Study On The Fabrication Of Phenylboronic Acid-functionalized Ultra-pH-sensitive Micelle And Its Antitumor Activity

Chemotherapy is still an essential therapeutic strategy for clinical cancer therapy.However,the clinical therapeutic efficacy of most antitumor drugs is severely restricted by modest water solubility,poor cancer selectivity and severe side toxicity.With the development of the technology,drug delivery systems(DDS)including liposomes.nanoparticles and polymeric micelles have been paid great efforts to developed as new methods for cancer treatment.They can improve drug’s water solubility,extend circulation time and enhance drug accumulation at the tumor tissues by the enhanced permeability and retention(EPR)effect,leading to the increased drug bioavailability and reduced off-target toxicity.Polymeric micelles,as a hopeful drug delivery system based on amphiphilic copolymers,have attracted considerable attention for excellent stability and high drug loading efficiency.Nevertheless,achieving tumor targeting only through EPR effect is very difficult because of complicated tumor microenvironments such as high interstitial fluid pressure(IFP)and dense extracellular matrix(ECM).In order to improve the targeting ability of micelles and enhance the internalization by cancer cells,a number of active targeting ligands such as biotin,phenylboronic acid(PBA)and folic acid(FA)can be effectively grafted onto the surface of nanocarriers to bind with their receptors,which were overexpressed on the tumor cell membrane.However,even if the nanocarriers pass through the barriers exist in the blood and enter into tumor cells,the therapeutic efficacy are still far from reaching the expectations due to sluggish drug release in tumor tissue and/or inside the tumor cells.Based on the study of tumors,there exists an evident pH gradient in tumor extracellular environment(pH 6.5-7.2)and endosomes/lysosomes compartments(pH 4.5-5.5).Therefore,a series of pH-sensitive nanocarriers have been developed and studied for accelerating drug release.While,most of the drug carriers mainly aim at the design of acid environment in tumor cells,and often fail to accurately distinguish extracellular environment and normal physiological environment(pH 7.4),resulting in lower tumor targeting and severe side effects on normal tissues.So,it is necessary to design a ultra-pH-sensitive drug carrier that can stably exist in blood vessels,achieve a more accurate response to the slightly acidic environment of the tumor,improve the targeting of tumors to realize more ideal killing effect by using inexpensive and available raw materials,as well as a simple preparation process.Based on the above research,a type of novel dextran-based tumor-targeted ultra-pH-sensitive micelles(PBA/Dex-g-OE)were prepared with a facile strategy by grafting acid-degradable orthoester(OE)monomer and tumor-targeting ligands 3-aminophenylboronic acid(3-APBA)to the hydrophilic backbone of dextran,and in vitro antitumor ability have been evaluated.The main research contents are as follows:1.An acid-labiled orthoester monomer(OE)was obtained by previous reported work.Then,dextran was grafted with orthoester monomer and 3-Aminophenylboronic acid to prepared a new type of PBA-functionalized ultra-pH-sensitive conjugates(PBA/Dex-g-OE)with the help of CDI.Otherwise,a non-targeted conjugates(Dex-g-OE)was also prepared as for control.The structure of two conjugates were verified by 1H NMR,and the graft ratio of OE and PBA was determined by elementary analysis and ICP-MS,respectively.2.The two amphiphilic copolymers PBA/Dex-g-OE and Dex-g-OE were self-assemble into micelles.The diameter of two micelles were 160.2 nm(PBA/Dex-g-OE)and 139.7 nm(Dex-g-OE),respectively.The morphologies of two micelles were determined by TEM with regular spherical shape and well distribution.The stability test proved that both micelles have desirable physiological and long-term stability.DLS and 1HNMR analysis were utilized to study the hydrolysis ability of two micelles in different acid conditions,the results showed that both micelles with ultra-pH-sensitivity and the dynamic change of particle size as follows:keep stable in neutral(pH 7.4)environment;initially become larger and then smaller at pH 6.5;rapid dissolve at pH 5.5,with the possibility of rapid drug release.3.The model antitumor drug doxorubicin(D OX)was effectively loaded into micelles with the drug loading content(DLC)of 17.1%(PBA/Dex-g-OE-DOX)and 11.8%(Dex-g-OE-DOX),respectively.The different of DLC between two micelles may because of the coordination between the amino of DOX and the boron of phenylboronic acid.In vitro drug release result revealed that the release profiles of two DOX-loaded micelles with similar drug release behavior at same acidic pH conditions,showing the acid-responsive and ultra-pH-sensitivity.As the pH value decreased,the accumulative released drug and release rate increased obviously;and at pH 6.5,the drug was released slowly about 38～46%in 24 h.The MTT assays result revealed that blank micelles with excellent biocompatibility,and DOX-micelles showed a dose-dependent cytotoxicity towards HepG2 and H22 cells.Otherwise,PBA/Dex-g-OE-DOX-displayed higher cytotoxicity than Dex-g-OE-DOX at all tested concentrations.DOX-loaded micelles and free DOX were incubated with HepG2 multicellular tumor spheres(MCTS)to evaluate the ability of different formulation in tumor penetration and inhibition.Compare to free DOX,DOX-loaded micelles could be more effectively penetrate into MCTS leading to destruction of spheroids and most of tumor cells were killed on day 5.It is worth noting that the PBA/Dex-g-OE-DOX with highest penetration and inhibition ability at all tested time.In a word,PBA-modified ultra-pH-sensitive PBA/Dex-g-OE micelles provide great potential for tumor target drug delivery and acid-triggered release of sufficient anticancer drugs in tumor tissues,thus leads to the superior therapeutic efficacy.