Steve Ng

Double walled POE/PLGA microspheres: encapsulation of water-soluble and water-insoluble proteins and their release properties.

The poly(orthoester) (POE)-poly(D,L-lactide-co-glycolide) (50:50) (PLGA) double-walled microspheres with 50% POE in weight were loaded with hydrophilic bovine serum albumin (BSA) and hydrophobic cyclosporin A (CyA). Most of the BSA and CyA was entrapped within the shell and core, respectively, because of the difference in their hydrophilicity. The morphologies and release mechanisms of proteins-loaded double-walled POE/PLGA microspheres were investigated. Scanning electron microscope studies revealed that the CyA-BSA-loaded double-walled POE/PLGA microspheres yielded a more porous surface and PLGA shell than those without BSA. The neat POE and PLGA yielded slow and incomplete CyA and BSA release. In contrast, nearly complete BSA and more than 95% CyA were released in a sustained manner from the double-walled POE/PLGA microspheres. Both the BSA- and CyA-BSA-loaded POE/PLGA microspheres yielded a sustained BSA release over 5 days. The CyA release pattern of the CyA-loaded double-walled POE/PLGA microspheres was biphasic, characterized by a slow release over 15 days followed by a sustained release over 27 days. However, the CyA-BSA-loaded double-walled POE/PLGA microspheres provided a more constant and faster CyA release due to their more porous shell. In the CyA-BSA-loaded double-walled POE/PLGA microspheres system, the PLGA layer acted as a carrier for BSA and mild reservoir for CyA. During the first 5 days, most BSA was released from the shell but only 14% CyA was left from the microspheres. Subsequently, more than 80% CyA were released in the next 25 days. The distinct structure of double-walled POE/PLGA microspheres would make an interesting device for controlled delivery of therapeutic agents.

POE/PLGA composite microspheres: formation and in vitro behavior of double walled microspheres.

The poly(ortho ester) (POE) and poly(D,L-lactide-co-glycolide) 50:50 (PLGA) composite microspheres were fabricated by a water-in-oil-in-water (w/o/w) double emulsion process. The morphology of the composite microspheres varied depending on POE content. When the POE content was 50, 60 or 70% in weight, the double walled microspheres with a dense core of POE and a porous shell of PLGA were formed. The formation of the double walled POE/PLGA microspheres was analysed. Their in vitro degradation behavior was characterized by scanning electron microscopy, gel permeation chromatography, Fourier-transform infrared microscopy and nuclear magnetic resonance spectroscopy (NMR). It was found that compared to the neat POE or PLGA microspheres, distinct degradation mechanism was achieved in the double walled POE/PLGA microspheres system. The degradation of the POE core was accelerated due to the acidic microenvironment produced by the hydrolysis of the outer PLGA layer. The formation of hollow microspheres became pronounced after the first week in vitro. 1H NMR spectra showed that the POE core was completely degraded after 4 weeks. On the other hand, the outer PLGA layer experienced slightly retarded degradation after the POE core disappeared. PLGA in the double walled microspheres kept more than 32% of its initial molecular weight over a period of 7 weeks.

POE-PEG-POE triblock copolymeric microspheres containing protein. I. Preparation and characterization.

Poly(ortho ester) (POE)-poly (ethylene glycol) (PEG) triblock copolymers (POE-PEG-POE) with different PEG contents were synthesised as carriers for controlled protein delivery. POE-PEG-POE microspheres containing bovine serum albumin (BSA) were prepared using a double-emulsion (water-in-oil-in-water) process. In this first paper of a two-part series, we report the fundamentals of the fabrication and characterization of POE-PEG-POE microspheres. Because the triblock copolymer is more hydrophilic than neat poly(ortho ester), the triblock copolymer yields a more stable first emulsion (water-in-oil) and a greater BSA encapsulation efficiency (90% vs. 30%). No BSA is found on POE-PEG-POE microsphere surfaces measured by X-ray photoelectron spectroscopy, while uniform BSA distributions are observed within the microspheres by confocal microscopy. SEM pictures show that an increase in PEG content results in microspheres with a denser cross-section because of a more stable first emulsion and better affinity between the copolymer and water. POE-PEG(20%)-POE suffers significant swelling during the fabrication process and yields the biggest microspheres. However, the POE-PEG(30%)-POE microspheres are much smaller since the dissolution loss of POE-PEG(30%)-POE in the external water phase may be much higher than that of POE-PEG(20%)-POE. The salt concentration in the external water phase significantly affects the morphology of the resultant microspheres. Microspheres with a dense wall are produced when using pure water as the external water phase. Polymer concentration has less impact on BSA encapsulation efficiency but has a considerable effect on microsphere size and morphology. Increasing the concentration of the polyvinyl alcohol emulsifier does not cause an obvious decrease in microsphere size. However, increased BSA loading results in bigger microspheres.

