Recent Developments in CaCO3 Nano-Drug Delivery Systems: Advancing Biomedicine in Tumor Diagnosis and Treatment

Calcium carbonate (CaCO3), a natural common inorganic material with good biocompatibility, low toxicity, pH sensitivity, and low cost, has a widespread use in the pharmaceutical and chemical industries. In recent years, an increasing number of CaCO3-based nano-drug delivery systems have been developed. CaCO3 as a drug carrier and the utilization of CaCO3 as an efficient Ca2+ and CO2 donor have played a critical role in tumor diagnosis and treatment and have been explored in increasing depth and breadth. Starting from the CaCO3-based nano-drug delivery system, this paper systematically reviews the preparation of CaCO3 nanoparticles and the mechanisms of CaCO3-based therapeutic effects in the internal and external tumor environments and summarizes the latest advances in the application of CaCO3-based nano-drug delivery systems in tumor therapy. In view of the good biocompatibility and in vivo therapeutic mechanisms, they are expected to become an advancing biomedicine in the field of tumor diagnosis and treatment.


Introduction
Cancer, a serious health problem that threatens life and health security, has a perennial high incidence and mortality rate, resulting in nearly 10 million deaths worldwide in 2020 [1].Many different therapeutic approaches have been proposed and tested for the treatment of cancer [2].In the last few decades, nanoparticle-based drug delivery systems have gained great attention in chemotherapy due to their distinctive properties and potential applications in various cancers [3,4].Nano-drug delivery systems consist of a combination of drug and medicinal materials with sizes in the range from 1 to 100 nm [5].Nano-drug delivery systems can effectively improve the bioavailability, stability, and in vivo pharmacokinetic properties of drugs with high drug loading and can control the process of drug release [6,7].The traditional theory of passive targeting is dominated by the enhanced permeability and retention (EPR) effect, which suggests that nano-drug delivery systems are able to enrich tumor tissues by virtue of their specific sizes.This is due to the specific physiological characteristics of the tumor region.The hyperpermeability of tumor neovascularization results in enhanced permeability of nanoparticles into the tumor mesenchyme, while impaired lymphatic drainage limits nanoparticle clearance, leading to enhanced retention [8,9].As more and more novel targeting strategies have been proposed, researchers are now able to perform specific biologically or chemically targeted modifications on the surface of the nano-drug delivery system, which is referred to as the active targeting strategy, allowing the nanoparticles to be specifically targeted to the tumor site, enhancing therapeutic efficacy and reducing toxicities [10].
Based on the classification of different types of medicinal materials, we can classify the existing nanomedicine delivery systems into two categories: organic and inorganic, depending on whether they contain organic components or not [11].Inorganic nanomaterials, including gold nanoparticles [12], silicon-based nanoparticles [13], iron-based nanoparticles [14], and calcium-based nanoparticles [15], have shown promising applications in tumor treatment and diagnosis due to their ease of preparation, high drug delivery rate, good biocompatibility, and easy surface modification compared to other nanomaterials.
Calcium carbonate (CaCO 3 ) is a very common inorganic compound in nature and has wide applications in the pharmaceutical and chemical industries [16,17].CaCO 3 exists in nature mainly in crystalline and amorphous forms, and the crystals include aragonite, vaterite, and calcite, while the amorphous type is divided into the unstable amorphous calcium carbonate (ACC) phase and hydrated metastable forms [18][19][20].In cancer treatment, CaCO 3 nanoparticles have shown outstanding advantages due to their high biocompatibility, low toxicity, low-pH responsiveness, and low production cost [21].Due to their good drug encapsulation rate, CaCO 3 nanoparticles can effectively load small molecules and biomolecules for cancer therapy [22,23].As shown in Figure 1, CaCO 3 nanoparticles prepared by a series of different physical and chemical methods can decompose efficiently and releases the encapsulated drugs into the tumor tissues in a targeted manner, thus achieving targeted drug delivery and minimizing the leakage of drugs in the physiological environment of the organism [24][25][26].Meanwhile, the surface mineralization strategy of CaCO 3 nanoparticles has also shown great application value [27].In addition, Ca 2+ in CaCO 3 and the CO 2 generated from its decomposition have also shown important roles in the field of tumor therapy.The "calcium overload" effect [28] based on the significant increase in Ca 2+ concentration in tumor cells can disrupt the normal mitochondrial function of tumor cells.CaCO 3 is also critical for the regulation of acidity in the tumor microenvironment and CO 2 release for drug release as well as imaging enhancement.Given the outstanding characteristics of CaCO 3 nanoparticles in tumor therapy and the advantages of their nano-drug delivery system, we will explore and discuss the mechanisms and application progress of the CaCO 3 -based nano-drug delivery system in tumor therapy in more detail here.

The Synthesis Methods for CaCO3 Nanoparticles
For the preparation of CaCO3 nanoparticles, the main principle is to generate CaCO3 precipitates by mixing solutions containing Ca 2+ (e.g., CaCl2 and Ca(NO3)2) with solutions containing CO3 2− (e.g., Na2CO3 and NaHCO3) [26].By related particle size control methods, the prepared CaCO3 nanoparticles all showed a good nanoscale range.Based on this, the In this review, we first summarize several major methods for synthesizing CaCO 3 nanoparticles, such as chemical precipitation, gas diffusion, and microemulsion.Through these methods, CaCO 3 nanoparticles with certain morphological characteristics and particle size distribution can be successfully synthesized, and different drug molecules or nanostructures can be added during the synthesis process, which can be efficiently encapsulated at the same time with the help of reactions, thus constructing CaCO 3 -based nano-drug delivery systems.Next, we analyzed the mechanisms of CaCO 3 nanoparticles in tumor therapy and diagnosis in detail in combination with the existing constructed nano-drug delivery systems.Finally, we summarize and conclude the current CaCO 3 -based nano-drug delivery systems from the perspectives of encapsulated small molecules, biomolecules, and surface mineralization and provide an outlook on future development.We hope that this review will provide a more systematic overview of CaCO 3 -based nano-drug delivery systems and inspire researchers to design and develop novel related systems for safer and more efficient treatment of tumors.

The Synthesis Methods for CaCO 3 Nanoparticles
For the preparation of CaCO 3 nanoparticles, the main principle is to generate CaCO 3 precipitates by mixing solutions containing Ca 2+ (e.g., CaCl 2 and Ca(NO 3 ) 2 ) with solutions containing CO 3 2− (e.g., Na 2 CO 3 and NaHCO 3 ) [26].By related particle size control methods, the prepared CaCO 3 nanoparticles all showed a good nanoscale range.Based on this, the nano-drug delivery system based on CaCO 3 can be obtained by further drug encapsulation and surface modification [29].In addition, it is also possible to directly use bio-based materials containing CaCO 3 such as cockle shells present in nature for the preparation of CaCO 3 nanoparticles [30].Given below is the description of the synthesis process of the CaCO 3 nanoparticles in terms of different preparation methods.

