Preparation Of Pingyangmycin Quantum Dot Polylactic-co-glycolic Acid Nanoparticle And The Targeting To Cervical Lymph Node Metastasis Of Oral Squamous Cell Carcinoma

A primary cause leads patient with oral squamous cell carcinoma(OSCC) to treat difficultly even death is cervical lymphatic metastasis. With the development of OSCC, increasing tissue pressure in tumor resulted from a large number of neovascularization and the absence of lymphatic vessel transmitted to peri cancerous tissure by the exudate, as a result, an increasing number of expansive peri cancerous lymphatic vessel and openning endothelial channel emerged in order to meet the functional compensation resulted from tissue edema aggravated gradually. Following the different hydraulic pressure between internal and external peri cancerous lymphatic capillaries, plenty of interstitial fluid with macromolecules and particles such as exfoliated tumor cell enterred into lymphatic capillaries through the endothelial channel, this is the course of lymphatic metastasis.5years of survival rate will be reduced by half once oral-maxillofacial cancer has metastasized to cervical lymph node, it is important realistic meaning, therefore, to find effective method of diagnosis and treatment for cervical lymph node metastasis of OSCC. There is still no effective method to identify whether OSCC has been metastasized to cervical lymph node, up-to-date, therapies of OSCC emphasized the multidisciplinary treatment, the essential was surgical operation. Because of anatomical position and functional feature of oral-maxillofacial, thorough resection of tumor and metastasis would reduce the life quality by damaged the critical function of appearance, language, eating and swallowing. Utilization of the characteristics of lymphatic capillaries after carcinogenesis, anticancer drug combined with macromolecules or particles enterred into peri cancerous lymphatic capillaries through endothelial channel and the phagocytosis of endothelial cell, and then, regional lymph node metastasis would be targeted chemotherapied with lymphatic drainage.The primary foci of oral cancer is superficial relatively, it is benefit of submucous injection or subcutaneous injection under direct vision. On the one hand, drug can be used for treating metastatic oral-maxillofacial cancer, there were advantages such as high local dosage, little side effect, avoiding damage by enzyme in blood and hepatic first pass effect, prolonging the drug action. On the orther hand, it is benefit of surgical navigation if diagnostic reagent would be chosen in localization of cervical lymph node metastasis.Pingyangmycin(PYM) is an antitumor antibiotic developed and researched in China, it is used in head and neck squamous cell carcinoma commonly and confirmed in inhibiting growth of OSCC cell lines(Tca8113) in vitro. Quantum dots(QDs) have unique optical and electronic features as following:1) QDs have broad excitation spectra together with narrow and symmetric emission spectra;2) QDs have been found to be10-20times brighter than organic dyes;3) QDs can effectively eliminate the light absorbance and background interference of tissue and body fluid;4) The stability with regard to photobleaching is much more superior to conventional fluorophores. Lactic-co-glycolic acid(PLGA) is a biomaterial that can be used in human body approved by American Food and Drug Administration (FDA) due to its excellent biocompatibility, biodegradability and mechanical strength.PYM was selected as the model drug, QDs was used as fluorescent probe and PLGA was served as the carrier material. A nanoparticle with combination of targeted chemotherapy, localization and real-time monitoring was designed and synthesized, and its targeting ability to cervical lymph node metastasis resulted from OSCC was explored preliminarily.Chapter one Preparation of pingyangmycin quantum dot polylactic-co-glycolic acid nanoparticleObjectsA nanoparticle with combination of targeted chemotherapy, localization and real-time monitoring for cervical lymph node metastasis resulted from OSCC was designed and synthesized. Methods1. PYM was selected as the model drug, QDs was used as fluorescent probe and PLGA was served as the carrier material, pingyangmycin quantum dot lactic-co-glycolic acid nanoparticles (PYM-QD-PLGA-NPs) were prepared with the method of multiple emulsion solvent evaporation and optimized through orthogonal experimental design.2. Drug loading (DL) and embedding ratio (ER) of PYM-QD-PLGA-NPs was measured.3. Size and Zeta potential of PYM-QD-PLGA-NPs was detected.4. To analysis PYM-QD-PLGA-NPs, PYM, QDs and PLGA by infrared spectroscopy technology. 