Application prospect of calcium peroxide nanoparticles in biomedical ﬁ eld

: In recent years, calcium peroxide ( CaO 2 ) has attracted widespread attention in the medical community due to its excellent antitumor and antibacterial proper - ties, and has gradually become a hot research topic in the biomedical ﬁ eld. CaO 2 reacts with water ( H 2 O ) to produce calcium ion ( Ca 2 + ) , oxygen ( O 2 ) , and hydrogen peroxide ( H 2 O 2 ) , where Ca 2 + is suitable for calcium death caused by calcium overload, O 2 is suitable for O 2 - dependent anticancer therapy, and H 2 O 2 is suitable for H 2 O 2 - depen - dent anticancer therapy. In addition, H 2 O 2 can also be used in the antibacterial ﬁ eld to treat bacterial infections. All these make the CaO 2 to become a kind of excellent antitumor and antibacterial drug. This study mainly reviews the preparation and surface modi ﬁ cation of CaO 2 , probes into the latest progress about CaO 2 nanoparticles in the ﬁ eld of tumor treatment and antimicrobial therapy. Finally, the challenges that CaO 2 still faces in the future research ﬁ eld are clari ﬁ ed, and its prospects are forecasted.


Introduction
So far, cancer is still a major risk factor threatening human health. The traditional methods of cancer treatment mainly include surgery, radiotherapy, and chemotherapy. Although it has a certain therapeutic effect, the unavoidable side effects and poor specificity of cancer cells, so far, cannot meet the needs of cancer treatment.
With the progress and development of modern science and technology, new tumor treatment methods such as chemodynamic therapy (CDT) [1,2], photodynamic therapy (PDT) [3][4][5], photothermal therapy (PTT) [6,7], sonodynamic therapy (SDT) [8], and calcium overload [9] emerge as the times requirement, which broadens the mode of diagnosis and treatment related to cancer. It brings more hope for cancer patients. However, affected by the tumor microenvironment (TME), the emerging tumor treatment methods are also difficult to play effectively. For bacterial infection, antibiotics are the most commonly used method to treat bacterial infection [10]. Although it reduces the morbidity and mortality of human beings, its long-term and overuse makes multidrug-resistant bacterial infection one of the world's public health threats [11,12]. At present, oxidative stress induced by reactive oxygen species (ROS) can effectively treat bacterial infection, which is favored by researchers because of its destructive effect on bacteria.
Under acidic conditions, CaO 2 with structural peroxy bond reacts with H 2 O to form a large amount of H 2 O 2 (0.47 g H 2 O 2 /g CaO 2 ) and a small amount of O 2 (0.2222 O 2 /g CaO 2 ) [13,14], accompanied by the release of Ca 2+ . Therefore, the introduction of CaO 2 , on the one hand, reshape the TME to ensure the effective play of anticancer therapy such as O 2dependent (chemotherapy, PDT), H 2 O 2 -dependent (CDT), and calcium overload, while, on the other hand, effectively inhibit bacterial infection.
This article mainly summarized the preparation and surface modification of CaO 2 , probes into the latest progress about CaO 2 nanoparticles in the field of tumor treatment and antimicrobial therapy. The schematic diagram of the review is shown in Figure 1. At present, there are three main methods to prepare CaO 2 at home and abroad: CaCl 2 method, CaO method, and Ca(OH) 2 method. Ca(OH) 2 method is divided into traditional method, spray drying method, and air cathode method. Among them, CaCl 2 method and traditional Ca(OH) 2 method belong to hydrolytic precipitation method and are the most commonly used methods for preparing CaO 2 [20].

CaCl 2 method
General preparation process: first of all, ammonia water is injected into an alkaline solution of metal chloride (such as CaCl 2 ), and in the agitation state, H 2 O 2 containing a stabilizer is injected to activate the reaction [21]. The reaction equation is as follows: (2) Without the addition of stabilizer, low temperature reaction is needed, and the preparation process is complicated and the cost is high. At present, the method of preparing CaO 2 at room temperature by introducing stabilizer is widely used to improve the utilization rate of H 2 O 2 and the yield of CaO 2 , and to overcome the problems of complex preparation process and high cost under low temperature production conditions.
The injecting of ammonia provides the required alkaline environment for the synthesis of CaO 2 material and neutralizes the by-product hydrochloric acid (HCl), which promotes the forward progress of the reaction [22]. Similarly, sodium hydroxide as an alkaline substance, cannot replace ammonia to provide a suitable alkaline environment for the synthesis of CaO 2 NPs. The reason might be that sodium hydroxide is a strong base environment which can easily generate Ca(OH) 2 precipitation and excessive hydroxyl ions to promote H 2 O 2 decomposition.
CaCl 2 method has the advantages of simple preparation process, low cost, mature technology, and suitable for small-scale production. However, due to the use of dilute solution production, the decomposition loss of H 2 O 2 in mother liquor is large, and energy consumption is large, resulting in low product content of CaO 2 , generally 50-60%.

CaO method
The general preparation process: CaO is dissolved in H 2 O to generate Ca(OH) 2 solution, and after the temperature of the reaction solution is stable, H 2 O 2 is added to obtain CaO 2 ·8H 2 O. After filtering and drying the filter cake, anhydrous calcium peroxide (CaO 2) is obtained. The reaction equation is as follows: (5) Figure 1: Schematic diagram of the review: Preparation, surface modification, and application of CaO 2 NPs in tumor and bacteria. Ion interference therapy: reproduced with permission from previous study [15]. © 2019 Elsevier Inc. O 2 dependence therapy: reproduced with permission from previous study [16]. Copyright © 2021, American Chemical Society. H 2 O 2 dependence therapy (the field of tumor therapy): reproduced with permission from previous study [17]. © 2021 Elsevier B.V. All rights reserved. H 2 O 2 dependence therapy (the field of bacterial therapy): reproduced with permission from previous study [18]. © 2021 Wiley-VCH GmbH; Surface modification: reproduced with permission from previous study [19]. © 2021 Elsevier B.V. All rights reserved. Preparation method: reproduced with permission from previous study [18]. © 2021 Wiley-VCH GmbH. Notes: polyethylene glycol (PEG), polyethylene pyrrolidone (PVP), hyaluronic acid (HA), sodium hyaluronate (SH), tannic acid (TA), calcium chloride (CaCl 2 ), calcium oxide (CaO), calcium hydroxide (Ca(OH) 2 ).
