imaging (MRI) for continuous monitoring, controlled anti-inflammatory drug delivery and imaging, respectively. A glucose biosensor, diclofenac sodium (Diclo) and magnetic nanoparticles (MNP) were used as the biosensor component, anti-inflammatory agent and MRI contrast agent, respectively. MNP were synthesized by the co-precipitation technique and loaded with the sensor and drug components into alginate microspheres using a commercial droplet generator. The multifunctional system was then characterized using optical microscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, vibrating sample magnetometry (VSM) and MRI. The MNP were found to be in the size range of 5-15 nm. The final system, comprising the biosensor, drug and MNP loaded inside alginate microspheres, was found to be in the size range of 10-60 μm. Biosensing studies indicated an excellent glucose response curve, with a regression coefficient of 0.974 (0-10mM of glucose, response time: 4 min). In vitro Diclo release shows that MNP loading in alginate microspheres increases the burst release percentage by 11-12% in both 60 and 10 μm particles. However, the duration of release for 85% drug release decreases with MNP loading by 7 and 6 days for 39 the 60 and 10 μm particles, respectively. Super-paramagnetism was confirmed by VSM, with 2.09 and 1.368 emu g(-1), respectively, for the 60 and 10 μm particles, with no hysteresis. MRI showed significant contrast for both sizes. The particles showed an excellent biocompatibility (>80%) for all combinations of formulations. The system shows a great potential for biosensing with concurrent drug delivery and visualization for biomedical applications.     

Intraseptal implantation of NGF-releasing microspheres promote the survival of axotomized cholinergic neurons

Neurotrophic factors therapy requires their precise delivery to the targeted neuronal population. For this purpose, a wide range of strategies have been developed, and among them the stereotaxic implantation of biodegradable microparticles. To assess the in vivo activity of NGF-releasing PLGA microspheres, unloaded and NGF-loaded microparticles were implanted in the rat brain, near the septal cholinergic neurons, axotomized by an unilateral transection of the fornix-fimbria. Histological analysis at two and six weeks after implantation revealed a non-specific astro- and micro-glial reaction around the microspheres, identical for both unloaded and NGF-loaded microspheres. No neuronal toxicity was noticed, and healthy looking neurons were observed in contact with the microspheres. In the non-treated animals, the percentage of axotomized surviving neurons, when compared to the contralateral intact side, was 31 +/- 2 and 27 +/- 1% at two and six weeks, respectively. Unloaded microspheres caused no protective nor neurotoxic effects (40 +/- 9 and 39 +/- 6% of surviving cholinergic neurons at two and six weeks, respectively). In contrast, NGF-loaded microspheres showed a significant effect on the survival of axotomized cholinergic neurons at two and six weeks after implantation (66 +/- 9 and 61 +/- 5% when compared to the contralateral intact side, respectively). These results show that PLGA microparticles present no neurotoxicity and release sufficient amounts of bioactive NGF to significantly limit the lesion-induced disappearance of cholinergic neurons in the septum during at least six weeks. PLGA microparticles can be used in the future to administer neurotrophic factors in central nervous system disorders.     

A drug delivery system based on alginate microspheres: Mass-transport test and <Emphasis Type="Italic">in vitro</Emphasis> validation

This paper presents a drug delivery system based on alginate microspheres. The biocompatibility, the flexibility in size and shape, the ability to entrap biomolecules as well as cells make alginate based systems ideal for in vivo drug delivery. Specifically, considering the target application of neural regeneration and the issue of neuroprotection for the development of innovative neuroprostheses, the authors describe a system for controlled release of Netrin-1, an axonal guidance protein. Microspheres dimensioning (based on specifications of drug release time and release modality), microspheres realization, and mass transport tests are described. The release efficiency is finally assessed by in vitro experiments of axonal guidance performed on embryonic neuronal cells. Preliminary results show that neuronal axons grow approaching the Netrin-1 source, thus indicating an efficient entrapment and release of the protein in the microspheres, in agreement with the microsphere modelling described before.     