5. To test the fluorescent property of PYM-QD-PLGA-NPs.6. To study the drug-release behavior of PYM-QD-PLGA-NPs in vitro.Results1. The PYM-QD-PLGA-NPs of orthogonal experimental design NO.4had the highest DL (5.9±0.3%) and ER (78.1±2.1%), which was selected for the following studies.2. The average size, partical size dispersibility index (PDI) and Zeta potential of PYM-QD-PLGA-NPs was245.4nm,0.375±0.009and-6.68±4.11mV respectively.3. Infrared spectrum indicated that PLGA located at the outmost layer of PYM-QD-PLGA-NPs, PYM and QDs were encapsulated.4. PYM-QD-PLGA-NPs shared the similar red fluorescence with QDs under fluorescence microscope, the fact nanoparticles include QDs was confirmed in reverse.5. In vitro, the drug cumulative release ratio of PYM-QD-PLGA-NPs met with the Equation "Higuchi", it had excellent control release effect.Chapter two Sensitivity test of pingyangmycin quantum dot polylactic-co-glycolic acid nanoparticles by oral squamous cell carcinoma cell lines(Tca8113) in vitroObjectsTo investigate the uptake and sensitivity of PYM-QD-PLGA-NPs by OSCC cell lines (Tca8113) in vitro.Methods1. The proliferation of Tca8113cellTca8113cells were inoculated into96well culture plate with culture medium 190μL per well, the density was1000,2000,4000,6000,8000and10000cell per well respectively. MTS tested the proliferation ability of Tca8113cells each day to the day7after inoculation.2. The uptake of PYM-QD-PLGA-NPs by Tca8113cell in vitroTca8113cells were inoculated into12well culture plate with culture medium2mL,20000cells per well, QDs and PYM-QD-PLGA-NPs were respectively added into6well after two days. Observating under fluorescence and natural light in the same field of view each hour to the hour12.3. The sensitivity of PYM-QD-PLGA-NPs by Tca8113cell in vitroGroups:PYM, QDs, PLGA, PYM-QD-PLGA-NPs, negative control and blank control, each treatment group divided into5groups according as drug concentration:0.1μg/mL,1μg/mL,10μg/mL,100μg/mL,1000μg/mL. After addition of samples, optical density(OD) was measured by enzyme mark instrument each day to the day7.4. Statistical analysisSPSS16.0and Mcrosoft Excel software was used for statistical analyses. An ANOVA by two factors of design was selected as comparation with drug groups, One-Way ANOVA was used to determine the significance of differences in means, Bonferroni was permitted as comparation between two groups. There was statistical significance when P<0.05occurred with two-sided test.Results1. On NO.7day,1000and2000cells per well keep proliferating under96well culture plate with190μL10%serum culture medium per well.2. Under the same culture condition and time, compared with QDs, although both QDs and PYM-QD-PLGA-NPs could be swallowed by Tca8113cell in vitro, PYM-QD-PLGA-NPs had a significant depression of uptake amount.3. The inhibition ability of PYM-QD-PLGA-NPs in Tca8113cell was similar to PYM in vitro, both of them had concentration and time dependent manner. PLGA had no effect on Tca8113cell almostly, the inhibition ability of QDs was obviously weaker than PYM-QD-PLGA-NPs, this further proved that the inhibition ability of PYM-QD-PLGA-NPs in Tca8113cell belong to PYM majorly.Chapter3targeting ability of PYM-QD-PLGA-NP to cervical lymph node metastasis resulted from OSCCObjectTo preliminarily evaluate targeting ability of PYM-QD-PLGA-NP to cervical lymph node metastasis resulted from OSCC.Methods1. Tumor-bearing animal modelOSCC cell lines Tca8113cells (107/mL) were submucously injected into pars buccalis of nude mice with0.2mL, the growth of tumor was observed regularly.2. Live little animal imaging in vivo0.2mL PYM-QD-PLGA-NPs with drug concentration (100μg/mL) were submucously injected into peri cancerous tissue in symmetric four sites. The distribution of nanoparticles in vivo were observed under live little animal imaging system per10minutes, the excitation wavelength was488nm, the emission wavelength was610nm.3. Tissue mass (Organ) and tissue sectionThe nude mice,12hour after injection of PYM-QD-PLGA-NPs, were killed, peri cancerous tissue and ipsilateral cervical lymph node was selected as fixed tissue mass (Organ) or wax-tissue section, which were observated under fluorescence and natural light in the same field of view.Results 1. The day from10to15after injection, the diameter of buccal tumor was more than0.5cm and cervical lymph node metastasis has formed2.2hours after buccal submucous injection of PYM-QD-PLGA-NPs, cervical lymph node had the similar fluorescence as injected area of peri cancer. This indicated some of PYM-QD-PLGA-NPs have transferred from peri cancer to cervical lymph node.3. Cervical lymph node had the similar red fluorescence as peri cancerous tissure in organic pictures. Small spherical red fluorescence, in wax-tissure sections, was observed not only at striated muscle’s edge of peri cancerous tissure but also at medullary cord and medullary sinus of cervical lymph node. It further proved that PYM-QD-PLGA-NPs have transferred from peri cancer to cervical lymph node.Conclusions1. The average diameter of optimized PYM-QD-PLGA-NPs was245.4nm, zeta potential was-6.68±4.11mV, DL and ER was5.9%±0.29%and78.1%±2.1%%respectively. The nanoparticle shared the similar fluorescence with QDs and the excellent control release effect in vitro.2. Under the same culture condition and time, compared with QDs, although both QDs and PYM-QD-PLGA-NPs could be swallowed by Tca8113cell in vitro, PYM-QD-PLGA-NPs had a significant depression of uptake amount.3. The inhibition ability of PYM-QD-PLGA-NPs in Tca8113cell was similar to PYM in vitro, both of them had concentration and time dependent manner. PLGA had no effect on Tca8113cell almostly, the inhibition ability of QDs was obviously weaker than PYM-QD-PLGA-NPs.4. PYM-QD-PLGA-NPs could be transferred from peri cancerous tissure to cervical lymph node have been proved by means of live little animal imaging system, tissue mass (Organ) pictures or tissue slice photographs. The information of this study may be useful to relative animal and clinical experiment.