Construction Of Core-shell Glycogen Nanodelivery Systems For Enhancing Tumor Penetration And Gene Delivery

Nanocarriers used for delivery of chemotherapeutic drugs and gene drugs have brought tumor treatment research into a new stage.They can prolong drug circulation,increase cellular uptake,reduce toxic and side effects and improve efficacy.However,the impenetrability of solid tumor and low efficiency of gene nanocarriers are still difficult problems in the further development of nano drug delivery systems.Glycogen（Gly）is a dendritic polymer with spherical dendritic nanostructure,and it is a natural advantage of being a drug carrier due to the high safety,easy availability and easy functionalization.Therefore,two core-shell glycogen based delivery systems were constructed in this paper through structural modification and surface modification,which performed excellently in enhancing solid tumor penetration and gene delivery efficiency.The main research contents are as follows:（1）Construction and anti-tumor effect of DOX/HAase/GTPP nanoparticles.Firstly,the cationic glycogen derivative GLy-TPP（GT）was obtained by modifying triphenylphosphine（TPP）onto the glycogen through mild chemical reaction.In addition,polyethylene glycol（PEG）with matrix metalloproteinase-2（MMP-2）recognizes fragments was modified to synthesize cationic glycogen derivative Gly-TPP-pep-PEG（GTPP）.Their structures were confirmed by ¹H NMR and IR spectra.Doxorubicin（DOX）was encapsulated in the cavity of glycogen structure,and hyaluronidase（HAase）was loaded through electrostatic interaction to construct the nanodelivery system DOX/HAase/GTPP for enhancing drug penetration.The drug loadings of DOX and HAase were（22.42±0.47）%and（51.65±2.23）%,respectively,and the particle size and Zeta potential of DOX/HAase/GTPP nanoparticles were（137.33±9.18）nm and（-2.21±0.77）m V,respectively.In vitro drug release results showed that the cumulative drug release of DOX/HAase/GTPP in the simulated tumor tissue environment was（55.83±1.90）%,which was higher than that in normal physiological environment of（31.31±1.98）%,indicating the nanoparticles had p H dependent and MMP-2 responsive feature.In vitro protein adsorption experiments,hemolysis experiments and stability experiments proved that DOX/HAase/GTPP had good biocompatibility and stability.In vitro cell evaluation showed that the glycogen carrier was safe and non-toxic to cells.DOX/HAase/GTPP was taken up by cells through a variety of ways,and actively targeted to mitochondria.Tumor penetration experiments showed that after the drug-loaded nanoparticles reached the tumor site,they could actively degrade hyaluronic acid（HA）and promote the penetration of the drug deep into the tumor,and had the best penetration effect in the presence of MMP-2.In vivo anti-tumor experiments,DOX/HAase/GTPP showed the best anti-tumor effect and was safer than the free DOX.The above results showed that the synergistic delivery of DOX and HAase by biocompatible GTPP can effectively promote the penetration of drugs into deep tumor,which provide a new way to enhance tumor therapeutic effect and reduce side effects.（2）Construction and anti-tumor effect of of si RNA/GT@Lip complex.Based on the excellent drug-loading properties of the cationic glycogen derivative GT,in this chapter,survivin si RNA was combined with GT through electrostatic interaction.Afterwards,distearoyl phosphatidylcholine（DSPC）and distearyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG_2K)were coated on the GT surface to construct a gene delivery system si RNA/GT@Lip.Agarose gel electrophoresis was used to investigate the loading capacity of GT to si RNA and the effect of lipid coating on the combination of GT and si RNA.The results showed that when the positive/negative ratio was 18:1,si RNA could be completely loaded by GT,and lipid didn’t affect the adsorption of si RNA.The particle size of the si RNA/GT@Lip complex was（130-140）nm,and the Zeta potential was（-1.39±0.33）m V.In vitro protein adsorption experiments showed that the presence of lipids could effectively avoid the adsorption of proteins resulting in improving the stability of the system.In the hemolysis experiment,si RNA/GT@Lip showed good biocompatibility and hardly caused hemolysis.The particle size monitoring results showed that the si RNA/GT@Lip complex had strong storage stability and did not aggregate in a simulated in vivo environment.Agarose gel electrophoresis showed that si RNA/GT@Lip complex could effectively avoid the degradation of si RNA by RNase.Compared to the commercially available transfection reagent Lip2000,the cytotoxicity and cell transfection experiments showed that si RNA/GT@Lip had good gene transfection efficiency and promoted cell apoptosis in vitro.Lysosomes staining results showed that si RNA/GT@Lip could escape from the lysosomes and enter the cytoplasm to exert gene silencing effect.The above results indicated that si RNA/GT@Lip had a good si RNA loading efficiency and could effectively improve the stability and anti-tumor effect,which can be used as a good application prospect in si RNA delivery.