In vitro and in vivo release properties of brilliant blue and tumour necrosis factor-alpha (TNF-alpha) from poly(D,L-lactic-co-glycolic acid) multiphase microspheres

The dissolution properties of two model compounds, brilliant blue and tumour necrosis factor (TNF-alpha), from poly(D,L-lactic-co-glycolic acid) (PLGA) multiphase microspheres were investigated. In addition, the in vivo release of TNF-alpha from the microspheres, in mice, was studied. The microspheres were prepared by an anhydrous multiple emulsion solvent evaporation method. Multiphase microspheres containing brilliant blue exhibited a three phase release profile in vitro, and displayed a significantly lower level of dye released during the initial phase compared to conventional matrix-type microspheres. Slow release of the dye was observed during the second phase, which was followed by a disintegration of the polymer wall during the third phase of the release process. In vitro dissolution profiles of TNF-alpha were calculated by compensation for the simultaneous degradation of the protein in the dissolution medium. The initial burst release of TNF-alpha was significantly reduced with the multiphase microspheres. The three phase release profile, as seen with the dye, was not observed for the microspheres containing the TNF-alpha. The rate of release of the protein from the microspheres was determined in vivo by analysing the residual level of TNF-alpha remaining in the particles following intraperitoneal administration of the microspheres to mice. The release of the protein from the microspheres in vivo was significantly faster than predicted from the results of the in vitro studies. The absence of an initial burst release of TNF-alpha from the multiphase microspheres was reflected in a significant reduction in the plasma level of TNF-alpha when compared to the matrix-type microspheres and a solution of the protein. The controlled release property of the multiphase microspheres is expected to overcome the adverse reactions due to dose dumping that occurs following the local administration of TNF-alpha.     

Dissolution,Stability, and Morphological Properties of Conventional and Multiphase Poly(DL-Lactic-Co-Glycolic Acid) Microspheres Containing Water-Soluble Compounds

Multiphase microspheres of poly(DL-lactic-co-glycolic acid) (PLGA) containing water-soluble compounds were prepared by a multiple-emulsion solvent evaporation technique. These compounds were dissolved in the aqueous phase of a W/O emulsion with soybean oil as the oil phase. This emulsion was dispersed throughout the matrix of the microsphere. The morphological properties of the multiphase microspheres during in vitro dissolution studies were compared to those of conventional microspheres prepared from the same polymer. Drug release from the multiphase microspheres was characterized by an initial uniform release for the first 20 days followed by a more rapid phase of drug release. Chlorpheniramine maleate (CPM) and brilliant blue (BB) were the soluble model compounds investigated. The release rates of these agents from the multiphase microspheres were independent of the drug content in the microspheres. The release profiles from the conventional microspheres showed a lag time of 10 and 16 days for the CPM and BB, respectively. The dissolution rate of the model soluble compounds from the conventional microspheres increased as the loading in the microspheres increased. No differences in the degradation rate of the PLGA from the multiphase and the conventional microspheres were seen during the dissolution studies.     

Particle size and loading efficiency of poly(D,L-lactic-coglycolic acid) multiphase microspheres containing water soluble substances prepared by the hydrous and anhydrous solvent evaporation methods

PLGA multiphase microspheres were prepared by the multiple emulsion solvent evaporation method using acetonitrile as the polymer solvent and mineral oil as the evaporation medium. The preparation process was further developed in the present study to reduce the particle size and to increase the loading capacity of brilliant blue, bovine serum albumin (BSA) and tumour necrosis factor-alpha (TNF-alpha) which were used as water soluble model drug substances. Sorbitan sesqui-oleate (SO-15EX), present at the 1% w/w level in the evaporation medium, prevented agglomeration of the microspheres containing a solid-in-oil (S/O) suspension as the core phase. This S/O suspension core provided significantly higher loading efficiency of the proteins to the W/O emulsion core. The W/O emulsion system resulted in agglomeration of the protein-loaded microspheres and the loading efficiency decreased significantly. When brilliant blue was included as the model compound, the loading efficiencies were not influenced by the core type. Heavy mineral oil was employed to stabilize the dispersed unhardened microspheres rather than light mineral oil that was reported previously. This anhydrous emulsion system employing the S/O suspension core and containing a dispersion of TNF-alpha enabled the encapsulation of this protein without loss of activity. It was concluded that the anhydrous emulsion system is asuitable approach toprepare multiple microspheres as an alternative to the W/O emulsion system, especially when solvent sensitive proteins are incorporated into the microspheres.     