The preparation of CaO 2 by CaO method has the advantages of cheap raw materials, simple preparation process, simple equipment, no need to add ammonia and other substances, and basically no problem of discharge of the three wastes. General preparation process: under the action of stabilizer, Ca(OH) 2 is slowly added to H 2 O 2 to get CaO 2 . After further drying anhydrous, CaO 2 material can be obtained [23]. The reaction equation is as follows: The purpose of introducing stabilizer is to make the reaction proceed at room temperature and improve the utilization rate of H 2 O 2 and the stability of the product. When no stabilizer is added, low temperature reaction is required where the reaction temperature should be controlled below 5℃, which is of high cost and high energy consumption.
Because CaO 2 is slightly soluble in H 2 O, most of the domestic Ca(OH) 2 is dissolved in ammonium solution to generate ammonium complex, and then the free Ca 2+ in the complex reacts with H 2 O 2 to prepare CaO 2 substance. The reaction equation is as follows: The preparation of CaO 2 by Ca(OH) 2 method simplifies the production and preparation process, reduces energy consumption and production cost, and the technology is mature. However, because it is also produced by dilute solution, the product yield and the content of CaO 2 are not high, and the content of CaO 2 is generally 50-60%.

Spray drying method
Under the condition of cooling and constant stirring, the concentrated suspension of Ca(OH) 2 water reacted with the concentrated H 2 O 2 , and the resulting mixture was spray dried directly after the reaction [24]. This method can obtain anhydrous CaO 2 with good dispersion and uniform particle size without separation and purification. The reaction equation is as follows: Compared with the traditional preparation method, spray drying method has the following advantages: (a) H 2 O 2 and Ca(OH) 2 are cheap and easy to obtain. (b) It saves time and cost without separation and purification. (c) It improves the utilization rate of H 2 O 2 and the yield of CaO 2 . (d) Energy saving. (e) Continuous production and intermittent production can be carried out, so that the product has good uniformity.
However, this method is also faced with problems such as heavy equipment investment, easy blockage of pipes, high energy consumption, and explosive risk. In addition, high concentration of H 2 O 2 is lacking in China.

Air cathode method
In order to further solve the problem of high cost of H 2 O 2 , this method is a low-cost H 2 O 2 production process after the industrialization of producing H 2 O 2 by anthraquinone method. Ca(OH) 2 reacts with diluted H 2 O 2 to generate CaO 2 ·8H 2 O. The mother liquor is removed after centrifugation, and the obtained NaOH is recycled or discharged in time. CaO 2 product can be obtained by grinding the product after vacuum drying of filter cake. The reaction equation is as follows: Compared with traditional preparation methods, CaO 2 prepared by air cathode method can reduce production cost, but it still has the following limitations: (a) The utilization rate of H 2 O 2 and the yield of CaO 2 are low. (b) High energy consumption. (c) The separation of CaO 2 is difficult and the production cost is increased due to its fine particles and colloids. (d) Additional crushing device is required. (e) CaO 2 products obtained are heterogeneous. Table 1 presents a comparison of advantages and disadvantages between different preparation methods.

Surface modifiers of CaO 2
CaO 2 NPs prepared by traditional methods often has some phenomena such as different particle size, different morphology, and poor stability. Therefore, surface modifiers are often used to regulate the particle size, morphology, and stability of CaO 2 NPs. In addition, some surface modifiers can endow CaO 2 NPs with different functions.
Zheng [28] prepared CaO 2 NPs without adding surface modifier and with adding surface modifier, and the results are as follows: 1) Pure CaO 2 NPs: the particle size is different and the dispersion is poor, showing agglomerate morphology; 2) PDA-CaO 2 NPs: the particle size is different, about ∼ 150 nm, and the agglomeration is serious; 3) PDA-SC-CaO 2 NPs: the particle size is small, about 30 nm, with a filamentous structure; 4) SC-CaO 2 NPs: uniform particle size, monodisperse spherical structure, and good dispersibility; 5) PAA-CaO 2 NPs: the particle size is different, and the phenomenon of agglomeration is serious; 6) EDTA·2Na-CaO 2 NPs: different particle size, showing cross-linked reticular structure.
Park et al. [29] successfully synthesized/CaO 2 NPs by introducing TA in the process of CaO 2 NPs synthesis. Figure 2A shows the particle size distribution and scanning electron microscope (SEM) images of three kinds of TA/CaO 2 NPs. It can be seen from the diagram that the three kinds of TA/CaO 2 NPs have the problems of relatively uniform particle size, relatively regular morphology, and poor dispersion.
Khodaveisi et al. [21], Yin et al. [30], and Liu et al. [31] successfully prepared PEG-modified CaO 2 NPs ( Figure  2B-D). Transmission electron microscopy (TEM) images showed that although PEG as a surface modifier could alleviate the agglomeration problem of CaO 2 NPs in aqueous solution [21], NPs often showed irregular size and morphology. In addition, the method requires a timeconsuming washing process to stabilize pH [29].
He et al. [26] successfully prepared CaO 2 NPs modified by CO-520 by reverse microemulsion method. The introduction of CO-520 gives CaO 2 NPs a good dispersion, but it still faces the problems of different size and irregular morphology.  Shen et al. [32] successfully synthesized CaO 2 nanocrystals and spherical aggregates with controllable size and uniform morphology by adjusting the concentration of CaCl 2 and PVP on the basis of solving the CaO 2 NPs agglomeration problem ( Figure 2F and G). Most of all, CaO 2 NPs modified by PVP should be dispersed in ethanol for preservation after synthesis, as drying might promote CaO 2 NPs irreversible aggregation [32].
