Effect of the molecular weight of poly(ethylene glycol) used as emulsifier on α-chymotrypsin stability upon encapsulation in PLGA microspheres (original) (raw)
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Development of polylactide microspheres for protein encapsulation and delivery
Journal of Applied Polymer Science, 2002
The development of injectable microparticles for protein delivery is a major challenge. We demonstrated the possibility of entrapping human serum albumin (HSA) and thrombin (Thr) in poly(ethylene glycol) (PEG)-coated, monodisperse, biodegradable microspheres with a mean diameter of about 10 m. In our earlier studies, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) analysis was used to characterize the surface of PEG-coated, taxol-loaded poly(lactic acid) (PLA) microspheres. An analysis by DRIFTS revealed that PEG was incorporated well on the PLA microsphere surface. An emulsion of protein (in water) and PLA dissolved in an acetone-dichloromethane (or acetone-chloroform) mixture were poured into an aqueous solution of PEG [or poly(vinyl alcohol) (PVA)] with stirring with a high-speed homogenizer for the formation of microparticles. HSA recovery in microspheres ranged from 13 to 40%, depending on the solvent and emulsification systems used for the preparation. PLA dissolved in a dichloromethane/acetone system and albumin loaded via a PEG emulsification solution (PLA-PEG-HSA) showed maximum drug recovery (39.5%) and drug content (9.9%). Scanning electron microscopy revealed that PEG-coated microspheres had less surface micropores than PVA-based preparations. The drug-release behavior of microspheres suspended in phosphate-buffered saline exhibited a biphasic pattern. An initial burst release (30%) followed by a constant slow release for 20 days was observed for HSA and Thr from PLA-PEG microspheres. PEG-coated PLA microspheres show great potential for protein-based drug delivery.
The AAPS Journal, 2009
The reduced injection frequency and more nearly constant serum concentrations afforded by sustained release devices have been exploited for the chronic delivery of several therapeutic peptides via poly(lactide-co-glycolide) (PLG) microspheres. The clinical success of these formulations has motivated the exploration of similar depot systems for chronic protein delivery; however, this application has not been fully realized in practice. Problems with the delivery of unmodified proteins in PLG depot systems include high initial "burst" release and irreversible adsorption of protein to the biodegradable polymer. Further, protein activity may be lost due to the damaging effects of protein-interface and protein-surface interactions that occur during both microsphere formation and release. Several techniques are discussed in this review that may improve the performance of PLG depot delivery systems for proteins. One promising approach is the covalent attachment of poly(ethylene glycol) (PEG) to the protein prior to encapsulation in the PLG microspheres. The combination of the extended circulation time of PEGylated proteins and the shielding and potential stabilizing effects of the attached PEG may lead to improved release kinetics from PLG microsphere system and more complete release of the active conjugate.
Effect of additives on the release of a model protein from PLGA microspheres
AAPS PharmSciTech, 2001
The purpose of this study was to investigate the effect of 2 additives, poly(ethylene glycol) (PEG) 1000 and 1,2,3-tridecanoyl glycerol (tricaprin), on the physico-chemical characteristics and in vitro release of a model protein, bovine serum albumin (BSA), form poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres. BSA-loaded microspheres were prepared by the double emulsion solvent evaporation method. Additives were incorporated into microspheres to modify the release of protein. The addition of PEG 1000 and tricaprin changed the surface characteristics of microspheres from smooth and nonporous to porous and dimpled, respectively. The in vitro release profiles showed that the additives significantly (P < 0.05) increased the early-stage release of BSA from microspheres.
Journal of Controlled Release, 2005
Encapsulation of proteins in poly(lactide-co-glycolide) microspheres via emulsion is known to cause insoluble protein aggregates. Following protein emulsification and encapsulation in PLGA microspheres, we used circular dichroism to show that the recoverable soluble protein fraction also suffers subtle conformational changes. For a panel of proteins selected on the basis of molecular size and structural class, conformational stability measured by chemical denaturation was not indicative of stability during emulsion-encapsulation. Partial loss of structure was observed for a-helical proteins released from freeze-dried microspheres in aqueous buffer, with dramatic loss of structure for a h-sandwich protein. The addition of sucrose (a lyoprotectant) did not prevent the loss of protein conformation upon encapsulation. Therefore, the conformational changes seen for the released soluble protein fraction originates during emulsification rather than microsphere freeze-drying. Analysis of the burst release for all proteins in buffer containing denaturant or surfactant showed that the degree of protein solubilisation was the dominant factor in determining the initial rate and extent of release. Our data for protein release into increasing concentrations of denaturing buffer suggest that the emulsion-denatured protein fraction remains insoluble; this fraction may represent the protein loss encountered upon comparison of protein encapsulated versus protein released.