Development of poly(ortho esters) and their application for bovine serum albumin and bupivacaine delivery

The preparation of drug delivery devices using solventless fabrication procedures is of significant interest and two such procedures are described. In one such procedure, powdered polymer and micronized protein are intimately mixed and then extruded into 1 mm strands that are cut to the desired length. The polymers used were specifically designed to allow extrusion at temperatures where proteins maintain activity in the dry state. In vitro erosion and BSA release show that BSA release and polymer erosion occur concomitantly indicating an erosion-controlled process. There is a lag-time, but that can be eliminated by the addition to the mixture prior to extrusion small amounts of poly(ethylene glycol) or its methoxy derivatives. The lag-time could also be eliminated by using an AB-block copolymer where A is poly(ortho ester) and B is poly(ethylene glycol). Another means of using solventless fabrication methods is to use a semi-solid material into which drugs can be mixed at room temperature and the semi-solid injected. Data on BSA and bupivacaine release are presented.

Development and applications of injectable poly(ortho esters) for pain control and periodontal treatment.

Poly(ortho esters) with a low glass transition temperature are semi-solid materials so that therapeutic agents can be incorporated at room temperature, without the use of solvents, by a simple mixing procedure. When molecular weights are limited to < 5 kDa, such materials are directly injectable using a needle size no larger than 22 gauge. Somewhat hydrophilic polymers can be produced by using the diketene acetal 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane and triethylene glycol (TEG), while hydrophobic materials can be produced by using the diketene acetal and 1,10-decanediol. Molecular weight can be reproducibly controlled by using an excess of the diol, or by use of an alcohol that acts as a chain-stopper. Erosion rates can be controlled by varying the amount of latent acid incorporated into the polymer backbone. Toxicology studies using the TEG polymer have been completed and have shown that the polymer is non-toxic. Toxicology studies using the decanediol polymer are underway. Development studies using the TEG polymer aimed at providing a sustained delivery of an analgesic agent to control post-surgical pain are under development and human clinical trials using the decanediol polymer for the treatment of periodontitis are also underway.

Auto-catalyzed poly(ortho ester) microspheres: a study of their erosion and drug release mechanism.

A study has been carried out to investigate the degradation and protein release mechanisms of BSA-loaded microspheres made with auto-catalyzed poly(ortho esters) (POEs) of varying diol composition and molecular weights. Due to the instability of the POE/dichloromethane primary emulsion, microspheres made using the W/O/W double emulsion solvent extraction/evaporation method showed a multivesicular internal structure. An O/W single emulsion process yielded dense POE microspheres. Using electron scanning microscopy, the microspheres were observed to erode throughout their matrices with increasing internal pore sizes and a steady loss of mass. However, despite a substantial weight loss of almost 80% after an in vitro period of 129 days, the molecular weight of the polymer remained relatively unchanged with loss averaging about 18 and 32% for low- and high-molecular-weight POEs, respectively. Such constancy in molecular weight was similarly reflected in the glass transition temperature of the degrading microspheres. The differences in both the molecular weight loss and polydispersity index changes depended largely on the molecular weight of the polymer. For protein release of POE microspheres, an induction period followed by BSA release for a period of 3 to 10 days was observed. The lag time depended on the hydrophilicity and the molecular weight of the polymer as well as the morphology of the microspheres. Protein release was incomplete, possibly due to the slow degradation of the POE polymers, protein aggregation and protein degradation.

