Solvent hydrolysis rate determines critical quality attributes of PLGA microspheres prepared using non-volatile green solvent.

This study aimed to develop a solvent hydrolysis-based microencapsulation technique that could fabricate PLGA microspheres, while using dimethyl carbonate as a green dispersed solvent. Instead of existing physical solvent removal techniques, a strategy was derived to use a chemical reaction that could transform oil droplets into microspheres. An oil-in-water emulsion was first produced by emulsifying a PLGA/Nile red/progesterone/dimethyl carbonate dispersed phase in an aqueous phase. Adding a NaOH solution into the emulsion led to the decomposition of dimethyl carbonate that partitioned to the water phase. This chemical reaction allowed the continuous diffusion of dimethyl carbonate existing in emulsion droplets into the aqueous phase and its complete removal. The solvent hydrolysis rate was identified as the most important process parameter affecting the major quality attributes of PLGA microspheres. For instance, it was shown through a 3D analysis that Nile red was uniformly dispersed across the microsphere matrix at a fast solvent hydrolysis rate. In comparison, a slow solvent hydrolysis rate allowed the dye to disperse heterogeneously in the microsphere matrix. A drug crystallization phenomenon, being commonly observed in conventional emulsion-templated processes, was inhibited by increasing the hydrolysis rate of dimethyl carbonate. Furthermore, adjusting the solvent hydrolysis rate made it possible to improve drug encapsulation efficiency, to disperse drug homogeneously across microsphere matrix, and to reduce variations in size distribution. The green solvent hydrolysis-based microencapsulation technique could be a promising alternative to conventional microencapsulation methods using toxic halogenated organic solvents.

Stabilization of proteins against methylene chloride/water interface-induced denaturation and aggregation.

The purpose of this study was to stabilize proteins during the commonly used microencapsulation process of methylene chloride/water emulsification. The model proteins used in this study included bovine serum albumin (BSA), S-carboxymethylated BSA (CM-BSA), reduced-S-carboxymethylated BSA (RCM-BSA), ovalbumin, and lysozyme. Emulsification of a 0.5-mg/ml protein solution in methylene chloride brought about changes in the composition of water-soluble species and interfacial coagulation. As a result, 37.8, 71.8 and 98.7% of ovalbumin, lysozyme and BSA were recovered from the aqueous phase after emulsification, respectively. Experiments with BSA, CM-BSA, and RCM-BSA demonstrated that a free thiol group and/or disulfide bond participated in interfacially induced dimerization and polymerization of proteins. Interfacial reactions that led to the aggregation of ovalbumin and lysozyme were inhibited by adding hydroxypropyl-beta-cyclodextrin or BSA into their aqueous solutions. Moreover, such beneficial effect of the excipients was observed to be concentration dependent. Under our experimental conditions, the recovery of ovalbumin and lysozyme was improved up to 97.7 and 95.6%, respectively. This study substantiated that an adequate formulation could overcome denaturing effects of the methylene chloride/water interface upon a protein of interest to be encapsulated into microspheres.

Protein behavior at the water/methylene chloride interface.

The objective of this study was to investigate the behaviors of proteins at the water/methylene chloride interface to better understand denaturing effects of emulsification upon proteins. Ribonuclease A (RNase) and human serum albumin (HSA) were used as model proteins throughout this study. Their behaviors at the interface were studied in terms of protein recovery after emulsification, interfacial protein aggregation, and dynamic interfacial tension. This study demonstrated that protein instability during emulsification was traced to consequences of the adsorption and conformational rearrangements of proteins at the water/methylene chloride interface. Compared to HSA, RNase was much more vulnerable to the interface-induced aggregation reactions that led to formation of water-insoluble aggregates upon emulsification. Even though HSA was almost completely recovered from the emulsified aqueous phase, the protein underwent dimerization and oligomerization reactions to some extent. The results also demonstrated that the extent of interfacial RNase aggregation was affected by its aqueous concentration and the presence of HSA. Interestingly, RNase stability during emulsification was almost achieved by dissolving an adequate quantity of HSA in the RNase solution. HSA seemed to compete for the interface site and to effectively keep RNase out the interface, minimizing the likelihood of the interface-induced RNase aggregation. These results indicated that competitive adsorption modes of proteins could be used to stabilize a protein of interest against the denaturing effects of emulsification.

