Experimental Study On Enhancement Of Gene Transfection And Drug Delivery Using Ultrasound-Targeted Microbubble Destruction

PART IExperimental study on enhancement of gene transfection using ultrasound-targeted microbubble destructionChapter 1:Evaluation of viability of HepG2 cell after ultrasound exposure:an in vitro studyPurpose:To evaluate the viability of HepG2 cell after ultrasound (US) exposure in vitro.Methods:Ninety-six well cell culture plate was divided into 6 areas (4 wells each area), HepG2 cells were cultured in 5% CO2 at 37?. After 12 hours'culture, the cells were irradiated by US from the bottom of the cell culture plate. The overall protocol was performed by orthogonal design with 3 factors and 5 levels (acoustic intensity: 0.5 W/cm2,1.0 W/cm2,1.5 W/cm2,2.0 W/cm2,2.5 W/cm2; duty cycle:10%,20%, 40%,60%,80%; duration:20 s,40 s,60 s,120 s,180 s). After US irradiation, the cells were cultured for another 24h, then the shape, distribution and growth situation were observed by optical microscope, and MTT essays were performed to assess the cell viability.Results:After US irradiation, when the acoustic intensity was greater than 2 W/cm2 with any cycle duty or the intensity was 1.5 W/cm2 with the cycle duty greater than 60%, the viability of HepG2 was significantly decreased, and the cells detachment and aggregation like crater, cell fragmentation could be observed by optical microscopy. When the intensity was ranged from 0.5 W/cm2 to 1.0 W/cm2 and the duty cycle was 10%-20%, the rate of cell viability would be more than 90% immovably.Conclusion:The acoustic intensity and cycle duty were the most important parameters for the HepG2 cell viability irradiated by therapeutic US in vitro.Chapter 2:Evaluation of cell viability of HepG2 cell after ultrasound-targeted microbubble destruction:an in vitro studyPurpose:To evaluate the viability of HepG2 cell after ultrasound (US)-targeted microbubble (MB) destruction (UTMD).Methods:Ninety-six well cell culture plate was divided into 6 areas (4 wells each area), HepG2 cells were cultured in 5% CO2 at 37?. After 12 hours' culture, the cells were irradiated by US from the bottom of the cell culture plate. The overall protocol was the orthogonal design with 4 factors and 4 levels(US power:0.5 W/cm2,0.75 W/cm2,1.0 W/cm2,1.25 W/cm2, duty cycle:10%,20%,40%,60%; duration:5 s,10 s, 20 s,40 s; concentration of the SonoVue MB (v/v):5%,10%,20%,30%). After UTMD, the cells were cultured for another 24 hours, then the shape, distribution and growth of cells were observed by optical microscope, and MTT essays were performed to assess the cell viability.Results:When the acoustic intensity was at 0.5?0.75 W/cm2 and other factors were at any levels, no significant change was found in the morphology of cells. The concave surface could be observed on the cells when the cells were exposed to UTMD with 1 W/cm2 acoustic intensity,40% duty cycle,30% (v/v) MB and 40 s duration. Part of cells detached from the plate when the acoustic intensity, the duty cycle, the duration and the MB concentration were at 1.0 W/cm2,60%,40s and 30%, respectively. The areas of the detached cells were enlarged when the acoustic intensity was up to 1.25 W/cm2. The statistical analysis showed that the acoustic intensity, the duty cycle and the MB concentration were the main factors that influenced the cell viability. The cell viability could be up to 90% when the acoustic intensity, the duty cycle, the duration and MB concentration were no more than 1.0 W/cm2,20%,20s and 30%, respectively.Conclusion:The acoustic intensity, the cycle duty and the MB concentration were the main factors influencing the viability of HepG2 cell exposed under UTMD.Chapter 3:Enhancement of gene transfection of HepG2 cells combining ultrasound-targeted microbubble destruction with polyethylenimine:an in vitro studyPurpose:To investigate the effect of gene transfection of HepG2 cells combining ultrasound (US)-targeted microbubble (MB) destruction (UTMD) with polyethylenimine (PEI).Methods:MB concentration and duration were selected as influencing factors (4 levels each) for investigating the efficiency of gene transfection according to factorial design. GFP reporter plasmid was transferred to HepG2 cell line by combination of UTMD and PEI at varied levels of MB concentration (10%,20%,30% and 40%) and duration (10,20,30,40 s). The acoustic intensity and duty cycle were set at 1.0 W/cm2 and 20% respectively. Forty-eight hours after transfection with UTMD, the cells were harvested to measured the efficiency of GFP transfection and cell viability with flow cytometry. Then the optimized parameters