Synthesis and characterization of self-catalyzed poly(ortho ester)

Poly(ortho esters) are currently under investigation as a carrier system for an antiproliferative agent in glaucoma filtering surgery. The present investigation illustrates the development of a series of self-catalyzed poly(ortho ester). These polymers contain short dimer segments of alpha-hydroxy acids in their backbone and are prepared by the addition of different polyols to the diketene acetal 3,9-diethylidene-2,4,8,10-tetra-oxaspiro-[5.5]-undecane. The structures were confirmed by NMR- and FT-IR-spectroscopy. The polymers were characterized by determination of the molecular weight, the glass transition temperature and the rheological behavior. The amount of residual solvents was also analyzed. The characteristics of the polymer can be varied by the type of polyol incorporated in its backbone. Since poly(ortho ester) is susceptible to acid-catalyzed degradation, the polymer hydrolysis can be controlled by the amount of incorporated portion of alpha-hydroxy acid. Due to the high hydrophobicity of the polymer structure, the ester bonds are more susceptible to hydrolysis than the ortho ester bonds in the polymer backbone. The hydrolysis proceeds via initial protonation of the exocyclic alkoxy group to yield pentaerythritol dipropionate and the free diol. In a next step, the pentaerythritol dipropionate hydrolysis to pentaerythritol and propionic acid. The molecular weight decrease, weight loss and the pH profile of the polymer in aqueous medium were monitored during the degradation.

POE–PEG–POE triblock copolymeric microspheres containing protein: II. Polymer erosion and protein release mechanism

The first paper of this series presented the fabrication and characterization of POE-PEG-POE triblock copolymeric microspheres containing protein. In this paper, we focus on the polymer erosion and the mechanism of protein release. Fourteen-week in vitro behaviors of POE-PEG-POE microspheres loaded with bovine serum albumin (BSA) have been monitored. SEM micrographs reveal that after 14-week incubation in PBS buffer, pH 7.4, 37 degrees C, the polymeric particles remain spherical despite mass loss of almost 90%. On the other hand, molecular weight undergoes a high initial loss of 38% and 44% during the first 2-week incubation for POE-PEG(5%)-POE and POE-PEG(10%)-POE, respectively. Then, it keeps relatively unchanged over 12 weeks. However, POE-PEG(20%)-POE copolymer provides a better compatibility between the POE and PEG blocks. Hydrolysis is homogeneous through the polymer backbone. Thus, its molecular weight remains relatively constant and mass loss shows quite sustained over the 14-week in vitro release. The similar phenomena are observed in the polydispersity index of the degrading copolymers. SDS-PAGE of the encapsulated BSA within the POE-PEG(5%)-POE microspheres displays that the structural integrity of BSA is intact for at least 8 weeks due to a mild environment provided by the copolymer. In addition, XPS and FTIR are utilized to investigate protein behaviors in the degrading microspheres. Protein release from the POE-PEG-POE microspheres shows a biphasic pattern, characterized by an initial stage followed by a non-detectable release. The non-release phase is dominated by either slow polymer degradation or dense microsphere matrix structures. The microsphere formulation is optimized and a sustained protein release over 2 weeks is achieved by using POE-PEG(20%)-POE at a high protein loading.

In vitro drug release from self-catalyzed poly(ortho ester): case study of 5-fluorouracil.

Self-catalyzed poly(ortho esters) are a new variation of linear poly(ortho esters) prepared by the addition of diols to the diketene acetal 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5,5]undecane where dimer segments of lactic acid or glycolic acid are built into the polymer backbone. By varying the concentration of these segments, polymer erosion rate can be controlled. The present investigation describes the in vitro drug release characteristics from these new polymers. Because poly(ortho esters) have potential applications for the delivery of antifibroblastic agents for example after glaucoma-filtering surgery, the in vitro release studies were evaluated using 5-fluorouracil as the active compound. It was shown that a mole ratio of 90/10 or 80/20 diol/diol-lactate incorporated into the polymer lead to a release of 5-fluorouracil by an erosion process. Smaller amounts of diol-lactate lead to a concomitant drug release by diffusion and erosion. It was also shown that the release rate depends on the alkyl chain length of the diol in the polymer backbone but it does not depend on the drug loading.