Bovine serum albumin/chitosan-nanoparticle bio-complex; spectroscopic study and in vivo toxicological – Hypersensitivity evaluation

doi:10.1016/j.saa.2021.119582

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Volume 253, 15 May 2021, 119582

https://doi.org/10.1016/j.saa.2021.119582 Get rights and content

Highlights

•
We demonstrated an in vitro loading interaction of CsNPs with BSA as immunogenic proteins.
•
The loading interaction occurs via static and dynamic quenching mechanisms.
•
BSA-CsNPs nano-formulations exhibit no hypersensitivity or allergic.
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The results might be relevant to the pharmaceutical use of CsNPs.

Abstract

This study, investigates the interaction of bovine serum albumin (BSA) with synthesized chitosan nanoparticles (CSNPs) using steady-state fluorescence and UV–vis absorbance spectroscopy as well as picosecond time-resolved fluorescence technique. The fluorescence quenching mechanism of BSA by CSNPs indicates the presence of both static and dynamic mechanism. The loading efficiency of BSA-CSNPs exhibited a decrease by about 6% in neutral pH under physiological temperature. Transmission electron microcopy (TEM) images revealed the Synthesized CSNPs were irregular in shape with size of ~42 nm. The safety and biocompatibility of BSA-CSNPs inside the body was investigated after intraperitoneal (IP) injection of male mice for nine days, analysis of in vivo results, revealed no toxicity with a hypocholesterolemic effect and a predicted mild activation of WBCs due to CSNPs adjuvant and immunogenic peptides in BSA. Accordingly, no signs of hypersensitivity were observed due to the administration of such formulations. The results can be used for a better understanding the interaction of CSNPs within biological protein environment.

Graphical abstract

Introduction

Recently, interest in the Nanomaterials for delivery of therapeutic agents is growing very rapidly because of their high drug encapsulation/loading capacity, tunable size, ease of synthesis, low toxicity, stability in the presence of serum, and stimuli-responsiveness [1], [2]. Chitosan nanoparticles (CSNPs) represents one of the most innovative polymeric nanoparticles that have attracted significant attention in the biomedical applications, as a result of their unique chemical properties, biological effects, such as good biocompatibility, nontoxicity, biodegradability and antibacterial properties [3], [4].

The use of CSNPs in biological applications as bio-imaging, drug/gene delivery approaches, tissue engineering and in the pharmaceutical field, [5], [6] as well as cleaner of harmful water pollutants [7] has increased significantly. It has been widely employed for disease treatment, due to its antioxidant, anti-allergic, anti-inflammatory, anticoagulant, anti-bacterial, anti-HIV, anti-hypertensive, anti-Alzheimer’s, anti-diabetic, anti-obesity, and anti-cancer properties [8], [9].

In addition, Mariam et al. [10] showed that bovine serum albumin (BSA)-derived nanoparticles have sustained release properties which can increase the half-life of drug and therefore decrease the frequency of administration and improves patient compliance.

Protein / nanoparticle (NP) complexes have considered as one of the most promising vehicles for targeted drug delivery, due to enhanced stability, half-life of drug, ease of administration, and reducing the side effects [11]. Studying the binding of CSNPs to protein on an atomic level is crucial for understanding the biological effects and functions of drugs in the body. It has reported that the ligand binding to protein is usually weak and noncovalent interactions (hydrophobic, electrostatic, van der Waals, and hydrogen bonding) [12].

BSA (~66.4 KDa molecular weight) is a serum albumin protein has a single polypeptide chain consisting of about 583 amino acid residues organized in a single polypeptide chain of 3 domains. Each domain has two subdomains (A and B). It is characterized with negatively charged Lysine amino acids that aid in its binding with charged ligands and it has two Tryptophan residues with its intrinsic fluorescence unlike HSA that has only one Trp residue. Trp-212 is buried in the hydrophobic pocket of subdomain IIA and Trp-134 is at the surface of domain I. The ligand binding sites on BSA molecule are localized at subdomain IIA (site I) and subdomain IIIA (site II) [13], [14].

