| Polyphosphoesters(PPEs)are biodegradable polymers made of phosphoester repeating units,which can be completely degraded into small molecules by hydrolysis,enzymatic degradation and autocatalytic degradation under physiological conditions.PPEs not only have good biocompatibility and biodegradability,but also have non-specific protein resistance,so that they can be the alternative of PEG as "stealth" materials to protect nanocarriers in vivo.In addition,their pentavalent phosphorus atoms allow high-density and modular functional modifications,which is essential for the development of new multifunctional polymers.Therefore,the performance advantages of PPEs make them a good candidate of drug delivery materials,and have attracted a great attention in the field of biomedical materials research.Up to now,although the multifunctional drug carriers based on PPEs have been widely studied,most of the carriers are assembled micellar systems formed by linear PPEs.However,the critical micelle concentration(CMC)brings them an inherent drawback of dissociation in the highly diluted biotic environment.The dissociation caused by the low thermodynamic stability of micellar structures limits their probability in clinical application.Therefore,in order to improve the structural stability and optimize the performance of PPE-based carriers,this study constructed a kind of multi-arm star phosphoester copolymer with dendritic polyaminoamine(PAMAM)as the core and biodegradable PPEs as the arms.The carriers based on this copolymer fully combines the homogeneous stability of the multi-arm star structure and the excellent biocompatibility,complete degradability and multi-functional modification of PPEs,which can be used for efficient drug delivery and anti-tumor treatment.Various and multi-functional targeted drug delivery systems were constructed through the regulation of PAMAM generation and the ratio of hydrophilic/hydrophobic chain segments,and the terminal modification of copolymers.Then,we explored their potential application in targeted therapy of tumor.The main research contents and results are summarized as follows:(1)The functional cyclic phosphate monomers BYP and MP that separately containing butyne side chains and methoxy side chains were firstly prepared by a substitution reaction from 2-chloro-2-oxy-1,3,2-dioxyo-heterocyclopentane(COP).Then,the multi-arm star phosphoester copolymers were synthetized by the ring-opening polymerization(ROP)of cyclic phosphoester monomers,initiated by the hydroxyl-terminated first generation PAMAM dendrimer.The controllable synthesis was realized by the control of monomer feed ratio and reaction time.On the basis of controllable synthesis,an 8-arm star copolymer PAMAM-PBYP-PMP was prepared with definite structure,narrow molecular weight distribution and adjustable ratio of hydrophilic and hydrophobic chain segments.The hydrophobic anticancer drug doxorubicin(DOX)and the obtained copolymers were co-assembled into drug-loaded supramolecular micelles,and DOX was physically encapsulated into the inner core and hydrophobic inner shell of the micelles.The prepared micelles have spherical structure with smooth surface and uniform size.They also possess high drug loading capacity and load efficiency(up to 90.68 wt%).In vitro drug release experiments were conducted,and the results showed that the release amount of DOX was very low in the neutral release medium,while DOX was released sustainably in the acid enzymatic condition.In vitro cytotoxicity experiments showed that the drug-loading micelles could effectively inhibit the growth of Hela cells,so that they could be used as a drug delivery system.(2)Based on the controllable synthesis of 8-arm star amphiphilic phosphoester copolymers,folic acid(FA),an active targeting molecule,was introduced at the end of the copolymer chains through the chemical reaction between the terminated hydroxyl groups and carboxyl groups.In this case,the FA-modified amphiphilic polyphosphate copolymer PAMAM-PBEP-PMP-FA was prepared.Afterwards,DOX was physically encapsulated in the hydrophobic segment of the copolymers by self-assembly to get the FA-targeted and DOX-loaded supramolecular micelles.Supramolecular micelles have uniform spherical structure,stable hydrodynamic size,high drug load efficiency and low non-specific protein adsorption.In vitro drug release experiments showed that the DOX release amount was very low in the neutral medium(pH 7.4),while gradually increased along with time in the acid enzymatic environment(pH 5.0 and phosphatase),which benefits from the combined effect of an pH-responsive and an enzyme-responsive release behavior.In vivo and in vitro cytotoxicity tests showed that the drug-loaded supramolecular micelles displayed non-toxic to normal cells and organs,and had an effective inhibitory effect on the growth of Hep G2 cells and tissues.(3)In order to improve the structural stability of the nanocarriers and simplify the preparation process,a multi-arm random copolymer PAMAM-P(BEP-co-MP)was prepared by a simple "one-pot" ring-opening polymerization.First,the fourth-generation 64-arm dendritic PAMAM was used as the macromolecular initiator to initiate the functionalized phosphoester monomers.Then,the PBA targeting group was conjugated to the end of the polymer chain to prepare the PBA-modified multi-arm random copolymer PAMAM-P(BEP-co-MP)-PBA.Subsequently,the drug-loaded unimolecular micelles(UMs)PAMAM-P(BEP-co-MP)-PBA/DOX were prepared by physical encapsulation of DOX.The prepared UMs have uniform spherical structures and stable hydrodynamic sizes.Besides,the information of the number of copolymer repeat units was theoretically calculated by comparison of hydrodynamic sizes and the contour lengths.The obtained DOX-loaded UMs exhibited a slow and sustainable release behavior in an intracellular simulated environment.Furthermore,in vitro cytotoxicity and cellular uptake studies demonstrated that the DOX-loaded UMs could specifically recognize and accumulate in HepG2 cells,and then inhibit tumor cell growth effectively.Therefore,this PBA-functionalized unimolecular micelle could be a promising candidate for targeted drug delivery.(4)In order to further improve the circulation stability of DOX-loaded UMs in vivo and avoid the premature release of DOX,a multi-arm block copolymer drug prodrug PAMAM-(PBEP-g-DOX)-b-PMP-PBA was prepared by covalently binding DOX to the nanocarrier framework material.The copolymer prodrugs existed in the form of monomolecular micelles in aqueous solution,and have a uniform spherical structure with uniform size.In vitro drug release experiments showed that they keep stable in the neutral fluid environment and DOX was almost not released,while DOX was sustainably released in the lysosome enzymatic environment.In vitro cytotoxicity results showed that the multi-arm star copolymer PAMAM-PBEP-b-PMP exhibited non-toxic to NIH/3T3 cells,and the PBA-modified prodrug PAMAM-(PBEP-g-DOX)-b-PMP-PBA had obvious cytotoxicity and growth inhibition effects toward HepG2 cells.Cell uptake results showed that PAMAM-(PBEP-g-DOX)-b-PMP-PBA was rapidly and effectively internalized in HepG2 cells with an active targeting ability,which has a great potential in the targeted tumor treatment. |
