Experimental Study On Load-Drug Hollow Periodic Mesoporous Organosilicas Ultrasound Contrast Agent Targeting Tumor Therapy

PART ⅠOrganic-Inorganic Hybrid Hollow Mesoporous Organosilica Nanoparticles for Efficient Ultrasound-Based Imaging and Ultrasound-mediated TherapyChapter 1:Synthesis of HPMOs, the physical and chemical properties of HPMOs and the capacity of drug loadingPurpose:Study the Synthesis, physical and chemical properties of HPMOs and drug loading.Methods:The synthetic procedure for benzene-bridged HPMOs was based on our previous developed silica etching chemistry via a structure difference-based selective etching mechanism. Firstly, Stober method-based monodispersed silica nanoparticles (NPs) were coated with a periodic mesoporous organosilica (PMOs) layer to form a special SiO2@PMOs core/shell nanostructures. 1,4-bis(triethoxysilyl)benzene (BTEB) was employed as the organosilica precursors and cetyltrimethyl ammonium bromide (CTAB) was introduced as the structure-directing agents to form the well-defined mesopores. Then, the solid silica core of SiO2@PMOs was removed by etching mechanism in mild alkaline solution (0.6M Na2CO3,80℃,1 h) and the CTAB surfactants were extracted in HCl ethanol solution. The microstructures of HPMOs can be easily observed in TEM images and STEM image. The drug loading efficiency of DOX was tested by UV-vis.Results:The average particle size is 450.2 nm; Specific surface area:1029.4 m2/g, pore volume:0.66 cm3/g, aperture:3.8 nm; Drug loadings of PTX-HPMOs is 91 mg/g.Conclusion:It can be found that the obtained HPMOs with nanoscale particle size, the hollow mesoporous structure and the spherical morphology exhibiting the high dispersity without aggregation, which is the prerequisite for further in vivo applications to avoid the blocking of blood vessels. HPMOs has high drug loading efficiency and unique drug encapsulation and release characteristics.Chapter 2:Contrast-enhanced ultrasonography of HPMOs in vitro Purpose:Study the effect of HPMOs as ultrasound contrast agent.Methods:The in vitro contrast-enhanced ultrasound imaging was conducted using HPMOs/PBS solutions with different concentration by wrapping them into a small sac, which was further infused into a big sac pre-filled with PBS solution. The performance of HPMOs as contrast agents for ultrasonography was compared with commercial SonoVue in different imaging modalities and different mechanical index.Results:The introduction of HPMOs exhibits the obvious contrast enhancement under both harmonic- and B-modes, and this special grayscale imaging enhancement is strongly dependent on the HPMOs concentrations and mechanical indexes. The quantitative measurement of the gray values of ultrasound imaging using HPMOs as the CAs further demonstrates that the HPMOs can significantly enhance the ultrasound imaging.Conclusion:The contrast enhancement generated by the introduction of HPMOs is very stable under high mechanical indexes.Chapter 3:The drug release kinetics of ultrasound mediated PTX-HPMOs in vitroPurpose:Study the drug release kinetics of ultrasound mediated PTX-HPMOs in vitro.Methods:PTX-loaded HPMOs (5 mg) were added into the dialysis bag and then introduced into a vial with PBS (50 ml) with or without ultrasound irradiation. The absorbance intensity of the released PTX in the replaced media was detected by an UV spectrophotometer at wavelength of 233 nm. Then ultrasound irritation was applied at 1 h,3.5 h and 5.5 h to the PTX-loaded HPMOs respectively.Results:After 12 h,40% of the loaded PTX was still enveloped within HPMOs and the release enters into the plateau. Comparatively, Ultrasound irradiation can enhance about 18% the PTX release every time. After 3 times of ultrasound irradiation, the release of PTX reached 90% at 5.5 h.Conclusion:PTX-loaded HPMOs exhibit ultrasound-triggered drug releasing performances and could be used in ultrasonic-assisted chemotherapy for cancer.Chapter 4:The antitumor activity of ultrasound mediated PTX-HPMOs in vitroPurpose:Evaluation of the antitumor activity of ultrasound mediated PTX-HPMOs in vitro.Methods:The evaluation is based on the typical CCK-8 assay. Hela cells were seeded in 96-well plate. The experiment was divided into three groups:free PTX group; PTX-HPMOs group and PTX-HPMOs+US group. CCK-8 assay was used to determine the cell survival.Results:the