Journal of Applied Polymer Science, 2001
Eight kinds of self-catalyzed poly(ortho-esters) (POEs) are used to fabricate bovine serum albumin (BSA)-containing microspheres using a W/O/W double-emulsion solvent extraction/evaporation method. All eight kinds of POE polymers used in this study are shown able to form microspheres under proposed fabrication conditions. The surface morphology and inner structure of the microspheres are analyzed using scanning electron microscopy (SEM). The microspheres have a size range from 64.7 to 120.2 m. POE with a higher viscosity leads to bigger microspheres. It was found that the POE composition has a significant effect on BSA release profiles. POEs, which are more hydrophilic and contain a greater amount of glycolide or lactate (latent acid), yield higher BSA release rates. Specifically, POE containing 1,6-hexanediol diglycolide (HD-diGL) microspheres have the highest BSA release rate after a 20-day test through a combination of surface erosion and diffusion mechanisms. POE containing a high percentage of the trans-cyclohexanedimethanol (CDM) segment tends to yield microspheres with a lower release rate because of its hydrophobic nature. It was also found that the BSA release rate is more rapid at 37°C than at 22°C because of faster polymer degradation and water penetration at 37°C. Experimental results suggest that various protein release rates can be achieved by using different compositions of POEs.
Journal of Biomedical Materials Research, 1999
The development of injectable nanoparticulate "stealth" carriers for protein delivery is a major challenge. The aim of this work was to investigate the possibility of achieving the controlled release of a model protein, human serum albumin (HSA), from poly(ethylene glycol) (PEG)coated biodegradable nanospheres (mean diameter of about 200 nm) prepared from amphiphilic diblock PEG-poly(lactic acid) (PLA) copolymers. HSA was efficiently incorporated into the nanospheres, reaching loadings as high as 9% (w/ w). Results of the in vitro release studies showed that it is possible to control the HSA release by choosing the appropriate nanosphere size, loading, and composition. These results also revealed that, following their release, HSA molecules readsorbed onto the nanospheres surfaces when they were not protected by a PEG coating. We were surprised to observe that in spite of the water uptake of the PLA-PEG nanospheres [11-29% (w/w)], the copolymer did not significantly degrade after a 15-day incubation period. Therefore, we concluded that during this time HSA release from PLA-PEG nanospheres followed a diffusion mechanism where bulk erosion and surface desorption were negligible.
Journal of Controlled Release, 1991
Poly(o~-lactide/glycolide, 50: 50) microspheres containing bovine serum albumin (RSA) were prepared with and without Carbopol" 95 I (a potential adjuvant agent) by o/o, o/w and (w/o)lw emulsion methods. The protein loading of the microspheres reached 50-700/o of the theoretical amount of protein put into the formulation medium. The microsphere particle size was approximately 500 pm, 2%IOOpm, IO-20pm usingojo, o/w, or (w/o)/ w emulsion techniques. respectively. The release of BSA was dependent on the preparation method. The greatest burst of release was found for vacuumdried microspheres formulated using the (w/0)/w method. This burst effect could be eliminated by lyophilizing the microspheres following their preparation. BSA was released at a higher initial rate from microspheres prepared by the o/w emulsion method that contained Carbopol" 95 I than from microspheres not containing CarbopolO 951. Release studies also suggested that the release of SSA could be sustained for 54, 36, or 34 days pa! microspheres prepared by o/o. o/w, or (w/o)@ methods, respectively. I 2 3 4 5 6 7 8 9 10 efemnces R.J. Linhardt, Bvadegradable polymers for the controlled release ofdrugs. In: M. Roroff (Ed. ), Controlled Releaseof Drugs: Po,ymersand Aggrcgatc Systems. VCH Publishers, New York, 1989, pp. 53-95. K. Junl and M. Nakano, Poly(hydroxy acids) in drug delivery. Crit. Rev. Ther. Drug Carrier Syst., 3 (1987) X9-232. J.P. Kilchell and D.L. Wise, Poly(lactide/glycolide) biodegradable drug-polymer matrix system. In: K.J. Widderand R. Green (Eds.), Methods in Enzymology, Vol. 112. Academic Press, Orlando, FL, 1985, pp. 436-448. J. Hcller. Zero order drug release from biocrodibk polp men. In: J.M. Anderson and S.W. Kim (Eds.), Recent Advancer in Drug Dclivcv Systems, Plenum Press New York. 1984,~~. 