Zhang et al. [15] successfully prepared SH-CaO 2 NPs by using SH as surface modifiers ( Figure 2E). In addition, Han et al. [25] successfully prepared HA-CaO 2 NPs by using HA as surface modifiers. The introduction of SH and HA not only gives CaO 2 NPs the advantages of good dispersion, uniform size, and uniform morphology but also gives it the function of active targeting to improve its enrichment rate in tumor tissue. Table 2 describes the effects of the presence or absence of surface modifiers on the morphology, particle size, and stability of CaO 2 NPs, and the potential application of partially modified NPs to cancer cells.

Application of CaO 2 in tumor therapy
CaO 2 as a promising anticancer material, introduced into the nanodrugs delivery system might regulate the TME. The construction of a multi-functional nano-therapy platform based on CaO 2 NPs can achieve a single treatment for tumors, and even achieve a combined antitumor effect. This chapter mainly focuses on several anticancer therapies based on CaO 2 NPs.  [32]. The inset in (a) shows an image at a higher magnification [32]. (A) Reproduced with permission from a previous study [29]. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. (B) Reproduced with permission from a previous study [21]. Copyright © 2011 Elsevier B.V. All rights reserved. (C) Reproduced with permission from a previous study [30]. © 2021 Elsevier B.V. All rights reserved. (D) Reproduced with permission from a previous study [31]. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (E) Reproduced with permission from a previous study [15]. © 2019 Elsevier Inc. (F and G) Reproduced with permission from a previous study [32]. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced chemotherapy
Chemotherapy refers to the use of highly cytotoxic chemotherapeutic drugs to interfere with the proliferation of cancer cells and then kill cancer cells to achieve the purpose of tumor treatment, independent of external energy, and also suitable for the treatment of deep tumors that are difficult to reach by laser. The permeability of nanodrugs in the tumor site is an important factor affecting the effectiveness of chemotherapy. The poor permeability of nanodrugs in tumor site is mainly affected by TME, including heterogeneous blood supply, interstitial fluid pressure (IFP), and extracellular matrix (ECM) [33]. First of all, because the heterogeneous blood supply of tumor tissue is mainly distributed around the tissue, coupled with the high oxygen consumption of tumor cells, hypoxia has become a prominent feature of advanced solid tumors. Compared with normal physiological blood vessels, hypoxia up-regulates hypoxia inducible factor-1alpha (HIF-1a) and multidrug resistance protein 2 (MRP2), inducing immunosuppression and immune escape of tumor cells, resulting in multidrug resistance and rendering tumor-related chemotherapy drugs ineffective [34][35][36]. Therefore, hypoxia poses a great challenge to O 2 -dependent chemotherapy. Second, in tumor tissue, IFP increases gradually from outside to inside, which hinders the further spread of nanodrugs to deep tumors. In addition, ECM further hinders the penetration of nanodrugs in the tumor site, due to the fact that collagen is the main component of tumor tissue and is expressed in a HIF-1a-dependent manner. In the hypoxic environment of tumor tissue, collagen deposition increases the density of ECM, which affects the penetration of nanodrugs in the tumor site. This section mainly discusses how to improve the antitumor effect of chemotherapy drugs from the point of view of hypoxia and ECM. CaO 2 has more advantages than other O 2 producing materials, and its biocompatibility and biodegradability are better.
CaO 2 can not only produce O 2 in situ but also provide reaction substrate for other O 2 -producing materials (for example, provide raw material for manganese dioxide (MnO 2 ) to produce O 2 under acidic TME), as shown in equation (14) [37].
In situ O 2 production of CaO 2 can regulate TME, downregulate HIF-1a and MRP2, reverse multidrug resistance of tumor cells, overcome hypoxia-induced chemotherapy limitation etc., and improve the therapeutic effect of chemotherapy drugs. Zhang et al. [38] constructed an intelligent O 2 nanocarrier coated with solid lipid monostearin (MS) for CaO 2 /MnO 2 to comprehensively optimize doxorubicin (DOX) transport and enhance chemotherapy effect. Due to the high expression of lipase in tumor cells, NPs are passively accumulated in tumor tissues through enhanced permeability and retention (EPR) effect and ingested by tumor cells [39]. The MS shell of NPs is degraded by lipase, exposing core NPs (CaO 2 /MnO 2 ) and releasing O 2 and DOX. After cancer cells death, the incomplete reaction core NPs are  2 and Fe 2+ , the former downregulated the expression of HIF-1a and P-glycoprotein (P-gp) and enhanced the chemotherapy effect of DOX, while the latter continued to react with H 2 O 2 to produce hydroxyl radical (·OH) and enhance the effect of CDT. It has been proved that nanomaterials have excellent anticancer effect and good biosafety through in vivo and in vitro experiments.
Wang et al. [41] used CaO 2 NPs as a synergist in cooperation with transcatheter arterial chemoembolization (TACE) to improve its antitumor effect. After intratumoral injection, CaO 2 NPs react with H 2 O to form O 2 , H 2 O 2 , hydroxyl ions (OH − ), and Ca 2+ , to reshape TME and induce calcium overload. In addition, due to the regulation of TME, the therapeutic effect of chemotherapy drugs in TACE has been effectively improved. The results in vitro and in vivo showed that the synergistic antitumor effect of CaO 2 NPs + TACE group was significant.
