The Diagnostic And Therapeutic Application Of Multifuctional Gold Nanords-based Nanoplex In Oral Cancer

As the one of the most common malignancies in the word, oral cancer consists the 5%-6% of the cancer in the whole body, with only 50% of the 5-year survival rate. The tumors always involve the important ograns and tissues in the head and neck as their sepecific anatomical positions, therefore is usually difficult to cure. The early diagnosis of oral cancer can significantly improve the 5-year survival rate and the life quality of patients. However, the diagnosis of oral cancer in early phase depends on the sensitivity and specificity of diagnostic techniques as well as expensive equipments in most cases. Therefore, to develop the diagonstic technique with high sensitivity and specificity is urgently needed. The major treatment of oral cancers is combined therapy based on surgery, radiotherapy and chemotherapy. These tranditional treatments always cause severe side effects and disfigurement of visage, leading the psychological problems of the patients and their quantity of life. Therefore, a novel treatment with proven curative effects and less adverse effects is also needed.Gold nanorods (GNRs), as a novel nanomaterial, are now widely used in bio-medical fields, such as biosensing, bioimaging, durg delivery and photothermal therapy, with their unique surface plasmon resonance (SPR) effects. Meanwhile, as GNRs is easy to synthesize and to be surface modified with good biocompatibility, it is possible to combine GNRs with other diagnosis and treatment methods through multifunctional surface modification. Moreover, as the excellent optical and physical properties, GNRs are now widely studied for the diagnosis and treatment of cancers. In the present study, we created a novel GNRs-based nanomaterial by conjugating with rose bengal (RB), a specific dye for oral cancer, for diagnosis and rapid screening for oral cancer with the high sensitivity. In addition, the nanomaterial was also used for oral cancer therapy in photodynamic and photothermal dependent manners. Besides, another GNRs-siRNA conjugating nanomaterial was synthesized to improve the curative effects of GNRs mediated photothermal therapy in oral cancer.Part1. Preparation and physicochemical property test of GNRs based nanomaterialsObjective:The advantage of GNRs as multifunctional nanomaterial is the diverse surface modifications for different purpose. In this part, rose Bengal (RB), a dye that could specifically bind to oral cancer cells, was conjugated to the surface of GNRs, therefore to prepare the RB-GNRs nanoplex, which was then used for the diagnosis and treatment of oral caner. In addition, the GNRs-siRNA complex was prepared by their electrostatic interaction.Methods:The pristine CTAB-coated gold nanorods (GNRs) were synthesized by a seed-mediated growth method, which was then coated with PAH layer by layer. RB-GNRs were generated by bingding PAH-coated GNRs with rose bengal (RB) via electrostatic interaction. The characterization of RB-GNRs was confirmed by UV-VIS-NIR absorption spectra, Zeta potential and TEM images. The GNRs-siRNA complex was also prepared via electrostatic interaction between them, and the characterization of this complex was then analyzed. Agarose gel electrophoresis was performed to seize the best ratio between GNRs and siRNA. Finally, the biocompatibility of the complexes was measured by MTT assays.Results:The RB-GNRs were synthesized according to the methods described above, and the results from UV-VIS-NIR absorption sepctra showed the specific absorption peak of RB and GNRs in the absorption spectra of RB-GNRs, suggesting the formation of stable complex. The results of Zeta potential showed the change of surface charge of the nanomaterials during the systhesis process. The images from TEM revealed the length and width of RB-GNRs were 52±4 nm and 13±2 nm, respectively, with a rato of 4.2, indicating their excellent cellular uptake properties. As the negative charge of siRNA, during the systhesis of GNRs-siRNA, the results from Zeta potential showed the transversion of the surface potentials. The results from UV-VIS-NIR absorption sepctra showed the red shift of the GNRs absorption peak. The best ratio between siRNA and GNRs was 24 μg/pM (siRNA:GNRs)according to the agarose gel electrophoresis. MTT assays were then performed, and the results suggested the good biocompatibility of the both nanomaterials.Conclusions:The nanoplex, RB-GNRs and GNRs-siRNA, with strong optical absorption in the near-infrared (NIR) region, good stability in aqueous solution, low