A new strategy to determine the actual protein content of poly(lactide-co-glycolide) microspheres.

Bovine serum albumin, lysozyme, and trypsin inhibitor were first encapsulated into poly-d,l-lactide-co-glycolide (PLGA) microspheres and then a new strategy was used to quantitate the actual levels of proteins in the microspheres. The proper combination of water-miscible dimethyl sulfoxide and 0.05 N-NaOH containing 0.5% sodium dodecyl sulfate (SDS) made it possible to solubilize both PLGA microspheres and proteins in a single phase. A total protein assay conveniently provided accurate information on the amount of protein encapsulated into the microspheres. In contrast to conventional techniques making use of acetonitrile, dichloromethane, and SDS extraction methods, this new method did not necessitate polymer precipitation, filtration, and protein extraction into other phases. These features were a great advantage in recovering proteins without any loss due to experimental processes. As a consequence, the new method reported in this study provided accurate data for the actual level of protein in PLGA microspheres, regardless of the pattern of protein distribution inside microspheres or the characteristics of microspheres. The experiment relying on the use of a radiolabeled protein also validated the reliability of this new method.

Effects of H+ liberated from hydrolytic cleavage of polyester microcapsules on their permeability and degradability.

Microcapsules prepared from blends of poly(d,l-lactide-coglycolide) with a lactide:glycolide ratio of 75:25 (PLGA75:25) and poly-(d,l-lactide) (PLA5000) or poly(d,l-lactic acid-co-glycolic acid) (PLGA5000) were dispersed in phosphate-buffered saline, and their hydrolytic rates were investigated. Using the Henderson-Hasselbalch equation and an L-lactic acid experiment, the concentration of hydrogen ions released into the bulk medium was calculated from the change in buffer pH. The rate of H+ formation was found to be dependent upon the polymer composition of the microcapsules. The incorporation of PLGA5000 or PLA5000 into PLGA75:25 microcapsules drastically enhanced hydrolytic rates of microcapsules and resulted in controlled release of hydrogen ions generated from carboxyl end groups of both intact and degrading polymers. In contrast to microcapsules prepared with PLGA75:25 only, which liberated a negligible amount of H+ ions after a 21-day incubation, the microcapsules prepared from polymer blends released approximately (19.2-42.0) (x10(-3)) mmol of H+ ions. It has been found that the amount of hydrogen ions liberated into the bulk can be used as a qualitative indicator to monitor the change in microcapsule permeability to protein as well as polymer degradation.

Ethyl formate - alternative dispersed solvent useful in preparing PLGA microspheres.

In an effort to substitute methylene chloride with a less toxic solvent, this study was aimed at developing new ethyl formate-based emulsion processes to fabricate poly-D,L-lactide-co-glycolide (PLGA) microspheres. To do so, a polymeric dispersed phase was emulsified in a 1% polyvinyl alcohol aqueous solution at an ethyl formate to aqueous volume ratio of 8:20. Microsphere hardening was then achieved by solvent evaporation and quenching techniques. The average encapsulation efficiency of a model drug progesterone amounted to 95.2+/-2.7%. When the tendency of ethyl formate and methylene chloride to evaporate to air was compared, the evaporation rate of ethyl formate was 2.1 times faster than that of methylene chloride. The ease with which ethyl formate evaporated to air was beneficial in shortening the microsphere hardening step. For the solvent quenching process, only 80 ml of additional water was required to extract ethyl formate to the aqueous phase, due to its considerable water miscibility. In particular, the timing of ethyl formate quenching affected to a great extent dynamic processes of the breakup of elementary microdroplets into smaller ones. Therefore, variations in quenching time affected microsphere characteristics such as the degree of solvation, size distribution, and tendency to aggregate on drying. The results of this study showed that PLGA microspheres were successfully prepared using the new ethyl formate-based processes.

Prolonged immune response evoked by a single subcutaneous injection of microcapsules having a monophasic antigen release.

