Protein nanoparticles with ligand-binding and enzymatic activities

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Protein nanoparticles with ligand-binding and enzymatic activities

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Author(s): Olga V Morozova ¹ ^, ² , Elizaveta R Pavlova ¹ ^, ³ , Dmitry V Bagrov ¹ ^, ⁴ , Nikolay A Barinov ¹ , Kirill A Prusakov ¹ ^, ³ , Elena I Isaeva ² , Victor V Podgorsky ¹ , Dmitry V Basmanov ¹ , Dmitry V Klinov ¹

Publication date (Electronic): 18 October 2018

Journal: International Journal of Nanomedicine

Publisher: Dove Medical Press

Keywords: protein nanostructures, nanoprecipitation, atomic force and electron microscopy, energy-dispersive X-ray spectroscopy, ultraviolet and circular dichroism spectroscopy, ELISA, lysozyme activity assay

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Abstract

Purpose

To develop a general method for NP fabrication from various proteins with maintenance of biological activity.

Methods

A novel general approach for producing protein nanoparticles (NP) by nanoprecipitation of the protein solutions in 1,1,1,3,3,3-hexafluoroisopropanol is described. Protein NP sizes and shapes were analyzed by dynamic light scattering, scanning electron and atomic force microscopy (SEM and AFM). Chemical composition of the NP was confirmed using ultraviolet (UV) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and circular dichroism (CD). Biological properties of the NP were analyzed in ELISA, immunofluorescent analysis and lysozyme activity assay.

Results

Water-insoluble NP were constructed from globular (bovine serum albumin (BSA), lysozyme, immunoglobulins), fibrillar (fibrinogen) proteins and linear polylysines by means of nanoprecipitation of protein solutions in fluoroalcohols. AFM and SEM revealed NP sizes of 20–250 nm. The NP chemical structure was confirmed by UV spectroscopy, protease digestion and EDX spectroscopy. CD spectra revealed a stable secondary structure of proteins in NP. The UV spectra, microscopy and SDS-PAA gel electrophoresis (PAGE) proved the NP stability at +4°C for 7 months. Co-precipitation of proteins with fluorophores or nanoprecipitation of pre-labeled BSA resulted in fluorescent NP that retained antigenic structures as shown by their binding with specific antibodies. Moreover, NP from monoclonal antibodies could bind with the hepatitis B virus antigen S. Besides that, lysozyme NP could digest bacterial cellular walls.

Conclusion

Thus, the water-insoluble, stable protein NP were produced by nanoprecipitation without cross-linking and retained ligand-binding and enzymatic activities.

Most cited references 21

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Nano spray drying: a novel method for preparing protein nanoparticles for protein therapy.

Hak-Kim Chan, D YC Heng, S Lee … (2011)

There has been an increasing interest in the development of protein nanotherapeutics for diseases such as cancer, diabetes and asthma. Spray drying with prior micro mixing is commonly used to obtain these powders. However, the separation and collection of protein nanoparticles with conventional spray dryer setups has been known to be extremely challenging due to its typical low collection efficiency for fine particles less than 2μm. To date, there has been no feasible approach to produce these protein nanoparticles in a single step and with high yield (>70%). In this study, we explored the feasibility of the novel Nano Spray Dryer B-90 (equipped with a vibrating mesh spray technology and an electrostatic particle collector) for the production of bovine serum albumin (BSA) nanoparticles. A statistical experimental design method (Taguchi method based on three levels, five variables L(18) orthogonal array robust design) was implemented to study the effect of and optimize the experimental conditions of: (1) spray mesh size, (2) BSA solution concentration, (3) surfactant concentration, (4) drying air flow rate and (5) inlet temperature on: (1) size and (2) morphology (axial ratio). Particle size and morphology were predominantly influenced by the spray mesh size and surfactant concentration, respectively. The drying air flow rate and inlet temperature had minimal impact. Optimized production of smooth spherical nanoparticles (median size: 460±10nm, axial ratio: 1.03±0.00, span 1.03±0.03, yield: 72±4%) was achieved using the 4μm spray mesh at BSA concentration of 0.1% (w/v), surfactant concentration of 0.05% (w/v), drying flow rate of 150L/min and inlet temperature of 120°C. The Nano Spray Dryer B-90 thus offers a new, simple and alternative approach for the production of protein nanoparticles suited for a variety of drug delivery applications. Copyright © 2010 Elsevier B.V. All rights reserved.

