Evaluation of Poly (1, 6-bis-(p-carboxyphenoxy) Hexane-co-sebacic Acid Microspheres for Controlled Basal Insulin Delivery

Purpose

To develop poly 1,3-bis-(p-carboxyphenoxy) hexane-co-sebacic acid (p(CPH/SA)) microspheres for controlled basal insulin delivery and evaluate their in vivo efficacy and toxicity.

Methods

A series of CPH/SA copolymers with molar ratios 20/80, 40/60, and 50/50 were synthesized and characterized. The stability of encapsulated insulin and the fraction of insulin released from microspheres were assessed by different analytical techniques. The skin from the injection site of rats was examined microscopically for histomorphological changes.

Results

Increasing the molar ratio of CPH/SA significantly (p?<?0.05) improved insulin loading and controlled insulin release. However, dimer aggregates of insulin were observed as CPH/SA molar ratio increased. Co-encapsulation of zinc oxide with insulin inhibited dimer aggregate formation and further controlled insulin release. Insulin was stable after entrapment into microspheres and during in vitro release studies. Administration of microsphere formulations CPH/SA 40/60 and 50/50 with zinc oxide controlled insulin release and maintained basal insulin levels for 42 days in rats. Skin sections showed minimal inflammation with no evidence for histomorphological changes and toxicity.

Conclusions

Insulin-loaded CPH/SA microspheres demonstrated considerable potential as controlled delivery system for insulin. Copolymer microspheres maintained basal insulin levels for 42 days and were biodegradable and biocompatible.     

The Effect of Glutathione as Chain Transfer Agent in PNIPAAm-Based Thermo-responsive Hydrogels for Controlled Release of Proteins

Purpose

To control degradation and protein release using thermo-responsive hydrogels for localized delivery of anti-angiogenic proteins.

Methods

Thermo-responsive hydrogels derived from N-isopropylacrylamide (NIPAAm) and crosslinked with poly(ethylene glycol)-co-(L-lactic acid) diacrylate (Acry-PLLA-b-PEG-b-PLLA-Acry) were synthesized via free radical polymerization in the presence of glutathione, a chain transfer agent (CTA) added to modulate their degradation and release properties. Immunoglobulin G (IgG) and the recombinant proteins Avastin® and Lucentis® were encapsulated in these hydrogels and their release was studied.

Results

The encapsulation efficiency of IgG was high (75–87%) and decreased with CTA concentration. The transition temperature of these hydrogels was below physiological temperature, which is important for minimally invasive therapies involving these materials. The toxicity from unreacted monomers and free radical initiators was eliminated with a minimum of three buffer extractions. Addition of CTA accelerated degradation and resulted in complete protein release. Glutathione caused the degradation products to become solubilized even at 37°C. Hydrogels prepared without glutathione did not disintegrate nor released protein completely after 3 weeks at 37°C. PEGylation of IgG postponed the burst release effect. Avastin® and Lucentis® released from degraded hydrogels retained their biological activity.

Conclusions

These systems offer a promising platform for the localized delivery of proteins.     

Gamma Irradiation of Active Self-Healing PLGA Microspheres for Efficient Aqueous Encapsulation of Vaccine Antigens

Purpose

To investigate the effect of γ-irradiation of poly(lactic-co-glycolic acid) (PLGA)/Al(OH)₃/0 or 5 wt% diethyl phthalate (DEP) microspheres for active self-healing encapsulation of vaccine antigens.

Methods

Microspheres were irradiated with ⁶⁰Co at 2.5 and 1.8 MRad and 0.37 and 0.20 MRad/h. Encapsulation of tetanus toxoid (TT) was achieved by mixing Al(OH)₃-PLGA microspheres with TT solution at 10–38°C. Electron paramagnetic resonance (EPR) spectroscopy was used to examine free radical formation. Glass transition temperature (T_g) and molecular weight of PLGA was measured by differential scanning calorimetry and gel permeation chromatography, respectively. Loading and release of TT were examined by modified Bradford, amino acid analysis, and ELISA assays.

