Effect Of CaCO₃ Nanoparticles On Intracellular Ca²⁺ Concentration And Function Of Immune Cells

Background and objective:As an essential component of human life,metal elements have been used in many ways to regulate mmunological function in recent years.Calcium is one of the essential metal elements in human body,mostly in solid form.In the body,free Ca²⁺controls intracellular signal transduction and regulation,also known as the"second messenger".Many recent studies have demonstrated that the immune environment can be regulated by changing the concentration of Ca²⁺in cells.Due to the existence of numerous pathways on the plasma membrane of cell membrane and organelles,it is difficult to artificially regulate the concentration of calcium ions in cells by maintaining the concentration difference of nearly 10,000 times inside and outside cells,preserving Ca²⁺homeostasis and ensuring the normal activity and function of different cell groups.The rapid development of nanocarriers technology provides a new way for the treatment of diseases and drug delivery,which is expected to make up many shortcomings of traditional therapeutic drugs,such as poor water solubility of most anticancer drugs,lack of targeting ability,non-specific distribution,systemic toxicity and therapeutic index defects.Meanwhile,the size effect and targeting potential of nanomaterials provide a feasible strategy for regulating intracellular calcium concentration.Calcium carbonate is one of the most abundant substances in the earth's crust and one of the most basic inorganic materials in nature.It is mainly divided into four forms:the crystalline form of calcite,spheroidal aragonite,spheroidal aragonite and amorphous calcium carbonate.Amorphous calcium carbonate has been widely used in biological mineralization,biocatalysis and biopharmaceuticals due to its advantages of easy to obtain,simple preparation process,excellent application performance and good biocompatibility.As a kind of inorganic nano delivery system,Ca CO₃ nanoparticles have the characteristics of availability,safety,low cost,biocompatibility,p H sensitivity,bone conductivity and slow biodegradation.At the same time,the slow degradation of the Ca CO₃ matrix can prolong the retention time of the drug and decompose into water and carbon dioxide.In addition,it has the potential to target tumors.Nowadays,Ca CO₃nanoparticles have been widely used in the construction of drug carriers,such as transdermal transportation through gastrointestinal pathway as antibacterial carriers,delivery of tumor contrast agent components,vaccine adjuvant,anti-cancer genes,etc.,and also used in tooth and bone joint repair.However,it has not been reported that Ca CO₃ nanoparticles are used to regulate the immune microenvironment by changing intracellular calcium ion concentration,and their effects on immune cells and tissues have not been clearly explained.Therefore,exploring the above problems can provide new methods and strategies for disease prevention and treatment using Ca CO₃nanoparticles,and improve the effect of combination therapy or emerging therapy.Methods:Firstly,three kinds of Ca NPs with different particle sizes were prepared using the Nanosystem Platform.The particle size of the three particles were characterized with the sizes of 260 nm,370 nm and 530 nm,and the electric potentials were-36 m V,-34m V and-36 m V.The morphology of the three particles was observed by transmission electron microscope.In vitro experiments,particles were labeled with fluorescence to detect the uptake rate of particles by cells.Secondly,cell lines were incubated with particles of different sizes and dosages to determine the ratio of apoptosis and Ca²⁺content in cells and mitochondria.Finally,confocal microscopy was applied to observe the mitochondria and their damage.In vivo experiment was performed as following:Ca NP530 was administrated intravenously through the tail vein,then sorting out the different immune cells were sorted out and the intracellular Ca²⁺concentration changes in the body were determined.Flow cytometry was applied to analyze immune cells in the spleen based on the change of the quantity and the total number of cells.Finally,preliminary exploration of the expression of activated surface markers of macrophages and DC cells were preformed,as well as the effect and influence of Ca NPs on immune cells was also explored.Results:1.Three sizes of CaNPs with diameters of 248.2±1.6 nm,370.3±1.9 nm and 534.3 ±3.7 nm were prepared by using Nano Assemblr^TM Platform,and spherical particles were obtained.2.In vitro experiments:CaNPs with different sizes and dosages could induce apoptosis of cells to different degrees,and Ca²⁺oscillations in cells and mitochondria were observed after incubating with those nanoparticles.3.In vitro cell lines:after incubated with CaNP530,mitochondria were damaged by the increased Ca²⁺concentration.4.In vivo experiments:the immune cell population in whole spleen cells of mice was separated by flow cytometry after a large dose of Ca NP530 injected through the tail vein.Significant change in Ca²⁺concentration was detected in the population of immune cell from whole spleen cells of mice.5.In vivo experiments:the proportion and number of immune cells in whole spleen cells were changed and the total number of cells was significantly increased after injection of Ca NP530 through vein injection and the expression of CD86,a surface activation marker,was significantly increased in macrophages and DCs.6.Security verification:continuous injection of large doses of CaNP530 through the tail vein showed that there were no obvious toxic and side effects,indicating that CaCO₃ nanoparticles have a good safety.Conclusion:In this study,it was found that the size of CaNPs affected the degree of inducing cell apoptosis by causing intracellular Ca²⁺overload,leading to calcium shock in mitochondria and inducing mitochondrial damage.In vivo,by changing the content of Ca²⁺in immune cell population,the proportion and number of whole spleen immune cell population can be affected,and macrophages and DC cells can be activated.This study provides new research ideas for the role of Ca CO₃nanoparticles in immunotherapy,and provides new opportunities for future disease treatment and prevention.