Facile and Controllable Fabrication of Protein-Only Nanoparticles through Photo-Induced Crosslinking of Albumin and Their Application as DOX Carriers

Abstract

Protein-based nanoparticles, as an alternative to conventional polymer-based nanoparticles, offer great advantages in biomedical applications owing to their functional and biocompatible characteristics. However, the route of fabrication towards protein-based nanoparticles faces substantial challenges, including limitations in size control and unavoidable usage of toxic crosslinkers or organic solvents, which may raise safety concerns related to products and their degradation components. In the present study, a photo-induced crosslinking approach was developed to prepare stable, size-controlled protein-only nanoparticles. The facile one-step reaction irradiated by visible light enables the formation of monodispersed bovine serum albumin nanoparticles (BSA NPs) within several minutes through a tyrosine photo-redox reaction, requiring no cross-linking agents. The size of the BSA NPs could be precisely manipulated (from 20 to 100 nm) by controlling the duration time of illumination. The resultant BSA NPs exhibited spherical morphology, and the α-helix structure in BSA was preserved. Further study demonstrated that the 35 nm doxorubicin (DOX)-loaded BSA NPs achieved a drug loading content of 6.3%, encapsulation efficiency of 70.7%, and a controlled release profile with responsivity to both pH and reducing conditions. Importantly, the in vitro drug delivery experiment demonstrated efficient cellular internalizations of the DOX-loaded BSA NPs and inhibitory activities on MCF-7 and HeLa cells. This method shows the promise of being a platform for the green synthesis of protein-only nanoparticles for biomedical applications.

Keywords: BSA; biomedical application; drug delivery; photo-induced crosslinking; protein-only nanoparticles.

Scheme 1

Illustration of the fabrication of drug-loaded bovine serum albumin nanoparticles (BSA NPs) and their application as a doxorubicin (DOX) vehicle.

Figure 1

Transmission electron microscope (TEM) images (A–C) and hydrodynamic diameters determined with dynamic light scattering (DLS) of BSA NPs (D–F) at 20, 40, and 100 nm. The scale bar represents 100 nm.

Figure 2

Secondary structure, stability property, and Fourier transform infrared spectroscopy (FTIR) characterization of BSA NPs. (A) Far-UV circular dichroism (CD) spectra of native BSA and BSA NPs of different sizes. Representative spectra are the average of three consecutive scans and smoothed with a Savitzy−Golay least-squares fit. (B) Change of the particle size of BSA NPs over a period of 7 d according to DLS measurements. The sample was stored in PBS (pH 7.4, 150 mM NaCl) at 4 °C. (C) FTIR spectra of native BSA and BSA NPs.

Figure 3

Characterization of secondary structure and particle size of DOX/BSA NPs. (A) TEM images of DOX/BSA NPs and the particle size of DOX/BSA NPs measured by DLS. (B) Far-UV CD spectra of DOX/BSA NPs. Representative spectra are the average of three consecutive scans and smoothed with a Savitzy−Golay least-squares fit.

Figure 4

In vitro DOX release profiles from DOX/BSA NPs under different conditions at 37 °C.

Figure 5

Confocal images of MCF-7 cells after being treated by DOX/BSA NPs for 4 and 8 h. The blue fluorescence showed cell nuclei were stained with Hoechst 33342. The green fluorescence represents FITC, which was labeled on BSA.

Figure 6

Cytotoxicity of empty BSA NPs and DOX/BSA NPs against cancer cells. The cell viability of HeLa cells (A) and MCF-7 cells (B) after 48 h incubation with free DOX and DOX/BSA NPs. (C) HeLa and MCF-7 cell viability after incubated (48 h) with BSA NPs. Data are presented as mean ± SD, n = 6.