Microsphere Release Research Articles

Iron Chelators Augment Large Osteochondral Allograft Osseointegration in a Preclinical Canine Model.

Osteochondral allograft transplantation (OCAT) can be a successful joint restoration treatment option for large post-traumatic articular defects but is still associated with significant revision and failure rates. Despite recent advances that have improved OCAT success, insufficient osteochondral allograft (OCA) osseointegration remains a major cause of failure. Deferoxamine (DFO) is an effective angiogenic and osteo-anabolic iron chelator that consistently promotes bone neovascularization and regeneration. This study was designed to investigate local delivery of DFO for augmenting OCA osseointegration using a preclinical canine model for OCAT in the knee and hip as commonly affected joints. On Institutional Animal Care and Use Committee (IACUC) approval, 12 purpose-bred dogs underwent OCAT of the femoral head or femoral condyles with DFO or DFO-free (controls) microspheres in recipient sites. OCA revascularization, cellular repopulation, and integration were evaluated based on functional, diagnostic imaging, microcomputed tomography, histology, and immunohistochemistry outcome measures. Local delivery of DFO into OCAT recipient sites was associated with maintained or improved joint function, superior radiographic appearance, significantly greater trabecular thickness, higher bone volume, and new bone ingrowth compared with DFO-free controls. OCA osseointegration is dependent on cellular repopulation and neovascularization, resulting in new bone ingrowth through creeping substitution, and insufficient osseointegration with resorption and subsidence of the OCA remains a major cause of failure after transplantation. The results of this study suggest that local delivery of DFO using a controlled microsphere release system may reduce resorption and improve revascularization and cellular repopulation to increase new bone ingrowth, potentially expediting OCA osseointegration after transplantation.

Performance assessment of a modular device for micro-sphere singularization

A wide variety of mechatronic products undergoes major changes to miniaturize them and integrate a larger number of functions. They require advanced technologies for their manipulation and assembly, that become even more challenging due to the different properties and tiny size of their components. Innovative devices are necessary to enable the automation of such operations, such as sorting and feeding systems able to singularize and deliver micro-parts precisely and reliably at high-speed. To this aim, an original “Store&Place device” was developed for micro-sphere singularization and deposition (Fontana et al., 2018) [3], based on the novel operating principle proposed in Fontana et al. (2017) [1]. A potential application of this device concerns the automatic rework of electronic components, more specifically the reballing of Ball Grid Array packages, that basically consists in restoring their electrical connection to the board by attaching new solder balls. When the release of a single solder ball is commanded, the device singularizes it from a storage of identical balls and places it onto the corresponding circular land on the package.The paper presents the assessment of the device performance resulting from a structured experimental analysis on the manipulation of micro-spheres with diameter of 300 μm. It was applied to highlight the main process parameters influencing its performance, optimize them, and enhance the tool use. The performance indices to quantitatively evaluate the precision of the micro-sphere release were calculated according to Ruggeri et al. (2019) [5], and merged with other indices related to the reliability of the release and the time needed to execute it.

Design of novel orotransmucosal vaccine-delivery platforms using artificial intelligence

The linings of the oral cavity are excellent needle-free vaccination sites, able to induce immune responses at distal sites and confer systemic protection. However, owing to the mucosal tissues’ intrinsic characteristics, the design of effective antigen-delivery systems is not an easy task. In the present work, we propose to develop and characterize thermosensitive and mucoadhesive hydrogels for orotransmucosal vaccination taking advantage of artificial intelligence tools (AIT). Hydrogels of variable composition were obtained combining Pluronic® F127 (PF127), Hybrane® S1200 (HS1200) and Gantrez® AN119 (AN119) or S97 (S97). Systems were characterized in terms of physicochemical properties, adhesion capacity to mucosal tissues and antigen-like microspheres release. Additionally, polymers biocompatibility and their immune-stimulation capacity was assessed in human macrophages. Interestingly, cells treated with HS1200 exhibited a significant proliferation enhancement compared to control. The use of AIT allowed to determine the effect of each polymer on formulations properties. The proportions of PF127 and Gantrez® are mainly the factors controlling gelation temperature, mucoadhesion, adhesion work and gel strength. Meanwhile, cohesion and short-term microsphere release are dependent on the PF127 concentration. However, long-term microsphere release varies depending on the Gantrez® variety and the PF127 concentration used. Hydrogels prepared with S97 showed slower microsphere release. The use of AIT allowed to establish the conditions able to produce ternary hydrogels with immune-stimulatory properties together with adequate mucoadhesion capacity and antigen-like microspheres release.

