Experimental Studies Of The Effect On Tumor Neovascularization After TACE On Rabbit VX2 Liver Neoplasms By HIF-1α Antisense Oligonucleotides Loaded Nanoparticles

PurposeTranscatheter arterial chemoembolization(TACE) has been one of the most common used method to treat liver cancer in clinic,unfortunately the long-term outcome for patients with HCC is still very poor.Complete tumor necrosis is rarely seen after TACE. One of the major reason is that a broad range of ischemic, hypoxic conditions after embolization promote the angiogenesis in the residual tumor tissue. Nowadays, anti-angiogenic gene therapy, especially antisense oligodeoxynucleotide, becomes the focus of tumor treatment, but the gene delivery vectors and vectors associated immune response, cell toxicity and safety are bottleneck of research and clinic application of gene therapy.Therefore,to study the vector of gene becomes the key tache of whether the gene therapy can be successful. In recent years, nanoparticle is developing as a new gene delivery vector, it can protect oligodeoxynucleotide and then the effective target gene therapy can be achieved.Hypoxia-inducible factor-1(HIF-1α), which was found in 1992, give us a new insight to modulate hypoxia-inducible gene for anti-angiogenic therapy. In the present study, it is proved that HIF-1αis an important transcription factor in hypoxic response and pathophysiological activation,it can control the up-regulation of a number of angiogenic factors important for solid tumor expansion,enhance the ability of anaerobic glycolysis and play an important role in tumor growth, invasion and metastasis. Given the multiplicity of mechanisms activating the HIF-1αsystem in cancer after TACE, and the many HIF-1αtarget genes involved in the angiogenic process, it is tempting to propose a simple system whereby activation of HIF-1αin HCC after TACE promotes angiogenesis and hence tumor growth.If true, such a model would strongly support the HIF-1αsystem as an anti-angiogenic target.Our work includes three parts. The first part is to investigate the effect on expression of HIF-1α,VEGF and their correlation with neovascularization after TACE on rabbit VX2 liver neoplasms,and further insight into the mechanism of tumor neovascularization after TACE.The second one is to prepare the PLGA nanoparticles carrying HIF-1αantisense oligodeoxynucleotides with W/O/W double emulsion-solvent evaporation method and investigate their general properties,drug release characteristics in vitro,so that to explore the feasibility of PLGA nanoparticles of HIF-1α-ASODN by PLGA as the carrier materials.The third one is to study the effect on tumor neovascularization after TACE on rabbit VX2 liver neoplasms by HtF-la antisense oligonucleotides loaded nanoparticles,so that to put forward a new insight in tumor therapy.Materials and Methods1. Tumor implantationAdult New Zealand white rabbits(60 total)weighing 2.1-3.5kg were used. The rabbit VX2 tumor was selected for implantation in the liver because of its blood supply similar to that of human hepatomas.Other characters of this tumor include rapid tumor growth, development of a sizable tumor that can be readily identified by X-ray imaging, in addition, the rabbit is large enough that selective manipulation of catheters in the hepatic artery for delivery of agents is possible.For successful implantation of the VX2 tumor into the liver, the tumor was first grown for 2 weeks on the hind leg of a carrier rabbit. Each carrier rabbit was used to supply tumor cells for implantation into the left central lobe of the liver of rabbit.All of the animals, carriers and recipients, were anesthetized with "pentobarbital sodium" administered i. v.. The VX2 tumor was then excised from the carrier rabbit and placed in RPMI-1640 solution.The tumor tissue were minced into small cubes as 1 mm~3 size.Then the abdomens of the recipient rabbits were shaved and layed on the bed,after which a midline subxyphoid incision was made.The anterior surface of the liver was exposed and tumor cubes were directly implanted into the left central lobe of the liver. This method allows the growth of a single solitary, well demarcated tumor in the liver of each recipient rabbit. The abdomen was closed in two layers. Proper aseptic technique was rigorously observed during each implantation. After surgery, animals were returned to their cages, and monitored in the animal laboratory. The tumors were allowed to grow for another 2 weeks,at which time they received enhanced CT examination and common reached an ellipsoidal shape, recorded the maximum and minimum diameter.2. Prepare HIF-1α-ASODN loaded PLGA nanoparticlesBiodegradable polylactide-co-glycolide(PLGA)was used as the carrier material to prepare HIF-la-ASODN loaded PLGA nanoparticles using an emulsification/solvent evaporation technique, while fixed other factors, choosing different PVA concentration and emulsifying speed, the effect on drug loading and diameter of nanoparticles has been observed.This kind of nanoparticles was characterized. The particle morphology and nanometer grains size were measured by scanning gelectron microscopy and granularity calcimeter, respectively. The drug loading and embedding rate, as well as the drug release characteristic in vitro were investigated with High Performance Liquid Chromatography.The physical stability of nanoparticles and UFL? suspension was observed by the naked eye and microscopy.3. Animals group and preparation of embolic agentsExperiment 1: 24 rabbits with implanted VX2 tumor were randomly divided into 3 groups: Control group(Each rabbit used 2ml normal saline); groups of UFLP alone and UFLP combined with THP. Each rabbit used 0.5～0.8ml UFLP, 2mg THP. Experiment 3.32 rabbits with implanted VX2 tumor were randomly divided into 4 groups: TACE group(UFLP+THP); PLGA group(UFLP+THP+PLGA); ASODN group(UFLP+THP+HIF-1α-ASOND); NP group(UFLP+THP+HIF-1α-ASODN-NP). Each rabbit with TACE used 0.5～0.8ml UFLP, 2mg