Design Multifunctional Delivery Systems For Synergistic Chemodynamic Therapy And Study Their Anti-cervical Cancer Activity

Cervical cancer has always been one of the malignant tumors that seriously threaten women’s health.Although HPV vaccination has been introduced,its preventive effect may not appear until 20 years later.For patients who already have cervical cancer,the main treatment for progressive cervical cancer is still chemotherapy,except for early stage cervical cancer which can be treated by surgical excision and radiotherapy.Since single-drug chemotherapy is highly toxic and prone to multidrug resistance,the main form of chemotherapy used in clinical practice is a two-drug or three-drug combination based on cisplatin,such as cisplatin and paclitaxel,or a three-drug combination based on cisplatin,paclitaxel and bevacizumab.In actual clinical application multi-drug combinations still have many side effects,such as nephrotoxicity of cisplatin,gastrointestinal toxicity of paclitaxel and haematotoxicity of bevacizumab.To overcome these disadvantages,some researchers have developed a number of nano-delivery systems to improve the efficiency of drug delivery and reduce drug side effects.Recently,many researchers have sought to develop new techniques and methods for synergistic therapy with drugs in order to improve the treatment quality.This study aimed to develop nano-sized,tumor-targeting,tumor microenvironment（TME）responsive,and multi-method integrated delivery systems for the synergistic therapy of cervical cancer.The main results of this research are listed as follows:（1）A hyaluronic acid（HA）and ferrocene acid（Fc）based micelle was prepared with the properties of GSH sensitive,CD44 receptor targeting for synergistic drug-chemodynamic therapy（CDT）.The natural polymer HA,which can specifically recognize CD44 receptor,was selected as the hydrophilic end,and Fc,which has good biocompatibility and can catalyze fenton reaction,was selected as the hydrophobic end,to form reduction-sensitive amphipathic polymeric FCH,and then loaded with doxorubicin（DOX）to produce multifunctional micellar DOX/FCH.The results showed that DOX/FCH has a suitable particle size（190.14±7.76 nm）and electronegative charge.The particle size was stable in complete medium at room temperature for 48 h.DOX released from DOX/FCH reached about 50%in the medium simulating intracellular environment of tumour cells within 2 h,indicating good reduction-sensitive characteristics.Electron spin-resonance spectroscopy（ESR）and 3,3,5,5’-tetramethylbenzidine（TMB）experiments confirmed that FCH has significant catalytic properties for the Fenton reaction.In vitro cellular assays showed that DOX/FCH could escape from the lysosomes to the cytoplasm after being uptaken by HeLa cells for 8 h.In presence of GSH,DOX and Fc were released,increasing intracellular oxidative stress levels,damaging mitochondria and synergistically exerting a chemical-CDT effect to inhibit cell proliferation with IC₅₀ 4.57μg/mL.（2）A host-guest interacion based prodrug nanoparticle was constructed for co-delivery of DOX,CaO₂ and Fc,which showed calcium death-chemotherapy-CDT synergistic effect driven by a triple-cascade reaction.In order to address the problem that the function of CDT was limited by the low level of hydrogen peroxide（H₂O₂）in tumour cells,this chapter proposed to introduce the exogenous H₂O₂ donor CaO₂ to increase the level of H₂O₂ in tumours.The decomposition of CaO₂ under the acidic conditions of TME not only provided H₂O₂,but the decomposition product Ca²⁺could induce tumour cells with Ca²⁺overload,leading to cell death.We chosed Fc as a catalyst for CDT and combined it with DOX to made a hydrophobic functional prodrug FD through amide bonds.β-cyclodextrin（β-CD）was grafted onto the main chain of HA to obtain the polymer HC.FD and HC could be prepared as amphiphilic polymers FDHC by intermolecular force between the host and guest.Both ESR and TMB verified that FDHC could catalyse the decomposition of H₂O₂ to produce·OH.The results of drug release showed that the content of Ca²⁺released from CaO₂@FDHC was up to 73.57%in 48 h under TME condition,while only 36%was released under physiological conditions.In vitro experiments showed that CaO₂@FDHC could escape from lysosome to cytoplasm after being endocytosed.Then Fc was oxidised to Fc⁺by H₂O₂and separated from host molecules releasing CaO₂.CaO₂ was broken down to Ca²⁺and H₂O₂under acidic condition,providing 2raw material for calcium death and CDT.The prodrug FD separated from host molecules could synergistically exert the chemotherapy-CDT effect to inhibit tumor cells proliferation with IC₅₀ 4.75μg/mL.The results of animal experiments showed that CaO₂@FDHC had good tumor targeting and in vivo anti-tumor activity,significantly reduced the toxic effects of DOX,and showed better synergistic treatment of CDT with chemotherapy and calcium death.（3）A glycogen based nanoparticle was designed for co-delivery of GOx and Fc,which showed starvation therapy（ST）and CDT synergistic effect and significant anti-cervical cancer activity.In order to further validate the anti-tumour effects of CDT,reduce the potential side effects of chemotherapeutic drugs and avoid accumulation of drugs and carriers in the body,this chapter used glucose oxidase（GOx）that exerts ST and CDT against cervical cancer.The electropositive amino glycogen（GN）aminated from the electronegative Gly was bonded to Fc to obtain the positively charged GF by amide bond.GF adsorbed GOx electrostatically was binded to HC by host-guest forces to gain GOx@GF/HC with a particle size of（170±1.27）nm.Cyclic voltammetry（CV）,ESR and TMB methods verified the cyclic redox characteristic and the catalytic property of Fenton reaction of GF/HC,respectively.The results of gel electrophoresis showed that GOx@GF/HC was successfully loaded with GOx at a loading rate of（7.24±0.18）%.Determinations of pH and H₂O₂ indicated that GOx@GF/HC retained the activity of GOx.The accumulate release rates of GOx within 24 h were 80%and 30%under（pH 6.8+100μM H₂O₂）and pH 7.4 conditions,respectively,indicating that Fc molecules could be oxdiated to separate fromβ-CD rapidly under H₂O₂conditions.Then GOx and Fc released from GOx@GF/HC acted synergistically as ST and CDT.In vitro cellular assays showed that GOx@GF/HC taken up by HeLa cells,could exert a cascade catalytic reaction,resulting in an obvious increase of intracellular ROS and a high level of oxidative stress of HeLa cells,following cell apoptosis and increased cytotoxicity,significant anti-cervical cancer effects.In vivo experiments showed that GOx@GF/HC had a good biosafety profile without causing changes in blood glucose in vivo,while being able to specifically target tumour tissue and exert tumour suppressive effects.