Extended and sequential delivery of protein from injectable thermoresponsive hydrogels

Thermoresponsive hydrogels are attractive for their injectability and retention in tissue sites where they may serve as a mechanical support and as a scaffold to guide tissue remodeling. Our objective in this report was to develop a thermoresponsive, biodegradable hydrogel system that would be capable of protein release from two distinct reservoirs--one where protein was attached to the hydrogel backbone, and one where protein was loaded into biodegradable microparticles mixed into the network. Thermoresponsive hydrogels consisting of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), and biodegradable methacrylate polylactide were synthesized along with modified copolymers incorporating 1 mol % protein-reactive methacryloxy N-hydroxysuccinimide (MANHS), hydrophilic acrylic acid (AAc), or both. In vitro bovine serum albumin (BSA) release was studied from hydrogels, poly(lactide-co-glycolide) microparticles, or microparticles mixed into the hydrogels. The synthesized copolymers were able to gel below 37°C and release protein in excess of 3 months. The presence of MANHS and AAc in the copolymers was associated with higher loaded protein retention during thermal transition (45% vs. 22%) and faster release (2 months), respectively. Microspheres entrapped in the hydrogel released protein in a delayed fashion relative to microspheres in saline. The combination of a protein-reactive hydrogel mixed with protein-loaded microspheres demonstrated a sequential release of specific BSA populations. Overall the described drug delivery system combines the advantages of injectability, degradability, extended release, and sequential release, which may be useful in tissue engineering applications.     

Delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders

Nerve growth factor (NGF) may enhance axonal regeneration following injury to the central nervous system (CNS), such as after spinal cord injury. The release profile of NGF, co-encapsulated with ovalbumin, was tailored from biodegradable polymeric microspheres using both polymer degradation and protein loading. Biodegradable polymeric microspheres were prepared from PLGA 50/50, PLGA 85/15, PCL and a blend of PCL/PLGA 50/50 (1:1, w/w), where the latter was used to further tailor the degradation rate. The amount of protein loaded in the microspheres was varied, with PCL encapsulating the greatest amount of protein and PLGA 50/50 encapsulating the least. A two-phase release profile was observed for all polymers where the first phase resulted from release of surface proteins and the second phase resulted predominantly from polymer degradation. Polymer degradation influenced the release profile most notably from PLGA 50/50 and PLGA 85/15 microspheres. The amount and bioactivity of released NGF was followed over a 91 d period using a NGF-ELISA and PC12 cells, respectively. NGF was found to be bioactive for 91 d, which is longer than previously reported.     

Promotion of peripheral nerve growth by collagen scaffolds loaded with collagen-targeting human nerve growth factor-beta

Nerve growth factor (NGF) plays a critical role in neuronal development and regeneration. However, the lack of efficient NGF delivery system limits its clinical application. We reported that a peptide deduced from collagenase, TKKTLRT, fused with NGF-beta could develop a collagen based NGF targeting delivery system. Our results showed that this peptide could allow fused NGF-beta bind to collagen specifically. In addition, we found that the polypeptide could result in a 2.3-fold increase in the expression level and a significant improvement of bioactivity of fused NGF-beta. In the in vivo function study, collagen membranes loaded with the collagen binding NGF enhanced the nerve growth. Thus, the targeting wound repair system could be important for the repair of peripheral nerve injury.     

Nerve guidance channels as drug delivery vehicles

Nerve guidance channels (NGCs) have been shown to facilitate regeneration after transection injury to the peripheral nerve or spinal cord. Various therapeutic molecules, including neurotrophic factors, have improved regeneration and functional recovery after injury when combined with NGCs; however, their impact has not been maximized partly due to the lack of an appropriate drug delivery system. To address this limitation, nerve growth factor (NGF) was incorporated into NGCs of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate), P(HEMA-co-MMA). The NGCs were synthesized by a liquid-liquid centrifugal casting process and three different methods of protein incorporation were compared in terms of protein distribution and NGF release profile: (1) NGF was encapsulated (with BSA) in biodegradable poly(d,l-lactide-co-glycolide) 85/15 microspheres, which were combined with a PHEMA polymerization formulation and coated on the inside of pre-formed NGCs by a second liquid-liquid centrifugal casting technique; (2) pre-formed NGCs were imbibed with a solution of NGF/BSA and (3) NGF/BSA alone was combined with a PHEMA formulation and coated on the inside of pre-formed NGCs by a second liquid-liquid centrifugal casting technique. Using a fluorescently labelled model protein, the distribution of proteins in NGCs prepared with a coating of either protein-loaded microspheres or protein alone was found to be confined to the inner PHEMA layer. Sustained release of NGF was achieved from NGCs with either NGF-loaded microspheres or NGF alone incorporated into the inner layer, but not from channels imbibed with NGF. By day 28, NGCs with microspheres released a total of 220 pg NGF/cm of channel whereas those NGCs imbibed with NGF released 1040 pg/cm and those NGCs with NGF incorporated directly in a PHEMA layer released 8624 pg/cm. The release of NGF from NGCs with microspheres was limited by a slow-degrading microsphere formulation and by the maximum amount of microspheres that could be incorporated into the NGCs structure. Notwithstanding, the liquid-liquid centrifugal casting process is promising for localized and controlled release of multiple factors that are key to tissue regeneration.     

