Rational Design Of Active Transcytosis Polymers For Effective Cancer Drug Delivery

Recent years witness significant advances in the field of nanomedicines with several nanomedicines clinically translated and many in the pipeline of clinical trials.However,current nanomedicines have not met the expected clinical therapeutic efficacy.An effective nanomedicine must overcome biological barriers to go through at least five steps in the CAPIR cascade(including blood Circulation,tumor Accumulation and Penetration,then tumor cell Internalization and finally drug Release)to deliver the drug into the cytosol of tumor cells.As a result of the inherent size dilemma of nanoparticles and the complexity of tumor microenvironment barriers,traditional approaches are still inefficient to augment tumor accumulation(A)and penetration(P)of nanomedicines in solid tumors.Our previous work shows that the active transcellular delivery(transcytosis)enables efficient extravasation and infiltration of nanomedicines,which opens a new way to overcome the passive diffusion obstacles of the tumor microenvironment and the inherent size limitation of nanomedicines,thus simultaneously breaks the above-mentioned A and P bottlenecks in drug delivery.However,our understanding of the mechanism of active transcytosis is still limited;therefore,this thesis will focus on the design,synthesis and evaluation of active transcytosis-polymer carriers for effective tumor accumulation and penetration.The first part presents a polyzwitterion whose protein non-sticking but phospholipid-binding ability makes it simultaneously capable of long blood circulating and cell-membrane binding,thus quickly transcytosis across endothelial cells for highly effective tumor penetration and infiltration.The zwitterionic polymer,poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate](OPDEA)with a molecular weight of 20 kDa was synthesized.Briefly,living atom-transfer radical polymerization(ATRP)of 2-(N,N-diethylamino)ethyl methacrylate(DEA)was used to prepare PDEA,then PDEA was oxidized into OPDEA.OPDEA was very water-soluble and nearly neutral at the physiological pH(pH 4～10)and had low cytotoxicity both in vitro and vivo.The SDS-PAGE assay demonstrated that OPDEA was poly zwitterionic and thus antifouling to proteins;the simulation,surface plasmon resonance(SPR)and isothermal titration calorimetry(ITC)assay showed that OPDEA bound on red blood cells(RBCs),endothelial cells(ECs),and tumor cells(TCs).The mechanisms of OPDEA’s cellular endo-/exocytosis pathways and active transcytosis process were illustrated via confocal laser scanning microscopy(CLSM),flow cytometry(FCM)and Transwell assays.Finally,the in vitro and in vivo tumor-penetration of OPDEA were observed via multicellular tumor spheroids(MTSs)and tumor-bearing mouse models.In conclusion,once injected,the protein non-sticking and binding on red blood cells enabled OPDEA to circulate long in the blood;its adsorption on the tumor endothelial cells triggered fast transcytosis-mediated active extravasation into tumor interstitium;its subsequent binding on cancer cells triggered macropinocytosis-mediated endocytosis and Golgi apparatus-mediated exocytosis,thus achieved transcytosis-mediated active tumor infiltration.A potent anticancer drug,7-ethyl-10-hydroxycamptothecin(SN3 8),was then introduced to OPDEA.We synthesized the amphiphilic block copolymer of OPDEA with an SN38-containing polymer chain as the hydrophobic block to fabricate the drug-conjugate micelles(OPDEA-PSN38).We used SN38’s phenol(10-OH)ester as the conjugation site because the phenol ester was readily hydrolyzed and released free SN38 in the presence of high levels of glutathione in the tumor.The SN38 moieties were conjugated to the hydrophobic block and buried in the hydrophobic micelle core.The OPDEA-PSN38 formed well-defined micelles of about 51 nm in diameter and negatively charged at the physiological pH(pH 5-8).In general,1)once injected,the RBC-binding ability enabled OPDEA-PSN38 to circulate long in the blood,and had a half-life of 8.2 h.2)Its adsorption on the tumoral ECs triggered fast transcytosis-mediated active extravasation into tumor interstitium,thus accumulated in the tumor site.3)Its subsequent binding on cancer cells triggered macropinocytosis-mediated endocytosis and Golgi apparatus-mediated exocytosis,thus achieved highly effective tumor penetration,even infiltrated in avascular and hypoxic regions.4)The internalized OPDEA-PSN38 was hydrolyzed by GSH to release SN38,thereby induced the death of the tumor cells throughout the three-dimensional tumor spheroids.5)The antitumor experiments demonstrated the OPDEA-PSN38 had radically increased anticancer activity than CPT-11 in a variety of tumor-bearing mouse models(including human xenograft tumor models).At regular administrating dose,OPDEA-PSN38 eradicated not only small tumors(70 mm3)but also the large tumors(500 mm3)at exponential growth.Therefore,the OPDEA’s protein non-sticking and weak phospholipid-binding abilities created a structurally simple but highly effective cancer drug delivery platform with high clinical translation potential.In the last part,we systematically investigated the effect of cationic charge density on the adsorption-mediated transcytosis behavior of polyethylenimine(PEI)in tumor spheroids.Commercially available PEI was amidized with acetic anhydride(Ac)to obtain AcPEI with different charge densities.1)As the amidization ratios increased,the zeta-potentials of AcPEI decreased,and the cytotoxicity against HepG2 cells of AcPEI decreased.2)The PEI and AcPEI24%with relatively higher cationic density could interact with the cell membrane for fast endocytosis.3)AcPEI24%had high transcytosis efficacy,whose binding on tumor cells triggered adsorption-mediated endocytosis and Golgi apparatus-mediated exocytosis,thus achieving highly effective tumor penetration.This fundamental study provided a guideline for engineering the nano-delivery systems with fast adsorption-mediated transcytosis,enhanced tumor extravasation and infiltration abilities.