Preparation Of Polymer Ultrasound Contrast Agents Targeting Tongue Squamous Cell And Targeting Study In Vitro

The patient of tongue squamous cell carcinoma(TSCC) is prone to cervical lymph node metastasis in early time,which affect the prognosis significantly and decide the tumor stage and treatment options.So diagnosis and treatment of cervical lymph node metastasis in patients with tongue cancer timely has become the urgent need to improve the survival rate.Based on none of a reliably noninvasive and objective inspection and treatment strategies in clinic yet,the fourth generation ultrasound contrast agent(UCA) has become the exploration direction of diagnosis and treatment of primary tumour and cervical lymph rode metastasis because of the function of targeted diagnosis and targeted drug delivery. High molecular polymer Poly Lactic-co-glycolic acid(PLGA) as one of the hull material of the fourth generation ultrasound contrast agent possesses the advantage function of excellent biocompatibility,controllable biodegradeability,goodball or film-forming property,enhancing drug-loaded and DNA fixed delivery.As one of cell penetrating peptides,TATp(transactivating transcriptional activator protein from human immunodeficiency virus type 1, HIV-1 TAT) can carry drug-loaded ultrasound microbubble into and remain in the interior of the cell promptly, efficiently and securely.But TATp lack cell specificity. Stromal cell-deried factor 1 also has been known as CXCL12.The receptor that It solely can bind to and activate is CXCR4,which is far higher expression on tongue squamous cell membrane appearing metastatic lymph node(MLN) and the positive expression rate is up to 72.3%～89.6% in different oral squamous carcinoma cell.While the apoptin protein(VP3) which is encoded by chichen anemia virus have tumor cell-specificity,non-toxic to normal cell and also has a certain extent targeting.Due to limitations of the multi-step in preparation formulations,the targeted drug-loaded PLGA UM which is prepared by the method of w/o/w double emulsion has still existed lower drug loading (DL) problems.So,it is indispensable to optimize the preparation.In summary, this study is mainly aimed to prepare a innovative VP3 gene-loaded polymer UM decorated with TATp and SDF-1,and optimize the preparation process and increase DL.To verify the targeting of the obtained UCA,SCC-15cell was targeted in vitro. Finally the study provides a new ideas and new methods for noninvasive early-stage diagnosis and non-surgical treatment of primary tumour and metastatic cervical lymph rode of tongue cancer. The experiment was divided into two parts: PART I PREPARATION AND OPTIMIZATION FOR FORMATION OF GENE-LOADED ULTRASOUND MICROBUBBLE TARGETING TONGUE SQUAMOUS CELLSObjective:Ⅰ.To prepare a innovative VP3 gene-loaded polymer UCA decorated with TATp and SDF-1, and to explore the influence of preparation factors to its particle size, drug loading and encapsulation efficiency,etc for selecting the best combination of preparation of UM.2.To evaluate the basic characteristics,DNA protection,release effect in vitro, molecular imaging effect of the targeted UCA as well as the conjugation status of TATp and SDF-1 Ton the surface of the UM.Methods:1.VP3 gene-loaded PLGA UM were prepared with the method of w/o/w double emulsion.Single factor and orthogonal analysis was used to optimize the preparation conditions and increase drug loading. SDF-1 and TATp were covalently congjugated to the surface of UM though thioether bonds to prepare VP3 gene-loaded PLGA UM targeting tongue squamous cell(TATp-SDF-1-VP3-PLGA-UM,TSVP-UM).2.The shape and dispersion of UM was observed by ordinary optical microscope;The drug loading and encapsulation efficiency was assessed by Nano-Drop ultraviolet specrrophotometer;The internal structure was observed by transmission electron microscope; The particle size,distribution and surface potential were determined by Malvern measuring instrument; The conjugation status of TATp and SDF-1 were detected by flow cytometry(FC) and laser scanning confocal microscopy(LSCM); Their DNA protection were identified by digestion reaction test; Drug release properties in vitro was tested by Nano-Drop ultraviolet spectrophotometer; The diagnostic imaging results in vitro was evaluated by Color Doppler ultrasound imaging.Results:The best preparation projection adopted by univariate and orthogonal analysis was that the mass ratio of VP3 gene/PLGA was 1.6%, VP3 gene concentration was 8ug/ul, the water phase/oil phase volume ratio is 1:15. The gene-loaded targeted UM showed regularly sphericity,good dispersion,and the concentrated particle size distribution with primarily between 490.2-522.6nm.The zeta potential was-17-Omv.The average gene loading was 0.86% with the average rate of 40.47% gene encapsulation efficiency. The DNA protective effection sustained 60min without damage. VP3 genes were released slowly and persist up to 24 days. Alone connection rates of TATp and SDF-1 with PLGA microbubbles surface were 98.37% and 97.07% respectively.The connection rate of TATp and SDF-1 loaded on PLGA microbubbles surface at the same time was 69.84% and 30.04%. Ultrasound imaging in vitro is better.Conclusions:TSVP-UM are successfully prepared by optimizing the preparation process and chemical thioether linkage.The particle size of UCA meet the needs of intravenous administration with the property of high drug loading, long-acting release, better imaging characteristics in vitro. Collectively,the prepared UM embrace the basic function of ideal drug-loaded ultrasound contrast agent system bearing both diagnosis and treatmentPART Ⅱ PREPARATION OF GENE-LOADED ULTRASOUND MICROBUBBLES TARGETING TONGUE SQUAMOUS CELL AND TARGETING STUDY IN VITROObjective:To prepare TSVP-UM according to optimization preparation projection and preliminarily explore targeting properties in vitro. Methods 1. SCC-15 cells were cultured in vitro,6-well plate was inoculated and then cell climbing films were prepared.2.TSVP-UM of targeted groups and VPP-UM of non-targeted groups were prepared according to optimization preparation projection,followed by incubation with cell climbing films about 30-60 minuts.3.The binding situation of two sets of UM and SCC-15 cell was observed by light microscope and the association rate was measured by flow cytometry targeted.Results:1.Light microscopy showed that TSVP-UM was clustered around SCC-15 cell membranes in targeted groups and a small amount had been passed through the cell membrane into the cytoplasm.while a few of VPP-UM had less adhered to the cell membrane of SCC-15 cell.2.Flow cytometry semi-quantitative results indicated that the connection rate of targeted microbubble was 91.44% instead of 12.96% connection rate in non-targeted groups.Conclusions:The experiment of targeting in vitro confirmed that TSVP-UM has the strong function of targeting sec-15 cell,in the meanwhile it is also found that a few of TSVP-UM has been penetrated into the interior of SCC-15 cells in a short time.It is preliminarily validated the biological activity of TATp which connected with the shell of TSVP-UM.TSVP-UNM as a non-viral gene vector has the potentiality to be a safe and effective gene delivery systems to target tongue TSCC and metastasis lymph rode.