Effect of cyclodextrins on α-chymotrypsin stability and loading in PLGA microspheres upon S/O/W encapsulation

Castellanos, Ingrid J.; Flores, Giselle; Griebenow, Kai

doi:10.1002/jps.20512

Cited by 35 publications

(24 citation statements)

References 33 publications

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“…However, while lysozyme encapsulation afforded a highly active enzyme, substantial enzyme inactivation and formation of buffer-insoluble aggregates were observed for α-chymotrypsin. The formation of buffer-insoluble aggregates and loss in specific activity found for α-chymotrypsin is similar to results obtained before upon α-chymotrypsin encapsulation in PLGA microspheres using a s/o/w technique [27,28,34–36]. The use of stabilizing additives (e.g., methyl-β-cyclodextrin or poly(ethylene glycol)) was necessary in the latter case to preserve protein integrity.…”

Section: Resultssupporting

confidence: 82%

Two-step nanoprecipitation for the production of protein-loaded PLGA nanospheres

Morales‐Cruz

Flores-Fernández

Morales-Cruz

et al. 2012

Results in Pharma Sciences

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One of the first methods to encapsulate drugs within polymer nanospheres was developed by Fessi and coworkers in 1989 and consisted of one-step nanoprecipitation based on solvent displacement. However, proteins are poorly encapsulated within polymer nanoparticles using this method because of their limited solubility in organic solvents. To overcome this limitation, we developed a two-step nanoprecipitation method and encapsulated various proteins with high efficiency into poly(lactic-co-glycolic)acid (PLGA) nanospheres (NP). In this method, a protein nanoprecipitation step is used first followed by a second polymer nanoprecipitation step. Two model enzymes, lysozyme and α-chymotrypsin, were used for the optimization of the method. We obtained encapsulation efficiencies of >70%, an amount of buffer-insoluble protein aggregates of typically <2%, and a high residual activity of typically >90%. The optimum conditions identified for lysozyme were used to successfully encapsulate cytochrome c(Cyt-c), an apoptosis-initiating basic protein of similar size, to verify reproducibility of the encapsulation procedure. The size of the Cyt-c loaded-PLGA nanospheres was around 300–400 nm indicating the potential of the delivery system to passively target tumors. Cell viability studies, using a human cervical cancer cell line (HeLa), demonstrate excellent biocompatibility of the PLGA nanoparticles. PLGA nanoparticles carrying encapsulated Cyt-c were not efficient in causing apoptosis presumably because PLGA nanoparticles are not efficiently taken up by the cells. Future systems will have to be optimized to ascertain efficient cellular uptake of the nanoparticles by, e.g., surface modification with receptor ligands.

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Section: Resultssupporting

confidence: 82%

Two-step nanoprecipitation for the production of protein-loaded PLGA nanospheres

Morales‐Cruz

Flores-Fernández

Morales-Cruz

et al. 2012

Results in Pharma Sciences

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“…The amount of non-covalent buffer-insoluble aggregates formed during encapsulation was 24% for the non-modified α-CT formulated as nanoparticles (Table 2) which is comparable to 18% reported for lyophilized α-CT [2,12]. A substantial reduction was noted for the encapsulated glycoconjugates: only 2% aggregates were found for encapsulated Lac 4 -α-CT bound and 8% for Lac 7 -α-CT.…”

Section: Resultssupporting

confidence: 54%

“…Some research has been performed focusing on eradicating protein aggregation and inactivation during the harsh encapsulation procedures and during release caused by PLGA-degradation produced acidification [1,7,12,13]. However, protein inactivation, aggregation, and unfolding during encapsulation are still issues severely hampering the application of sustained protein release PLGA microparticles [9].…”

Section: Introductionmentioning

confidence: 99%

Glycosylation improves α-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic)acid microspheres

Flores-Fernández

Griebenow

2012

Results in Pharma Sciences

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Enhancing protein stability upon encapsulation and release from polymers is a key issue in sustained release applications. In addition, optimum drug dispersion in the polymer particles is critical for achieving release profiles with low unwanted initial “burst” release. Herein, we address both issues by formulating the model enzyme α-chymotrypsin (α-CT) as nanoparticles to improve drug dispersion and by covalently modifying it with glycans to afford improved stability during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres. α-CT was chemically modified with activated lactose (500 Da) to achieve molar ratios of 4.5 and 7.1 lactose-to-protein. The bioconjugates were co-lyophilized with methyl-β-cyclodextrin followed by suspension in ethyl acetate to afford nanoparticles. Nanoparticle formation did not significantly impact protein stability; less than 5% of the protein was aggregated and the residual activity remained above 90% for all formulations. Using a solid-in-oil-in-water (s/o/w) methodology developed in our laboratory for nanoparticles, we obtained a maximum encapsulation efficiency of 61%. Glycosylation completely prevented otherwise substantial protein aggregation and activity loss during encapsulation of the non-modified enzyme. Moreover, in vitro protein release was improved for glycosylated formulations. These results highlight the potential of chemical glycosylation to improve the stability of pharmaceutical proteins in sustained release applications.

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“…Use of ion-pairing excipient to increase peptide hydrophobicity was found to be an effective strategy to reduce initial burst [91, 92]. Complexing therapeutic protein with other proteins, polysaccharide or cyclodextrin, was also utilized to reduce bursts and improve stability of encapsulated agents [15,93]. The third category involves optimizing the polymer microstructure by optimizing process variables.…”

Section: Approaches To Overcome Issues Impeding Depot Developmentmentioning

confidence: 99%

Injectable controlled release depots for large molecules

Schwendeman

Shah

Bailey

et al. 2014

Journal of Controlled Release

168

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Biodegradable, injectable depot formulations for long-term controlled drug release have improved therapy for a number of drug molecules and led to over a dozen highly successful pharmaceutical products. Until now, success has been limited to several small molecules and peptides, although remarkable improvements have been accomplished in some of these cases. For example, twice-a-year depot injections with leuprolide are available compared to the once-a-day injection of the solution dosage form. Injectable depots are typically prepared by encapsulation of the drug in poly(lactic-co-glycolic acid) (PLGA), a polymer that is used in children every day as a resorbable suture material, and therefore, highly biocompatible. PLGAs remain today as one of the few “real world” biodegradable synthetic biomaterials used in US FDA-approved parenteral long-acting-release (LAR) products. Despite their success, there remain critical barriers to the more widespread use of PLGA LAR products, particularly for delivery of more peptides and other large molecular drugs, namely proteins. In this review, we describe key concepts in the development of injectable PLGA controlled-release depots for peptides and proteins, and then use this information to identify key issues impeding greater widespread use of PLGA depots for this class of drugs. Finally, we examine important approaches, particularly those developed in our research laboratory, toward overcoming these barriers to advance commercial LAR development.

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Effect of cyclodextrins on α-chymotrypsin stability and loading in PLGA microspheres upon S/O/W encapsulation

Cited by 35 publications

References 33 publications

Two-step nanoprecipitation for the production of protein-loaded PLGA nanospheres

Two-step nanoprecipitation for the production of protein-loaded PLGA nanospheres

Glycosylation improves α-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic)acid microspheres

Injectable controlled release depots for large molecules

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