Results

EPR spectroscopy results indicated absence of free radicals in PLGA microspheres after γ-irradiation. Antigen-sorbing capacity, encapsulation efficiency, and T_g of the polymer were also not adversely affected. When DEP-loaded microspheres were irradiated at 0.2 MRad/h, some PLGA pores healed during irradiation and PLGA healing during encapsulation was suppressed. The molecular weight of PLGA was slightly reduced when DEP-loaded microspheres were irradiated at the same dose rate. At the 0.37 MRad/h dose rate, these trends were not observed and the full immunoreactivity of TT was preserved during encapsulation and 1-month release. Gamma irradiation slightly increased TT initial burst release. The small increase in total irradiation dose from 1.8 to 2.5 MRad had insignificant effect on the polymer and microspheres properties analyzed.

Conclusions

Gamma irradiation is a plausible approach to provide a terminally sterilized, self-healing encapsulation PLGA excipient for vaccine delivery.     

Co-delivery of Vascular Endothelial Growth Factor and Angiopoietin-1 Using Injectable Microsphere/Hydrogel Hybrid Systems for Therapeutic Angiogenesis

Purpose

We hypothesized that combined delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) using microsphere/hydrogel hybrid systems could enhance mature vessel formation compared with administration of each factor alone.

Methods

Hybrid delivery systems composed of alginate hydrogels and poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres containing angiogenic factors were prepared. The release behavior of angiogenic factors from hybrid systems was monitored in vitro. The hybrid systems were injected into an ischemic rodent model, and blood vessel formation at the ischemic site was evaluated.

Results

The sustained release over 4 weeks of both VEGF and Ang-1 from hybrid systems was achieved in vitro. Co-delivery of VEGF and Ang-1 was advantageous to retain muscle tissues and significantly induced vessel enlargement at the ischemic site, compared to mice treated with either VEGF or Ang-1 alone.

Conclusions

Sustained and combined delivery of VEGF and Ang-1 significantly enhances vessel enlargement at the ischemic site, compared with sustained delivery of either factor alone. Microsphere/hydrogel hybrid systems may be a promising vehicle for delivery of multiple drugs for many therapeutic applications.     

Microspheres Prepared with PLGA Blends for Delivery of Dexamethasone for Implantable Medical Devices

Purpose

To develop and characterize microspheres using poly (lactic-co-glycolic acid) (PLGA) blends (PLGA5050 (25 KD) and PLGA6535 (70 KD)) for dexamethasone delivery to prevent foreign body response to implantable biosensors.

Methods

A single emulsion based oil/water solvent evaporation/extraction method was used to prepare microspheres.

Results

All the microspheres prepared exhibited the typical triphasic release profile, but with different initial burst release, lag phase and zero order release rates. The burst release was reduced when the two PLGA were mixed at a molecular level, whereas increase in burst release was observed when phase separation occurred. Microspheres prepared using PLGA blends had significantly shorter lag phase. The activation energy (Ea) of dexamethasone release from microspheres was similar to the Ea value of PLGA degradation. The release kinetics were significantly enhanced under accelerated conditions (45 and 53°C) without altering the release mechanism of the post-burst phase. A rank order correlation between accelerated and “real-time” release kinetics was observed.

Conclusions

Polymer blends of PLGA can produce microspheres with reduced lag time. The accelerated release testing conditions investigated can discriminate the formulations and predict “real-time” release. Such accelerated release testing can be used as a rapid screening method to facilitate formulation development.     

Bone Morphogenetic Protein-2 Release from Composite Hydrogels of Oligo(poly(ethylene glycol) fumarate) and Gelatin

Purpose

Hydrogel composites of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (GMs) were investigated as carriers of bone morphogenetic protein-2 (BMP-2) for bone tissue engineering applications.

Methods

Hydrogel composites with different physical characteristics were prepared by changing the amount and type (acidic vs. basic) of gelatin incorporated in the OPF bulk phase. Composites with differing physical properties (degradation, swelling, and mechanical properties) and differing BMP-2 loading phase were investigated to determine the effect of these factors on BMP-2 release profiles over 28 days.

Results

Overall, higher gelatin amount increased the degradation and swelling of composites, and acidic GMs further increased the degradation and swelling and reduced the compressive modulus of the composites. The most significant factor affecting the release of BMP-2 from composites was the loading phase of the growth factor: GM loading reduced the burst release, increased BMP-2 release during the later phases of the experiment, and increased the cumulative release in faster degrading samples.

