Green synthesis of collagen nanoparticles by Streptomyces xinghaiensis NEAA-1, statistical optimization, characterization, and evaluation of their anticancer potential

Abstract

Collagen nanoparticles (collagen-NPs) are promising biopolymeric nanoparticles due to their superior biodegradability and biocompatibility. The low immunogenicity and non-toxicity of collagen-NPs makes it preferable for a wide range of applications. A total of eight morphologically distinct actinomycetes strains were newly isolated from various soil samples in Egypt. The cell-free supernatants of these strains were tested for their ability. These strains' cell-free supernatants were tested for their ability to synthesize collagen-NPs. Five isolates had the ability to biosynthesize collagen-NPs. Among these, a potential culture, Streptomyces sp. NEAA-1, was chosen and identified as Streptomyces xinghaiensis NEAA-1 based on 16S rRNA sequence analysis as well as morphological, cultural and physiological properties. The sequence data has been deposited at the GenBank database under the accession No. OQ652077.1. Face-centered central composite design (FCCD) has been conducted to maximize collagen-NPs biosynthesis. Maximum collagen-NPs was 8.92 mg/mL under the condition of 10 mg/mL of collagen concentration, initial pH 7, incubation time of 48 h and temperature of 35 °C. The yield of collagen-NPs obtained via FCCD optimization (8.92 mg/mL) was 3.32-fold compared to the yield obtained under non-optimized conditions (2.5 mg/mL). TEM analysis of collagen-NPs showed hollow sphere nanoscale particles with mean of 32.63 ± 14.59 nm in diameter. FTIR spectra showed major peaks of amide I, amide II and amide III of collagen and also the cell-free supernatant involved in effective capping of collagen-NPs. The biosynthesized collagen-NPs exhibited anti-hemolytic, antioxidant and cytotoxic activities. The inhibitory concentrations (IC₅₀) against MCF-7, HeP-G2 and HCT116 cell lines were 11.62 ± 0.8, 19.60 ± 1.2 and 41.67 ± 2.2 µg/mL; respectively. The in-vivo investigation showed that collagen-NPs can suppress Ehrlich ascites carcinoma (EAC) growth in mice. The collagen-NPs/DOX combination treatment showed considerable tumor growth suppression (95.58%). Collagen-NPs evaluated as nanocarrier with a chemotherapeutic agent, methotrexate (MTX). The average size of MTX loaded collagen-NPs was 42.73 ± 3.5 nm. Encapsulation efficiency percentage (EE %) was 48.91% and drug loading percentage (DL %) was 24.45%.