were used to compare the efficiency of gene transfection and cell viability by six different methods:(1) plasmid only, (2) plasmid plus US, (3) plasmid plus US and MB, (4) plasmid plus PEI, (5) plasmid plus PEI and US, (6) plasmid plus PEI, US and MB.Results:The maximal efficiency of gene transfection (39.5%±5.8%) was acquired when the MB concentration and the duration were set at 30% and 20 seconds, and the cell viability could be up to 91%±2.7%. The efficiency of GFP transfection of plasmid only, Plasmid+US, Plasmid+US+MB, plasmid+PEI, plasmid+PEI+US and plasmid+PEI+US+MB were 0.08%±0.01%,1.7%±0.71%,8.36%±3.62%, 25.63±4.84%,29.41±4.91% and 39.5±5.8%, respectively. There was significant difference among groups (P<0.05). The cell viability in each group was more than 90%, and had no significant difference in statistics (P>0.05).Conclusion:The combination of UTMD and PEI could increase the efficiency of gene transfection with higher cell viability.Chapter 4:HepG2 cell apoptosis induced by survivin small interfering RNA plasmid transfection using ultrasound-targeted microbubble destruction combined with polyethylenimine:an in vitro studyPurpose:To investigate whether survivin small interfering RNA (siRNA) plasmid transfection could better induce HepG2 cell apoptosis by combining ultrasound (US)-targeted microbubble (MB) destruction (UTMD) with polyethylenimine (PEI).Methods:Two survivin siRNA plasmids (psiRNA-S-55 and siRNA-S-387) were constructed with pRNAT-U6.1/Neo plasmid according to the principle of siRNA design. The recombinant siRNA plasmids were delivered into HepG2 cells by combining UTMD with PEI. Expression of survivin mRNA and protein were assessed by reverse transcription-polymerse chain reaction (RT-PCR) and western blotting. Apoptosis ratio marked with annexin V-PE and 7-AAD was quantified by flow cytometry.Results:The recombinant siRNA plasmids were constructed successfully and confirmed by DNA sequencing. Forty-eight hours after transfection, the expression of survivin mRNA and protein in psiRNA-S-55 and psiRNA-S-387 group was significantly lower than that of untreated control group and negative plasmid group (P<0.05). The cell apoptosis ratio in psiRNA-S-55 group and psiRNA-S-387 group was significantly higher than that of untreated control group and negative plasmid group (P<0.05). The siRNA-S-387 group had the lowest level of survivin expression and the highest cell apoptosis ratio (P<0.05).Conclusion:Recombinant expression plasmid of siRNA targeting survivin gene combining UTMD with PEI technique could effectively inhibit the expression of target gene and induce HepG2 cell apoptosis.Chapter 5:Enhancement of gene transfection to muscle cell combining ultrasound-targeted microbubble destruction with polyethyleneimine:An in vivo studyPurpose:To investigate the effect of gene transfection to mice's muscle combining polyethyleneimine (PEI) with ultrasound (US)-targeted microbubble (MB) destruction (UTMD).Methods:After optimizing the parameters of US exposion in mice in vivo, the acoustic intensity and duty cycle were set at 2.0 W/cm2 and 50% respectively to perform the gene transfection. Twenty-five female Balb/c mice were divided into 5 groups (5 mice per group) randomly. Plasmid DNA (20?g) encoding green fluorescent protein (GFP) was used as expresion vector. The transfection materials were injected into the bilateral tibialis anterior muscles of mice respectively and delivered as follows:(a) untreated control, (b) plasmid+US, (c) plasmid+US+MB, (d) plasmid+PEI, (e) plasmid+PEI+US, (f) plasmid+PEI+MB+US. SonoVue MB was used at a final concentration of 30% (v/v). Transfection efficiency was assessed by counting the number of GFP-positive fibers under fluorescence microscopy 10 days after gene delivery. Hematoxylin-eosin staining was performed on separate specimen and examined with light microscopy for tissue damage.Results:No GFP-positive fiber was found in the untreated control group. There were 14±3.3 58±6.2,96±7.1,119±10.5 and 158±18.4 GFP-positive fibers respectively found in plasmid+US, plasmid+MB+US, plasmid+PEI, plasmid+PEI+US, and plasmid+PEI+MB+US groups. The plasmid+PEI+MB+US group had the maximal GFP gene delivery, the differences were significant compared with other groups (P<0.05). No evidence of inflammation or necrosis was revealed in histological examination with hematoxylin-eosin staining.Conclusions:PEI combining with UTMD can significantly improve efficiency of gene transfection in vivo and has great potential in gene therapy. Chapter 6:Induction of tumor apoptosis by survivin small interfering plasmid transfection combining