Construction And Antitumor Application Of Hybrid Lipid Nanoparticles Based On Amorphous Calcium Carbonate

Although nanostructured lipid carriers have irreplaceable advantages over other counterparts,such as high biocompatibility and cell affinity,they have failed to meet the various demands in cancer therapy due to the limitation of lipids,such as low drug loading capacity and lack of responsiveness.As a result,the development of a hybrid form of nanostructured lipid carriers,which maintains the advantages of lipids,but also have the ability to accommodate different types of drugs with controllable behaviors for responsive drug release in cancer site,is the emerging orientation.It is well established that calcium carbonate,a naturally nontoxic biomineral,has broad biomedical utilizations owing to its intrinsic advantages of biocompatibility,simple chemical composition,low cost as well as ease of large-scale production Although numerous drug delivery systems have been developed based on crystalize calcium carbonate(CCC)nanoparticles,their slow drug release is not suitable for controlled drug delivery.Amorphous calcium carbonate(ACC)is a special member of calcium carbonate family.Despite from the common advantages of calcium carbonate,the phenomenon that ACC will dissolve and/or transfer polymorph in water,which will in turn lead to burst release of its payload,is considered as a effective approach to solve the release dilemma of CCC.However,the aqueous instability of ACC also severely hinder its extended application in nanomedicine.Therefore,a hybrid ACC carrier should be developed to retain and protect ACC from the hostile aqueous environment during delivery while capable of exposing ACC within cancer cells to exert its water responsive nature to effectively release the payload.As a result,the combination of nanostructured lipid carriers and calcium carbonate would be a preferable choice.We firstly prepared ACC by vapor diffusion method and study the influence of different factors.Phospholipids(PL)was applied to modify ACC to construct hybrid lipid nanoparticles(PL/ACC).The optimal PL:ACC ratio was obtained and size-controlled PL/ACC were prepared.The in vitro cellular uptake,endocytosis mechanism,in vitro and in vivo localization and distribution of PL/ACC were explored.It provides theoretical guidance for formulation design and structural modification of hybrid lipid nanoparticles based on ACC and lipid materials.Subsequently,doxorubicin hydrochloride(DOX)was selected as a model drug and loaded into ACC during synthesis to prepare ACC-DOX.Subsequently,the obtained ACC-DOX nanoparticles were further modified with PL and targeting group folic acid(FA).The tumor targeted and drug-loaded hybrid lipid nanoparticle PL/ACC-DOX with diameter of around 100 nm was constructed.We found that PL/ACC-DOX was stable in the systemic circulation and was capable of specifically targeting tumor tissue overexpressing folate receptors.The accumulation of nanoparticles in tumor tissue overexpressing folate receptors was 2.11-fold of that in tumor tissue underexpressing folate receptors.Upon reaching the tumor tissue,with the degradation of the protective PL layer,ACC could fully and efficiently release the encapsulated DOX in both water-sensitive and acid-sensitive ways within cancer cells.PL/ACC-DOX achieved comparable cytotoxicity to free DOX in in vitro antitumor assays,and was superior to free DOX in in vivo antitumor assays.The tumor inhibition rate of PL/ACC-DOX was 68.3%,which was 21.1%higher to that of free DOX without significant cytotoxicity on main organs.Finally,ACC-DOX was subjected to hydrophobic modification and loaded into glyceryl monostearate(MS)to prepare MS/ACC-DOX.MS is one of the substrates of monoacylglycerol lipase(MAGL),which is highly expressed in tumor tissues.Therefore,MS/ACC.-DOX could achieve MAGL-sensitive drug release.The viability of MS/ACC-DOX(DOX concentration:2.g/mL,48 h)treated SKOV3 cells was merely 35.7%of L02 cells subjected to the same treatment.Studies have shown that MS/ACC-DOX,which is excessively taken up in host cells,is released from the dead host cells after being degraded to expose ACC-DOX,and is transformed into a"Pandora's Box"to continue its water-sensitive ability and release drug.The sustained release of the drug triggers"neighbouring effect"that effectively infects peripheral cells,causing 76.0%of peripheral cells to develop apoptotic symptoms.At the same time,this effect also promotes deep penetration of MS/ACC-DOX into tumor tissue.As a result,MS/ACC-DOX achieved superior in vivo antitumor assays with the tumor inhinition rate of 73.1%,which was 21.1%higher to that of free DOX.In adddtion,the main organ toxicity caused by DOX chemotherapy was overcame with no significant side effects on the surrounding tissues of tumors and normal tissues rich in lipase.