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A one-step in vitro continuous flow assessment of protein release from core-shell polymer microcapsules designed for therapeutic protein delivery

Kupikowska-Stobba Barbara, Grzeczkowicz Marcin, Lewińska Dorota

Biocybernetics and Biomedical Engineering

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2021

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Vol. 41, no. 4

1347--1364

EN

Developing accurate methods for the assessment of therapeutic protein release from polymer drug delivery systems (microcapsules, microspheres, nanoparticles, 3D-printed systems) is of paramount importance for new formulation development. The most straightforward method for protein release assessment is spectrophotometric analysis of the release medium surrounding the formulation. However, direct spectrophotometric analysis is inapplicable to formulations releasing interfering compounds (coencapsulated drugs, additives) absorbing light in the same spectrum as proteins. Conventional protein release assays also require frequent release medium sampling and replacement, which reduces their accuracy. We propose a one-step method to assess protein release from core/shell microcapsules eliminating the need for sampling and allowing selective real-time protein quantitation in the release medium. To prevent spectral interferences, released protein is differentiated from interfering compounds by employing a colorimetric protein assay reagent, forming a colour complex selectively with the protein, as the release medium. To eliminate sampling, we employed a continuous flow closed loop set-up, where the release medium is constantly circulating between microcapsule-containing tank and spectrophotometer. A series of colorimetric protein assay reagents (bromocresol green, tetrabromophenol blue, eosin B, eosin Y, biuret) were evaluated in terms of their applicability as the release medium in described system. Only biuret reagent was found compatible with proposed method due to formation of color complex stable over extended period of time and low adsorption to microcapsules. Presented method allowed effective evaluation of albumin release from alginate-polyethersulfone microcapsules with accuracy equal to conventional ‘sample and separate’ technique. Albumin release followed first-order kinetics with plateau reached after 19 h.

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Effect of sonication reactor geometry on cell disruption and protein release from yeast cells

Bałdyga J., Jasińska M., Dzięgielewska M., Żochowska M.

Chemical and Process Engineering

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2018

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Vol. 39, nr 4

475-–489

EN

The measured rate of release of intercellular protein from yeast cells by ultrasonication was applied for evaluating the effects of sonication reactor geometry on cell disruption rate and for validation of the simulation method. Disintegration of two strains of Saccharomyces cerevisiae has been investigated experimentally using a batch sonication reactor equipped with a horn type sonicator and an ultrasonic processor operating at the ultrasound frequency of 20 kHz. The results have shown that the rate of release of protein is directly proportional to the frequency of the emitter surface and the square of the amplitude of oscillations and strongly depends on the sonication reactor geometry. The model based on the Helmholtz equation has been used to predict spatial distribution of acoustic pressure in the sonication reactor. Effects of suspension volume, horn tip position, vessel diameter and amplitude of ultrasound waves on the spatial distribution of pressure amplitude have been simulated. A strong correlation between the rate of protein release and the magnitude of acoustic pressure and its spatial distribution has been observed. This shows that modeling of acoustic pressure is useful for optimization of sonication reactor geometry.

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Uwalnianie białka z komórek drożdży Saccharomyces cerevisiae

Heim A., Kamionowska U., Solecki M.

Inżynieria Rolnicza

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2005

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R. 9, nr 9

133--142

PL

Przedstawiono wyniki doświadczeń dezintegracji drożdży Saccharomyces cerevisiae w klasycznym młynie perełkowym. Badano efekt niszczenia komórek w zależności od stężenia zawiesiny zmienianego w zakresie od 0,05 do 0,20 g suchej masy/cm3 i prędkości obrotowej mieszadła zmienianej w zakresie od 1000 do 3500 obr/min. Stopień dezintegracji określano dwoma metodami: na podstawie absorbancji mierzonej przy długości fali λ = 260 nm i na podstawie ilości uwolnionego białka określanego metodą Lowry’ego. Wykazano zwiększanie się stałej szybkości procesu wraz ze zwiększaniem koncentracji początkowej komórek w zawiesinie.

EN

Results of disintegration of yeast Saccharomyces cerevisiae in the classical bead mill are presented. The disintegration effect was investigated depending on the concentration of cell suspension ranging from 0.05 to 0.20 g d.m./cm3 and rotational speed of the impeller ranging from 1000 to 3500 rpm. The disintegration was determined by two methods: on the basis of the measurement of absorbance (λ = 260 nm) of supernatant samples of the processed suspension, and on the basis of the quantity of released protein determined by the Lowry method. The process rate constant was shown to increase with the increase of the initial concentration of cells.