Construction And Properties Of Stimuli-responsive Liver-targeting Nanoparticles

Globally,Primary liver cancer is the second leading cause of death from cancer.However,there is still a lack of effective methods for the treatment of liver cancer.Due to the long incubation period of liver cancer and the fact that most patients are found at an advanced or middle-late stage,chemotherapy is usually the treatment of choice.However,these chemotherapeutic drugs often kill tumor cells and damage healthy cells at the same time,resulting in great toxic side effects.Nano drug delivery system（NDDS）has many advantages.Targeted modification of NDDS system can change the biodistribution,solubility and bioavailability of drugs,and effectively improve the safety and efficacy of drugs,which makes it become a research hotspot in cancer diagnosis and treatment.Asialoglycoprotein receptor（ASGPR）is expressed on the surface of hepatic parenchymal cells,but hardly expressed on extrahepatic cells.It can specifically recognize and bind to the terminal non-reducing galactose and acetylgalactosamine in blood glycoproteins,sugar receptors,and drugs or drug carriers.Liver-targeted drug delivery systems can utilize this receptor to achieve liver-targeted drug delivery.However,despite the use of nanocarriers to load chemotherapeutic drugs,it is still difficult to achieve the desired release effect.Nanocarriers must meet certain requirements in order to better treat cancer and reduce the side effects of drugs.Compared with normal tissues and cells,the cellular environment of tumor tissues is weakly acidic and the concentration of glutathione is higher.People use these properties to continuously develop nano-drug delivery systems that can achieve responsive release.Based on this,this thesis uses galactose as the target to construct a reduction-sensitive and pH-responsive nano-drug delivery system.This research contents include the following two aspects:（1）Preparation and properties of reduction-sensitive liver-targeting polymeric micellesFirst,using hepta（6-azido-6-deoxy）-β-cyclodextrin and butyl-3-alkynyl-β-d-galactoside as raw materials,the liver-targeting function Galactose-β-cyclodextrin（Gal₇-CD）was synthesized by Click reaction.Secondly,using cystamine dihydrochloride and octadecanoic acid as raw materials,through amino protection,acylation reaction,deprotection and other steps to obtain cystamine octadecyl amide（NH₂-SS-SA）.Again,using adamantane carboxylic acid and double-end amino polyethylene glycol as raw materials,through acylation reaction to obtain adamantyl polyethylene glycol amine(Ad-PEG₁₀₀₀-NH₂),and then reacted with succinic anhydride to obtain Ad-PEG₁₀₀₀-COOH.Finally,NH₂-SS-SA reacted with Ad-PEG₁₀₀₀-COOH to obtain Ad-PEG₁₀₀₀-SS-C₁₈.Then,using dialysis method,Gal₇-CD and Ad-PEG₁₀₀₀-SS-C₁₈formed amphiphilic polymers through host-guest self-assembly,and then formed polymer micelles Gal-SS-C₁₈.Using doxorubicin as a model drug,the corresponding drug-loaded polymer micelles Gal-SS-C₁₈-DOX were prepared.The particle sizes of blank micelles and drug-loaded micelles were determined by Dynamic Light Scattering（DLS）to be（169.2±1.3）nm and（177.9±3.0）nm,respectively.The drug-loaded micelles Gal-SS-C₁₈-DOX encapsulation efficiency was 70.1%±0.7%,and the drug loading was 21.2%±0.5%.In the in vitro drug release test,the drug-loaded polymer micelles released slowly and had a low drug release rate in a physiological environment,but could be released rapidly in a simulated high GSH environment of cancer cells,reaching 82.4%within 48 h.The cytotoxicity and antitumor activity of polymer micelles were evaluated by using the high expression（ASGPR）hepatoma cell HepG2 and normal tissue cell HEK-293 as cell models.The research results showed that the polymer micelles had good biocompatibility,and the drug-loaded polymer micelles showed good targeting and controllable release properties,which were stronger than free doxorubicin on liver cancer cells.It showed low toxicity to normal cells and good therapeutic selectivity.（2）Construction and Properties of pH-Sensitive Liver-targeted Gold NanoparticlesBased on the previous chapter,this chapter used lipoic acid,4-aminomethyl-2-aminoethyl-[1,3]-dioxolane and 1,6-hexanediamine as raw materials,and LA-OE-NH₂ and LA-C₆-NH₂were obtained by acylation reaction.Then Ad-PEG₁₀₀₀-COOH reacted with LA-OE-NH₂ and LA-C₆-NH₂ by acid-amine to obtain Ad-PEG-OE-LA and Ad-PEG-LA,which were characterized by ¹H NMR.Next,the ligand exchange method was used to construct pH-sensitive liver-targeted gold nanoparticles Gal-CD-OE@AuNPs and control group Gal-CD@AuNPs and CD-OE@AuNPs,and using DOX as a model drug,the corresponding drug-loaded gold nanoparticles Gal-CD-OE-DOX@AuNPs,Gal-CD-DOX@AuNPs,CD-OE-DOX@AuNPs were prepared.The particle size of Gal-CD-OE-@AuNPs was determined by dynamic light scattering to be（40.7±3.1）nm.The drug loading of Gal-CD-OE-DOX@AuNPs was 41.4%±0.1%,and the encapsulation efficiency was 88.1%±0.6%.In the in vitro drug release test,the drug-loaded gold nanoparticles Gal-CD-OE-DOX@AuNPs released the drug slowly in the physiological environment,with a drug release rate of only 13%,while the drug-loaded gold nanoparticles had a faster release rate in the acidic environment of simulated cancer cells,75%within 48 h.The cytotoxicity and cellular uptake efficiency of drug-loaded gold nanoparticles were evaluated by HepG2 hepatoma cells with high expression（ASGPR）and HEK-293 normal tissue cells.The results showed that Gal-CD-OE-DOX@AuNPs could rapidly enter HepG2 cells.It showed obvious inhibitory effect on it,and had a good controlled release effect,but had less inhibitory effect on HEK-293 cells.Although Gal-CD-DOX@AuNPs entered cells quickly,they could not produce response release behavior,and there was no significant difference in inhibitory effect on HepG2 cells and HEK-293 cells.For CD-OE-DOX@AuNPs,HepG2 cells had lower uptake,and its inhibitory effect on cells was less.