cytotoxicity, and high specificity to oral cancer cells were successfully systhesized, which were further studied for their diagnosis and treatment applications. and treatment of oral caner. In addition, the GNRs-siRNA complex was prepared by their electrostatic interaction.Part2. Diagnostic application of RB-GNRs nanocomplex in oral cancerObjective:As the specific combination of rose bengal in RB-GNRs nanocomplex to oral cancers caused the shift of surface plasmon resonance band of GNRs as well as the absorption change in near infrared ray (NIR) intensity, the RB-GNRs could be used as a diagnositic platform and rapid screening for oral cancer cells with the high sensitivity.Methods:The lysates of Cal-27 and normal oral epithelial cells were added into the RB-GNRs solution, the shift of longitudinal surface plasmon resonance (LSPR) peak was then detected by UV-VIS-NIR spectrophotometer, which could sense the combination between Cal-27 cells and the nanoplex. The oral cancer cells, Cal-27, were incubated with RB-GNRs with concentration of 0.45 nM. After 2 hours, the uptake of RB-GNRs by Cal-27 was analzyed by the specific absorption of RB-GNRs in NIR spectra. The absorption was then quantified and expressed as heat map images using home-made NIR absorption detection and imaging system.Results:The results from the UV-VIS-NIR spectrophotometer suggested the great red-shift of LSPR peak after combination with the Cal-27 and normal oral epethelial cells lysates induced the aggregation of RB-GNRs. Moreover, the red-shift amounts had a significant positive relationship with the amounts of cancer cells in the range of 2.2×103-30.3×103 cells/ml with a detection limit down to 2,000 cells/ml, suggesting the great diagnositic sensitivity of RB-GNRs. No red-shift in the NIR absorption wavelength was found when RB-GNRs were incubated with the lysates of normal epithelial cells, indicating the high specificity of RB-GNRs to oral cancer cells. By employing the RB-GNRs as an imaging probe, an imaging assay was established on a homemade NIR absorption imaging system. The results demonstrated the NIR absorption of RB-GNRs was higher in cancer cells than that in normal epithelial cells, which was also postively related with the cell numbers.Conclusion:The high sensitivity and specificity of the RB-GNR platform as demonstrated by the two complementary assays provide non-invasive optical diagnostics of oral cancer cells enabling convenient screening and monitoring. used as a diagnositic platform and rapid screening for oral cancer cells with the high sensitivity.Part3. RB-GNRs nanocomplex mediated combined photodynamic and photothermal therapeutics for oral cancerObjective:Rose bengal (RB), as a classical photosensitizer, could generate singlet oxygen under the irradiation of 532nm light, while gold nanorods (GNRs) had extremely high photo-heat conversion rate under the 810nm laser irradiation for their strong absorption in the near-infraed region. Based on the photodynamic effects of RB and photothermal effects of GNRs, we speculated that RB-GNRs might have both effects under the combined light, therefore achieving better therapeutic outcome in oral cancer.Methods:The photo-heat conversion rate of GNRs was measured by a home-made device under the irradiation of 810 nm light, and the photo-degradation assays of ABDA was used to measure the singlet oxygen generation of RBs under 532 nm light. The photodynamic effect of RB-GNRs on cell viability and apoptosis was detected by MTT assay and Annexin-V/PI double staning under the irradiation of 532nm light. By the flow cytometry analysis, we investigated the ROS generation ability of RB-GNRs irradiated by the 532nm light. The photothermal effect of RB-GNRs on cell viability was determined with MTT and typan blue assay. The photothermal effect of RB-GNRs on tumors from DMBA induced hamaster model by near-infraed heat map images. In vivo experiments were conducted on hamster cheek pouches to resemble the human oral cancer conditions more accurately to assess the therapeutic effectiveness.Results:The results measured by the home-made device showed that the significant increase of temperature induced by GNRs under the 810 nm light, suggesting the high photothermal efficiency of GNRs. The results from ABDA degration assays demonstrated that RB-GNRs remarkably improved the degration of ABDA under 532 nm light, indicating the efficient singlet oxygen generation. The reactive oxygen species (ROS) were measured by DCFH fluorescence using FACS, which were further casued significant cell death via MTT and Annexin V/PI double staining assays. The