A model antigen, bovine serum albumin (BSA), was successfully incorporated into microcapsules fabricated from blends of poly(d,l-lactide-co-glycolide) and poly(d,l-lactide). The microcapsules possessed diameters ranging from 10 to 100 microns and exhibited a continuous monophasic release of the protein in-vitro for 3 weeks. They were found to enhance its immunogenicity, thereby potentiating the anti-BSA antibody response following a single subcutaneous injection in mice and rabbits. Enzyme-linked immunosorbent assays demonstrated that the microcapsules provoked high-titre and long-lived immunoglobulin G immune responses over a period of 192 days in mice. When rabbits were immunized by a single subcutaneous injection of BSA-loaded microcapsules, a high level of anti-BSA antibody was still present in the sera obtained at 17 weeks post-injection. The immunization protocol using the BSA-loaded microcapsules was superior to that using BSA dissolved in saline or adsorbed to alum. These microcapsules providing the controlled release of antigens may be valuable in designing better vaccine formulations.

Dynamic changes in size distribution of emulsion droplets during ethyl acetate-based microencapsulation process.

This study investigated the dynamic effect of the emulsification process on emulsion droplet size in manufacturing microspheres using ethyl acetate as an organic solvent. A dispersed phase consisting of poly(lactide-co-glycolide) and ethyl acetate was emulsified in a poly(vinyl alcohol) aqueous solution for a predetermined time ranging from 2 to 9, 16, 23, 30, 40, 50, or 60 minutes. Ethyl acetate was then quickly extracted to transform emulsion droplets into solidified microspheres, and their size distribution was determined. This experimental design allowed quantification of the size distribution of emulsion droplets over the course of emulsification. When emulsification time was extended from 2 to 60 minutes, the emulsion droplets decreased in size from 98.1 to 50.3 microm and their surface area increased from 0.07 to 0.29 m2/g. Overall, prolonging emulsification time up to 60 minutes resulted in the progressive evolution of smaller emulsion droplets (1-60 microm) and the simultaneous disappearance of larger ones (> 81 microm). Increases in the total number of microspheres and their surface area were caused mainly by continuous fragmentation of emulsion droplets before ethyl acetate extraction. The increase in the smaller microsphere population might also be due in part to shrinkage of microspheres. These results show that the onset of ethyl acetate extraction influenced the kinetics of the breakup and formation of emulsion droplets, thereby affecting to a great extent the size distribution of microspheres.

Protein instability toward organic solvent/water emulsification: implications for protein microencapsulation into microspheres.

The objective of this study was to investigate the behavior of three proteins at an organic solvent/water interface. To simulate the first microencapsulation step of a water-in-oil-in-water emulsion technique, a water-in-oil emulsion was prepared by emulsifying an aqueous protein solution in either methylene chloride or ethyl acetate. Phase separation was then followed to collect protein samples from the aqueous phase and the organic solvent/water interface. Their properties were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion-HPLC. Bovine serum albumin was relatively unharmed during emulsification, compared to other proteins such as ovalbumin and lysozyme. In particular, the methylene chloride treatment on ovalbumin led to the formation of a large quantity of water-insoluble, solid-like aggregates and changes in the composition of monomeric and dimeric ovalbumin species. With regard to the question of ovalbumin recovery, only 9.74 approximately 37.72% of the used ovalbumin was present in the aqueous phases after emulsification. Similar penchant was noted with lysozyme. Water-insoluble aggregates brought with by emulsification were found to be covalently bound. Interestingly, less emulsification-induced denaturing effects were observed with ethyl acetate. Our study clearly demonstrated the emulsification-induced adverse events that were detrimental to the integrity of proteins and the importance of preserving protein stability toward microencapsulation.

Formulation and process parameters affecting protein encapsulation into PLGA microspheres during ethyl acetate-based microencapsulation process.