Microspheres scar formation glaucoma filtration surgery area therapeutic effect—Based on rapamycin-chitosan-calcium alginate sustained-release

To study the efficacy of rapamycin (RAPA)-chitosan (CS)-calcium alginate (CA) sustained-release microspheres on scar formation in a rabbit model of glaucoma filtration surgery. Eighty New Zealand white rabbits were randomly divided into four groups and a glaucoma filtration model was established by scleral bite through the eyes. RAPA-CS-CA sustained-release microspheres were implanted in the right eye of 40 rabbits (Group A) and CS blank sustained-release microspheres were implanted in the left eye (Group B). Another 40 rabbits were treated with a 0.2 g·L-1 RAPA cotton sheet in the right eye (Group C) and the left eye underwent a simple sclerotomy (Group D). The intraocular pressure, filter bleb, anterior chamber inflammation, complications, and corneal endothelial cell density were observed after the operation. Rabbits were euthanized for pathological examination 7 days, 14 days, and 21 days after the operation. The drug loading rate of RAPA-CS-CA sustainedrelease microspheres was (34.58±1.47)% and the encapsulation rate was (56.23±1.55)%. The microsphere release in vitro was relatively stable. The release rate of the microspheres during the burst was only 16.54%. After 49 days, the cumulative release rate of the microspheres reached 94.07% and the sustained release effect was significant within 45 days. Group A maintained low-level intraocular pressure for the longest period of time, followed by Group C, and then Group B and D. The survival time of filter vesicles in Group A was longer than that in other groups. There were no postoperative complications in each group. The conjunctival epithelium of Group A had better integrity and fewer subconjunctival fibroblasts than other groups. There was no obvious inflammation or infiltration around the filtering mouth and there were fewer new collagen fibers. RAPA-CS-CA slow-release microspheres safely and effectively inhibited the proliferation of fibroblasts and neonatal collagen fibers in rabbit glaucoma filtration surgery and significantly improved the success rate of glaucoma filtration surgery.

Development of Level A in vitro-in vivo correlations for peptide loaded PLGA microspheres

Peptide loaded PLGA microsphere products are more complex in terms of manufacturing, drug release characteristics as well as release mechanism compared to small molecule loaded PLGA microsphere products. This is due to the complex structure of peptides, their hydrophilicity, charged state, large size and potential for instability. Moreover, therapeutic peptides are highly potent and therefore, any unintended change in the microsphere release profile may lead to undesirable side effects and toxicity. Accordingly, the objectives of the present work were: 1) to evaluate the impact of minor manufacturing changes on the quality and performance of peptide microspheres; and 2) to investigate the feasibility of developing Level A in vitro-in vivo correlations (IVIVCs) for peptide microspheres. Compositionally equivalent leuprolide acetate (LA) microspheres prepared with minor manufacturing changes (solvent system/homogenization speed) showed significant differences in their physicochemical properties (such as pore size, total porosity, particle size and surface distribution of peptide on the prepared microspheres). This, in turn, resulted in significant alteration in the release characteristics. Peptide-polymer interaction, in vitro degradation and microsphere morphology studies were conducted to facilitate understanding of the differences in the drug release characteristics. A rabbit model was used to determine the pharmacokinetic profiles of all the prepared formulations. The obtained in vivo release profiles showed the same rank order as the in vitro release profiles but with low burst release and overall faster in vivo release rates. The low in vivo burst release is considered to be due to the masking effect of the absorption phase from the intramuscular site, and this complicated the development of an IVIVC. Despite these challenges, an affirmative Level A IVIVC over the entire release profile was successfully developed in a rabbit model for peptide microspheres for the first time. The developed IVIVC was also predictive of the RLD product, Lupron Depot®. This work highlights the feasibility of developing IVIVCs for complex parenteral drug products such as peptide microspheres. In conclusion, these results indicate the sensitivity of peptide release, and hence, the safety and efficacy of highly potent peptide microspheres, to minor manufacturing changes. Accordingly, development of IVIVCs for such complex drug products is highly desirable.

