Fabrication Of Protein-only Nanoparticles As Drug Delivery Systems Via Photo-induced Cross-linking

Protein-based nanoparticles hold inherent advantages over their synthetic polymer-based counterparts and have great promise in biomedical applications.Albumin nanoparticles have shown promising potential in drug delivery.Conventional preparation methods like desolvation exists toxic reagents residual,difficult size control and protein denaturation issues.In addition,the fabrication processes might involve the usage of toxic crosslinkers or organic solvents and control of size is poor.Therefore,developing a novel mild,facile,and size-controlled all-protein nanoparticles preparation strategy in clinical cancer treatment is of great significance.In this paper,a photo-induced crosslinking approach was developed to prepare all-protein nanoparticles through tyrosine photo-redox reaction.The advantages of this method include mild conditions,without addition of organic reagents,fast speed within minutes.The albumin nanoparticles exhibited regular shape with uniform size distribution and are size-controlled,as well as maintaining protein bioactivity.The research contents of this paper include the following aspects:(1)Human IgG and BSA nanoparticles,protein hydrogels were successfully prepared using one-step photo-induced crosslinking in illumination of visible light and physiological conditions.The size of the BSA NPs could be precisely manipulated from 20 to 100 nanometers within minutes,and exhibited spherical morphology.The secondary structures of protein were preserved.The results of in vitro cytotoxicity of BSA NPs in HeLa and MCF-7 cells confirm that BSA NPs have good biocompatibility and stability.The storage modulus and the loss modulus of the human IgG hydrogel can reach 500 Pa and 50 Pa respectively.The photo-crosslinking method brings a novel strategy for fabrication all-protein nanoparticles and hydrogel.(2)A green method for fabrication of albumin nano drug delivery system was developed.In addition to electrostatic interaction,hydrophobic interaction should be mainly accountable for the binding of anticancer drug DOX molecules to BSA NPs which obtained through photo-crosslinking.The 35 nm-sized DOX/BSA NPs were prepared,which achieved a drug loading content of 6.3%,encapsulation efficiency of 70.7% and zeta potential of-12.5 ± 0.6 mV in 1:10 drug/carrier ratio(m/m).DOX/BSA NPs showed slow drug release behaviour.DOX release observed from DOX/BSA NPs in physiological pH 7.4 and acidic pH 5.0 over a period of 20 h was 40% and 60%,respectively.The release of DOX was significantly accelerated under reductive conditions at 10 mM DTT.Confocal microscopy was used to observe the cellular internalization of DOX/BSA NPs by MCF-7 cells.The results indicated that by prolonging the incubating time,the intracellular concentration of DOX/BSA NPs in MCF-7 cells enhanced.In vitro cytotoxicity results confirm that DOX/BSA NPs still hold cancer cell killing ability within the intracellular condition and the activity of drug could be maintained maximumly.(3)Explore the feasibility of using photo-induced cross-linking method to prepare eGFP nanoparticles and maintain their bioactivity.eGFP NPs of about 20 nm in average size were successfully achieved and the fluorescent activity of eGFP NPs can be maintained at 40%.Meanwhile,eGFP NPs have good stability and the secondary structures can be preserved.In vitro cytotoxicity results confirm that nanoparticles do not have unfavorable effects on the viability for MCF-7 cells.The cellular internalization results showed that eGFP NPs can reach the cytoplasm through endocytosis and maintain the fluorescent activity.Using eGFP mixed with BSA to synthesize BSA/eGFP NPs can enhance the cellular uptake of nanoparticles.