Chemical Precipitation Method
Chemical precipitation is the direct reaction of Ca 2+ and CO 3  2− in solution under stirring conditions to produce CaCO 3 nanoparticles [31].Compared to other methods, chemical precipitation is simple and has a shorter reaction time.However, it should be noted that when CaCO 3 nanoparticles of a specific size and shape need to be obtained, polyelectrolyte is usually added to interact with Ca 2+ ions to control the nucleation and growth process of CaCO 3 .At the same time, for the reaction conditions, including the initial concentration of reactants, pH, and temperature, different choices will lead to the production of different CaCO 3 crystals [32].Therefore, although the chemical precipitation method is simpler than others, it has many influencing factors.In order to obtain the ideal nanoparticles, special attention should be paid to the changes in nucleation and crystal growth during the reaction.
Table 1 summarizes some of the representative literature on the preparation of CaCO 3 nanoparticles using chemical precipitation methods, including information on raw materials, brief methods, and characterizations.By the direct chemical precipitation process, this method can achieve effective drug loading and surface mineralization modification for some drug molecules and even nanoparticles.
Zhang and Hou et al. [33] used soluble starch as a growth platform for CaCO 3 nanoparticles by adding a mixture of CaCl 2 and soluble starch solution in a flask and stirring.Then, the Na 2 CO 3 solution was quickly added to it, and the reaction was stirred vigorously.Finally, the resulting nanoparticles were collected by centrifugation.Ca 2+ caused different changes in conformation of the added soluble starch.At this time, the starch with different conformation can in turn direct the arrangement of CaCO 3 crystals after the addition of CO 3 2− .The X-ray diffraction pattern results showed that CaCO 3 nanoparticles were a calcite/vaterite mixture, which was more soluble than other polymorphs and suitable for degradation in an acidic environment.Gu and Lu et al. [34] used PEG-P(Glu) copolymers to interact with Ca 2+ and CO 3 2− to prepare CaCO 3 nanoparticles loaded with anti-PD1 antibodies (aPD1).The carboxyl group on glutamic acid (Glu) was able to interact with Ca 2+ to prevent the generation of oversized CaCO 3 nanoparticles, while the PEGylated structure was able to avoid the inter-agglomeration of nanoparticles.The average size of the nanoparticles was ~100 nm, and the aPD1 encapsulation efficiency was ~50% (Figure 2A,B).CM@CaCO 3 @SAF NPs CaCl 2 and Na 2 CO 3 solution SAF NPs@DOX #* 150~160 nm (CaCO 3 @SAF NPs) #* ~200 nm (CM@CaCO 3 @SAF NPs) [35] SH@FP@CaCO Zhang et al. [35] successfully prepared SAF NPs@DOX nanoparticles covered with CaCO 3 by surface mineralization modification of the synthesized CaCO 3 @SAF NPs@DOX by chemical precipitation (Figure 2C).The aqueous SAF NPs@DOX solution was mixed with a CaCl 2 solution and stirred, and then the Na 2 CO 3 solution was mixed to the above reaction system with continuous stirring.After that, the CaCO 3 -coated SAF NPs@DOX were finally obtained by separating the mixture by centrifugation.Transmission electron microscopy (TEM) imaging results showed that the CaCO 3 @SAF NPs@DOX display a homogeneous polygonal morphology with a diameter of ~120 nm.The CaCO 3 -mineralized shell can be detected from the TEM images clearly, and the layer of mineralization has a thickness of about 15 nm (Figure 2D,E).
reaction system with continuous stirring.After that, the CaCO3-coated SAF NPs@DOX were finally obtained by separating the mixture by centrifugation.Transmission electron microscopy (TEM) imaging results showed that the CaCO3@SAF NPs@DOX display a homogeneous polygonal morphology with a diameter of ~120 nm.The CaCO3mineralized shell can be detected from the TEM images clearly, and the layer of mineralization has a thickness of about 15 nm (Figure 2D,E).(E) The TEM image of CaCO 3 @SAF NPs@DOX.Scale bars: 100 nm.Adopted with permission from Ref. [35].2023, Chenteng Lin.

Gas Diffusion Method
The preparation of CaCO 3 nanoparticles by a gas diffusion reaction [24] is based on the volatilization and diffusion of CO 2 in a vacuum-closed system and dissolution in the solution containing Ca 2+ .The precursors used in this method are mainly ethanolic solutions of CaCl 2 and (NH 4 ) 2 CO 3 or NH 4 HCO 3 solid particles; the latter decompose under vacuum to generate CO 2 .The gas diffusion method is also relatively simple to operate.The thermodynamically unstable amorphous calcium carbonate (ACC) phase can be prepared efficiently by the gas diffusion method and by controlling the water content in the reactive organic solvent [48].In contrast to the traditional crystalline form of CaCO 3 , ACC can be easily obtained on a nanoscale.ACC nanoparticles have active acid responsiveness and thermal instability and are easily hydrolyzed in the intracellular environment, which has profound implications for the preparation of a preloaded drug delivery vector and therapeutic system for biomedical applications [48,49].
Table 2 summarizes some of the representative literature on the preparation of CaCO 3 nanoparticles using the gas diffusion method.By adding different drug molecules or nanoparticles to the CaCl 2 solution and mixing them well, it is possible to generate CaCO 3 nanoparticles while achieving efficient loading of the drug.Moreover, by adjusting the temperature and time, CaCO 3 nanoparticles generated by this method have a desirable particle size distribution and excellent morphological characteristics.Han et al. [74] dissolved CaCl 2 in a beaker containing anhydrous ethanol and covered the beaker with a thin film with holes on its surface.They added NH 4 HCO 3 into another beaker and dried them in a vacuum environment for 24 h to prepare spherical CaCO 3 nanoparticles with diameters of about 100 nm.On this basis, by adding different compound molecules, such as doxorubicin (DOX), curcumin (Cur), tizaramine (TPZ), indole green (ICG), and glucose oxidase (GOD) to the CaCl 2 anhydrous ethanol solution, the researchers achieved efficient loading of these molecules by CaCO 3 nanoparticles with the help of gas diffusion-reaction, using CO 2 decomposed from ammonium bicarbonate to bind with Ca 2+ in the solution.
Chu and Zheng et al. [61] added kaempferol-3-O-Rutinoside (KAE), a biosafety flavonoid that can effectively promote Ca 2+ influx and disrupt calcium homeostasis regulation of tumor cells, into a CaCl 2 anhydrous ethanol solution.CaCO 3 @KAE nanoparticles were prepared after being placed together with NH 4 HCO 3 in a vacuum-drying environment for 4 days (Figure 3A).The diameter of CaCO 3 @KAE nanoparticles is ~100 nm and has a uniform morphology with good dispersion (Figure 3B).Calculating the drug-loading amount of KAE in the nanoparticles through UV-vis absorbance spectra, the drug content in the nanoparticles is 18.57 ± 1.34%, which indicates that CaCO 3 nanoparticles can achieve the effective load of KAE (Figure 3C).
nanoparticles were prepared after being placed together with NH4HCO3 in a vacuumdrying environment for 4 days (Figure 3A).The diameter of CaCO3@KAE nanoparticles is ~100 nm and has a uniform morphology with good dispersion (Figure 3B).Calculating the drug-loading amount of KAE in the nanoparticles through UV-vis absorbance spectra, the drug content in the nanoparticles is 18.57 ± 1.34%, which indicates that CaCO3 nanoparticles can achieve the effective load of KAE (Figure 3C).

