Functionalized Calcium Carbonate–Based Microparticles as a Versatile Tool for Targeted Cancer Treatment

1. Introduction

Microparticles are widely used in various fields of research and drug delivery applications [1,2]. Among the various materials used for microparticle fabrication, silica has long been considered the best candidate, but it has several disadvantages that limit its clinical potential, especially in preventing the spontaneous drug release [3]. Calcium carbonate (CaCO₃) is an abundant, inexpensive, biocompatible material with suitable chemical and physical properties, such as a small size of the particles with a large surface area [4]. These properties make it an attractive material for numerous biomedical applications and an ideal choice for targeted cancer immunotherapy [5]. There are three polymorphs of CaCO₃ particles: calcite, aragonite, and vaterite crystals. Though less thermodynamically stable than the others, vaterite crystals are spherical, composed of nanodomains, and highly porous, which makes them a good candidate for using in drug delivery systems [6,7].

The most common methods of synthesis of CaCO₃ microparticles are solid–liquid–gas carbonation [8] and chemical precipitation through the reaction of CaCl₂ with Na₂CO₃ in an aqueous medium [9]. There are also other methods of synthesis of CaCO₃ microparticles [10], such as the supercritical fluid technology [11] and the emulsion techniques [12,13]. In the course of synthesis, the temperature, pH, reagent concentrations, and other parameters can be controlled to optimize the size, morphology, and composition of the microparticles. It has been shown that gradual addition of a calcium nitrate solution to the sodium carbonate solution allows controlling the saturation of the reaction medium and obtaining smaller CaCO₃ particles after prolonged agitation. Overall, temperature influences particle morphology and polymorphism, whereas the calcium and carbonate ion concentrations determine their size [14]. These different techniques of synthesis offer flexible approaches for obtaining CaCO₃ particles suitable for various therapeutic applications. CaCO₃-based microparticles have a wide range of potential applications, particularly in targeted drug delivery. Their use can offer significant advantages in terms of efficiency, cost-effectiveness, and sustainability compared to existing materials.

Three main types of CaCO₃-based microparticles with sizes ranging from about 0.2 to 6 µm have been extensively studied: core-only microparticles, polymer-coated cores (or core/shell microparticles), and hollow (shell) polymer capsules, for which CaCO₃ particles are used as sacrificial templates [2,15,16] (Figure 1). Each of these types possesses unique characteristics suitable for specific applications in cancer treatment.

Core-only microparticles are primarily used due to their capacity for absorbing and carrying therapeutic agents. Their simple, porous structure also ensures drug release. However, their use is limited by the lack of targeting specificity and insufficient resistance to potentially aggressive factors of biological microenvironment. Additional strategies may be necessary to prevent their degradation or aggregation during the delivery [17].

Core/shell structures are considerably more advantageous, because their polyelectrolyte shell provides enhanced protection of the encapsulated compound compared to core-only systems and can be functionalized to ensure specific targeting. Current research focuses on developing new strategies to enhance stability, targeting, and release control by coating microparticles with polymers [18] or lipids [19]. These microparticles can be designed to respond to specific stimuli, such as changes in pH [20,21], or temperature [22], by releasing their contents. They are commonly fabricated by means of layer-by-layer (LbL) deposition of alternating anionic and cationic polyelectrolytes, depending on the charge of the template microparticle [23,24].

Polymer microcapsules [16,25] are particularly interesting because of their capacity for encapsulating therapeutic agents while avoiding the adverse effect of CaCO₃ on the cellular calcium balance. They also can be designed to respond to specific stimuli, allowing for targeted drug release within tumors [26,27]. Polymeric microcapsules are synthesized on the basis of CaCO₃ templates, which are usually dissolved by ethylenediaminetetraacetic acid (EDTA) after LbL assembly of polyelectrolytes [28]. The EDTA concentration determines the dissolution rate and the final properties of the microparticles, including size, porosity, and stability.

Various therapeutic agents, including low-molecular-weight drugs, proteins, and nucleic acids, can be encapsulated by loading into CaCO₃ cores through absorption or chemical coprecipitation during the formation of the cores [29]. The loading capacity of these systems depends on several factors, such as the porosity and specific surface area of the CaCO₃ particles and the chemical properties of the drug. Studies have shown significant effectiveness of low-molecular-weight drug encapsulation [30] and their controlled release from CaCO₃ cores [31], sometimes with a reduced cytotoxicity [32]. The efficiency of encapsulation and stability of encapsulated molecules have been also demonstrated for proteins [33] and nucleic acids [34]. Drug release from delivery systems based on CaCO₃ microparticles can be activated by external stimuli, such as a change in pH [35] (slightly acidic in tumors) or temperature [22]. For targeted drug delivery, CaCO₃ microparticles can be functionalized with recognition molecules, usually antibodies, interacting with specific receptors on target cells [36]. Moreover, in vivo studies of a nasal drug delivery system based on CaCO₃ microparticles has shown improved bioavailability of the active substance [37]. Recently, in vivo applications of CaCO₃ particles using various administration routes have been intensely studied and proven to be promising [38].

