Design And In Vitro/in Vivo Antitumor Evaluation Of Redox Sensitive Amphiphilic Small Molecule Drug Delivery Systems

Malignancy is one of the major public health problems in the world and is one of the major causes of human death.Chemotherapy is the most important method to treat malignant tumors clinically.However,the use of single chemotherapy is prone to produce multi-drug resistance problems.Photothermal therapy is an emerging and highly selective and minimally invasive technique for the treatment of tumors in the 1980s.In order to further improve the therapeutic effect of tumors,the combination of chemotherapy and photothermal therapy has become a hot research topic.Traditional chemotherapy and photothermal therapy drugs generally have poor water solubility,poor tumor targeting and serious side effects,causing great pain to patients.Since the EPR(Enhanced Permeability and Retention)effect was discovered,it is realized that nano-drug carriers can penetrate out of tumor blood vessels at a high rate due to their proper nano-size,thus allowing the drugs in the carriers to enter into the tumor tissues,improving the drug efficacy and reducing the side effects.Therefore,to overcome the shortcomings of traditional chemotherapeutic and photothermal agents,nano-drug delivery systems are widely studied,among which amphiphilic small molecule nano-drug delivery systems have attracted much attention of the researchers due to their advantages of simple structure,high drug loading and easy formation of abundant nanostructures.Based on the above research,we selected the chemotherapy drug cytarabine(Ara-C)which is widely used clinically,and camptothecin(CPT)which has a promising clinical application,and the organic near infrared photothermal molecule IR-780 as the model drug molecules,to build redox-sensitive amphiphilic small molecule nano-drug delivery systems.The contents of this subject involved two parts:(1)We use the redox-sensitive disulfide bond to link a hydrophobic antitumor drug and a hydrophilic antitumor drug to construct an amphiphilic small molecule drug-drug conjugate system.(2)We employ a targeting group to modify the organic near-infrared photothermal molecule,to construct a redox sensitive amphiphilic small molecule prodrug system combining with chemotherapy and photothermal therapy.The specific research contents,methods and conclusions are as follows:1.Synthesis,assembly and biological evaluation of the redox-sensitive camptothecin-cytarabine conjugatesCPT is hydrophobic and it is a potent broad-spectrum DNA topoisomerase I inhibitor which shows a strong cytotoxic activity against gastric carcinoma,rectal carcinoma,leukemia and non-small cell lung cancer.Nonetheless,many limitations such as low aqueous solubility,severe side effects,and rapid hydrolysis of the active lactone form into an inactive carboxylate form under physiological conditions have restricted the clinical use of CPT.Many articles showed that the modification of the hydroxyl group of CPT molecule can effectively inhibit the deactivation of CPT.Ara-C is a hydrophilic antimetabolism anticancer drug extensively used for the treatment of both acute and chronic myeloid leukemias and other malignancies.Nevertheless,there exist several limitations of Ara-C in drug application including weak lipophilicity,poor membrane permeability as well as short plasma half-life because of the deamination of the amino group.Modification of the amino group of Ara-C can effectively avoid the inactivation of Ara-C.Herein,we use a redox sensitive disulfide bond to link the hydrophobic anticancer drug CPT with the hydrophilic anticancer drug Ara-C to form an amphiphilic drug-drug conjugate CPT-ss-Ara.]H-NMR,FT-IR and ESI-MS confirmed the successful synthesis of the product.CPT-ss-Ara nanoparticles were prepared by dialysis,the CAC value of CPT-ss-Ara conjugate constructed nanoparticles can be determined by using pyrene as a fluorescence hydrophobic probe,the small CAC guarantees its diluted stability in the blood cycle.The TEM image demonstrated that the nanoparticles were spherical and the DLS measurement indicated that the average diameter of the nanoparticles was about(78.3 ± 1.4)nm.The in vitro release behavior of the drugs from CPT-ss-Ara nanoparticles was investigated by the dialysis,the results indicated that the drug release had a redox-sensitive property.The CPT-ss-Ara nanoparticles had good biocompatibility judging from the hemolysis experiment results.The in vitro anticancer effect of CPT-ss-Ara nanoparticles was evaluated using MTT assay.The human umbilical vein endothelial cells(HUVEC)were selected as the normal cells to evaluate the cytotoxicity of CPT-ss-Ara nanoparticles;the results indicated that CPT-ss-Ara nanoparticles had lower cytotoxicity to normal cells compared with free drugs.The in vitro anticancer effect of CPT-ss-Ara nanoparticles was evaluated against the B16F10 and HT-29 cancer cell lines,the results showed that CPT-ss-Ara nanoparticles had higher cytotoxicity to cancer cells compared with free drugs.The in vitro anticancer experiments further demonstrated the