| In tumor treatment,anti-angiogenesis drugs can inhibit angiogenesis,thereby inhibiting tumor growth and metastasis.However,as the sole use of anti-angiogenesis drugs cannot meet the needs of tumor treatment,combining them with chemotherapeutic drugs with different anti-tumor mechanisms is often considered as an effective strategy to improve treatment efficiency.In addition,owing to the specific microenvironment of tumor tissue,how to achieve effective drug delivery in the deep tumor layers is also crucial to improving the effect of tumor treatment.Based on the combined application of anti-angiogenesis drugs combined with chemotherapeutic drugs and the specific size characteristics of polyamidoamine dendrimer(PAMAM),this study used p H-responsive liposomes and PAMAM as drug carriers,cyclic arginine-glycine-aspartic acid peptide(c RGD)as the target,fruquinitib(FRU)and doxorubicin(DOX)as model drugs to construct a p H-responsive size-converted targeted nano-drug delivery system(PRL-PD/FRU-c RGD).The research content of this thsis is mainly divided into the following three parts:Part I: Preparation and Characterization of PRL-PD/FRU-c RGDFirst,the DOX-loaded small particle size complex(PD)was prepared via physical stirring method,and then the “reverse evaporation method” was used to encapsulate the prepared hydrophilic PD and hydrophobic drug FRU into p H-responsive liposomes with surface-modified c RGD peptides to construct the targeted nano-drug delivery system PRL-PD/FRU-c RGD.(1)Characterization of nanoformulation.The characterization results of particle size,potential,and morphology showed that the PD had good dispersibility,the particle size was approximately 15.0 nm,and the zeta potential was approximately +11.9 m V.The PRL-PD/FRU-c RGD encapsulated with FRU and PD was regular and nearly circular,the particle size was approximately 120.0 nm,and the zeta potential was approximately-18.2 m V.The drug loading and encapsulation efficiency of PRL-PD/FRU-c RGD for FRU were 5.74 ± 0.21% and 86.75 ± 0.16%,respectively;for DOX,they were 4.99 ± 0.28% and 70.29 ± 0.28%,respectively.(2)In vitro drug release.The results of the in vitro release efficiency of FRU and DOX in PRL-PD/FRU-c RGD showed a significant difference in drug release under different p H conditions.Among them,at 72 h,the cumulative release of FRU at p H 6.5 and 5.0was approximately 2.11-fold and 2.78-fold higher than that at physiological conditions.The cumulative release of DOX at p H 5.0 was approximately 2.28-fold higher than that at physiological conditions,and the release efficiency was not obvious at p H 6.5.These results indicated that PRL-PD/FRU-c RGD achieved the purpose of controlled drug release in the tumor microenvironment.(3)Haemolysis assay.Further results showed that the haemolysis rate of PRL-P-c RGD was within the range of 0.0625 ～ 4.5 mg/m L and did not exceed 3%.This showed that the PRL-P-c RGD nanoformulation is biocompatible.Part II: In vitro antitumor activity of PRL-PD/FRU-c RGDThe 4T1 breast cancer cells and human umbilical vein endothelial cells(HUVEC)were used as models to preliminarily investigate the antitumor activity of PRL-PD/FRU-c RGD in vitro at the cellular level.(1)Cellualr uptake assay.The results showed that the uptake of drugs by 4T1 cells can be effectively increased under the targeting of nanoformulation.(2)MTT experiments.The results showed that within the experimental concentration range of 5 ～ 640 μg/m L,the blank carrier(PRL-P-c RGD)did not show obvious inhibitory effects on cells,pointing to the safety of the blank carrier.Under the combined effect of FRU and DOX,PRL-PD/FRU-c RGD could significantly inhibit the proliferation of 4T1 cells,and the inhibitory ability towards 4T1 cells was significantly higher than that in the single-drug nanoformulations.(3)In vitro penetration study using the 3D-tumor spheroid model.The results showed that PRL-PD/FRU-c RGD could release PD by responding to the tumor microenvironment,and had stronger tumor penetration ability than the control group,resulting in deep penetration of the drug in 4T1 tumor spheroid.