Design and delivery of silver sulfadiazine from alginate microspheres-impregnated collagen scaffold

A reconstituted collagen scaffold impregnated with silver sulfadiazine (SSD) loaded alginate microspheres, capable of delivering the drug in a controlled manner has been developed. SSD-loaded alginate microspheres were prepared by modified water-in-oil emulsion technique through interfacial ionic gelation of alginate using CaCl2. The SSD-loaded microspheres were impregnated in pepsin-solubilized collagen, in situ, while inducing fibrillation and cast as thin scaffold. Morphological features of microspheres and microsphere-impregnated collagen were analyzed through SEM. Distribution homogeneity of impregnated microspheres, their in vitro behavior in (Dulbecco's modified minimal essential media) DMEM, and antibacterial efficiency against ATCC pathogens were determined. Initial drug load of 20% (w/w) with respect to alginate and 40% (v/v) of 2% alginate with respect to oil phase were found to produce microspheres of optimum drug entrapment (3%) and required size range (300-370 microm). In vitro drug release studies from the scaffold showed an initial burst release of 47.5% and a controlled release for 72 h with equilibrium concentration of 68.8%. SSD-loaded microspheres exhibited minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) levels of 32 and 40.2 microg/mL to both K. pneumoniae and E. coli respectively. P. aeruginosa showed MIC and MBC levels of 44.8 and 51.2 microg/mL respectively, while Staphylococcus aureus exhibited MIC and MBC at the same concentration range (57.6 microg/mL). The collagen-based scaffold impregnated with SSD-loaded alginate microspheres can deliver SSD in a controlled fashion, can control infection for extended time period with lesser dressing frequencies, and will enable easier assessment of wound.     

Characterization of holmium loaded alginate microspheres for multimodality imaging and therapeutic applications

In this paper the preparation and characterization of holmium-loaded alginate microspheres is described. The rapid development of medical imaging techniques offers new opportunities for the visualisation of (drug-loaded) microparticles. Therefore, suitable imaging agents have to be incorporated into these particles. For this reason, the element holmium was used in this study in order to utilize its unique imaging characteristics. The paramagnetic behaviour of this element allows visualisation with MRI and holmium can also be neutron-activated resulting in the emission of gamma-radiation, allowing visualisation with gamma cameras, and beta-radiation, suitable for therapeutic applications. Almost monodisperse alginate microspheres were obtained by JetCutter technology where alginate droplets of a uniform size were hardened in an aqueous holmium chloride solution. Ho(3+) binds via electrostatic interactions to the carboxylate groups of the alginate polymer and as a result alginate microspheres loaded with holmium were obtained. The microspheres had a mean size of 159 microm and a holmium loading of 1.3 +/- 0.1% (w/w) (corresponding with a holmium content based on dry alginate of 18.3 +/- 0.3% (w/w)). The binding capacity of the alginate polymer for Ho(3+) (expressed in molar amounts) is equal to that for Ca(2+), which is commonly used for the hardening of alginate. This indicates that Ho(3+) has the same binding affinity as Ca(2+). In line herewith, dynamic mechanical analyses demonstrated that alginate gels hardened with Ca(2+) or Ho(3+) had similar viscoelastic properties. The MRI relaxation properties of the microspheres were determined by a MRI phantom experiment, demonstrating a strong R(2)* effect of the particles. Alginate microspheres could also be labelled with radioactive holmium by adding holmium-166 to alginate microspheres, previously hardened with calcium (labelling efficiency 96%). The labelled microspheres had a high radiochemical stability (94% after 48 h incubation in human serum), allowing therapeutic applications for treatment of cancer. The potential in vivo application of the microspheres for a MR-guided renal embolization procedure was illustrated by selective administration of microspheres to the left kidney of a pig. Anatomic MR-imaging showed the presence of holmium-loaded microspheres in the kidney. In conclusion, this study demonstrates that the incorporation of holmium into alginate microspheres allows their visualisation with a gamma camera and MRI. Holmium-loaded alginate microspheres can be used therapeutically for embolization and, when radioactive, for local radiotherapy of tumours.     