In conclusion, the loading of drugs into calcium carbonate cores, core/shell microparticles based on them, or microcapsules is a promising technique in the field of drug delivery, especially for cancer therapy. CaCO₃-based microparticles efficiently encapsulate various therapeutic agents, improving their bioavailability and specifically targeting cancer cells while reducing side effects. In this review, we will first discuss the methods of synthesis of calcium carbonate cores and fabrication of CaCO₃-based microparticles and microcapsules, then explore the systems for delivery small-molecule drugs, proteins, and DNAs based on each of these structures, and finally address the potential uses and key challenges of these microstructures in cancer treatment.

2. Core-Only CaCO₃ Microparticles

Calcium carbonate cores have been used as containers over the past two decades [39] and offer numerous advantages for the delivery of pharmacological compounds, such as biocompatibility, a high loading capacity, and maintenance of the properties of the loaded molecules [40]. Their size and shape vary depending on synthesis conditions, including temperature, reactant concentrations, viscosity of the medium, and reaction time, which allows obtaining cores with desired properties [1,6,7]. The internal porous structure of functionalized calcium carbonate cores is also an important factor influencing drug loading, which has recently been elucidated by mercury intrusion porosimetry and scanning electron microscopy with a focused ion beam [41]. A reduced pore size has been found to be associated with an increased maximum payload, i.e., a higher capacity for retaining compounds within the particles.

2.1. Loading Methods

Calcium carbonate cores are used for the loading of small molecules [21,42], proteins [43,44], nucleic acids [34], and radionuclides [45,46]. The substances are loaded into the CaCO₃ cores either by co-synthesis, when the proteins are captured by the CaCO₃ cores during their growth, or by adsorption of loading molecules onto the matrix surface of preformed CaCO₃ cores [47]. An alternative drug loading method by solvent evaporation is suitable for small molecules with different solubilities [42]. The adsorption of poorly soluble drugs onto the CaCO₃ particles may help overcome the low bioavailability of drugs [48], whereas loading during co-synthesis leads to aggregation of proteins [43]. The co-precipitation method has proven to have a high loading efficiency for both small-molecule drugs and proteins [18,49]. The loading efficiency depends on the drug diffusion through the pores at the pH and ionic strength suited to each particular compound, while ensuring preservation of its bioactivity. For example, the loading of superoxide dismutase into vaterite CaCO₃ crystals at pH 8.5 was highly efficient, with its activity retained, whereas at pH 9.5, only a 30% retention was achieved [43].

Enhancement of protein encapsulation into 6.9 µm CaCO₃ microparticles using protein–polysaccharide interactions has been shown [50]. The chitin-binding domain (ChBD) was inserted into a β-lactamase protein (BlaP) to obtain a chimeric protein, BlaPChBD, exhibiting affinity for hyaluronic acid (HA). In the presence of HA, the particle size was decreased to 4.5 µm, which indicated a templating effect of HA on CaCO₃. The chitin-binding domain (ChBD) ensured a more stable interaction between the protein and HA, reducing aggregation and decreasing the particle size. The use of supercritical CO₂ (ScCO₂) technology in the presence of HA ensured successful encapsulation of BlaPChBD in vaterite CaCO₃ microparticles, increasing protein encapsulation sixfold compared to BlaP alone. In addition, thrombin cleavage sites were introduced to facilitate protein release by protease cleavage, the release rate being increased from less than 20% to 87% within 36 h. The β-lactamase encapsulation rate was below 1%, apparently, due to unfavorable electrostatic interactions at pH 6.5, and was slightly increased (to 1.2%) after the insertion of the chitin-binding domain. The use of HA significantly increased the encapsulation of BlaPChBD (to 6.27%) due to protein–polysaccharide interactions. The results demonstrate the efficacy of using HA for enhancing encapsulation and controlled release of proteins in CaCO₃-based delivery systems, offering a promising approach to the development of biodegradable and targeted drug delivery systems.

2.2. Demonstration and Limitations

Loading of three therapeutic proteins (insulin, catalase, and aprotinin) into vaterite CaCO₃ cores has shown that the protein loading capacity is independent of their molecular weight and depends only on inter-protein interactions [44]. The tested proteins differ from one another in adsorption kinetics, which indicates differences in the adsorption mechanisms.

The efficiencies of loading catalase into CaCO₃ vaterite crystals by means of absorption into preformed crystals (ADS) and co-synthesis (COS) [51] have been compared. COS has been shown to be more efficient, as in the case of the loading of small molecules [18], with a protein content of 20.3% versus 3.5% loaded by the ADS method. The high loading capacity of COS, with a local protein concentration of about 550 mg/mL, was due to CaCl₂-induced interprotein interactions resulting in aggregation. The adsorption isotherms better fitted the Langmuir and Brunauer–Emmett–Teller (BET) models than the Freundlich model, which indicated aggregation in solution followed by absorption of aggregates into the crystals. Furthermore, catalase was found to retain about 79% of its specific activity after ADS loading. The stability of the aggregates in the crystals was confirmed by that catalase loaded by the COS method could not be effectively removed by a single washing, unlike catalase loaded by the ADS method. This study highlights the high potential of the COS method for loading large amounts of active proteins into CaCO₃ crystals, offering a new approach to the encapsulation of therapeutic proteins [51]. One of the main problems with vaterite CaCO₃ particles is their aggregation [25]. However, stabilizers, such as SDS, successfully overcome this problem [21].