The objective of this study was to investigate formulation and process parameters affecting protein encapsulation into PLGA microspheres during an ethyl acetate-based double emulsion microencapsulation process. Lysozyme was used as a model protein throughout this study. An aqueous lysozyme solution was emulsified in ethyl acetate containing 0.6 approximately 1.2 g PLGA75 : 25. The primary emulsion was then transferred quickly to an aqueous phase to make a water-in-oil-in-water emulsion. Ethyl acetate quenching was performed on the double emulsion stirred for 5, 15, 30 or 45 min. The resultant microspheres were further hardened, collected and dried overnight under vacuum. The bicinchoninic acid assay was carried out to determine the quantity of lysozyme present in the aqueous continuous phase and inside the microspheres. While the primary emulsion was stirred without quenching, lysozyme in the inner water phase continued diffusing across the ethyl acetate phase into the aqueous continuous phase. Emulsion droplets were also broken into smaller ones with ongoing stirring; this event also contributed to lysozyme leaking out of the inner water phase. The amount of lysozyme leaching to the aqueous continuous phase ranged from 4.79 +/- 2.1 to 51.9 +/- 5.3% under the experimental condition. Ethyl acetate quenching stopped the primary emulsion droplets from being fragmented into smaller ones and caused PLA75 : 25 precipitation to form microspheres. As a result, the rate of ethyl acetate removal influenced lysozyme encapsulation efficiency, as well as microsphere size. Depending on the timing of ethyl acetate quenching, lysozyme encapsulation efficiencies were found to be 9.89 +/- 4.53 approximately 75.82 +/- 6.55%. Optimization of the onset of ethyl acetate quenching and formulations could permit attainment of a desirable protein encapsulation efficiency.

A novel method of preparing PLGA microcapsules utilizing methylethyl ketone.

PURPOSE: To substitute dichloromethane with a safer solvent, a solvent extraction process using methylethyl ketone (MEK) was developed to prepare poly(d,l-lactide-co-glycolide) microcapsules. METHODS: The MEK dispersed phase containing PLGA and progesterone was emulsified in the MEK-saturated aqueous phase (W1) to make a transient oil-in-water (O/W1) emulsion. It was then transferred to a sufficient amount of water (W2) so that MEK residing in polymeric droplets could be extracted effectively into the continuous phase. RESULTS: This solvent extraction process provided the encapsulation efficiency for progesterone ranging from 77 to 60%. The amount of MEK predissolved in W1, as well as the degree of progesterone payload, influenced the encapsulation efficiency. The leaching profile of MEK analyzed by GC substantiated that, upon dispersion of O/W1, to W2, MEK quickly diffused into the continuous phase. Such a rapid diffusion of MEK from and the ingression of water into polymeric droplets produced hollow microcapsules, as evidenced by their SEM micrographs. CONCLUSIONS: When solvent extraction/evaporation techniques are employed for preparing PLGA microcapsules, water-immiscibility of a dispersed phase is not an absolute prerequisite to the successful microencapsulation. Adjustment of an initial extraction rate of MEK and formation of a primary transient O/W1 emulsion are found to be very crucial not only for the success of microencapsulation but also for the determination of microcapsule morphology.

Biodegradable microcapsules prepared by a w/o/w technique: effects of shear force to make a primary w/o emulsion on their morphology and protein release.

A water-in-oil-in-water (w/o/w) technique, sometimes known as in-water drying method, was used to prepare microcapsules consisting of polylactic acid and poly(lactide-co-glycolide). The influence of shear force to produce an initial water-in-oil (w/o) emulsion on the characteristics of microcapsules and protein release was investigated. Bovine serum albumin (BSA) was used as the model protein drug for encapsulation. The initial w/o emulsion was prepared by a Polytron homogenizer. The shear rate was varied from 11 to 23 krpm to produce w/o emulsions with different shear forces. This study revealed pronounced effects of shear force on the characteristics of microcapsules and release profiles of BSA. Depending on the degree of the shear applied, the inner structure of microcapsules showed very different morphology, which was responsible for different release patterns. A low shear produced microcapsules with a high initial burst release of BSA, whereas microcapsules using a high shear exhibited a controlled release of protein without any initial burst release. Also, at a given shear force, a variation in polymer composition of microcapsules was found to be effective in controlling the release characteristics of protein. Thus, the homogenization technique should be carefully considered in designing microcapsules with desirable release profiles of proteins and an adequate period of protein delivery.