Polylactic acid microspheres containing quinidine base and quinidine sulphate prepared by the solvent evaporation method. III. Morphology of the microspheres during dissolution studies

Poly(dl-lactide) (PLA) microspheres containing quinidine or quinidine sulphate were prepared by the emulsification-solvent evaporation technique. The in vitro release profile of quinidine or quinidine sulphate from the microspheres was characterized by three phases: a lag time, a rapid release phase (burst), and a slow release phase. Drug release was studied as a function of the ionic strength of the dissolution medium, to demonstrate the importance of the water imbition into the microspheres which induced the drug release. The lag time increased with increasing ionic strength. The microspheres stayed intact during the dissolution study as shown by scanning electron microscopy (SEM). Disintegration of microspheres which was initially observed was an artifact introduced during the SEM procedure. The high vacuum applied either during the coating of the microspheres with gold-palladium or during the actual observation in the scanning electron microscope caused the microspheres to collapse or rupture.     

Comparison of various injectable protein-loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices: in-situ-formed implant versus in-situ-formed microspheres versus isolated microspheres

The purpose of this research was to prepare various injectable, protein (cytochrome c)-loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices by a novel microencapsulation method and to compare their characteristics. Syringeable mixtures of polymer and protein solidified upon injection when coming in contact with water, and formed a solid matrix-type implant or microspheres (in-situ-formed implant or in-situ-formed microspheres, respectively) with cytochrome c entrapped. These devices exhibited different characteristics in terms of in vitro cytochrome c release profile, percentage cytochrome c encapsulation efficiency, and particle size. The burst effect from these devices exhibited the following trend: in-situ-formed implant > in-situ-formed microspheres > isolated microspheres. The in-situ-formed microspheres were larger in size than the isolated microspheres. Also, the isolated microspheres exhibited the slowest release of cytochrome c, whereas the in-situ-formed implant exhibited the fastest release. The microencapsulation process can produce various drug-loaded injectable biodegradable PLGA devices having different characteristics.     

Evaluation and preparation of controlled release lipid micropheres of sulphamethizole by a congealable disperse phase encapsulation method

A congealable disperse phase encapsulation method was used to prepare controlled release lipid microspheres of sulphamethizole as a model drug. Hydrogenated cotton seed oil (HCSO) and stearic acid were employed as the lipid matrix materials. Tween 60 was the droplet stabilizer used to form microspheres. In in vitro dissolution tests, the drug release was found to be affected by the type of lipid material depending on hydrophilicity. Generally, an initial rapid release followed by a slower release of the drug from the lipid microspheres was observed. Lipid microspheres were also compressed in the tablet form to prevent the initial rapid release of the drug. But the drug release drastically decreased. To achieve a controlled release of the drug. Eudragit L as a channeling agent was added internally to HCSO-microspheres. Although the drug release increased, the controlled release pattern was not achieved. The external addition of polyethyleneglycole 4000 to HCSO-microspheres before compressing tablets, did not produce an affirmative change in the release profile. The lipid microspheres prepared by stearic acid released all of the drug within 1 h. Upon compression, the drug release was very low. Therefore, stearic acid-microspheres were compressed in the tablet form adding disintegrating agents, sodium alginate and Ac-Di-Sol (cross-linked sodium carboxymethylcellulose). A pH-dependent drug release was obtained from the tablets containing sodium alginate. With the tablets of stearic acid-microspheres containing Ac-Di-Sol, the controlled release could be achieved due to gradual disintegration from the tablet to aggregates, and to individual microspheres. Furthermore, in vivo study on 6 healthy volunteers confirmed the controlled release pattern of this dosage form.     