The CaCO₃-based delivery systems are often designed to be pH-dependent. Calcium carbonate/hyaluronate/glutamate submicron hollow spheres loaded with doxorubicin (DOX) [52] released 59.97% of DOX within 14 days at pH 7.4, 87.89% at pH 6.0, and 99.15% at pH 5.0, with a loading efficiency of 85%. Specific binding of these particles to cancer cells was provided by the ligand–receptor interaction between HA and CD44 receptors, overexpressed on cancer cells. The IC₅₀ of DOX-loaded microspheres was much lower than that of free DOX when tested on HeLa cancer cells. At the same time, in tests on V79-4 normal cells, the IC₅₀ was significantly lower for free DOX than for DOX-loaded microspheres, thus confirming the enhanced specificity of treatment with microspheres (Figure 2).

Pneumolysin (PLY)-loaded CaCO3 particles (0.95 µm) containing ovalbumin as a model antigen have been developed as a multimodal antigen delivery system for antitumor vaccines. OVA/CaCO3/PLY nanoparticles obtained by physical adsorption of OVA and PLY on CaCO3 promoted lysosomal degradation, cytoplasmic release, and cross-presentation of antigens, enhancing cellular immunity. The OVA/CaCO3/PLY system induced efficient lysosomal leakage and cytoplasmic delivery of OVA in vitro [53].

The kinetics of drug release from the systems based on CaCO3 cores is often bimodal, with initially rapid release due to the dissolution of aggregates followed by sustained release [54]. As the vaterite crystals destabilize, their morphology changes into the calcite one, making the release irreversible. The presence of aggregates within the matrix and the high loading rate by the co-synthesis method, especially for proteins, indicate the limitations of the application of the loading method by adsorption [55]. Nevertheless, other CaCO3-based particle systems are being developed and exhibit a high efficiency in substance delivery. Table 1 summarizes the characteristics of the systems based on CaCO3 cores as vehicles for the delivery of small molecules, proteins, DNAs, and radionuclides.

3. CaCO3-Based Core/Shell Systems

3.1. Methods of Fabrication

The CaCO3 particles coated with a polyelectrolyte shell are better suited for the delivery of drugs and proteins. Polyelectrolytes are deposited onto the cores by the LbL method [56,57] or by electrospray [58]. Variation of the number of cationic/anionic bilayers deposited on the particle surface allows better control of the kinetics of substance delivery to the target. Application of these polymers is driven by electrostatic interaction, through covalent or hydrogen bonds, which explains how the release of loaded molecules can be induced by different stimuli, such as pH, ionic strength, temperature change, or ultrasound.

For encapsulation of therapeutic agents, adsorption or co-synthesis can be used, which determines their location: on the surface, between the layers, or within the matrix. Figure 3 summarizes the data on fabrication of CaCO3 core-only and core/shell microparticles.

3.2. Delivery of Small Molecules

In the development of systems for small-molecule delivery based on microparticles, DOX is often used as a model anticancer drug. Efficient loading of DOX has been shown for core/shell microparticles composed of ∼2-µm CaCO3 cores coated with poly-L-ornithine and fucoidan. The release of DOX from these particles was confirmed by a significant antiproliferative effect on MCF-7 breast cancer cells [59]. DOX-loaded CaCO3 microparticles modified with oleic acid (OA) and polyethylene glycol (PEG) exhibited a 70% drug release within 2 h in cancer cells in response to specific environment, whereas their stability and drug retention in various other aqueous media were enhanced. Hybrid CaCO3 microspheres have also been obtained using yeast cells as the organic matrix and the polyelectrolytes poly(diallyldimethylammonium chloride) (PDDA) and sodium poly(styrene sulfonate) (PSS) as shell components, with subsequent calcination and DOX loading [60]. Drug release tests showed accelerated release of DOX in an acidic environment (pH 4.8) typical of cancer tissues compared with a neutral medium (pH 7). Cytotoxicity tests have shown a good biocompatibility of CaCO3 microparticles 3 µm in diameter loaded with herbal medicinal products (HMPs) (Figure 4). Gradual decomposition of the coated particles in the acidic microenvironment of tumors ensures the targeted release of the drug directly into the cancer cells, thereby improving the efficacy of the treatment and minimizing the side effects on the surrounding healthy tissue. Thus, the feasibility of delivery of small molecules using the core/shell system has been demonstrated.

3.3. Delivery of Proteins

Calcium carbonate microparticles containing cancer cell lysate and coated with polymer substituted with the low-molecular-weight TLR7/8 agonist have been developed, which could serve as novel personalized anticancer vaccines [64].

Solid-in-oil-in-water emulsion method for the manufacture of CaCO₃/polylactic acid core/shell microparticles about 1.11 µm in size have been designed as tools for the controlled transport and release of water-soluble bioactive molecules. This technology could be used for developing more effective drug delivery systems [65].

The biomimetic approach has been used to obtain core/shell microparticles with a liquid core consisting of charged emulsion droplets or liposomes and a CaCO₃ shell, which also can be used as delivery vehicles [66].

Overall, these techniques improve the encapsulation and release of proteins, offering promising advances for drug delivery systems.

3.4. Delivery of Nucleic Acids

Although encapsulation of nucleic acids in core/shell systems has not yet been reported, some studies envisage it. For example, Bewernitz et al. [66] explore the manufacture of liquid-core/solid-shell microcapsules representing CaCO₃-coated emulsions and liposomes. These microcapsules, ranging in size from 2 to 10 µm, have been designed for potential applications in controlled release of substances, including DNA molecules. The method relies on the precipitation of CaCO₃ to form a shell around emulsion droplets or liposomes. This approach could be used to engineer a promising system for the protection and targeted delivery and release of DNA in biomedical applications due to the possibility of controlling the permeability and degradation of the CaCO₃ shell.