Conclusions

The results indicate that the physical properties and the BMP-2 release kinetics of hydrogel composites can be controlled by adjusting multiple parameters at the time of the hydrogel composite fabrication.     

Evaluation of Wound Healing Potential of β-Chitin Hydrogel/Nano Zinc Oxide Composite Bandage

Purpose

β-chitin hydrogel/nZnO composite bandage was fabricated and evaluated in detail as an alternative to existing bandages.

Methods

β-chitin hydrogel was synthesized by dissolving β-chitin powder in Methanol/CaCl₂ solvent, followed by the addition of distilled water. ZnO nanoparticles were added to the β-chitin hydrogel and stirred for homogenized distribution. The resultant slurry was frozen at 0°C for 12 h. The frozen samples were lyophilized for 24 h to obtain porous composite bandages.

Results

The bandages showed controlled swelling and degradation. The composite bandages showed blood clotting ability as well as platelet activation, which was higher when compared to the control. The antibacterial activity of the bandages were proven against Staphylococcus aureus (S. aureus) and Escherichia coli (E.coli). Cytocompatibility of the composite bandages were assessed using human dermal fibroblast cells (HDF) and these cells on the composite bandages were viable similar to the Kaltostat control bandages and bare β-chitin hydrogel based bandages. The viability was reduced to 50–60% in bandages with higher concentration of zinc oxide nanoparticles (nZnO) and showed 80–90% viability with lower concentration of nZnO. In vivo evaluation in Sprague Dawley rats (S.D. rats) showed faster healing and higher collagen deposition ability of composite bandages when compared to the control.

Conclusions

The prepared bandages can be used on various types of infected wounds with large volume of exudates.     

Functionalized three-arm poly(ε-caprolactone) maleic acid microspheres for controlled protein release

Introduction

The objective of this work was to fabricate a novel class of protein carriers from double-bond-functionalized multiarm poly(ε-caprolactone) maleic acid (PGCLM) microspheres and to examine protein sustained-release profiles in vitro over a period of a few months.

Methods

The double-emulsion technique was used to formulate terminal functionalized three-arm PGCLM microspheres having three different types of functional groups (-OH, -COOH, and -C = C-), and one of the functional groups (>C = C< bonds) was used to formulate surface-crosslinked microspheres (NPGCLM). Ovalbumin (OVA) was used as a model protein for examining its release profiles from PGCLM and NPGCLM microspheres in 0.1M phosphate-buffered saline (PBS) at 37°C. These microspheres were also characterized in terms of their morphology, size distribution, and stability.

Results

The mean size of fabricated microspheres ranged from 21.9µm to 51.1µm. An OVA protein was successfully encapsulated into these biodegradable PGCLM microspheres with loading efficiency ranging from 34.2% to 46% (w/w), depending on the ratio of PGCLM to polyvinyl alcohol (PVA) stabilizer. Depending on the polymer to PVA stabilizer ratio, the cumulative OVA release % (w/w) in 0.1M PBS at 37°C ranged from 30% to 40% within 50 days. We further demonstrated the availability of the functional >C = C< bonds on the surface of PGCLM microspheres, which we expect could be used for either covalent binding of bioactive agents or imparting different chemical characteristics onto the surface of the microspheres for broadening of their applications.

Discussion/Conclusion

A method for the preparation of biodegradable microspheres from water/oil emulsion of multiarm and functionalized poly(ε-caprolactone) [PGCL, PGCLM, and NPGCLM] was reported as a potential means of developing injectable therapeutic formulations for drugs. The most unique aspect of these biodegradable microspheres is the availability of two functional groups (>C = C< bonds and -COOH pendant group) on the microsphere surface for potential additional chemical or/and biochemical modifications, such as the formation of crosslinked network surface structure. The incorporation of terminal hydrophilic functional maleic acid into hydrophobic three-arm PGCL chains significantly improved the loading efficiency of OVA and its release profiles. These functionalized biodegradable microspheres may provide a good alternative to linear aliphatic polyester-based drug delivery systems and resulted in development of a long-acting, injectable drug carrier for sustained drug release over a period of few months.     

