Preparation And Anti-Tumor Study Of Gambogenic Acid Nanodrug For Self-Enhanced Tumor Accumulation

Achieving effective drug accumulation while reducing tumor vascular density is a huge challenge,but it is one of the keys to breakthrough the effect of anti-vascular therapy.Here,we prepared a surface-charged acid nanoparticle（CRNP-GNA）that activates a positive feedback loop between improved tumor vascular permeability and increased drug accumulation.This positive feedback loop would significantly enhance the permeability of tumor blood vessels and increase the effective accumulation of drugs in tumor tissue.Compared with another CINP-GNA group,the accumulation of tumor vascular permeability and retention index（VPRI）increased from 8.32-fold and 60-fold increase,respectively,while the tumor microvessel density decreased to 7%.The accumulation of self-enhancement amplified the antitumor efficacy through antiangiogenesis,vascular destruction,ameliorated tumor hypoxia,activation of autoimmune responses,and multiple proapoptotic pathways,and five of the six transplanted mice in the CRNP-GNA group had tumors completely cured.This study confirms that antivascular therapy can induce a potential positive feedback loop through a charge-reversal drug delivery nanosystem,enabling efficient and self-enhanced drug accumulation even in tumors with few blood vessels.This provides a new strategy to overcome the dilemma between anti-vascular efficacy and drug accumulation.This paper mainly consists of the following parts:Part Ⅰ Preparatio)n and characterization of nanoformulation for self-enhanced tumor accumulation.Poly（ethylene glycol）₄₅-polycaprolactone₄₀-poly[2-（2′,3′-dime-th ylmaleic acid amide）ethyl acrylate]₃₃(PEG₄₅-PCL₄₀-PAEA₃₃-DMMA)was prepared with polymalonic acid（GNA）for nanocharged drugs（CRNP-GNA）.The surface Zeta potential is determined to be about-10 m V,and the particle size is about100 nm.By transmission electron microscope（TEM）observation,the nanoparticl es are spherical,and the structure is evenly dispersed,and the surface is smooth.The amount of the prepared nanodrugs is about 8.1%,and the encapsulation de gree is about 89.4%.Using the amphiphilic three-block copolymer poly（ethylene glycol）_45-polycaprolactone₄₀-poly[2-（succinic acid amide）ethyl acrylate]₃₃(PEG₄₅-PCL₄₀-PAEA₃₃-SA)was used to control.When p H from p H7.4 to p H6.8,the charge r eversal nanoparticles（CRNP）underwent charge reversion behavior,and the Zeta potential changed from-10 m V to about+10 m V,while the CINP potential hard ly changed.Part Ⅱ In vitro antiangiogenesis and anti-tumor study of self-enhanced tumor accumulation.In vitro tube formation experiments showed that under p H6.8,CRNP-GNA can not only inhibit the tube formation process of human umbilical vein endothelial cells（HUVECs）,but also destroy the formed lumen,indicating that CRNP-GNA has significant anti-angiogenesis and vascular destruction effects.The uptake of human s-derived Hep G2 cells and HUVECs cells showed that the cellular uptake of CRNP-GNA group was about 3 times that of CINP-GNA group.The 3-（4,5dimethylthiazole-2）-2,5-diphenyltetrazole bromine salt（MTT）method was used to detect drug cytotoxicity on Hep G2 cells.The results showed that CRNP-GNA had no significant inhibitory effect on cells at p H=7.4,but at p H=6.8,IC₅₀was about4.137μg/m L,which was comparable to the cytotoxicity of free drug.Similarly,the MTT experiment of Hepa1-6 cells was consistent with that of Hep G2,which showed a strong inhibitory effect on tumor cells.The pro-apoptotic results showed that CRNP-GNA had p H-dependent pro-apoptotic effect on both Hep G2 cells and HUVECs cells,namely non-apoptotic effect at p H7.4 but pro-apoptotic effect at p H6.8.Part Ⅲ In vivo anti-tumor study of tumor accumulation.The results of in vivo pharmacokinetic experiments showed that when the free drug extended the half-life of free GNA from t_1/2=80min to t_1/2=215min of CINP-GNA,and t_1/2=249min of CRNP-GNA,the extended drug half-life can improve the bioavailability of the drug and reduce the side effects of the drug.The vascular irritation experiment of the ear edge vein in rabbits showed that the damage of GNA to normal blood vessels could be alleviated by the improvement of the dosage form.The results of the pharmacodynamic experiments showed that,comparing the efficacy of the first-line bevacizumab and sorafenib for liver cancer,mice in the CRNP-GNA group exhibited up to 69%tumor suppression rates,While bevacizumab and sorafenib exhibited only 11%and 32%,combining the histochemical results of tumor microvessel density（MVD）,vascular endothelial growth factor（VEGF）,hypoxia-induced factor-1（HIF-1α）,and the polarization of immune cells（M1,M2）in tumors,CRNP-GNA can not only significantly inhibit tumor growth but also reduce the microvascular compaction in tumor tissue,destroy tumor blood vessels,relieve tumor hypoxia,activate autoimmune response,and induce the polarization of M2 type macrophages to M1 type macrophages.The survival results of mice showed that all the other groups died within 37,while the CRNP-GNA group had no mice died within 75 days,and the tumors of 5 of 6 mice disappeared,indicating that the nanodrug can significantly prolong the overall survival of mice,and has a high cure rate.Part Ⅳ Study on the mechanism of self-enhanced tumor accumulation of neooic acid.In order to verify that CRNP-GNA nanodrugs can exert anti-angiogenesis and increase the accumulation of drugs in tumor in tumor tissue,this section uses small animal in vitro imaging combined with immunohistochemistry and immunofluorescence analysis in isolated tumor.The results showed that the mean fluorescence intensity（ATFI）was not significantly different from CINP-GNA 1h after the CRNP-GNA injection,but the ATFI was 5.26 and 2.76 times that of CINP-GNA after the fourth and seventh injection,respectively.This implies that the tumor vascular permeability and microenvironment undergo remodeling in the CRNP-GNA group,thus enhancing the vascular extravasation of GNA nanopreparations.When the time was extended to 6 h,the ATFI in the CRNP-GNA group was 2.16 times that in the CINP-GNA group,indicating that its charge-inversible could favored the tumor accumulation of GNA nanopreparations.Six hours after the fourth and seventh injections,the ATFI ratio between CRNP-GNA to CINP-GNA groups increased to 8.32 and 4.28,respectively.Combined with the immunohistochemical results of MVD,with the increasing number of injections,drug accumulation in tumors has also increased,at the same time,the blood vessel density is decreasing.This suggests that CRNP-GNA achieves a highly efficient anti-tumor effect by activating the tumor positive feedback loop.It also proves that nanomedicine can increase drug accumulation in tumors while exerting anti-vascular effects.Combined with the analysis of the tumor immunofluorescence results,this accumulation of drugs may result from the gaps of plasma perfusion present in the tumor.These gaps contain large amounts of fluorescently labeled nanodrugs,acting as a drug reservoir,Large accumulation of drugs can further damage tumor blood vessels,make more drugs accumulate at the tumor site,resulting in a positive feedback loop.