Alginate microspheres prepared by internal gelation: development and effect on insulin stability

Recombinant human insulin was encapsulated within alginate microspheres by the emulsification/internal gelation technique with the objective of preserving protein stability during encapsulation procedure. The influence of process and formulation parameters was evaluated on the morphology and encapsulation efficiency of insulin. The in vitro release of insulin from microspheres was studied under simulated gastrointestinal conditions and the in vivo activity of protein after processing was assessed by subcutaneous administration of extracted insulin from microspheres to streptozotocin-induced diabetic rats. Microspheres mean diameter, ranging from 21 to 287 microm, decreased with the internal phase ratio, emulsifier concentration, mixer rotational speed and increased with alginate concentration. Insulin encapsulation efficiency, near 75%, was not affected by emulsifier concentration, mixer rotational speed and zinc/insulin hexamer molar ratio but decreased either by increasing internal phase ratio and calcium/alginate mass ratio or by decreasing acid/calcium molar ratio and alginate concentration. A high insulin release, above 75%, was obtained at pH 1.2 and under simulated intestinal pH a complete dissolution of microspheres occurred. Extracted insulin from microspheres decreased hyperglycemia of diabetic rats proving to be bioactive and showing that encapsulation in alginate microspheres using the emulsification/internal gelation is an appropriate method for protein encapsulation.     

微球的制备和表征

目的制备葡激酶突变体(K35R，DGR)的聚乳酸-羟基乙酸(PLGA)微球，使其在包封和释放过程中都能保持活性。方法使用复乳溶剂挥发法制备DGR的PLGA微球，研究了搅拌速度、PLGA浓度、内水相和外水相中的添加剂对蛋白包封率以及微球性质的影响，并进行了DGR微球的体外和体内释放试验。结果2%聚乙烯醇可以有效抑制超声乳化时DGR在水/二氯甲烷界面上的变性，将DGR的活性回收率从16%提高到几乎100%。在外水相中加入NaCl可以显著提高蛋白包封率，同时对微球的粒径分布和表面形态也产生了重要影响。DGR微球的体外释放呈现两个时相，15 d释放大约DGR总活性的50%。DGR微球在体内持续释放5 d。结论制备的PLGA微球，DGR包封率高，稳定性较好，是DGR的良好载药系统。     

尼群地平缓释微球的制备及其体内外相关性的研究

目的制备具有固体分散体结构的尼群地平缓释微球 ,并筛选具有良好体内外相关性的释放介质。方法采用球晶造粒法制备尼群地平缓释微球 ,考察微球的粒径、载药量、包封率及释放行为 ,并根据 6只试验犬体内药物动力学试验结果 ,将不同时间的吸收分数与不同释放介质的相应时间点的体外累积释放百分数作线性回归 ,筛选具有良好体内外相关性的释放介质。结果制备的微球的粒径随搅拌速度的增加而减少 ,包封率均在 96 80 %以上 ,药物从微球中的释放速度随处方中固体分散体载体量的增加而增加 ,随阻滞剂量的增加而减小。以 1 7 4mmol L十二烷基硫酸钠为释放介质时 ,体外累积释放百分数与体内吸收分数相关系数较好 (r =0 985 1 ) ,方程为Fa =1 64 5 8ft-2 7 64 2。结论该方法较适用于难溶性药物制备缓释微球。以 1 7 4mmol L十二烷基硫酸钠水溶液为释放介质可作为控制微球内在质量的标准     

In Vitro/in Vivo Correlation for 14C-Methylated Lysozyme Release from Poly(Ether-Ester) Microspheres