Computer Modeling Assisted Design of Monodisperse PLGA Microspheres with Controlled Porosity Affords Zero Order Release of an Encapsulated Macromolecule for 3 Months

Purpose

The aim of this study was the development of poly(D,L-lactide-co-glycolide) (PLGA) microspheres with controlled porosity, to obtain microspheres that afford continuous release of a macromolecular model compound (blue dextran).

Methods

PLGA microspheres with a size of around 40 μm and narrow size distribution (span value of 0.3) were prepared with a double emulsion membrane emulsification method. Gene expression programming (GEP) analysis was applied to design and formulate a batch of microspheres with controlled porosity that shows continuous release of blue dextran.

Results

Low porous microspheres with a high loading efficiency were formed at high polymer concentrations (30% w/w in the oil phase) and were characterized with a burst release <10% and a three-phasic release profile of blue dextran. Increasing porosity (10% w/w polymer concentrations), a sustained release of blue dextran was obtained albeit with up to 40% of burst release. The desired formulation, calculated by GEP, resulted in microspheres with 72% loading efficiency and intermediate porosity. Blue dextran was indeed released continuously in almost a zero order manner over a period of 3 months after an initial small burst release of 9%.

Conclusions

By fine-tuning the porosity, the release profile of PLGA microspheres for macromolecules can be predicted and changed from a three-phasic to a continuous release.     

A Floating Hydrogel System Capable of Generating CO2 Bubbles to Diminish Urinary Obstruction After Intravesical Instillation

Aim

Intravesical instillation is commonly used to decrease the tumor recurrence after transurethral resection. However, most drug solutions would be eliminated from bladder after the first voiding of urine, so its clinical efficacy is limited. To overcome this obstacle, we developed a floating hydrogel system for controlled delivery of antitumor drugs.

Methods

The floating hydrogel was made of Adriamycin, thermo-sensitive polymer (Poloxamer 407) and NH₄HCO₃, which was liquid at low temperature while forming hydrogel at high temperature. Meanwhile, at high temperature, NH₄HCO₃ decomposed to produce CO₂ bubbles, which helped hydrogel float in bladder without urinary obstruction.

Results

The mixture containing 45% P407 and 6% NH₄HCO₃ was selected as the optimal formulation. At 37°C, the mixture formed hydrogel and produced many bubbles which could be observed by B ultrasound. The vitro study showed that the antitumor drug Doxorubicin was released in a controlled manner. After the mixture was instilled into rabbit bladder, it formed hydrogel and floated in the bladder. The bladder stimuli was reduced and antitumor drugs could be released continuously in the bladder.

Conclusion

Our results suggested that the floating hydrogel was a feasible intravesical drug delivery system and may have application prospects in intravesical therapy for bladder cancer.     

Comparative Studies on the Influences of Primary Emulsion Preparation on Properties of Uniform-Sized Exenatide-Loaded PLGA Microspheres

Purpose

It is well known that primary emulsion (W₁/O) preparation process (by ultrasonication or homogenization) plays an important role in the properties of drug-loaded microspheres, such as encapsulation efficiency, release behavior and pharmacodynamics. However, its involved mechanism has not been intensively and systematically studied, partly because that broad size distribution of the resultant particles prepared by conventional preparation can greatly disturb the analysis and reliability of the results. Here, we focused on the relevant studies.

Methods

In order to eliminate the disturbance caused by broad size distribution, uniform-sized exenatide-loaded poly(DL-lactic-co-glycolic acid) (PLGA) microspheres were prepared by Shirasu Porous Glass (SPG) premix membrane emulsification. The properties of microspheres whose W₁/O was formed by ultrasonication (UMS) and homogenization (HMS) were compared including in vitro release, pharmacology and so forth.

Results

HMS exhibited fast release rate and hyperglycemic efficacy within first 14 days, but declined afterwards. Comparatively, UMS showed slower polymer degradation, more acidic microclimate pH (μpH) in vitro, and stable drug release with sustained efficacy during 1 month in vivo.

Conclusions

HMS was desirable for the 2-week-sustained release in vivo, while UMS was more appropriate for the longer time release (about 1 month). These comparative researches can provide guidance for emulsion-microsphere preparation routs in pharmaceutics.     

