ARFI Microbubble Destruction Improves Targeted Delivery Of Fe₃O₄ Nanoparticles On SD Rats Subcutaneous Transplantation Tumor

Background and objectIn recent years, tumor targeting drug delivery system based on nano engineering developed rapidly. A conventional idea is that the chance of administered nanoparticles to reach the target would increase by prolonging the blood circulation time and escaping the reticuloendothelial system, Most solid tumors show a higher vascular density, and the neovasculature formed in solid tumor had an abnormal architecture, including wide fenestrations in the defective endothelial cells and lack effective lymphatic drainage. Therefore macromolecular drugs and lipidic particles can accumulated and remained in solid tumor, the phenomenon was known as high permeability and retention effect of solid tumors. EPR effect is an important basis for targeted therapy of solid tumors, drug-loaded nanoparticles were intravenously(iv) administered into the blood circulation, the nanoparticles circulate in blood to reach target tumors by extravasation. Theoretically, nanoparticles combined with ligand or antibody can delivery chemotherapy drugs selectively to the targeted tumor. But the biological distribution of targeted nanoparticles in tumor tissue after intravenous injection is affected by many factors, The actual accumulation of the administered dose varies depending on the nanoparticle formulation, drug, drug assay method, and other experimental arameters, but usually it is less than 5%, more than 90% of the nanoparticles are cleared in the blood circulation instead of end up in targeted organs. Nanoparticles extravasated from blood vessel to the interstitium of tumors is thought to be achieved by EPR effect, even if nanoparticles reach the tumor site, it needs to penetrate tumor microenvironment obstacles which is different from that of normal tissues. Tumor microenvironment has denser extracellular matrix(ECM) compares to normal tissues, which makes it hard for drug penetration, because of vascular permeability and lacking of well developed lymphatic vessels, the elevated tumor interstitial fluid pressure(IFP) in tumor increased significantly, which is bad for drug delivery. These physiological barriers has to be overcome to achieve successful interstitial drug transport in tumor tissues.Studies have shown that microbubble ultrasound contrast agents in cultured cells suspension, with ultrasonic irradiation the cell membrane can be temporarily open and closed to macromolecules, so macromoleculars can get into cells and be captured by the cells, this is called sonoporation. Sonoporation can increase the efficiency of gene transfection and this could change the phenotype of cultured cells. Sonoporation is the results of acoustic cavitation caused to cells. Ultrasound targeted microbubble destruction(UTMD) is to use ultrasonic irradiation microbubbles in the tumor site, then microvascular permeability will increase for a short time, marked red blood cells, colloidal particles and plasmid DNA will be delivered into the tumor tissues which was treated with ultrasonic irradiation. UTMD can obviously improve the targeting delivery of macromolecular in tumor tissues. Tumor angiogenesis is not only increase in number, but there is also significant abnormal structure which is different from normal tissue. the abnormal structure including vascular expansion, distortion, abnormal anastomosis of microvascular and abnormal distribution of three-dimensional spatial microvascular, reducing number of supporting endothelial cells, thin and discontinuous basement membrane, increasing permeability of the blood vessel wall. These changes is advantageous to the particles to transfer from blood vessels into tumor tissues.Traditional UTMD use the typically ultrasonic diagnostic instrument with large acoustic field, microbubbles in the large acoustic field will all be irradiated without precise positioning in the tumor tissues. Meanwhile typically ultrasonic diagnostic apparatus with continuous wave is not conducive to microbubbles reperfusion in target tumor site. Acoustic radiation force imaging(ARFI) can provides information about local variations of the mechanical properties of soft tissue in response of acoustic pulses. ARFI imaging has been demonstrated in many clinical applications, these include the assessment of liver fibrosis, monitoring thermal radiofrequency and distinguishing between benign and malignant tumors. On the other hand, ARFI using commercial diagnostic ultrasound scanners not only to monitor the transient, dynamic displacement tissue response within the excited tissues but also to generate localized, impulsive acoustic radiation forces in tissues. As a low intensive focused acoustic beam with small volume and geometry of the tissue that is excited, ARFI has smaller excited district than traditional ultrasound imaging, so it can accurately locate the scope of the microbubbles irradiation within the excited tissues, ARFI have stronger local effects for drug-loading nanoparticles targeting delivery. Some