Applications of CaCO₃ core-based core/shell microparticles are summarized in Table 1.

4. CaCO3-Based Hollow Microcapsules

4.1. Methods of Fabrication

Calcium carbonate-based hollow (or shell) microcapsules represent a fascinating area of research in medical nanotechnology, providing unique opportunities for targeted cancer treatment [29]. These microcapsules with encapsulated therapeutic agents are often designed to interact directly with tumors by functionalization of their surface with antibodies, peptides, proteins, hyaluronic acid, or nucleic acids to ensure controlled, targeted drug delivery [67].

The fabrication of these microcarriers is based on the LbL assembly of polyelectrolytes, a technique first tested on metformin particles [24], which allows the construction of multilayer films with nanometric precision by alternating the immersion of a substrate in solutions of polyelectrolytes of opposite charges. CaCO₃ cores, whose synthesis was considered above, are used as templates for the fabrication of microcapsules. Then, the cores are dissolved with a chelating agent, e.g., EDTA and washed, and hollow spherical polyelectrolyte capsules are thus formed. Polyelectrolytes in different combinations, such as poly(allylamine hydrochloride) (PAH) and PSS [18,68], PAH and poly(vinyl sulfate) (PVS) [20], chitosan (Chi) and alginate (Alg) [69], HA and PAH/poly-L-lysine (PLL) [70], and poly-L-arginine (pArg) and dextran sulfate (DS) [71], are particularly effective in forming these multilayers on vaterite CaCO₃ cores. Detailed comparison of the stabilities, shrinkabilities, and internal structures of capsules made of different biopolymers have been performed [15]. These polymers, selected for their capability for self-assembling, ensure high stability and functionality of the microcapsules, making it possible to modulate their properties, such as solubility, reactivity and biological compatibility, for the purposes of biomedical engineering and formation of protective coatings and sensors [72]. Figure 5 summarizes the methods for obtaining shell microcapsules.

4.2. Delivery of Small Molecules

The capability of multilayered polyelectrolyte capsules to host low-molecular-weight drugs for cancer targeting has been recently demonstrated [30]. These smart polymer capsules exhibit considerable versatility, paving the way for future developments in medical nanotechnology and personalized medicine [94]. In recent years, uniformly sized microcapsules obtained on the basis of CaCO₃ cores as removable templates, have been loaded with gemcitabine and clodronate [95], DOX [18], apigenin and ascorbic acid [96], curcumin and ciprofloxacin [22], and Gratiola officinalis extract [97] as model drugs for cancer and other diseases.

Different encapsulation approaches are used with small-molecule drugs. Microcapsules fabricated using the PAH and PSS polyelectrolytes on CaCO₃ cores have exhibited efficiencies of DOX loading by coprecipitation and spontaneous loading of about 73 and 65%, respectively, due to optimized pH and salt concentration [18]. PAH/dextran sulfate (DS) polymer microcapsules designed for the delivery of apigenin and ascorbic acid exhibited a loading efficiency of about 20% for each substance after incubation of the microcapsules in the presence of the drugs [96]. The gemcitabine loading efficiency of submicron pArg/DS microcapsules was about 47% [95].

The microcapsules are so designed as to release the loaded drugs in response to specific stimuli. In the case of PAH/DS capsules containing apigenin and ascorbic acid, in vitro release was 45% and 40%, respectively, after 2 h at the physiological pH [96]. This study has also shown that the chemical composition of the capsules strongly affects the drug solubility and rate of its release. The release of DOX by diffusion from PAH/PSS microcapsules was prolonged at pH 6.0 and 7.4, corresponding to the pH values in tumor and normal tissues, respectively. The cumulative release of DOX within 48 h did not exceed 70% [18].

In in vitro experiments, pArg/DS microcapsules loaded with gemcitabine were internalized at a rate higher than 75% by macrophages and lung and liver epithelial cells [95]. Experiments in mouse models showed specificity of microcapsule delivery: they were better retained by lung tumor than by healthy lung tissue. The efficiency of encapsulated gemcitabine estimated by the MTT assay was lower than that of the free drug after 24 and 48 h of incubation and equal to it after 72 h of incubation, which confirmed prolonged, gradual release of the drug (Figure 6).

Microcapsules are commonly developed to reduce the side effects of drugs and to allow a more prolonged and targeted action of, e.g., DOX, thereby improving the efficacy of the treatment. It can be co-administered, thus compensating for the rapidity of elimination from the body [32]. Also, the microcapsules containing Gratiola officinalis extract were shown to effectively release the drug, causing death of 100% of cultured cancer cells through overcoming protective autophagy [97].

Hollow polymeric microcapsules are also used for encapsulation of live E. coli cells. CaCO₃ cores containing E. coli cells were obtained by co-precipitation and coated with different polyelectrolytes. Then, CaCO₃ cores were dissolved in EDTA to obtain capsules with a size of about 5 μm. Encapsulation reduced cell viability, the effect being mainly accounted for by PAH, with only minor contribution of the other components. The encapsulated cells exhibited a prolonged lag phase of growth while retaining the ability to produce green fluorescent protein. About 40% of cells were alive after the encapsulation. This method has potential applications in high-throughput screening of biocatalyst libraries, requiring optimization to improve cell survival [98].