ultrasound-targeted microbubble destruction with polyethyleniminePurpose:To induce tumor apoptosis by survivin small interfering RNA plasmid transfection combining polyethylenimine (PEI) with ultrasound (US)-targeted microbubble (MB) destruction (UTMD).Methods:Twenty-four nude mice with subcutaneous tumors induced by inoculation of HepG2 cells were divided randomly into four groups (6 mice per group) as follows: (1) untreated control, (2) negative psiRNA-N, (3) psiRNA-S-387 plus UTMD, (4) psiRNA-S-387 plus PEI and UTMD. When the diameters of tumors were up to 0.5 cm, the recombinant psiRNA-S-387 plasmids were delivered into the tumors as described previously. The acoustic intensity and duty cycle were set at 2.0 W/cm2 and 50% respectively. The duration was 2 minutes. SonoVue MB was used and at a final concentration of 30% (v/v). Thirty-one day after the first gene transfection, the nude mice were euthanized. Expressions of survivin mRNA and protein were assessed by reverse transcription-polymerse chain reaction (RT-PCR) and western blotting. Apoptosis ratio was tested by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL).Results:The size of tumors of the untreated control group and the negative psiRNA-N group were significantly bigger than those of the psiRNA-S-387 plus UTMD group and the psiRNA-S-387 plus PEI and UTMD group (P<0.05). There was no significant difference between the untreated control group and the negative psiRNA-N group. However, the sizes of tumors of the psiRNA-S-387 plus PEI and UTMD group were significantly decreased than those of other groups. The expression of survivin mRNA and protein in the psiRNA-S-387 plus UTMD group and the psiRNA-S-387 plus PEI and UTMD group were significantly lower than those of untreated control group and negative plasmid group (P<0.05). The cell apoptosis ratio in the psiRNA-S-387 plus UTMD group and the psiRNA-S-387 plus PEI and UTMD group were significantly higher than those of untreated control group and negative plasmid group (P<0.05). The psiRNA-S-387 plus PEI and UTMD group had the lowest level of survivin expression and highest cell apoptosis ratio (P<0.05).Conclusion:Recombinant expression plasmid of siRNA targeting survivin gene mediated by combining UTMD with PEI technique could effectively inhibit the expression of target gene and induce HepG2 cell apoptosis.PART II Experimental study on enhancement of antibiotic activity against biofilm of Staphylococcus epidermidis using ultrasound-targeted microbubble destructionChapter 7:Influence of ultrasound-targeted microbubble destruction on biofilm.Purpose:To investigate whether ultrasound (US)-targeted microbubble (MB) destruction (UTMD) can influence the biofilm and bacteria in morphology.Methods:Twenty-hour biofilms of Staphylococcus epidermidis RP62A were treated by the following 3 experimental conditions with and without microbubbles:(a) untreated control, (b) acoustic intensity at 0.5 W/cm, (c) acoustic intensity at 1.0 W/cm, (d) acoustic intensity at 1.5 W/cm2. The duty cycle was 50% and the duration was 10 minutes. After treatment, the absorbance values (A570) of biofilms stained with the crystal violet were measured to assess the biofilm density. The biofilms were observed with macroscopy and light microscopy. In addition, the biofilms were treated with following 3 conditions:a) untreated control, b) ultrasound alone and c) UTMD. The acoustic intensity, duty cycle and duration were 0.5 W/cm2,50% and 30 minutes, respectively. After treatment, the biofilms were examined by confocal laser-scanning microscopy (CLSM) and scanning electron microscopy (SEM).Results:A thick and compact biofilm was observed in the untreated control group, and there were no obvious micropores in biofilms under macrology and light micrology. Although there were no significant changes under macroscopy in both biofilms treated with US only and UTMD with 0.5 W/cm2 acoustic intensity, interestingly, many micropores could be found under microscopy. The diameters of micropores increased with increasing acoustic intensity, and the micropores in biofilms treated with UTMD were bigger than those treated with US-only in the same condition of acoustic intensity (P<0.05). The micropores in the biofilms treated with US and UTMD of 1.0-1.5 W/cm2 acoustic intensity could be observed under macroscopy. The largest diameters of micropores were up to 1 mm in biofilms treated with UTMD using 1.5 W/cm2 (P<0.05). The biofilm density (A570 value) decreased with increasing of the acoustic intensity, and the values in UTMD group of 1.5 W/cm2 were the lowest (P<0.05). Micropores also could be observed