Microemulsion Method
The precipitation in nano-droplets of microemulsions is one of the common methods for the synthesis of CaCO 3 nanoparticles.Researchers have studied the preparation of CaCO 3 nanoparticles by microemulsion methods such as reversed-phase microemulsion and biphasic microemulsion.Typically, in water-in-oil (W/O) systems, the precipitation of CaCO 3 begins with the mixing of two micellar solutions containing Ca 2+ and CO 3 2− .In this microreactor, the nucleation and growth of CaCO 3 are limited, and the agglomeration of CaCO 3 particles is effectively inhibited.Therefore, CaCO 3 nanocrystals with particularly narrow size distribution, polycrystalline content, and morphology can be synthesized [75,76].At the same time, the supported drug is dissolved in the corresponding water phase/oil phase, which can achieve the efficient loading of the drug while synthesizing CaCO 3 nanoparticles.Compared with the previous methods, the operation of the microemulsion method is more complex, but its requirements for reaction conditions are not as strict.And there are more possibilities for the load selection of water-soluble/waterinsoluble drugs when constructing a nano-drug delivery system.
Table 3 summarizes some of the representative literature on the preparation of CaCO 3 nanoparticles by the microemulsion method, which lists the raw materials, the specific microemulsion method, and the size of the synthesized nanoparticles.Khan et al. [80] prepared CaCO 3 nanoparticles encapsulated with cisplatin by the water-in-oil (W/O) microemulsion method.First, they prepared two types of water-in-oil microemulsions.In brief, the CaCl 2 solution was dispersed in the oil phase to produce an oil-in-water calcium inverse microemulsion.A carbonate phase emulsion was prepared by a dispersive Na 2 CO 3 aqueous solution in an oil phase.A prodrug solution of cisplatin dissolved in chloroform and 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA) were simultaneously added to the carbonate phase.After 20 min of mixing alone, the two phases were mixed and after 30 min, anhydrous ethanol was mixed to disrupt the microemulsion system.The nanoparticles were then centrifuged, washed with anhydrous ethanol to remove surfactant and cyclohexane, and collected.The average diameter of the nanoparticles as measured by dynamic light scattering (DLS) was 217 ± 20 nm; the zeta potential was −23.7 ± 2 mV, and the average polydispersity index was 0.187.The transmission electron microscope (TEM) results indicated that the nanoparticles (NPs) were spherical, with a core of CaCO 3 nanoparticles.
In addition to the W/O microemulsion method, other methods generally based on emulsion techniques including the water-in-oil-in-water (W1/O/W2) double emulsion [86] and oil-in-water (O/W) microemulsion method by using a High-Pressure Homogenization (HPH) [29] have also been used for the preparation of CaCO 3 nanoparticles.
Liu and Feng et al. [83] adopted an improved W1/O/W2 double emulsification method to prepare CaCO 3 nanoparticles (Figure 4A).Briefly, to prepare emulsions A (CaCl 2 phase) and B (NaHCO 3 phase), first, a CaCl 2 aqueous solution (containing DOX) and a NaHCO 3 aqueous solution were added to the dichloromethane solution containing alkylated NLG919 (Anlg919), poly(lactic-co-glycolic acid) (PLGA), and poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) followed by 99 pulses using the probe sonicator for sonication.Then, emulsions A and B were mixed and 99 pulses were performed using the probe sonicator to obtain emulsion C. Emulsion C was added dropwise to the polyvinyl alcohol (PVA) solution under sonication in a water bath and left overnight to remove the dichloromethane by evaporation.Afterward, the obtained DNCaNPs were washed by centrifugation to remove excess PVA, free drug, and large particles.With the help of TEM, it was observed that there were obvious dark spots of CaCO 3 nanoparticles in the DNCaNPs, and the content of CaCO 3 was 17.6% as measured by a commercial calcium colorimetric assay kit (Figure 4B-D).

Bio-Based Preparation Method
Bio-based materials with CaCO3 as the main component are widely distributed in nature, so the preparation of CaCO3 nanoparticles from biobased CaCO3 materials is a cheap and environmentally friendly method.Through multi-step physical grinding, the original biobased material can be sorted into powders at different micron/nanometer levels for further applications.The production cost of this method is the lowest of all methods and can allow industrial scale-up production.

Bio-Based Preparation Method
Bio-based materials with CaCO 3 as the main component are widely distributed in nature, so the preparation of CaCO 3 nanoparticles from biobased CaCO 3 materials is a cheap and environmentally friendly method.Through multi-step physical grinding, the original biobased material can be sorted into powders at different micron/nanometer levels for further applications.The production cost of this method is the lowest of all methods and can allow industrial scale-up production.
Hamidu et al. [87] have successfully prepared 30 nm CaCO 3 nanoparticles using commercially available shells (Anadara granosa) and showed uniform spherical shape.They first washed the shells with water and then boiled and dried them.The dried shells were further cleaned in water with a squeezed banana pelt agent, removing the impurities from the shells.Subsequently, the shells were crushed into powder by a rotary crushing mixer and sieved with a pore size of 75 µm.Then, the powder was mixed to deionized water with dodecyl dimethyl betaine (BS-12) and stirred.After cooling, the solution was filtered and precipitated and dried.The high-resolution transmission electron microscopy (HRTEM) results showed that the CaCO 3 nanoparticles display a homogeneous spherical morphology, with a size of 35.5 nm.

The Effects and Mechanisms of CaCO 3 Nanoparticles in Cancer Diagnosis and Treatment
CaCO 3 nanoparticles have significant advantages such as pH sensitivity and a high Ca 2+ content.When the nanomaterials containing CaCO 3 enter the low-pH environment of the tumor tissue, CaCO 3 decomposes, neutralizes acidic H + , generates large amounts of Ca 2+ , and generates CO 2 .When these substances enter tumor cells, they can specifically regulate the metabolic process of tumor cells by their special properties.Below, we will discuss some of the effects and mechanisms of CaCO 3 nanoparticles in cancer diagnosis and treatment from different aspects.

Acidity Modulation
The tumor tissue microenvironment has a lower pH compared to normal physiological tissues, which allows acid-sensitive nanomaterials to specifically respond to the high proton concentrations within the tumor tissue, thus enabling the neutralization of H + in the tumor microenvironment (TME) and the pH-responsive release of the loaded drugs.CaCO 3 , a classical high-pH sensitivity compound, plays an essential factor in reprogramming the acidic environment of the TME, and that is why CaCO 3 -based nanoparticles have a modulating effect on the immune environment of tumors [88,89].Their acidic degradation products include only Ca 2+ and CO 2 [27,43].While significantly enhancing the efficacy of other oncology therapies, it is unlikely to have adverse effects [57,62,90].
Liu and Feng et al. [91] constructed AIM NPs (acidity-IDO1-modulation nanoparticles, 4PI-Zn@CaCO 3 ) based on CaCO 3 nanoparticles.To investigate the modulatory effect of AIM NPs on the TME, the investigators used commercial pH microelectrodes to directly measure the pH in mouse CT26 tumors and assess the neutralizing efficacy of CaCO 3 , 4PI-Zn, and 4PI-Zn@CaCO 3 on the acidity in the TME.The results indicated that the pH of these tumors increased significantly 24 h after intravenous injection of AIM and CaCO 3 nanoparticles, from pH 6.6 at about 0 h to pH 7.0 at 24 h, compared with that before the corresponding pre-injection period.In contrast, the intravenous injection of 4PI-Zn nanoparticles had a minimal effect on tumor pH, and even the pH at 0 h dropped from 6.6 to 6.5.The results suggested that both AIM and blank CaCO 3 nanoparticles are effective tumor acid regulators.Gu et al. [92] prepared an aCD47@CaCO 3 nanoparticles-based bioresponsive fibrin gel for in situ spraying at postoperative tumor resection sites, and after spraying the gel, was able to remove H + from surgical wounds and polarize tumor-associated macrophages (TAMs) to an M1-like phenotype, while inducing macrophage phagocytosis of tumor cells through the blockade of the interaction between CD47 and SIRPα to enhance anti-tumor T cell responses.The researchers investigated the effect of CaCO 3 as a proton scavenger in aCD47@CaCO 3 nanoparticles on the pH change in phosphate buffer solution (PBS) in vitro.After the addition of aCD47@CaCO 3 nanoparticles to PBS solutions with original pH values of 6.0 and 6.5, the pH of the solution progressively increased to about 7.4 as time progressed from 0 h to 24 h.

