Adjuvants, delivery systems and TLR ligands are considered three important components for an effective vaccine (Malyala et al., 2009). Non-living vaccine antigens, particularly recombinant subunit vaccines, are in general weakly immunogenic (Mohan et al., 2013). Natural/synthetic molecules such as alum and calcium mineral salts act as immune-stimulators, i.e., adjuvants, that induce the desired potent immune responses to the absorbed/conjugated antigen (Egli et al., 2014). An adjuvant is used as a component of the vaccine that can boost the immunogenicity of vaccine antigens (García and De Sanctis, 2014), while a delivery system ensures optimal delivery of antigen and adjuvant cargo to the desired site or immune cell (Skwarczynski and Toth, 2016). Vaccine adjuvant and delivery systems are not mutually exclusive, and can both act to prevent the antigen from degradation while it is carried to the desired site (Skwarczynski and Toth, 2016). Appropriate and optimised adjuvants play a central role in vaccine design as they can overcome immunosuppression and help induce a strong immune response (Kano et al., 2016).

At present, only a few adjuvants (such as mineral salts, aluminium salts (alum), calcium phosphate, and oil-in-water emulsions (MF59, AS03), cholesterol, phospholipid and polyethylene glycol (PEG) 2000 lipid) have been widely approved for human use (Del Giudice et al., 2018; Thi et al., 2021). These adjuvants are effective at inducing humoral immune response but, with the exception of calcium phosphate materials, have a poor ability to stimulate a cellular immune response, which is critical for viral and tumour eradication (García and De Sanctis, 2014; Skwarczynski and Toth, 2016). With the exception of calcium phosphate, they also have several drawbacks such as severe local tissue irritation, longer inflammatory response and induction of allergic responses in some people (Sharma et al., 2015a). Consequently, there is an urgent need for a novel delivery vehicle and adjuvant to increase cellular and humoral immunogenicity in vaccine design.

Various types of nanocarriers have been used for vaccination, and the most widely studied include polymeric NPs, lipid NPs, inorganic NPs (calcium phosphate, silicate, gold), and their applications in immunotherapy have been widely reviewed in the published literature (Peek et al., 2008b; Park et al., 2013; Smith et al., 2013; Zhao et al., 2014; Zhu et al., 2014; Irvine et al., 2015; Torres-Sangiao et al., 2016; Khalifehzadeh and Arami, 2020a; Mao et al., 2021). Compared with other polymeric NPs, poly (lactic-co-glycolic acid) (PLGA) NPs are the most extensively investigated NPs as vaccine carriers, largely because they have previously been approved by FDA and licensed for human use in medical applications such as bone implants and sutures (Pavot et al., 2014; Vartak and Sucheck, 2016). A number of studies have shown that PLGA NP are effective antigen delivery systems and when co-administered with an adjuvant can be effective vaccine formulations (Gu et al., 2019a; Gu et al., 2019b). However, PLGA NPs vaccines have a short half-life as they are often degraded rapidly, thus losing immunogenicity and effectiveness. Immunogenicity loss during storage has adverse effects on the encapsulated protein antigen that makes the vaccine less effective (Bolhassani et al., 2014). Gold NPs are also suggested as vaccine carriers as their surface can be highly modified with diverse ligands, they are biocompatible, physiologically stable, their size and shape can be easily controlled, and because they are easy to synthesis (Smith et al., 2015). However, there are several disadvantages that limit the potential use of Gold NPs as a cancer therapy. They are non-porous and non-biodegradable, and thus they are not suitable for the time-release of small molecules (Almeida et al., 2014). Although nanocarriers can enhance vaccine efficacy, there remain some potential problems that require further research to overcome.

