The Novel Therapy Of Melanoma By Nanoparticle Drug Delivery System With Light-Switchable Gene Expression

Melanoma threatens the longevity of patients,because of the increasing incidence rate,low survival rate and high recurrence and metastasis rate of melanoma worldwide.To cope with this problem,a novel nanoparticle drug delivery system with light-switchable gene expression was proposed.Vitamin E succinate(VES)and polyethyleneimine(PEI)were adopted to synthesize the amphiphilic polymer VES-ss-PEI polymer by disulfide bond(-ss-)and then self-assembly cationic micelles were prepared in water.Ovalbumin(OVA)or genes were loaded through electrostatic adsorption.The drug loaded micelle was still positively electrical,RGD-modified PEGlyate hyaluronic acid(HA)shell(HA-PEG-RGD)was coated outside the micelle via electrostatic interaction.Light-switchable gene expression technology was applied to control the expression of diphtheria toxin in cancer cells to kill tumor cells,OVA was marked on tumor cells and generate antigenicity,the specific immune cell was activated to suppress tumor growth.In vivo and in vitro studies were preformed to illstrate the melanoma inhibition effect.This research will lay the experimental foundation and theoretical basis for the treatment of melanoma.VES-ss-PEI and HA-PEG-RGD were synthesized and characterised by 1H NMR,13C NMR,infrared spectrum,etc.Cationic micelle VES-ss-PEI was prepared by ultrasonic,with particle size ranging from 50.8 to 70.4 nm and zeta potential between 48.8 and 53.4 mV.VES-ss-PEI/HA-PEG-RGD micelle was prepared by coating the surface with a multifunctional shell.The micelle possesses good stability both in vitro and in vivo.The distribution of micelle in vivo was investigated by IR-775 dye.The results showed that,compared with the free IR-775 group,the AUC0-2h in blood of the IR-775@VES-ss-PEI/HA-PEG-RGD micellar group increases by 4.4 times,and the AUC0-24h in the liver and spleen increased by 3.3 times and 1.5 times,respectively.These results indicated that the micelle had the ability of long circulation in blood and could enrich in the liver and spleen while targeted tumor.OVA@mPEG-g-HA/PEI-g-VES micelle was prepared by ultrasonic,with particle size at 55.9 nm and loading effienicy of OVA at 28.8%.OVA@mPEG-g-HAA/VES-g-PEI was prepared by coating multifunctional shell outside catanic micelle with particle size at 72.1 nm.OVA@mPEG-g-HA/PEI-g-VES micelle had sustained release profile in the medium of PBS at pH 7.4.In vitro experiments showed that compared with the free OVA group,OVA@mPEG-g-HA/PEI-g-VES micelle group could significantly increase the ability of cells uptake,endosome escape ability,the expression of MHC I-OVA257-264 peptide markers on tumor cell surface,and the expression of dendritic cell mutation markers CD80 and CD86,indicating micelle can effectively stimulate dendritic cells.In vivo experiment observed the inhibition effect of tumor after immunotherapy,pDNA@VES-ss-PEI/HA-PEG-RGD micelle was prepared at the N/P ratio of 10,optimized by investigating particle size,zeta potential and gene loading ability.The micelle could improve gene stability,and the cumulative release amount of gene in 72 h at the normal physiological environment and the high concentration reductase glutathione(GSH)environment mimicking the tumor area was 14.3%and 62.0%,respectively,indicating the reduction sensitivity release profile.In vitro studies showed that light-switchable gene loaded VES-ss-PEI/HA-PEG-RGD micelle could significantly improve gene uptake and transfection ability.More importantly,it could regulate the expression of target protein in cells by controlling the intensity of blue light.pDNA@VES-ss-PEI/HA-PEG-RGD micelle could inhibit cell growth effectively according to MTT assay,3D tumor sphere assay,cell scratch assay and cell apoptosis assay.In vivo tumor inhibition experiment results showed that compared with the control group,the tumor size of tumor-bearing mice treated with pDNA@VES-ss-PEI/HA-PEG-RGD micelle was inhibited by 79.2%,while the surviving rate increased from 10%to 100%,indicating the significant tumor inhibition ability.The results showed that the tumor cells were significantly damaged,and the number of neovascularization cells were also significantly reduced,which could prevent the growth of tumor.The staining of organs results indicated no toxicity and side effects on tissues not exposed to blue light.The tumor inhibition effect of light induced diphtheria toxin pDNA and ovalbumin immunotherapy was investigated,which provided theoretical basis for a new melanoma treatment strategy that combined immunotherapy and light-switchable gene expression system expressing diphtheria toxin.