Drug carrier systems based on collagen–alginate composite structures for improving the performance of GDNF-secreting HEK293 cells

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor. Development of drug delivery technologies facilitating controlled release of GDNF is critical to applying GDNF in treating neurodegenerative diseases. We previously developed 3D collagen microspheres and demonstrated enhanced GDNF secretion after encapsulation of HEK293 cells, which were transduced to overexpress GDNF in these microspheres. However, the entrapped HEK293 cells were able to migrate out of the collagen microspheres, making it undesirable for clinical applications. In this report, we investigate two new carrier designs, namely collagen–alginate composite gel and collagen microspheres embedded in alginate gel in preventing cell leakage, maintaining cell growth and controlling GDNF secretion in the HEK293 cells. We demonstrated that inclusion of alginate gel in both designs is efficient in preventing cell leakage to the surrounding yet permitting the GDNF secretion, although the cellular growth rate is reduced in an alginate concentration dependent manner. Differential patterns of GDNF secretion in the two designs were demonstrated. The collagen–alginate composite gel maintains a more or less constant GDNF secretion over time while the collagen microspheres embedded in alginate gel continue to increase the secretion level of GDNF over time. This study contributes towards the development of cell-based GDNF delivery devices for the future therapeutics of neurodegenerative diseases.     

Microfluidic fabrication of 6-methoxyethylamino numonafide-eluting magnetic microspheres

Recently, 6-methoxyethylamino numonafide (MEAN) exhibited potent inhibition of hepatocellular carcinoma (HCC) cell growth and less systemic toxicity than amonafide. MEAN may serve as an ideal candidate for the treatment of HCC; however, liver-directed, selective infusion methods may be critical to maximize the MEAN dose delivered to the targeted tumors. This study describes the microfluidic fabrication of MEAN-eluting ultrasmall superparamagnetic iron oxide (USPIO) nanocluster-containing alginate microspheres (MEAN-magnetic microspheres) intended for selective transcatheter delivery to HCC. The resulting drug delivery platform was mono-disperse, microsphere sizes were readily controlled based on channel flow rates during synthesis procedures, and drug release rates from the microspheres could be readily controlled with the introduction of USPIO nanoclusters. The MR relaxivity properties of the microspheres suggest the feasibility of in vivo imaging after administration, and these microspheres exhibited potent therapeutic effects significantly inhibiting cell growth inducing apoptosis in hepatoma cells.     

Novel thermosensitive calcium alginate microspheres: Physico-chemical characterization and delivery properties

The system described in this paper was obtained by soaking calcium alginate (CaAlg) microspheres in a water solution of poly-[(3-acrylamidopropyl)-trimethylammonium chloride-b-N-isopropylacrylamide] [poly(AMPTMA-b-NIPAAM)], a new block co-polymer recently synthesized by atom transfer radical polymerization (ATRP). The block co-polymer is characterized by a lower critical solution temperature (LCST) of 41 °C in aqueous 0.1 M NaCl solution, and can be anchored on the CaAlg microspheres by means of polyion interactions. Polycations (permanently positively charged blocks) and polyanions (free alginate carboxylic groups) interact, leading to microspheres with thermosensitive properties. As an effect of interaction with the microspheres the LCST of the co-polymer is lowered to 36–38 °C. In this temperature range a colloidal water suspension of the microspheres collapses, forming macroscopic aggregates. The new system shows, at human body temperature, an improved ability to carry and deliver both hydrophobic and hydrophilic molecules in comparison with unmodified CaAlg microspheres. The release properties of the microspheres loaded with different model drugs can be appropriately modulated by the amount of the poly(AMPTMA-b-NIPAAM). Furthermore, the microspheres show the interesting capability of retaining the activity of a loaded enzyme (horseradish peroxidase), used as a model protein. The results obtained indicate that the proposed drug delivery system may be suitable for drug depot applications.     

