Design,Preparation And Properties Of PH-Responsive Co-assembly Nanoprodrugs Based On Tetraphenylethene And Doxorubicin Modified Glycopolypeptide Analogues

Nowdays,polymeric micelles（PMs）loading chemotherapeutic drugs have been widely used to inhibit tumor growth,however,their therapeutical efficacy is still modest in clinical applications.This problem is caused by the five cascading steps encounted by the PMs including the circulation（C）in blood stream,the accumulation（A）in tumor sites,the penetration（P）into deep tumor tissue,and the internalization and the drug release（R）inside the cells after the PMs are intravenously injected.Hence,complexity of the CAPIR cascade results in low efficiency of overally delivered drug content within the tumor cells,which leads to the weak therapeutical efficacy of the PMs.Such complexity is really reflected by contradictory requirements for physicochemical properties of the PMs in each step of the CAPIR cascade.Typically,the PMs should stay stable to avoid premature drug release in the alkalescent blood circulation,but they should have capacity to release plenty of drugs inside the tumor cells within a short time to stop the cell growth efficiently.In response to such needs in drug delivery,p H-sensitive PMs are employed to overcome the incompatibility of the CAPIR cascade.This kind of delivery system can inhibit the drug burst at physiological p H but can release most of the encapsulated drugs due to the p H-sensitive triggered by the more acidic extracellular matrix（ECM）and the lysosome/endsome（p H 4.0-7.0）.Even though this feature of the p H-sensitive PMs can be simulated by extracellular modeling experinents through p H-dependent drug release dynamics,it can’t reflect the real intracellular fate of the PMs in response to the p H alteration.Particularly,it is still a challenge to visulize the p H-dependent drug release process inside the tumor cells.Moreover,precise manipulation of drug release dynamics of the p H-sensitive PMs under higher drug content is still quite difficult.In this work,in order to obtain p H-sensitive titumor PMs with ability to visually monitor intracellular dynamic drug release as well as to control drug release behaviors,we prepare tetraphenylethene（TPE）/doxorubicin（DOX）functionalized p H-sensitive nanoprodrugs formed by co-assembly glycopeptide analogues.This work begins with a novel amphiphilic glycopeptide analog namely HA-PFLG,in which the hydrophilic segment of HA is hyaluronic acid with cancer cell overexpression receptor targeting capicity,and the hydrophobic segment of N₃-PFLG is a polyamino acid with furan ring at its side chains.After that,the antitumor drug of doxorubicin（DOX）is linked to the side chains of the N₃-PFLG through a p H-sensitive acylhydrazone bond to obtain the first glycopeptide analogue of HA-PFLG-DOX;Then,the TPE having aggregation-induced luminescence effect（AIE）is coupled with the N₃-PFLG side chain to obtain another glycopeptide analogue of HA-PFLG-TPE.Among them,the DOX and the TPE are grafted onto the N₃-PFLG side chains through Diels-Alder（D-A）coupling reaction.The advantage of this method is that the D-A coupling does’t require any reactive additives,which avoids sequent purification steps of the polymers,thus improving of final nanoparticle preparation efficiency greatly.The HA-PFLG-TPE and the HA-PFLG-DOX are then co-dissolved in dimethyl sulfoxide（DMSO）with the mass ratio of 0:1,1:1 and 2:1.The corresponding HA-PFLG-DOX and its co-assembled micelles are preparaed by dialysis in aqueous solution.With the mass ratio of 0:1,1:1 and 2:1,the resulting drug loading/encapsulation ratios are 24.9%/86.4%,11.2%/90.0%,7.6%/96.7%,respectively;Average particle size measured by dynamic light scattering（DLS）of the four micelles including the HA-PFLAG,the HA-PFLAG-TPE,the HA-PFLG-DOX and HA-PFLG-DOX/HA-PFLG-TPE（1/2）is 119.1,142.6,194.6 and 202.2 nm,respectively;Their average potential is-19.9,-19.1,-18.9 and-19.7 m V,respectively;Transmission electron microscopy（TEM）shows that the corresponding particles are spherical micelles,olive-like micelles,uniform spherical micelles and irregular spherical micelles,respectively;In addition,in vitro drug release of DLS and DOX is used to study the effect of the acid sensitivity of micelles and the TPE content changes on the release behavior of the DOX.The results show that the DOX can release more and faster DOX under higher acid condition;With increasing TPE content,the DOX releases less and more slowly at each p H value.These results indicate that the micelles will have p H-responsiveness in the tumor microenvironment,and both the release rate and the amount of the drug can be controlled by changing the TPE content;Meanwhile,due to the AIE feature of the TPE and the fluorescence energy resonance transfer（FRET）effect between DOX/TPE,the visual monitoring of the DOX dynamic delivery process can be achieved within Hela celles for the HA-PFLG-DOX/HA-PFLG-TPE（1/2）micelles.MTT cytotoxicity assays show that the micelles can inhibit proliferation of the Hela cells.By using flow cytometry（FCM）to study the endocytosis of micelles,we have found that endocytosis pathway of the micelles is determined by type of the micelles:the HA-PFLG-DOX micelles depend on clathrin-mediated endocytosis,while the co-assembled micelles depend on both the clathrin and the caveolin-mediated endocytosis.Results of laser scanning confocal microscopy（CLSM）indicate that the micelles can deliver DOX into cell nucleus successfully after being endocytosed.In short,in this work we have provided an effective method to prepare a multifunctional antitumor nanodrug treatment platform that has the ability to visually track intracellular drug release,control the drug release,ensure high-efficiency drug loading,reduce burst release and achieve actively targeting at the same time.