Exploring the Solid-Form Landscape of Pharmaceutical Hydrates: Transformation Pathways of the Sodium Naproxen Anhydrate-Hydrate System

Purpose

To understand the transformation pathways amongst anhydrate/hydrate solid forms of sodium naproxen and to highlight the importance of a polymorphic dihydrate within this context.

Methods

Multi-temperature dynamic vapour sorption (DVS) analysis combined with variable-humidity X-ray powder diffraction (XRPD) to establish the transformation pathways as a function of temperature and humidity. XRPD and thermogravimetric analysis (TGA) to characterise bulk samples. Monitoring of in-situ dehydration using solid-state ¹³C CP/MAS spectroscopy.

Results

At 25°C, anhydrous sodium naproxen (AH) transforms directly to one dihydrate polymorph (DH-II). At 50°C, AH transforms stepwise to a monohydrate (MH) then to the other dihydrate polymorph (DH-I). DH-II transforms to a tetrahydrate (TH) more readily than DH-I transforms to TH. Both dihydrate polymorphs transform to the same MH.

Conclusions

The properties of the polymorphic dihydrate control the transformation pathways of sodium naproxen.     

Combination of Hyaluronic Acid Hydrogel Scaffold and PLGA Microspheres for Supporting Survival of Neural Stem Cells

Purpose

To develop a biomaterial composite for promoting proliferation and migration of neural stem cells (NSCs), as well as angiogenesis on the materials, to rescue central nervous system (CNS) injuries.

Methods

A delivery system was constructed based on cross-linked hyaluronic acid (HA) hydrogels, containing embedded BDNF and VEGF-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for controlled delivery and support for NSCs in the CNS. The surface morphologies were evaluated by SEM and AFM, mechanical property was investigated by rheological tests, and release kinetics were performed by ELISA. Bioactivity of released BDNF and VEGF was assessed by neuron and endothelial cell culture, respectively. Compatibility with NSCs was studied by immunofluorescent staining.

Results

Release kinetics showed the delivery of BDNF and VEGF from PLGA microspheres and HA hydrogel composite were sustainable and stable, releasing ~20–30% within 150 h. The bioactivities preserved well to promote survival and growth of the cells. Evaluation of structure and mechanical properties showed the hydrogel composite possessed an elastic scaffold structure. Biocompatibility assay showed NSCs adhered and proliferated well on the hydrogel.

Conclusions

Our created HA hydrogel/PLGA microsphere systems have a good potential for controlled delivery of varied biofactors and supporting NSCs for brain repair and implantation.

     

Formulating Weakly Basic HCl Salts: Relative Ability of Common Excipients to Induce Disproportionation and the Unique Deleterious Effects of Magnesium Stearate

Purpose

Nine common excipients were examined to determine their ability to cause disproportionation of the HCl salt of a a weakly basic compound. The goal was to determine which excipients were problematic and correlate the results to known properties such as surface pH, slurry pH, or molecular structure. Such a correlation enables a general, simple excipient selection process.

Methods

Binary compacts and “pseudo formulations” are studied after stressing at 40°C/75%RH and 40°C/35% RH for up to 28 days. Near-Infrared (NIR) and X-Ray powder diffraction (XRPD) measurements monitored the conversion of the HCl salt to the free base.

Results

The excipients which induced measureable disproportionation were magnesium stearate, sodium croscarmellose, and sodium stearyl fumarate. Magnesium stearate induced the most extensive and rapid disproportionation at 40°C/75%RH and 40°C/35%RH. Samples containing magnesium stearate showed a unique and significant water uptake above 31%RH.

Conclusions

The problematic excipients are best explained by the proton accepting capacity of excipient carboxylate groups which have pK_a’s higher than the pH_max of the drug salt. Alternative lubricants and disintegrants are suggested and a simple excipient screening process is proposed. Magnesium stearate was the most deleterious excipient for HCl salts due to the formation of the deliquescent salt magnesium chloride.     

Effects of Ionic Strength and Sugars on the Aggregation Propensity of Monoclonal Antibodies: Influence of Colloidal and Conformational Stabilities

Purpose

To develop a general strategy for optimizing monoclonal antibody (MAb) formulations.