THP, 2mg PLGA, HIF-1α-ASOND 50D.4. TACE procedureAfter 2 weeks of tumor implantment and CT examinations, the animals with liver tumor were treated with different methods. Administration of anesthesia access as described above, transcatheter hepatic artery injection of chemoembolic agents was performed. The animals were brought to the angiography room and subxyphoid incision was made to expose and separate the femoral artery.Puncture needle of 18G(Terumo, Japan) was used to puncture the femoral artery, after which a 4F sheath was performed using Seldinger technique.The nutrition artery, which ususlly provides most of the blood flow to the tumor, was selectively catherterized by 4F Cobra and 3F SP catheter. After having adequately positioned the catheter within the nutrition artery, the chemoembolic agents were infused directly into the artery. The procedure was considered successful when forward flow was no longer demonstrated within the nutrition artery. In addition, an intense tumor stain was identified in each case, which suggested a successful embolization procedure.5. Sample observationThe animals were examined with CT scaning after 2 weeks of TACE procedure. Then the animals were killed and the tumor isolated and measured,measure its maximum and minimum diameter to evaluate the tumor volume. Then isolated the liver, sliced at 3 mm intervals for gross examination,the tissue fixed in 10% formalin solution.6. HistopathologyTumor tisse were selected and embedded completely in paraffin, after which 3μm section were stained with HE and immunohistochemical staining,including HIF-1α, VEGFand CD34. The positive cells were appeared as brown and yellow staining in nuclear or cytoplasm. Then calculated the positive cells of HIF-1α, VEGFand CD34 according to the methods of Birner, Park and Weidner, respectively.ResultsExperiment 1: The implanted VX2 tumor grew in round or oval shape in the left central lobe.There is no significant difference in the volume size at the end of 2 weeks(P＞0.05). While after the different treatments of the 3 groups, the tumor growth appeared very different. Comparing with the Control group, the volume size of experimental groups were suppressed obviously(P＜0.01); especially in the TACE group, but there was no significant difference(P＞0.05) between TAE and TACE group. Expression of HIF-1αenhanced apparently in TAE and TACE groups comparing with the Control group, The strong positive rate of HIF-1αis 12.5%, 62.5%, 50%, respectively, there was no significant difference in TACE group versus Control group(P＞0.05), but there was statistically significant difference in TAE group versus Control group(P＜0.05). Expression of VEGF enhanced apparently in TAE and TACE groups comparing with the Control group, but there was no statistically significant(P＞0.05). The value of MVD was 51.63±4.90, 67.08±4.57 and 63.50±4.93 respectively in these groups, MVD was higher in TAE and TACE groups comparing with that in Control group(P＜0.05).There was positive correlation between HIF-1α, VEGF expression level and the value of MVD(r_s=0.537, P＜0.01; r_s=0.486, P＜0.05; r_s=0.423, P＜0.05).Experiment 2: The HIF-1α-ASOND loaded PLGA nanoparticles were spherical or oval shape, uniform, with mean diameter of 320±50 nm. The drug loading efficiency and encapsulation rate of the HIF-1α-ASOND nanoparticles was (0.93±0.015)% and (79.14±1.78)% respectively. In the drug release test in vitro, the drug release rate was 31.2% during the burst release phase and rose to 81.5% at the end of 10th day. Suspension of UFLP with HIF-1α-ASOND loaded PLGA nanoparticles had good physical stability.Experiment 3: After the different treatments of the 4 groups, the volume size of NP group was the smallest, Comparing with TACE group, ASODN group and NP group were statistically significant(P＜0.05), PLGA group was not statistically significant(P＞0.05), and there was statistically significant difference(P＜0.05) in ASODN group versus NP group. Serum AST and ALT level markedly increased 3 days and gradually decreased 7 days after embolization treatment,there was no significant difference(P＞0.05) in these groups. The strong positive rate of HIF-1αis 50%, 62.5%, 25%, 0, respectively, there was statistically significant difference(P＜0.05) in NP group versus TACE group. The strong positive rate of VEGF is 50%, 50%, 25%, 0, respectively, there was statistically significant difference(P＜0.05) in NP group versus TACE group. The value of MVD was higher in ASODN and NP groups comparing with that in TACE and PLGA groups, comparing with TACE group, ASODN group and NP group were statistically significant(P＜0.05), and there was statistically significant difference(P＜0.05) in ASODN group versus NP group. There was positive correlation between HIF-1α, VEGF expression level and the value of MVD(r_s=0.473, P＜0.01; r_s=0.406, P＜0.05; r_s=0.374, P＜0.05).Conclusion1. Subxyphoid incision was made to expose and separate the femoral artery, and then seldinger technique was performed using 18G puncture needle and 4F sheath, TACE on rabbit VX2 liver neoplasms was successful.2. Significant overexpression of HIF-1αwas found after TACE, HIF-1αplays an important role in the prognosis of liver neoplasms by up-regulating the expression of VEGF and increasing MVD.3. The preparation technology of the HIF-1α-ASOND loaded PLGA nanoparticles using an emulsification/solvent evaporation technique have satisfactory pharmaceutical properties and good repetition. 4. The HIF-1α-ASOND PLGA nanoparticles were spherical or oval shape, uniform, with mean diameter of 320+50nm and sustained release effect in vitro.5. HIF-1α-ASOND can suppress angiogenesis after TACE in the treatment of rabbit VX2 liver neoplasms,the anti-angiogenesis by HIF-1α-ASOND loaded PLGA nanoparticles is far more effective than that by HIF-1α-ASOND alone.6. The combination of antiangiogenesis therapy and routine TACE could enhance the clinic effect and will be a new way in liver cancer therapy.