Calcium Overload
Calcium overload, as the name implies, is the accumulation of free Ca 2+ in the cell cytoplasm resulting in an abnormally high concentration of Ca 2+ , which can lead to severe cellular damage or even cell death [93].Normally, calcium signaling acts as a second messenger of cellular signaling to control important physiological processes by modulating the activity of specific targets, which are essentially achieved in response to fluctuations in local calcium concentrations [94].However, any changes in intracellular Ca 2+ concentrations induced by external factors can disrupt normal calcium signaling and thus affect various cellular activities.For example, under oxidative stress, the cellular regulation of Ca 2+ is reduced, as evidenced by abnormal calcium channel functions such as calcium pumps, and intracellular calcium ion concentrations are difficult to reregulate to a normal state, leading to a calcium overload [95].Once intracellular calcium overload occurs, it will trigger the disruption of Ca 2+ homeostasis in the mitochondria, resulting in decreased mitochondrial membrane potential and adenosine triphosphate (ATP) levels, osmotic swelling, and mitochondrial respiratory disorder and eventually lead to mitochondria-related cell damage and apoptosis [96].
As an ideal Ca 2+ donor, CaCO 3 -based intracellular calcium nanoreactors have good low-pH responsiveness in the tumor microenvironment and have been exploited for calcium overload-mediated therapy.Ding et al. [65] prepared a multichannel Ca 2+ nanomodulator (CaNM CUR+CDDP ), loaded with cisplatin (CDDP) and curcumin (CUR) to enhance calcium overload-induced therapy.After administration, PEGylated CaNM CUR+CDDP ( PEG CaNM CUR+CDDP ) aggregates in the tumor and enters into tumor cells, inducing multilevel mitochondrial disruption under the synergistic effects of massive Ca 2+ burst release, CUR inhibition of calcium efflux, and chemotherapy of CDDP, thereby significantly enhancing mitochondria-targeted suppression of the tumor.
Dong and Song et al. [97] designed a CaCO 3 -based nanoplatform (CaNP CAT+BSO @Ce6-PEG) for the synergistic treatment of enhanced photodynamic therapy (PDT) and calcium overload (Figure 5A).In acidic TME, CaCO 3 decomposes and releases loaded drugs, including catalase (CAT), buthionine sulfoximine (BSO), the photosensitizer Chlorin e6 (Ce6), and Ca 2+ .CAT and BSO significantly reverse the hypoxic microenvironment of tumors, significantly enhancing the PDT effect.The 1 O 2 produced by PDT kills tumor cells directly and disrupts the mitochondrial calcium homeostasis, leading to a mitochondrial calcium overload.The increased concentration of Ca 2+ inhibits the production of ATP in mitochondria, triggering mitochondrial dysfunction and thus accelerating cell death (Figure 5B,C).With the combined effect of enhanced PDT and calcium overload, the nanoparticles exhibited a significant synergistic tumor-suppressive effect.
tumors, significantly enhancing the PDT effect.The 1 O2 produced by PDT kills tumor cells directly and disrupts the mitochondrial calcium homeostasis, leading to a mitochondrial calcium overload.The increased concentration of Ca 2+ inhibits the production of ATP in mitochondria, triggering mitochondrial dysfunction and thus accelerating cell death (Figure 5B,C).With the combined effect of enhanced PDT and calcium overload, the nanoparticles exhibited a significant synergistic tumor-suppressive effect.

Facilitating Lysosome Escape and Intracellular Drug Release
When a nano-drug delivery system is uptaken by a cell, how to achieve efficient lysosomal/endosomal escape is a critical step for its function [98].Currently, the proton sponge theory has a place in the research of the mechanism of lysosomal/endosomal escape [99].Researchers suggest that when substances with high buffering capacity and adaptability enter the endosome, in order to buffer the acidic environment of the endosome, a large number of protons will be taken up, resulting in a difference in transmembrane voltage and a large amount of Cl-and H 2 O in the cytoplasm influx, which ultimately leads to the lysosomal swelling and rupture [100].This is referred to as the proton sponge effect.CaCO 3 nanoparticles are widely regarded as a class of proton nanosponge materials due to their efficient acid response properties [101].
Peng et al. [79] prepared a CaCO 3 liposome nanoparticle (LCC) loaded with curcumin (CUR), which has a high sensitivity to the lysosomal low-pH environment.Because of the pH sensitivity of CaCO 3 , the LCC swelled in the lysosomal acidic environment and rapidly released the encapsulated CUR.Further studies showed that the accumulation of CUR in the cytoplasm of LCC was due to the liposomes' pH sensitivity, leading to effective lysosomal escape and the release of CUR.The breakdown of CaCO 3 resulted in the presence of higher concentrations of Ca 2+ in the lysosomes and an increase in osmolality and allowed the influx of water from the cytoplasm into the lysosome.Eventually, CUR, which is rapidly released in an acidic environment, is transported into the cytoplasm after lysosome rupture.Thus, LCC can effectively promoted the accumulation of CUR in the cytoplasm.
In the low-pH environment, CaCO 3 decomposition is not only able to produce Ca 2+ but also to generate CO 2 gas for gas-driven reactions in tumor cells.When the nanodrug delivery system based on CaCO 3 enters the lysosome, CaCO 3 decomposes into large volumes of CO 2 in the low-pH environment, which drives the platforms to penetrate the lysosome barrier and release the drugs.Moreover, because of the driving effect of gas, the drug release can be accelerated [102].Jiao and Zhang et al. [102] designed a cascaded Near-Infrared (NIR) light/gas-driven Janus CaCO 3 particle micromotor (JCPM) to overcome the biological barrier at various stages after the system enters the body to achieve active drug delivery targeting tumor cells (Figure 6A).When the JCPM enters the lysosome, large amounts of CO 2 are rapidly produced during CaCO 3 decomposition in an acidic lysosomal microenvironment, and the pressure in the lysosome increases dramatically, promoting gas-driven lysosomal escape and DOX release induced by CaCO 3 decomposition (Figure 6B).

Tumor Immunomodulation
Previous studies have shown that calcium-based materials can enhance anticancer immunity [103,104].Ca 2+ has significant advantages in tumor immunotherapy such as (i) inducing immunogenic cell death (ICD) in tumor cells [64]; (ii) increasing autophagic efficiency [41]; and (iii) promoting the polarization of M2 macrophages to M1 macrophages [105].Many CaCO3-based nanosystems have been constructed to activate immunotherapy against cancer.
Tumor immunogenic cell death (ICD) activates damage-associated molecular

Tumor Immunomodulation
Previous studies have shown that calcium-based materials can enhance anticancer immunity [103,104].Ca 2+ has significant advantages in tumor immunotherapy such as (i) inducing immunogenic cell death (ICD) in tumor cells [64]; (ii) increasing autophagic efficiency [41]; and (iii) promoting the polarization of M2 macrophages to M1 macrophages [105].Many CaCO 3 -based nanosystems have been constructed to activate immunotherapy against cancer.
Tumor immunogenic cell death (ICD) activates damage-associated molecular patterns (DAMPs) that promote the maturation of dendritic cells (DCs) and proliferation of cytotoxic T lymphocytes, thereby activating anti-tumor immune responses [106].Recently, Ca 2+ has been shown as a novel ICD-inducing factor [107], and imbalances of mitochondrial calcium homeostasis can modulate reactive oxygen species (ROS) production, which stimulates damage-associated molecular patterns (DAMPs) to cause ICD and finally activate defensive anti-tumor immunity.Zheng et al. [64] synthesized a multifunctional Ca 2+ nanoregulator, PEG CaCUR, which is a pH-sensitive PEG-modified CaCO 3 nanoparticle loaded with curcumin (CUR).PEG CaCUR is capable of releasing Ca 2+ and CUR in a low-pH intracellular environment.CUR promotes Ca 2+ release from the endoplasmic reticulum to the cytoplasm and inhibits Ca 2+ efflux, inducing calcium overload, which in turn leads to apoptosis.After combining with the effect of ultrasound (US), it not only enhanced cellular uptake, but also promoted the influx of extracellular Ca 2+ , leading to an enhanced calcium overload and the upregulation of ROS levels.
Autophagy promotes phagocytosis and the presentation of antigen by dendritic cells during antigen processing [108,109].However, the autophagic capacity of dendritic cells is frequently inhibited in TME [110], causing a severe reduction in the intensity of antigen presentation.It has been shown that the homeostasis of different intracellular ions controls the activity of a wide variety of enzymes/proteins, such as Ca 2+ , which play a key role in antigen presentation [111].Thus, modulating calcium levels in dendritic cells has the promise of improving autophagy and thus enhancing immunotherapy.Ma and Wei et al. [46] developed a bionic approach to prepare graded ovalbumin@CaCO 3 nanoparticles (OVA@NP) in the presence of a template of ovalbumin antigen.OVA@NP was able to efficiently transport antigen into dendritic cells and primitive lysosomes.Once OVA@NP entered the lysosome, the environment caused the rapid disintegration of CaCO 3 material and was accompanied by bursts of CO 2 production and a dramatic increase in pressure within the lysosome, leading to lysosomal disintegration.Moreover, the researchers also observed the formation of isolated/extended bowl-shaped phagosomes and typical double-membrane autophagosomes near the disintegrated lysosomes in NP-treated DCs.The bowl-shaped phagosomes wrapped around the cytoplasmic components to form an intact autophagic vesicle.Based on this phenomenon, the researchers presented the first evidence that physical stress generated by carbon dioxide induces autophagy via the Lc3/Beclin 1 pathway.In this process, residual OVA in lysosomes, together with cytoplasmic proteasomes, were wrapped by autophagosomes, which together promoted antigen cross-presentation in DC cells, induced the proliferation of CD8+ T cell, triggered a vigorous specific cytotoxic T lymphocyte (CTL) response, and ultimately led to significant tumor therapeutic effects.
Macrophages are involved in a multitude of biological processes, encompassing stimulation of infection, pathological progression, and maintenance of homeostasis [112,113], and are expressed in two main phenotypes, inflammation-promoting M1-type macrophages and anti-inflammation-promoting M2-type macrophages.Significantly, TAMs, the most abundant tumor-expanding immune cells, are often expressed as M2-type and promote immune escape and tumor metastasis [114,115].So, regulating the polarization of macrophage phenotypes from M2 to M1 is essential for immunotherapy.The function of Ca 2+ in regulating the macrophage polarization from M2 to M1 has been well reported [116,117], not the least of which is the construction of nano-drug delivery systems based on CaCO 3 .For example, Huo and colleagues [36] synthesized a CaCO 3 -DC biomineralized hydrogel vaccine by immobilizing membrane proteins of 4T1 cell-DC fusion cells (FPs) in a hydrogel.The addition of CaCO 3 increased the pH of TME and facilitated the polarization from M2 to M1, which in turn reversed the immune-suppressive microenvironment and mitigated the immune-suppressive effects on T cells.In the experiments, the researchers observed an upregulation of CD80 (M1-type marker) expression and a downregulation of CD206 (M2type marker) expression after IL-4+CaCO 3 treatment.Similarly, Yang et al. [40] synthesized CaCO 3 -encapsulated Au nanoparticles (Au@CaCO 3 NPs) as a stimulus for macrophage regulation.In contrast to AuNPs, which polarized macrophages to the M2-type, this study showed that coincubation of Au@CaCO 3 NPs with macrophages resulted in cell rounding and induced secretion of the M1-type biomarker nitric oxide (NO), as well as TNF-α and IL-1β.A change in the macrophage polarization phenotype from M2 to M1 was observed after the coincubation of Au@CaCO 3 NPs with M2-type macrophages formed by IL-10 induction.In summary, CaCO 3 nanoparticles play a key role in polarizing to the M1type macrophages, reversing the suppressive immune-microenvironment and facilitating tumor killing.