Methods

Colloidal stabilities of four representative MAbs solutions were assessed based on the second virial coefficient (B ₂) at 20°C and 40°C, and net charges at different NaCl concentrations, and/or in the presence of sugars. Conformational stabilities were evaluated from the unfolding temperatures. The aggregation propensities were determined at 40°C and after freeze–thawing. The electrostatic potential of antibody surfaces was simulated for the development of rational formulations.

Results

Similar B ₂ values were obtained at 20°C and 40°C, implying little dependence on temperature. B ₂ correlated quantitatively with aggregation propensities at 40°C. The net charge partly correlated with colloidal stability. Salts stabilized or destabilized MAbs, depending on repulsive or attractive interactions. Sugars improved the aggregation propensity under freeze–thaw stress through improved conformational stability. Uneven and even distributions of potential surfaces were attributed to attractive and strong repulsive electrostatic interactions.

Conclusions

Assessment of colloidal stability at the lowest ionic strength is particularly effective for the development of formulations. If necessary, salts are added to enhance the colloidal stability. Sugars further improved aggregation propensities by enhancing conformational stability. These behaviors are rationally predictable according to the surface potentials of MAbs.     

Spatial Characterization of Hot Melt Extruded Dispersion Systems Using Thermal Atomic Force Microscopy Methods: The Effects of Processing Parameters on Phase Separation

Purpose

In this study we explore the use of nano-scale localized thermal analysis (LTA) and transition temperature microcopy (TTM) as a novel combined approach to studying phase separation in HME dispersions of cyclosporine A in Eudragit EPO.

Methods

Modulated temperature differential scanning calorimetry (MTDSC), attenuated total reflectance FTIR spectroscopy, nano-LTA and TTM were performed on raw materials and dispersions prepared by hot melt extrusion (HME) and spin coating. For samples prepared by HME, two mixing temperatures (110°C and 150°C) and residence times (5 and 15 min) were investigated.

Results

Spin coated samples showed an intermediate T _g for the mixed systems consistent with molecular dispersion formation. The HME samples prepared at 110°C showed evidence of inhomogeneity using MTDSC and FTIR, while those produced at 150°C h showed evidence for the formation of a single phase system using MTDSC. The nanothermal methods, however, indicated the presence of phase separated cyclosporine A at the higher preparation temperature while the TTM was able to map regions of differing penetration temperatures, indicating the presence of compositionally inhomogeneous regions in all but the high processing temperature/high residence time samples.

Conclusions

TTM is a potentially important new method for studying phase separation and that such separation may remain undetected or poorly understood using conventional bulk analytical techniques.     

Toxicity Studies of Poly(Anhydride) Nanoparticles as Carriers for Oral Drug Delivery

Purpose

To evaluate the acute and subacute toxicity of poly(anhydride) nanoparticles as carriers for oral drug/antigen delivery.

Methods

Three types of poly(anhydride) nanoparticles were assayed: conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) and nanoparticles coated with poly(ethylene glycol) 6000 (PEG-NP). Nanoparticles were prepared by a desolvation method and characterized in terms of size, zeta potential and morphology. For in vivo oral studies, acute and sub-acute toxicity studies were performed in rats in accordance to the OECD 425 and 407 guidelines respectively. Finally, biodistribution studies were carried out after radiolabelling nanoparticles with ^99mtechnetium.

Results

Nanoparticle formulations displayed a homogeneous size of about 180 nm and a negative zeta potential. The LD₅₀ for all the nanoparticles tested was established to be higher than 2000 mg/kg bw. In the sub-chronic oral toxicity studies at two different doses (30 and 300 mg/kg bw), no evident signs of toxicity were found. Lastly, biodistribution studies demonstrated that these carriers remained in the gut with no evidences of particle translocation or distribution to other organs.

Conclusions

Poly(anhydride) nanoparticles (either conventional or modified with HPCD or PEG6000) showed no toxic effects, indicating that these carriers might be a safe strategy for oral delivery of therapeutics.     

Enhancing Effect of Glucose Microspheres in the Viability of Human Mesenchymal Stem Cell Suspensions for Clinical Administration

Purpose

A critical limiting factor of cell therapy is the short life of the stem cells. In this study, glucose containing alginate microspheres were developed and characterized to provide a sustained release system prolonging the viability of human mesenchymal stem cells (hMSCs) in a suspension for clinical application.

