Self-Assembled Dimer Prodrug Nanoparticles For Hypoxia-Activated Prodrug Combination Therapy

Hypoxia-activated prodrugs(HAPs)are a class of drugs that are non-toxic or have low toxicity to normal tissues,and can be activated by highly expressed reductase in the hypoxic area of the tumor to produce toxicity.Compared with traditional chemotherapy drugs,HAPs have unique advantages in in vivo safety due to their special activation mechanism.HAPs are currently in the clinical research stage,the rapid elimination from the body and the difficulty of spreading to the hypoxic area of the tumor are important factors that restrict their clinical application.The therapeutic effect of HAPs alone is limited,and current research focuses on the combined application of HAPs with traditional chemotherapy,radiotherapy,tumor embolotherapy,and immune checkpoint inhibitors.The application of nano-drug delivery systems(nano-DDSs)in combination therapy has been a research hotspot in recent years.However,the traditional combination strategy of non-covalent co-loading two or more drugs in the same carrier does not work well,and its drawbacks include low drug loading,poor stability,and premature drug leakage.The self-assembled prodrug nano-DDSs are formed by the prodrug conjugates themselves,which show the dual advantages of prodrug strategy and nano-DDS strategy.So,self-assembled prodrug nano-DDSs have great potential in the application of combination therapy.Photodynamic therapy(PDT)refers to a treatment in which a photosensitizer is excited under the irradiation of light,and then reacts with oxygen,water or other molecules to generate reactive oxygen species(ROS)to destroy tumors.However,in the process of PDT,oxygen is a major factor restricting its therapeutic effect.Because the tumor is hypoxic as a whole,and the oxygen content continues to decrease with the distance from the surface of the solid tumor,the anti-tumor efficacy of PDT is greatly limited.Studies have shown that PDT will further aggravate the hypoxic state of tumors after it takes effect,and when combined with HAPs,it will promote the activation of HAPs and achieve synergistic therapeutic effects.Herein,we have designed three dimer prodrug compounds.PR104A and pyropheophorbide a(PPa)are bridged by thioether,thioketal or non-sensitive carbon chain,respectively,to obtain PR104A-S-PPa,PR104A-TK-PPa and PR104A-PPa.All three dimer prodrugs could self-assemble to form nanoparticles(NPs)(PSP NPs,PTKP NPs and PP NPs)under the stabilization of DSPE-PEG2k.The prodrug-NPs had uniform particle size and good stability.Under laser irradiation,PSP NPs and PTKPNPs could achieve PR 104 A release triggered by both photoinduced electron transfer(PET)and ROS.Prodrug-NPs exhibit a molecular aggregation state,so they suffer from aggregation-induced quenching(ACQ)effects,resulting in low ROS generation efficiency.It is precisely because of the stacking of prodrug molecules that the PET reaction is possible.After laser irradiation,the excited PPa preferentially reacts with the sulfur atoms in the connecting chain to generate sulfur radical cations.The sulfur radical cations can promote the cleavage of the C-S bond and promote the release of PR104A.When the PET reaction causes the self-assembled prodrug-NPs to depolymerize,the ACQ effect disappears,and the excited PPa restores the ability to generate ROS through PDT with the surrounding oxygen.The generated ROS could further promote the response of ROS-sensitive thioether and thioketal,releasing free PR104A again.The light-triggered dual-modality responsive prodrug-NPs with different release characteristics according to the change of nanoparticle aggregation morphology could release free PR104A to the maximum.The released PR104A could be effectively activated under the action of oxygen consumption by PDT,thereby exerting a synergistic anti-tumor effect.The research results proved that compared with PSP NPs,PTKP NPs had better circulation stability,more tumor accumulation,and faster PET-triggered and ROS-triggered dual-modality PR104A release.Therefore,PTKP NPs showed the best anti-tumor and anti-metastasis abilities.Traditional chemotherapeutics were reported to be able to amplify their tumor penetration and tumor killing capabilities through neighboring effects.Neighboring effect means that after the chemotherapeutics-loaded NPs are taken up by tumor cells,the tumor cells become drug reservoirs.When the tumor cells die,the remaining drugs will be released to continue to infect other neighboring tumor cells.The specific mechanism of the neighboring effect is still not very clear.We speculate that this effect may be mediated by apoptotic bodies.The neighboring effect of chemotherapy is not obvious in practice,which may be due to the consumption of chemotherapeutic drugs in the process of drug delivery to the deep part of the solid tumor,and the internal hypoxic tumor cells are resistant to traditional chemotherapy,leading to penetration limitations of traditional chemotherapeutic drugs.HAPs are inactive in normoxic cells and can selectively kill hypoxic tumor cells.Therefore,the combined use of HAPs and traditional chemotherapeutic drugs may overcome the delivery barrier and promote neighboring effect-mediated tumor penetration and comprehensive tumor killing effect.Herein,we bridged camptothecin(CPT)and PR104A through disulfide bond or non-sensitive carbon chain to obtain the dimer prodrug compounds CPT-SS-PR104A and CPT-CC-PR104A,respectively.Both prodrugs could self-assemble to form prodrug-NPs(CSSP NPs and CP NPs)under the stabilization of egg yolk lecithin and DSPE-PEG2k.When the self-assembled NPs accumulate in the tumor site,the outer normoxic tumor cells take up the NPs,and under the action of high levels of intracellular GSH,the disulfide bond breaks and two free drugs can be released simultaneously.CPT acts on normoxic cells to cause apoptosis,while PR104A remains non-toxic.The apoptotic bodies released by the apoptotic cells encapsulate the remaining drugs and deliver them to the interior through the neighboring effect.After reaching the tumor hypoxic zone,because CPT is consumed by normoxic tumor cells during the infiltration process,the content of active CPT is reduced,and hypoxic tumor cells are also resistant to CPT,so CPT toxicity decreases.However,PR104A can be activated in hypoxic tumor cells to produce toxicity,killing hypoxic tumor cells to achieve continued delivery to the deep tumor mediated by the neighboring effect.The results of the study proved that CSSP NPs exhibited good reduction-responsive dual-drug release ability,stable systemic circulation,high tumor accumulation,good toxicity to hypoxic and normoxic cells,good penetration and tumor killing in 3D cell sphere model,and good anti-tumor and anti-metastasis abilities.