BSA used in this work as a model of carrier immunogenic proteins that help in low molecular weight peptides (Mwt < 20 KDa) delivery to increase their immunogenicity inside the body. BSA contains some long immunogenic epitopes that can be detected by macrophages and induce the proper immune response [15]. Otherwise, it was reported that the most critical antigenic sequence in BSA is involved in the peptide sequence of amino acids 524–542 that is must be in a longer sequence to be recognized by antibodies [16].

In this study we attempt to understand the interaction mechanism of CSNPs as a model drug with biological environment as BSA for biomedical applications, particularly drug and peptide delivery. The nature of quenching and binding parameters was evaluated using steady-state and time-resolved fluorescence spectroscopic approaches. Moreover, the safety and biocompatibility of BSA-CSNPs were also investigated by in vivo methods.

Section snippets

Materials

Low Molecular Weight Chitosan (50 KDa, 90–95% deacetylation) was purchased from Oxford, Mumbai, India. Sodium tripolyphosphate (TPP) (Mw = 367.86 g/mol) and Bovine serum albumin (BSA) were purchased from Sigma Aldrich. Phenylbutazone (Mw = 308.374 g/mol) was provided from Global Nabi Pharmaceuticals company, Egypt. Ibuprofen (Mw = 206.29 g/mol) was provided from the Egyptian Drug Authority. Cholesterol, triglycerides, creatinine, albumin, globulin, total protein kits were obtained from

Characterization of CSNPs

Fig. 1 shows the SEM, (A) and TEM (B) micrographs of the synthesized CSNPs. It exhibits an irregular shape with uniform dispersion as a result crosslinking of TPP and CS due to the ionic interaction of negatively charged phosphoric ions and positively charged chitosan as shown in Scheme 1 [22]. Based on TEM Image, an average size of ≈ 42 ± 6 nm was observed for CSNPs.

To gain information about the surface charge and homogeneity of CSNPs, which would consequently lead to gain insight about its

Conclusion

In the present study, 42 nm diameter CSNPs have been synthesized with an irregular shape. The synthesized CSNPs were loaded with BSA via its binding site I. The experimental results revealed that NPs were stable in normal saline at 4 °C for 2 months. The fluorescence quenching of BSA by CSNPs occurs via both static and dynamic mechanisms. The interaction of BSA with CSNPs is favored in acidic media and low temperatures. Furthermore, BSA-CSNPs exhibited proper nontoxic and biocompatibility with

CRediT authorship contribution statement

Moataz M. Rashad: Methodology, Writing - original draft. Nesma M. El-Kemary: Investigation, Visualization, Methodology. Said Amer: Conceptualization, Methodology. Maged El-Kemary: Conceptualization, Funding acquisition, Supervision, Resources.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The study was financially supported by the office of vice-President for research of Kafrelsheikh University.

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Bovine serum albumin/chitosan-nanoparticle bio-complex; spectroscopic study and in vivo toxicological – Hypersensitivity evaluation

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Characterization of CSNPs

Conclusion

CRediT authorship contribution statement

Declaration of Competing Interest

Acknowledgment

Carbohyd. Polym.

Carbohydr. Polym.

J. Control. Release

Carbohydr. Polym.

Int. J. Biol. Macromol.

Acta Pharm. Sin. B.

Adv. Drug Deliv. Rev.

J. Mol. Liqui.

Mol. Immunol.

Nano Energy

Colloids Surf. B

Biologicals

Int. J. Biol. Macromol.

Biomaterials

J. Control. Release

Mater. Sci. Eng., C

J. Pharmaceut. Biomed.

Spectrochim. Acta. A.

J. Colloid Interface Sci.

Carbohydr. Polym.

Int. J. Pharm.

Nano-encapsulated metformin-curcumin in PLGA/PEG inhibits synergistically growth and hTERT gene expression in human breast cancer cells

Artif. Cells Nanomed. Biotechnol.

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ACS Nano

Chitosan based nanoparticles towards biomedical applications

J. Nanomed. Res.