PURPOSE: The purpose of this study was to obtain an in vitro/in vivo correlation for the sustained release of a protein from poly(ethylene glycol) terephthalate (PEGT)/poly(butylene terephthalate) (PBT) microspheres. METHODS: Radiolabeled lysozyme was encapsulated in PEGT/PBT microspheres via a water-in-oil-in-water emulsion. Three microsphere formulations varying in copolymer composition were administered subcutaneously to rats. The blood plasma was analyzed for radioactivity content representing released lysozyme at various time points post-dose. The in vitro release was studied in phosphate-buffered saline. RESULTS: The encapsulation efficiency, calculated from the radioactivity in the outer water phase of the emulsion, varied from 60-87%. Depending on the PEG segment length and wt% PEGT, the lysozyme was released completely in vitro within 14 to 28 days without initial burst. 14C-methylated lysozyme could be detected in the plasma over the same time courses. The in vitro/in vivo correlation coefficients obtained from point-to-point analysis were greater than 0.96 for all microsphere formulations. In addition, less then 10% of administered radioactivity remained at dose site at 28 days for the microsphere formulations, indicating no notable retention of the protein at the injection site. CONCLUSION: The in vitro release in phosphate-buffered saline and the in vivo release in rats showed an excellent congruence independent of the release rate of 14C-methylated lysozyme from PEGT/PBT microspheres.     

Sodium alginate microspheres of metformin HCl: formulation and in vitro evaluation

Metformin microspheres with sodium alginate alone and in combination with gellan were prepared using an emulsion-cross linking method. The prepared microspheres were evaluated for their physico-chemical characteristics like particle size, morphology using SEM, incorporation efficiency, equilibrium water content (swelling) and in vitro drug release. The effect of various formulation variables like polymer concentration (sodium alginate; and proportion of gellan in microspheres prepared by a combination of sodium alginate and gellan), drug loading, crosslinking agent concentration and cross-linking time on the in vitro dissolution of the prepared microspheres were evaluated. The results showed that both the particle size and the incorporation efficiency were proportional to the polymer concentration. In case of microspheres containing both sodium alginate and gellan, the mean diameter and the incorporation efficiency were higher than the corresponding microspheres containing only alginate, both increasing with an increase in proportion of gellan. The prepared microspheres were found to be discrete and spherical in shape and were successful in sustaining the drug release for 8 hours. Incorporation of gellan caused a significant decrease in drug release. The release followed a biphasic profile, in all cases, characterized by an initial phase of moderate drug release followed by a phase of higher release. Further, the kinetic treatment of the dissolution data revealed the prevalence of matrix diffusion kinetics.     

Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(D,L-lactic-co-glycolic acid) versus chitosan microspheres

Stable polymeric microspheres capable of controlled release of tetanus toxoid (TT) for periods ranging from days to over months were developed. TT was stabilized, encapsulated in microspheres prepared from poly(D,L)-lactide-co-glycolide (PLGA) and chitosan by using protein stabilizer (trehalose) and its immune response was compared. The influence of co-encapsulated protein stabilizer on tetanus toxoid's stability and release from the microspheres was studied. The protein stabilizer (trehalose) prevented structural losses and aggregation of microencapsulated TT. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polymer, we also co-incorporated into the polymer an antacid, (Mg(OH)2), which neutralized the acidity during degradation of the polymer and also prevented TT structural losses and aggregation. The in vitro release experiments with PLGA and chitosan microspheres were performed and the release of TT was increased up to 80-90%. The antigen integrity was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie brilliant blue staining. The SDS-PAGE analysis confirmed that antigen integrity was not affected by the encapsulation procedure. In addition, the immunogenicity of PLGA and chitosan microspheres based single dose vaccine was evaluated in guinea pigs and compared with multiple doses of alum adsorbed TT. Results indicated that a single injection of PLGA and chitosan microspheres containing TT could maintain the antibody response at a level comparable to the booster injections of conventional alum adsorbed vaccines. The both PLGA and chitosan based stable vaccine formulations produced an equal immune response. Hence chitosan can be used to replace the expensive polymer PLGA. This approach should have potential application in the field of vaccine delivery.     

Preliminary evaluation of the correlation between in vitro release and in vivo bioavailability of two aminophylline slow-release tablets

An in vitro dissolution study has been carried out with a USP rotating paddle apparatus using the stepwise pH change method. The dissolution kinetics of two different matrix-type aminophylline slow-release (SR) tablets were compared. both aminophylline SR tablets show similar release rates and slow release behavior. The bioavailability of these two aminophylline SR tablets was also evaluated in eight healthy male Chinese volunteers and correlated with in vitro dissolution results by moment analysis. The preliminary results suggest that there is no evidence indicating that the two different matrix types of aminophylline SR tablets are not bioequivalent, and a good in vitro-in vivo correlation for these aminophylline SR tablets may therefore be presumed.     

