Ultrasound Mediated Oxygen And Paclitaxel Loaded Microbubbles For Synergistic Paclitaxel Treatment Hypoxia Ovrian Cancer Cells In Vitro

PART ONEPREPARATION AND DETECTION OF OXYGEN ANDPACLITAXEL LOADED LIPID MICROBUBBLESObjective To prepare oxygen and paclitaxel loaded lipidmicrobubble(OPLMBs) and compare its characteristics with paclitaxelloaded lipid microbubbles(PLMBs).Methods The OPLMBs and PLMBs were prepared by mechanicvibration technique. The morphology was observed by a bright fieldmicroscopy, size and zeta potential were detected by a Malvern ZeasizerNano ZS unit. The PTX loading rate was measured by HPLC. The oxygenloading characteristics was determined by dissolved oxygen analyzer.Results The synthesized OPLMBs have a size distribution of(1688.70±107.00)nm, a mean Zeta potential of-(10.32±0.31)mV, a drugentrapment efficiency of (97.71±1.13)%and a drug-loading rate of(22.70±0.82)%. In comparision, the PLMBs have a size distribution of(2159±144.70)nm, a mean Zeta potential of-(17.58±0.25)mv, a drugentrapment efficiency of (98.63±0.94)%and a drug-loading rateof(26.28±0.59)%, respectively. No significant morphological difference is observed between the OPLMBs and PLMBs, but the aggregationphenomenon was observed for both microbubbles at4℃for14days.OPLMBs has the ability to release oxygen after ultrasound exposure.Conclusions Oxygen and paclitaxel microbubbles(OPLMBs) weresuccessfully prepared. The size was well-ditributed. This kind ofmicrobubbles had high drug entrapment efficiency and ultrasoundmediated oxygen release ability.PART TWOESTABLISH HYPOXIA INDUCED DRUGRESISTANCE MODEL OF HUMAN OVARIAN CANCERCELLSObjective To establish hypoxia induced drug resistance model(HDRM)of human ovarian cancer SKOV3cell linesMethods Cobalt Chloride(CoCl2)with different concentrations wasadded to SKOV3cells for12,24,48,72h. Methyl thiazolyl tetrazolium(MTT) was used to detect the cell proliferation activity and drug resistancemultiple (DRM) of paclitaxel.Results No significant cell growth inhibitory was detected with theconcentration of CoCl2was less than150μmol/L, while obvious cellgrowth inhibition was detected with the concentration of CoCl2no lessthan200μmol/L. The proliferation activity of SKOV3cells was correlatedwith CoCl2in a dose and time dependent manner (F=2802.394, P<0.05).The DRM of SKOV3increased with the increase of CoCl2concentration(P <0.05).Conclusions Hypoxia indiced paclitaxel resistance model of ovariancancer SKOV3(SKOV3/PTXR) cells has been successfully established by CoCl2induction method,the suitable condition to establish SKOV3HDRM is150μmol/L CoCl2for24h.PART THREEULTRASOUND MEDIATED OXYGEN AND PACLITAXELLOADED MICROBUBBLES FOR SYNERGISTICPACLITAXEL TREATMENT HYPOXIA OVRIANCANCER CELLS SKOV3IN VITROObjective To investigate the effection of ultrasound mediated oxygenand paclitaxel loaded lipid microbubbles on the proliferation and apoptosisof SKOV3/PTXR cells.Method Exponentially growing SKOV3/PTXR were divided into thefollowing seven treatment groups:(a) applying PBS only (i.e.,‘‘negativecontrol’’);(b)applying PTX only (i.e.,“PTX only”);(c) applying PLMBsonly (i.e.,“PLMBs only”);(d) applying OPLMBs only (i.e.,“OPLMBsonly”);(e) applying PTX followed by ultrasound mediation (i.e.,“PTX+US”);(f) applying PLMBs followed by ultrasound destruction (i.e.,“PLMBs+US”);(g) applying OPLMBs followed by ultrasound destruction(i.e.,“OPLMBs+US”). For treatment groups (b)-(g), PTX wasadministered at a dose of6μg/ml. For treatment groups (e),(f), and (g),ultrasound pulses with an average intensity of0.5W/cm2were applied tothe medium for15seconds after PTX, PLMBs or OPLMBs were added tothe cell medium.24h after different treatment,the morphology of cellswere observed by optical microscope.The ratio of proliferation inhibitionwas detected by MTT.The cell apoptosis was determined by flowcytometry. The expression of HIF-1α and MDR-1mRNA were analysed by RT-PCR. The expression of HIF-1α and P-gp protein werecharacterized by western blot analysis.Results Treatment groups of (a),(c) and (d) exhibited normalmorphology. Treatment groups (b) exhibit a slight morphology change,only a few round translucent cells were observed. Treatment groups (e)exhibit moderate morphology change and some round translucent cellswere observed.Treatment groups(f) and (g) exhibit severe morphologychange, many round translucent cells were observed. OPLMBs incombination with ultrasound demonstrated anti-proliferative activities of(52.8±2.75)%and cell apoptosis ratio of (32.05±0.336)%at24hoursafter the treatment, which is significantly higher than other treatmentgroups such as PTX only and PTX-loaded MBs (PLMBs) with or withoutultrasound mediation(P＜0.05). RT-PCR and Western Blot tests furtherconfirmed the decreased HIF-1α, MDR-1mRNA and HIF-1α protein andP-gp in OPLMBs plus ultrasound group(P＜0.05).Conclusions Ultrasound mediated OPLMBs could inhibit proliferationand induce apoptsis of SKOV3/PTXR. The mechanism may be related toreduced HIF-1α and MDR-1mRNA expression,thereby inhibiting itscoding HIF-1α protein and P-gp expression.