Biosynthetic Nanobubbles For Targeted Gene Delivery By Focused Ultrasound

Objective: To evaluate the tansfection efficiency of a novel cationic biosynthetic nanobubbles(CBNBs),which is fabricated through co-culture of biosynthetic nanobubbles(BNBs)and polyethyleneimine(PEI),under the aid of UTMD both in vitro and in vivo.Methods: 1.Firstly,Halobacterium NRC-1(Halo)was cultured for a certain period of time.Then culture medium was transferd to a separatory funnel that is left undisturbed for up to a week to allow the buoyant cells producing BNBs to float to the top and separate from media.Next,Buoyant cells are lysed using hyperosmotic shock.Subsequently,centrifugally assisted floatation is used to isolate and purify BNBs and yield a concentrated,milky white solution of BNBs in deionized water.Then,the morphology of the BNBs was directly determined using a transmission electron microscope.The particle size,surface charge and distribution of BNBs were analyzed using a Zetasizer NANO ZS system at room temperature.The concentration of BNBs was determined by measuring the optical density at 500 nm(OD500).Capacity of BNBs to generate ultrasound contrast signal was evaluated through Vevo2100? High-Resolution Micro-Imaging System in vitro.Cytotoxicity of BNBs in different concentration was analysed using CCK8 kit.Self-assemble microbubbles(MBs)was introduced as positive control and the general properties of MBs were assessed.2.First of all,stability of BNBs in room temperature was determined.Next,CBNBs were fabricated by modifying the protein shell of BNBs with a increasing amount of PEI.Then,the zeta potentials were measured using a Zetasizer NANO ZS system at room temperature after unbound PEI was discarded,and the optimal ratio between BNBs and PEI was determined according to the data of zeta potentials.To determine the appropriate ultrasound parameters to destruct BNBs,BNBs solution(OD500=0.5,200 ?l)in a 96-well plate were exposed to various acoustic pressures ranging from 0 MPa to 0.8 MPa.Then,ultrasound contrast images of BNBs that received ultrasound irradiation was record.To determine the plasmid loading capacity of CBNBs,4 ?g of plasmid DNA was mixed with CBNBs containing different amounts of PEI at room temperature for 20 min by gentle rotation.Then,the unbound DNA was removed by centrifugation through washing thrice in an Eppendorf tube.The amount of plasmid DNA bound onto CBNBs was evaluated to measure the DNA loading capacity of CBNBs using gel electrophoresis.Cytotoxicity of CBNBs with different amount of PEI was analysed using CCK8 kit.3.Ultrasound mediate gene transfection efficiency was evaluated both in vitro and in vivo study using 293 T cell and 4T1 cell.Then,the effect of different ultrasound exposure conditions on transfection efficiency was analysed.Hemolysis assay and in vivo biocompatibility was assessed.Then,tumor-bearing mice model was established.In brief,approximately 1×107 4T1 cells in 200 ?l PBS were injected subcutaneously into the right rear flank of each mouse.When the tumors reached 100 mm3,various concentrations of BNBs ranging from OD500 = 0.5 to 1.5 were injected intratumorally into the tumors,followed by imaging with Vevo2100 ultrasound imaging system.Finally,UTMD and CBNBs mediated gene transfection efficiency were evaluated after intratumoral and tail vein injection.The in vivo gene expression efficiency was assessed through bioluminescence imaging using an IVIS Spectrum imaging system.Moreover,cationic microbubble(CMBs)and Lipofectine 2000 was introduced as positive control to highlight the underlying advantages of CBNBs.Results: The CBNBs have a nanoscale size,giving them the potential to extravasate out of the leaky capillary network of tumors and to get fully attached to targeted cells when applied in vivo.Also,we revealed the optimal ratio between BNBs(OD500)and PEI(?g),which is 0.1?8,to guarantee that the plasmid DNA can be effectively coupled onto the surface of the CBNBs.Furthermore,the best match between(OD500),PEI(?g),DNA(?g)was also revealed in current study.Additionally,CBNBs could produce stable contrast signals both in vitro and in vivo.Transfection efficiency was promoted significantly in the DNA/CBNBs group both for 293 T cells and 4T1 cells and in the allograft tumor model.Conlusion: To our knowledge,this is the first report to apply biosynthetic bubbles as non-viral gene carriers which can effectively deliver genes into tumor cells with the aid of ultrasound cavitation.Our study provides a powerful tool for image-guided and efficient gene delivery using biosynthetic nanoscale contrast agents.