photothermal effects of RB-GNRs under the irradiation of 810nm light caused the increased cell death via MTT and typan blue assays. By injecting the RB-GNRs into the tumors of hamaster models and followed by NIR imaging, the photothermal effects were also confirmed in vivo. Most importantly, the combined photodynamic and photothermal effects induced by RB-GNRs under combined irradiation of 810 nm and 532nm lights significantly suppressed the tumor growth and even cured the tumor in the models.Conclusions:Compared to the single photodynamic therapy or photothermal therapy, the RB-GNRs with combined PDT-PTT capabilities provide better therapeutic effects against oral cancer and have large potential in cancer treatment.Part3. GNRs-BAG3 siRNA nanoplex increased the sensitivity of GNRs mediated photothermal therapy by gene silencingObjective:In order to eliminate the themral resistance of the cancer cells and improve the efficacy of the photothermal therapy mediated by GNRs, the siRNA against thermal resistance related genes were combined to the GNRs via electrostatic interaction, therefore increasing the sensitivity of GNRs mediated photothermal therapy.Methods:The moderate irradiation power used for further experiment was selected by MTT assay. And the heat shock response related gene and proteins were then detected by real-time PCR and western blots after photothermal therapy of GNRs incubated cancer cells irradiated at moderate laser power. Fluorescence microscopy and flow cytometry were used to determine the cell uptake of GNRs-siRNAFAM. Real time PCR and western blots were used to detect the silencing efficiency of GNRs-BAG3 siRNA for BAG3 in Cal-27 cells before and after the laser irradiation and then compared the silencing efficiency among naked BAG3 siRNA, Lipofectamine-BAG3 siRNA and GNRs-BAG3 siRNA. The therapeutic efficacy of GNRs-BAG3 siRNA in the photothermal therapy was determined by MTT assays, apoptosis assays and the protein expression of cleaved caspase-3. The tumor tissue uptake of GNRs-siRNAFAM was investigated by fluorescence microscopy. The combined therapeutic efficacy of GNRs-BAG3 siRNA for tumor ablation was confirmed in the xenograft tumor model at the moderate irradiation laser power.Results:The results from real time PCR and western blots showed that during the GNRs mediated photothermal therapy, the expression levels of HSP27, HSP60, HSP70, HSP90 and BAG3 were significantly increased. Accroding to the results, BAG3 was selected as the target gene. GNRs succeeded in deliverying the siRNAFAM into Cal-27 cells. In addition, GNRs-BAG3 siRNA decreased the expression of BAG3 during the photothermal therapy on both mRNA and protein levels. Moreover, MTT assays and Annexin V/PI double staining assays suggested that GNRs-BAG siRNA caused more cell death in Cal-27 cells than pure GNRs, which was further confirmed by the increased expression of cleaved-caspase3. By injecting GNRs-siRNAFAM into the xenograft tumor, the delivery ability of GNRs was also confirmed in vivo. Most importantly, the results from the tumor growth curve and tumor weight suggested the better therepeutic effects of GNRs-BAG3 than that of GNRs alone with down-regulation of BAG3 and up-regulation of apoptosis.Conclusions:The present GNRs-siRNA complex can enhance the therapeutic efficiency of photothermal therapy by inhibiting the increased expression of BAG3 induced by heat stress, offering a safer strategy for photothermal therapy of oral cancer. The gene therapy only exerts its therapeutic effect in BAG3 overexpressing cancer cells, expecially in photothermal activating cancer cells, attenuating their therathermal resistance and more sensitivity to photothermal therapy.The preesent research systematically studied the diagnostic and therapeutic applications of GNRs based nanoplex in oral cancer. In the diagnosis of oral cancer, the prepared RB-GNRs nanoplex was used for diagnosis and rapid screening with the high sensitivity due to its unique surface plasmon resonance (SPR) effects and the high absorption in NIR field. In the therapeutics of oral cancer, RB-GNRs showed combined photodynamic and photothermal therapy under the combined irradiation of both 532 nm and 810 nm lights. Furthermore, GNRs-BAG3 siRNA nanoplex was prepared to conquer the thermal resistance of oral cancer during the GNRs mediated photothermal therapy, therefore improving the efficacy of GNRs induced photothermal therapy. In summary, the present study demonstrated the advantages of GNRs based nanoplex as the promosing integrated theranostic platform for both diagnosis and therapeutics of oral cancers.