Study Of The Anti-Tumor Targeting Nanoparticles Co-Loaded With Ag/QUE/siRNA

This article aims to prepare targeted nanoparticles of quercetin（QUE）and PD-L1 siRNA（siP）in silver nanoparticles（AgNPs）（cRGD-modified targeting liposomes co-loaded with Ag/QUE/siRNA,Ag/QUE/siP-c-L）and study its anti-tumor effect.It uses siP as a bridge when AgNPs absorb graphene quantum dots（GQDs）as inner core,with modified phospholipid materials of different functions as outer layer.QUE was delivered in the hydrophobic bilayer to achieve combined anti-tumor effect and real-time tracking.Firstly,we used the oxidation reaction to obtain AgNPs,and the interaction between π-π bonds to adsorb siP and GQDs on the AgNPs surface.After that,the ethanol injection method is used to cover phospholipids and load QUE on its surface to obtain the preparation Ag/QUE/siP-c-L.Its phospholipid composition includes 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-（polyethyleneglycol）-2000（DSPE-PEG2000）,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N（polyethyleneglycol）-2000-cRGD（DSPE-PEG2000-cRGD）,2,3-dioleoyloxy-propyltrimethylammonium-chloride（DOTAP）and dipalmitoyl-phosphatidyl choline（DPPC）.By evaluating the factors that affect the preparations,such as phospholipid ratio and ratio of drug to lipid,the optimal formulation is screened;the nano-preparation has been examined through the agarose gel retard experiment.The concentration of siP in Ag/QUE/siP-c-L is 0.23nmol/mL（N:P=500:1）.Using particle size analyzer,scanned electronic microscopy and transmission electron microscope,Ag/QUE/siP-c-L is observed in a spherical or spherical shape,with particle size at 90-110nm.Surface element analysis results indicated that the Ag content on the surface of the nanoparticles is significantly reduced.Inductively coupled plasma optical emission spectrometry（ICP-OES）showed that silver contents of AgNPs,Ag/QUE/siP-L and Ag/QUE/siP-c-L are（211±3.21）μg/mL,（99.9±1.70）μg/mL,and（100.52±0.97）μg/mL,respectively.By establishing a HPLC method of QUE,the QUE content is determined to be（0.80±0.01）mg/mL.Stability experiment proves that the siP in Ag/QUE/siP-c-L can be stable within 6h under simulating serum environment.The IC₅₀ values of AgNPs,Ag/QUE/siP-L and Ag/QUE/siP-c-L were measured by CCK-8 method.The cellar uptake of preparations was evaluated by cofocal laser scanning microscope.Compared with GQDs group,the cellular uptake of Ag/QUE/siP-L and Ag/QUE/siP-c-L are improved.The dialysis method is used to study the QUE release behaviors in release medium of different pH values.The results show that compared with QUE suspension,the QUE encapsulated in the nanopreparation is sustained-released.The mouse breast tumor model was established.The anti-tumor effect,immune biomarkers,blood biochemical indicators,and H&E staining of Ag/QUE/siP-c-L were studied.The body weight,tumor volume and tumor weight of mice of different groups indicated that Ag/QUE/siP-c-L exhibited good safety and tumor inhibition effect.Compared with QUE suspension,the tumor suppression rate of Ag/QUE/siP-c-L was increased by 59.5%.The regulation of Ag/QUE/siP-c-L on the immune system was explored by flow cytometry.The H&E staining experiment proved that no obvious toxic damage was observed for Ag/QUE/siP-c-L to major organs,and it exhibited a therapeutic effect on tumor tissues.The blood biochemical test results showed that compared with the model group,there was no significant difference in liver and kidney biomarkers of mice in each treatment group.The Ag content of different tissues at different time points is determined by ICP-OES to investigate the biodistribution of preparations in mice.The obvious tumor targeted effect of Ag/QUE/siP-c-L was proved with increased accumulation.The Ag content in mice blood after treatment indicated that the AgNPs was widely distributed in mice and cleared from the body quickly,while the retention time of Ag in Ag/QUE/siP-c-L is significantly extended.This article has successfully prepared tumor-targeted Ag/QUE/siP-c-L nanoparticles and proves that it has good safety and stability at the cellular and animal levels.It can successfully achieve tumor targeting effect and exhibit an anti-tumor effect.