Improved  -Chymotrypsin Stability Upon Encapsulation in PLGA Microspheres by Solvent Replacement

Castellanos, Ingrid J.; Griebenow, Kai

doi:10.1023/b:pham.0000003388.59659.fa

Cited by 23 publications

(27 citation statements)

References 19 publications

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“…Activity of α-chymotrypsin was determined using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate [28]. The reaction was carried out in 1 ml of 0.1 M Tris-HCl buffer containing 0.05 mg/ml enzyme, 0.35 mM substrate, and 10 mM CaCl 2 at pH 7.8 and our data for α-chymotrypsin as purchased are comparable to those reported [28].…”

Section: Methodssupporting

confidence: 68%

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: Methodssupporting

confidence: 68%

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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“…α-CT was chosen as the model protein since it has been employed prior to this investigation by us in solid-phase stability studies and also as model protein to test drug- delivery systems [1,30,31]. α-CT is prone to undergo aggregation and thus is an excellent sensor for aggregation-related instability events.…”

Section: Resultsmentioning

confidence: 99%

Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

et al. 2010

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BackgroundProtein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.ResultsFirst, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme α-chymotrypsin (α-CT) under controlled humidity conditions. Results showed that α-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, α-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that α-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size.ConclusionWater sorption leads to aggregation, inactivation, and structural changes of α-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.

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“…By delivering drug at a controlled rate over a prolonged time, such particles can maintain optimal drug concentrations [1e4], target specific organs and tissues [5], protect and stabilize the drug [6] and aid patient compliance by reducing the frequency of administration. Furthermore, particles with desired size are easily administered by injection.…”

Section: Introductionmentioning

confidence: 99%

Formation of poly(l,d-lactide) spheres with controlled size by direct dialysis

Zhou

Yang

et al. 2007

Polymer

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Improved -Chymotrypsin Stability Upon Encapsulation in PLGA Microspheres by Solvent Replacement

Abstract: The s/o/w technique was successfully improved by replacing the ICH class 2 solvent CH2Cl2 with the class 3 solvent butyl acetate without compromising alpha-chymotrypsin stability.

Cited by 23 publications

References 19 publications

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

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

Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

Formation of poly(l,d-lactide) spheres with controlled size by direct dialysis

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