loaded PTX with ultrasound irradiation can exhibit higher cytotoxicity at PTX concentrations of 2.5,5 and 10 μg/mL. When the concentration of PTX was 10μug/mL, more than 75.69% Hela cells were killed. However, pure ultrasound irradiation has no obvious cytotoxicity.Conclusion:ultrasound irradiation could enhance the release of PTX from PTX-containing nanocapsules, which should be responsible for the higher concentration of PTX in the cell, leading to the enhanced PTX cytotoxicity and cell death. Moreover, the cavitation effect of ultrasound can also promote the tumor cell death.Chapter 5:The antitumor activity of ultrasound-mediated PTX-HPMOs in vivoPurpose:Evaluation of the antitumor activity of ultrasound-mediated PTX-HPMOs in vivoMethods:The antitumor activity of ultrasound-mediated PTX-HPMOs-treated tumor was evaluated using nude mice implanted with a human cervical cancer Hela cell line. Mice with tumor were randomly divided into four groups:(ⅰ)PTX administration group, (ⅱ)PTX-HPMOs administration group, (ⅲ)PTX-HPMOs+US administration group, (ⅳ)control administration group. Mice were treated weekly for 3 weeks. The antitumor effect was assessed by measuring the tumor size at two day intervals.Results:PTX-HPMOs with ultrasound irradiation exhibit significantly higher tumor inhibition rate (61%) compared to free PTX (42%) and PTX-HPMOs (36%). We also observed the large necrosis of tumor tissues in PTX-HPMOs combined with ultrasound group..Conclusion:The combined ultrasound-triggered drug releasing and ultrasound-based cavitation effect cause this high therapeutic efficiency.Chapter 6:Contrast-enhanced ultrasonography of PTX-HPMOs in vivoPurpose:Evaluation of the effect of HPMOs as ultrasound contrast agent in vivoMethods:The experiment using nude mice implanted with a human cervical cancer Hela cell line. Mice with tumor were randomly divided into three groups:(ⅰ) The control was injected at a dose of 200μl saline; (ⅱ) The second group was given SonoVue; (ⅲ) The third group was injected PTX-HPMOs. The ultrasound imaging of tumor tissues was obtained immediately,30s and 7days respectively.Results:The tumor tissues exhibited obviously contrast enhancement in ultrasonography image after the administration of HPMOs and it could diffuse rapidly within tumor tissues. Importantly, HPMOs exhibit higher in vivo stability than commercial clinically used SonoVue.Conclusion:HPMOs can be used as the CAs for ultrasound imaging during the chemotherapeutic process.PARTⅡHollow periodic mesoporous organosilicass for Highly Efficient HIFU-Based Synergistic TherapyChapter 1:DOX-loading into HPMOsPurpose:Study the capacity of DOX loading into HPMOs.Methods:HPMOs (50 mg) were dispersed into DOX PBS solution (0.5 mg/mL,20 mL) by mild ultrasound treatment. After stirring for 24 h in the dark, DOX-loaded HPMOs were collected by centrifugation. The obtained products were freeze dried for further use. The supernatant was collected and tested by UV-vis to determine the remaining DOX amount.Results:The DOX loading amount is 94.2 mg/g.Conclusion:The capacity of drug loading of HPMOs is highChapter 2:HIFU-triggered drug releasePurpose:Study the capacity of HIFU-triggered drug release.Methods:DOX-HPMOs were subjected to different temperatures (25,37,42 and 60℃) up to 60 min. The released DOX at the exposure temperature was monitored by an UV-vis spectrophotometer. A clinical HIFU system was used, which consists of an ultrasound generator and a single-element transducer placed inside a water bath. The beams of ultrasound were pointed upwards with the sample placed at the focus of the ultrasound. The absorbance intensity of samples was detected by an UV-vis spectrophotometer, and the released DOX in these replaced media was calculated. The temperature at the position of the focal point was measured with a thermocouple.Results:The releasing percentage can reach 44% and 70% under the condition of 100 W/60s and 200 W/60s, respectively. It is found that the DOX releasing profiles exhibit the similar patterns at different temperatures. TEM re-test of HPMOs after HIFU exposure shows that the organic-inorganic hybrid framework of HPMOs is very stable under the HIFU irradiation.Conclusion:The DOX releasing speeds up once the HIFU irradiation was employed. It is found that HIFU-triggered DOX releasing is caused by the mechanical