Antitumor Efficacy And Mechanisms Of Vefg-targeted Paclitaxel-loaded Lipid Microbubbles Combinded With Ultrasound Targeted Microbublle Destruction On Laryngeal Carcinnoma

Laryngeal squamous cell carcinoma(LSCC),is the third most common malignancy in head-neck region.Surgical therapy of LSCC has a history of more than 100 years,but the curative effect of routine operations is unsatisfactory for patients at middle or advanced stage or with reoccurrence.In this case,chemotherapy becomes very useful.However,most anti-cancer drugs do not show much targetability,and their use will cause irreversible damages to normal tissues and reduce human immune ability,thereby delaying therapy.Thus,the concerns in malignant-tumor treatment include how to improve the drug sensitivity of tumor tissues,enhance the curative effect of anti-cancer drugs,and reduce the toxicity and side effect of targeted therapy.With the deepened research on LSCC and its molecular biological mechanism,thus,the molecular targeted therapy with vascular factors provides opportunities for LSCC patients at late stage or with postoperative reoccurrence or metastasis.Tumor targeting therapy is a kind of method that can kill cancer cells via transporting the anti-cancer drug to the targeted tissue or organs by certain specific carrier.And this method will not affect the normal cell,tissue or organs,which can improve curative effect,reduce side effects.Recently,with the deepened research on ultrasound contrast agents(UCAs),the targeted ultrasonography with the carry of specific ligand allows the targeted microbubbles to identify and bind with the target at molecular level,gather at the targeted tissues via blood circulation,and observe the specific imaging of targeted tissues at molecular or cellular level.Molecular ultrasonography becomes a hotspot in targeted tumor diagnosis and treatment with drug carriers,offering new clues for treatment of LSCC.Angiogenesis is so important that the tumor can't grow and metastasize without it.And tumor's growth and survival depends on the generation of blood vessels as it provides oxygen and nutrients.Studies shown that there were many different factors and genes involved in the progress of regulation.There are more than 30 kinds of factors related to angiogenesis have been found.And cell division,proliferation,migration,and angiogenesis have an inseparable relationship with vascular endothelial growth factor(VEGF)and its receptors.Paclitaxel is extracted from yew which is used for anti-tumor therapy.Currently,the PTX has been used in the treatment of advanced head and neck squamous cell carcinomas need to keep throat.Ultrasound microbubble has been widely used in gene therapy,drug delivery,cancer treatment and so on,because of its advantages of safety,effective and noninvasive.Targeted microbubble can selectively bind to targeted tissues or organs,in view of the specific antigen antibody on the surface,and it can realize the target tissue or target organs,thus reduce retention in the systemic circulation and reducing systemic side effects.In this study,we use targeted ultrasound molecular probe preparation technology and biotin avidin method to connect VEGF and paclitaxel-loaded liposome microbubbles.We detected the physical and chemical properties of VEGF-targeted paclitaxel-loaded liposome microbubbles.Effect and mechanisms of VEGF-targeted paclitaxel-loaded liposome microbubbles combined with ultrasound targeted microbubble destruction(UTMD)on laryngeal squamous cell carcinoma were investigated through cytological experiments and animal models,which may provide new experimental evidence for treatment of laryngeal cancer.PART ? PREPARTION OF VEGF-TARGETED PACLITAXEL-LOADED MICROBUBBLES AND DETCTION OF THE SISE AND COMBINING CAPACITY IN VITROObjective: To prepare a VEGF-targeted paclitaxel-loaded microbubble and test its physical properties and ability of targeted adhesion to laryngeal squamous cell carcinoma Hep-2 cells.Methods Paclitaxel-loadedlipid microbubbles(PLLMs)and biotinylated lipid microbubble(MB-BS)were prepared via rotary evaporation + mechanical vibration.VEGF-targeted microbubbles(VTPLLM)were prepared by binding biotinylated VEGF to the surfaces of PLLM via avidin-biotin system.We use immunofluorescent test and observe the dispersion and appearance of VTPLLM and PLLM.The properties such as size,concentration,zeta potential,drug loadings,coating rate were determined.We observed the connection of VTPLLMs and Hep-2 cells,and compared with non-targeted PLLMs group.Results The average size of VTPLLM was 960±20nm,and the concentration was 4.07?109 /ml.The average drug loading amount was 33.2% and the average coating rate was 81.6%.We found there was a green ring around the surface of VTPLLM under the fluorescence