Enzyme-responsive Smart Nano-drug Delivery System For Combined Antitumor Therapy

The morbidity and mortality rates of lung cancer have increased in recent years,seriously jeopardizing people's live and health.Chemotherapy is one of the main therapeutic methods for lung cancer,but it has the defects of big side effects,low drug accumulation and weak drug permeability in tumor cells,and multidrug resistance.There is an urgent need to find new drug carrier systems and treatments to enhance drug bioavailability and achieve better anti-cancer efficacy.To solve those problems,this project proposed a matrix metalloproteinase?MMPs?that is highly expressed in tumor,and designed and prepared an MMPs enzyme response smart nano-drug delivery system to improve specificity.On this basis,combined with cell-penetrating peptides to solve the problem of low internalization of cells.At the same time,curcumin and losartan were combined antitumor therapy.Among them,curcumin has the anti-tumor ability and reversal of multidrug resistance;Losartan can increase the permeability inside solid tumors and works mainly as follows:?1?Synthesis of star-shaped copolymers and polyethylene glycol grafted Peptide:Star-shaped copolymers?DPE-PCL?were synthesized by ring-opening polymerization with dipentaerythritol and cyclocaprolactone.And its structural formula and molecular weight were characterized by ¹H-NMR,FT-IR and GPC.The results showed that DPE-PCL was successfully synthesized with a number-averaged molecular weight of 9440g/mol,a weight-averaged molecular weight of 10253 g/mol,and a PDI of 1.08.Polyethylene glycol?molecular weight 10 k Da?was modified with succinic anhydride and N-hydroxysuccinimide,and then grafted with peptide.According to the results of¹H-NMR and FT-IR,the polyethylene glycol grafted peptide?PEG-Peptide?was successfully synthesized.?2?Preparation and characterization of nanoparticles:Enzyme-responsive nanoparticles?Cur-P-NPs?were prepared by the solvent evaporation method and ion cross-linking method.?1?The effects of four types of surfactants in the water phase,the ratio of medicinal materials,drug concentration,and volume ratio of water phase and organic phase on the characteristics of nanoparticles were investigated.The optimal formula for the preparation of nanoparticles was obtained:1 mg/m L of P188?surfactant?as the aqueous phase,acetone as the organic phase,and the medicinal material ratio?Cur:DPE-PCL:PEG-Peptide?was 1:7:7?w/w?,Cur concentration is 2 mg/m L,and the volume ratio of water phase and organic phase was 4:1?v/v?.The diameter of Cur-P-NPs prepared under these conditions was?135.82±1.62?nm,the potential was?7.49±1.01?m V,and the drug loading and encapsulation efficiency were?16.0±0.79?%and?93.5±0.42?%,respectively.?2?Microstructures of the composites were investigated by transmission electron microscopy?TEM?.The results show that the nanoparticles have good uniformity and dispersibility.?3?Microstructures of the composites were also investigated by X ray diffraction?XRD?.The results showed that curcumin was successfully encapsulated inside the nanoparticles.?4?The in vitro release experiment showed that Cur-P-NPs has a sustained release ability,and in the environment of MMPs,the release rate and amount of the drug are increased.?5?Stability results showed that Cur-P-NPs has good storage stability and kinetic stability in vitro.?3?Nanoparticle safety and in vitro anti-tumor studies:MTT method was used to study the safety of nanoparticles.The results showed that blank nanoparticles have good biological safety for both L929 and A549 cells;The results of cell anti-proliferation and drug uptake experiments showed that the peptide fragments in Cur-P-NPs can be cleaved in the environment of MMPs,and after part of the group was shed,the cell-penetrating peptide was effectively"activated",allowing more nanoparticles to be delivered to tumor cells.It makes Cur-P-NPs have stronger anti-proliferation effect in vitro;Flow cytometry was used to investigate the effect of nanoparticles on the A549cell cycle.The experimental results showed that Cur-P-NPs can effectively stop the cell cycle in the S and G₂/M phases,thereby causing cell apoptosis.?4?Targeted distribution of nanoparticles in vivo and anti-tumor study:A549tumor-bearing mice were injected intraperitoneally with losartan solution daily for three weeks,to investigate the effect of losartan on tumor permeability using two methods:injecting Evans blue in the tail vein and taking tumors for fluorescent sections.The results showed that losartan can increase the permeability of solid tumors by reducing collagenase I in tumors.In vivo imaging experiments showed that nanoparticles co-modified with targeting peptides and penetrating peptides have a better ability to target tumor sites.The in vivo antitumor activity research results showed that the combined use of Cur-P-NPs and Losartan has the best effect on inhibiting tumor growth.Tissue section results showed that this combined administration mode has low damage to normal organs and strong killing ability to tumor cells.All in all,this subject successfully prepared an enzyme-responsive nano-drug delivery system with suitable particle size,Zeta potential,high drug loading,good biocompatibility,and biological stability.Cellular and animal experiments showed that the peptide contributes to the success of active tumor?targeting and the enhancement of the penetration of the nanoparticles in tumor tissue,and effectively reduce the effects on normal tissues.Besides,the combined use of curcumin-nano drug delivery system can effectively improve the permeability of solid tumors and enhance the inhibition of solid tumor growth.It has a good application prospect in clinical application.