and/or cavitation effect of HIFU rather than the temperature effect and the drugs can be released from HPMOs with a sustained matters rather than the explosive release caused by the broken of nanospheres.Chapter 3:CLSM observations of the uptake of free DOX and DOX-HPMOsPurpose:observe the uptake of DOX-HPMOs of Hela cell.Methods:The intracellular uptake and release of DOX from DOX-loaded HPMOs were observed by confocal laser scanning microscopy (CLSM). HeLa cancer cells were seeded in the Petri-dishes. DOX-HPMOs dispersed within culture medium was added into the incubation medium. After the medium was removed, the cells was visualized under CLSM. Control group containing free DOX with the same DOX concentration of DOX-HPMOs and culture conditions were employed for the comparisons.Results:It is found that DOX could be effectively delivered within cancer cells mediated by DOX-HPMOs demonstrated by the presence of strong red fluorescence. Importantly, the fluorescent intensity of HeLa cells after co-incubation with DOX-HPMOs is significantly higher than cells after co-incubation with free DOX.Conclusion:The in vitro CLSM observations give the direct evidences that DOX-loaded HPMOs can deliver high amounts of DOX molecules into cancer cells with sustained intracellular releasing performances, which guarantee the further HIFU-based ablation and combined chemotherapy.Chapter 4:Ex vivo evaluations of HPMOs-based HIFU synergistic ablation using degassed bovine liver as the model tissuePurpose:Ex vivo evaluations of HPMOs-based HIFU synergistic ablation.Methods:The effectiveness of HPMOs as the synergistic agent for HIFU-enhanced therapy was evaluated ex vivo by choosing the typical degassed bovine liver as the model tissue under the HIFU exposure after the injection of HPMOs in PBS solution or of PBS as the control. HIFU was applied on the injection site with the desired power and time durations. After the treatment, the degassed bovine livers were dissected to measure the ablated volume.Results:It is found that the injection of HPMOs could cause the higher gray value change under either 100 W/7 s or 200 W/4 s compared to the injection of PBS. The HPMOs group exhibits much larger ablated volume of bovine liver than PBS group after the HIFU irradiation. In the group that received HPMOs, the 100 W/7s HIFU exposure caused the same coagulated volume as the high exposure ultrasound power (150 W/4 s) caused in the group receiving only PBS.Conclusion:This result indicates that the introduction of HPMOs can cause higher tissue ablation under HIFU irradiation. The higher necrosis efficiency by HPMOs could be ascribed to the change of acoustic environment of tissues, which enhances ultrasonic energy deposition in tissues and corresponding intensified mechanical/cavitation effect.Chapter 5:In vivo therapeutic evaluation of antitumor activity of HIFU-based synergistic therapy combined with chemotherapyPurpose:In vivo therapeutic evaluation of antitumor activity of HIFU-based synergistic therapy combined with chemotherapyMethods:The antitumor activity of HIFU-mediated DOX-HPMOs-treated tumor was evaluated using ICR mice implanted with rat sarcoma S-180. Mice were randomly divided into four groups:(i)HIFU group, (ii)DOX-HPMOs group, (iii)DOX-HPMOs +HIFU group and (iv)control group. Mice were treated weekly for 3 weeks. The treatment effect was assessed by measuring the tumor size every two days. All mic^ were sacrificed at end of the test, and the tumor block was separated. Tumor volume and the inhibition rates of tumor weight were calculated. The histological observations was using TUNEL staining.Results:the group of DOX-HPMOs combined with HIFU irritation exhibits the highest antitumor activity compared with control group, DOX-HPMOs group and HIFU group. The antitumor efficiency can reach 80% while the HIFU group and-DOX-HPMOs are only 51.10% and 69.22%, respectively. The remarkably destructed cells, large vacuoles and irregular widening of tumor tissues could be found in DOX-HPMOs combined with HIFU irradiation groups compared to DOX-HPMOs groups, HIFU groups and saline control groups.Conclusion:The in vivo therapeutic outcome indicates that the elaborately designed HPMOs-based nanoplatform provides an excellent synergistic agent and intelligent drug delivery nanosystem for highly efficient HIFU-based cancer surgery.