Magnetic Resonance Imaging and Ultrasound Contrast Enhancers
CaCO 3 has been extensively studied as a pH-responsive material not only because of its suitability for targeted delivery of active drugs but also because of its ability to generate CO 2 in the low-pH environment of typical tumor microenvironments, which provides a potential ultrasound contrast agent for detecting and imaging tumors [27,118].
Luo and Zhang et al. [119] constructed CaCO 3 /pul-PCB (CPP) hybrid nanoparticles by using pul-PCB copolymer as a surface modifier, and the surface of the nanoparticles was modified by mineralization of Pullulan polysaccharides (Figure 7A).After injection, the nanoparticles can achieve efficient accumulation in the tumor tissue and decompose under acidic conditions, producing CO 2 bubbles with echogenic effects that enhance ultrasonic signals.It was demonstrated that the contrast of the ultrasonic signal was strengthened 6 times at the tumor site within 35 min in tumor-bearing mice.In contrast, there is almost no change in signal in normal mice (Figure 7B,C).So, CPP nanoparticles were considered a prospective contrast material for the imaging of liver cancer.
Guo and Zhang et al. [120] developed a CaCO 3 -based diagnostic nanosystem for ultrasound and fluorescence dual-mode imaging.Under acidic conditions, the nanoparticles were able to generate CO 2 to strengthen ultrasound imaging, and a large number of CO 2 bubbles were generated in the tumor tissue after injecting CaCO 3 -DOX NPs intravenously into tumor-bearing mice under the effect of echo reflection from the ultrasound field.To investigate the possibility of CaCO 3 -DOX nanoparticles for in vivo ultrasound imaging of tumors, the researchers injected the nanoparticles by tail vein injection and subsequently performed ultrasound imaging.The results showed that the enhancement of ultrasound images at the tumor site was clearly observed after CaCO 3 -DOX nanoparticles injection, and this signal contrast enhancement lasted for nearly 100 min.Compared with conventional contrast agents (sulfur hexafluoride microbubbles for injection), CaCO 3 nanoparticles can enter the tumor through the EPR effect and release CO 2 in the low-pH microenvironment of the tumor for a longer duration, thus obtaining more accurate diagnosis results.
In addition to applications in ultrasonography, CaCO 3 -based nanosystems are also of use in magnetic resonance imaging (MRI) contrast.Liu et al. [121] reported a new type of contrast agent consisting of NaGdF 4 and CaCO 3 nanoparticles (Figure 7D).In the study, a "turn down" in MRI signal resulted from spatial confinement of the T1 source (Gd 3+ ions) due to insufficient interaction between the plasma and the lattice.However, upon entering a low-pH environment, CaCO 3 nanoparticles can produce CO 2 , which breaks the binding between NaGdF 4 and CaCO 3 , allowing the MRI signal to be "turned on".As shown in the figure, NaGdF 4 -CaCO 3 nanoconjugates showed a significant enhancement of the contrast between the tumor site and the background tissue before and after intravenous injection, with the tumor-to-background (T/B) ratio reaching 48.Compared with the commercially available contrast agent, Magnevist, it provided a significant enhancement of the imaging contrast for the tumor site.(Figure 7E-G).This research represents remarkable progress in the creation of intelligent MRI nanoprobes, with key implications for the imaging of deep tissues and the diagnosis of specific cancers.
ultrasound field.To investigate the possibility of CaCO3-DOX nanoparticles for in vivo ultrasound imaging of tumors, the researchers injected the nanoparticles by tail vein injection and subsequently performed ultrasound imaging.The results showed that the enhancement of ultrasound images at the tumor site was clearly observed after CaCO3-DOX nanoparticles injection, and this signal contrast enhancement lasted for nearly 100 min.Compared with conventional contrast agents (sulfur hexafluoride microbubbles for injection), CaCO3 nanoparticles can enter the tumor through the EPR effect and release CO2 in the low-pH microenvironment of the tumor for a longer duration, thus obtaining more accurate diagnosis results.