The induction of a robust and specific cytotoxic T-lymphocyte (CTL) response by vaccination is a challenging goal in viral immunity and also cancer immunotherapy that has the added complication that the tumour microenvironment induces immunosuppression of T cells via a number of pathways (Butterfield, 2015). A key factor in enhancing the efficacy of a vaccine is to increase its binding to host cells and to deliver the antigen cargo to the endocytic pathway of APCs, thus initiating a strong immune response (Zhao et al., 2014). TLRs are a group of transmembrane protein complexes that recognise microbial components called pathogen-associated molecular patterns (PAMPs) in order to initiate an immune response (Akira et al., 2001). The use of TLR agonists as adjuvants has gained increasing interest due to their ability to improve the efficacy and immunogenicity of vaccines formulations (Hansen, 2011; Rahimian et al., 2015; Bruno et al., 2016; Gutjahr et al., 2016; Halliday et al., 2016; Zilker et al., 2016; Gutjahr et al., 2017; Kasturi et al., 2017; Sokolova et al., 2017). Pattern recognition receptors (PRRs), such as TLRs, are expressed on APCs (dendritic cells and macrophages) and mucosal/oral epithelial cells. PRRs play a significant role in inducing and enhancing both innate and adaptive immune responses and are thus being targeted in vaccine design and formulations (Halliday et al., 2016). TLRs facilitate the recognition of pathogens by immune cells, resulting in increased antigen uptake and processing, expression of pro-inflammatory cytokines, chemokines, and co-stimulatory molecules such as CD80, 86 and 40, leading to the induction of a robust innate and adaptive immune response (Blander and Medzhitov, 2006; Temizoz et al., 2016). Several different TLR ligands have been investigated for use as vaccine adjuvants, including ligands for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 (Kawai and Akira, 2007; Coffman et al., 2010; Duthie et al., 2011), with the selection of the ligand often dependent on the disease being targeted (Kaur et al., 2018). One of the main challenges in cancer immunotherapy is the difficulty to abrogate the immunosuppressive tumour environment. However, immunosuppression can be overcome by co-immunising an antigen with a TLR ligand, with the benefit of combining mechanisms of up-regulating the production of co-stimulatory molecules, and induction of pro-inflammatory cytokines (Dewitte et al., 2014). Ligation of TLRs 2, 3, 4, 7, and 9 enhances dendritic cells (DCs) activation and antigen phagocytosis, which in turn improves CTL priming (Johnson et al., 2009; Watts et al., 2010). Moreover, the characteristics of CaP NPs and their immunostimulatory activity are controlled by different types of TLR molecular structure. The composition, backbone, sequencing, and concentrations of CpGs all influence the mineralization of CaP NPs (Khalifehzadeh and Arami, 2020b).

Compared with conventional adjuvants such as aluminium (alum) salts, CaP NPs display higher adjuvanticity and greater affinity to biological materials including proteins, cells and enzymes (Kalita et al., 2007). CaP materials used as adjuvants and have been shown to induce high titre immunoglobulin G1 (IgG1) to absorbed vaccine antigens for diphtheria, tetanus, pertussis, meningococcal, polio, rabies and hepatitis-B (Relyveld, 1986). In comparison with alum, CaP promoted a higher level of antibody response to Bothrops asper snake venom (Olmedo et al., 2014). CaP NPs were found to be more potent compared to alum for inducing a high IgG2a neutralising antibody against Epstein-Barr (EBV) infections (He et al., 2000). In addition, CaP NPs have been shown to be efficient mucosal adjuvants. He et al. reported that herpes simplex virus type 2 (HSV-2) vaccine using CaP NPs as an adjuvant elicited a stronger mucosal and systemic immune response when administered intravaginally and intranasally as compared to Alum (He et al., 2002). In a recent study, Cao et al. found that after 35 days immunization in mice, OVA administered orally via polymer-coated CaP nanocomposites significantly increased systemic and mucosal immune responses compared to antigen OVA alone in vivo (Cao et al., 2021). After 35 days, ELISA results demonstrated that mice vaccinated with CaP-based nanocomposites-OVA had considerably greater faecal antibody levels (about 1.57 fold higher than free OVA), suggesting that CaP has a significant impact on mucosal immunity (Cao et al., 2021). A study by Saeed et al. also showed that the human Enterovirus-71 vaccine encapsulated in CaP NPs had an enhanced antibody responses (IgA and IgG) in rabbits as compared to the standard vaccine (Saeed et al., 2015a). These studies suggested that CaP NPs have great potential as a vaccine adjuvant inducing cellular as well as humoral immunity (He et al., 2000; Lin et al., 2017). CaP, particularly in nanoform, has been suggested to be used instead of alum as a potent adjuvant (Johnson et al., 2020). In addition, CaP can be used as a nanocarrier for antigen delivery. Cao et al. demonstrated that when compared to naked OVA, oral administration of alginate-chitosan-coated CaP@OVA NPs dramatically increased mucosal IgA and serum IgG antibody responses, showing that the CaP-Chi-Alg NP might be a viable oral vaccine delivery strategy (Cao et al., 2020).

