Biodegradable poly (lactic acid) microspheres for drug delivery systems

In connection with aim of maximizing the bio-availability of conventional drugs with minimum side-effects, new drug delivery systems (DDS) continue to attracted much attention. The controlled or sustained release of drugs represents one such approach, and in this regard report upon a study of DDS using biodegradable polymers which include poly (lactic acid) (PLA), poly (glycolic acid), and their copolymers (PLGA). Much attention is being paid to the controlled release of bio-active agents from microcapsules and microspheres made of biodegradable polymers, such as lactic acid homopolymers, as well as copolymers of glycolic acid. (11-21) Microcapsules or microspheres are injectable and able to provide pre-programmed durations of action, offering several advantages over the conventional dosage forms. This article reviews the results of a work program conducted in collaboration with a medical doctor upon DDS using biodegradable microspheres, such as PLA and PLGA.     

Reduction of inflammatory reaction of poly(d,l-lactic-co-glycolic Acid) using demineralized bone particles

Poly(lactide-co-glycolic acid) (PLGA) has been widely applied to tissue engineering as a good biocompatible material because of its biodegradability and nontoxic metabolites, but how the inflammatory reaction of PLGA on the surrounding tissue in vivo is reduced has not been discussed sufficiently. We hypothesized that the cells neighboring the PLGA implant might have an inflammatory response that could be reduced by impregnating demineralized bone particles (DBPs) into the PLGA. We manufactured five different ratios of DBP/PLGA hybrid materials, with each material containing 0, 10, 20, 40, and 80 wt% of DBPs of PLGA. For biocompatibility test, NIH/3T3 mouse fibroblasts were cultured on the DBP/PLGA scaffold for 3 days. The inflammatory potential of PLGA was evaluated using messenger ribonucleic acid expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 1-beta (IL-1beta) on a human acute promyelocytic leukemic cell (HL-60). The in vivo response of DBP/PLGA film was compared with that of PLGA film implanted subcutaneously; the local inflammatory response was observed according to histology. The DBP/PLGA scaffold had no adverse effect on NIH/3T3 initial cell attachment and did not affect cell viability. DBP/PLGA films, especially PLGA films containing 80% DBP, elicited a significantly lower expression of IL-1beta and TNF-alpha from HL-60 cells than PLGA film alone. In vivo, DBP/PLGA film demonstrated a more favorable tissue response profile than PLGA film, with significantly less inflammation and fibrous capsule formation as below only 20% of DBP in PLGA film during implantation. This study shows that application of DBPs reduces the fibrous tissue encapsulation and foreign body giant cell response that commonly occurs at the interface of PLGA.     

Controlled delivery of taxol from poly(ethylene glycol)-coated poly(lactic acid) microspheres.

The development of injectable microspheres for sustained drug delivery to the arterial wall is a major challenge. We demonstrated the possibility of entrapping an antiproliferative agent, taxol, in poly(ethylene glycol) (PEG)-coated biodegradable poly(lactic acid) (PLA) microspheres with a mean diameter of 2-6 microm. A solution of taxol and PLA dissolved in an acetone/dichloromethane mixture was poured into an aqueous solution of PEG [or poly(vinyl alcohol) (PVA] with stirring with a high-speed homogenizer for the formation of microspheres. Taxol recovery in PLA-PEG microspheres was higher (61.2 +/- 2.3%) than with PVA-based (41.6 +/- 1.8%) preparations. An analysis by diffuse reflectance infrared Fourier transform spectroscopy revealed that PEG was incorporated well on the PLA microsphere surface. Scanning electron microscopy revealed that the PEG-coated PLA microspheres were spherical in shape and had a smooth surface texture like those of PVA-based preparations. The amount of drug release was much higher initially (25-30%); this was followed by a constant slow-release profile for a 30-day period of study. This PEG-coated PLA microsphere formulation may have potential for the targeted delivery of antiproliferative agents to treat restenosis.     

