Self-assembled Nanoparticles Of Chondroitin Sulfate/poly(D,L-lactide-co-glycolide)Copolymers For The Improved Delivery Of Doxorubicin

Doxorubicin(DOX)is one of the most widely used anticancer drugs in clinic,which has strong potency and a broad spectrum of activity against variety types of cancer.However,the clinical use of DOX is often restricted with its short half-life and severe adverse reactions such as cardiotoxicity,kidney toxicity and bone marrow suppression and so on.Therefore,it is of great significance to prolong the acting time of DOX and reduce its side effects.Self-assembled polymeric nanoparticles are composed of amphiphilic polymers,which could self-assemble into nanoparticles with core-shell structure via hydrophobic interactions.The hydrophobic segments of the polymer aggregates to form the core,serving as a reservoir of poorly soluble drugs.The hydrophilic skeleton wraps around the outer layer to form a hydrophilic shell.Recently,self-assembled polymeric nanoparticles received much attention because of their suitable particle size,good stability,high drug loading and sustained-release property.In the past decades,naturally polymers like polysaccharides have been widely researched for drug delivery due to their outstanding physical and biological properties.Chondroitin sulfate(Chs),a natural anionic glycosaminoglycan,has excellent biocompatibility and biodegradability,which also can be readily modified with hydrophobical moieties because of its derivable groups,such as carbolic groups and hydroxyl groups.In this paper,chondroitin sulfate/poly(D,L-lactideco-glycolide)graft copolymers(Chs-g-PLGA)were synthesized by using hydrophilic chondroitin sulfate and hydrophobic poly(lactic co glycolic acid)(PLGA),which were further used to constructed self-assembled nanoparticles.DOX was encapsulated by the copolymers to improve its anti-tumor effect and reduce its adverse reactions.The synthesis of Chs-g-PLGA copolymer included two steps of amide reaction and adipic acid hydrazide(ADH)was selected as the linker.First,ADH was grafted with the carboxyl in chondroitin sulfate.Then,activated PLGA was reacted with the amino of ADH and finally Chs-g-PLGA was obtained.The structure of the copolymer was confirmed by 1H NMR,and the critical aggregation concentration(CAC)was measured with pyrene fluorescence probe method.Chs-g-PLGA self-assembled nanoparticles were prepared by ultrasonic method and their properties were investigated.With the increase of PLGA molecular weight,the particle size and CAC of the nanoparticles both decreased.Copolymers with PLGA molecular weight of 2K were selected to load DOX.The drug loaded nanoparticles were spherical solid with a particle size around 160 nm,and the drug loading was 11.4%.The release behavior of DOX/Chs-g-PLGA2K nanoparticles in release media with two different pH values was investigated by dialysis method.The results showed that the nanoparticles had sustained release property and pH sensitivity(fast release under low pH).SRB was used to evaluate the cytotoxicity of Chs-g-PLGA2K on two cells(L929 and MCF-7)and the inhibitory effect of DOX/Chs-g-PLGA2K nanoparticles on MCF-7 cells.The results demonstrated that the polymer had no toxicity to L929 and MCF-7 cells,showing good safety.The cytotoxicity of DOX loaded nanoparticles had similar cytotoxicity to MCF-7 cells with DOX solution.The uptake of DOX loaded nanoparticles by MCF-7 cells was observed by fluorescence microscopy and the results showed that the nanoparticles could be successfully absorbed by MCF-7 cells and released into the nucleus.The method for determination the concentration of DOX in rat plasma by fluorescence spectrophotometry was established.The pharmacokinetic parameters of DOX/Chs-g-PLGA2K including maximum concentration,area-under-the-curve and mean residence time were all improved significantly,indicating the blood circulation time of DOX was prolonged after being encapsulated with the copolymer,which could accelerate DOX to accumulate at the tumor site.The blood compatibility of the polymer was determined and the results showed that Chs-g-PLGA2K had good blood compatibility and no hemolytic risk.Kunming mice were used to investigate the maximum tolerated dose(MTD)of the DOX loaded nanoparticles,and the MTD of DOX/Chs-g-PLGA2K nanoparticles was about 2 times of that of free DOX,indicating the DOX/Chs-g-PLGA2K nanoparticles effectively reduced the toxicity of DOX.A model of subcutaneous liver cancer in mice was established and used for evaluating the in vivo distribution and anti-cancer effect of DOX/Chs-g-PLGA2K nanoparticles.The results of distribution showed the nanoparticles enhanced the accumulation of DOX in the tumor site.The tumor-bearing mice were treated with free DOX and DOX/Chs-g-PLGA2K nanoparticles by intravenous administration,respectively.The tumor volume of the mice was monitored and the results demonstrated that the anti-tumor effect of DOX/Chs-g-PLGA2K was significantly higher than that of free DOX.At the end of the treatment,the mice were sacrificed and the heart,liver,spleen,lung,kidney and tumor tissues were separated and stained withhematoxylin and eosin to evaluate the organ injuries and necrosis of the tumor tissue.The results further demonstrated the advantages of the nanoparticles,which caused massive necrosis of tumor tissue.In this paper,novel amphiphilic copolymer Chs-g-PLGA was synthesized and DOX/Chs-g-PLGA2K self-assembled nanoparticles were prepared for DOX delivery.The nanoparticles can significantly enhance the anti-tumor effect and reduce the side effects of DOX,which has excellent medical application prospects.