Fabrication Of Dual PH-Responsive Doxorubicin-Silk Sericin-based Nanoparticles Drug Delivery System For Tumor Inhibition

"Smart" stimuli-responsive nanoparticles-based drug delivery systems(NDDSs)are designed to cross multiple physiological barriers to deliver their loads to target sites by responding to internal and external stimuli,which could maximize therapeutic effect with minimized side effects.It is a promising strategy to trigger chemotherapy drug to targeted release by making use of tumor mild acidic environment.Featured by excellent biocompatibility and biodegradability,silk-based nanoparticles shows a great potential in the field of NDDSs.To extend applications of silk sericin in nanomedicine,silk sericin was selected in this study.The ratio of amine to carboxyl controlled with the help of chitosan,a physical and chemical crosslinking method was attempted to fabricate silk sericin-based nanoparticles(SSC@NPs),which reversed its surface charge when exposed to a mild acidic pH.An anti-cancer drug,doxorubicin(DOX),was readily loaded on the SSC@NPs and released differently by responding to pH.Afterwards,the ablity of reversing surface charge favored the uptake of tumor cells.Furthermore,DOX-SSC@NPs was demonstrated to surpress the tumor proliferation without side effects.The study was divided by three parts.Part one is about the fabrication and characterization of SSC@NPs.Primitive SSC@NPs was first developed by the electrostatic interaction of sericin and chitosan.Then EDC-induced crosslinking of amines and carboxyls in both sericin and chitosan lead to the formation of stable SSC@NPs.The preparation of SSC@NPs was carried out throughout the aqueous phase,avoiding the use and residual problems of organic solvents,initiators and surfactants.By adjusting the mass ratio of sericin and chitosan(20:1),the corss-linking dose(15 mg)and the crosslinking time(2 h)of the carbodiimide,SSC@NPs with an average size of 200 nm were screened.The SSC@NPs had good dispersibility(PDI:0.033)and negative surface potential(-10.1 mV).Because sericin is rich in a large number of hydrophilic amino acids,SSC@NPs can achieve excellent reconstitution stability without adding any cryoprotectants,and resist the non-specific adsorption of serum protein.The hydrophilic amino acids in the sericin are responsible for the dispersion stability.The introduction of chitosan in the preparation process increased the isoelectric point(?6.3)of SSC@NPs to the interval of tumor acidic microenvironment,realizing that the SSC@NPs carried the negative charge under physiological condition,and reversed the surface charge in the acidic environment.The second part deals with the construction of the drug system of sericin nanoparticles and the interaction between nanoparticles and tumor cells.In this part,DOX can be adsorbed on SSC@NPs by electrostatic interaction,and the drug loading reached 9.9%when the drug feeding ratio was set as 1:5.At the same time,acidic conditions promoted the release of DOX.For example,the cumulative release of doxorubicin at pH 7.4 within 48 h was 27%,while the release was about 80%at pH 5.0.Similarly,the surface charge of the DOX-sericin nanoparticle complex in the acidic environment underwent a transition from positive to negative.The surface charge reversal of sericin nanoparticles under acidic conditions improved the uptake of tumor cells,where the fluorescence intensity of intracellular SSC@NPs at pH 6.0 was about 6 times than that of pH 7.4 In the slightly acidic environment of the tumor cells,weakly alkaline DOX can not effectively enter the cell inside,while DOX-SSC@NPs can be uptaken into the tumor cells through endocytosis,improving the inhibition of tumor cells.The third part is the evaluation of tumor targeting and antitumor effect of sericin nanoparticle drug delivery system.The preliminary evaluation of the biosafety of SSC@NPs showed that it was harmless at an intravenous injection of 100 mg/kg of SSC@NPs in mice.Fluorescent dye-labeled SSC@NPs can be targeted to tumor tissue through blood circulation and EPR effect.When DOX was loaded on SSC@NPs,the distribution of DOX in the heart was significantly reduced,compared with free DOX.The inhibition effect of DOX-SSC@NPs on HepG2 subcutaneous tumor growth was comparable to free DOX at a dose of 4 mg/kg.At the same time,DOX-SSC@NPs significantly reduced cardiotoxicity and systemic toxicity.