Enhancement of poly(orthoester) microspheres for DNA vaccine delivery by blending with poly(ethylenimine)

Poly(orthoester) (POE) microspheres have been previously shown to possess certain advantages for the in vivo delivery of DNA vaccines. In particular, timing of DNA release from POE microspheres in response to acidic phagosomal pH was shown to be an important factor in determining immunogenicity, which was hypothesized to be linked to the natural progression of antigen-presenting cell uptake, transfection, maturation, and antigen presentation. Here we report in vitro characterization of the enhanced efficacy of POE microspheres by blending poly(ethylenimine) (PEI), a well-characterized cationic transfection agent, into the POE matrix. Blending of a tiny amount of PEI (approximately 0.04 wt%) with POE caused large alterations in POE microsphere properties. PEI provided greater control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA and enhanced gene transfection efficiency of the microspheres up to 50-fold without any significant cytotoxicity. Confocal microscopy results of labeled PEI and DNA plasmids revealed that PEI caused a surface-localizing distribution of DNA and PEI within the POE microsphere as well as focal co-localization of PEI with DNA. We provide evidence that upon degradation, the microspheres of POE-PEI blends released electrostatic complexes of DNA and PEI, which are responsible for the enhanced gene transfection. Furthermore, blending PEI into the POE microsphere induced 50-60% greater phenotypic maturation and activation of bone marrow-derived dendritic cells in vitro, judged by the up-regulation of co-stimulatory markers on the cell surface. Physically blending PEI with POE is a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Combined with the ability to induce maturation of antigen-presenting cells, POE-PEI blended microspheres may be excellent carriers for DNA vaccines.     

Allergen-loaded biodegradable poly(d,l-lactic-co-glycolic) acid nanoparticles down-regulate an ongoing Th2 response in the BALB/c mouse model

BACKGROUND AND OBJECTIVE: Biocompatible and biodegradable microparticles have gained interest as antigen delivery systems during the recent years. We investigated whether biodegradable poly(d,l-lactic-co-glycolic) acid (PLGA) nanospheres could be used as allergen vehicles for few-shot therapy of type I allergy. METHODS: The major birch pollen allergen Bet v 1 was encapsulated in PLGA nanospheres (PLGA-Bet v 1). We examined the antigenicity and the immune response to PLGA-Bet v 1 in a BALB/c mouse model. RESULTS: The antigenicity of Bet v 1 was largely unaffected by PLGA entrapment. When BALB/c mice were immunized subcutaneously with PLGA-Bet v 1, they formed allergen-specific IgG antibodies, but did not develop hypersensitivity to Bet v 1, as shown by type I skin tests. To evaluate their therapeutic potential, PLGA-Bet v 1 with or without Al(OH)3 or non-entrapped Bet v 1 with Al(OH)3 were used for single-shot treatment of sensitized mice. Both groups treated with PLGA-Bet v 1 developed high levels of Bet v 1-specific IgG2a antibodies (P<0.01), whereas IgG1 levels decreased significantly (P<0.01). Moreover, T cells from mice treated with PLGA-Bet v 1 showed IFN-gamma and IL-10 production. The synthesis of these cytokines was enhanced in the groups where Al(OH)3 had been added to the vaccine formulation. CONCLUSION: Allergen-loaded PLGA nanoparticles modulate an ongoing Th2 response in the BALB/c mouse model, as demonstrated by down-regulation of IgG1 and production of IFN-gamma and IL-10. Our data strongly suggest that PLGA nanospheres can advantageously be used for formulations of allergen extracts or allergen derivatives for the few-shot treatment of type I allergy.     

Changes in cisplatin delivery due to surface-coated poly (lactic acid)-poly(epsilon-caprolactone) microspheres

