Preparation Of Stimuli-responsive Polyethylene Glycol-based Polymer Micelles For Antitumor Activity

Doxorubicin is a commonly used chemotherapy drug in clinical practice.It has a killing effect on tumor cells in various growth cycles.However,doxorubicin has problems such as poor water solubility,short half-life and strong side effects.Polyethylene glycol?PEG?is a commonly used polymer in the construction of polymeric micelles which has the advantages of being non-toxic and avoiding protein adsorption and prolonging the circulation time of nanoparticles.Compared with normal physiological tissues,tumor microenvironment has the characteristics of low pH,low oxygen and high blood pressure,etc.In view of the deficiencies in the physical properties of doxorubicin,a pH-sensitive polymeric micelle system based on polyethylene glycol was constructed.The pH-sensitive structure of the orthoester was used to achieve the purpose of releasing DOX from drug-loaded micelles to the microenvironment of the tumor site,and the effects of different molecular weight PEG on cellular uptake of polymeric micelles were explored.Furthermore,disulfide bond was introduced to the pH-sensitive polymeric micelle to endow it with reduction sensitivity,and PEG-CYS-?12-HDA?-TDA?PCT?was synthesized to enhance the sensitivity of pH-sensitive polymer micelles containing orthoester to the tumor microenvironment and accelerate drug release.Using pH-sensitive PEG-ADH-?12-HDA?-TDA?PAT?as control,the model drug doxorubicin?DOX?was loaded into micelles to form DOX/PCT and DOX/PAT micelles,and their pharmaceutical properties,cytotoxicity,cellular uptake,cellular uptake mechanism and anti-tumor effects in vivo were further studied.?1?Effect of PEG Molecular Weight on pH-sensitive polymeric micellesA simple method was used to prepare pH-sensitive doxorubicin?DOX?-loaded micelles?DOX/POD?based on polyethylene glycol-2-?octadecyloxy?-1,3-dioxan-5-amine?POD?polymers,and the influence of PEG molecular weights?1 K,2 K,and 5 K?on in vitro drug release and antitumor effect was further studied.All three POD polymers can self-assemble to form micelles with a particle size of 100?300 nm and the micelles containing smaller molecular weight PEG form denser micelles.In the in vitro drug release experiment,the release behavior of the three drug-loaded micelles showed significant pH sensitivity,and the cumulative release of DOX/POD_5K micelles containing the largest molecular weight PEG was the highest,reaching?75.9±1.8?%at 48 h.In the cytotoxicity test,as the molecular weight of PEG increased,the toxicity of the drug-loaded micelles decreased.The IC₅₀ of DOX/POD_1KK is?0.196±0.024??g·mL^-1,which has the strongest cytotoxicity;the cytotoxicity of DOX/POD_2K is the second,of which IC₅₀ is?0.404±0.038??g·mL^-1;DOX/POD_5K toxicity is the weakest,of which IC₅₀ is?0.651±0.071??g·mL^-1.Cell uptake experiments showed that as the molecular weight of PEG decreased,the uptake of cells in-creased,thus resulting in enhanced anti-tumor effect.The above results indicate that low molecular weight pH-sensitive POD micelles can achieve more efficient drug delivery,leading to the conclusion that PEG molecular weight can surely affect anti-tumor effects of pH-sensitive nanocarriers.?2?Construction and evaluation of pH/reduction dual-sensitive rapid drug release systemIn order to promote the rapid release of polymer micelles at the tumor site,a reduction-sensitive and pH-sensitive polymer PCT containing disulfide bonds and orthoester groups was synthesized.The PCT conjugates self-assembled in water and entrapped doxorubicin to form drug-loaded micelles having an average particle size of?214.2±9.8?nm and a zeta potential of?-2.390±0.305?mV.The encapsulation efficiency of DOX/PCT was?95.4±1.97?%,and the drug loading was?11.4±1.74?%,indicating that PCT can effectively encapsulate doxorubicin.The particle size of PCT micelles increased sharply under low pH and reduction?high glutathione GSH concentration?condition,and the average particle size increased from about 200 nm to more than 1?m,indicating that it has pH-sensitive and reduction-sensitive properties.In the in vitro drug release experiment,the cumulative release of DOX/PCT micelles in the simulated tumor environment of PBS?pH 5.0,GSH concentration of 20 mM?reached?89.7±11.7?%at 72 h,while in simulated the normal physiological environment of PBS?pH 7.4,GSH concentration of 0 mM?was only?16.7±6.1?%,indicating that drug release behavior of DOX/PCT micelles showed a difference between normal physiological environment and tumor environment;meanwhile,compared with the cumulative release of pH-sensitive DOX/PAT control group being?57.3±4.7?%,DOX/PCT micelles were able to release drugs more quickly.In cytotoxicity experiments,compared with DOX/PAT micelles,DOX/PCT micelles can rapidly release drugs and kill tumor cells in a low pH and reducing environment in tumor cells to achieve an effective anti-tumor effect.DOX/PCT micelles entered tumor cells through clathrin-mediated endocytosis,caveolin-mediated endocytosis and macropinocytosis,and then depolymerized in a lysosomal acidic environment and an intracellular reducing environment,allowing DOX to rapidly enter the nucleus and exert cell killing effect.In vivo antitumor activity studies showed that DOX/PCT micelles had a better inhibitory effect on tumor growth than DOX/PAT micelles.Therefore,pH/reduction dual-sensitive PCT conjugates are potential to be used for the delivery of antitumor drugs.