CaCO₃ nanoparticles pH-sensitively induce blood coagulation as a potential strategy for starving tumor therapy

Based on the concept of starving tumor therapy, in this study we put forward a new idea that the pH-sensitive Ca²⁺ delivery of calcium carbonate nanoparticles (CaCO₃ NPs) induced blood coagulation of tumor vessels, and first explored the effect of CaCO₃ NPs on the in vitro and in vivo blood coagulation by acid stimulus. CaCO₃ NPs with a size of about 100 nm and a porous structure of several nanometers were synthesized in an emulsion system, which showed a high loading capacity (49%) of doxorubicin hydrochloride (DOX) with an encapsulation efficiency of 98% and a pH-sensitive drug delivery. The hemolysis test showed that CaCO₃ NPs were blood compatible. The in vitro Ca²⁺ delivery and blood clotting tests indicated that CaCO₃ NPs pH-sensitively released Ca²⁺, and caused rapid blood coagulation at pH 5.0 but no thrombus at pH 7.4. Confocal laser scanning microscopy showed that after uptake by MCF-7 or MDA-MB-231 breast cancer cells, CaCO₃ NPs mainly distributed in endosomes/lysosomes within the initial 2 h and then decomposed by acid stimulus, leading to the intracellular delivery of Ca²⁺ that subsequently migrated outside the cells. CaCO₃ NPs were nontoxic to NIH3T3 mouse fibroblasts, but highly toxic to both MCF-7 and MDA-MB-231 cells after loading DOX. After topical administration into the breast tumors of mice, CaCO₃ NPs evoked significant thrombosis and hemorrhage of tumor vasculature by hematoxylin-eosin and Masson's trichrome staining. These results indicated that CaCO₃ NPs could induce blood coagulation via acid stimulus, showing potential applications in blocking tumor vessels for starving tumor therapy.