Preparation,characterization and in vitro release study of BSA-loaded double-walled glucose-poly(lactide-co-glycolide) microspheres

The aim of this study was to prepare a model protein, bovine serum albumin (BSA) loaded double-walled microspheres using a fast degrading glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA) and a moderate-degrading carboxyl-terminated PLGA polymers to reduce the initial burst release and to eliminate the lag phase from the release profile of PLGA microspheres. The double-walled microspheres were prepared using a modified water-in-oil-in-oil-in-water (w/o/o/w) method and single-polymer microspheres were prepared using a conventional water-in-oil-in-water (w/o/w) emulsion solvent evaporation method. The particle size, morphology, encapsulation efficiency, thermal properties, in vitro drug release and structural integrity of BSA were evaluated in this study. Double-walled microspheres prepared with Glu-PLGA and PLGA polymers with a mass ratio of 1:1 were non-porous, smooth-surfaced, and spherical in shape. A significant reduction of initial burst release was achieved for the double-walled microspheres compared to single-polymer microspheres. In addition, microspheres prepared using Glu-PLGA and PLGA polymers in a mass ratio of 1:1 exhibited continuous BSA release after the small initial burst without any lag phase. It can be concluded that the double-walled microspheres made of Glu-PLGA and PLGA polymers in a mass ratio of 1:1 can be a potential delivery system for pharmaceutical proteins.     

微球内部结构对体外释放的影响初步研究

目的 考察微球内部结构对其体外释放是否存在影响。方法 内水相中加入Na Cl-BSA比例不同的氯化钠,将微球切片考察其内部结构差异,通过摄取实验考察微球孔洞连通情况;研究不同处方体外释放曲线,并采用扫描电镜对释放过程不同时间点微球外观变化进行观察。结果 内水相中加入氯化钠后,微球内部结构有明显差异。微球内部结构会影响其体外释放曲线,且不同处方微球释放过程形态差异较大。结论 微球内部结构会显著影响其体外释放速率。     

Release of bioactive BMP from dextran-derived microspheres: a novel delivery concept

Recent developments of biotechnology have produced a great variety of protein and bioactive drugs. For these drugs to be used therapeutically, suitable drug delivery systems have become increasingly essential. Dextran-derived biomaterials have been considered to be compatible matrices for protein and bioactive drugs because of their hydrophilic properties and ability to control drug dissolution and permeability. A novel class of dextran-glycidylmethacrylate (Dex-GMA)/poly(ethylene glycol) (PEG) microspheres were designed and synthesized by polymerization of Dex-GMA emulsified in an aqueous PEG solution. Dex-GMA was prepared by substituting the hydroxyl groups in Dex by GMA. The drug loading and in vitro drug release was evaluated by routine procedure and the biological activity of BMP-loaded microspheres was studied by experimental cytology methods. Recombinant human bone morphogenetic protein-2 (rhBMP-2) were entrapped in dextran-derived microspheres quantitatively and with full preservation of their biological activity. In vitro release kinetics indicated that dextran-derived microspheres could retain rhBMP-2 in a variable manner depending on the preparation and degradation of the microspheres. The release profiles of rhBMP-2 from microspheres as a function of time showed that rhBMP-2 releasing kinetics in vitro fitted to first-order and Higuchi equations. The release profile in vitro was in accord with two phases kinetics law and more than 60% drug were released during 20 days. Cytology studies showed rhBMP-2 microspheres have good biological effects on cultured periodontal ligament cells, and could achieve a longer action time than concentration of rhBMP-2 solution. These properties make those microspheres interesting osteo-conductive BMP carriers, allowing to decrease the amount of implanted factor required for tissue regeneration.     

