Targeted CRISPR/Cas9-loaded Lipid-polymer Hybrid Nanoparticles Combined With Focused Ultrasound For Gene Therapy For TMZ-resistant Glioblastoma

Part I Preparation and characterization of CRISPR/Cas9-loaded lipid-polymer hybrid nanoparticlesObjective1.To prepare the gene-loaded lipid-polymer hybrid nanoparticles(LPHNs_pCas9/MGMT-cRGD)and test the characterization of nanoparticles;2.To test the structural integrity of pCas9 in nanoparticles and the protective effect of LPHNs-cRGD from DNase I.Method1.Constructed three pairs of dual-g RNA/Cas9 plasmids targeting the MGMT gene,plasmids with effective editing efficiency were selected for the subsequent experiments.2.LPHNs-cRGD were prepared via nanoprecipitation method,the particles size and zeta potential were determined using the dynamic light scattering method.The encapsulation efficiency and release curve of the nanoparticles were measured.Fluorescence microscope and transmission electron microscope(TEM)were used to observe the morphology and distribution of nanoparticles,3.The structural integrity of the plasmid in the nanoparticles and the protective effect of LPHNs-cRGD from DNase I were detected by agarose gel electrophoresis.Result1.LPHNs_pCas9/MGMT-cRGD were successfully prepared.The particles size and zeta potential of optimized nanoparticles were 179.6±44.82nm,29.6±4.33 m V,respectively.The encapsulation efficiency was 76.5±7.2%,the release curve showed a slow and controlled release.Fluorescence microscope and TEM showed that the LPHNs-cRGD had uniform spherical shapes and typical core-shell structures;2.The structure of the pCas9 in the nanoparticles remained intact,and LPHNs-cRGD could protect the pCas9 from DNase I degradation.ConclusionGene-loadedlipid-polymerhybridnanoparticles(LPHNs_pCas9/MGMT-cRGD)were successfully prepared.The nanoparticles could encapsulate the pCas9 and protect it from DNase I degradation.Part II Effect of gene-loaded LPHNs-cRGD on TMZ-resistant glioblastoma cells in vitroObjective1.To screened out GBM cells with high expression of MGMT and observe the potential cytotoxicity of hybrid nanoparticles on GBM cells in vitro;2.To confirm that LPHNs_pCas9/MGMT-cRGD nanoparticles have active targeting effect,and to prove that it can mediate the gene transfection on GBM cells;3.To observe the gene editing effects of LPHNs_pCas9/MGMT-cRGD in vitro and whether it can increase the sensitivity of GBM cells to TMZ.Method1.WB assay was used to selected out GBM cells with high MGMT expression.And CCK8 assay was used to detect the cell viability of GBM cells after treated with different concentrations of nanoparticles;2.Di I-labeled LPHNs and LPHNs-cRGD were prepared.T98G cells were divided into groups(control,LPHNs,LPHNs-cRGD),and fluorescence microscope and flow cytometry were used to observe the distribution of nanoparticles in cells;3.Fluorescence microscope and flow cytometry were used to observe the transfection efficiency under different transfection conditions(nude pCas9?LPHNs_pCas9/MGMT,LPHNs_pCas9/MGMT-cRGD and Lip3000+pCas9);4.WB assay was used to detect the expression of MGMT in different groups,and CCK8 assay and flow cytometry were used to detect the cell viability and apoptosis rate after different treatments(control group,TMZ group,LPHNs_pCas9/MGMT-cRGD group,LPHNs_pCas9/MGMT+TMZ group,LPHNs_pCas9/MGMT-cRGD+TMZ group);Result1.The expression of MGMT protein in T98G and U251-R cells was higher than that in other cell lines.With different concentrations of nanoparticles,the cell viability remained above 80%,indicating that nanoparticles had little effect on cell viability and had no obvious cytotoxicity;2.In the LPHNs-cRGD group,the intracellular nanoparticle concentration was significantly higher than the other groups,indicating that LPHNs-cRGD had active targeting ability;3.LPHNs_pCas9/MGMT-cRGD could transfected GBM cells successfully,48h after transfection,the transfection efficiency of the LPHNs-cRGD group was 36.39%in T98G cells,which was slightly higher than other groups;4.Compared with other groups,LPHNs_pCas9/MGMT-cRGD could down-regulate the expression of MGMT,and enhance the treatment effects of TMZ on glioblastoma cells,inducing apoptosis and inhibition of cell viability.Among the T98G and U251-R cells,LPHNs_pCas9/MGMT-cRGD+TMZ treatment showed the lowest cell viability and the highest apoptosis rate,followed by the LPHNs_pCas9/MGMT+TMZ group.The cell viability and apoptosis rate in the other groups showed no significant