Validation of a cage implant system for assessing invivo performance of long-acting release microspheres.

Here we describe development of a silicone rubber/stainless steel mesh cage implant system, much like that used to assess biocompatibility of biomaterials [1], for easy removal of injectable polymer microspheres invivo. The sterile cage has a type 316 stainless steel mesh size (38μm) large enough for cell penetration and free fluid flow invivo but small enough for microsphere retention, and a silicone rubber shell for injection of the microspheres. Two model drugs, the poorly soluble steroid, triamcinolone acetonide, and the highly water-soluble luteinizing hormone-releasing hormone (LHRH) peptide superagonist, leuprolide, were encapsulated in PLGA microspheres large enough (63-90μm) to be restrained by the cage implant invivo. The invitro release from both formulations was followed by ultra-performance liquid chromatography (UPLC) with and without the cage in a standard release media, PBS pH 7.4+0.02% Tween 80+0.05% sodium azide, at 37°C. Pharmacokinetics (PK) in rats was assessed after SC injection or SC in-cage implantation of microspheres with plasma analysis by LC-MS/MS or EIA. Tr-A and leuprolide invitro release was largely unaffected after the initial burst irrespective of the cage or test tube incubation vessel and release was much slower than observed invivo for both drugs. Moreover, Tr-A and leuprolide pharmacokinetics with and without the cage were highly similar during the 2-3 week release duration before a significant inflammatory response was caused by the cage implant. Hence, the PK-validated cage implant provides a simple means to recover and evaluate the microsphere drug carriers invivo during a time window of at least a few weeks in order to characterize the polymer microsphere release and erosion behavior invivo. This approach may facilitate development of mechanism-based invitro/invivo correlations and enable development of more accurate and useful invitro release tests.

To which extent do rain interruption periods affect colloid retention in macroporous soils?

Colloid retention in soils has mainly been studied in model homogeneous porous media, and during single irrigation events. However, in the field, colloidal suspensions flow through a small number of preferential flow paths, and the soil experiences succession of rainfalls, interrupted by dry periods. We therefore performed series of successive rainfalls (21mmh−1) on three decimetric undisturbed luvisol E-horizon cores to systematically study the impact of the rainfall interruption duration (RID) on retention. Successive rains were doped with 0.5μm fluorescent microspheres of different colors. Microsphere release at the base of the soil column was monitored by flow cytometry. When preferential macropore flow was preponderant, increasing the RID from 5 to 1200h led to the retention of up to an additional 27% of the microspheres rained onto the soil. This increased retention was ascribed to a higher absorption of the colloidal suspension into the macropore walls for longer RIDs, when water redistribution far from the macropores was more important. When matrix flow was preponderant, RID effect on retention was overwhelmed and massive microspheres retention was observed. These results are useful to provide guidelines for the safe use of reclaimed water in agriculture, and to estimate the importance of clay translocation (a colloid transfer based soil formation mechanism).

Functionalized PLGA-doped zirconium oxide ceramics for bone tissue regeneration

Bone tissue engineering is an alternative approach to bone grafts. In our study we aim to develop a composite scaffold for bone regeneration made of doped zirconium oxide (ZrO2) conjugated with poly(lactic-co-glycolic acid) (PLGA) particles for the delivery of growth factors. In this composite, the PLGA microspheres are designed to release a crucial growth factor for bone formation, bone morphogenetic protein-2 (BMP2). We found that by changing the polymer's molecular weight and composition, we could control microsphere loading, release and size. The BMP2 released from PLGA microspheres retained its biological activity and increased osteoblastic marker expression in human mesenchymal stem cells (hMSCs). Uncapped PLGA microspheres were conjugated to ZrO2 scaffolds using carbodiimide chemistry, and the composite scaffold was shown to support hMSCs growth. We also demonstrated that human umbilical vein endothelial cells (HUVECs) can be co-cultured with hMSCs on the ZrO2 scaffold for future vascularization of the scaffold. The ZrO2 composite scaffold could serve as a bone substitute for bone grafting applications with the added ability of releasing different growth factors needed for bone regeneration.

