A Novel in Vitro Delivery System for Assessing the Biological Integrity of Protein upon Release from PLGA Microspheres (original) (raw)
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Preparation and in vitro release profiling of PLGA microspheres containing BSA as a model protein
Brazilian Journal of Pharmaceutical Sciences, 2020
Conventional drug formulations are incapable of adequate delivery of proteins and peptides for therapeutic purposes. As these molecules have very short biological half-life, multiple dosing is required to achieve the desirable therapeutic effects. Microspheres are able to encapsulate proteins and peptide in the polymeric matrix while protecting them from enzymatic degradation. In this study Bovine Serum Albumin (BSA) matrix type microspheres were fabricated using Polylactideco-glycolide (PLGA) by double emulsion solvent evaporation method. The effects of variables such as homogenizer speed, molecular weight of polymer and the effect of pH of the water phases, were investigated against factors such as drug loading, encapsulation efficiency, morphology, size, drug distribution and release profile of the microspheres. Results, suggested that an increase in homogenization speed leads to a decrease in microsphere size. The increase in homogenization speed also caused a significant effect on the release profile only when higher molecular weight of polymer had been used.. The pH change of the internal aqueous phase led to modification of surface morphology of spheres to a porous structure that significantly increased the total amount of released protein. Integrity of protein structure was intact as shown by SDS-PAGE. According to the results, it can be concluded that we achieved a reproducible method regarding controlled protein delivery for different sizes of particles.
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.
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.
European Journal of Pharmaceutics and Biopharmaceutics, 2010
Incomplete protein release from PLGA-based microspheres due to protein interactions with the polymer is one of the main issues in the development of PLGA protein-loaded microspheres. In this study, a twodimensional adsorption model was designed to rapidly assess the anti-adsorption effect of formulation components (additives, additives blended with the polymer or modified polymers). Lysozyme was chosen as a model protein because of its strong, non-specific adsorption on the PLGA surface. This study showed that PEGs, poloxamer 188 and BSA totally inhibited protein adsorption onto the PLGA37.5/25 layer. Similarly, it was emphasised that more hydrophilic polymers were less prone to protein adsorption. The correlation between this model and the in vitro release profile was made by microencapsulating lysozyme with a low loading in the presence of these excipients by a non-denaturing s/o/w encapsulation technique. The precipitation of lysozyme with the amphiphilic poloxamer 188 prior to encapsulation exhibited continuous release of active lysozyme over 3 weeks without any burst effect. To promote lysozyme release in the latter stage of release, a PLGA-PEG-PLGA tribloc copolymer was used; lysozyme was continuously released over 45 days in a biologically active form.
Controlled Release of Lysozyme from Double-Walled Poly(Lactide-Co-Glycolide) (PLGA) Microspheres
Polymers
Double-walled microspheres based on poly(lactide-co-glycolide) (PLGA) are potential delivery systems for reducing a very high initial burst release of encapsulated protein and peptide drugs. In this study, double-walled microspheres made of glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA), and carboxyl-terminated PLGA were fabricated using a modified water-in-oil-in-oil-in-water (w 1 /o/o/w 2) emulsion solvent evaporation technique for the controlled release of a model protein, lysozyme. Microspheres size, morphology, encapsulation efficiency, lysozyme in vitro release profiles, bioactivity, and structural integrity, were evaluated. Scanning electron microscopy (SEM) images revealed that double-walled microspheres comprising of Glu-PLGA and PLGA with a mass ratio of 1:1 have a spherical shape and smooth surfaces. A statistically significant increase in the encapsulation efficiency (82.52 ± 3.28%) was achieved when 1% (w/v) polyvinyl alcohol (PVA) and 2.5% (w/v) trehalose were incorporated in the internal and external aqueous phase, respectively, during emulsification. Double-walled microspheres prepared together with excipients (PVA and trehalose) showed a better control release of lysozyme. The released lysozyme was fully bioactive, and its structural integrity was slightly affected during microspheres fabrication and in vitro release studies. Therefore, double-walled microspheres made of Glu-PLGA and PLGA together with excipients (PVA and trehalose) provide a controlled and sustained release for lysozyme.
International Journal of Molecular Sciences, 2021
Over the past few decades, long acting injectable (LAI) depots of polylactide-co-glycolide (PLGA) or polylactic acid (PLA) based microspheres have been developed for controlled drug delivery to reduce dosing frequency and to improve the therapeutic effects. Biopharmaceuticals such as proteins and peptides are encapsulated in the microspheres to increase their bioavailability and provide a long release period (days or months) with constant drug plasma concentration. The biodegradable and biocompatible properties of PLGA/PLA polymers, including but not limited to molecular weight, end group, lactide to glycolide ratio, and minor manufacturing changes, could greatly affect the quality attributes of microsphere formulations such as release profile, size, encapsulation efficiency, and bioactivity of biopharmaceuticals. Besides, the encapsulated proteins/peptides are susceptible to harsh processing conditions associated with microsphere fabrication methods, including exposure to organic s...
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.
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.
A potential approach for decreasing the burst effect of protein from PLGA microspheres
Journal of Pharmaceutical Sciences, 2003
A central issue in controlled delivery of therapeutics from biodegradable microspheres is the immediate burst of drug release upon injection. This burst is often observed with microsphere systems made by the double emulsion (w/o/w) technique, and may be prevented by improving the drug distribution throughout the polymer matrix. To this end, protein and polymer (poly-lactide-co-glycolide or PLGA) were dissolved within the same solvent system, and micron-sized microspheres were created from this solution by spontaneous emulsification. Improved protein loading was achieved by ion-pairing the protein with charged surfactants to increase solubility in the single-phase solvent system. Both in vitro and in vivo results showed a much diminished burst: compared to microspheres made by double emulsion, it was reduced over 10-fold. ß
Development and Scale-up of a Microsphere Protein Delivery System
Biotechnology Progress, 1998
This paper reviews the development path for the ProLease injectable microsphere delivery system for proteins using human growth hormone as an example. The process consists of four stages, the selection of a lead formulation for clinical testing, the preclinical evaluation of the lead formulation including toxicology and stability studies, the manufacture of phase I clinical supplies, and the scale-up for phase II and phase III clinical trials. The approaches used to overcome obstacles during each stage are summarized including ways of stabilizing the protein, obtaining desirable release kinetics, and manufacturing sterile batches for clinical testing. Stability, release, toxicology, and scale-up results for ProLease recombinant human growth hormone (rhGH) are given. The phase I clinical data show that bioactive rhGH was released for about 1 month in humans. This work shows that processes and procedures have been developed that enable the production of microsphere sustained release formulations for proteins suitable for clinical trails and commercialization.