Methods

The glucose microspheres were satisfactorily elaborated with alginate by emulsification/internal gelation method. Particle size was evaluated by light diffraction and optical microscopy. Shape and surface texture by scanning electron microscopy (SEM). Zeta potential, infrared spectra and release studies were also conducted. Also, rheological properties and stability of hMSCs suspensions with microspheres were tested. The viability of hMSCs was determined by trypan blue dye exclusion staining.

Results

Microspheres of 86.62 μm, spherical shaped and ?32.54 mV zeta potential with excellent stability, good encapsulation efficiency and providing an exponential release of glucose were obtained. hMSCs had better survival rate when they were packed with glucose microspheres. Microspheres maintained the aseptic conditions of the cell suspension without rheological, morphological or immunophenotypic disturbances on hMSCs.

Conclusions

Developed microspheres were able to enhance the functionality of hMSC suspension. This strategy could be broadly applied to various therapeutic approaches in which prolonged viability of cells is necessary.     

Bioactive Long-Term Release from Biodegradable Microspheres Preserves Implanted ALG-PLO-ALG Microcapsules from In Vivo Response to Purified Alginate

Purpose

To assess whether prevention of unexpected in vivo adverse inflammatory and immune responses to biohybrid organ grafts for the treatment of Type I Diabetes Mellitus (T1DM) is possible by superoxide dismutase and ketoprofen controlled release.

Methods

Superoxide dismutase and ketoprofen-loaded polyester microspheres were prepared by W/O/W and O/W methods, embodied into purified alginate-poly-L-ornithine-alginate microcapsules and intraperitoneally implanted into CD1 mice. The microspheres were characterized for morphology, size, encapsulation efficiency, enzyme activity and in vitro release. Purified alginate contaminants were assayed, and the obtained microcapsules were investigated for size and morphology before and after implantation over 30 days. Cell pericapsular overgrowth and expression were evaluated by optical microscopy and flow cytometry.

Results

Superoxide dismutase and ketoprofen sustained release reduced cell pericapsular overgrowth in comparison to the control. Superoxide dismutase release allowed preserving the microcapsules over 30 days. Ketoprofen-loaded microspheres showed some effect in the immediate post-grafting period. A higher macrophage and T-cell expression was observed for the control group.

Conclusions

Microspheres containing superoxide dismutase and ketoprofen may represent novel tools to limit or prevent unpredictable adverse in vivo response to alginate, thus contributing to improve cell transplantation success rates in T1DM treatment.     

Lysozyme Release and Polymer Erosion Behavior of Injectable Implants Prepared from PLGA-PEG Block Copolymers and PLGA/PLGA-PEG Blends

Purpose

We evaluated the controlled release of lysozyme from various poly(D,L-lactic-co-glycolic acid) (PLGA) 50/50-polyethylene glycol (PEG) block copolymers relative to PLGA 50/50.

Methods

Lysozyme was encapsulated in cylindrical implants (0.8 mm diameter) by a solvent extrusion method. Release studies were conducted in phosphate buffered saline +0.02% Tween 80 (PBST) at 37°C. Lysozyme activity was measured by a fluorescence-based assay. Implant erosion was evaluated by kinetics of polymer molecular weight decline, water uptake, and mass loss.

Results

Lysozyme release from an AB15 di-block copolymer (15% 5 kDa PEG, PLGA 28 kDa) was very fast, whereas an AB10 di-block copolymer (with 10% 5 kDa PEG, PLGA 45 kDa) and ABA10 tri-block copolymer (with 10% 6 kDa PEG, PLGA 27 kDa) showed release profiles similar to PLGA. We achieved continuous lysozyme release for up to 4 weeks from AB10 and ABA10 by lysozyme co-encapsulation with the pore-forming and acid-neutralizing MgCO₃, and from AB15 by co-encapsulation of MgCO₃ and blending AB15 with PLGA. Lysozyme activity was mostly recovered during 4 weeks.

Conclusions

These block co-polymers may have utility either alone or as PLGA blends for the controlled release of proteins.