Fabrication and characterization of monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells for controlled drug release

Monodisperse PLGA–alginate core–shell microspheres with controlled size and homogeneous shells were first fabricated using capillary microfluidic devices for the purpose of controlling drug release kinetics. Sizes of PLGA cores were readily controlled by the geometries of microfluidic devices and the fluid flow rates. PLGA microspheres with sizes ranging from 15 to 50 μm were fabricated to investigate the influence of the core size on the release kinetics. Rifampicin was loaded into both monodisperse PLGA microspheres and PLGA–alginate core–shell microspheres as a model drug for the release kinetics studies. The in vitro release of rifampicin showed that the PLGA core of all sizes exhibited sigmoid release patterns, although smaller PLGA cores had a higher release rate and a shorter lag phase. The shell could modulate the drug release kinetics as a buffer layer and a near-zero-order release pattern was observed when the drug release rate of the PLGA core was high enough. The biocompatibility of PLGA–alginate core–shell microspheres was assessed by MTT assay on L929 mouse fibroblasts cell line and no obvious cytotoxicity was found. This technique provides a convenient method to control the drug release kinetics of the PLGA microsphere by delicately controlling the microstructures. The obtained monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells could be a promising device for controlled drug release.     

The effect of the controlled release of nerve growth factor from collagen gel on the efficiency of neural cell culture

In this study, we tested the hypothesis that the amount of nerve growth factor (NGF) required for pheochromocytoma (PC12) cell culture can be dramatically reduced by controlled release of NGF from a collagen gel coating on the culture surface. Cells were cultured on collagen gels loaded with various amounts of NGF. As a control, PC12 cells were cultured on collagen gels with daily addition of various amounts of NGF to the culture medium. After an initial 12h burst, NGF was steadily released from the gels for 4 days. Apoptotic activity and cell viability were determined using terminal uridine nick end labeling and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Neuronal differentiation was determined using immunocytochemistry and Western blot analysis. Compared to 100 ng NGF daily addition (300 ng over 3 days), 1 ng NGF daily addition showed dramatically decreased cell viability and neuronal differentiation and increased apoptotic activity. In contrast, collagen gels loaded with 10 ng NGF yielded cell viability, apoptotic activity, and neuronal differentiation similar to those of culture with 100 ng NGF daily addition. Our method reduced the amount of NGF required for PC12 cell culture to 1/3th of that used in daily addition without affecting cell viability, apoptosis, or differentiation. This method could economize large-scale culture of stem cells by reducing the amount of costly growth factors needed.     

Polyphosphoester microspheres for sustained release of biologically active nerve growth factor

Controlled delivery of neurotrophic proteins to a target tissue by biodegradable polymer microspheres has been explored widely for its potential applications in the treatment of various disorders in the nervous system. We investigated in this study the potential of polyphosphoester microspheres as carriers for the sustained release of nerve growth factor (NGF), a water-soluble neurotrophic protein. Two polyphosphoesters (PPEs), P(BHET-EOP/TC) and P(DAPG-EOP), as well as poly(lactide/glycolic acid) (PLGA), were used to fabricate microspheres by a W/O/W emulsion and solvent evaporation/extraction method. With bovine serum albumin as a model protein to optimize processing parameters. P(DAPG-EOP) microspheres exhibited a lower burst effect but similar protein entrapment levels and efficiencies when compared with those made of PLGA. Bioactive NGF could be released for at least 10 weeks from the P(DAPG-EOP) microspheres, as confirmed by a neurite outgrowth assay of the PC12 cells. These NGF containing microspheres were incorporated into the nerve guide conduits that were implanted to bridge a 10 mm gap in a rat sciatic nerve model. Two weeks after implantation, immunostaining with an antibody against the neurofilament protein confirmed the presence of axons at the distal end of regenerated cables within the NGF microsphere-loaded conduits. These results demonstrated the feasibility of using biodegradable PPEs for microencapsulation of NGF and provided a basis for future therapeutic application of the microspheres.     

Therapeutic effect of orally administered microencapsulated oxaliplatin for colorectal cancer

Colorectal cancer is a significant source of morbidity and mortality in the United States and other Western countries. Oral delivery of therapeutics remains the most patient accepted form of medication. The development of an oral delivery formulation for local delivery of chemotherapeutics in the gastrointestinal tract can potentially alleviate the adverse side effects including systemic cytotoxicity, as well as focus therapy to the lesions. Here we develop an oral formulation of the chemotherapeutic drug oxaliplatin for the treatment of colorectal cancer. Oxaliplatin was encapsulated in pH sensitive, mucoadhesive chitosan-coated alginate microspheres. The microparticles were formulated to release the chemotherapeutics after passing through the acidic gastric environment thus targeting the intestinal tract. In vivo, these particles substantially reduced the tumor burden in an orthotopic mouse model of colorectal cancer, and reduced mortality.     