Zhang et al. [42] constructed a nanohybrid material (CaO 2 @FePt-DOX@PDA@CM) camouflaged by the membrane of 4T1 cancer cells, which enhances the therapeutic effect of cancer by cooperating with chemotherapy, CDT, Ca 2+ overload, and PTT. 4T1 cancer cell membrane has the ability of immune escape and homologous targeting to tumor cells. After the nanohybrid material was internalized by cancer cells, the O 2 -producing characteristics of CaO 2 were used to alleviate the hypoxia of tumor cells, downregulate the expression of HIF-1a and P-gp, and enhance the chemotherapeutic effect of DOX, the H 2 O 2producing characteristics of CaO 2 were used to enhance the CDT effect of FePt, and the Ca 2+ -releasing characteristics of CaO 2 were used to induce mitochondrial damage, upregulate Cytochrome C and Caspace-3, and achieve calcium overload. In addition, the existence of PDA gives nanohybrid materials excellent photothermal conversion properties, which can enhance the efficiency of Fenton reaction while giving full play to PTT. In vitro and in vivo studies have shown that nanohybrid materials can cooperate with a variety of anticancer therapies and effectively improve the therapeutic effect by optimizing individual advantages.

Enhanced PDT
PDT is one of the effective antitumor therapies approved by the US Food and Drug Administration (FDA), which is a promising antitumor strategy. PDT consists of three basic components: light, photosensitizer (PS), and oxygen source [43]. The antitumor mechanism of PDT is that PS reacts with molecular oxygen under laser irradiation at a specific wavelength to produce efficient singlet oxygen ( 1 O 2 ), which causes irreversible damage to tumor cells and blood vessels and leads to photoinduced tumor cell death. Figure 3 illustrates the basic principles of PDT, PS change from ground state to instantaneous singlet state ( 1 PS * ) under the irradiation of light source. There are two alternative paths for instantaneous singlet states: (1) Transition from instantaneous singlet state to ground state. (2) The transition from instantaneous singlet state to long-lived excited triplet state ( 3 PS * ). The excitation triplet also includes type I and type II reaction processes: (1) Type I: The cation or anion radical is formed by   [45]. Type I and Type II photochemical reactions can occur simultaneously, and the ratio between the two is determined by the type of PS and the concentration of O 2 [23,46]. According to the latest research progress, compared with O 2 -dependent type II PDT, type I PDT is not O 2 -dependent, ROS is more toxic, and has a broader prospect in the treatment of hypoxic tumors [44,47]. However, for most PDT photosensitizers, type II photochemical processes dominate [48]. Therefore, this chapter mainly discusses the application of CaO 2 NPs in enhancing O 2 -dependent type II PDT.
However, whether PDT effect can play effectively is mainly limited by O 2 . The consumption of O 2 in tumor cells by PDT will further aggravate the hypoxia of tumor cells which will lead to further weakening of PDT treatment effect or even treatment failure and tumor cells metastasis which form a vicious cycle. The effective way to improve the PDT effect of O 2 consumption is to establish an O 2 supply system to reduce O 2 consumption and improve the utilization rate of O 2 . CaO 2 NPs as an O 2 supply system is a promising material for regulating acid TME and enhancing PDT. After introducing CaO 2 NPs into tumor cells, O 2 generated through disproportionation reaction with H 2 O compensated for the O 2 consumed during PDT treatment and promoted the effective play of PDT effect (Figure 3).
Sun et al. [49] loaded CaO 2 NPs (PCN-224-CaO 2 -HA) modified by HA on the basis of porphyrin metal-organic frameworks (MOFs) to target and enhance PDT (Figure 4) Although the above cases partly overcome the problem of insufficient oxygen source of PDT, the consumption of ROS by the highly expressed glutathione (GSH) Figure 4: Schematic representation of PCN-224 combined with HA-modified CaO 2 NPs for enhanced PDT [49]. Reproduced with permission from a previous study [49]. © The Royal Society of Chemistry.
in tumor cells still limits the antitumor effect of PDT. Therefore, the effect of single treatment is limited after all, and combined antitumor therapy is necessary.
Gulzar et al. [51] constructed a multi-mode imageguided nanocomposite CaO 2 -MnO 2 -UCNPs-Ce6/DOX (Ca-Mn-NUC). When the nanocomposite is endocytosed by tumor cells, CaO 2 generates O 2 in situ under acidic TME, providing sufficient oxygen source for PDT. After MnO 2 converts GSH to oxidized glutathione (GSSH), it can reduce the consumption of ROS, further enhance the oxidative stress of tumor cells, and finally enhance PDT. In addition, the O 2 generated can also improve chemotherapy, and ultimately achieve chemotherapy/ PDT combined treatment, which is highly anticancer. The nanocomposite can also provide computed tomography (CT) and magnetic resonance imaging (MRI) dual-mode imaging for real-time monitoring of anticancer effects.
Chen et al. [52] developed a TME-responsive CaO 2based nanosystem (CF@CO@HC) using a bottom-up approach, which has the ability of GSH consumption and ROS self-amplification, and can cooperate with PDT and CDT for cancer treatment. The nanosystem consists of CaO 2 (CaO 2 -FM) doped with PS as core, hybrid silicone skeleton (Cu-ONS), and local hydrophobic cage (HC) as shell. After the nanosystem was internalized by cancer cells, the hybrid organosilicon skeleton was cleaved under the reduction of high concentration of GSH to release Cu + . CaO 2 reacts with H 2 O in acidic environment to form H 2 O 2 and O 2 , and releases the doped PS derivative 4-FM. Subsequently, the generation of H 2 O 2 enhances Cu + -mediated CDT. The generation of O 2 enhances 4-FM-mediated PDT. In addition, GSH consumption further increases the production of ROS. Therefore, the nanosystem provides a strategy to increase revenue and reduce expenditure for cancer treatment, and effectively enhances cancer treatment.
In addition, Yan et al. [53] designed and synthesized a multistimulus TME response nanoplatform composed of MCMnH NPs and CaO 2 NPs to improve the antitumor effect of the guidance of MRI and photothermal imaging. HA can target tumor cells with overexpression of CD44+ receptor and induce tumor cells to uptake MCMn NPs. Subsequently, MNP realizes PTT under the irradiation of 808 nm laser, and PDT is realized through energy transfer between PTT and Ce6. MnO CDT works independent of O 2 and external energy compared to other cancer treatments [59,60]. Therefore, CDT is more suitable for deep tumors with hypoxia or difficult to reach by laser.
