Construction And Evaluation Of Bifunctional Targeting Nano Drug Delivery System Based On Cellulose Nanocrystals

Background and Objective At present,the incidence of cancer in China is high,and the incidence of cervical cancer ranks first among gynecological malignancies.Chemisotherapy is one of the main treatment methods for cervical cancer,but its clinical effects are reduced by its toxic and side effects and multidrug resistance of tumor.PD-L1,which is highly expressed on the surface of tumor cells,combined with PD-1 can inhibit the activation and proliferation of T cells,causing T cells to lose their killing effect on tumor cells,thus causing tumor immune escape,promoting tumor metastasis and drug resistance to chemotherapy.RNAinterference(mainly si RNA)can directly degrade PD-L1 m RNA and down regulate its expression;however,the application of synthetic si RNA is limited due to its easy degradation,difficulty in penetrating cell membrane and poor targeting.Functionalized nanoparticles can significantly improve the penetration and stability of drugs to cell membranes,among which cellulose nanocrystalline(CNC)is an ideal drug carrier due to its unique rod-like structure,excellent physicochemical properties and biological affinity.Hyaluronic acid(HA)can specifically bind to CD44 receptors overexpressed on the surface of tumor cells,thus achieving tumor targeting.Polyethyleneimine(PEI)is an excellent cationic transfection reagent that can deliver RNAinto cells and protect it from lysosomal degradation.Therefore,this study attempted to construct a a bifunctional targeted nano drug delivery system(NDSS)based on cellulose nanocrystals to achieve co-delivery of PD-L1 si RNA and paclitaxel(PTX),improve the drug loading efficiency and transfection efficiency,followed by the investigation of its targeting ability and synergistic therapeutic efficacy on cervical cancer cells.Methods 1.1 HA@PTX@HA@PEI@CNC,HA/(PD-L1 si RNA)@ PEI@CNC,HA@PTX@HA/(PD-L1 si RNA)@PEI@CNC and other targeting nanodrugs were constructed by electrostatic self-assembly method using CNC as the carrier and HA as the targeting ligand.2.The physicochemical properties such as particle size and morphology of newly prepared nanomedicines were determined by laser nanoparticle sizer,transmission electron microscopy,etc.3.The drug loading and encapsulation efficiency of nanomedicines were determined by UV spectrophotometry and other methods.4.CCK-8 method was used to study the proliferation toxicity of different concentrations of drug-loaded nanoparticles against Hela cells and normal cervical epithelial cells.5.Western Blot method and fluorescence quantitative PCR method were used to study the silencing effect of HA/(PD-L1 si RNA)@PEI@CNC on PD-L1.6.Scratch assay was used to study the inhibitory effect of HA/(PD-L1 si RNA)@PEI@CNC on the proliferation and migration ability of Hela cells.7.Fluorescently labeled nanodrugs were constructed by the near-infrared fluorescent dye IR780.8.The targeting ability of the targeted nanodrug was investigated by inverted fluorescence microscopy using fluorescently labeled nanodrug.Results1.The nanodrugs HA@PTX@HA@PEI@CNC,HA/(PD-L1 si RNA)@PEI@CNC,HA@PTX@HA/(PD-L1 si RNA)@PEI@CNC were successfully prepared with uniform milk-white appearance.2.The particle size lengths of the above nanoparticles were measured by laser nanoparticle sizer as 181.6 ± 1.97 nm,184.4 ± 4.3 nm,199.9 ± 8.4 nm,respectively,and the zeta potential of HA@PTX@HA@PEI@CNC in water during the preparation process flipped sequentially from-49 ± 1.4 m V to 36.4 ± 3.3 m V,-31.7 ±2.8 m V,12.2 ± 0.2 m V,-35 ± 1.0 m V;CNC was seen as rod-like nanoparticles with a length of 50-200 nm under transmission electron microscopy,and the rod-like structure was still clear after modification with PEI and HA.3.Ultraviolet spectrophotometer showed that the drug loading content and drug loading efficiency of HA@PTX@HA@PEI@CNC were 6.85% and 17.0%,respectively;the encapsulation rate of HA/(PD-L1 si RNA)@PEI@CNC reached 80% when the feeding ratio of CNC and PD-L1 si RNA was 35:1.4.The results of the proliferation toxicity test showed that compared with PTX alone,the proliferation toxicity of nanodrugs containing the same concentration of PTX was stronger on He La cells,and the toxicity of nanodrugs co-delivered PTX and PD-L1 si RNA was stronger than that of any single drug;the half inhibit concentration(IC50)at 48 h was 5.22 ng/ml.Meanwhile,the survival rate of normal cervical epithelial cells was above 80%,indicating that the nanocarriers were basically non-toxic to normal cells.5.Western Blot showed that PD-L1 protein expression was significantly down-regulated after He La cells were transfected with HA/(PD-L1 si RNA)@PEI@CNC;the silencing efficiency of m RNA level was up to 93% as determined by fluorescence quantitative PCR.6.The results of scratch assay showed that HA/(PD-L1 si RNA)@PEI@CNC had a significant inhibitory effect on the proliferation and migration of Hela cells.7.NMR spectra showed that HA was successfully prepared and nanocarriers were successfully labeled.8.The results of fluorescence inversion microscopy showed that Hela cells successfully absorbed the nanocarriers and accumulated them in the cytoplasm,and the HA-modified nanocarriers enhanced the uptake by Hela cells.Conclusion1.A bifunctional targeted nano-drug codelivery system with cellulose nanocrystals as the core and HA as the target ligand was successfully constructed and fluorescently labeled.2.The fluorescent labeling method proved that the prepared nano delivery system had targeting ability to He La cells.3.The targeted delivery of PD-L1 si RNA by the nano-drug delivery system can efficiently silence PD-L1 and reduce its expression.4.The nanodrug delivery system co-delivered PD-L1 si RNA and PTX,which could synergistically inhibit the proliferation of cervical cancer cells.