Preparation Of Gene-loaded Cationic Nanobubbles And Their Inhibition On The Growth Of AIPC Transplanted Tumor

Background:With the development of biological technology, gene therapy for cancer has becomethe focus of cancer treatment study. Gene therapy is an experimental treatment that involvesintroducing genetic materials into a person’s cells to fight or prevent disease and has madesignificant progress. The vector, which could insert the desired genes into the target cellssuccessfully, is the bottleneck problem of gene therapy. The gene delivery vectors aremainly divided into viral vectors and non viral vectors. Non viral vectors, such asliposomes, plasmids, have good biocompatibility, while the transfection efficiency isrelatively low. Viral vectors, such as adenovirus and lentivirus, usually have a hightransfection efficiency, but the toxicity and immunogenicity of them remain major obstaclesfor their translation to the clinics successfully. Therefore, it is important to develop a newkind of gene vectors, which has few adverse effects and high transfection efficiency.Ultrasound microbubble-mediated gene transfection (UMGT) has become one of the mostpromising methods for gene transfection in recent years. UMGT has a characteristics ofsecurity, stability and convenience, so that it is an ideal transmission system for genetherapy. The main problems associated with the use of ultrasound microbubbles(MBs)as agene vector include their low permeability, low stability and low gene-loading dosage, onthe other hand, the MBs are also unstable in the blood circulation and are easily engulfed bythe reticuloendothelial system.Currently, there are no effective long-term therapies for prostate cancer, especially forandrogen-independent prostate cancer (AIPC). In our preliminary studies, nanobubbles(NBs) carrying ARsiRNA were prepared using poly-L-lysine (PLL) and electrostaticadsorption methods, and the NBs carrying ARsiRNA could transfect AIPC cells effectivelyin combination with ultrasound radiation and significantly inhibit the growth of AIPC cells.PLL contains a polycation chain with a positively charged surface, and the prepared NBs have a negatively charged surface. After PLL binding via static electricity, the surface of theNB has a positive charge and can combine with the negatively charged siRNA viaelectrostatic adsorption, which can be regarded as an indirect electrostatic binding method.However, the PLL method is not suitable for wide use due to its instability. It is importantto develop a novel kind of NBs, which exhibits evenly distributed positive charges anduniform size. Luckily, the positive charge on the surface of the cationic NBs could attractedthe negatively charged substance (DNA, siRNA, plasmid,) to the NB surface throughelectrostatic attractions easily, which increase the stability of the gene-carrying NBs. Thisnovel kind of NBs could build a foundation for future experimental studies on the treatmentof transplanted prostate tumors using gene-carrying cationic NBs under ultrasoundirradiation.Objectives:A novel kind of cationic NBs is prepared, and then is detected its in vitro and in vivocontrast enhancements compared with those of SonoVue. To investigate cationic NBsgene-carrying ability of different therapeutic genes (siRNA, plasmid, antibody), we preparethe ARsiRNA-loaded cationic NBs for AIPC and detect the gene-carrying capacity ofARsiRNA. The AIPC cells and its xenografted tumor could be investigated to clarify theeffects on the growth of AIPC under ultrasonic irradiation combined with the cationic NBscarrying ARsiRNA, and to observe the microstructure changes of the cell membrane byscanning electron microscope.Methods:1. Cationic NBs with protamine and other lipid components were prepared usingmechanical oscillation and freeze drying method, and their size, Zeta potential,concentration, stability, contrast enhancement ability in vitro and in vivo were assayedcompared with those of SonoVue.2. The biosafety of cationic NBs was detected in vitro, their gene-carrying capacity ofdifferent therapeutic genes (siRNA, plasmid, antibody) was tested with a fluorescentmicroscope. Additionally, the ARsiRNA-loaded dosage and encapsulation efficiency of theNBs was measured at260nm using a spectrophotometer. We used the cationic NBscombined with ultrasound radiation and the liposome to carry the ARsiRNA to transfectAIPC cells, and then used CCK-8method to detect the growth inhibition of cells with two methods.3. The cationic NBs carrying the ARsiRNA combined with ultrasound irradiationwere transfected into AIPC tumors and then we used qRT-PCR and WB methods to detectthe expression level of ARmRNA and AR protein in xenografted tumors and observed themicrostructure changes of the cell membrane by scanning electron microscope.Results:1. The prepared protamine cationic NBs appeared to have a uniform size, regularmorphologies, and an even distribution without obvious aggregation. The mean diameter ofthe NBs was (521±37.57) nm, the mean concentration was (1.26±0.37) x108/ml, the zetapotential of the NB surface was (18.5±5.13) mV, and their physical properties did notchange significantly after one week. The cationic NBs exhibited superior contrastenhancement for in vivo imaging compared with SonoVue, and the protamine cationic NBwas found to have a stronger ability to penetrate the tumor tissue with a larger filling areathan that obtained from SonoVue. Additionally, the protamine cationic NBs has a longerimaging duration time than SonoVue, which lasted approximately20min in the livers,xenografted tumors and kidneys of the nude mice.2. Based on the results of fluorescent microscope, cationic NBs could carry siRNA,plasmid and antibody effectively, and could also transfect tumor cells, primary cells andstem cells efficiently. Red fluorescence was observed in the cationic NBs carryingCy3ARsiRNA under a fluorescence microscope, and some of the NBs were ring-shaped.The gene-loading dosage was15.69mg per108NBs, the cationic NBs carrying ARsiRNAcould transfect AIPC cells in vitro in combination with ultrasound irradiation, and thegrowth curve of CCK-8demonstrated that the cationic NBs could inhibit more effectivelythan liposomes based on the transfection assays.3. Ultrasound irradiated ARsiRNA-loaded cationic NBs could inhibit the growth ofAIPC tumors in the in vivo experiment, and the expression levels of AR protein andARmRNA in nude mice were obviously inhibited. The cryosections of xenografted tumorsshowed that Cy3ARsiRNA could penetrate the blood vessels and aggregate in the tumortissue. And both in vitro and in vivo experiments could find the formation of pores on thesurface of cell membrane through the scanning electron microscope (SEM). Conclusions:1. The protamine cationic NBs could appear to have a uniform size and distributionand exhibit superior contrast enhancement for in vitro and in vivo imaging.2. The protamine cationic NBs could carry different kinds of therapeutic geneseffectively, and could inhibit the growth of C4-2cells efficiently. The cationic NBs carryingARsiRNA could transfect AIPC cells effectively in combination with ultrasound irradiationand significantly inhibit AIPC cells growth.3. Ultrasonic irradiation with cationic NBs could promote ARsiRNA entry into cellsefficiently, and thus suppress the expression of ARmRNA and protein and ultimatelyinhibiting cell growth by silencing the AR receptor. The microstructure of the cellmembrane demonstrate that ultrasound irradiated ARsiRNA-loaded cationic NBs couldenhance the permeability of cell membrane both in vitro and in vivo.