Ultrasound-mediated Long-circulating Nanopolymer Delivery Of SIK2 Sirna And Anti-miR21 Leads To Enhanced Paclitaxel Sensitivity In Epithelial Ovarian Cancert Chemotherapy

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SYNTHESIS AND CHARACTERIZATION OFFOLATE-TARGETED LONG-CIRCULATINGNANOPOLYMERObjects To synthesize a SIK2 si RNA/antisense-micro RNA21 loaded lipid-glycolic acid poly(lactic acid)(PLGA)polymer nanoparticles(Fa LPHNP @ si SIK2 / anti-mi R21),characterize their physical and chemical properties,stability,and RNAs loading ability and RNAs release profle in vitro.Methods Folate targeting lipid-poly(lactic acid glycolic acid)nanocomplex(Fa LPHNP)was prepared and its physicochemical properties were identified.Folic acid targeted nanopolymer Fa LPNP(NC-NPs,without RNAs),untargeted nanopolymer(LPHNP),and targeted nanopolymer containing two nucleic acids: SIK2 si RNA and anti-mi R21(si SIK2 /antimi R21)were prepared by optimized two-step self-assembly method.At the same time,the physical and chemical properties of three kinds of nanopolymers were tested,including morphology,particle size and potential.At room temperature,the morphology,particle size and potential of nanoparticles were observed at different time points in PBS,which indicating their stability.RNA loading efficiency and its sustained release profile was measured.Using agarose gel electrophoresis,Fa LPHNP was evaluated to protect RNA from rapid degradation by RNA enzymes in the presence of RNA enzymes.The self-assembled nanopolymer was a milky white solution.Scanning Electron Microscope(SEM)observed that the nanopolymer had a spherical structure,uniform size and good dispersion.The nanoparticles were observed by Transmission Electron Microscope(TEM)with a double lipid shell.Fa LPNP @si SIK2 /anti-mi R21 average particle size of 169.25 ± 21 nm and average zeta potential of 5.28 ± 3 m V were measured by Marvin.After being dissolved in PBS at 4°C for 15 days,the particle size and potential of Fa LPHNP@si SIK2 /anti-mi R21 did not change significantly,which proved that the nanoparticles had better stability.Agarose gel electrophoresis showed that in the presence of RNA enzyme(RNase),Fa LPHNP can prevent RNA from being rapidly degraded by RNA enzyme.The RNAs loading efficiency was measured at the encapsulation rate of si SIK2/ anti-mir21 :82.33 ± 4/81.27 ± 7%.In vitro drug release shows a sustained release profile.Conclusion: the Fa LPHNP@si SIK2 / anti-mi R21 polymer nanoparticle was prepared by the improved two-step self-assembly method in this study,with regular morphology,uniform size distribution,stable in vitro properties,loaded with SIK2 si RNA and anti-mi R21,with high RNAs loading efficiency and in vitro sustained release capacity.mi RNA,sustained release.PART ? EVALUATION OF THE THERAPEUTIC EFFECT OF SIK2 SIRNA AND ANTI-MIR21 COMPOSITE NANOPARTICLES ON OVARIAN CELL TARGETING UNDER ULTRASOUND-MICROVESICLE MEDIATION(IN VITRO ASSAY)Purpose To observe the targeting of Fa LPHNP in three epithelial ovarian cancer cells: OVCAR3,SKOV3 and A2780,and the effects of Fa LPHNP@si SIK2/anti-mi R21 combined with paclitaxel(PTX)on ovarian cancer cell viability,apoptosis and migration under ultrasound-microbubble mediation.Methods Folic acid modified nanoparticles(Fa LPHNP)and non-folic acid modified nanoparticles(LPHNP)were first prepared.Confocal laser scanning microscopy(CLSM)was used to observe the co-localization of nanoparticles containing Cy3-labeled si RNA with DAPI-stained epithelial ovarian cancer cells(OVCAR3,SKOV3 and A2780),respectively,while flow cytometry was used to examine the transfection efficiency of each experimental group: a).ultrasound-microbubble-mediated transfection of Fa LPHNP to EOC cells;b).transfection of Fa LPHNP to EOC cells without ultrasound-microbubble;c).transfection of non-targeted LPHNP to EOC cells.The CCK8 method was used to observe changes in cell viability after co-incubation of Fa LPHNP with OVCAR3,SKOV3 and A2780 cells at different concentrations of SIK2 si RNA(5n M,10 n M,20 