Preparation And Properties Of Intelligent Carboxymethyl Chitosan Nanogel Drug Delivery System

Chemotherapy is still a common method for clinical cancer treatment,even though most chemotherapeutic agents often result in severe side effects and systemic toxicity.During the past decades,nano-scale carriers such as liposomes,nanoparticles,micelles,etc.have been designed as drug delivery systems(DDS)to improve drug efficacy and reduce side effects.In a wide variety of nano-carriers,nanogels have higher serum stability and greater drug loading than other nano-carriers.The drug release of common nanogels is usually passive diffusion with swelling of nanogels,leading to non-specific release and cytotoxicity to normal tissues.Therefore,the design of smart nanogels with specific tumor targeting ability and stimulus-responsive behavior(pH,heat or enzyme)is specifically imperative.As well known,several active targeting ligands such as RGD peptide,phenylboronic acid(PBA),lactobionic acid(LA)and folic acid(FA)can be effectively incorporated into nanogels to improve the tumor-homing capacity and drug accumulation.In addition,the presence of pH gradients in the tumor physiological environment has made acid-sensitivity widely used in the design of a series of nanogels.Thus,a pH-sensitive nanogel can be prepared by using an acid-labile cross-linker or selecting a polymer comprising an acid-degradable backbone.The objective of the present work is to prepare acid-degradable and tumor-targeted nanogels by copolymerization of lactobionic acid-modified methacrylated carboxymethyl chitosan with acid-labile methacrylate orthoester-based monomer.First,an acid-labile methacrylated orthoester monomer(OEAM)was synthesized as a cross-linker based on the previous reported work.Subsequently,methacrylated carboxymethyl chitosan(MCMCS),i.e.,double bonded carboxymethyl chitosan was prepared by the reaction of carboxymethyl chitosan(CMCS)with methacrylic anhydride.Lactobionic acid(LA),as a tumor-homing ligand,was then reacted with the amino groups of MCMCS by NHS/EDC to give tumor-targeted polymer(LA-MCMCS).The structures of OEAM,MCMCS were confirmed by NMR and FTIR.Then,the acid-labile cross-linker(OEAM)was co-polymerized with MCMCS or LA-MCMCS at 80℃ to obtain pH-sensitive nanogels,which is designed as NG1 and NG2,respectively.The diameter of NGI and NG2 was 197.8 nm and 168.2 nm,respectively.Moreover,both NG1 and NG2 had a regular spherical shape with a homogeneous size and distribution,which was calculated by the transmission electron microscopy(TEM)and scanning electron microscopy(SEM).The stability of NG1 and NG2 were evaluated in different conditions and the results exhibited excellent time and physiological stability.Acid degradation at different pH values confirmed that NG1 and NG2 had acid-labile property.At acidic pH conditions,the average diameter continued to increase and the light scattering intensity consistent to decrease.On the contrary,no obvious change of NG1 and NG2 was observed at pH 7.4.The outcome illustrates substantially that the acid-labile ortho ester bonds inside nanogels can be gradually hydrolyzed in mildly acid environment.The model drug(doxorubicin hydrochloride,DOX)was loaded into NG1 and NG2 to give NG1/DOX and NG2/DOX,respectively.The drug loading content(DLC)of NG1/DOX and NG2/DOX was 16.39%and 18.17%,respectively,according to the standard curve of doxorubicin hydrochloride.Also,in vitro drug release experiments displayed that the release profiles of NGI/DOX and NG2/DOX had similar trends at same conditions.With the pH decreased,the cumulative percent and release rate increased rigorously,and at pH 4.0,the cumulative percent of NG1/DOX and NG2/DOX reached to 84.13%and 88.21%in 160 h.Besides,in vitro cytotoxicity,cell uptake and inhibitory effects were then examined by a variety of experiments at 2D and 3D cell levels.The vitro cytotoxicity was measured by MTT assay with SH-SY5Y(human neuroblastoma cells),HepG2(human hepatoma cells)and H22(murine hepatoma cells)for 24 h.All cell viability is higher than 95%after incubating with NG1 and NG2,suggesting that these CMCS-based nanogels have no cytotoxicity to tested cells,in other words,the nanogels have a good biocompatibility.Meanwhile,NG2/DOX possesses higher cytotoxicity than NG1/DOX at all tested concentrations against HepG2 cells and H22 cells.For SH-SY5Y cells,NG1/DOX and NG2/DOX display similar cytotoxicities.In addition,qualitative and quantitative experiments of cell uptake by confocal laser scanning microscope(CLSM)and flow cytometry confirmed that NG2/DOX was superior to NG1/DOX in two liver cancer cells as above mentioned.Then,NG1/DOX and NG2/DOX were co-cultured with the 3D models established by SH-SY5Y and HepG2 cell lines.In HepG2 multicellular spheres,the penetration of NG2/DOX was highest at all experimental time points,and the red fluorescence signal was completely filled with each cell spheres.In addition,the strongest inhibitory effect of NG2/DOX was shown in 7 days.However,there was no significant difference about the results of NG1/DOX and NG2/DOX in SH-SY5Y multicellular spheres.In conclusion,acid-degradable and tumor-targeted drug-loaded nanogels were named as NG2/DOX,which has broad application prospects in drug delivery system basing on strong stability,high biosafety,significant targeting specificity and drug-release controllability of hepatocarcinoma cells.