Preparation And Characterization Of PH/redox Dual-sensitive Platinum(Ⅳ)Polymeric Prodrug-based Synergetic Drug Delivery System

In recent decades,great efforts have been made in the progress of nano-drug delivery systems(Nano-DDS)in cancer therapy,and a few of them have undergone clinical trials.However,the efficacy of these DDS is compromised in clinical trials,which is due to the lack of an optimized carrier with precise structure and composition,low drug loading capacity and pre-mature drug releasing during circulation as well as batch-to-batch differences in pharmacokinetics and pharmacodynamics.To address this,various prodrug-based Nano-DDS were developed to integrate the superiority of prodrug and nanotechnology.Among them,polymer prodrug conjugates self-assembled nanosystems,in which chemotherapeutic drugs are covalently conjugated to polymers by cleavable linkers(pH or redox sensitive bonds,for example),have shown great potential to realize more efficient and precise drug delivery and controlled drug release.The common polymers used in the preparation of prodrug generally include liner polymers,amphiphilic block copolymers,dendrimers,and comb-like polymers,in which have plentiful active functional groups for drug loading and surface modifications.Although these polymers have precise architecture and provide a simple route for preparing Nano-DDS,their utilization is limited by the aspects such as complex synthesis and purification procedure,carrier-induced toxicity and immunogenicity.Besides,the nanosystems need to release loaded drugs as soon as they accumulated in tumor sites or internalized by tumor cells,however,most hydrophobic drugs are located in the core of the self-assembled nanocarriers,and their drug release rate and amount is restrained by the breakage of cleavable bonds between drug molecular and polymer skeleton.In response to such a situation,liner polymer prodrug containing drugs in the main chain,that is,drugs or their derivatives acting as constitutional unit of the polymer backbone instead of coupled onto polymer chains as side groups,were developed to achieve the precise control over the composition of Nano-DDS as well as increase the drug loading content without causing burst releaseHerein,we focus on the investigation of multi-functional Pt(Ⅳ)based polymers using the small molecular antitumor drug cisplatin and acid-labile ortho ester contained compounds,and then the polymer could self-assemble with another antitumor drug Doxorubicin(DOX)in aqueous solution to give a synergetic drug delivery system.The Pt(Ⅳ)monomer with two carboxyl groups(DS-CP)was readily synthesized using cisplatin after oxidizing and further modification.Afterwards,the DS-CP was polymerized with 2,2’,((4,4’-(Oxybis(methylene))bis(1,3-dioxo-lane-4,2-diyl))bis(Oxy))(OE monomer)which was previously obtained,and then successfully prepared a pH and reduction dual stimuli-sensitive polymer,designed as P(OE DS-CP).Nuclear magnetic resonance(NMR)and gel permeation chromatography(GPC)were used to conform the structure and the molecular weight of P(OE DS-CP),the results show that the polymer has the right structure and the number-average molecular weight(Mn)is 1.04×104The prepared amphipathic polymers P(OE DS-CP),could easily self-assemble into micelles in water(designed as OCM).Considering that doxorubicin(DOX)can restrain the DNA remodeling through intercalating duplex DNA and further inhibit the repairing of cisplatin-damaged DNA by inhibiting the activity of topoisomerase Ⅱ(TOP2),overcome cancer multidrug resistance(MDR)to a certain degree.Thus,DOX was loaded into the micelles to establish a combination drug delivery system,designed as OCM/D.The size and micromorphology of micelles were observed by dynamic light scattering(DLS)and transmission electron microscopy(TEM),the results show that the OCM and OCM/D was around 190 nm and 210 nm with regular morphology and uniform distribution.Furthermore,the CMC value of OCM was 1.12×10-5 mg/mL,have long blood circulation.And the OCM micelles have excellent stability in various physiological environments(saline,PBS(pH 7.4),FBS or RPMI 1640)according to the micellar stability experiments.The content of doxorubicin in OCM/D micelles was determined by microplate reader.The drug loading rate and encapsulation rate of OCM/D was about 10%and 70%respectively.In vitro drug release experiments were carried out under different pH environments and reduction conditions to verify the pH/redox-triggered drug release behaviors.The results demonstrate that both OCM and OCM/D had excellent acid and reduction sensitivity,moreover,the drug release rate increased under the acid and reduction conditions.The in vitro cytotoxicity tests were examined via MTT assay after co-culture of OCM and OCM/D with cancer cells for 24 and 48 hours.The results show that both micelles could effectively kill cancer cells and exhibited a dose and time-dependent cytotoxicity.That is,the cell viability rate decreased with the increase of the corresponding drug concentration,and the cytotoxicity after co-culture for 48 hours was higher than that of 24 hours at the same concentration.The cellular uptake of OCM/D was qualitatived and quantitatived by a confocal laser scanning microscope(CLSM)and flow cytometry.The results demonstrated that DOX-loaded micelle OCM/D could easily internalized by cancer cells and released DOX quickly in acidic and reductive environment.To study the tumor-penetrating ability and growth inhibition of the prepared micelles,three-dimensional tumor-like multicellular spheroids(MCs)are used as an in vitro tumor model to co-cultured with micelles OCM/D.The results demonstrate that OCM/D showed better penetration and accumulation ability to MCs than free DOX with the prolongation of co-culture time.In addition,micelles OCM and OCM/D has greater growth inhibition efficiency than free DOX and cisplatin,and the DOX-loaded micelles OCM/D was more effective than free drug or single drug formulation,which further proves the advantages of the synergistic drug delivery system.H22 subcutaneous tumor-bearing mice were established to investigate the antitumor activity and drug biodistribution in various organs and tumor tissues in vivo The results of drug biodistribution assay show that micelles OCM and OCM/D possess a relative long circulating time and effectively improve the drug accumulating ability in tumor tissues.In vivo antitumor activity results show that the tumor volume of mice in saline group increased rapidly after injection for 9 days.Whereas,the other groups have different antitumor effect,the tumor growth inhibitions ratio of these group was 28.36%(CP),55.48%(CP+DOX),62.82%(OCM)and 85.90%(OCM/D).Finally,we use the histological section to further examine the toxicity and antitumor efficacy of micelles OCM and OCM/D.The results show that there was significant damage in different treatments group compared with the saline-treated group,more importantly,the tumor section in micelles OCM/D the higher degree of injury than other groups.Whereas,it was found some damage in kidney and heart and there was no obvious damage in micelles-treated groups.In summary,this platinum(Ⅳ)polymeric prodrug-based synergetic drug delivery system with pH/redox dual-sensitive has great potential in the field of cancer therapy.