制备工艺对石杉碱甲微球体外释药机制的影响

目的考察制备工艺对石杉碱甲(Hup)乳酸-羟基乙酸共聚物(PLGA)微球体外释药机制的影响。方法 采用两种O/O型乳化溶剂挥发法工艺(A法和B法)制备Hup微球。考察微球的体外释药曲线，结合微球在释放介质中的降解速度和溶胀速度曲线以及微球的形态和微球中药物的分布情况阐述微球的释药机制。结果采用A法制备的微球包封率为47.60%，体外无明显突释现象，可缓释35 d，符合零级动力学方程，通过扩散和降解两种机制释药。采用B法制备的微球包封率为83.50%，体外可缓释21 d，整体释药曲线符合Higuchi方程，主要以扩散机制释药。结论采用A法制备的微球具有更理想的缓释效果。     

Dexamethasone-loaded magnetic albumin microspheres: Preparation and in vitro release

Magnetic albumin microspheres containing nearly 13% w/w dexamethasone were prepared. Two separate studies were carried out to investigate: (a) the responsivity of these microspheres in a 8000 G magnetic field in a flow rate (0.5 cm/s) equal to that of the blood flow rate in capillaries; (b) the in vitro release profile of dexamethasone from magnetic albumin microspheres up to 7 h after dispersion in normal saline medium, using a USP dissolution apparatus. The results obtained suggest that the retention of microspheres in the presence of the magnetic field for 15 min was significantly (P < 0.05) more than those in the absence of the magnetic field. Drug release in the first hour was found to increase and then reached a maximum. After 7 h, approximately 30% of the total drug content of microspheres was released. A third order equation for the drug release was also calculated. From this study, it is suggested that magnetic albumin microspheres could be retained at their target site in vivo, following the application of a magnetic field, and are capable of releasing their drug content for an extended period of time. This would make them a suitable depot for delivering chemotherapeutic agent(s) in vivo.     

In vitro performance of carbamazepine loaded to various molecular weights of poly (D,L-lactide-co-glycolide)

The purpose of this study was to develop and assess the in vitro characteristics of carbamazepine-loaded microspheres. A solvent evaporation method was used to incorporate carbamazepine (CBZ) into poly (D,L-lactide-co-glycolide) (PLGA) with different molecular weights. The optimum conditions for CBZ-PLGA microspheres preparation were considered and the in vitro release of CBZ of PLGA microspheres were followed up to 24 hr in USP dissolution medium. The effect of using different ratios of PLGA microspheres, prepared with different molecular weights, for optimizing CBZ release also was investigated. CBZ encapsulation efficiency was 68 to 82% for all prepared formulations. Thermograms of CBZ-PLGA microspheres suggest that CBZ was totally entrapped with the PLGA polymer. The presence of Pluronic F-68 has improved the encapsulation of CBZ, resulted in better and smoother microspheres surfaces and enhanced its release pattern. CBZ release profiles were biphasic patterns; after an initial burst, a constant CBZ release rate was observed up to 24 hr. The release from these PLGA-based spherical matrices was consistent with the diffusion mechanism. CBZ dissolution T(50%) was significantly affected (> 3-fold) by increasing the lactide percent from 33.3 to 66.6% from different microspheres mixtures. The present study provides evidence that the encapsulation of CBZ to PLGA microspheres, either as a single polymer or mixture of two, was a successful attempt to control the release of CBZ.     

Comparison of in vitro-in vivo release of Risperdal(®) Consta(®) microspheres

The objective was to investigate the relationship between in vitro and in vivo release of commercial Risperdal(?) Consta(?) microspheres. A modified USP apparatus 4 method was used for accelerated and real-time in vitro release testing. The in vivo plasma profile (clinical data) reported for the product was deconvoluted for comparison with the in vitro release profiles. The in vivo profile differed from the real-time in vitro profile and was faster initially and then slower after approximately 30 days. This effect is considered to be due to differences in the in vivo conditions such as small interstitial volume, low pH and immune response. Accelerated in vitro release profiles obtained at temperatures (50°C and 54.5°C) above the microsphere glass transition temperature (Tg～48°C) overlapped with the in vivo profile after time scaling. A linear in vitro-in vivo relationship was observed with correlation coefficients of 0.97 and 0.99 at 50°C and 54.5°C, respectively. The accelerated test performed below the Tg had a similar release profile to that of the real-time in vitro test. The accelerated tests performed above the Tg of the microspheres showed the potential to be used for in vivo performance prediction as well as for quality control purposes.