101-121. T.R. Tice and D.R. Cowsar. Biodegradable mntrolledrclcasc parenteral systems, Pharm. Tech., 8( I I ) ( ,984, 26-35. R.S. Langcr and N.A. Pcppas. Prcscnt and future applicauons of biomatcrials in controlled drug delivery systems. Biomalerials, 2 (1981 ) 201-214. D.A. Wood, Biodegmdable drug d&very systems, Jnt. J. Phsrm..7 (1980, l-18. S. Yolbs and M.F. Sanon, Dcgradablc polymers for sustained drug release. In: R.L. Juliano (Ed. ). Drug DP livery Systems, Oxford University Press, New York, ,980, pp. 84-1 IO. A. Schindler, R. Jeffcoat,G.L. Kimmel. C.G. Pit1.M.E. Wall and R. Zweldmger. Biodegradable polymers for sustained drug delivery. In: E.M. Pearce and J.R. Schaeffgrn (Eds ). Conrcmporary Topics in Polymer scwnce. "cd. 2. Acnum Press, New York, 1977, pp. 25 I-286. N. Marcotlc, A. Polk and M.F.A. Gooaen. Kinetics of protcm release from a poly(w-lactide) reservoir systcm.J.Pharm.Sci.. 79(5) (1990) 407-410. 26 R.J Llnhaidt, DR. Fhagim. E. SEhrnl,, and H.T. Wang. Blodcgradable poly(eners, and [he debvwy of bloacwc Bg",s. Pobm. Prcpr., 31(,) (1990, x9-259. ConwAled release of prowm and vatcmes from poly(e9er) microsphcrcs in vitro. In: G. 00. b&in (Ed 1. Polymers for Cosmeuc and Pharmaceuucal Appbcauons. Plenum, New York, I991 ~ in press. 29 J Kreuler and E. Liehl. Long-term studies of nucroenprolidc accLatc into microcapwles of polylactlc or copoly(lacuc,Slycol,c, acid. Cbem. Pbarm. Bull.. 36( 3, (1988) 1095-1103 35 M. Bradford, A raped and scnsilw method for the quanl~tation of microgram qu3nIilies of protein utiliring the principle of pmlem-dye bindtng. Anal. Biochem.. 72 (1976) 248-254. 36 G.L. Gualandi. NM Losio. C. Muratori and E. Font. The ab~bty by dxfferem preparations of porcine parve-+I~P to enhance humoral immunity m swine and guinea p,gs, MIcroblolag,ca. I I t 1988) 363-369.
Journal of Controlled Release, 1997
The water soluble peptide, pBC 264, derived from cholecystokinin and composed of seven aminoacids was encapsulated in poly(lactide-co-glycolide) (PLG) microspheres prepared by a multiple emulsion [(W1/O)W2] solvent evaporation method. The encapsulation efficiency of pBC 264 was very low when the inner emulsion contained no stabilizing agent. However, the encapsulation rate was improved by the addition of ovalbumin (OVA) used as stabilizer of the inner emulsion. In addition, the presence of a pH gradient between the inner and the outer aqueous phases of the multiple emulsion led to an increase of the retention of the peptide within the microspheres. Nevertheless, the molecular weight of the polymer did not have any influence on the encapsulation efficiency of the peptide. All microspheres batches showed a mean diameter below 10 μm. In vitro release studies were carried out in both phosphate (pH 7.4) and TRIS buffer (pH 8), with microspheres made from several copolymers differing in their molecular weight. Release kinetics were characterized by a dramatic burst effect corresponding to the release of the major part of the entrapped pBC 264. DSC analysis were able to show that there was no interaction between OVA and the polymer suggesting that the protein is not dissolved in the polymer network but mainly present at the interfaces of the inner aqueous phase/polymer and microsphere surface/outer aqueous phase. The OVA that is present on microspheres might create a porous structure that was observed by electron microscopy. These pores would be responsible for the fast release of pBC 264. These results allow better comprehension of the effect of pH gradient and the use of OVA as a stabilizer on the encapsulation and release pattern of PLG microsphere-loaded small peptides.
Journal of Pharmaceutical Investigation, 2012
Spray-dried microspheres based on polysaccharides were developed and the conformational stability and controlled release of incorporated protein were evaluated using bovine serum albumin (BSA) as a model protein. Microspheres composed of water soluble chitosan (WCS), hydroxypropyl-b-cyclodextrin (HP-b-CD) and polyethylene glycol (PEG) were prepared by spray dying. WCS was used as a mucoadhesive and biocompatible polymer. HP-b-CD and PEG were used as protein stabilizer during the spray drying process. Microspheres with 6-7 lm of mean diameter were successfully developed. Encapsulation efficiency of BSA in microsphere was over 70 %. Primary, secondary and tertiary structure of incorporated BSA in microsphere was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism, and fluorescence intensity measurement, respectively. Conformational stability of BSA was maintained during the spray drying process. BSA release from microspheres was evaluated in in vitro model using the Transwell Ò insert, and showed a sustained release profile compared to naive BSA. Thus, these microspheres could possibly serve as an optimized delivery system for preserved stability and sustained release of protein.