Construction Of Thermal And PH Dual-sensitive Nanogel And Its Synergistic Mechanisms Against Breast Cancer

5-fluorouracil(5-FU)is an analogue of uracil,by inhibiting deoxy thymidylate synthase and being incorporated into RNA and DNA,it interferes with nucleoside metabolism,leading to cytotoxicity and cell death.5-FU is widely used in breast cancer treatment.However,the emergence of severe side effects greatly limits the clinical efficacy of 5-FU.Therefore,it is urgent to explore new therapeutic strategies.With the rise of nanomedicine,it is expected to resolve the existing problems of traditonal chemotherapeutics.As one of the most widely employed nanomaterials,nanogels are three-dimensional hydrogel particles in a nanoscale size range formed by crosslinked swellable networks with a high capacity of holding water.The porous crosslinked network endows nanogel systems with superior in vitro/in vivo stability and allows for incorporation of a plethora of hydrophilic bioactive drugs.As a result,nanogels become an optimal delivery system for the hydrophilic chemotherapeutic drug 5-FU.Moreover,by introduction of functional monomers,stimuli-responsive nanogels can be obtained,namely smart nanogels.Among them,poly N-isopropyl acrylamide(PNIPAM)is by far the most extensively used nanomaterial to construct thermal-responsive nanocarriers.The volume phase transition temperature(VPTT)of PNIPAM was approximately 32 ?,the microstructures and properties of PNIPAM-based nanogels will shift as the temperature changes.However,there have been few mechanism studies of the thermal-sensitive nanogels at the cellular level.As we all know,due to the reversible protonation ability of cationic polyethyleneimine(PEI),it can not only effectively pass through the bilayer phospholipid membrane structure,but also responsive to pH variations,making it suitable for intracellular mechanism investigation of nano systems.So in this study,we synthesized smart nanogels with thermosensitive PNIPAM as its core and pH-responsive PEI as its shell [Poly(N-isopropyl acrylamide-g-ethylene amine),PNIPAM-g-PEI] via radical grafting copolymerization and in-situ self-assembly.Auxiliary data received from the dynamic and static light scattering instrument,zeta potential instrument and transmission electron microscope further confirmed the well-defined core-shell structure and the finely tunable surface physicochemical properties of the nanogels.With the increase of PEI chains,the VPTT of the nanogels rose from 32 ? to 39 ?.In vivo and in vitro studies indicated that the smart nanogels was an effective carrier for the water-soluble drug 5-FU.The 5-FU-loaded nanogels(5-FU-gel)could be specifically and efficiently accumulated at the tumor site.As a result,the intracellular drug concentration increased and the therapeutic efficacy against breast cancer was significant and persistent.MDA-MB-231 breast cancer cell line was used as the target cells for further exploring the death way of cancer cells induced by drug loaded nanogels as well as its related intracellular mechanisms.Cell Counting Kit-8(CCK-8)assay showed that 5-FU-gel could greatly inhibit the proliferation of MDA-MB-231 cells;with the incubation temperature rose from 37 ?(T<VPTT)to 40 ?(T>VPTT),the endocytosed nanogels were significantly increased,which confirmed the passive targeting effect by the thermosensitive polymers.Studies showed that the main intracellular pathway of nanocarriers was through endosome-autophagosome-lysosome.After endocytosis,the nanogels were tracked by confocal laser scanning microscopy(CLSM)through fluorescence colocalization and the autophagy associated protein LC-3 detection,3-methyladenine(3-MA)and chloroquine(CQ)were added to regulate the formation of these organelles.The results showed that nanogels were mainly accumulated in lysosomes;with combination of 3-MA to suppress the initiation step of autophagy,the nanogels were mainly resided in endosomes;with combination of CQ to block the downstream step of autophagy,the nanogels were mainly located in autophagosomes.Furthermore,CCK-8 assay revealed that the cytotoxicity of 5-FU-gel,5-FU-gel+CQ,5-FU-gel+3-MA towards MDA-MB-231 cells gradually decreased,indicating that nanogels mainly played its role in lysosomes.Then we checked whether PEI chains that can be protonated would trigger lysosome-dependent mechanisms.By detecting lysosomal membrane-associated protein LAMP-2 and lysosomal enzyme cathepsin B,we found that the PEI chains of 5-FU-gel would be protonated in the acidic lysosomal lumen,and the nanogels swelled through the proton sponge mechanism,resulting in the release of vast amount of the encapsulated drug 5-FU to interfere with nucleotide metabolism in the target cells,thereby killing them.Meanwhile,the strongly protonated PEI could disrupt the lysosomal membrane,so that cathepsin B was disseminated to cytocol and activated the downstream signaling pathways.Then the apoptosis-inducing ability of 5-FU-gel was evaluated by FITC-Annexin V and PI double staining and was visualized by flow cytometry.The results demonstrated that 5-FU-gel could unexpectedly induce apoptosis of MDA-MB-231 cells,and the apoptosis degree was greater at 40 ? than that at 37 ?.Then mitochondrial membrane potential change and cytochrome c release were observed by CLSM,and caspase-9 protein expression was examined by western blot.We discovered that after incubation with 5-FU-gel,the mitochondrial membrane potential decreased,which promoted release of cytochrome c and increased caspase-9 protein expression level.These fingings together confirmed that 5-FU-gel induced cancer cell apoptosis via a mitochondrial-mediated caspase-dependent pathway.In conclusion,the thermal and pH dual-sensitive nanogels can facilitate its uptake by cancer cells due to its passive-targeting effect of the thermosensitive core.Meanwhile,we are the first to discover that the protonated PEI shell can not only promote release of the loaded hydrophilic drug 5-FU,but also synergistically induce caspase-dependent apoptosis by lysosomal and mitochondrial membrane destruction.Our research represents an enormous advancement in the study of the synergistic mechanisms of cancer cell death induced by 5-FU loaded smart nanogels and its potential value in clinical application.We anticipate our study will provide theoretical basis for better design of organelle-targeted nanomedicne.