The Preparation Of PH-sensitive Chitosan Nanogel Drug Carriers And Its Anti-tumor Activity

Chemotherapy is a routine treatment for much malignant cancer in clinical usage.However,most of conventional anti-cancer drugs,such as paclitaxel,cisplatin and doxorubicin are still practically limited by some serious' side effects,which is due to the lack of tumor-targeted specificity and nonspecific biodistribution in the body.Nanotechnology provides an effective method to cover the shortcomings of antitumor drugs.Nanotechnology-based drug delivery systems(DDS)such as nanoparticles,micelles,vesicles and nanogels have shown promising results in controlled drug release and tumor-specific drug delivery.DDS can markedly prolong drug's circulation time in the blood and improve intratumoral drug's concentration on account of the enhanced permeability and retention(EPR)effect,thus increasing the therapeutic effect and reducing side effects.Among them,nanogels as a type of cross-linked polymeric nanoparticles are promising candidates for anti-cancer drug delivery.The excellent drug loading efficiency and controlled release capability benefits from tunable and stable interior three-dimensional networks.Appropriate biocompatibility is fatal for drug delivery systems.Various biodegradable and biocompatible natural polymers such as hyaluronic acid,alginic acid and chitosan(CS)are used to develop nanogels.As well-known,chitosan is widely applied in many fields including drug delivery and gene transfer,which is attributed to its good biocompatibility,nonpoisonous,biodegradability and low immunogenicity.However,the low solubility in water of high molecular weight chitosan in neutral or alkaline environments limits its potential applications as drug carriers.In this work,succinic anhydride-modified chitosan(SCS)was chosen as an initial material to prepare nanogel-based drug delivery systems.To introduce carboxyl group into chitosan can distinctly improve its solubility in neutral and alkaline environment,as well as provide a negatively charged surface.These nanogels with a negatively charged surface can avoid aggregation and possess a longer blood circulation time,thus increasing their opportunity to pass through the leaky vasculature,and enhancing accumulation in tumor areas.Although longer circulation time and EPR effect can improve nanogels' accumulation in tumor tissues,another challenge is how to trigger drug release at tumor area.To solve this issue,a lot of efforts have been made in development of stimuli-responsive(reduction,enzyme,temperature and pH)drug delivery systems.Compared with normal tissues,solid tumors display lower extracellular pH values(6.8-7.2),and pH values of endosomes and lysosomes are even lower(5.0-6.5).Therefore,various pH-sensitive nano-carriers via acid-sensitive linkages or groups including acetal,ketal or ortho ester have been developed to trigger drug release.For example,pH-sensitive nanogels with ortho ester linkages in side-chains are labile in mildly acidic conditions,and can release the encapsulated hydrophobic drugs.Compared to acetals and ketals,ortho ester-based nanocarriers can be hydrolyzed more quickly in response to mildly acid conditions,and the kinetics of drug release can be controlled by the hydrophobicity of its surrounding environments at pH 5-6.In addition,ortho ester-based nanocarriers have some advantages such as excellent biocompatibility and greater stability in neutral environment.So,the strategy of developing pH-triggered nanogels via acid-labile ortho ester linkages is especially attractive.In conclusion,this work have designed and prepared a new type of acid-sensitive chitosan nanaogels.In vivo and in vitro evaluation has been performed as anti-tumor drug carriers,and the main contents are as follows:(1)Synthesis of a new acid-labile cross-linker and chitosan derivative:Acid-labile cross-linker(OEAM)was obtain by ortho ester-based diamine reacted with methacrylic anhydride,and the structure of OEAM was confirmed by 1H NMR and 13C NMR.Then,the succinyl-chitosan(SCS)was obtain by succinic anhydride reacted with the chitosan(CS),and the structure was confirmed by FTIR spectra and XRD spectra.Finally,the methacrylated succinyl-chitosan(MASCS)was readily obtained by methacrylation strategy to functionalize SCS with vinyl groups,and the structure was confirmed by 1H NMR.(2)Fabrication of CS nanogels:CS nanogels were prepared via aqueous dispersion polymerization with OEAM and MASCS.The particle size,light scattering intensity and zeta potential of CS nanogels were measured using a dynamic light scattering(DLS)particle sizer.The morphologies of nanogels were observed by transmission electron microscopy(TEM)and scanning electronic microscopy(SEM).(3)CS nanogels characterization in vitro:Doxorubicin(DOX)as a model anticancer drug was loaded into CS nanogels,and exhibited drug loading content of 16.5%.As expected,with the incorporation of ortho ester linkages,these nanogels showed pH-triggered degradation and drug release at acidic pH values.The cytotoxicities showed the excellent biocompatibility of the empty nanogels,and the DOX-loaded nanogels showed a dose-dependent growth inhibitory.In vitro cellular uptake showed that the DOX-loaded nanogels could be preferentially internalized by two-dimensional(2D)cells and three-dimensional(3-D)multicellular spheroids(MCs),resulting higher inhibition on the proliferation of tumor cells.(4)CS nanogels characterization in vivo:In vivo biodistribution and anti-tumor effect were determined in H22 tumor-bearing mice,and the results demonstrate that the acid-labile CS nanogels can significantly prolong the blood circulation time of DOX,and improve the accumulation in tumor areas,leading to higher therapeutic efficacy.