At present, antitumor research on CDT is still in the initial stage. However, there are many factors affecting the efficiency of CDT. In order to improve the therapeutic effect of CDT, researchers mainly proposed the following methods: Change TME (increase H 2 O 2 concentration, decrease GSH level, decrease pH value) and select suitable catalyst and combine with other anticancer therapy effectively. In addition, in order to improve the efficacy of CDT, current studies mostly focus on the catalytic activity of nanomaterials, but ignore their biocompatibility and biodegradation.
CaO 2 NPs have good biocompatibility and biodegradability. CaO 2 NPs and H 2 O generate H 2 O 2 and O 2 through disproportionation reaction (equations (15) and (16)), in which the former provides sufficient substrates for subsequent CDT, thus ensuring the effective play of CDT effect and enhancing the antitumor effect of CDT. The latter regulates TME and reverses hypoxia in tumor cells [61][62][63]. Therefore, considering the advantages of CaO 2 NPs, it is particularly important to construct a nanotherapy platform based on CaO 2 NPs.
Due to the limited H 2 O 2 concentration (∼100 µM) in tumor cells, ROS production is difficult to maintain. Based on this, Han et al. [25] prepared a H 2 O 2 self-supporting nanotubule platform CaO 2 -Fe 3 O 4 @HA-Cy7 NPs for guiding targeted and imaging CDT. The HA can target the tumor cell receptor over-expressed by CD44+ and reach the cell through endocytosis, which is hydrolyzed by hyaluronidase in the cytoplasm and releases nearinfrared (NIR) fluorescent group markers, CaO 2 , and Fe 3 O 4 . Through further reaction, the H 2 O 2 consumed by CDT can be compensated and the therapeutic effect of CDT can be enhanced. NIR fluorescence and magnetic resonance bimodal imaging provide timely CDT therapeutic effect of NPs in vivo. In vivo antitumor studies have shown that CaO 2 -Fe 3 O 4 @HA-Cy7 NPs have excellent tumor specificity, enhanced CDT efficacy, and good biocompatibility.
Mamat et al. [64] constructed CaO 2 /Fe 3 O 4 nanocomposites coated with oleyl-amine (OA). The NPs inhibited the premature reaction of CaO 2 , overcame the deficiency of H 2 O 2 in tumor tissues, and realized the non-oxidative generation of ROS and efficient CDT. The results show that the nanocomposites material has significant tumor growth inhibition ability and good biocompatibility, and has great clinical transformation value in tumor therapy.
GSH is another important factor that limits the efficacy of CDT therapy. Therefore, reducing GSH expression level in cancer cells to reduce ROS consumption is an effective way to improve the efficiency of CDT treatment. In addition, the commonly used Fenton and Fenton-like reaction catalysts (Fe 2+ and Fe 3+ ) have high catalytic efficiency only under strong acidic conditions (pH 2-4), maximizing the therapeutic effect of CDT, on the contrary, the catalytic efficiency is relatively low under neutral and weak acidic conditions. Therefore, Kong et al. [65] explored a new combination of antitumor therapy by designing and synthesizing a Cu-ferrocene modified CaO 2 NPs (CaO 2 /Cu-ferrocene). In terms of CDT enhancement, this scheme not only solves the problem of insufficient H 2 O 2 in tumor cells, but also overcomes the problems of high expression of GSH and low efficiency of Fenton and Fenton-like reaction catalysts in tumor cells. The NPs remain stable under neutral conditions and rapidly release H 2 0 2 under acidic TME. Among them, H 2 O 2 and ferrocene generate ·OH through Fenton reaction, which achieves CDT, while Cu 2+ reacts with GSH to generate GSSH and Cu + , which reduces the consumption of ROS by GSH and improves the catalytic efficiency of Fenton and Fenton-like reaction catalysts, thus greatly improving the therapeutic effect of CDT. In vitro and in vivo experimental results showed that the consumption of GSH and the production of ·OH significantly enhanced the therapeutic effect of CDT.
In addition, in order to improve the killing effect of NPs on tumor cells, the combined antitumor application based on CDT has also been reported one after another [66][67][68]. Compared with single anticancer therapy, two or more CDT-based treatment modes can achieve better anticancer effects.
The combination of chemotherapeutic drugs and ROS for tumor therapy is also an effective treatment strategy to improve the efficiency of CDT [69][70][71][72]. Gao et al. [73] designed and synthesized a self-supplied O 2 /H 2 O 2 nanocatalysis drug CaO 2 @DOX@ZIF-67, and constructed a pH responsive CDT/chemotherapy combined antitumor treatment platform (Figure 5a). The NPs are taken up by acid lysosomes after entering the tumor cells. Subsequently, the MOFs decomposes and releases Co 2+ , CaO 2 and DOX. The Co 2+ reacts with H 2 O 2 to achieve CDT, and DOX to achieve chemotherapy. In addition, CaO 2 further reacts to generate H 2 O 2 and O 2 to improve the TME, reverse hypoxia of tumor cells, avoid multidrug resistance of cancer cells, enhance CDT and chemotherapy, and enhance the combination therapy of the two. The experimental results show that CaO 2 @DOX@ZIF-67 had good antitumor activity. Therefore, the nanocomposite is expected to be a candidate for pH-responsive chemotherapy/CDT combined antitumor therapy, and has great clinical transformation value.
