PH Sensitive,Sequential Drug Delivery Nanosystem For Cancer Therapy

Malignant tumors are a major disease which seriously threatens human health.Exploring new anti-tumor treatment targets and strategies is a hot spot in field of life science research today.The occurrence and development of tumor is a complex biological process driven by multiple factors.Therefore,conventional model of clinical anti-tumor therapy is to combine multiple treatment methods or multiple drugs.Although combination of multiple drugs can improve anti-tumor effect,conventional combination of drugs can improve anti-tumor effect.However,due to untargeted distribution of pharmacokinetics and untargeted cells at pharmacodynamic level offtarget effects have significant toxicity in vivo which cause significantly limited efficacy in therapy.Because of good tumor targeting properties,nano drug delivery system has obvious advantages for combined treatment with anti-tumor drugs.At present,nano delivery systems for drug combination therapy are always foucus on deliveing drugs with pharmacological synergy to tumor cells.However,when targets of combined drugs are located in outside of tumor cells(such as intracellular microtubules)and tumor cells(such as tumor cell membrane receptor extracellular domain,tumor extracellular matrix,etc.)respectively.The sequential release delivery system should be developed to solve problem.After CS NP has been administered intravenously and enriched at tumor site through enhanced penetration and retention effects.One drug is first released that acts on the extracellular target of tumor and then nanocarrier is loaded with another one via endocytosis which introduced into tumor cells and acts on.The advantage of this delivery system is that drug acting on extracellular target of tumor can be released quickly before nanocarriers are taken up into tumor cells as whole to avoid off target.We used advantage of weak acidity tumor microenvironment.Two micell nanoparticles which loaded with different drugs were connected through chemical bonds have been constructed a p H-sensitive sequential drug delivery system.In this study,PEG-PLA and PEG-C7A-MA which with p H sensitivity polymer materials were synthesized and nanoparticles were prepared by emulsification respectively.Transmission electron microscopy,DLS and small animal imaging instruments were used to measure the particle size and physics of p H sensitive satellite nanoparticles(Satellite NP),cation lipid doped core nanoparticles(Core NP)and core-satellite nanoparticles(CS NP).Nanopartilces chemical properties are characterized as well.The reaction rate of maleimide-mercapto covalent bond linking Core NP and Satellite NP was investigated by quantification of thiol and maleimide functional groups.Furthermore,drug loadings of cabotinib(XL184)and paclitaxel(PTX)were determined by high performance liquid chromatography and drug release proflie of CS NP under different p H conditions was investigated.Then,CS NP and 4T1 tumor cells were co-incubated in vitro in p H 7.4 and p H 6.6values PBS respectively.Using a multifunctional microplate reader and a laser confocal microscope to observe cytotoxicity,cell uptake,and tumor multicellular sphere experiment results and investigate CS NP penetration ability.Subsequently,CS NP and HUVEC cells were co-incubated and inhibition of cell migration and tube formation was investigated by using a microscope and a multifunctional microplate reader.Finally,CS NP was injected into orthotopic breast tumor-inoculated mice through tail vein.The anti-tumor effect of the nanomedicine was evaluated by examining the tumor tissue volume,mouse survival time and body weight.It was further evaluated by immunohistochemical staining of tumor tissue sections.The experimental results show that particle sizes of Core NP,Satellite NP,and CS NP are 111.9 nm,30.9 nm and 189.5 nm respectively.After mixing Core NP and Satellite NP in a weight ratio of 1: 3 that 95 % of final product can be obtained in 30 minutes.The drug loadings of PTX,XL184 in CS NP were 3.01 %,0.81 % respectively.At p H 7.4,the cumulative release of PTX and XL184 in 24 h was 15 % and 10 %.At p H 6.6,the cumulative release of PTX and XL184 was 18 % and 95 %.Because under p H 6.6 value condition that Satellite NP which at periphery around CS NP can burst within 3 minutes.This program not only release XL184 and also cause nanosystem sliding interface displacement as well.The particle size of delivery system decreases and positively charged Core NP is exposed.The rapid flip from-1.5 m V to20.1 m V which can promote more Core NP adhere to negatively charged cell membranes and enter cells through adsorption mediated endocytosis and promotes penetration into deep side of tumor shpere.In vitro cell experiment results show that compared to in p H 7.4,CS NP and 4T1 co-inhibition in p H 6.6 value was similar to weak acid value in tumors(p H 6.5-6.8).Using advantage of sequential drug delivery that 4T1 tumor cells can take up Core NP and increase by 30 % cytotoxicity than control.Compared with XL184 loaded in CS NP,prototype XL184 which released rapidly from Satellite NP can increase inhibition rates in HUVEC tube formation and migration by25 % and 35 % respectively.The results of in vivo pharmacodynamic experiments showed that compared with saline group and CS NP(empty)group,survival time of mice in each CS NP group was prolonged to varying degrees,but CS NP(XL184 + PTX)experimental group had shown best tumor suppression effect.Tumor tissue immunohistochemical section results further show that compared with other experimental groups CS NP(XL 184 +PTX)can not only significantly inhibit tumor cell proliferation in tissue but also reduce microvessel density.CS NP with sequential drug delivery has a safe and effective antitumor treatment effect and also has no toxic side effects.pH sensitive sequential delivery Core-Satellite nanosystem in this study can provide new theoretical ideas and guidance for design sequential delivery systems.