Smooth muscle cell proliferation plays a major role in the genesis of restenosis after angioplasty or vascular injury. Local delivery of agents capable of modulating vascular responses, have the potential to prevent restenosis. However, the development of injectable microspheres for sustained drug delivery to the arterial wall is a major challenge. We demonstrated the possibility of entrapping an antiproliferative agent, cisplatin, in a series of surface coated biodegradable microspheres composed of poly(lactic acid)poly(caprolactone) blends, with a mean diameter of 2-10 pm. The microspheres were surface coated with poly ethylene glycol (PEG), chitosan (Chit), or alginate (Alg). A solution of cisplatin and a 50:50 blend of polylactic acid (PLA)-polycaprolactone (PCL) dissolved in acetone-dichloromethane mixture was poured into an aqueous solution of PEG (or polyvinyl alcohol or Chit or Alg) with stirring using a high speed homogenizer, for the formation of microspheres. Cisplatin recovery in microspheres ranged from 25-45% depending on the emulsification system used for the preparations. Scanning electron microscopy revealed that the PLA-PCL microspheres were spherical in shape and had a smooth surface texture. The amount of drug release was much higher initially (20-30%), this was followed by a constant slow-release profile for a 30-day period of study. It has been found that drug release depends on the amount of entrapped drug, on the presence of extra cisplatin in the dispensing phase, and on the polymer coatings. This PEG or Alg-coated PLA/PCL microsphere formulation may have potential for the targeted delivery of antiproliferative agents to treat restenosis.     

Uptake of poly(D,L-lactic-co-glycolic acid) microspheres by antigen-presenting cells in vivo

Dendritic cells are the most potent antigen-presenting cells (APC) and the most effective stimulators of primary T cell responses. Based on the strong influence of the APC on the immune response, we investigated cellular uptake of a biodegradable antigen delivery system, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, at two sites of injection: intraperitoneal and intradermal. We hypothesized that a fluorescent probe, tetramethylrhodamine labeled dextran, loaded in PLGA microspheres would be taken up by APCs and thereby provide a means for studying cellular uptake of PLGA microspheres in vivo. Phagocytic load and cell phenotype were determined using flow cytometry and confocal laser scanning microscopy. The results revealed cellular uptake of tetramethylrhodamine dextran loaded PLGA microspheres at both injection sites. After intraperitoneal immunization, the predominant cell phagocytosing PLGA microspheres in the peritoneal cavity was the macrophage whereas the intradermal immunization resulted in uptake of PLGA microspheres by dendritic cells. Hence, these results suggest that the profile for cellular uptake varies with the site of injection. More importantly, this study provides direct and conclusive evidence of uptake of PLGA microspheres by the most potent APC, the dendritic cell.     

In vivo bone formation from human embryonic stem cell-derived osteogenic cells in poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffolds

We have previously reported the efficient osteogenic differentiation of human embryonic stem cells (hESCs) by co-culture with primary human bone-derived cells (hPBDs) without the use of exogenous factors. In the present study, we explored whether osteogenic cells derived from hESCs (OC-hESCs) using the previously reported method would be capable of regenerating bone tissue in vivo. A three-dimensional porous poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffold was used as a cell delivery vehicle. In vivo implantation of OC-hESC-seeded scaffolds showed significant bone formation in the subcutaneous sites of immunodeficient mice at 4 and 8 weeks after implantation (n=5 for each time point). Meanwhile, implantation of the control no cell-seeded scaffolds or human dermal fibroblast-seeded scaffolds did not show any new bone formation. In addition, the presence of BMP-2 (1 microg/scaffold) enhanced new bone tissue formation in terms of mineralization and the expression of bone-specific genetic markers. According to FISH analysis, implanted OC-hESCs remained in the regeneration sites, which suggested that the implanted cells participated in the formation of new bone. In conclusion, OC-hESCs successfully regenerated bone tissue upon in vivo implantation, and this regeneration can be further enhanced by the administration of BMP-2. These results suggest the clinical feasibility of OC-hESCs as a good alternative source of cells for bone regeneration.     

The microclimate pH in poly(d,l-lactide-co-hydroxymethyl glycolide) microspheres during biodegradation