Starvation therapy (ST) is to starve the tumor cells by cutting off the blood supply to the tumor tissue [74] or cutting off the nutrition source of the tumor cells [75]. Glucose is the energy source for the growth and proliferation of tumor cells. Reducing the concentration of glucose in tumor cells can effectively cut off the energy supply of cancer cells and inhibit or kill cancer cells. Glucose oxidase (GOx) is an endogenous redox enzyme that specifically oxidizes glucose to gluconic acid and H 2 O 2 . In recent years, GOx has been widely used in the treatment of tumor starvation, but its anticancer effect is limited by the hypoxic condition of tumor. Based on this, Zhang et al. [76] coated CaO 2 @Fe(OH) 3 nanometer composites and GOx in bio-compatible liposomes to prepare nanometer composite drugs, and their synthesis steps and antitumor mechanism are shown in Figure 5b. When the NPs entered the tumor cells, the degradation of CaO 2 @Fe(OH) 3  by GOx in the process of oxidizing glucose, cut off the energy material (glucose) of tumor tissue, realize the purpose of starving tumor cells, and strengthen ST, the latter triggers Fenton reaction to produce ·OH with strong oxidation capacity, which realizes CDT. In addition, the products gluconic acid and H 2 O 2 obtained by GOx in the process of glucose oxidation reduce the pH value of tumor tissue, provide the best reaction conditions for CDT, and further supplement its required substrate (H 2 O 2 ) and improve the effect of CDT. The experimental results confirmed that the anticancer effect of CDT/ST combination was significant.
PDT is an anticancer therapy based on ROS. Compared with CDT, both of them have the common goal of achieving tumor therapy by means of ROS. Liu et al. [77] designed a thermosensitive nano-system of H 2 O 2 /O 2 MSNs@CaO 2 -ICG@LA for CDT/PDT combination therapy of tumor cells (Figure 5c). The NPs were prepared by loading CaO 2 NPs and indocyanine green (ICG) with manganese silicate nanoparticles (MSNs) and further  [76]. © 2021 Elsevier Ltd. All rights reserved. (c) Reproduced with permission from a previous study [77]. modifying lauric acid (LA) on their surface. When the NPs reached the tumor cells through the endocytic pathway, ICG generated heat and 1 O 2 under the irradiation of 808 nm laser, the former caused the phase transformation of LA (from solid phase to liquid phase), and the latter realized PDT. In addition, exposed CaO 2 NPs further react with H 2 O to generate H 2 O 2 and O 2 . The production of H 2 O 2 provides sufficient substrates for subsequent CDT and enhances CDT. The production of O 2 alleviates tumor hypoxia and enhances PDT. MSNs are gradually exposed with further consumption of CaO 2 NPs, and react with GSH in cancer cells to release a large amount of Fenton-like reaction catalyst Mn 2+ , which is used to self-supply CDT of H 2 O 2 . The consumption of GSH by MSNs further promotes the continuous production of ROS in cancer cells. In conclusion, the team utilized PDT/CDT combined anticancer therapy to increase ROS sources in tumor cells while maintaining ROS production in tumor cells through GSH consumption. Experimental results in vivo and in vitro show that the strategy has remarkable anticancer effect and can effectively inhibit the growth of cancer cells.

Calcium overload
Calcium overload belongs to ion interference therapy, which is a new and efficient antitumor technology and has attracted more and more attention in recent years [17,78]. Calcium overload refers to the dysfunction of calcium balance system and disorder of calcium distribution under the action of some factors, resulting in abnormal increase in intracellular calcium concentration. Calcium overload has been linked to tumor death, but the root cause of cancer cell death remains unclear. Therefore, cancer cell death caused by calcium overload may be related to the following factors or the result of a combination of factors: 1) Induction of tumor cell death by activating mitochondrial apoptosis pathway [15,[79][80][81][82][83].
2) It is related to macroscopic calcification induced by microscopic calcium overload, leading to the death of cancer cells [15] (it is worth noting that tumor cell calcification can assist CT imaging and monitor the treatment process [84]).
3) It is related to interfering the process of mitochondrial oxidative phosphorylation, damaging mitochondria and inactivating phospholipase in cytoplasm, thus causing irreversible damage to tumor cells [85].
Ca 2+ are mainly stored in organelles such as cytoplasm, mitochondria, and endoplasmic reticulum, and are endogenous substances necessary for the maintenance of organisms in vivo. As one of the second messengers, Ca 2+ play an important regulatory role in the proliferation and migration of tumor cells [86,87]. As tumor cells are characterized by abnormal differentiation and proliferation, reduced apoptosis, and high metastasis, low concentration of Ca 2+ in the cytoplasm is a necessary condition to avoid apoptosis of tumor cells when cancer occurs [88,89]. As a result, improving the Ca 2+ concentration in the cytoplasm of tumor is an effective treatment strategy for antitumor. Unfortunately, due to the long neglect of Ca 2+ -induced cell damage, there have been few reports on the antitumor application of calcium overload when designing anticancer drugs based on calcium nanomaterials. This section introduces the strategy of calcium overload in the treatment of tumors [30].
Zhang et al. [15] designed and synthesized a kind of ultra-small SH-CaO 2 NPs by taking advantage of the special cellular biological effect of Ca 2+ and its enhancement effect on H 2 O 2 oxidative stress, and systematically studied the anticancer effect induced by Ca 2+ . Due to the pH responsiveness of CaO 2 NPs, under acidic TME, CaO 2 slowly decomposes into free Ca 2+ and H 2 O 2 , leading to calcium overload and oxidative stress, respectively. In addition, the low expression of catalase (CAT) in tumor cells [90] and continuous oxidative stress can lead to the functional disorder of protein, trigger desensitization of calcium-related channels, and lead to uncontrolled accumulation of Ca 2+ in tumor cells [91]. The anticancer mechanism of the nanocomposite is shown in Figure  6a. The experimental results showed that the survival rate of tumor cells decreased with the increase in SH-CaO 2 NPs concentration, (Figure 6b). The significant killing effect of SH-CaO 2 NPs was not limited by tumor type, and it could effectively kill multiple types of cancer cells (Figure 6c). In addition, Alizarin Bordeaux staining and von Kossa staining could clearly identify the calcification area caused by calcium overload in Figure 6d. According to the above results, SH-CaO 2 NPs have significant anticancer effect.
However, compared with most solid tumors, the antitumor effects of monotherapy are limited. Therefore, researchers have successively developed combined therapies based on calcium overload to improve the therapeutic effect of tumors.