Composite microcapsules based on CaCO₃ have been developed that contain various types of pectin with different degrees of methylation and amide content, as well as mixtures of polyelectrolyte complexes, including poly(allylamine) hydrochloride. These CaCO₃/pectin capsules were used as matrices for the loading of tetracycline hydrochloride (TCH), with analysis of drug release kinetics using the Higuchi and Korsmeyer–Peppas models. In vitro assays demonstrated the influence of CaCO₃ polymorphs on the drug release process, with 22–27% of TCH released within 10 h at pH 7.4 [99].

The potential of using CaCO₃-templated PAH/PSS polymer capsules for the targeted delivery of vitamin B12 has also been demonstrated [100]. Successful encapsulation of vitamin B12 was confirmed by optical absorption spectroscopy, transmission electron microscopy, and atomic force microscopy data. Experimental data on the specific encapsulation capacity of these polymer capsules for vitamin B12 show their potential as targeted vectors for nutrient delivery, highlighting the effectiveness of the PAH/PSS system in developing biocompatible and stable drug-delivery vectors.

4.3. Delivery of Proteins

Proteins can also be transported and released by polyelectrolyte capsule systems assembled on CaCO₃ cores [101]. The chemical methods for the fabrication and post-modification of hollow polymer capsules for proteins delivery, including covalent bonding, electrostatic attachment, and hydrogen bonding, have been described [102]. Proteins can be encapsulated by physical adsorption on preformed CaCO₃ cores or by co-precipitation during the CaCO₃ particle synthesis. The latter approach has been shown to be five times more efficient [103]. Horseradish peroxidase (HRP) and ovalbumin serving as model antigens have been encapsulated in CaCO₃-based pArgt/DS polymer capsules by co-precipitation. After lyophilization in the presence of polyols, HRP retained up to 70% of its enzymatic activity. Ovalbumin-loaded microcapsules were used as model vaccine formulation. Ovalbumin encapsulated in polyelectrolyte microcapsules caused enhanced antigen presentation and amplification of T-cell proliferation compared to soluble ovalbumin. The immunological activity of lyophilized microcapsules was preserved, according to the results of in vitro T-cell proliferation assay [104].

The effect of pH on the degradation of polyelectrolyte microcapsules formed on CaCO₃ particles with proteins encapsulated by adsorption was also studied [105]. An increase in pH led to an increase in protein yield and PAH detachment, apparently because the acidity of the medium (pH 7) was close to the charge exchange point of the PAH amino group. A high concentration of NaCl (2 M) caused considerable PAH dissociation and release of the protein.

4.4. Delivery of Nucleic Acids

Studies using polymeric capsules for delivering genetic material into cells are also carried out. CaCO₃-based microcapsules made from biodegradable biopolymers were used for the delivery of all CRISPR-Cas9 components to cells [106]. The efficiency of transfection indicated by loss of red fluorescence in dTomato-expressing HEK293T reached 70%. Submicro- and microcapsules with pArg/DS shells were successfully used as carriers for messenger RNA (mRNA) and small interfering RNA (siRNA) [107]. This study demonstrated that the package efficiency of RNA molecules, delivery efficiency, and biodistribution strongly depended on the size of the capsules. Both studies highlight the importance of developing safe and effective delivery systems for gene therapy and genome editing. The use of microcarriers offers a promising alternative to viral vectors, reducing the associated risks and potentially enhancing the clinical acceptance of these technologies. The delivery systems based on microcapsules are summarized in Table 1.

Finally, the use of CaCO₃-based microcapsules in various medical applications, especially in immunotherapy and targeted cancer treatment, appears a promising approach. Ongoing research and innovations in this field could transform cancer treatment, offering more effective and less invasive solutions, notably through the release of small molecules, proteins, and nucleic acids encapsulated in these polyelectrolyte capsules by physical absorption or coprecipitation, thus marking a significant evolution in therapeutic strategies.

5. Conclusion

CaCO₃ submicro- and microparticles have a considerable potential as vectors for targeted drug delivery, particularly in cancer treatment. Their controlled dissolution depending on pH ensures targeted drug release in the acidic areas of tumors while maintaining stability in the more neutral circulatory system. Different configurations of the delivery system, core-only and core/shell microparticles and microcapsules, offer solutions for the transport and controlled release of various therapeutic substances, including small molecules, proteins, and nucleic acids.

Vaterite CaCO₃ cores are effective for loading small molecules through techniques such as coprecipitation, allowing for subsequent controlled release. However, their rapid degradation in vivo can lead to premature release and disrupt cellular calcium balance. To address this issue, core/shell particles have been developed, where the CaCO₃ core is coated with a shell of polyelectrolytes, which regulates its degradation, thus allowing sustained and controlled drug release while minimizing cell damage. This system can also be modified to specifically target cells or tissues, improving therapeutic efficacy and reducing side effects.