A New Catheter for Tumor-Targeting With Radioactive Microspheres in Representative Hepatic Artery Systems—Part II: Solid Tumor-Targeting in a Patient-Inspired Hepatic Artery System

In this second part, the methodology for optimal tumor-targeting is further explored, employing a patient-inspired hepatic artery system which differs significantly from the idealized configuration discussed in Part I. Furthermore, the fluid dynamics of a microsphere supply apparatus is also analyzed. The best radial catheter positions and particle-release intervals for tumor targeting were determined for both the idealized and patient-inspired configurations. This was accomplished by numerically analyzing generated particle release maps (PRMs) for ten equally spaced intervals throughout the pulse. As in Part I, the effects of introducing a catheter were also investigated. In addition to the determination of micro-catheter positioning and, hence, optimal microsphere release, a microsphere-supply apparatus (MSA) was analyzed, which transports the particles to the catheter-nozzle, considering different axial particle injection functions, i.e., step, ramp, and S-curve. A refined targeting methodology was developed which demonstrates how the optimal injection region and interval can be determined with the presence of a catheter for any geometric configuration. Additionally, the less abrupt injection functions (i.e., ramp and S-curve) were shown to provide a more compact particle stream, making them better choices for targeting. The results of this study aid in designing the smart micro-catheter (SMC) in conjunction with the MSA, bringing this innovative treatment procedure one step closer to implementation in clinical practice.

Computer Modeling of Controlled Microsphere Release and Targeting in a Representative Hepatic Artery System

Combating liver tumors via yttrium-90 ((90)Y) radioembolization is a viable treatment option of nonresectable liver tumors. Employing clinical (90)Y microparticles (i.e., SIR-Spheres and TheraSpheres) in a computational model of a representative hepatic artery system, laminar transient 3D particle-hemodynamics were simulated. Specifically, optimal particle release positions in the right hepatic (parent) artery as well as the best temporal release window were determined for the microspheres to exit specific outlet daughter vessels, potentially connected to liver tumors. The results illustrate the influence of a curved geometry on the velocity field and the particle trajectory dependence on the spatial and temporal particle injection conditions. The differing physical particle characteristics of the SIR-Spheres and the TheraSpheres had a subtle impact on particle trajectories in the decelerating portion of the arterial pulse, i.e., when the inertial forces on the particles are weaker. Conversely, particle characteristics and inelastic wall collisions had little effect on particles released during the accelerating phase of the arterial pulse, i.e., both types of microspheres followed organized paths to predetermined outlets. Such results begin paving the way towards directing 100% of the released microspheres to specific daughter vessels (e.g., those connected to tumors) under transient flow conditions in realistic geometries via a novel drug-particle targeting methodology.

Computer Modeling of Yttrium-90–Microsphere Transport in the Hepatic Arterial Tree to Improve Clinical Outcomes

Radioembolization (RE) via yttrium-90 ((90)Y) microspheres is an effective and safe treatment for unresectable liver malignancies. However, no data are available regarding the impact of local blood flow dynamics on (90)Y-microsphere transport and distribution in the human hepatic arterial system. A three-dimensional (3-D) computer model was developed to analyze and simulate blood-microsphere flow dynamics in the hepatic arterial system with tumor. Supplemental geometric and flow data sets from patients undergoing RE were also available to validate the accuracy of the computer simulation model. Specifically, vessel diameters, curvatures, and branching patterns, as well as blood flow velocities/pressures and microsphere characteristics (i.e., diameter and specific gravity), were measured. Three-dimensional computer-aided design software was used to create the vessel geometries. Initial trials, with 10,000 noninteracting microspheres released into the hepatic artery, used resin spheres 32-microm in diameter with a density twice that of blood. Simulations of blood flow subject to different branch-outlet pressures as well as blood-microsphere transport were successfully carried out, allowing testing of two types of microsphere release distributions in the inlet plane of the main hepatic artery. If the inlet distribution of microspheres was uniform (evenly spaced particles), a greater percentage would exit into the vessel branch feeding the tumor. Conversely, a parabolic inlet distribution of microspheres (more particles around the vessel center) showed a high percentage of microspheres exiting the branch vessel leading to the normal liver. Computer simulations of both blood flow patterns and microsphere dynamics have the potential to provide valuable insight on how to optimize (90)Y-microsphere implantation into hepatic tumors while sparing normal tissue.