Liu et al. [84] used dendritic mesoporous organosilica (DMOS) bound by tetra-sulfide bonds as nanometer carriers, loaded with chloroperoxidase (CPO) and sodium-hyaluronate-modified CaO 2 NPs (CaO 2 -HA NPs), and improved the stability of CPO and the limited H 2 O 2 level in tumor cells. A novel combined treatment strategy of hydrogen sulfide (H 2 S) gas, enzyme dynamic therapy (EDT), and Ca 2+ interference was developed, as shown in Figure 7A. The NPs are enriched in tumor cells through enhanced EPR effect and specific targeting effect of HA on tumor cells. Subsequently, HA is degraded by hyaluronidase and CaO 2 further reacts with H 2 O to generate H 2 O 2 and Ca 2+ . In addition, DMOS have GSH responsiveness, releasing CPO, consuming GSH, and generating H 2 S gas under high GSH environment in tumor cells, and finally realizing multi-modal antitumor therapy under CT imaging. In vitro experiment results confirmed that when DCC-HA NPs were co-cultured with mouse fibroblasts (L929 cells) for a certain period of time, the survival rate of L929 cells remained high even at a high concentration of NPs, indicating that DCC-HA NPs had good biocompatibility ( Figure 7B). Subsequently, DCC-HA NPs were co-cultured with mouse 4T1 cells for a period of time. It was found that the survival rate of 4T1 cells not only decreased with the increase in NPs concentration, but also decreased much more than the survival rate of tumor cells under other conditions ( Figure 7C). In vivo results confirmed that 4T1 tumor cells were transplanted into mice and significantly inhibited after 14 days of NPs treatment ( Figure 7D). All the above results indicate that the TME responsive nanocomposite has significant anticancer effect.
Jiang et al. [92] constructed a nanoplatform containing UCNPs-Ce6@RuR@mSiO 2 @PL-HA NPs (UCRSPH) and SA-CaO 2 NPs. After entering tumor cells, the composite nanometer platform was irradiated by NIR (980 nm) to alter TME, reverse tumor hypoxia, and achieve Ca 2+ interference therapy and PDT self-enhancement. In addition, the nanoplatform provides fluorescence imaging and CT imaging to further guide in vivo antitumor therapy. In conclusion, the nanometer platform shows a potent combined anticancer potential with minimal toxic side effects on normal cells.
Chen et al. [93] designed and synthesized a new CaO 2 @TA-FE III nanodrug delivery system for enhancing  [15]. Reproduced with permission from a previous study [15]. © 2019 Elsevier Inc.
CDT. Its anticancer mechanism is: TA and Fe III form TA-Fe nanocoating on the surface of spherical CaO 2 nanoaggregate. When the nanocomposite enters tumor cells, H 2 O 2 is generated through degradation, which solves the problem of insufficient H 2 O 2 in CDT. In addition, TA of nanomaterials reduced Fe III to Fe II , improved the catalytic efficiency of Fenton reaction, generated ·OH, induced irreversible oxidative damage to tumor cells, promoted calcium overload, and accelerated the death of tumor cells. The experimental results in vitro and in vivo indicate that the nanocomposite is a promising novel and highly effective antitumor nanoplatform with good tumor treatment effect.
In addition to the anticancer therapies mentioned above, other anticancer therapies based on CaO 2 NPs are shown in Table 3.

Antimicrobial therapy
Bacterial infection has become one of the main problems threatening human health [107][108][109]. CaO 2 NPs have attracted much attention due to their small size, increased surface contact area with bacteria and damage to bacteria [110]. The antibacterial mechanism of CaO 2 NPs is related to the production of ROS ( 1 O 2 , O 2 ·-, H 2 O 2 and ·OH). Relevant literature has shown that ROS induced oxidative stress is one of the important antibacterial mechanisms for the construction of ROS, which can effectively treat bacterial infection [111]. ROS treats bacterial infections by destroying cell membranes [112], and lipid peroxidation caused by free radicals is one of the ways to change cell membranes [113]. However, due to the thicker cell wall structure of Gram-positive bacteria, the lipids seen in Staphylococcus aureus (S. aureus) are not affected. In addition, negatively charged ROS cannot easily penetrate negatively charged cell membranes, but H 2 O 2 , as a type of ROS, can easily penetrate cell membranes for bacteriostatic purposes [114].
ROS on cell walls are produced by negatively charged cell membranes interacting with positively charged NPs [115]. ROS can damage the cell membrane by interacting with the cell wall of bacteria, inhibit further growth of cells, leak the internal cell components, and finally lead  [84]. Reproduced with permission from a previous study [84]. © 2021 Wiley-VCH GmbH.
to the death of bacteria. Therefore, it is necessary to develop a new kind of biological material with high antibacterial ability. Shen et al. [32] synthesized CaO 2 nanocrystals and spherical aggregates with controllable size and uniform morphology using CaCl 2 as precursor by wet chemical process. By changing the concentration of CaCl 2 and PVP, the particle size of CaO 2 nanocrystals and their spherical aggregates can be adjusted in the range of 15-100 nm. The results of bacteriostatic experiment showed that the spherical aggregates showed obvious size dependent bacteriostatic effect. In addition, CaO 2 NPs synthesized with Ca(NO 3 ) 2 as the precursor showed similar antibacterial activity ( Table 4). It can be seen that the smallsized CaO 2 nanocrystals and their spherical aggregates have great potential in antibacterial applications.
Moreover, Thi et al. [116] developed a new hydrogel preparation method for antimicrobial therapy by promoting in situ cross-linking hydrogel formation mediated by horseradish peroxidase (HRP) using CaO 2 . General preparation process: Gelatin-hydroxyphenyl propionic acid (GH) polymer, HRP, and CaO 2 were used as raw materials, and the GH/C hydrogel was prepared by mixing the polymer solution with different concentrations of HRP and CaO 2 solution (volume ratios of GH:HRP:CaO 2 = 8:1:1). The introduction of CaO 2 can give hydrogel system the following two advantages: 1) On the premise of not affecting biocompatibility, H 2 O 2 is gradually generated to form hydrogel system. 2) Dynamic hydrogel matrix was constructed to stimulate surrounding tissues by continuously releasing Ca 2+ , H 2 O 2 , and O 2 .