The microclimate pH (μpH) in biodegradable polymers, such as poly(d,l-lactic-co-glycolic acid) (PLGA) 50/50, commonly falls to deleterious acidic levels during biodegradation, resulting in instability of encapsulated acid-labile molecules. The μpH distribution in microspheres of a more hydrophilic polyester, poly(d,l-lactide-co-hydroxymethyl glycolide) (PLHMGA), was measured and compared to that in PLGA 50/50 of similar molecular weight and degradation time scales. pH mapping in the polymers was performed after incubation under physiological conditions by using a previously validated ratiometric method employing confocal laser scanning microscopy (CLSM). Confocal μpH maps revealed that PLHMGA microspheres, regardless of copolymer composition, developed a far less acidic μpH during 4 weeks of incubation compared with microspheres from PLGA. A pH-independent fluorescent probe marker of polymer matrix diffusion of μpH-controlling water-soluble acid degradation products, bodipy, was observed by CLSM to diffuse ～3-7 fold more rapidly in PLHMGA compared to PLGA microspheres, consistent with much more rapid release of acids observed from the hydrophilic polymer during bioerosion. Hence, PLHMGA microspheres are less susceptible to acidification during degradation as compared to similar PLGA formulations, and therefore, PLHMGA may be more suitable to deliver acid labile molecules such as proteins.     

Surface characteristics of holmium-loaded poly(L-lactic acid) microspheres

Radioactive holmium-166-loaded poly(L-lactic acid) microspheres (Ho-PLLA-MS) are promising systems for the treatment of liver malignancies. The surface characteristics of Ho-PLLA-MS before and after both neutron and gamma irradiation were investigated in order to get insight into their suspending behaviour and to identify suitable surfactants for clinical application of these systems. X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used for surface characterization. The residual amounts of poly(vinyl alcohol) (PVA) of the microspheres, which was used as an emulsifier during the solvent evaporation process, were determined using a colorimetric iodine-borate method and the wettability of microspheres and PLLA films with and without holmium (Ho) loading was tested using suspending experiments and contact angle measurements. XPS showed that the surface of Ho-PLLA-MS mainly consisted of PLLA, less than 10% of the surface was covered with PVA after several washing and sieving steps. A colorimetric assay showed that the microspheres contained 0.2-0.3% (w/w) PVA. Combined with XPS data, this assay demonstrates that the PVA is likely dissolved in the core of the microspheres. XPS analysis also showed that after neutron irradiation, some holmium appeared on the surface. Moreover, Ho-loaded PLLA films had a much higher contact angle (85 degrees) than non-loaded films (70 degrees). Therefore, the Ho on the surface of neutron-irradiated Ho-PLLA-MS is probably the reason for their poor suspending behaviour in saline. No surface changes were seen with XPS after gamma irradiation. Based on their surface characteristics, a pharmaceutically acceptable solvent (1% Pluronic F68 or F127 in 10% ethanol) was formulated with which a homogeneous suspension of radioactive Ho-PLLA-MS could be easily obtained, making these systems feasible for further clinical evaluation.     

利福平/聚乳酸-聚羟基乙酸缓释微球的制备及特性

背景：虽然国内外有很多制备利福平/聚乳酸-聚羟基乙酸共聚物(poly lactic acid-glycolic acid copolymer,PLGA)微球的报道,但这些微球粒径多在10 μm左右,不适合与磷酸钙骨水泥复合制备成具有良好降解性的抗结核修复材料。 目的：制备大粒径利福平/PLGA缓释微球,观察其理化特性和体外缓释特性。 方法：以PLGA为载体,将利福平分散于PLGA的有机溶剂中,采用复乳溶剂挥发法制备利福平/ PLGA缓释微球。光镜和扫描电镜下观察微球的形态特征,测定微球平均直径和跨距,高效液相色谱法测定载药量和包封率,以溶出法和高效液相色谱法观察其体外释药特性,并拟合药物体外释放曲线建立曲线方程。 结果与结论：利福平/PLGA微球电镜观察呈圆球形,分散性好,粘连少,粒径分布集中,平均粒径(80.0±9.4) μm。载药量、包封率分别为(33.18±1.36)%,(54.79±1.13)%。体外缓释试验显示突释期内微球释放度为(14.66±0.18)%,前3 d累计释放度(18.09±0.45)%,到42 d体外累积释放度达到(92.17±1.23)%。提示利福平/PLGA微球具有良好的缓释效果,是一种较为理想的抗结核药物的载体材料和释放系统;PLGA是良好的药物缓释载体,可以用来制备载药缓释微球。     

