Research On Polymer Anti-tumor Nano-drugs For Combination Therapy Of Cancer

Cancer has been a extremely serious threat to human health,and people are terrified to hear about it.In addition to the three traditional therapies with surgery,radiotherapy and chemotherapy,there are many emerging therapies for cancer treatment,especially tumor vascular blocking therapy.The application of nanotechnology in vascular blockade therapy can improve the tumor vascular targeting and the therapeutic efficacy of vascular disrupting agents.Since single therapy cannot achieve ideal antitumor effect,it is necessary to carry out reasonable combination therapy to improve the anti-tumor effect and even to inhibit tumor recurrence and metastasis.Nano-drugs based on polymer can prolong the circulation time in the body and passively target tumor tissues through the"enhanced permeability and retention effect"?EPR effect?.Besides,the in vivo bioavailability of drugs can be improved after nanometerization.In this paper,the optimization of polymer carriers,the construction of polymeric vascular disrupting agents?VDAs?combretastatin A4?CA4?nano drug,and the development of combination therapy were studied systematically.The internal and external killing strategies based on vascular disrupting-photodynamic therapy and the anti-metastasis strategies based on vascular disrupting-hypoxia-induced factor inhibition were well designed,respectively.The antitumor effect and mechanism of combined therapy were further investigated.The research contents and conclusions of this paper are as follows:?1?A series of poly?L-glutamic acid?grafted polyethylene glycol monomethyl ether polymers?PLG-g-mPEG?were prepared.Specifically,the influences of grafted mPEG with different molecular weight?550,2 K,5 K and 10 K?and different mass ratio of PLG to mPEG?PLG/mPEG=1/1,PLG/mPEG=1/2 and PLG/mPEG=1/4?were investigated.Cisplatin?CDDP?was used as the model drug.The CDDP nanoparticles?CDDP-NPs?were prepared by loading CDDP with PLG-g-mPEG.The carriers were screened through the pharmacokinetics,solubility and the drug loading capacity of PLG-g-mPEG.The results showed that blood circulation time of CDDP-NPs was prolonged with the mass ratio of PLG/mPEG decreasing and the molecular weight of mPEG increasing.And the DLC of PLG-g-mPEG increased with the PLG/mPEG mass ratio increasing.And the optimal carrier of PLG₁₆₀-g-mPEG5k?mass ratio of PLG/mPEG was 1:2?was used for further study.?2?The PLG-g-mPEG-CA4 nanoparticles?PGC?were prepared by Yamaguchi reaction where PLG-g-mPEG with good biocompatibility and biodegradability was used as the carrier.And PLG-g-mPEG-TPP nanoparticles?PGT?were constructed using PLG-g-mPEG and photosensitizer amine porphyrin through condensation reaction between carboxyl group of PLG-g-mPEG and amino group of porphyrin.PGC was pH sensitive and can be released quickly under alkaline conditions.PGT coould produce singlet oxygen?¹O₂?to kill tumor cells under the light irradiation.The cytotoxicity of PGC,PGT and combination of PGC and PGT on 4T1,HeLa and MCF-7 cells were determined and the results showed that the combination of PGC and PGT had obvious cell proliferation inhibition effects under the light irradiation.The combinated therapy not only showed good anti-tumor effect in vitro,but also indicated a potential advantage in treating solid tumors.In a word,the tumor cells in the center of the tumor could be"starve to death"due to lack of oxygen and nutrients after the blood vessels were blockaded.Meanwhile the tumor edge cells would be killed by ¹O₂ produced by photodynamic therapy.Therefore,the combination of photodynamic therapy and vascular blockade therapy was finally realized to improve antitumor effect.?3?The favorable results were obtained based on the combination of vascular disrupting agents?VDAs?and photosensitizers.The drug loaded system was further optimized,such as the two drugs were loaded together on the same carrier to realize the combination of the two cancer treatments.Simplifying the drug delivery process will improve the quality of life of patients.The VDAs CA4 and the photosensitizer TPP-NH₂ were simultaneously bonded to PLG-g-mPEG by Yamaguchi reaction and the condensation of carboxy-amino group.The dual-drug nanometer system?PGCT?with CA4 and TPP realized the combined treatment of photodynamic therapy and vascular blockade therapy.The results of CA4 drug release in vitro showed that the PGCT was pH sensitive and the drug was released rapidly under alkaline conditions.Meanwhile,the PGCT could produce singlet oxygen?¹O₂?and kill tumor cells under the laser irradiation.The cytotoxicities of 4T1 cells,HeLa cells and MCF-7 cells were evaluated.The results showed that PGCT had certain cytotoxicity on HeLa cells and MCF-7 cells,but PGCT had good cytocompatibility to 4T1 cells without light irradiation.However,the PGCT dual-drug nanosystem has a good inhibited effect on tumor cell proliferation under the light condition.The two drugs are co-loaded on the same carrier,which still maintains the effect of vascular blockage and photodynamic therapy,that is,cutting off the supply of nutrients in the center of the tumor and producing ¹O₂ to kill the tumor marginal cells,achieving the effect of internal and external killing.This work achieved a combined use of dual-drug nanosystems containing two kinds of tumor therapeutic drugs?photosensitizers and vascular blockers?,which provideed a favorable basis for the diversification of tumor treatment methods for dual-drug nanosystems and even multi-drug nanosystems.?4?In order to further improve the solubility of PGC,the PGC was treated by NaHCO₃,and finally obtained better solubility of CA4 nano-drug?CA4-NPs?.Although the CA4-NPs had great potential to inhibit cancer growth,it was still a challenge to avert tumor recurrence and metastasis after treatment.It was mainly tightly associated with hypoxia induced by CA4-NPs,which could activate many downstream genes regulating tumor growth and metastasis.Herein,to relieve a tumor hypoxia microenvironment,the mTOR inhibitor Temsirolimus was employed to modulate the tumor microenvironment after CA4-NPs treatment.In vitro MTT experiments strongly verified that the combination of Temsirolimus with polymeric CA4-NPs exhibited an additive toxicity to 4T1 cells.An in vivo study with the 4T1 mammary adenocarcinoma model revealed that the result was consistent with the proposed scenario,combination therapy with CA4-NPs and Temsirolimus significantly suppressed tumor growth,compared to either CA4-NPs or Temsirolimus,and the inhibition rate to 4T1 tumor with a volume of 300 mm³ was 71%.The mechanism underling combination treatment was confirmed by western blotting and immunofluorescence staining,and the results demonstrated that Temsirolimus could inhibit HIF1?expression.Thus,this work provided a mechanistic rationale for the use of VDAs and mTOR inhibitor to enhance antitumor efficacy,delay tumor recurrence and inhibit tumor metastasis.The related research results in this paper provided a theoretical and experimental basis for the research and development of VDAs nano-drug and the combined treatment system.Meanwhile,it provided a foundation and insights into the strategy based on the combination treatments of vascular blockade therapy and tumor influencing factor inhibition.