n M,50 n M,100 n M,150 n M)and in the presence of anti-mi R21.and optimize the SIK2 si RNA treatment concentration according to this trial.Further validation by the CCK8 method was performed on OVCAR3,SKOV3,and A2780 cells by co-incubation with combined Fa LPNP@si SIK2/anti-mi R21 and paclitaxel under ultrasound-microbubble mediation to observe the effect on cell viability for 48 hours.The changes in SIK2 m RNA and mi R21 expression levels in each cell were also observed using rt PCR.As well as changes in the expression levels of SIK2 protein in OVCAR3,SKOV3 and A2780 cells treated as described above,and the downstream protein PDCD4,which is inhibited by mi R21,were observed by Western Blot test.The effects of the above experiments on the migratory properties of three epithelial ovarian cancer cells were observed by cell wound healing assays,and the effects of ultrasound-microbubble mediated Fa LPHNP@si SIK2/anti-mi R21 nanoparticle combined PTX treatment on the apoptosis of ovarian cancer cells by flow cytometry.Results Laser confocal scanning microscopy(CLSM)was observed for a).si RNA nanoparticles containing the same concentration of red fluorescent Cy3-labeled(Fa LPHNP@Cy3-si RNA)in the ultrasound-microvesiclemediated transfection group,compared to b).Non-Ultrasound-microvesiclemediated(Fa LPHNP@Cy3-si RNA)group as well as c).The non-targeted(LPHNP@Cy3-si RNA)group had more red fluorescence localized in the perinuclear cytoplasm compared to the a).The transfection efficiency was significantly higher(P < 0.01)than that of the other experimental groups,as measured by flow cytometry.A concentration-dependent decrease in OVCAR3,SKOV3 and A2780 cell activity was observed after co-incubation of si RNA containing different treatment concentrations of SIK2 with Fa LPHNP.Following the synthesis of Fa LPHNP@si SIK2/anti-mi R21 by optimizing the potent therapeutic concentration of SIK2 si RNA+anti-mi R21,a significant reduction in the activity of ultrasound-microbubble mediated Fa LPHNP@si SIK2/anti-mi R21 combined PTX-treated OVCAR3,SKOV3,and A2780 cells was observed compared to other experimental groups(P < 0.01).Ultrasound-microbubble-mediated reduction of Fa LPHNP@si SIK2/anti-mi R21 in OVCAR3,SKOV3 and A2780 cells was observed by rt PCR assay 48 hours after transfection,which was most significant(P < 0.01)for endogenous SIK2 m RNA and endogenous mi R21 levels in these three cells.Meanwhile,it was observed by Western Blotting test that 48 hours after transfection with Fa LPHNP@si SIK2/anti-mi R21 mediated by ultrasound-microbubble,the levels of SIK2 protein in OVCAR3,SKOV3 and A2780 cells were significantly reduced and the expression level of PDCD4,a downstream protein inhibited by mi R21,was significantly increased(P < 0.01).The results of the cell scratch test(cell wound healing test)showed that the smallest area of cell scratch healing(P < 0.01)was observed from 0 to 48 hours in the experimental group transfected with ultrasound-microbubble mediated Fa LPHNP@si SIK2/anti-mi R21 cells in combination with PTX,while the most significant early apoptosis(P < 0.01)was observed in the experimental group transfected with ultrasoundmicrobubble-mediated Fa LPHNP@si SIK2/anti-mi R21 cells in combination with PTX.Conclusion The prepared Fa LPHNP@si SIK2/anti-mi R21 nanoparticles have the ability to actively target ovarian cancer cells(OVCAR3,SKOV3 and A2780)under ultrasound-microbubble-mediated targeting of RNA-like drugs to ovarian cancer cells.It also has a significant interfering effect on the level of target genes and the synthesis of corresponding proteins in ovarian cancer cells.Compared with the PTX-treated cell group alone,the ultrasound-microbubble mediated Fa LPHNP@si SIK2/anti-mi R21 combined paclitaxel(PTX)-treated group significantly reduced the migration and apoptosis of the above three epithelial ovarian cancers,and preliminarily realized the sensitizing effect of this RNA drug delivery platform on the chemotherapy drug paclitaxel(PTX)on OVCAR3,SKOV3 and A2780 in ovarian cancer