CaCO 3 -Based Nano-Drug Delivery Systems for Tumor Diagnosis and Treatment
Currently, CaCO 3 -based nano-drug delivery therapeutic systems have formed a relatively mature system and have shown their unique and extensive use in the delivery of various small-molecule drugs and biomolecules.In addition, CaCO 3 can be covered on the surface of other nanostructures such as carbon quantum dots (CQDs) and metal-organic frameworks (MOFs) by surface mineralization to achieve CaCO 3 -based nano-medical delivery.In the following, we will introduce the construction of some recent CaCO 3 -based nano-drug delivery systems and their important roles in oncology therapy from three dimensions: small-molecule drugs, biomolecules, and surface mineralization.The preparation methods, physicochemical properties, and main findings of a part of CaCO 3 -based nano-drug delivery systems loaded with small-molecule drugs, biomolecule drugs, and surface mineralization are shown in Table 4.
Yu and Luo et al. [90] designed a tumor-targeting amorphous calcium carbonate (ACC)-based nanoparticle for tumor-targeted iron death therapy (Figure 8A).DOX was first chelated with Fe 2+ and then ACC encapsulated with Fe 2+ -DOX molecules was synthesized in one step by the gas diffusion method.Subsequently, an additional layer of SiO 2 -CaCO 3 (CaSi) was modified on the ACC surface and continued to be conjugated with folic acid-modified and PEGylated polyamide (PAMAM) dendrimers to confer tumor targeting to the nanoparticles.After the nanoparticles were successfully intercalated into tumor cells, the ACC was hydrolyzed in the low-pH environment of lysosomes.Meanwhile, PAMAM dendritic macromolecules promoted the lysosome escape of Fe 2+ -DOX nanoparticles and the release of Fe 2+ and DOX into the cytoplasm through the proton sponge effect.Compared with other iron death strategies that simply increase intracellular iron accumulation, the simultaneous release of DOX can induce nitrogen oxide (NOX) activation for further ROS production in addition to its cytotoxic effect, which synergizes the Fe 2+ -induced oxidative stress response, thus enhancing the iron death effect in tumor cells.In vivo anti-tumor evaluation demonstrated that ACC@DOX.Fe 2+ -CaSi-PAMAM-FA/Mpeg significantly reduced tumor volume by more than 5 times compared with the control and free DOX groups and prolonged the median survival time of the tumor-bearing mice to more than 50 days.In conclusion, the nanoformulation can effectively inhibit tumor growth in the hormonal animal model via complementary ferroptosis and chemotherapy.
Based on the chemical structures of ICG and Ce6, Yuan et al. [54] chose to use a coreshell material (HM) composed of an ACC core and a phospholipid shell as a carrier to construct a core-shell-structured nano-drug delivery system HM-I&C encapsulating ICG-Ca 2+ -Ce6 molecules (Figure 8B).The researchers creatively linked two conventional photothermal/photodynamic agents, ICG and Ce6, through Ca 2+ .Through the Förster resonance energy transfer (FRET) effect satisfied by the linkage of Ca 2+ , the ICG in the ICG-Ca 2+ -Ce6 molecule can achieve the physical quenching of Ce6 phototoxicity.When the nanoparticles enter the tumor tissue, ACC degrades under acidic conditions, and the association among Ca 2+ , ICG, and Ce6 is weakened, resulting in increased intermolecular distance, and the fluorescence property of Ce6 is restored, enabling the ce6-mediated tumor photodynamic therapy (PDT) strategy.Through the ingenious combination of ICG and Ce6, the researchers achieved a combination of photothermal therapy (PTT) and PDT in low-temperature synergistic therapy.In vivo and in vitro anti-tumor evaluation, researchers used the combination of 808/680 nm laser irradiation to compare with high-temperature PTT and PDT alone.The results showed that the efficacy of low-temperature synergistic therapy was significantly better than that of high-temperature PTT and PDT alone, and the tumor volume of tumor-bearing mice decreased significantly.Moreover, Western blotting analysis and TUNEL assay also showed that the apoptosis of tumor cells was particularly remarkable.
Natural active anti-tumor agents have an outstanding role in modulating cellular channel activity.For intracellular calcium overload, a tacit cooperation with Ca 2+ -related channels is required to achieve it, which makes many related natural active agents become potential targets for CaCO 3 -based nano-drug delivery systems.Xu and Han et al. loaded capsaicin (CAP) into CaCO 3 nanoparticles [71].As a natural TRPV1 channel agonist, CAP can effectively regulate the opening of the TRPV1 channel to allow Ca 2+ to influx, which has a significant promotion effect on the influx of exogenous Ca 2+ .In combination with the release of Ca 2+ from CaCO 3 , CaCO 3 @CAP nanoparticles effectively achieve the calcium overload effect.The results of anti-tumor evaluation in vivo showed that the tumor mass and density of tumor-bearing mice were significantly decreased, and the tumor growth was significantly inhibited.The naturalness and non-toxicity of capsaicin make the nanoparticles have good biocompatibility and good development prospects and clinical application potential.Based on the chemical structures of ICG and Ce6, Yuan et al. [54] chose to use a coreshell material (HM) composed of an ACC core and a phospholipid shell as a carrier to

Biomolecular Drug Nano-Drug Delivery Systems
Lee and Xiang et al. [78] co-loaded miR-375 and Sorafenib into CaCO 3 nanoparticles (miR-375/Sf-LCC NPs) with a lipid coating for liver cancer therapy.The results showed that the miR-375/Sf-LCC nanoparticles had a pH-dependent release of the drug and effective cytotoxicity.And in vivo experiments demonstrated that NPs by injection increased the concentration of miR-375 and Sorafenib in tumors and prolonged the time of retention of both drugs in the tumor site compared to direct injection of the two free drugs.In addition, it was demonstrated that co-delivery of miR-375 and Sorafenib significantly inhibited Sorafenib-induced cellular autophagy, thereby reducing Sorafenib resistance in liver cancer cells.The NPs showed significant tumor volume suppression in a xenograft tumor suppression assay in nude mice, demonstrating that NPs can promote the efficient delivery of the drugs and show effective liver cancer treatment.
Hu, Chen, and Zheng et al. [70] constructed an efficient pH-sensitive nanocatalyst (G/A@CaCO 3 -PEG) composed of CaCO 3 -loaded glucose oxidase (GOD) and 2D antimony quantum dots (AQD) (Figure 9).Afterward, the emitted GOD can effectively consume endogenous glucose and the concentration-dependent decrease in intracellular ATP levels with the increase in GOD concentration.The decrease in ATP levels also reverses the thermotolerance of tumor cells by down-regulating the expression of heat shock protein (HSP).This effect can enhance the therapeutic effect of 2D AQD-induced photothermal therapy under NIR irradiation.The results showed that G/A@CaCO 3 -PEG combined with laser irradiation induced significant apoptosis of tumor cells through mild PTT-induced HSP down-regulation and GOD-mediated glucose exhaustion and through ATP inhibition.
The in vivo experiments also demonstrated that G/A@CaCO 3 -PEG in conjunction with laser effectively inhibited tumor growth in mice.This work provides an effective way to enhance photothermal-based tumor therapies by limiting ATP production in tumor cells.Gu et al. [92] encapsulated CaCO3 nanoparticles containing anti-CD47 antibodies in fibrin gels and sprayed them in situ at the tumor excision area.It was demonstrated that CaCO3 nanoparticles could gradually lyse and free aCD47 encapsulated in specific TME and promote the revitalization of M1-type macrophages.By blocking CD47 and SIRPα interactions, macrophages and dendritic cells enhanced the phagocytosis of cancer cells and activated the natural immune system.In addition to this, the proportion of CD8+ and CD4+ T cells in the tumor was significantly increased, and the production of cytokines (including IFN-γ, IL-6, and IL-12p70) was also significantly increased after CD47@CaCO3@fibronectin treatment.The experimental results suggested that immunotherapeutic fibronectin gel can activate the innate and acquired immune system of the host to inhibit the local recurrence of the tumor after surgery and reduce the risk of metastatic spread.Gu et al. [92] encapsulated CaCO 3 nanoparticles containing anti-CD47 antibodies in fibrin gels and sprayed them in situ at the tumor excision area.It was demonstrated that CaCO 3 nanoparticles could gradually lyse and free aCD47 encapsulated in specific TME and promote the revitalization of M1-type macrophages.By blocking CD47 and SIRPα interactions, macrophages and dendritic cells enhanced the phagocytosis of cancer cells and activated the natural immune system.In addition to this, the proportion of CD8+ and CD4+ T cells in the tumor was significantly increased, and the production of cytokines (including IFN-γ, IL-6, and IL-12p70) was also significantly increased after CD47@CaCO 3 @fibronectin treatment.The experimental results suggested that immunotherapeutic fibronectin gel can activate the innate and acquired immune system of the host to inhibit the local recurrence of the tumor after surgery and reduce the risk of metastatic spread.