Formulation, characterization and release studies of alginate microspheres encapsulated with tetanus toxoid

Alginate is a safe, non-immunogenic and inexpensive natural polymer with high mucoadhesive properties. Alginate microspheres can be used as a delivery system for antigens to mucosal surfaces. In the present study alginate microspheres were prepared by an emulsification technique. The effects of sonication time, concentration of alginate, emulsifier and calcium chloride, and also the volume of calcium solution, were evaluated on mean size, size range, surface roughness and porosity, sphericity and clumping of microspheres using an optical microscope and particle size analyzer. The most desirable conditions were 90 s sonication, 3% alginate solution, 2% surfactant and 60 ml of 0.33% CaCl2 in octanol. The resulting microspheres had a mean size of 1.34 +/- 0.3 microm and size range of 0.3 +/- 2.0 microm, with no surface roughness and porosity, low clumping and high sphericity. The encapsulation efficiency was about 47.7%. All batches showed nearly the same release profiles with a low burst release. The stability of the model antigen (tetanus toxoid (TT)) extracted from microspheres was confirmed by SDS-PAGE; and the antigenicity of TT was studied by ELISA and found to be 91 +/- 5% of the original TT. It can be concluded that, with regard to the size and morphological characteristics of the prepared microspheres and their ability in preserving the antigenicity of the encapsulated TT, they could be used as a delivery system for mucosal delivery of TT.     

In vitro and in vivo release of nerve growth factor from biodegradable poly-lactic-co-glycolic-acid microspheres

Regeneration of peripheral nerves after injury is suboptimal. We now report the long term delivery of nerve growth factor (NGF) by biodegradable poly-lactic-co-glycolic acid (PLGA) microspheres in vitro and in vivo. Lactic to glycolic acid ratios of 50:50 and 85:15 were fabricated using the double emulsion solvent, evaporation technique. Three different inherent viscosities (0.1 dL g(-1) : 1A, 0.4 dL g(-1) : 4A, 0.7 dL g(-1) : 7A) were analyzed. In vitro, release of NGF for 23 days was measured. Electron microscopy demonstrated intact spheres for at least 7 days (50:50 1A), 14 days (50:50 4A), or 35 days (50:50 7A and 85:15 7A). In vitro release kinetics was characterized by burst release, followed by release of NGF at a rate of 0.6-1.6% a day. Release curves for 50:50 1A and 85:15 7A differed significantly from other compositions (p < 0.01). In vivo, release was characterized by a novel radionuclide tracking assay. Release rates varied from 0.9 to 2.2% per day with linear kinetics. All but the 85:15 type of spheres showed different release profiles in vivo compared to in vitro conditions. On the basis of the surface morphology and release profiles, we found microspheres fabricated from 50:50 4A PLGA to be best suited for the use in a rat sciatic nerve injury model.     

Enhanced MSC chondrogenesis following delivery of TGF-β3 from alginate microspheres within hyaluronic acid hydrogels in vitro and in vivo

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair and members of the transforming growth factor-beta (TGF-β) superfamily are a key mediator of MSC chondrogenesis. While TGF-β mediated MSC chondrogenesis is well established in in vitro pellet or hydrogel cultures, clinical translation will require effective delivery of TGF-βs in vivo. Here, we investigated the co-encapsulation of TGF-β3 containing alginate microspheres with human MSCs in hyaluronic acid (HA) hydrogels towards the development of implantable constructs for cartilage repair. TGF-β3 encapsulated in alginate microspheres with nanofilm coatings showed significantly reduced initial burst release compared to uncoated microspheres, with release times extending up to 6 days. HA hydrogel constructs seeded with MSCs and TGF-β3 containing microspheres developed comparable mechanical properties and cartilage matrix content compared to constructs supplemented with TGF-β3 continuously in culture media, whereas constructs with TGF-β3 directly encapsulated in the gels without microspheres had inferior properties. When implanted subcutaneously in nude mice, constructs containing TGF-β3 microspheres resulted in superior cartilage matrix formation when compared to groups without TGF-β3 or with TGF-β3 added directly to the gel. However, calcification was observed in implanted constructs after 8 weeks of subcutaneous implantation. To prevent this, the co-delivery of parathyroid hormone-related protein (PTHrP) with TGF-β3 in alginate microspheres was pursued, resulting in partially reduced calcification. This study demonstrates that the controlled local delivery of TGF-β3 is essential to neocartilage formation by MSCs and that further optimization is needed to avert the differentiation of chondrogenically induced MSCs towards a hypertrophic phenotype.