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The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo.

J. Nash, Tiffany Nicole S Ballard, T. Weaver … (2006)

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.

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Poly-(lactic-co-glycolic-acid)-based particulate vaccines: particle uptake by dendritic cells is a key parameter for immune activation.

J. SILVA, R A Rosalia, E Varypataki … (2015)

Poly(lactic-co-glycolic acid) (PLGA) particles have been extensively studied as biodegradable delivery system to improve the potency and safety of protein-based vaccines. In this study we analyzed how the size of PLGA particles, and hence their ability to be engulfed by dendritic cells (DC), affects the type and magnitude of the immune response in comparison to sustained release from a local depot. PLGA microparticles (MP, volume mean diameter≈112 μm) and nanoparticles (NP, Z-average diameter≈350 nm) co-encapsulating ovalbumin (OVA) and poly(I:C), with comparable antigen (Ag) release characteristics, were prepared and characterized. The immunogenicity of these two distinct particulate vaccines was evaluated in vitro and in vivo. NP were efficiently taken up by DC and greatly facilitated MHC I Ag presentation in vitro, whereas DC cultured in the presence of MP failed to internalize significant amounts of Ag and hardly showed MHC I Ag presentation. Vaccination of mice with NP resulted in significantly better priming of Ag-specific CD8(+) T cells compared to MP and OVA emulsified with incomplete Freund's adjuvant (IFA). Moreover, NP induced a balanced TH1/TH2-type antibody response, compared to vaccinations with IFA which stimulated a predominant TH2-type response, whereas MP failed to increase antibody titers. In conclusion, we postulate that particle internalization is of crucial importance and therefore particulate vaccines should be formulated in the nano- but not micro-size range to achieve efficient uptake, significant MHC class I cross-presentation and effective T and B cell responses.

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Author and article information

Journal

Journal ID (nlm-ta): Int J Nanomedicine

Journal ID (iso-abbrev): Int J Nanomedicine

Journal ID (publisher-id): International Journal of Nanomedicine

Title: International Journal of Nanomedicine

Publisher: Dove Medical Press

ISSN (Print): 1176-9114

ISSN (Electronic): 1178-2013

Publication date Collection: 2018

Publication date (Electronic): 18 October 2018

Volume: 13

Pages: 6637-6646

Affiliations

[1 ]Department of Biophysics, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency (FRCC PCM), Moscow, Russia, omorozova2010@ 123456gmail.com

[2 ]Ivanovsky Institute of Virology of the National Research Center of Epidemiology and Microbiology of N.F. Gamaleya of the Russian Ministry of Health, Moscow, Russia, omorozova2010@ 123456gmail.com

[3 ]Moscow Institute of Physics and Technology, Moscow, Russia

[4 ]Department of Bioengineering, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia

Author notes

Correspondence: Olga V Morozova, Federal Research Clinical Center of Physico-Chemical Medicine of the Federal Medical and Biological Agency of Russia, 1a Malaya Pirogovskaya Street, Moscow 119435, Russia, Tel +7 916 421 2628, Fax +7 499 246 4409, Email omorozova2010@ 123456gmail.com

Article

Publisher ID: ijn-13-6637

DOI: 10.2147/IJN.S177627

PMC ID: 6202000

PubMed ID: 30425479

SO-VID: 833ce7ec-32ab-4dba-ba06-f0ad520b5cf1

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Protein nanoparticles with ligand-binding and enzymatic activities

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Abstract

Purpose

Methods

Results

Conclusion

Most cited references 21

Nano spray drying: a novel method for preparing protein nanoparticles for protein therapy.

The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo.

Poly-(lactic-co-glycolic-acid)-based particulate vaccines: particle uptake by dendritic cells is a key parameter for immune activation.

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