Finally, CaCO₃-based polyelectrolyte capsules overcome the issues entailed with CaCO₃ particles. Removal of the core through calcium chelation limits the destabilization of the tumor microenvironment by the increase in intracellular Ca²⁺ and ultimately controlling the pH. The capsules are particularly promising for the encapsulation and controlled release of small molecules, nucleic acids, and proteins, due to their ability to degrade under specific intracellular conditions. Although the delivery of biomacromolecular therapeutic agents presents a huge challenge compared to the delivery of small molecules due to both their high molecular weight and fragile structure, these problems can be solved by using polymer delivery systems [108]. In summary, CaCO₃-based particles offer a versatile platform for more effective therapeutic treatments, particularly for complex diseases, such as cancer, due to their adaptability and capability for targeted and controlled drug delivery and release.

6. Outlook: In Vivo Studies

6.1. Modulation of the pH of Tumor Environment

Submicron CaCO₃ particles offer a promising tool to counteract the characteristic acidity of tumors, a known factor in promoting their aggressiveness and metastatic potential. The targeting of tumors with 20- to 300-nm calcium carbonate particles allows gradually increasing the tumor pH to neutrality. This pH modulation is crucial, because a less acidic environment can inhibit the growth and spread of cancer cells, thereby reducing their virulence. Particularly, 100-nm particles stand out for their ability to sustain a prolonged pH elevation. This highlights the importance of the particle size optimization in maximizing the treatment efficacy. Tests on animal models have shown a significant reduction of tumor growth, attesting to the therapeutic potential of this method. However, further research is required to optimize the dosage, evaluate the synergy with other treatments, and predict side effects. This advancement shows a way for improving cancer treatment strategies by targeting a fundamental aspect of tumor biology [109].

6.2. Biodistribution and Biocompatibility

The in vivo biodistribution of capsules is a major issue for the development of safe and effective drug carriers. Fluorescent CaCO₃-based pArg/DS capsules have been developed for kidney targeting via the renal artery [110]. The high efficiency of delivery to the area of interest was provided by optimization of the administration protocol and dosage.

6.3. Retention, Stability, and Toxicity

CaCO₃ particles labeled with ²²⁴Ra were proposed for local therapy of disseminated tumors [46]. Biodistribution studies showed that radioactivity was primarily localized in the peritoneal area after administration, with the highest activity associated with intraperitoneal adipose tissue and the parietal peritoneum. The release of ²²⁴Ra from the particles was relatively limited, as evidenced by reduced absorption in the skeleton compared to the administration of free ²²⁴Ra. Non-abdominal organs, such as the heart, muscles, and brain, displayed radioactivity levels below 100 Bq/g, which indicated a limited radiation exposure outside the abdominal area. These results indicate that radiolabeled CaCO₃ particles possess a high retention capacity and targeted bioavailability, making them potentially useful for targeted medical applications, minimizing non-target tissue exposure to radiation. The antitumor effect of CaCO₃ microparticles labeled with the alpha-emitting ²²⁴Ra was shown in mice [45]. This study highlights the advantage of using CaCO₃ as carrier of therapeutic agents and shows a particularly promising therapeutic strategy for tumors located in the abdominal cavity.

CaCO₃ core/shell particles 0.8 µm in size were used for encapsulation of the alpha-emitting ²²⁵Ac in order to enhance its retention and reduce systemic toxicity during alpha therapy [111]. The study showed a 93–94% retention of ²²⁵Ac after 20 days, with the majority of ²²⁵Ac-microparticles localized in the lungs, which indicated a reduced renal toxicity potential. In vivo tests on Wistar rats confirmed the high retention efficiency of the particles, underscoring the effectiveness of ²²⁵Ac-doped core/shell particles in safely retaining alpha emitters used for cancer treatment.

The wide potential applications of CaCO₃ nanoparticles in various sectors, including medicine, calls for thorough evaluation of their toxicity. In vitro experiments on NIH 3T3 and MCF7 cells treated with CaCO₃ nanoparticles at different concentrations (1–50 μg/mL) for 12 to 72 hours showed no cytotoxicity, oxidative stress, or DNA damage, indicating excellent biocompatibility. In vivo studies with zebrafish treated with CaCO₃ nanoparticles at doses as high as 200 μg/mL showed an absence of significant toxic effects on embryonic development. These results underscore the safety of CaCO₃ nanoparticles, suggesting their applicability in medicine and other fields, without cytotoxic or genotoxic risks to biological systems [112].

6.4. Vaccinal Applications

Recent studies illustrate innovative use of vaccines in anticancer immunotherapy, highlighting the in vivo efficacy of formulations based on submicron- and micron-sized CaCO₃ particles. The physical adsorption of an antigen (ovalbumin) into CaCO₃ particles with adsorbed pneumolysin, the key virulence factor of Streptococcus pneumoniae, significantly amplified cellular and humoral immunity, demonstrating preventive and therapeutic antitumor efficacy [113]. The 0.95-µm CaCO₃ particles degraded into Ca²⁺ and CO₂ in the acidic lysosomal environment, promoting cross-presentation of antigens. This biodegradability of the particles was confirmed by the detection of intracellular Ca²⁺, with the highest levels observed for the ovalbumin/CaCO₃/pneumolysin group. This study illustrates the induction of a robust immune response, offering an effective platform based on submicron- and micron-sized CaCO3 particles for the development of anticancer immunotherapy through vaccination.

Table 1. CaCO₃ core-only and CaCO₃-based core/shell microparticles and shell-only microcapsules.

