Preparation Of PD-L1 Antibody Nanoparticles And Its Therapeutic Effect On Lung Cancer

In contemporary world,lung cancer is a very common malignant tumor,and it is also one of the main tumors death in urban population in China.Chemotherapy is one of the main methods for the modern treatment of cancer.Although widely used,this type of treatment may cause many toxic side effects to the patients' body.In recent years,targeted therapy has progressed rapidly,but to find suitable targets is still an issue that is currently focused on targeted therapy.A number of studies have found that tumor cells are capable of immune escape through sustained high expression of PD-L1.Therefore,PD-L1 is a hot spot in the treatment of cancer and has important clinical significance.The use of polymer nanotechnology to deliver drugs has shown great help in improving cancer treatment.Liposomes are highly biocompatible,capable of interacting with a variety of molecules,and the liposome surface is easily functionalized to facilitate the attachment of various targeted molecules.Combining both and further attaching the targeted molecules to the surface of the particle enables it to accumulate better at the tumor site.The application of small animal imaging methods can encapsulate fluorescent dyes in nanoparticles and observe the targeted and therapeutic effects at the living body level.This experiment utilized the advantages of both liposomes and polymer nanoparticles to synthesize doxorubicin hydrochloride-loaded liposome-polymer nanoparticles(abbreviated as nanoparticles)and couple it with PD-L1 antibody(abbreviated as PD-L1 antibody nanoparticles).The doxorubicin hydrochloride and near-infrared dye DIR are encapsulated in the nanoparticles,and the targeted and therapeutic effects will be evaluated by using animal imaging methods in animal experiments.The specific experimental program is as follows:1.Preparation of PD-L1 antibody nanoparticles: Double-emulsification method was used to synthesize PEG-PLGA nanoparticles encapsulating anti-tumor water-soluble drug doxorubicin hydrochloride and near-infrared fat-soluble dye DIR,and the supernatant was synthesized and retained.Doxorubicin hydrochloride is entrapped in the hydrophilic layer of the central core of the nanoparticles and the DIR dye is in the PLGA layer.Then,a layer of liposomes was coated on the surface of the polymer nanoparticles by a film dispersion method,and then a PD-L1 antibody targeted molecule was attached to the surface of the liposome.Appearance of pink colloidal particle which size of 100-200 nm nanoparticles were synthetized finally.2.Characterization determination,entrapment efficiency and drug loading calculation: Particle size potentiometer was used to determine the particle size of PD-L1 antibody nanoparticles.The results showed that the synthesized probe particle size was 100-200 nm,indicating that the synthesized probe has good stability;through electron microscopy observation of the probe morphology,the results showed that the synthesized particles were spherical,and we can clearly see the liposome membrane and nanomaterial stratification,and the diameter is 100-200nm;with dialysis in nanoparticle vitro release experiments,the results showed that nanoparticles have a certain ability to release;to dilute different concentrations of doxorubicin hydrochloride solution and establish standard curve of doxorubicin ultraviolet absorption,and measure the absorbance of the retained supernatant to determine the content of doxorubicin hydrochloride and the quality was determined from the standard curve to measure the encapsulation efficiency and drug loading.The calculation results showed that the encapsulation efficiency of nanoparticles was about 60.24% and the drug loading was about 5.62%.3.Vitro cell assay: MTS assay was used to detect cytotoxicity.Different concentrations of non-drug loaded nanoparticles were co-incubated with A549 lung cancer cells for 24 hours.Free drugs with different concentrations of doxorubicin hydrochloride and PD-L1 antibody nanoparticles were incubated with cells for 24 h.The results showed that there was no significant difference between the survival rate of non-loading nanoparticles and the normal group(0 mg/mL for doxorubicin),and there was no significant difference with the increase in concentration.The PD-L1 antibody nanoparticles group was compared with the free drug group in the concentration of 20?g/mL and 10?g/mL,the cell survival rate of PD-L1 antibody nanoparticles group was significantly lower than that of free drug group(P<0.05),indicating that PD-L1 antibody nanoparticles can better carry drugs into tumors.And cell uptake experiments were performed to investigate cellular uptake,and doxorubicin hydrochloride nanoparticles were co-incubated with PD-L1 antibody nanoparticles and cells for 4 h.The results showed that the fluorescence intensity of PD-L1 antibody nanoparticle group at the concentration of 20?g/mL and 10?g/mL was significantly stronger than that of the nanoparticle group(P<0.05);the incorporation of nanoparticles was observed by confocal microscopy,and the confocal petri dish was observed.Free doxorubicin hydrochloride and nanoparticles were co-incubated with A549 cells for 2h and 4h respectively.The results showed that the red fluorescence of doxorubicin hydrochloride entered the cells,and the red fluorescence of PD-L1 antibody nanoparticles in the 4h group was stronger than that of the 2h group.The intracellular red fluorescence of the PD-L1 antibody nanoparticles 4 h group was stronger than that of the free doxorubicin 4 h group.Flow and confocal experiments showed that A549 cells had more uptake of PD-L1 antibody nanoparticles.4.Targeted experiments in vivo: A549 lung cancer cells were used to establish a subcutaneous tumor model in nude mice.Nanoparticles and PD-L1 antibody nanoparticles were injected into the tail vein respectively.The live animal imaging system was taking photos before and after injection 1h,4h,12 h,and 24 h and so on.The results showed that the fluorescence intensity of PD-L1 antibody nanoparticles group at the tumor was significantly stronger than that of the nanoparticle group,and the tumor had a clear outline and the fluorescence intensity became stronger and stronger with time,indicating that the target PD-L1 antibody nanoparticles have a certain amount of targeted ability.In vivo treatment experiment: A nude mice subcutaneous tumor model was established using A549-luc lung cancer cells capable of expressing luciferase and divided into three groups of 5 mice each.PBS,doxorubicin,and PD-L1 antibody nanoparticles were injected into the tail vein,and to dose every 2 days with 4 mg/kg(in terms of doxorubicin hydrochloride)was administered.The weight of the mice was recorded every two days and the tumor size was measured.Autofluorescence images were taken every 2 days for four weeks.The experimental results showed that at the end of the fourth week,the tumor volume of the PD-L1 antibody nanoparticles group was significantly smaller than that of the free doxorubicin group and the PBS control group(P<0.05),and the free doxorubicin group was significantly smaller than the PBS control group(P <0.05),indicating that PD-L1 antibody nanoparticles have a better therapeutic effect.5.Conclusion: The synthesized PD-L1 antibody nanoparticle is a good drug carrier for targeted lung cancer cells and can be taken up by A549 lung cancer cells better.It is more effective in killing tumor cells and inhibiting tumor growth.Encapsulation of near-infrared dyes is more conducive to the imaging of animals in vivo and is beneficial to the observation of targeted therapeutic effects of lung cancer.