Surface Mineralization Nano-Drug Delivery Systems
Covering the surface by using CaCO 3 can effectively protect the nanoparticles to maintain stability during the process of in vivo circulation and avoid the leakage of the drug encapsulated in the nanoparticles.At the same time, CaCO 3 can decompose in the acidic environment of the tumor, so that the nanoparticles can be specifically released.In addition, the reverse osmotic pressure of Ca 2+ and HCO 3 − [137] and gas expansion of CO 2 [122] promote the lysosomal escape of the nanoparticles, making the drugs further enter the cytoplasm, realizing the therapeutic effect of drugs in tumor cells.
Xia and Xu et al. [138] reported mesoporous silica nanoparticles (MSNs) coated with CaCO 3 and lipid bilayers (MSNs@CaCO 3 @liposomes).The nanoparticles can achieve continuous drug release and enhance biocompatibility in the tumor microenvironment.The CaCO 3 -mineralized layer can cap the pore channels of MSNs, allowing the drug loaded in MSNs to be transported to the tumor site without leakage and achieving a pH-triggered drug release at the target site.In the experiments, doxorubicin (DOX) was effectively loaded into MSNs.The experiments also showed that nanoparticles exhibited a delayed release of DOX at a low pH, while no drug release occurred at a normal pH.
Lin and Hou et al. [56] have developed a "turn-on" therapeutic nanoplatform for the delivery of targeted therapy of H 2 S-rich colorectal cancer.The researchers first prepared hollow mesoporous Cu 2 O nanoparticles with a size of ~100 nm and subsequently coated them with a CaCO 3 shell (Cu 2 O@CaCO 3 ) by in situ surface mineralization.Finally, hyaluronic acid (HA) was functionalized onto Cu 2 O@CaCO 3 , dependent on its desirable targeting ability and biocompatibility, to form Cu 2 O@CaCO 3 @HA (CCH) nanoparticles (Figure 10A-C).When the nanoparticles reach the tumor site through HA targeting of colorectal cancer, the CaCO 3 -mineralized layer breaks down in the acidic tumor microenvironment to produce Ca 2+ , causing intracellular calcium overload, thus enabling Ca 2+ -based ion interference therapy (CIT).Subsequently, when exposed Cu 2 O comes in contact with high H 2 S in colon cancer, the nanocomposite can be decomposed into ultra-small Cu 31 S 16 nanoparticles, and the generated Cu 31 S 16 shows outstanding photothermal properties, photocatalytic properties, and Fenton-like activity toward PTT/PDT/chemodynamic therapy (CDT).
In addition to surface mineralization of mesoporous nanoparticles, CaCO 3 is also capable of achieving good surface wrapping for novel metal-organic framework (MOF) materials.Li and Tang et al. [55] reported that an Fe-based NMOF (nanoscale MOF) is used for the synergistic tumor treatment (Figure 10D).When the nanoplatforms reach the target site, the CaCO 3 -mineralized layer can begin to dissolve, resulting in the generation of NMOF@DHA and Ca 2+ .Subsequently, Ca 2+ acts synergistically with Fe 3+ in the NMOF@DHA structure, DHA, and the photosensitizer TCPP through Fe 2+ -DHA-mediated chemodynamic therapy, Ca 2+ -DHA-mediated tumor therapy (OT), and TCPP-mediated PDT showing a triple synergistic therapeutic effect and exhibiting high therapeutic efficiency, capable of achieving complete tumor ablation.
Zhang et al. [44] constructed a lysosome-targeted nanoparticle (LYS-NP) by combining a mineralized MOF with a targeted inducer (CD63-aptamer) to enhance the anti-tumor quality of T cells.CaCO 3 was used to induce the mineralization of Zn-based MOF, conferring good biocompatibility and acid degradation to the nanoparticles, while also improving the stability of the composite-encapsulated therapeutic proteins.Ca 2+ produced by the CaCO 3 mineralization layer can also enhance the function of perforin and granzyme B. Upon entry into the body, LYS-NPs are targeted to tumor cell lysosomes with the aid of CD63-aptamer.Under lysosomal acidic conditions, LYS-NPs are degraded and the degradation products, perforin, granzyme B, and Ca 2+ , are released into the lysosomes.Upon activation of the T cell receptor (TCR) by the major histocompatibility complex (MHC) of tumor cells, adoptive T cell vectors (ATVs) activate the under autonomous controlled release of perforin, granzyme B and Ca 2+ into lysosomes to treat tumors.This study constructed a "super cytotoxic T lymphocyte" that targets tumors and releases cytotoxic proteins, opening up a new strategy for immunotherapy of solid tumors.hyaluronic acid (HA) was functionalized onto Cu2O@CaCO3, dependent on its desirable targeting ability and biocompatibility, to form Cu2O@CaCO3@HA (CCH) nanoparticles (Figure 10A-C).When the nanoparticles reach the tumor site through HA targeting of colorectal cancer, the CaCO3-mineralized layer breaks down in the acidic tumor microenvironment to produce Ca 2+ , causing intracellular calcium overload, thus enabling Ca 2+ -based ion interference therapy (CIT).Subsequently, when exposed Cu2O comes in contact with high H2S in colon cancer, the nanocomposite can be decomposed into ultrasmall Cu31S16 nanoparticles, and the generated Cu31S16 shows outstanding photothermal properties, photocatalytic properties, and Fenton-like activity toward PTT/PDT/chemodynamic therapy (CDT).(D) Schematic illustration of the mechanism of NMOF@DHA@CaCO 3 for programmed drug release in cancer therapy.Adopted with permission from Ref. [55].2023, Chenteng Lin.

Conclusions and Perspective
In recent years, CaCO 3 -based nano-drug delivery systems have been widely used in the field of oncology diagnosis and treatment.This enables CaCO 3 -based nano-drug delivery systems as carriers or shell structures to maintain high structural integrity during in vivo circulation to ensure the least possible leakage of the drug molecules or nanostructures encapsulated within them, minimizing the systemic toxic effects of the drugs.Meanwhile, in the low-pH environment of tumor tissues, CaCO 3 can rapidly respond to degradation and release drug molecules and nanostructures in a targeted manner to achieve targeted drug accumulation and release in tumor sites.In addition, the degradation of CaCO 3 can also generate large amounts of Ca 2+ and CO 2 , the former of which has a key position in the regulation of cell metabolism, giving it special therapeutic effects in tumor therapy, such as calcium ion interference therapy.The rapid intracellular production of CO 2 leads to rapid gas expansion, which promotes lysosomal escape and the release of drugs and nanoparticles and plays a key role in tumor diagnosis as an enhancer for magnetic resonance imaging and ultrasound development.Therefore, with excellent biocompatibility, sensitive pH degradation, high Ca 2+ content, and CO 2 generation properties, CaCO 3 -based nano-drug delivery systems have shown significant advantages in drug delivery carriers, tumor therapy, and tumor diagnostics in vivo and in vitro.
At the same time, CaCO 3 -based nano-drug delivery systems, due to their good biocompatibility, are able to avoid many of the toxic side effects of other traditional inorganic nanomaterials when used as drug delivery carriers in vivo, which include long-term potential toxicity and low clearance in vivo.This advantage also implies that CaCO 3 -based nano-drug delivery systems are highly promising for clinical translation and are expected to become an advancing nanomedicine for tumor diagnosis and treatment.
However, it should also be noted that CaCO 3 has variable crystal types, which may undergo reversible/irreversible transitions between various crystal types under different external conditions and environments.This leads to the need to pay particular attention to the choice of reaction conditions and the control of nanoparticle dissolution-crystallization nucleation rates when synthesizing CaCO 3 nanoparticles, so that the CaCO 3 -based nanodrug delivery system is suitable for drug delivery.The long-range disorder and instability of amorphous calcium carbonate (ACC) make it promising for a wide range of applications in drug loading and delivery as a highly acid-responsive material, but its structure remains mysterious.Researchers have now studied the clustering and crystalline nucleation of ACC in some depth and discovered the key role played by water molecules, which is of great significance for the further application of ACC in the future.
Therefore, CaCO 3 -based nano-drug delivery systems with good biocompatibility, sensitive pH degradation, high Ca 2+ content, and CO 2 generation characteristics show significant advantages in the field of tumor diagnosis and therapy as carriers for in vivo drug delivery.It is believed that in the future, with the deepening research on CaCO 3 /Ca 2+ mode of action and tumor therapeutic methods, it is expected to become an advancing nanomedicine for clinical tumor diagnosis and treatment.

Figure 1 .
Figure 1.The synthetic methods, effects, and mechanisms of CaCO 3 nanoparticles in cancer diagnosis and treatment.

Figure 2 .
Figure 2. Example diagrams associated with the chemical precipitation method for the preparation of CaCO3 nanoparticles.(A) Schematic illustration of ROS/H + dual-bioresponsive drug delivery depot.(B) Particle size distribution of aPD1-loaded CaCO3 NPs measured by dynamic light scattering (DLS) and morphology observed by transmission electron microscopy (TEM), where the scale bar is 100 nm.Adopted with permission from Ref. [34].2023, Chenteng Lin.(C) The above figure reveals the preparation of CM@CaCO3@SAF NPs@DOX nanoplatforms.(D) The TEM image of SAF NPs.(E)The TEM image of CaCO3@SAF NPs@DOX.Scale bars: 100 nm.Adopted with permission from Ref.[35].2023, Chenteng Lin.