difference compared with the control group.ConclusionLPHNs_pCas9/MGMT-cRGD showed no obvious cytotoxicity on GBM cells,and could promote gene transfection in GBM cells with an active targeting ability,LPHNs_pCas9/MGMT-cRGD could reduce the expression of MGMT protein,and enhance the sensitivity of GBM cells to TMZ,promoted cell apoptosis and inhibited cell viability eventually.Part III gene-loaded LPHNs-cRGD combined with Ultrasound-Microbubbles for BBB-opening and inhibition glioblastoma growth in vivo.Objective1.Topreparemicrobubble-nanoparticlecomplexes(MBs-LPHNs-cRGD),test the characterization,and confirm the irradiation times and amounts of MBs-LPHNs-cRGD when combined with focused ultrasound(FUS)to open the blood-brain barrier(BBB);2.To clarify the biosafety and the active targeting ability of MBs-LPHNs-cRGD when combined with FUS to open BBB in orthotopic xenograft glioblastoma mice;3.Toobservethetherapeuticeffectof FUS+MBs-LPHNs_pCas9/MGMT-cRGD complexes combined with TMZ in vivo.Method1.Lipid microbubbles were prepared via lipid film hydration method,and MBs-LPHNs-cRGD complexes was obtained by connecting the microbubbles with hybrid nanoparticles by biotin-avidin linkage,and the characterization of the complexes was tested.The BBB-opening experiment was carried out with different amounts of MBs-LPHNs-cRGD(2 x 10⁶,4 x 10⁶,8 x10⁶)and FUS irradiation time(3 min,5 min)before injecting EB dye intravenously and assessed the BBB-opening by observing the EB dye permeation.HE staining were used to observe the damage of brain.2.Mice were injected with MBs-LPHNs-cRGD or normal saline,and their survival situation was observed.One week later,HE staining of the main organs were used to evaluate the biosafety.The orthotopic xenograft T98G GBM model was established in NOD-SCID mice.The tumor-bearing mice were divided into different groups(LPHNs-cRGD,MBs-LPHNs-cRGD,FUS+MBs-LPHNs,FUS+MBs-LPHNs-cRGD,LPHNs-cRGD were labeled by Di R),images were acquired at 2h and 24 h after treatment using a fluorescence imaging system.Then brains and major organs were collected for ex vivo imaging.3.Tumor-bearing mice were divided into five groups:Group 1 were injected saline as control group;Group 2 were injected TMZ alone;Group3 were injected TMZ and gene-loaded MBs-LPHNs-cRGD complexes;Group 4 were injected of gene-loaded MBs-LPHNs complexes before ultrasound irradiation,followed by TMZ treatment;Group 5 were treated with of gene-loaded MBs-LPHNs-cRGD complexes before ultrasound irradiation and TMZ treatment.In the group treated with TMZ,TMZ(50mg/kg)was given intraperitoneally two days after the nanoparticle or(and)ultrasound irradiation for continuously 5 days.Changes in tumor volume were observed using T2 MRI after every treatment cycle(7 days).When developed severe neurological symptoms,the mice were sacrificed and their survival time was recorded.Result1.When the amounts of MBs-LPHNs-cRGD were 4 x 10⁶ and the ultrasound irradiation time was 3 min,the BBB can be successfully opened,and no erythrocyte extravasation or pathological changes were observed in HE staining.In other groups,BBB cannot be successfully opened or brain tissue was damaged;2.After the injection of MBs-LPHNs-cRGD complexes,the weight and activity of the mice were normal.One week later,no significant damage was observed in the main organs of the mice,confirming the biosafety of MBs-LPHNs-cRGD complexes in vivo.In the orthotopic glioblastoma model,the fluorescence intensity at the tumor site of the FUS+MBs-LPHNs-cRGD group was higher than that in the FUS+MBs-LPHNs group and other groups,indicating that FUS+MBs-LPHNs-cRGD could successfully deliver nanoparticles into tumor cells with an active targeting ability in vivo;3.Inthecombinedtreatmentof FUS+MBs-LPHNs_pCas9/MGMT-cRGD+TMZ,the tumor volume was smaller than that of other groups in the same period.At the end of treatment,the tumor volume of this group was also smaller than other groups.The median survival time of control group,TMZ group,MBs-LPHNs-cRGD+TMZ group,FUS+MBs-LPHNs+TMZ group,FUS+MBs-LPHNs-cRGD+TMZ group were 22,24,26,30,and 43 days,respectively.It showed that FUS+MBs-LPHNs-cRGD+TMZ could suppress tumor growth and prolong the survival time of mice.Conclusion:MBs-LPHNs_pCas9/MGMT-cRGD complexes combined with FUS could effectively,safely,and locally open the BBB,target tumor cells,enhance the sensitivity to TMZ,inhibit tumor growth,and prolong the survival time of tumor-bearing mice with high biosafety.