Development and characterization of wheat gluten microspheres for use in a controlled release system

The purpose of this study was the development and characterization of wheat gluten microspheres for use as controlled release devices, and the evaluation of the effect of the addition of poly (ethylene glycol) (PEG). Diltiazem hydrochloride was used as the model drug in the in vitro release essay. The physical–chemical and morphological properties of the microspheres were evaluated, as well as their encapsulation efficiency. Porosity varied with the presence or absence of PEG. The diltiazem encapsulation efficiency was 72.8% and 96.7% for wheat gluten and gluten/PEG 95/05 microspheres, respectively. The DSC and FTIR results indicated interactions between the microparticles and additives used. In the in vitro release tests it was observed that, for all the studied systems, the burst effect occurred in the first 2 h of release and the microspheres prepared with PEG had a faster release rate. In the attempt to elucidate the release mechanism, the systems were treated based on two well known mathematical models: the Higuchi and the power law. It was found that the microsphere release mechanism is not exclusively diffusion-controlled and, probably, the release occurs through a combination of partial diffusion through the swelling matrix and hydrophilic pores.

Laser-induced defect insertion in DNA-linked 2D colloidal crystal array

Insertion of vacancies at predetermined sites within the lattice of colloidal crystals is a prerequisite in order to realize high-quality, opaline-based photonic devices. In this contribution, we demonstrate a novel methodology to afford controlled insertion of vacancies within two-dimensional (2D) opaline arrays. These 2D opaline arrays have been substrate-anchored with the help of DNA hybridization. This provides a heat-sensitive ‘adhesive’ between substrate and microspheres within a surrounding aqueous medium that enables tuning the hybridization strength of DNA linker as well as a mechanism to facilitate the removal of unbound microspheres. Focusing a laser beam onto the substrate/microsphere interface induces a localized heating event that detaches the irradiated microspheres, leaving behind vacancies. By repeating this process, line vacancies were successfully obtained. The effects of salt concentration, laser power, light-absorbing dyes, DNA length and refractive-index mismatch were investigated and found to correlate with heat-induced microsphere release.

Novel scaffolds fabricated from protein-loaded microspheres for tissue engineering

Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly( l, d-lactic- co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3±58.0 μm) rather than smaller (11.15±11.08 μm) microspheres, generated pores on the order of 200 μm as compared to 20 μm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4 h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications.

Mathematical Modeling and Optimization of Drug Delivery from Intratumorally Injected Microspheres

Paclitaxel is a highly promising phase-sensitive antitumor drug that could conceivably be improved by extended lower dosing as opposed to intermittent higher dosing. Although intratumoral delivery of paclitaxel to the whole tumor at different loads and rates has already been achieved, determining an optimal release mode of paclitaxel for tumor eradication remains difficult. This study set out to rationally design such an optimal microsphere release mode based on mathematical modeling. A computational reaction-diffusion framework was used to model drug release from intratumorally injected microspheres, drug transport and binding in tumor interstitum, and drug clearance by microvasculature and intracellular uptake and binding. Numerical simulations suggest that interstitial drug concentration is characterized by a fast spatially inhomogeneous rise phase, during which interstitial and intracellular binding sites are saturated, followed by a slow spatially homogeneous phase that is governed by the rate of drug release from microspheres. For zero-order drug release, the slow phase corresponds to a plateau drug concentration that is proportional to the ratio of the rate of blood clearance of drug to the rate of drug release from microspheres. Consequently, increasing the duration of intratumoral drug release extends the duration of cell exposure to the drug but lowers the plateau drug concentration. This tradeoff implies that intratumoral drug release can be designed to optimize tumor cell kill. Synthesizing our modeling predictions with published dose-response data, we propose an optimal protocol for the delivery of paclitaxel-loaded microspheres to small solid tumors.

