Camptothecin Nanoprodrug Possessing Dual-responsiveness To Intracellular Microenvironment For Amplified Cytotoxic Potencies

Selective activation of prodrug nanomedicine in cell interiors is deemed to be crucial in pursuit of precision anti-tumor therapy.In the present study,we attempted to synthesize amphiphilic diblock copolymer of poly（ethylene glycol）-polylysine（PEG-PLys）based on ring-opening polymerization.Theγterminal amines of lysine units were conjugated with camptothecin through redox-responsive disulfide linkage,followed by conversion of the rest amines of PLys into carboxyl groups.Core-shell architectural nanoparticles could be achieved by self-assembly of the yielded amphiphiles,characterized to possess CPT-linked PLys segments as internal core and PEG segments as external shell.Furthermore,CaPO₃ was attempted to precipitate upon the yielded core with aids of the carboxyl groups.1.Synthesis and characterizations of PEG-PLys（ss-CPT&SAA）/CaPThe PEG-PLys diblock copolymer was prepared by ring-opening polymerization.The product was characterized by ¹H NMR for verification of the chemical structure and quantification of the degree of polymerization of PLys（approximately 60）.Subsequently,the redox responsive camptothecin prodrug（CPT-ss-OH）was conjugate at theγ-terminal amine of the lysine unit to prepare the amphiphilic polymeric PEG-PLys（ss-CPT&SAA）.The product was verified by ¹H NMR and MS,followed by precipitation of CaP onto the core compartment of PEG-PLys（ss-CPT&SAA）.Subsequent investigations confirmed the formation of uniform nano-particles,with a hydrodynamic diameter of approximately 63.0 nm,ζpotential close to neutrality,and excellent colloidal stability.The hemolysis experiment implied excellent its biocompatibility,as evidenced by its negligible hemolysis rate（less than 3%）at the concentration of 1 mg/m L.2.Dual-responsiveness of PEG-PLys（ss-CPT&SAA）/CaPThe prodrug nanomedicine was constructed to possess sequential responsiveness in the intracellular microenvironment:1)The inorganic CaP precipitate can not only exclude the internal payload from the premature reaction,but also rapidly dissolve in the acid lysosomal compartment,which elicits a rise in osmotic pressure and promotes the transportation of the prodrug into the cytoplasm.The intracellular trafficking studies verified the lower entrapment in lysosomal compartment with aids of CaP by CLSM,thereby facilitated trafficking into cytoplasm;2)The nanomedicine could not only respond to the acidic environment but also liberate redox gradient in cytoplasm,particularly GSH,wherein complete CPT liberation could be achieved in presence of 10 m M GSH;3)Excellent protection of the internal CPT payloads and facilitated cytoplasmic transportation owing to CaP demonstrated improved arrival of CPT at its pharmaceutic destination（nucleus）,wherein supplementation with CaP promoted approximate 4.9 fold accumulation of CPT in nuclei.3.Cytotoxic potenciesParticularly,significantly lower cell viabilities were observed for PEG-PLys（ss-CPT&SAA）/CaP,well consistent with the results in pertinent to the intranuclear CPT level.Note that IC50 of PEG-PLys（ss-CPT&SAA）/CaP was calculated to be approximately 1.1μM,significantly lower than PEG-PLys（ss-CPT&SAA）（IC50:approximately 4.4μM）and relatively higher than free CPT（IC50:approximately 0.5μM）.This augmented cytotoxic potencies from PEG-PLys（ss-CPT&SAA）/CaP should benefit from the improved translocation from endolysosomal entrapment by virtue of the functional components of CaP.