Particle type	Size	Cargo type	Encapsulated molecule	Shell composition	Ref.
Core	1 μm	Small molecule	Doxorubicin	Hyaluronate/glutamate	[52]
Core	0.6–3.2 µm	-	-	-	[6]
Core	0.43 µm	Fluorescent dye	Rhodamine 6G	-	[40]
Core	0.52 µm	-	-	-	[1]
Core	0.4–2.7 µm	-	-	-	[7]
Core	n/a	Protein	BSA	-	[41]
Core	17.9 μm	Small molecule	Ibuprofen, nifedipine, losartan potassium, and metronidazole benzoate	-	[48]
Core	3.1–23.5 µm	Small molecule	Aspirin, vanillin	-	[42]
Core	17.9 μm	Protein	Lysozyme, BSA	-	[49]
Core	3.4 μm	Protein	Superoxide dismutase	-	[43]
Core	10 μm	Protein	Catalase, insulin, aprotinin	-	[44]
Core	0.8-1.6 μm	Small molecule	Doxorubicin	-	[21]
Core	4–5 μm	Protein	Catalase	-	[51]
Core	1 μm	Protein	Ovalbumin, pneumolysin	-	[113]
Core	5.45 μm	Protein	β-lactamase	-	[50]
Core	1.3 μm	-	-	-	[114]
Core	4–7 μm	Radionuclide	224Ra	-	[45]
Core	1–3, 3–15 μm	Radionuclide	224Ra	-	[46]
Core	0.2-1.1 μm	Nucleic acid	DNA	-	[34]
Core/shell	2 μm	Small molecule	Doxorubicin	Poly-L-ornithine/ fucoidan	[115]
Core/shell	0.2 μm	Small molecule	Doxorubicin	Oleic acid/PEG	[116]
Core/shell	3 μm	Small molecule	Doxorubicin	PDDA/PSS	[63]
Core/shell	∼10 μm	Protein	Ovalbumin, cancer cell lysate	Poly(HPMA-APMA) with TLR7/8-agonists	[64]
Core/shell	0.65, 3.2 μm	Radionuclide	225Ac	HSA/TA	[111]
Core/shell	∼2 μm	Protein	BSA	PLA	[65]
Core/shell	2–4 μm	Fluorescent dye	Nile Red, rhodamine 110	CaCO3	[66]
Core/shell, shell	2–2.5 μm	Small molecule	Doxorubicin	PAH/PSS/QD	[18]
Shell	4.75 μm	Protein	Lactalbumine, lysozyme, horseradish peroxidase, chymotrypsin	-	[39]
Shell	5.4 μm	-	-	PAH/PSS	[117]
Shell	9 μm	-	-	PLL, PR, DA, COL/HA, CS, DS, HS	[15]
Shell	3–6 μm	Protein	Insulin	PAH/PSS, PVS, DS	[20]
Shell	5 μm	Fluorescent dye	FITC-dextran	HA/PAH, PLL	[70]
Shell	3 μm	Fluorescent dye	FITC-dextran	pARG/DS, p(HPMA-DMAE)/PSS, PAH/PSS	[71]
Shell	∼1 μm	Fluorescent dye, protein	Rhodamine B, methylene blue, insulin	Phenylboronic –modified alginate/PVPON	[94]
Shell	1.8–3.8 μm	-	-	pArg/DS	[118]
Shell	0.5 μm	Small molecule	Doxorubicin	pArg/DS	[32]
Shell	3–5 μm	Extract	Gratiola officinalis extract	PAH/PSS/DS	[97]
Shell	4 μm	Small molecule	Apigenin, ascorbic acid	PAH/DS	[96]
Shell	0.25–0.5 μm	Small molecule	Gemcitabine, clodronate	pArg/DS	[95]
Shell	3.3–4.8 μm	Protein	BSA, chymotrypsin, lysozyme	PAH/PSS	[103]
Shell	5.0–8.3 μm	Small molecule	Tetracycline hydrochloride	PAH/pectin	[99]
Shell	5.0 μm	Cells	Escherichia coli	PAH/PSS	[98]
Shell	4.5 μm	Small molecule	Doxorubicin, nimbin	PAH/PMA/NR	[119]
Shell	5.0 μm	Small molecule	Vitamin B12	PAH/PSS	[100]
Shell	5.0 μm	Protein	BSA	PAH/PSS	[105]
Shell	4.2–6.3 μm	-	-	PAH/PSS/QD	[36]
Shell	3–4 μm	Protein	Ovalbumin, horseradish peroxidase	pArg/DS	[104]
Shell	3 μm	Nucleic acid	G-quadruplex DNA, double stranded DNA	PMA/PVPON	[29]
Shell	0.65, 3.3 μm	Nucleic acid	mRNA, siRNA	pArg/DS	[107]
Shell	∼3 μm	Nucleic acid	mRNA, pDNA, plasmid	pArg/DS/SiO2	[106]
Shell	1–4 μm	Fluorescent dye	Tetramethylrhodamine dextran	PAH/DNA	[120]
Shell	2.84 μm	Labeled protein	BSA-Cy7	pArg/DS	[110]
Shell	3–5 μm	Small molecule	Doxorubicin	Chitosan/alginate	[69]
Shell	3–6 μm	Labeled protein	FITC-BSA	PLL/CS	[121]

Table 1. CaCO₃ core-only and CaCO₃-based core/shell microparticles and shell-only microcapsules.