乳酸-羟基乙酸共聚物缓控释微球的制备及突释成因与影响因素

背景：乳酸-羟基乙酸共聚物是一种生物可降解高分子材料,以乳酸-羟基乙酸共聚物为原料制备的载药微球和纳米粒既可提高药物的稳定性,又能实现缓释、控释和靶向释放。 目的：分析乳酸-羟基乙酸共聚物缓控释微球的制备方法以及突释的成因、影响因素和改进方法。 方法：应用计算机检索1990/2010中国期刊全文数据库和PubMed数据库与乳酸-羟基乙酸共聚物缓控释微球的制备及突释联系紧密的文章。 结果与结论：目前乳酸-羟基乙酸共聚物缓释微球制备方法主要有单凝聚法、乳化-固化法、喷雾干燥法。造成其突释的原因首先是药物分子和聚合物分子之间的相互作用太弱,导致药物很容易从微球进入释放递质中,其次是在微球释放初期,药物从微球中的孔洞和缝隙中释放出来导致突释。影响突释程度的具体因素有乳酸-羟基乙酸共聚物的相对分子质量、浓度、微球载药量、主药理化性质、微球制备方法及制备参数等。虽然国内外对突释机制以及控制突释措施的研究都还处于初步阶段,通过对各影响因素加以适当优化与控制,可在一定程度上减少微球的突释率,突释问题应该能够得到解决和控制。     

Biodegradable poly(dl-lactic acid) formulations in a calcitonin delivery system

A synthetic analogue of eel calcitonin, [Asu^1,7]-ECT, was incorporated into biodegradable poly(dl-lactic acids) with number-average molecular weights (M_n) of 1400–4400 by the meltpressing technique. The in vitro release of drug from a parabolically degradable poly(DL-lactic acid) with M_n = 1400 showed an initial burst release and completed the release in 3 d from the start of the test. The drug release from a M_n = 4400 polymer with an S-type degradation pattern was kept at 14 ± 5 units/d for an experimental period of 24 d.     

Silk fibroin/poly(vinyl alcohol) photocrosslinked hydrogels for delivery of macromolecular drugs

Hydrogels are three-dimensional polymer networks widely used in biomedical applications as drug delivery and tissue engineered scaffolds to effectively repair or replace damaged tissue. In this paper we demonstrate a newly synthesized cytocompatible and drug releasing photo-crosslinked hydrogel based on poly(vinyl alcohol) methacrylate and silk fibroin which possesses tailorable structural and biological properties. The initial silk fibroin content was 0%, 10%, 20%, 30%, 40% and 50% with respect to the weight of poly(vinyl alcohol) methacrylate. The prepared hydrogels were characterized with respect to morphology, crystallinity, stability, swelling, mass loss and cytotoxicity. FITC-dextrans of different molecular weights were chosen as model drugs molecules for release studies from the hydrogels. The hydrogels containing different silk fibroin percentages showed differences in pore size and distribution. X-ray diffraction analysis revealed that amorphous silk fibroin in poly(vinyl alcohol) methacrylate is crystallized to β-sheet secondary structure upon gelation. The sol fraction increased with increasing fibroin concentration in the co-polymer gel (from 18% to 45%), although the hydrogel extracts were non-cytotoxic. Similarly, the addition of silk fibroin increased water uptake by the gels (from 7% to 21%). FITC-dextran release from the hydrogels was dependent on the silk fibroin content and the molecular weight of encapsulated molecules. The study outlines a newer type of photo-crosslinked interpenetrating polymer network hydrogel that possess immense potential in drug delivery applications.     

Characterization of poly(L-lactic acid) microspheres loaded with holmium acetylacetonate

Holmium-loaded PLLA microspheres are useful systems in radioembolization therapy of liver metastases because of their low density, biodegradability and favourable radiation characteristics. Neutron activated Ho-loaded microspheres showed a surprisingly low release of the relatively small holmium complex. In this paper factors responsible for this behaviour are investigated, in particular by the use of differential scanning calorimetry, scanning electron microscopy, infrared spectroscopy and X-ray diffraction. The holmium complex is soluble in PLLA up to 8% in films and 17% in microspheres. Interactions between carbonyl groups of PLLA, and the Ho-ion in the HoAcAc complex, explain very satisfactorily the high stability of holmium-loaded microspheres.     

