Studies Of Thermosensitive Hydrogel Co-loaded With Gold Nanoparticles And Doxorubicin For Chemoradiotherapy

Cancer is among the greatest threats to human health, and chemoradiotherapy is a well-established paradigm to treat various cancers clinically. Novel strategies for chemoradiotherapy have been reported over the past few years to enhance the therapeutic effects. Recent advances in radiosensitizers exploration have provided new opportunities for constructing novel strategies for chemoradiotherapy.Various radiosensitizers have been investigated in the past decades. Significant efforts have been geared towards gold nanoparticles due to their biocompatibility, inert attributes and environmental-friendly synthesis procedures. Based on the guideline of combination therapy for cancers, combinations of gold nanoparticles and other therapeutic agents have drawn substantial attentions.As a broad-spectrum anti-cancer chemotherapeutics, doxorubicin (DOX) has been applied to treat several tumors, including leukaemia, malignant lymphadenoma, breast cancer and lung cancer. DOX is an anthracycline topoisomerase-II inhibitor, intercalating between base pairs in the DNA helix, and leading to damage to DNA replication, RNA transcription, and protein synthesis. In this study, DOX was used as the model anti-cancer drug according to its favorable features such as clarified anti-cancer mechanisims and broad applications. The combination of gold nanoparticles and DOX was studied as a novel strategy for chemoradiotherapy.In many of recent studies. DOX has been applied for melanoma treatments and yeilded favorable results. Being one of the highest-mortality skin cancers, melanoma could be hardly detected in its early stage. Many melanoma patients could not be diagnosed until its late stage, with many cancer cells invading into the deeper tissues. Chemoradiotherapy has been one of the important strategies to treat developed melanoma, making the issue of enhancing the melanoma chemoradiotherapy effect more and more important for many researchers. Guidelines for melanoma treatment from National Comprehensive Cancer Network indicates that intratumoral administration is one of the common administartion ways, due to its features of increasing intratumoral drug concentrations and thus enhancing therapeutic efficacy. Therefore the study of a novel melanoma treatment strategy through intratumoral administration is highly research-worthy and has high potential for clinics.Recently, in the search of proper intratumoral administration dosages, more and more attentions have been focused on thermosensitive hydrogel due to its advantageous features:being fluid at room temperature makes the hydrogel easy to handle for injections; being hydrogel at body temperature makes the hydrogel release drugs slowly and prolong the drug retention. Pluronic(?) F127 (F127) is one of the most commonly applied thermosensitive hydrogel materials. Approved by FDA, the F127 has been proved high safety, low prices as well as good biocompatibility. The research of thermosensitive hydrogel based on F127 is of high potential for clinical and commercial applications.In order to achieve enhanced chemoradiotherapy effects, a novel therapeutical strategy was proposed in this project, combining DOX and gold nanoparticles to enhance cancer chemoradiotherapy. By forming a depot of AuNPs and DOX at the tumor site, the F127-based thermosensitive hydrogel designed in this study was expected to enhance the effect of melanoma chemoradiotherapy. The main methods and results of this study were listed as follows:1. Gold nanoparticles were synthesized and modified by PEG successfully. The obtained AuNPs were at a maximum UV-vis absorption of 524 nm. The methods for DOX concentrations determination was constructed by UV-vis spectrum, under the conditions of pH6.8 and pH7.4 PBS. respectively. It showed that the concentration of DOX could be detected accurately within a range of 0.5～100μg/mL.2. Cold method was applied to prepare the blank hydrogel, mono-loaded hydrogel and co-loaded hydrogel. The formulation was optimized according to parameters of gelation temperature, gelation time as well as injectability. The optimized formulation (Au-DOX-Gel) was as follows:22% F127, DOX 0.5 mg/mL and AuNPs 