redox sensitive release characteristics of CPT-ss-Ara nanoparticles.To confrm the superiority of the redox-sensitive drug release of CPT-ss-Ara nanoparticles,the GSH insensitive control CPT-c-Ara conjugate was synthesized.The CPT-c-Ara nanoparticles were prepared in the same way as CPT-ss-Ara nanoparticles,however,the CPT-c-Ara nanoparticles could only be stable for dispersion for about two hours.In addition,compared with CPT-ss-Ara nanopaiticles,CPT-c-Ara conjugate exhibits significantly lower killing ability to the B16F10 cancer cells,indicating the superiority of redox-sensitive drug release and storage stability of CPT-ss-Ara nanoparticles.The cellular uptake behavior of CPT-ss-Ara nanoparticles was qualitatively and quantitatively investigated by fluorescent inverted microscope and flow cytometry.From the obtained results,we can conclude that the CPT-ss-Ara nanoparticles have much better cellular uptake than that of the free drugs.The flow cytometry analysis was used to monitor whether the cell death is caused by drug-induced apoptosis,the obtained results indicated that compared with free drugs,the CPT-ss-Ara nanoparticles induced much higher cell apoptosis rate of B16F10 cells.The in vivo biodistribution and anticancer experiment indicated that the CPT-ss-Ara nanoparticles showed tumor accumulation effect and antitumor effect.In addition,histology analysis showed that CPT-ss-Ara nanoparticles led to no signifcant cell necrosis in the major organ sections and could reduce the systemic toxicity of the free drugs.All the experimental results show that CPT-ss-Ara nanoparticles have great potential in the delivery of antitumor drugs.2.A redox-sensitive small molecule drug delivery system for chemotherapy and photothermal therapy of hepatic carcinomaIR-780 is a hydrophobic,near-infrared fluorescent dye for fluorescence imaging and photothermal therapy in vitro and in vivo.Lactobionic acid(LA)is a hydrophilic hepatic cell-targeting small molecule that binds to the asialoglycoprotein receptor on the surface of hepatic cells.And this kind of asialoglycoprotein receptor is over-expressed on the surface of the rapidly dividing hepatic carcinoma cells,therefore LA can be actively targeted to hepatic carcinoma cells.The hydrophobic near infrared photothermal molecule IR-780 is linked to the hydrophilic hepatic carcinoma cell-targeting molecule LA to form the amphiphilic conjugate IR780-LA.A disulfide bond was used to conjugate two CPT molecules to form CPT-ss-CPT prodrug.CPT-ss-CPT binds to IR780-LA via hydrophobic interactions and π-πinteractions to form IR780-LA/CPT-ss-CPT nanoparticles,this multi-functional nano-drug system can be used for the fluorescence imaging of tumors and the combination of chemotherapy and photothermal therapy.The 1H-NMR results confirmed the successful synthesis of the products.The self-assembled nanodrug system was prepared by the film-ultrasonication method,the TEM image demonstrated that the nanoparticles were spherical and the DLS measurement indicates that the average diameter of the nanoparticles was about(91.0 ± 1.8)nm.The in vitro temperature increasing experiment showed that the IR780-LA/CPT-ss-CPT nanoparticle solutions(irradiated with 660 nm)had a similar temperature increasing effect with the IR-780 solutions(irradiated with 808 nm).The in vitro release behavior of CPT from IR780-LA/CPT-ss-CPT nanoparticles was investigated by the dialysis method;the results indicated that the drug release had a redox-sensitive property.The hemolysis experiment initially determined the IR780-LA/CPT-ss-CPT nanoparticles had good biocompatibility.The human hepatoma cells(HepG2)and the murine hepatoma cells(Hep1-6)were selected as tumor cell models to study the cytotoxicity of the nanoparticles.The in vitro cytotoxicity experiments and cellular uptake experiments showed that IR780-LA/CPT-ss-CPT nanoparticles had higher cytotoxicity and cellular uptake rate than that of free drugs,and showed the combination effect of photothermal therapy and chemotherapy.The in vivo biodistribution experiment and antitumor experiment showed us the excellent tumor accumulation capacity and antitumor effect of the IR780-LA/CPT-ss-CPT nanoparticles.In addition,the histological sections of the major organs and tumor tissues showed that the IR780-LA/CPT-ss-CPT nanoparticles significantly reduced the systemic toxicity of free drugs.Therefore,it is one of the promising ways to use the amphiphilic small molecule nano-drug system to treat tumors with the combination of chemotherapy and photothermal therapy.In summary,two amphiphilic small molecule nano-drug delivery systems were studied in this paper for the combination of chemotherapy and photothermal therapy.The results showed that these two small molecules could self-assemble in water to form stable nanoparticles,which could realize drug self-delivery and reduce the use of inert non-pharmaceutical materials,thus improving the drug efficacy and reducing the side effects.This study laid the theoretical foundation for the clinical application of amphiphilic small molecule nano-drug delivery systems.