under CLSM, however, there were no obvious dead bacteria in biofilms treated with US and UTMD compared with untreated control group (P>0.05). Under SEM, the shape of bacteria in biofilms treated with US and UTMD became irregular, and many rounded projection could be observed in the surface of the bacteria treated with UTMD.Conclusion:US and UTMD can produce micropores in biofilms, which might help to promote antibiotic activity against biofilms.Chapter 8:Enhancement of vancomycin activity in biofilm using ultrasound-targeted microbubble destructionPurpose:To investigated whether ultrasound (US)-targeted microbubble (MB) destruction (UTMD) can enhance antibiotic action and reduce viable bacteria in biofilm.Methods:Twelve-hour biofilms of S. epidermidis RP62A were treated under the following 6 experimental conditions:a) untreated control, b) Vancomycin, c) US only, d) Vancomycin+US, e) MB+US, f) Vancomycin+MB+US. US irradiation (80 kHz, 1.0 W/cm2,50% duty cycle) was conducted from the bottom of 96-well planes for 10 minutes. The vancomycin and MB were used respectively at concentrations of 100?g/ml and of 30% (v/v). After the treatments, the biofilms were cultured for another 12h at 37?, then viable log10CFU among different groups were compared and absorbance values (A570) of biofilms stained with the crystal violet were measured to assess the density of biofilms. The biofilms were observed under macroscopy and light microscopy and the morphology of bilfilms were also examined and analyzed by confocal laser-scanning microscopy (CLSM).Results:The control group and the vancomycin group were found to form a thick and compact biofilm with maximum A570 values, and it was also observed that many micropores were in the biofilms of US-alone group and vancomycin+US group with smaller A570 values (P<0.01) as compared with the untreated control group and the vancomycin group. Even bigger micropores could be found in the biofilms of US+MB group and vancomycin+US+MB group with even smaller A570 values (P<0.01). The viable log10CFU counts of S. epidermidis recovered from the biofilms in the vancomycin+MB+US group decreased significantly (P<0.01) compared with other groups. The contrastive results of viable log10CFU obtained in the planktonic cells were similar to the results in the biofilm. The CLSM examination and quantitative analyses confirmed that the highest proportions of dead cells were found in the vancomycin+MB+US group.Conclusions:UTMD might have a great potential in increasing antibiotic activity and reducing viable bacteria in biofilm.Chapter 9:Enhancement of vancomycin activity against biofilm combining with ultrasound-targeted microbubble destruction in rabbits'modelPurpose:To investigate the enhancing effect of vancomycin against biofilm infection using ultrasound (US)-targeted microbubble (MB) destruction (UTMD) in rabbit.Methods:Biofilm-infected polyethylene disks were implanted subcutaneously in four New Zealand White rabbits, bilateral to the spine. The four rabbits were treated as follows:(a) untreated control, (b) vancomycin, (c) vancomycin plus US, (d) Vancomycin+US+MB. Vancomycin (50 mg/kg) was administered through a marginal vein in the appropriate rabbits'ears immediately after surgery and 3 times a day thereafter. Twenty-four hours after surgery, US exposure or UTMD were performed for 20 min immediately following the administration of vancomycin,3 times a day. SonoVue MB (200?L) was injected subcutaneously into the area of the disks at 5 minute intervals for a total 800?L per treatment. The acoustic intensity was set at 0.5 W/cm2 and the duty cycle 50%. The transducer was placed on the skin over the implanted biofilm-infected disks using an acoustically conductive gel adhesive. Three days after treatment, the rabbits were euthanized and the implants were removed. Biofilms were scraped from the disks and the viable bacteria were determined by CFU counting.Results:The log10 numbers of viable CFU from biofilms of the untreated control group, vancomycin group, vancomycin plus US group and vancomycin plus US and MB group were 5.18±0.25,5.15±0.20,4.68±0.18 and 3.75±0.19, respectively. There was no significant difference between the untreated control group and the vancomycin group (P>0.05). However, the log10 numbers of viable CFU decreased significantly in biofilms treated with vancomycin plus US or vancomycin plus US and MB (P< 0.05). The vancomycin plus US and MB group contained the smallest log10 number of CFU (P<0.05) compared with the other groups. Conclusion:UTMD can significantly increase the vancomycin action against biofilm-related infection in vivo.