cells.PART ? ULTRASOUND-MICROBUBBLE-MEDIATEDDISTRIBUTION,BIOSAFETY,TARGETING ANDTHERAPEUTIC EFFICACY OF SIK2 SIRNA/ANTI-MIR21-LOADED FALPHNP NANOPARTICLES INHORMONE MICEObjective To observe the therapeutic effects of ultrasound-microbubblemediated Fa LPHNP@si SIK2/anti-mi R21 in vivo delivery targeting efficiency,in vivo biodistribution,half-life in blood,biocompatibility and combined PTX on OVCAR3 hormone mice.Methods Immunogenicity of the nanoparticle was first evaluated by using human peripheral blood mononuclear cells(PBMC).After co-incubated with different concentrations of Fa LPHNPs(50n M,200 n M)and Fa LPHNP@si SIK2/anti-mi R21(50n M,200 n M)for 24 h.The cultural supernatant was collected and levels of cytokine IL-6 and TNF-? were evaluated by ELISA.Lipopolysaccharide(LPS)-treated PBMC cell group served as positive control.To in vestigate the biocompatibility of nanoparticles in vivo,different concentrations of nanoparticles(Fa LPHNP @si SIK2/anti-mi R21): 5mg/ml,10mg/ml were injected through the tail vein of Balb/c mice,respectively;liver and kidney tissues were collected for H&E staining on days 1,4 and 7 post injection;and blood was collected for CK,LDH-L(heart function),CRE,BUN(kidney function)and AST,ALT,TBi L(liver function),and cytokines IL-6,TNF-? on day 7,respectively.To verify the distribution of ultrasound microbubble-mediated nanoparticles in vivo and the accumulation of tumor site.We first prepared folic acid-modified Di R-tagged targeted nanoparticles(Fa LPHNP @si SIK2/anti-mi R21)and non-folic acid-modified nanoparticles(LPHNP @si SIK2/anti-mi R21).Balb/c-OVCAR3 mouse model were established subcutaneously,when tumors volume reached approximately 100 mm3,mice were injected separately via the tail vein with: a).Di RFa LPHNP@si SIK2/anti-mi R2 + Sono VueTM microbubbles,subcutaneous tumor ultrasonography presentation using a diagnostic ultrasound transducer at the tumor site to detect local sensitivity of the Sono VueTM microbubble,followed by irradiation of the tumor site with a therapeutic ultrasound probe for targeted destruction of lipid microbubbles;b).No ultrasoundmicrobbuble-mediated Fa LPHN@si SIK2/anti-mi R2;c).LPHN@si SIK2/anti-mi R2.The above three groups of Balb/c nude mice were injected by tail vein 24 hr and 48 hr,respectively,through Xenogen IVIS bioluminescence imaging system to analyze the fluorescence signal intensity of tumor and other organs.24 h later,some mice were killed,and the tumor,heart,liver,kidney,spleen and lung tissues were collected by Xenogen IVIS bioluminescence imaging system to analyze the fluorescence intensity of tumor and other important organs.To further verify the specific accumulation of nanoparticles at the tumor site,mice were divided into four groups and injected via tail vein with 1)ultrasound-microbubble-mediated Fa LPHNP@Cy3-si RNA;2)Fa LPHNP@Cy3-si RNA;3)LPHNP@Cy3-si RNA and 4)PBS as negative control.Mice were sacrificed 24 hours after treatment,and frozen sections of tumor tissue were taken and analyzed by fluorescence microscopy for red fluorescence intensity of tumor tissue from different experimental groups.To analyze the in vivo kinetic of nanoparticles,we compared Cy3-labeled si RNA nanoparticles(Fa LPHNP @Cy3-si RNA)and naked Cy3-labeled si RNA in mice after tail vein injection.Blood was withdrawn at different time points to detect Cy3 fluorescence using Bio Tek multifunctional microplate reader.Half-time period(t1/2)was calculated.To evaluate the efficacy of complementary delivery of Fa LPHNP @si SIK2/anti-mi R21 in the OVCAR3-xenograft mouse model.Firstly,targeted nanoparticle Fa LPHNP @si SIK2/anti-mi R21 and non-targeted nanoparticle Fa LPHNP @si SIK2/anti-mi R21 were prepared.OVCAR3-Balb/c-nu mouse model were established,when the tumor reached 75mm3.Mice were randomly divided into seven groups: 1.Control(saline);2.paclitaxel alone;3.LPHNP @si SIK2/anti-mi R21;4.LPHNP @si SIK2/anti-mi R21 combined with ultrasound-microbubble(US-MB)-mediated delivery;5.Fa LPHNP @si SIK2/anti-mi R21 alone;6.LPHNP @si