Long-term toxicity of holmium-loaded poly(L-lactic acid) microspheres in rats

The aim of this study was to get insight into the toxic effects of holmium-166-loaded poly(L-lactic acid) microspheres (Ho-PLLA-MS) which have very interesting features for treatment of liver malignancies. Acute, mid- and long-term effects were studied in healthy Wistar rats by evaluating clinical, biochemical and tissue response. Rats were divided into four treatment groups: sham, decayed neutron-irradiated Ho-PLLA-MS, non-irradiated Ho-PLLA-MS and PLLA-MS. After implantation of the microspheres into the liver of the rats, the animals were monitored (body weight, temperature and liver enzymes) for a period of 14-18 months. Some of the rats that received previously neutron-irradiated Ho-PLLA-MS were periodically scanned with magnetic resonance imaging (MRI) to see if holmium was released from the microspheres. After sacrifice, the liver tissue was histologically evaluated. Bone tissue was subjected to neutron-activation analysis in order to examine whether accumulation of released holmium in the bone had occurred. No measurable clinical and biochemical toxic effects were observed in any of the treatment groups. Furthermore, histological analyses of liver tissue samples only showed signs of a slight chronic inflammation and no significant differences in the tissue reaction between rats of the different treatment groups could be observed. The non-irradiated PLLA-MS and Ho-PLLA-MS stayed intact during the study. In contrast, 14 months after administration, the neutron-irradiated Ho-PLLA-MS was not completely spherical anymore, indicating that degradation had started. However, the holmium loading had not been released as was illustrated with MRI and affirmed by neutron-activation analysis of bone tissue. In conclusion, neutron-irradiated Ho-PLLA-MS does not provoke any toxic reaction and can be applied safely in vivo.     

乳酸-羟基乙酸共聚物多肽蛋白类药物微球控释系统的突释与控制

背景：突释问题是限制多肽蛋白类微球广泛应用的一个关键技术问题,已经成为PLGA微球控释系统面临的一个亟待解决的问题。 目的：分析近年来国内外对乳酸-羟基乙酸共聚物多肽蛋白类药物微球的突释与控制的研究,对突释的原因、影响突释的因素以及减少突释的方法与措施进行了详细的介绍。 方法：应用计算机检索CNKI和PubMed数据库中1999-01/2010-12关于乳酸-羟基乙酸共聚物多肽蛋白类药物微球控释系统研究的文章,在标题和摘要中以“聚乳酸-羟基乙酸;多肽;蛋白;微球;突释;控制”或“PLGA; peptide; protein ; microspheres; burst release; control”为检索词进行检索。通过阅读标题和摘要进行初选,排出较陈旧和重复研究文献,保留符合纳入标准的文献24篇。 结果与结论：对乳酸-羟基乙酸共聚物多肽蛋白类药物微球突释机制的理解,可以更好地实现对微球突释的控制,以扩大多肽蛋白类药物在临床上的应用。PLGA的性质、微球的制备方法、微球的制备参数都在不同程度上影响微球的突释,并且可能是多因素协同作用。通过对上述各种因素加以适当控制,可在一定程度上减少微球的突释率。通过该方面的机制研究对指导新药开发具有重要意义。     

Immune responses in mice of beta-galactosidase adsorbed or encapsulated in poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres

The immune response induced in mice by beta-galactosidase (beta-gal) adsorbed or encapsulated on poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) microspheres was investigated. The encapsulated protein elicited higher antibody response than the protein adsorbed on the microspheres in the case of the PLA microspheres. However, the encapsulated protein elicited weaker antibody response than the adsorbed protein in the case of the PLGA (50:50) microspheres, probably because, in this case, the encapsulation process adversely affected protein immunogenicity. In the case of adsorbed beta-gal, higher antibody response was obtained with the PLA microspheres than with the PLGA (50:50) microspheres. This may be related to the lower rate of beta-gal desorption from the PLA microspheres. Based on the immunoglobulin G1/immunoglobulin G2a ratios and the stimulation indices for interferon-gamma and interleukin-4, beta-gal encapsulated or adsorbed on PLA microspheres induced a Th(1)-biased immune response whereas beta-gal encapsulated or adsorbed on PLGA (50:50) microspheres induced a Th(2)-biased immune response. The results obtained indicate that more potent immune responses are obtained when the protein is encapsulated than adsorbed on the microspheres, providing that the encapsulation process does not adversely affect protein immunogenicity. Also, the type of polymer used to prepare the microspheres, but not the method of protein association with the microspheres, may affect the type of immune response.     

