Preparation And Antitumor Activity Of Drug-loaded Nanoparticles Based On Chitosan Quaternary Ammonium Salt

The ever-increasing morbidity and mortality induced by malignant tumors are constantly impacting the social medical security system of human society,which is a severe challenge all over the world.Currently,the main therapeutic modalities for cancer include surgical operation,chemotherapy,and radiotherapy.Thereinto,chemotherapy is one of the most important forms of cancer therapy.However,clinical trials showed that cancer chemotherapy could cause damage to healthy tissues or incomplete eradication of cancer cells.By developing new drugs,changing the route of drug administration and developing new drug dosage forms,people hope to achieve the role of anti-tumor drugs in targeting,high efficiency and low toxicity.As a new drug delivery method,nanoparticles have attracted more and more attention.It has shown good application value in improving drug utilization,enhancing tumor targeting,improving drug stability and realizing drug sustained release.As a kind of soluble polysaccharide polymer,chitosan quaternary ammonium salt possesses many biological properties,including biodegradability,biocompatibility,nontoxicity,antiinflammatory,and antioxidant activities,which can be widely used in the field of drug carriers in drug delivery system.In this thesis,four series of quaternary ammonium salt of chitosan nanoparticles,including chitosan quaternary ammonium salt/sodium tripolyphosphate nanoparticles,chitosan quaternary ammonium salt/carboxymethyl chitosan nanoparticles,chitosan quaternary ammonium salt/carboxymethyl cyclodextrin nanoparticles,and chitosan quaternary ammonium salt self-assembled nanoparticles,were prepared by ionic gelation and self-assembly method.The structures of chitosan derivatives were determined by FTIR and 1H NMR,and the particle size,zeta potential,apparent morphology,encapsulation and drug loading effect of the nanoparticles were determined by nanometer particle size measuring instrument,scanning electron microscopy,and UV spectrophotometer.The specific investigations on the drug release behavior of ADM from nanoparticles under three pH values(6.8,7.4,and 8.0)were undertaken by drug dissolution tester.The antioxidant activity of nanoparticles was studied by antioxidant activity test.In addition,CCK-8 method was used to test the effects of samples on the growth of four tumor cells(BGC-823,A549,MCF-7,HEPG2)and normal cells(L929)at different concentrations,and to evaluate the anti-tumor activity and biocompatibility of the nanoparticles.Firstly,chitosan quaternary ammonium salt/sodium tripolyphosphate nanoparticles were prepared using chitosan quaternary ammonium salt grafting folic acid as precursor,sodium tripolyphosphate as crosslinking agent,and adriamycin as model drug.The surface of the nanoparticles was spherical or nearly spherical,and the nanoparticles possessed small hydrodynamic diameter(85.67±2.04 nm),positive zeta potential(+21.06±0.96 mV),and good encapsulation and drug loading effect.The results of drug release test in vitro showed that the new nanoparticles were stable in the release medium and could control drug release and prolong drug efficacy.The antioxidant efficiency of nanoparticles was assayed,and nanoparticles represented significant enhancement in radicals scavenging activity.The results of antitumor activity showed that nanoparticles were inhibited the growth of cancer cells.When the concentration of the sample reached 25 μg/mL,the inhibition rate of BGC-823 tumor cells was up to 71.19%,which was much higher than the inhibitory effect of adriamycin.In addition,cytotoxicity results showed that chitosan quaternary ammonium salt/sodium tripolyphosphate nanoparticles as drug carriers could significantly reduce the toxicity of adriamycin to normal cells.Secondly,chitosan quaternary ammonium salt/carboxymethyl chitosan nanoparticles were prepared using chitosan quaternary ammonium salt grafting folic acid as precursor,negatively charged carboxymethyl chitosan as crosslinking agent,and adriamycin as model drug.The average particle size of the drug-loaded nanoparticles was 246.89±6.01 nm,the polydispersion coefficient was 22.20±1.40%,and the zeta potential was+24.57±0.69 mV.The specific investigations on the drug release behavior under three pH values were investigated.The results showed that the prepared