(4)The anti-metastasis study was investigated by wound healing assay and Transwell assay.The results showed that the anti-metastatic ability of PRL-PD/FRU-c RGD was significantly higher than that in the single-drug nanoformulations,and only 25.76% and 15.26% of 4T1 cells migrated and invaded,showing excellent anti-metastatic effect.(5)In vitro anti-angiogenesis experiments.The results of tubule formation assay and Western blot experiments showed that under the combined action of FRU and DOX,the PRL-PD/FRU-c RGD group had almost no complete tubular structures and showed the lowest level of vascular endothelial growth factor receptor(VEGFR-2)in each experimental group,indicating that PRL-PD/FRU-c RGD has excellent in vitro anti-angiogenesis ability and can significantly inhibit the expression of VEGFR-2.Part III: In vivo antitumor activity of PRL-PD/FRU-c RGDThe 4T1 tumor-bearing mice were used as a model to further investigate the antitumor activity of PRL-PD/FRU-c RGD in vivo.(1)In vivo targeting study.The distribution of PRL-PD/FRU-c RGD in various tissues of tumor-bearing mice was observed by fluorescence intravital imager.The results showed that the distribution of PRL-PD/FRU-c RGD in tumor tissues was significantly higher than that of free drugs,further confirming the targeting ability of nanoformulation.(2)Pharmacodynamic study.Compared with the other experimental groups,after PRL-PD/FRU-c RGD treatment,the mouse tumor tissue was smallest and the survival time was longest,showing the best tumor therapeutic effect and prognosis.Most importantly,compared with the control group,the mouse tissues in the PRL-PD/FRU-c RGD group did not show obvious pathological changes,and there were no significant differences in liver and kidney functions,which illustrated the safety of PRL-PD/FRU-c RGD to a certain extent.(3)In vivo anti-angiogenesis study.The expression of the tumor vascular density of mice in each experimental group was observed.The reduction in green blood vessels indensity was the most significant in the nanoformulation group loaded with the double drug,indicating that PRL-PD/FRU-c RGD can effectively inhibit angiogenesis.(4)In vivo deep tumor penetration study.The penetration of nanoformulation in tumor tissue was analyzed,and the red fluorescence of PRL-PD/FRU-c RGD was distributed on almost the entire tumor tissue.This result was consistent with that of in vitro permeation studies,further confirming that PRL-PD/FRU-c RGD released PD by responding to the tumor microenvironment,thereby achieving the deep penetration of drugs into tumor tissue.(5)Anti-lung metastatic study.The 4T1 lung metastasis mice were used as a model to investigate the combined anti-lung metastasis ability of FRU and DOX.The results showed that the PRL-PD/FRU-c RGD group had the least number of lung metastatic nodules in mice.Under the combined action of FRU and DOX,the inhibition rate of PRL-PD/FRU-c RGD on 4T1 lung metastasis was as high as 86.76 ± 1.80%,which was significantly higher than that in the single-drug nanoformulations,showing excellent anti-lung metastasis ability.Based on the particle size-converted strategy,this study successfully constructed a size-converted targeted nano-drug delivery system by combining anti-angiogenesis with chemotherapeutic drugs.In the acidic microenvironment of the tumor tissue,the system actively targeted the highly expressed integrin receptors on the surface of tumor cells.The released FRU can inhibit the expression of vascular endothelial growth factor receptor(VEGFR),thereby inhibiting the formation of new blood vessels and cut off the nutrients and oxygen that tumors rely on to survive,resulting in inhibiting the proliferation and metastasis of tumor cells.At the same time,particle size conversion occurs,releasing DOX-loaded small particle size complexes(PD),enhancing tumor tissue penetration of nanoformulation,achieving deep delivery of DOX.In summary,this system can achieve rapid tumor tissue enrichment and high-efficiency tumor tissue penetration and exhibits excellent anti-tumor effect under the combined effect of FRU and DOX.This provides practical strategies and expands new ideas for the combination of anti-angiogenesis drugs and chemotherapeutic drugs for tumor treatment. |
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