Particle type	Size	Cargo type	Encapsulated molecule	Shell composition	Ref.
Core	1 μm	Small molecule	Doxorubicin	Hyaluronate/glutamate	[52]
Core	0.6–3.2 µm	-	-	-	[6]
Core	0.43 µm	Fluorescent dye	Rhodamine 6G	-	[40]
Core	0.52 µm	-	-	-	[1]
Core	0.4–2.7 µm	-	-	-	[7]
Core	n/a	Protein	BSA	-	[41]
Core	17.9 μm	Small molecule	Ibuprofen, nifedipine, losartan potassium, and metronidazole benzoate	-	[48]
Core	3.1–23.5 µm	Small molecule	Aspirin, vanillin	-	[42]
Core	17.9 μm	Protein	Lysozyme, BSA	-	[49]
Core	3.4 μm	Protein	Superoxide dismutase	-	[43]
Core	10 μm	Protein	Catalase, insulin, aprotinin	-	[44]
Core	0.8-1.6 μm	Small molecule	Doxorubicin	-	[21]
Core	4–5 μm	Protein	Catalase	-	[51]
Core	1 μm	Protein	Ovalbumin, pneumolysin	-	[113]
Core	5.45 μm	Protein	β-lactamase	-	[50]
Core	1.3 μm	-	-	-	[114]
Core	4–7 μm	Radionuclide	224Ra	-	[45]
Core	1–3, 3–15 μm	Radionuclide	224Ra	-	[46]
Core	0.2-1.1 μm	Nucleic acid	DNA	-	[34]
Core/shell	2 μm	Small molecule	Doxorubicin	Poly-L-ornithine/ fucoidan	[115]
Core/shell	0.2 μm	Small molecule	Doxorubicin	Oleic acid/PEG	[116]
Core/shell	3 μm	Small molecule	Doxorubicin	PDDA/PSS	[63]
Core/shell	∼10 μm	Protein	Ovalbumin, cancer cell lysate	Poly(HPMA-APMA) with TLR7/8-agonists	[64]
Core/shell	0.65, 3.2 μm	Radionuclide	225Ac	HSA/TA	[111]
Core/shell	∼2 μm	Protein	BSA	PLA	[65]
Core/shell	2–4 μm	Fluorescent dye	Nile Red, rhodamine 110	CaCO3	[66]
Core/shell, shell	2–2.5 μm	Small molecule	Doxorubicin	PAH/PSS/QD	[18]
Shell	4.75 μm	Protein	Lactalbumine, lysozyme, horseradish peroxidase, chymotrypsin	-	[39]
Shell	5.4 μm	-	-	PAH/PSS	[117]
Shell	9 μm	-	-	PLL, PR, DA, COL/HA, CS, DS, HS	[15]
Shell	3–6 μm	Protein	Insulin	PAH/PSS, PVS, DS	[20]
Shell	5 μm	Fluorescent dye	FITC-dextran	HA/PAH, PLL	[70]
Shell	3 μm	Fluorescent dye	FITC-dextran	pARG/DS, p(HPMA-DMAE)/PSS, PAH/PSS	[71]
Shell	∼1 μm	Fluorescent dye, protein	Rhodamine B, methylene blue, insulin	Phenylboronic –modified alginate/PVPON	[94]
Shell	1.8–3.8 μm	-	-	pArg/DS	[118]
Shell	0.5 μm	Small molecule	Doxorubicin	pArg/DS	[32]
Shell	3–5 μm	Extract	Gratiola officinalis extract	PAH/PSS/DS	[97]
Shell	4 μm	Small molecule	Apigenin, ascorbic acid	PAH/DS	[96]
Shell	0.25–0.5 μm	Small molecule	Gemcitabine, clodronate	pArg/DS	[95]
Shell	3.3–4.8 μm	Protein	BSA, chymotrypsin, lysozyme	PAH/PSS	[103]
Shell	5.0–8.3 μm	Small molecule	Tetracycline hydrochloride	PAH/pectin	[99]
Shell	5.0 μm	Cells	Escherichia coli	PAH/PSS	[98]
Shell	4.5 μm	Small molecule	Doxorubicin, nimbin	PAH/PMA/NR	[119]
Shell	5.0 μm	Small molecule	Vitamin B12	PAH/PSS	[100]
Shell	5.0 μm	Protein	BSA	PAH/PSS	[105]
Shell	4.2–6.3 μm	-	-	PAH/PSS/QD	[36]
Shell	3–4 μm	Protein	Ovalbumin, horseradish peroxidase	pArg/DS	[104]
Shell	3 μm	Nucleic acid	G-quadruplex DNA, double stranded DNA	PMA/PVPON	[29]
Shell	0.65, 3.3 μm	Nucleic acid	mRNA, siRNA	pArg/DS	[107]
Shell	∼3 μm	Nucleic acid	mRNA, pDNA, plasmid	pArg/DS/SiO2	[106]
Shell	1–4 μm	Fluorescent dye	Tetramethylrhodamine dextran	PAH/DNA	[120]
Shell	2.84 μm	Labeled protein	BSA-Cy7	pArg/DS	[110]
Shell	3–5 μm	Small molecule	Doxorubicin	Chitosan/alginate	[69]
Shell	3–6 μm	Labeled protein	FITC-BSA	PLL/CS	[121]