nanoparticles had certain sustained release properties.Different from chitosan quaternary ammonium salt/sodium tripolyphosphate nanoparticles,chitosan quaternary ammonium salt/carboxymethyl chitosan drug-loaded nanoparticles showed obvious pH-sensitive drug release.When the pH values of dissolved medium were 6.8,7.4 and 8.0,the cumulative release rates were 53.14,48.65 and 42.38%,respectively.That is,the lower the pH of the release medium represented the higher the cumulative release rate.Compared with free adriamycin,nanoparticles showed higher antioxidant,antitumor activity,and significantly reduced cytotoxicity.In summary,the nanoparticles prepared in this chapter had uniform particle size,good dispersion and stability,good biocompatibility,high biological activity,and pH sensitive release characteristics,which had a good application prospect in the field of nano drug delivery system.Then,considering the unique structure of cyclodextrin with hydrophobic cavity and hydrophilic shell,it could encapsulate variety of hydrophobic molecules into hollow truncated conical cavity through non-covalent interaction.In order to improve the encapsulation and drug loading effects,chitosan quaternary ammonium salt/carboxymethyl cyclodextrin nanoparticles were prepared using chitosan quaternary ammonium salt grafting folic acid as precursor,carboxymethyl cyclodextrin as crosslinking agent,and adriamycin as model drug.The surface morphology of chitosan nanoparticles was spherical or nearly spherical,and the particle size distribution was narrow,the particle size was small,and the dispersion and stability were good.The encapsulation and drug loading rates of chitosan quaternary ammonium salt/carboxymethyl cyclodextrin nanoparticles were 31.25±0.59%and 75.75±0.57%,respectively.Compared with the chitosan nanoparticles prepared by sodium tripolyphosphate or carboxymethyl chitosan as crosslinking agent,the encapsulation and drug loading rates of chitosan nanoparticles prepared by carboxymethyl cyclodextrin were significantly improved.The results of drug release behavior in vitro showed that the prepared drug loaded nanoparticles had certain sustained release properties and showed obvious pH sensitive release characteristics.Similarly,nanoparticles had good antioxidant activity and significantly enhanced anti-tumor activity,which had a broad application prospect in the field of drug carrier.Finally,amphiphilic polymers bearing folic acid as the hydrophobic groups and pyridine quaternary ammonium salt as the hydrophilic group was prepared using the method of chemical modification.With adriamycin as model drug,amphiphilic polymers in aqueous solution under ultrasound assisted in spontaneously assembled into "shell/core structure" of self-assembled drug-loaded nanoparticles with chitosan quaternary ammonium salt.The preparation method of the drug-loaded nanoparticles is simple,with small particle size(163.79±7.31 nm)and narrow particle size distribution,and good dispersion and stability.The encapsulation and drug loading effects of chitosan quaternary ammonium salt self-assembled drug-loaded nanoparticles were 65.04%and 22.73%,respectively,indicating that nanoparticles had good encapsulation and drug loading capacity,which was suitable for drug delivery.In vitro release experiments showed that nanoparticles were a kind of drug delivery carrier system with sustained release and pH response.The antioxidant test results showed that the drugloaded nanoparticles had good antioxidant activity,and the free radical scavenging index was close to 100%at the highest concentration.Compared with free adriamycin,chitosan quaternary ammonium self-assembled nanoparticles showed significantly improved antitumor activity,which mainly depended on the size advantage and uptake advantage of chitosan nanoparticles.In addition,the carrier material has good biocompatibility,and self-assembled nanoparticles could significantly reduce the normal cytotoxicity.This thesis systematically studied the preparation method of chitosan quaternary ammonium salt nanoparticles by ionic crosslinking method and self-assembly method.The nanoparticles with small particle size,high potential,good drug loading effect,low toxicity and enhanced antitumor activity were screened by structure characterization and activity test,which might lay a foundation for the study of chitosan quaternary ammonium salt nanoparticles.