Application of USP Apparatus 4 and In Situ Fiber Optic Analysis to Microsphere Release Testing

Introduction In the past 15 years,the number of novel drug dosage forms such as controlled release (CR) parenterals (microspheres, liposomes,stents,and other implants) entering to the U.S. market has increased (1). These products can deliver drugs at a desired rate over periods of several days to months. It is essential to develop discriminatory in vitro release testing methods and standards for these delivery systems. In vitro release methods are necessary to monitor batch-to-batch variability,evaluate any process or manufacturing change,ensure the clinical performance of the drug i.e. safety and efficacy and determine a relationship between the in vitro and in vivo release data. Standard dissolution methods are inappropriate for CR parenterals since:these were designed for oral and transdermal products; they utilize large volumes of media,and often require separation of the delivery system from the release media for analysis. Methods of in vitro release testing that are currently used for CR parenterals include membrane diffusion,sample-and-separate, in situ and continuous flow (2). USP apparatus 1 (basket),2 (paddle) and 4 (flow through) have been used for microspheres and other dispersed systems. Sample-and-separate methods have been used where samples are taken from USP Apparatus 2 and the dispersed phase is isolated by centrifugation or filtration. In addition to the official USP Apparatus,other methods such as miniaturized methods and dialysis sacs have been used for CR dispersed systems. Since most of these products are small volume parenterals,miniaturized methods have been designed where the samples are placed into small vials instead of one-liter vessels. Problems with this method include violation of sink conditions,sample aggregation due to limited agitation,and disruption of the delivery system during centrifugation or filtration (e.g. coalescence of emulsion droplets,or aggregation of liposomes) in sample-and-separate methods leading to inaccuracy in the release data. A recent AAPS-EUFEPS workshop report (2003) on “Assuring Quality and Performance of Sustained and Controlled Release Parenterals”stated that there is a need for standards for in vitro release testing methods for controlled release parenterals with respect to apparatus, media,sampling interval,and temperature,and also acknowledged that guidance on in vivo release testing of these products and development of in vitro-in vivo relationships/prediction is necessary (1). In vitro release testing methods for poly (lactic-co-glycolic acid) (PLGA) microspheres are discussed herein. The 2003 AAPS-EUFEPS workshop report recommended the use of USP Apparatus 4 for in vitro release testing of CR microspheres (1). USP Apparatus 4,which is a flow through method that includes a pump,flow-through cells,water bath and media reservoir,was designed as an in vitro dissolution method for controlled release oral powders,granules,and solid dispersions. The specifications with respect to cell size of this apparatus are described in the US Pharmacopeia (3). USP Apparatus 4 can be operated under different conditions such as open or closed system mode;different flow rates and temperatures. The diversity of available cell types allows application to a wide range of dosage forms. Since the reservoir volume is not fixed,media volume can be decreased to accommodate systems where the concentration of drug released would otherwise be below the detection limit (e.g. systems with low drug loading) or increased to allow ease of maintenance of sink conditions for poorly soluble compounds. USP Apparatus 4 also offers flexibility in monitoring release via in situ UV analysis. Such analysis can be achieved without any correction for scattering by the dispersed system or particle accumulation on the probes since the microspheres or other delivery system are isolated Abstract The objective of this study was to determine the applicability of USP Apparatus 4 and in situ fiber optic UV analysis to in vitro release testing of poly (lactic-co-glycolic acid) (PLGA) microspheres. The release of dexamethasone from microspheres prepared with both high and low Mw PLGA was evaluated. Dexamethasone release profiles obtained using USP apparatus 4 were compared with those obtained using a sample-and-separate method. Cumulative release obtained using USP Apparatus 4 was approximately 16% higher over a 30-day period when compared to that obtained using the sample-and-separate method. This difference was attributed to loss of microspheres during separation as well as to microsphere aggregation in the sample-and-separate method. The versatility of USP Apparatus 4 with respect to alteration of flow rate allowed demonstration of diffusion-controlled release from low Mw PLGA microspheres. Fiber optic UV probes used in conjunction with USP Apparatus 4 enabled complete characterization of the initial burst release of drug from the microsphere systems.

In vitro study of GDNF release from biodegradable PLGA microspheres

Glial cell line-derived neurotrophic factor (GDNF) is a protein with potent trophic actions on dopaminergic neurons, which is under investigation as a therapeutic agent for the treatment of neurodegenerative disorders, including Parkinson's disease. The aim of this work was to develop GDNF-loaded microspheres, which could be implanted by stereotaxy in the brain and could offer an alternative strategy in the treatment of Parkinson's disease. A w/o/w extraction–evaporation technique was chosen to prepare protein-loaded microspheres. An in vitro release study of the protein was required to assess the retention of integrity and the performance of the microsphere formulation with regard to sustained release. In order to assess the in vitro release profile of the GDNF-loaded microspheres, a preliminary study was performed to select an appropriate buffer for GDNF stabilization, using experimental designs. GDNF was measured by both enzyme-linked immunosorbant assay (ELISA) and radioactivity using 125I-GDNF. The GDNF-loaded microsphere release profile was assessed in a low continuous flow system, and showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses.