Figure 2 .
Figure 2. Example diagrams associated with the chemical precipitation method for the preparation of CaCO 3 nanoparticles.(A) Schematic illustration of ROS/H + dual-bioresponsive drug delivery depot.(B) Particle size distribution of aPD1-loaded CaCO 3 NPs measured by dynamic light scattering (DLS) and morphology observed by transmission electron microscopy (TEM), where the scale bar is 100 nm.Adopted with permission from Ref. [34].2023, Chenteng Lin.(C) The above figure reveals the preparation of CM@CaCO 3 @SAF NPs@DOX nanoplatforms.(D) The TEM image of SAF NPs.(E)The TEM image of CaCO 3 @SAF NPs@DOX.Scale bars: 100 nm.Adopted with permission from Ref.[35].2023, Chenteng Lin.

Figure 4 .
Figure 4. Example diagrams associated with the microemulsion method for the preparation of CaCO3 nanoparticles.(A) Schematic illustration of the preparation process of DNCaNPs via double emulsion method.(B) TEM images of DNCaNPs.(C) TEM images of DNNPs.(D) DLS size distribution of DNNPs and DNCaNPs.Adopted with permission from Ref. [83].2023, Chenteng Lin.

Figure 4 .
Figure 4. Example diagrams associated with the microemulsion method for the preparation of CaCO 3 nanoparticles.(A) Schematic illustration of the preparation process of DNCaNPs via double emulsion method.(B) TEM images of DNCaNPs.(C) TEM images of DNNPs.(D) DLS size distribution of DNNPs and DNCaNPs.Adopted with permission from Ref. [83].2023, Chenteng Lin.

Figure 5 .
Figure 5. Example of nanosystems constructed from CaCO 3 nanoparticles for calcium overload of tumor cells.(A) Schematic illustration of the CaNP CAT-BSO @Ce6-PEG NPs for PDT-enhancing and mitochondrial Ca 2+ overload synergistic therapy.(B) The CLSM images demonstrate the difference in intracellular Ca 2+ content after different groups of drug treatments.(C) The CLSM images demonstrate the detection of intracellular Ca 2+ content after irradiation for different times.Adopted with permission from Ref. [97].2023, Chenteng Lin.

Figure 7 .
Figure 7. Examples of nanosystems constructed from CaCO3 nanoparticles for magnetic resonance imaging and ultrasound contrast enhancers.(A) Schematic illustration demonstrates the principle of CaCO3/pul-PCB (CPP) hybrid nanoparticles in contrast-enhanced US diagnosis of hepatocellular carcinoma.(B,C) show the difference in US imaging of tumor and liver before and after intravenous injection of CPP in vivo.Adopted with permission from Ref. [119].2023, Chenteng Lin.(D) Schematic illustration of the synthesis of MSNPs as smart contrast agents for T1-weighted MRI.(E-G) show T1-weighted MRI and corresponding pseudocolor images of tumor-bearing mice after intravenous injection of the same dose of MSNPs (E) and Magnevist (F).Tumor-to-background (T/B, (G), above) and tumor-to-muscle (T/M, (G), below) contrast based on the corresponding MRI images.The dotted circles represent the regions of interest: (1) tumor, (2) muscle, (3) background, and (4) bladder.Scale bars are 5 mm for all images.Adopted with permission from Ref. [121].2023, Chenteng Lin.

Figure 7 .
Figure 7. Examples of nanosystems constructed from CaCO 3 nanoparticles for magnetic resonance imaging and ultrasound contrast enhancers.(A) Schematic illustration demonstrates the principle of CaCO 3 /pul-PCB (CPP) hybrid nanoparticles in contrast-enhanced US diagnosis of hepatocellular carcinoma.(B,C) show the difference in US imaging of tumor and liver before and after intravenous injection of CPP in vivo.Adopted with permission from Ref. [119].2023, Chenteng Lin.(D) Schematic illustration of the synthesis of MSNPs as smart contrast agents for T1-weighted MRI.(E-G) show T1-weighted MRI and corresponding pseudocolor images of tumor-bearing mice after intravenous injection of the same dose of MSNPs (E) and Magnevist (F).Tumor-to-background (T/B, (G), above) and tumor-to-muscle (T/M, (G), below) contrast based on the corresponding MRI images.The dotted circles represent the regions of interest: (1) tumor, (2) muscle, (3) background, and (4) bladder.Scale bars are 5 mm for all images.Adopted with permission from Ref. [121].2023, Chenteng Lin.

Figure 8 .
Figure 8. Examples of small-molecule drug nano-drug delivery systems constructed based on CaCO3 nanoparticles.(A) Schematic illustration of the synthesis and mechanism of ACC@DOX.Fe 2+ -CaSi-PAMAM-FA/Mpeg and its complementary ferroptosis/apoptosis-based therapeutic action.Adopted with permission from Ref.[90].2023, Chenteng Lin.(B) Schematic illustration of the synthesis and mechanism of HM-I&C for dual-channel imaging and low-temperature synergistic therapy.Adopted with permission from Ref.[54].2023, Chenteng Lin.

Figure 8 .
Figure 8. Examples of small-molecule drug nano-drug delivery systems constructed based on CaCO 3 nanoparticles.(A) Schematic illustration of the synthesis and mechanism of ACC@DOX.Fe 2+ -CaSi-PAMAM-FA/Mpeg and its complementary ferroptosis/apoptosis-based therapeutic action.Adopted with permission from Ref. [90].2023, Chenteng Lin.(B) Schematic illustration of the synthesis and mechanism of HM-I&C for dual-channel imaging and low-temperature synergistic therapy.Adopted with permission from Ref. [54].2023, Chenteng Lin.
Pharmaceutics 2024, 16, 275 24 of 33 conjunction with laser effectively inhibited tumor growth in mice.This work provides an effective way to enhance photothermal-based tumor therapies by limiting ATP production in tumor cells.

Figure 9 .
Figure 9. Example of biomolecular drug nano-drug delivery systems constructed based on CaCO3 nanoparticles.The schematic illustration shows the mechanism of a pH-sensitive Ca 2+ -based nanocatalyst, G/A@CaCO3-PEG, which enhances the thermotherapy-based tumor treatment by limiting ATP supply.Adopted with permission from Ref. [70].2023, Chenteng Lin.

Figure 9 .
Figure 9. Example of biomolecular drug nano-drug delivery systems constructed based on CaCO 3 nanoparticles.The schematic illustration shows the mechanism of a pH-sensitive Ca 2+ -based nanocatalyst, G/A@CaCO 3 -PEG, which enhances the thermotherapy-based tumor treatment by limiting ATP supply.Adopted with permission from Ref. [70].2023, Chenteng Lin.

Figure 10 .
Figure 10.Examples of surface mineralization nano-drug delivery systems constructed based on CaCO 3 nanoparticles.(A) Cu 2 O@CaCO 3 @HA (CCH) synthesis route; CCH biodegradation, antitumor response, and renal clearance triggered by the tumor microenvironment in colorectal cancer (B) and its anti-tumor immune response activated by combined CD47 blockade in the tumor microenvironment in colorectal cancer (C).Adopted with permission from Ref. [56].2023, Chenteng Lin.(D) Schematic illustration of the mechanism of NMOF@DHA@CaCO 3 for programmed drug release in cancer therapy.Adopted with permission from Ref.[55].2023, Chenteng Lin.

Table 1 .
Representative CaCO 3 -based nano-drug delivery systems prepared by chemical precipitation method.

Table 2 .
Representative CaCO 3 -based nano-drug delivery systems prepared by gas diffusion method.

Table 3 .
Representative CaCO 3 -based nano-drug delivery systems prepared by microemulsion method.

Table 4 .
Summary of the basic characteristics of different types of CaCO 3 -based nano-drug delivery systems for tumor diagnosis and treatment.