Construction Of Lipid-coated Calcium Peroxide Nanocarriers For Cancer Therapeutic Enhancement

ObjectiveOxygen-containing active small molecules such as oxygen(O₂)and reactive oxygen species(ROS)in tumors are not only involved in normal life activities such as cell signal transduction,energy metabolism,proliferation and apoptosis,but also have a significant impact on the efficacy of anti-tumor strategies.For example,hypoxic cells in solid tumors express hypoxia-inducible factor-1(HIF-1)to upregulate some proteins that promote drug efflux,such as P-glycoprotein(P-gp)and multidrug resistance protein 2(MRP2),which reduce the antitumor efficacy of chemotherapy.Moreover,HIF-1 can hinder the penetration of drugs or nanodrugs in the tumor matrix,and lower their anti-tumor effect by increasing collagen levels in the tumor extracellular matrix(ECM).As another type of oxygen-containing small molecules,ROS such as hydrogen peroxide(H₂O₂)and hydroxyl radicals(·OH)possess the ability to directly destroy biological macromolecules and kill tumor cells due to their oxidizing properties.Based on the principle of Fenton reaction,the emerging chemodynamic therapy(CDT)catalyzes the H₂O₂ in tumor cells by exogenous delivery of metal ions(such as iron ions)to produce·OH to kill tumor cells.However,the content of H₂O₂ inside the tumor cells is also extremely limited,therefore the efficacy of CDT is compromised.Thus,it is of great significance to increase the content of such oxygen-active substances(such as O₂ and H₂O₂)in tumor cells for improving anti-tumor outcomes.MethodsCalcium peroxide(CaO₂)is a degradable material with good biocompatibility and p H sensitivity.Under acidic conditions,CaO₂can produce a large amount of H₂O₂.Under neutral conditions,it can slowly react with water to produce O₂ and H₂O₂.By the catalysis of catalase(CAT)and manganese dioxide(Mn O₂),H₂O₂ could be disproportionated to increase the O₂-producing efficiency of CaO₂.Based on the versatility of CaO₂,we have prepared three types of lipid-coated CaO₂ nanocarriers to deliver chemotherapeutic drugs to tumor sites,while achieving the supply of exogenous O₂ or H₂O₂ to enhance the antitumor efficacy of chemotherapy or CDT.ResultsFirstly,we prepared a lipid-coated CaO₂/cisplatin(DDP)nanoparticle(Lipo CaO₂/DDP)using a reverse microemulsion method,and explored the O₂production and glutathione(GSH)reduction by CaO₂,which synergistically enhanced the efficacy of cisplatin:(1)CaO₂reduced intracellular GSH levels and the formation of sulfhydryl-DDP complex;(2)the produced O₂ decreased the expression of HIF-1 and its downstream MRP2 protein,further inhibited the efflux of sulfhydryl-DDP complex.In both in vitro and in vivo experiments,we proved that CaO₂ could enhance the efficacy of DDP in a liver cancer cell model(Hep G2).However,this study also revealed the instability of CaO₂ and insufficient oxygen production of CaO₂without a catalyst.In order to improve the oxygen production efficiency of CaO₂,we designed a Mn O₂-doped CaO₂ nanoparticles as a carrier for the chemotherapeutic doxorubicin(DOX).Due to the water-instability of CaO₂,we introduced a solid lipid with tumor lipase selective degradation,monostearate(MS),as the outer layer of nanoparticles to improve their systemic stability and tumor selectivity.We also found that this kind of smart nanoparticles could release free DOX in the ECM to achieve a neighboring effect and enhanced drug penetration.Meanwhile,the produced O₂ further enhanced drug penetration by reducing the expression of collagen in the ECM.In addition,the water-sensitive disintegration of CaO₂ increased the release rate of DOX,and O₂ also effectively reduced the pumping of DOX by inhibiting P-gp.Taken together,the multiple processes of DOX transportation from nanoparticles to cells(release,permeation,and retention)had been comprehensively optimized,ultimately enhancing its anti-tumor effect.We selected the SKOV3 human ovarian cancer cell line with highly-expressed lipase and its nude mouse xenograft model as therapeutic objects,and the results showed that the efficacy and safety of DOX had been significantly improved.Finally,we used this solid lipid-coated CaO₂ nanocarrier to co-deliver DOX and a novel Fenton catalyst,iron oleate,for synergistically enhanced chemo-chemodynamic therapy.It is worth mentioning that,under the catalysis of iron ions,the H₂O₂ produced by CaO₂ could not only generate·OH to kill tumor cells,but also generate O₂ to enhance the chemotherapeutic effect of DOX towards hypoxic cells.The synergistic anti-tumor mechanism,efficacy and safety of the nanomedicine had also been verified in SKOV3 cells and animal models.ConclusionIn summary,we prepared three kinds of lipid-coated CaO₂ drug delivery systems to increase the levels of oxygen-containing active small molecules(O₂ or H₂O₂)in tumor microenvironment,which enhanced chemotherapy and chemodynamic therapy.This research not only clarifies a variety of novel and effective anti-tumor mechanisms,but also provides a safe and effective drug delivery platform for other O₂ or ROS-dependent anti-tumor strategies such as photodynamic therapy,radiotherapy,and immunotherapy.