SIK2/anti-mi R21 combined with ultrasound-microbubble(US-MB)-mediated delivery + paclitaxel.Group-1,2,3,and 5 were injected with saline,paclitaxel,non-targeted nanoparticles,and targeted nanoparticles in the tail vein of mice,respectively;groups 4,6,and 7 were injected with 200 ul Sono VueTM microbubble mixture.The ultrasound parameter used in the targeted destruction of microbubbles was: 1 MHz,1.8 W/cm2,50% duty cycle,duration = 10 s.The mice were monitored for body weight,tumor size,survival during the treatment.Mice were sacrificed at the end of treatment,and tumor tissues were taken for TUNEL and PCNA immunofluorescence staining to detect tumor cell proliferation and apoptosis.Results Human peripheral blood mononuclear cells were co-incubated with different concentrations of Fa LPHNP and Fa LPHNP@si SIK2/antimi R21 nanoparticles for 24 hours and cell cultures were collected for cytokine assay and found that the impact of concentration of 50 n M nanoparticles on IL-6,TNF-? was negligible and high concentration of nanoparticles(200 n M)caused mild-moderate IL-6,TNF-? elevation,but still significantly lower than positive control LPS-induced sharp IL-6 and TNF-? elevation(p < 0.001).H&E staining of liver and kidney tissues from mice taken on days 1,4,and 7 were not found to abnormal when different concentrations of Fa LPHNP @si SIK2/anti-mi R21(5mg/ml,10mg/ml)were injected into the tail veins of Balb/c mice.Which demonstrated a biological safety of this treatment.Also,there were no abnormalities found in the blood biochemistry assay of CK,LDH-L,CRE,BUN,and AST,ALT,and TBi L.For the in vivo nanoparticle kinetics assay.Naked Cy3-si RNA was almost undetectable in blood collected 30 min,whereas the t1/2 of the Fa LPHNP@Cy3-si RNA was ~8.5 h.The nanoparticle was shown to have the capability to protect nucleic acid from rapid degradation in the blood,providing the basis for gene therapy.In in vivo targeting experiments,the fluorescence intensity at the tumor site was observed to increase over time by ultrasound-mediated sonoporation,enhanced permeability and retention(EPR),and active targeting in the USMB-mediated Fa LPHNP@si SIK2/anti-mi R21 group.It peaked 24 hours after administration and then tapered off.The fluorescence signal was significantly lower in the non-US-MB-mediated Fa LPHNP @si SIK2/antimi R21 group nor non-targeted nanoparticle(LPHNP@si SIK2/anti-mi R21)group,(p<0.05).In the in vivo treatment efficacy evaluation.The US-MB-mediated targeted nanoparticle Fa LPHNP@si SIK2/anti-mi R21 combined PTX treatment group received complete inhibition of tumor growth and the total survival of mice in this treatment group exceeded 58 days.The PTX alone group,LPHNP@si SIK2/anti-mi R21 group had minimal tumor suppression;US-MB-mediated targeted nanoparticle Fa LPHNP@si SIK2/anti-mi R21 group,US-MB-mediated non-targeted nanoparticle LPHNP@si SIK2/anti-mi R21,non-US-MB targeted nanoparticle Fa LPHNP@si SIK2/anti-mi R21 group had a moderate tumor growth inhibition.TUNEL immunofluorescence staining of tumor tissues showed the highest fluorescence of apoptosis in the PTX + US-MB mediated Fa LPHNP@si SIK2/anti-mi R21 group.While the lowest fluorescence of PCNA in this group,which indicated tumor cell proliferation was significantly inhibited.The average survival of mice in the conrol group was 27 days;the average survival of PTX group and non-targeted nanoparticle group was 32 days and 29 days,respectively;the average survival of USMB-mediated targeted and non-targeted nanoparticle treatment group mice was 46 days and 35 days,respectively.Conclusion US-MB mediated in vivo delivery of Fa LPHNP@si SIK2/anti-mi R21 was improved by ultrasound-microbubble sonoporation,EPR effect,as well as active targeting of folic acid.Specific accumulation of the nanoparticle at the tumor site promotes the release of nucleic acid drugs.US-MB-mediated complementary delivery of Fa LPHNP@si SIK2/anti-mi R21 in vivo enhanced the sensitivity of PTX.This treatment has been shown to have good biological safety in mice.