microscopy.In cell experiments,we found that there were many green microbubbles surrounding Hep-2 cells in VTPLLM group while in PLLM group we found few around cells.Conclusion We prepared VTPLLM successfully which specifically adhered to Hep-2 cells firmly.VTPLLM is expected to become a new effective targeting drug carrier for therapy of laryngeal squamous carcinoma.PART ? EFFECT AND MECHANISMS OF VEGF-TARGETED PACLITAXEL-LOADED LIPID MICROBUBBLES COMBINED WITH UTRASOUND TARGETED MICROBUBBLE DESTRUCTION ON LARYNGEAL CANCER HEP-2 CELLS IN VITROObjective: To study the inhibition of proliferation,cell cycle arrest and apoptosis of VTPLLM combined with UTMD on Hep-2 cells,and test the m RNA and protein expression of MMP-9,VEGF and Caspase-3.Methods: According to ?Cell Culture Technology?,we cultivated human laryngeal squamous carcinoma cells(Hep-2)and divided them into six groups randomly,including the control group(Con)(this group was cultured normally,no special handled,only added PBS while processing),paclitaxel group(PTX),paclitaxel combined with ultrasound group(PTX+US),paclitaxel-loaded microbubbles combined with ultrasound group(PLLM+US),pure microbubbles with ultrasound group(MB+US),VEGF-targeted paclitaxel-loaded microbubbles combined with ultrasound group(VTPLLM+US).We determined the inhibition of Hep-2 cells by MTT,test the toxic effects of Hep-2 cells with CCK-8,while detected cell cycle arrest and induction of apoptosis via flow cytometry assay and at last we use RT-PCR and Western Blotting to teste the m RNA and protein expression levels of VEGF,MMP-9 and Caspase-3.Results: In MTT experiments,we found that PTX inhibited the proliferation of Hep-2 cells more when the drug concentration was increased.Under the same drug concentration,as the extension of culture,the inhibition rate raised.Of six groups,VTPLL+US group has the strongest proliferation inhibition.In flow cytometry assay: comparing with other treatment groups,apoptosis of Hep-2 was the strongest in VTPLLMs + US group(p < 0.01),it arrested cells in G2 / M phase.The m RNA and protein expression of Caspase-3 in this group were significantly up-regulated(p<0.01),while those of MMP-9 and VEGF decreased obviously(p<0.01).Conclusion: VTPLLMs have obvious inhibiting effect on cell growth and proliferation.And they can arrest cells in G2/M phase,induce apoptosis of Hep-2 cells.Compare with other groups,the VTPLLM one has stronger effect.Its mechanism may be associated with m RNA and protein expression of Caspase 3 genes,MMP-9,and VEGF.PART ? EFFECT AND MECHANISMS OF VEGF-TARGETED PACLITAXEL-LOADED LIPID MICROBUBBLES COMBINED WITH UTMD ON LARYNGEAL CANCER IN NUDE MICEObjective: To study the therapy effect and related mechanisms of VEGF-targeted paclitaxel-loaded microbubbles(VTPLLM)combined with UTMD on laryngeal squamous carcinoma in nude mice.Methods: Set up transplantation tumor models of laryngeal squamous carcinoma in nude mice.They were divided into five groups randomly,including the control group(Con),paclitaxel group(PTX),paclitaxel with US group(PTX+US),paclitaxel-loaded lipid microbubbles combined with US group(PLLM+US),VEGF-targeted paclitaxel-loaded lipid microbubble combined with US group(VTPLLM+US).Long diameters and short diameters of tumor were measured before and after treatment and the tumor volumes were calculated.We drew the growth curves of nude mice tumor,and calculated the tumor inhibition rate(TIR).We use the TUNEL assay to detect apoptosis of tumor cells,and expression of EGFR and VEGFR-2 by immunohistochemistry(IHC).RT-PCR and Western Blotting are used respectively to detect the m RNA and protein expression of Caspase-3,MMP-9 and VEGF.Results: Compared with other groups,the VTPLLM+US group had the highest inhibition rate of nude mice,the apoptosis index was significantly higher than other groups(p < 0.01),proliferation index significantly decreased(p < 0.01).Immunohistochemistry showed that the expression of MMP-9 and VEGF levels in VTPLLM+US group significantly lower than other groups(p < 0.01),and RT-PCR and WB results show: the m RNA and protein expression of Caspase-3 in VTPLLM+US group were significantly up-regulated(p<0.01),otherwise those of MMP-9 and VEGF were down-regulated(p<0.01).Conclusion: VTPLLM has obvious inhibitory effect on laryngeal squamous carcinoma in nude mice,and stronger antitumor effect than non-targeted PLLMs and PTX.The mechanism of this may relate with the stronger inhibition in tumor angiogenesis and invasion,raising Caspase-3,and decreasing MMP-9 and VEGF.VTPLLM combined with UTMD demonstrates a better targeted ability and stronger antitumor effect than non-targeted paclitaxel-loaded microbubbles and pure PTX,which provide the basis for targeting therapy of laryngeal squamous carcinoma.