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New Sunitinib-loaded Polymer Microbubbles for Targeted Renal Cell Carcinoma Therapy: in-vitro and in-vivo Experimental Study

Author: Hu
Tutor: SuHaiLi
School: Fourth Military Medical University
Course: Medical Imaging and Nuclear Medicine
Keywords: Targeted drug delivery Tumor therapy Microbubbles Sunitinib Renal cellcarcinoma
CLC: R737.11
Type: Master's thesis
Year: 2013
Downloads: 2
Quote: 0
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Abstract


Objective Chemotherapy is important for tumor treatment. But a variety ofchemotherapeutic drugs are stop using in medical treatment before to achieveconcentration because of significant systemic side effects, so that affects drugs clinicalcurative effect. The ultimate objective in tumor chemotherapy is choosing the rightchemotherapy drugs and bringing their effects into full play in vivo. In recent years, thedevelopment of ultrasound-triggered drug delivery for tumor therapy have beenbecoming one of the key researches.This subject planing to adopt the multipoly shape ofultrasound microbubble, combined with Sunitinib formation the new microbubble-drugscomplex,targeting at the tumor to crush when exposed to ultrasound, and explores thefunction of this microbubble-drugs complex in inhibiting the proliferation and apoptosisof human renal carcinoma GRC-1cells, and preliminarily investigates their inhibition onthe growth of renal transplanted tumor in nude mice. Methods Preparation of new Sunitinib-loaded polymer microbubbles. Microbubbleswere prepared by sonicating the mixture of nonionic surfactant Span60, Tween80andPEG with different polymerization degree suspended in PBS under SF6gas.Sunitinib-loaded liposomes were prepared by the transmembrane ammonium sulfategradients method. The new microbubble-drugs complex were obtained by mixing abovemicrobubbles and Sunitinib-loaded liposomes. The average size distribution ofmicrobubble and liposome were measured by Coulter Counter. And the concentration ofmicrobubbles were obtained by Photoshop software calculation, the Sunitinib entrapmentefficiency of liposome were measured by Spectrophotometer. To verify the adhesion ofliposome on microbubble surface, during the preparation of liposome,25-NBD-Cholesterol was added to the lipid solution to get fluorescence labeledliposomes. The high preload level of Sunitinib to the new microbubble-drugs complex isverified by the change of absorption curve of flow cytometry. In vitro experiments,Human renal carcinoma GRC-1cells were randomly divided into six groups: Control,MB, Liposome, Sunitinib, pMBS-US, and pMBS+US. The MTT method was used toobserve the cell survival rates of different treatment groups by changing the drugconcentration and acting duration. Caspase-3and FITC fluorescent staining wereemployed to detect apoptosis, DAPI fluorescent staining were employed to observe thechanges of apoptotic cell nucleus and transmission electron microscopy were used toobserve the change on the microscopic structure of apoptosis cells. In vivo experiments,subcutaneous transplanted models of nude mice renal tumors were established byprimary cultured cell suspension inoculation, and randomly divided into four groups:Control, pMB, Sunitinib and pMBS+US. Before the experiment, to confirm thecirculatory stability of the new microbubble-drugs complex. During the experiment,changes in nude mice tumor growth were real-time monitoring observed by ultrasonicexamination, and growth curves were illustrated. Micro-CT examination was used toobserve and assess tumor ’s development and metastasis. At the same time, occurrence ofSunitinib related adverse reactions were observed and recorded.Results The mean bubble diameter of microbubble was3.17μm, and the concentration was3×10~9/mL. The average diameter of liposome is165nm and Sunitinib entrapmentefficiency is about78%. The liposomes were visualized using the fluorescence ofNBD-cholesterol incorporated into the liposomes which surrounded the surface ofmicrobubbles. Flow cytometry analysis indicated that sunitinib successfully loaded onthe new microbubble-drugs complex. Results in vitro studies showed that the newmicrobubble-drugs complex has obvious growth inhibition and apoptosis-induced effectsfor human renal carcinoma GRC-1cells. Among all those groups, the pMBS+US groupexhibited greater inhibition and apoptosis-induced effects on this cells compared with theother treatment groups and the control group, and the inhibition degree increases asacting time and acting drug concentration increases. Results in vivo studies showed thatthe new microbubble-drugs complex has obvious growth inhibition effect for nudemice’s renal carcinoma. Among all those groups, pMBS+US group’s inhibition on tumorgrowth statistically differ from other groups. It manifests that this group’s treatmenteffect on this nude mice’s carcinoma is the best and even better than that of the single useof drugs. The experimental results in vivo and in vitro are consistent.Conclusions The experimental results prove that no matter in vivo or in vitroexperiments, the most effective therapeutic schedule of renal cell carcinoma requires newpolymer drug-carrying microbubble, effective sunitinib and designed irradiation ofultrasound. Furthermore, tumor growth in the pMBS+US group was slower in both invitro and in vivo studies. Therefore, the new Sunitinib-carrying polymer microbubblescan significantly promote drugs to function with tumor cells, remarkably increase theinhibition effect on nude mice renal transplanted tumors, and reduce side effects,providing a new and effect drug delivery method and enabling continuous drug deliveryfor targeted tumor therapy.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Kidney,renal pelvis tumor
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