| Oral squamous cell carcinoma is a highly malignant epithelial tumor of the head and neck.The occurrence of oral squamous cell carcinoma will cause serious damage to patients’ chewing,pronunciation,respiratory and psychological state.Intravenous or oral administration of small molecule chemotherapeutic drugs is a powerful mean to treat oral squamous cell carcinoma.Recently,the rapid development of nanotechnology has led to the emergence of numerous nano-sized drug delivery systems to alleviate the problems of low solubility and poor targeting for small molecule drugs.On this basis,more and more researchers begin to pay attention to the construction of intelligent carrier.Generally,intelligent carrier refers to a kind of carrier that can respond to the changes of tumor microenvironment or external stimulation and subsequently release drugs accurately,efficiently and sustainably.It can improve the therapeutic effect and reduce the toxic and side effects of drugs in vivo.In the design of intelligent carrier,chitosan(CTS),as a natural polymer,has the characteristics of acid sensitivity and enzyme degradation.It has been widely used in the construction of intelligent carrier.However,the poor water solubility of CTS makes the nanoparticles solely composed of CTS unstable in structure and prone to precipitation in aqueous solution.The hydrophilic materials(polyvinyl alcohol(PVA)and polyacrylic acid(PAA))were used to simply modify CTS nanoparticles to enhance their colloidal stability and give them higher drug loading efficiency.In addition,hybrid nanoparticles have unique advantages in drug delivery because they can combine the excellent properties of a variety of materials.As we know,nanosized noble metal materials have good biocompatibility,high surface activity,facil surface chemistry and certain imaging potential.Therefore,noble metal nanoparticles are combined with CTS to form multifunctional intelligent responsive hybrid nano drug carriers.Here,we introduced silver nanoparticles and gold nanoparticles respectively,and optimized the synthesis method and function of CTS-based nano drug carriers,in order to obtain stable drug delivery and accurate and efficient drug release.The specific works are as follows:Part Ⅰ: In order to improve the poor colloidal stability,low loading efficiency of cationic drugs and insufficient sensitivity to tumor microenvironment of CTS nanoparticles,CTS/silver nano drug carriers with variable size were synthesized by double emulsification(water/oil/water).Furthermore,the efficient loading and triple responsive drug release of doxorubicin hydrochloride(DOX)were also realized.Firstly,the disulfide bond in N,N’-bisacrylamide cystamine(BAC)broken under the action of silver nanoparticles.The broken segment containing terminal sulfhydryl group of BAC was grafted on silver nanoparticles through Ag-S bond to form bAG(nano crosslinker with double bond).Then,CTS and bAG were formed into hybrid nanoparticles through Michael addition reaction by using the method of water in oil in water.Finally,polyvinyl alcohol(PVA)in the aqueous phase was deposited on the surface of hybrid nanoparticles to form negatively charged CAB nanoparticles with core-shell structure.The results showed that the particle size and surface potential of CAB could be adjusted by changing the feed ratio of chitosan and bAG.The study of drug loading by dialysis showed that the entrapment efficiency of DOX by CAB could be as high as 80%.The stability experiment of nanoparticles showed that CAB had good colloidal stability in various mediums,and there was no agglomeration observed.In acidic PBS solution containing GSH and lysozyme,CAB could degrade into nano blocks with small particle size(10 nm-20 nm).Moreover,under the acid and reducing conditions,the release of DOX in CAB@DOX accelerated,and the drug release amount could reach 80% in the presence of lysozyme.MTT assays showed that CAB alone was non-toxic to human tongue squamous cell carcinoma cell line(CAL27 cell)and showed stronger antitumor activity while loading DOX than free DOX at the same concentration.The IC50 value of CAB@DOX toward CAL27 cells was only 1/2 of DOX.The results of laser confocal experiment showed that DOX was easier to enter the cell through CAB delivery.In vivo experiments showed that CAB@DOX possessed good antitumor effect on nude mice bearing squamous cell carcinoma and did not show obvious toxic and side effects.Part II: There are always such problems that the drug effect is not obvious due to the low utilization rate of nano drugs in vivo and the accumulation of nano drugs in tumor sites can not be observed effectively.Apart from improving the poor colloidal stability and low efficiency of cationic drug encapsulation of CTS nanoparticles,this part of the work taken advantage of the excellent sensivity of CTS nanoparticles and further optimized the imaging diagnostic function of CTS as nano carrier.As we all know,if we use image of the carrier as a reference and timely trace the accumulation of nano drugs in the tumor,we can report rich diagnostic information while precisely treating to provide guidance for the improvement of therapeutic effect.Therefore,we designed an intelligent CTS/gold nanocarrier with tracing function,in order to accurately release anticancer drugs in tumor tissues while using the CT imaging ability of gold nanoparticles.Firstly,CTS nanoparticles were formed by ionic crosslinking with anionic sodium tripolyphosphate(TPP)based on the protonation characteristics of CTS as cationic polysaccharide in weak acidic solution.Then,CTS/TPP/PAA nanoparticles with negative surface charge were formed by adding positively charged CTS nanoparticles to negatively charged polyacrylic acid(PAA)solution.Then,the gold nanoparticles functionalized with cysteine were combined with CTS/TPP/PAA nanoparticles activated with(1-ethyl-(3-dimethylaminopropyl)carboimide)/N-hydroxysuccinimide(EDC/NHS)to form CTPA nanoparticles with satellite structure.Through optimization,the size of CTPA nanoparticles was172.3±2.1 and the surface potential was-28.1±3.8 MV.Moreover,the entrapment efficiency of CTPA nanoparticles to DOX could be as high as 87.1±4.6%.Under the conditions of simulated tumor microenvironment(weak acid and overexpression of lysozyme)in vitro,CTPA could degrade and release smaller nano blocks to increase the permeability of tumor tissue.CTPA@DOX showed acid-triggering and lysozymetriggering drug release,which ensured the site specificity of drug release.In vitro CTPA showed X-ray attenuation ability dependent on the concentration of gold nanoparticles.Cell experiments showed that CTPA had good biocompatibility and significantly inhibited the growth of CAL27 cells after carrying DOX.From the laser confocal image,it can be found that the cell uptake efficiency of CTPA@DOX was better than that of free DOX.In the mouse model bearing squamous cell carcinoma,CTPA@DOX showed certain CT imaging potential and could report the distribution of drugs in tumors.In addition,compared with the same concentration of free DOX,CTPA@DOX showed better antitumor effect,and did not cause toxic effect to important organs.In this study,a series of intelligent degradable chitosan-based nano carriers were designed to carry DOX for the treatment of oral squamous cell carcinoma by using the sensitivity of CTS to tumor microenvironment featuring weak acid and highly expressed enzymes.In addition,silver nanoparticles and gold nanoparticles were introduced into the experiment and the synthesis method and function of chitosan particles were further optimized to overcome the problems of poor colloidal stability,low encapsulation efficiency of cationic drugs,insufficient stimulation response and lack of diagnostic function. |
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