Precise control of PLG microsphere size provides enhanced control of drug release rate

An important limitation in the development of biodegradable polymer microspheres for controlled-release drug delivery applications has been the difficulty of specifically designing systems exhibiting precisely controlled release rates. Because microparticle size is a primary determinant of drug release, we developed a methodology for controlling release kinetics employing monodisperse poly( d,l-lactide-co-glycolide) (PLG) microspheres. We fabricated 20-, 40- and 65-μm diameter rhodamine-containing microspheres and 10-, 50- and 100-μm diameter piroxicam-containing microspheres at various loadings from 1 to 20%. In vitro release kinetics were determined for each preparation. Drug release depended strongly on microsphere diameter with 10- and 20-μm particles exhibiting concave-downward release profiles while larger particles resulted in sigmoidal release profiles. Overall, the rate of release decreased and the duration increased with increasing microsphere size. Release kinetics from mixtures of uniform microspheres corresponded to mass-weighted averages of the individual microsphere release kinetics. Appropriate mixtures of uniform microspheres were identified that provided constant (zero-order) release of rhodamine and piroxicam for 8 and 14 days, respectively. Mixing of uniform microspheres, as well as control of microsphere size distribution, may provide an improved methodology to tailor small-molecule drug-release kinetics from simple, biodegradable-polymer microparticles.

A novel in vitro delivery system for assessing the biological integrity of protein upon release from PLGA microspheres.

The development of a novel in vitro system is required to assess the stability and release kinetics of a protein microsphere formulation used for drug delivery to the brain. Microspheres containing lysozyme as model protein were prepared using a (w/o/w) emulsion-solvent evaporation process. Both the active and total (active + inactive) encapsulation efficiencies and release profiles were determined. The biologic activity of lysozyme was measured using bacterial cell lysis; total protein content was measured using a 125I-radiolabel. A novel in vitro apparatus was developed to determine kinetics over a sustained time period (>30 days). The microencapsulation technique allowed an entrapment of active lysozyme at 80 +/- 4% and a sustained (>42 days) in vitro release. The kinetics study showed that the novel in vitro system was able to detect the release of low amounts (ng) of protein. To improve the stability of the protein within microspheres and allow the release of biologically active lysozyme, a basic additive ( Mg(OH)2 ) was successfully encapsulated. This novel in vitro system was appropriate to study protein microsphere release kinetics. In addition, the model is cost-effective and mimes brain physiological conditions more closely than previous models.

Preparation and characterization of a composite PLGA and poly(acryloyl hydroxyethyl starch) microsphere system for protein delivery.

To prepare and characterize a novel composite microsphere system based on poly(D,L-lactide-co-glycolide) (PLGA) and poly(acryloyl hydroxyethyl starch) (acHES) hydrogel for controlled protein delivery. Model proteins, bovine serum albumin, and horseradish peroxidase were encapsulated in the acHES hydrogel, and then the protein-containing acHES hydrogel particles were fabricated in the PLGA matrix by a solvent extraction or evaporation method. The protein-loaded PLGA-acHES composite microspheres were characterized for protein loading efficiency, particle size, and in vitro protein release. Protein stability was examined by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and monitoring the enzymatic activity. Scanning electron microscopy showed discrete PLGA microspheres containing many acHES particles. The composite microspheres were spherical and smooth in size range of 39-93 microm. The drug loading efficiency ranged from 51 to 101%. The composite microspheres showed more favorable in vitro release than conventional PLGA microspheres. The composite microspheres showed 20% less initial with a gradual sustained release compared to high burst (approximately 60%) followed by a very slow release with the conventional PLGA microspheres. The composite microspheres also stabilized encapsulated proteins from the loss of activity during the microsphere preparation and release. Proteins extracted from the composite microspheres showed good stability without protein degradation products and structural integrity changes in the size-exclusion chromatography and SDS-PAGE analyses. Horseradish peroxidase extracted from microspheres retained more than 81% enzymatic activity. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.