researches show that low-amplitude acoustic radiation as a mechanism can move circulating microbubbles toward targeted endothelium. With the acoustic radiation force treatment, microbubbles are forced to contact with the endothelium, and appreciable targeted microbubbles retention was observed in the microcirculation, which improve accuracy and efficiency of the gene and drug delivery in tumor tissues.Our study is to prepare a kind of Fe3O4 nanoparticles, and to improve Fe3O4 nanoparticles delivery in the blood circulation based on EPR effects in solid tumor tissue, the biological effects of ultrasound microbubble destruction can enhance the tumor hemoperfusion, increase the vascular permeability and increase the drug-loaded microbubble targeted delivery in tumor tissues. The ARFI microbubble destruction combines with nanoparticles provides a new strategy for tumor targeted delivery.Materials and Methods1. to prepare a Fe3O4 nanoparticles and to observe the physicochemical properties of the Fe3O4 nanoparticles.Hydrothermal reaction at high temperature was used to prepare Fe3O4 nanoparticles, all the materials are added in accordance with a certain proportion, then adjust the materials proportion to get different size of Fe3O4 nanoparticles, choose the proper Fe3O4 nanoparticles can meet our demand of study and the particle size and morphology are observed by transmission electron microscopy(SEM), with Malvern laser instrument for assessing the particle size distribution, surface zeta potential.2. explore Fe3O4 nanoparticles’ s effect for MR imaging in vitroFe3O4 nanoparticles were diluted to different concentrations of Fe in double steaming water, And mix them in centrifuge tube containing agarose gel, puts the centrifugal pipe on racks, and imaged on a 7.0 T MRI scanner, T2*-weighted images were acquired to observe the Fe3O4 nanoparticles effect of magnetic resonance imaging in vitro.3. To establish a subcutaneous transplantation tumor of W256 cell in SD ratsWalker256 cell was cultured with 1640 media under appropriate condition, adjusting the concentration of Walker256 cell to1×107 cells per mL in logarithmic phase of cell proliferation. Disinfecting rats with povidone iodine, then injecting 0.5 mL of the high concentration cell suspension in the left rear of rats subcutaneously, we observed that injection site skin of rats swelled, Subcutaneous nodules were observed after inoculation for 7 days, and then prepared for further experiments.4. investigate the impact of acoustic radiation force impulse(ARFI) microbubble destruction on targeted delivery of Fe3O4 nanoparticles on SD rats subcutaneous transplantation tumorSD rats were randomly divided into six groups, with 10 rats for each group. Group1 was treated with ARFI irradiation, group 2 with MBs injection, Group3 was ARFI microbubble destruction, Group4 was Fe3O4 nanoparticles injection, Group5 was Fe3O4 nanoparticles injection and ARFI irradiation, Group6 was Fe3O4 nanoparticles injection and ARFI microbubble destruction.Treatment: ARFI irradiation parameters: exposure duration 5s then pause 5s, total 5min with Low frequency pulsed focused ultrasound probe irradiation the tumor site. The MBs and Fe3O4 nanoparticles were given by intravenous injection. The dose of MBs was 0.2ml each rat. The dose of Fe3O4 nanoparticles for rat was 5mg/kg.Every group was given the corresponding treatment, then MRI was employed to observe the signal change of tumor on T2*WI before and after Fe3O4 nanoparticles injection and AFRI irradiation. The rats were killed for pathological assay after management.Results and conclusion1. The prepared Fe3O4 nanoparticles was observed with regular spherical morphology and narrow size distribution and it provided significant superparamagnetic properties for imaging.2. Prussian blue staining represent the distribution of Fe3O4 nanoparticles in tumor,ARFI irradiation group,MBs injection group and ARFI microbubble destruction group were similar with no blue stained particles, Fe3O4 nanoparticles injection group, Fe3O4 nanoparticles injection and ARFI irradiation group and Fe3O4 nanoparticles injection and ARFI microbubble destruction group were observed with blue stained particles. The count of blue stained particles in Fe3O4 nanoparticles injection and ARFI microbubble destruction group had statistically more than the other two groups. Maximum count of blue stained particles was Fe3O4 nanoparticles injection and ARFI microbubble destruction group with most of the blue stained particles were distributed close to vascular.3. The T2*WI signal was similar with no signal changed in ARFI irradiation group, MBs injection group and ARFI microbubble destruction group. Increased hypointense areas were observed inside the tumor in Fe3O4 nanoparticles injection group,Fe3O4 nanoparticles injection and ARFI irradiation group and Fe3O4 nanoparticles injection and ARFI microbubble destruction group.ARFI microbubble destruction improved Fe3O4 nanoparticles distribution on SD rats subcutaneous transplantation tumor,thus enhanced Fe3O4 nanoparticles in vivo tumor targeted delivery.