Cross-linked chitosan microspheres as carriers for prolonged delivery of macromolecular drugs

Bovine serum albumin (BSA) and diphtheria toxoid (DT) were loaded by passive absorption from aqueous solutions into preformed glutaraldehyde cross-linked chitosan microspheres. In vitro release of BSA under sink conditions at 37°C showed that even though there was a large burst effect, there was a more or less steady increase with time thereafter for several days. Coating the BSA-loaded particles with paraffin oil or with a polymer, such as polylactic acid, modulated drug release. After the initial burst from PLA coated particles, the release rate increased with time for nearly 2 months. Preliminary immunogenicity studies on Wistar rats using DT loaded chitosan spheres showed that the antibody titres were fairly constant over a 5-month period, although very low compared to DT given on alum as control. Histological studies of placebo microspheres intramuscularly injected into rats demonstrated their tissue compatibility. Biodegradation was not complete in 6 months demonstrating the potential of cross-linked chitosan spheres as a long-acting drug delivery vehicle. The study demonstrated the possibility of incorporating biological macromolecules which are very sensitive to organic solvents, pH, temperature, ultrasound, etc. by a passive absorption technique to degradable biopolymer matrices thereby preserving their biological integrity. It is also shown that drugs passively absorbed into such matrices by taking advantage of their swelling behaviour need not necessarily be released completely in the initial 'burst' and a sustained release may be possible for macromolecules thus incorporated.     

Biodegradable microspheres of novel segmented poly(ether-ester-amide)s based on poly(epsilon-caprolactone) for the delivery of bioactive compounds

A novel class of multiblock poly(epsilon-caprolactone)-based polymers containing hydrophilic trioxyethylene segments and potentially relevant to the delivery of drugs is described in this work. L-phenylalanine residues may also be inserted into the hydrophilic blocks to generate peptide bonds susceptible to enzymatic attack. The investigated polymers were poly(ether-ester-amide)s (PEEAs) obtained by a two-step polymerization procedure from OH-end capped low molecular weight poly(epsilon-caprolactone), sebacoyl chloride and either 4,7,10-trioxa-1,13-tridecanediamine (PEEA1) or 1,13-di(L-phenylalaninamido)-4,7,10-trioxatridecane (PEEA2). PEEAs were characterized by 1H-NMR spectroscopy, differential scanning calorimetry, gel permeation chromatography and were tested for their suitability in producing microspheres. Particles obtained by the single emulsion-solvent evaporation technique were regular and smooth (SEM analysis) showing a monomodal distribution of dimensions. To assess the potentiality of PEEAs in the oral delivery of drugs, three model compounds with different pKa and solubilities--diclofenac, nicardipine and dicumarol--were encapsulated within PEEA microspheres. For the sake of comparison, microspheres prepared from poly(epsilon-caprolactone) (PCL) with a molecular weight similar to PEEAs were also prepared and tested. The release of diclofenac from all the microspheres was very rapid (100% released within 2 h) whereas nicardipine release was slower and biphasic. The initial phase approximated a near zero-order release, being the fraction of nicardipine released after 8 h from PEEA microspheres higher with respect to PCL particles (about 70 vs. 30%). This result was ascribed to the lower crystallinity of PEEAs with respect to PCL which results in a facilitated access of water molecules through the polymer matrix. The lipophilic-unionizable dicumarol was released from PEEA microspheres at a very slow rate. Therefore, dicumarol-loaded PEEA2 microspheres allowed the study of the influence on the release rate of the insertion into the polymer chain of enzymatically degradable bonds. PEEA2 microspheres released dicumarol at the same rate in a medium with or without the proteolitic enzyme alpha-chymotrypsin. Although the insertion of an isolated amino acid was not sufficient to confer enzyme susceptibility to the polymer, the distinctive properties of PEEAs make their use very attractive in the field of controlled release.