| Oral squamous cell carcinoma(OSCC)often metastasizes from primary tumor to regional lymph node in the early stage of oral squamous cell carcinoma,which is the most important prognostic factor affecting the survival of patients.Lymph node metastasis is one of the causes of death in patients with oral squamous cell carcinoma.Current imaging examinations have proved to be unreliable for metastatic lymph nodes,especially for early metastatic lymph nodes.It is of great diagnostic and prognostic value for patients with oral cancer to find a reliable method for the detection and treatment of early lymph nodes.Photothermal therapy(PTT)is a new cancer treatment,which uses photothermal agents and lasers to convert laser energy into heat energy,that can directly ablation tumors.It is possible to overcome several shortcomings of traditional chemotherapeutic drugs,such as poor pharmacokinetics,low solubility,lack of ability to target tumor cells,and induction of multidrug resistance.As a kind of photothermal sensitizer approved by FDA,ICG has the ability of photothermal killing tumor cells under laser irradiation.It is considered to be a promising PTT agent because of its low cytotoxicity and excellent photothermal properties.In addition,ICG can also be used as a fluorescence imaging agent(its fluorescence absorption is about 800nm).Because of the poor optical stability and lack of targeting of free ICG,ICG is always encapsulated in nano-carrier to improve its performance as a PTT reagent and molecular imaging probe.We used PLGA as a carrier to co-encapsulate indocyanine green(ICG)and doxorubicin(DOX)to develop a multifunctional target Polymer nanoparticles(NPs)for targeted photoacoustic imaging and photothermal ablation of oral cancer cells.The chemokine SDF-1 is a specific antibody,and we coupled the chemokine SDF-1 to the nanoparticles using a carbodiimide method.Nanoparticles can automatically target tumor tissue in vitro and in vivo through the interaction of SDF-1-CXCR4.In this experiment,nanoparticle-targeted photoacoustic imaging and photothermal therapy for metastatic lymph nodes of tongue squamous cell carcinoma were studied in order to explore the feasibility of photoacoustic imaging combined with photothermal therapy combined with anti-tumor treatment of lymph nodes.In the first part,a molecular imaging delivery system modified by SDF-1 was constructed.We covalently link SDF-1 with COOH-PLGA-COOH containing a double-end carboxyl functional group to activate the carboxyl group with a carbodiimide(specific reaction of amino group with carboxyl group),thereby linking the chemokine SDF-1 to the shell membrane of the nanoparticle.We prepared the SDF-1 modified targeting nanoparticles by emulsification method.And the NPs were coated with doxorubicin(DOX),near-infrared dye and fluorocarbon gas(ICG/PFH)and simultaneously entrapped photoacoustic imaging materials,Drug delivery(DOX/ICG/PFH).The appearance,distribution and structure of the microbubbles were observed by optical microscopy and transmission electron microscopy.The particle size and surface potential of the nanoparticles were detected by Malvern particle size analyzer.The encapsulation efficiency and drug loading of the nanoparticles were determined by UV spectrophotometry.The first part of the experimental results show that the prepared nanoparticles are spherical,uniform in size and well dispersed.After the laser irradiation,the volume of the targeted nanoparticles becomes significantly larger.We suspect that it may be related to the phase transition of the wrapped PFH.The Malvern particle size analyzer detects that the size of the targeted multifunctional nanoparticles is about 195.3±52.64 nmnm,which can pass through the capillaries and meet the requirements of the lymphatic targeting carrier.The results of TEM characterization showed that ICG/PFH and DOX were encapsulated in the PLGA shell membrane,and the encapsulation efficiency of various nanoparticles was in accordance with the requirements.The in vitro imaging of nanoparticles was observed by acousto-gel model.The results showed that the SDF-1 modification had no significant effect on the physicochemical properties of the nanoparticles.The prepared nanoparticles had good imaging performance and could be used for the diagnosis of lymph nodes.The second part of the experiment is mainly to study the effect of targeted multi-functional nanoparticles on human tongue squamous cell carcinoma cells,and then evaluate the in vitro photoacoustic imaging effect and targeting of multi-functional targeting nanoparticles.The phagocytosis effect of the cells on the nanoparticles was observed by laser confocal microscopy by fluorescent labeling of CXCR4 on the surface of SCC-15 cells.The results of cellular immunofluorescence showed that CXCR4 was mainly located on the cell membrane and it could be used as a target protein.The phagocytosis experiments showed that the total amount of SDF-1d targeting nanoparticles was significantly higher than that of the other two groups.The combination of SDF-1 and CXCR4 could promote the entry of nanoparticles into cells and increase the local aggregation of NPs.Flow cytometry was used to detect the apoptosis of SCC-15 by laser irradiation after laser irradiation,and it was found that laser irradiation and targeted multi-functional nanoparticles could induce apoptosis,which was higher than other groups.The above studies provide a basis for the treatment of metastatic lymph nodes in vivo.The third part evaluates the in vivo targeting effect and therapeutic effect of SDF-1 modified targeting nanoparticles.Our research group has established a large animal model of lymph node metastasis of rabbit tongue cancer.In the experiment,we observed the in vivo photoacoustic imaging of targeted multi-functional nanoparticles and the effect of metastatic lymphatic therapy after multiple injections of DOX around the primary tumor,and initially examined the liver and kidney toxicity of New Zealand white rabbits.In vivo experiments we initially examined the liver and kidney toxicity of New Zealand white rabbits,and the results showed that the targeted multi-functional nanoparticle combined with photothermal therapy under the guidance of photoacoustic imaging played a good anti-tumor effect.In summary,vivo and vitro experiments show that the combination of photothermotherapy and targeted chemotherapy can produce greater cytotoxicity than chemotherapy,and multi-functional nanoparticles have good photoacoustic Imaging characteristics and photothermal therapy capabilities.The photothermal material changes after laser irradiation,and the generated heat causes the phase transformation of the nanoparticles,which increases the absorption of the nanoparticles by the cells.Under the laser irradiation,the PFH encapsulated inside the nanoparticle undergoes liquid phase gas change to convert the nanoparticle from the liquid core to the gas core,and the encapsulated drug(DOX)is released immediately after blasting.At the same time,the photoacoustic imaging system was used to observe strong photoacoustic signals in vitro and in vivo.Flow cytometry and histopathological detection showed that the nanoparticles had a killing effect on tumor cells.Therefore,we believe that the chemokine SDF-1 can be used for targeted chemotherapy of metastatic lymph nodes in OSCC,and combined photothermal therapy is more effective than single application. |
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