Then, the antibacterial activity of GH/C hydrogel was evaluated by measuring the optical density (OD) value of the bacterial suspension at 600 nm. The antibacterial activity of GH/C hydrogel is related to the content of CaO 2 , and its antibacterial activity increases with the increase in the dosage of CaO 2 , among which GH/C 0.5 shows the highest antibacterial effect (Figure 8). Therefore, although GH/C hydrogel could not kill bacteria, it could also effectively inhibit the growth rate of E. coli and S. aureus. We believe that the formation of in situ crosslinked hydrogels mediated by HRP/CaO 2 has potential application value in the treatment of bacterial infection.
Traditional titanium implants can effectively treat bacterial infection after modification, but the resulting    : Schematic illustration of the underlying antibacterial and anti-inflammatory mechanisms of the prepared samples [18]. Reproduced with permission from a previous study [18]. © 2021 Wiley-VCH GmbH.
anti-inflammatory response is also ignored. Therefore, He et al. [18] constructed a dual drug loading system (TNT@IL-4/GelMA@CaO 2 ) to treat bacterial infections while inhibiting the occurrence of pro-inflammatory reactions ( Figure 9). In vitro and in vivo experiments show that the drug loading system can effectively treat bacterial infection and regulate pro-inflammatory response. In short, the drug loading system provides a good example for the development of advanced titanium implants with anti-inflammatory and antibacterial properties. Zhang et al. [117] prepared a hydrogel library (OMCN) with self-activated nitric oxide (NO) release to cooperate with NO and PTT in the fight against bacterial infection. First of all, H 2 O 2 formed by the reaction of CaO 2 with H 2 O can oxidize L-arginine (L-arg) to form NO, which is used to treat bacterial infection. In addition, thanks to the excellent photothermal effect of OMCN, PTT can increase the production of NO gas and cooperate with sterilization at the same time. In vivo and in vitro studies showed that the material had good synergistic antibacterial activity under the laser irradiation of 808 nm, and there was no obvious toxicity. Therefore, the prepared hydrogel library provides a promising strategy for antibacterial in the future.
In addition, Table 5 describes other antibacterial applications based on CaO 2 NPs.

Biological effects and biosafety of CaO 2 NPs
Whether CaO 2 NPs can achieve further clinical progress is closely related to its biological effects and biosafety. The biological effects and biosafety of CaO 2 NPs have been preliminarily explored in the relevant data [124,125]. The relevant data are encouraging, but there is still a certain distance from further clinical conversion. For example, when CaO 2 NPs circulate in the vessel, it will react with H 2 O in advance, and its product H 2 O 2 is toxic and may affect normal tissues and cells. In the following studies, a more systematic in vitro and in vivo biosafety assessment should be conducted to further reveal the biological distribution and excretion of CaO 2 NPs in vivo, providing more powerful evidence for biological effects and biosafety.

Conclusion and future perspective
The preparation technology and surface modification of CaO 2 NPs have been relatively mature. Although CaO 2 NPs have achieved good results in tumor treatment and bacterial infection, there are still some challenges to be overcome in future research. If the following problems are solved, CaO 2 NPs are expected to continue to move forward in the field of biomedicine. 1) There are many factors affecting the effect of chemotherapy, including hypoxia, low pH value and overexpression of GSH etc. [126][127][128][129]. At present, most research are from the perspective of hypoxia, that is, through exogenous supply of O 2 and endogenous production of O 2 to alleviate the hypoxic environment in tumor cells, reduce MDR and enhance the effect of chemotherapy. However, there are few reports on how to overcome low pH value and overexpression of GSH to enhance the effect of chemotherapy. Therefore, in the future, it is necessary to study how to enhance the effect of chemotherapy from the aspects of low pH value and GSH overexpression.
2) The effect of PDT is affected by TME, the selection of effective PS and the management of light dose [130,131].
Up to now, great progress and achievements have been made in the study of PS selection and optical quantum management [132][133][134][135]. However, there are still many challenges in cancer treatment that regulates TME to enhance type II PDT. 3) As we all know, the best pH range to maximize the effect of CDT is 2-4 [136], while the pH value of tumor tissue is about 6.5 [54]. Although GOx and gold nanoparticles (GNPs) are often used to improve pH in tumor tissues, it is necessary to develop pH-independent CDT nanocatalysts. In addition, most of the previously reported CDT nanocatalysts are transition metal-based nanoparticles, which can cause acute inflammation and toxicity in vivo. Therefore, the development of CDT nanocatalyst with high biosafety and good biodegradability is a more feasible scheme for the development of CDT in future. 4) In a certain range, the smaller the size of NPs, the stronger the penetration of tumor tissue, and the better the therapeutic effect on bacterial infection. In addition, most studies have shown that spherical NPs are slightly less permeable in tumor tissue than other shapes [33]. Therefore, it is necessary to adjust the shape and size of NPs to improve the permeability and antibacterial activity of tumor tissues. 5) Oxidative stress induced by ROS is one of the effective bacteriostatic mechanisms. CaO 2 NPs can self-amplify ROS and effectively treat bacterial infection. Although the bacteriostatic effect is remarkable, the treatment mode is relatively simple. Therefore, it is necessary to use CaO 2 NPs to design a nanohybrid material based on CaO 2 and to develop a novel bacteriostatic mode with better therapeutic effect by using the products of CaO 2 NPs.
In a word, the reports of antitumor and antibacterial infection related to CaO 2 NPs have increased in recent years, but their application in clinical transformation still has a long way to go. Solving the above problems is helpful to speed up their application in clinical transformation. I believe that the hybrid nanomaterials related to CaO 2 NPs will provide a better way for tumor treatment and bacterial infection in the future through continuous optimization and improvement.
Funding information: This work was supported by the National Natural Science Foundation of China (no. 51873052).
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

Conflict of interest:
The authors state no conflict of interest.