1 mg/mL, with a gelation temperature of 27.2+0.3℃, gelation time of 170+9 s and a preferable injectability when under 20℃. The physicochemical characterization results showed that the AuNPs in the hydrogel were homogeneous sphere shapes with an average size of 13.09+1.77 nm. Average zeta potentials of AuNPs solutions and hydrogel were-13.5+2.93 and-14.9+2.20 mV, respectively. The rheology study showed that the viscosity of the hydrogel increased as the temperature increased from 10 to 27.5℃, then stayed stable at higher temperatures, proving the Au-DOX-Gel’s thermosensitivity attribute. Au-DOX-Gel was classified to pseudoplastic fluid at body temperature, according to a decreasing viscosity and increasing shear stress when the shear rate was increasing. Dialysis method was hired to run the in vitro release of DOX, and it turned out the DOX could release slower (p<0.05 at 8h) from the Au-DOX-Gel than from the mixed solution group.3. The cell viability was tested by MTT assay on the treatment of AuNPs and DOX. Results showed that the cell viabilities were higher than 80%, proving the safety of AuNPs towards HepG2 and B16 cell lines at concentrations of 1-20 nM. The average IC50 of DOX were 4.20±0.66 and 0.21±0.05μM on HepG2 and B16, respectively. The cellular uptake behavior of AuNPs by HepG2 and B16 was tested by ICP-AES. The maximum uptake time of HepG2 was at 36 h, with no statistical difference compared with that at 24 h. The maximum uptake time of B16 was at 24h. According to the cellular uptake results, the radiation time was set at 24 h after AuNPs were added. The radiosensitization effects of AuNPs and the combination effects were verified by MTT assay and colony formation assay. It showed that AuNPs with X-ray could effectively inhibit the cell viability compared to the radiation group (p<0.05), proving the radiosensitivity of AuNPs. Also, the combination of AuNPs and DOX under radiation could inhibit more cell viability compared to sole treatments (p<0.05), proving the favorable combination trategy of AuNPs and DOX.4. The radiosensitization effect of AuNPs and the combination treatment effect were tested by tumor inhibition experiments and Ki-67 staining on mice melanoma models. Results showed that AuNPs were non-toxic without X-ray radiation (p>0.05, vs. Saline), and then enhanced the anti-tumor effect under X-ray radiation (p<0.05). Moreover, the Au-DOX-Gel had a significant higher tumor inhibition rate than Au-Gel (p<0.01), DOX-Gel (p<0.01) as well as Au-DOX-H2O (p<0.01), proving the priority of Au-DOX-Gel. The sustained release of DOX and AuNPs from hydrogel was testified by determinating the DOX and Au concentrations inside the tumor by florospectrometer and ICP-AES, respectively. The mixed solution group released DOX significantly faster (p<0.01) than the hydrogel group, with a retention rate of 33.7±4.6% only at 8 h post administration. On the contrary, the hydrogel group had DOX retented at a rate of 58.7±6.6% and 15.7±2.1% at 24 h and 48 h, respectively. The retention of AuNPs at 48 h was as the similar situation as DOX:the hydrogel group and solution group had 35.8±3.42% and 14.2±3.43% of AuNPs retention, respectively, with p<0.05 between the two groups. The mice body weights, skin safety tests on rats’dorsal and the H&E staining of organs were designed for possible damage detection of the hydrogel. All these results showed that the Au-DOX-Gel was observed no apparent damage to mice body weights, rats’dorsal skin and mice organs.In summary, this study successfully synthesized and modified AuNPs. The F127 based Au-DOX-Gel was successfully constructed. The thermosensitivity, sustained release profile and the enhancement of chemoradiotherapy of Au-DOX-Gel were testified one by one. Au-DOX-Gel released AuNPs and DOX slower than the solutions in the in vitro release tests and the intratumoral retention tests. The enhancement of chemoradiotherapy of Au-DOX-Gel was verified, indicating the great potential of Au-DOX-Gel for enhanced chemoradiotherapy. The study of this project has provided a new therapeutic strategy of AuNPs and DOX co-delviery for cancer chemoradiotherapy. Also, the idea of combining the radiosensitizers and therapeutics could offer more therapeutic strategies for clinical applications.