Current strategies of CaP NP preparation are known to generate various sizes and shapes. Synthesis methodologies can be categorized into two groups, including the dry method (Zhang and Zhu, 2006; Fathi and Mohammadi Zahrani, 2009) and wet method chemical routes (Gentile et al., 2015; Sidane et al., 2015). More specifically, the dry method are involving solid-state (Zhang and Zhu, 2006) and mechanochemical (Fathi and Mohammadi Zahrani, 2009) techniques, while wet methods include chemical precipitation (Banik and Basu, 2014; Gentile et al., 2015; Andrade et al., 2016), sol-gel (Sidane et al., 2015), microemulsion (Saha et al., 2009; García et al., 2012; Yang et al., 2012; Hou et al., 2013), hydrothermal (Jin et al., 2015; Sarath Chandra et al., 2015), sonochemical (Utara and Klinkaewnarong, 2015), hydrolysis (Yang and Wang, 2016) and microwave methods (Hassan et al., 2016). Comparison of major methods for synthesis CaP NPs is summarised in Table 1. Various kinds of CaP NPs can be obtained via the different fabrication methods, such as particle size (10 nm–10 mm), size distribution (from narrow to broad), shape (from spherical to rod-shaped), crystallinities (low to high) and compositions (all CaP NPs forms).

CaP solubility is pH-dependent, it is stable at human blood pH of 7.4 and more alkaline environments but easily dissolves at low pH’s of <5.0. This physical property has specific advantages as a low pH is often found in tumour zones, endosome and lysosome, thus making CaP very suitable for delivery systems of drug and vaccine applications (Sokolova and Epple, 2021). Figure 2 shows a schematic representation of CaP NP cargo release in a low pH environment such as within an endosome/lysosome or at a tumour site. First, CaP NPs enter the tumour site or are endocytosed by the cell. Within the low pH environment of the endosome or tumour site the CaP NPs dissolve and disassemble releasing the cargo at the tumour site or within the endosome. It is proposed that the release of Ca²⁺ and PO₄ ^3- ions from the dissolved NPs within the endosome gives rise to high osmotic pressure, causing the endosome to expand and then rupture, releasing the antigen cargo, Ca²⁺, and PO₄ ^3- ions into the cytoplasm (Li et al., 2012) (Figure 2). This proposed mechanism is used as an explanation as to why CaP has the ability to act as an adjuvant to induce a cytotoxic T cell response.

It is well known that physicochemical properties such as size, shape and surface charge have a crucial role in the efficacy of vaccines (Zhao et al., 2014). Due to the importance of control of NP size and shape in vaccine formulations, different synthetic protocols have been developed to regulate the size and shape of CaP NPs (Sadat-Shojai et al., 2013). For example, Yan et al. (2006) showed that hydrothermal crystallisation of Ca(NO₃)₂ and (NH₄)₂HPO₄ in the presence of the poly (amidoamine) (PAMAM) dendrimers demonstrating that the size of CaP NPs fabricated decreased from 82, 77, 48 to 38 nm with increasing PAMAM dendrimer generation (G1.0 to G4.0, respectively). Hydrothermal crystallisation of Ca/P using CTAB (cetyltrimethylammonium bromide) as a template was found to result in the size and shape of the formed CaP NPs being able to be controlled by modifying the pH and reaction temperature (Wang et al., 2006).

The surface modification with either hydrophobic or hydrophilic molecules can enhance the versatility of CaP NPs in vaccine applications (Sharma et al., 2015a). The surface of HAP possesses positively charged Ca²⁺ and negatively charged PO₄ ^3- sites that provide binding sites for stabilisation as well as bio-conjugation to agents such as polymers (Kandori et al., 2009). For instance, polyethyleneimine (PEI) and poly (ethylene glycol) (PEG) have been added to the surface of formed CaP NPs to increase stabilisation in the physiological environment as well as altering the charge of the NP surface (Ramachandran et al., 2009; Kozlova et al., 2012). Furthermore, the polymers provide a chemical moiety such as an -NH₂, -OH or -COOH for conjugation or adsorption of additional agents on the NP surface such as peptide/protein, drugs, DNA/siRNA, or TLR ligands during the fabrication process, thus enhancing the scope of use and immunogenicity (Saeed et al., 2015b). This ability to use the layer-by-layer assembly approach with CaP was used in a multi-shell system to protect biomolecules (antigen and immunostimulants) within from degradation, increase transfection efficiency and targeting to B-cells (Temchura et al., 2014a).

CaP adjuvants, first reported by Relyveld and his colleagues in 1964 (Relyveld et al., 1964), have been used for many years as vaccine adjuvants (Relyveld et al., 1985). CaP adjuvants have been used in diphtheria, pertussis, tetanus and poliomyelitis vaccines in France (Coursaget et al., 1986; Gupta and Siber, 1995; Goto et al., 1997). Several recent papers have highlighted that CaP material should be considered as a replacement of alum salts in vaccine formulations (Sesardic et al., 2007; Issa et al., 2014) due to their distinct advantages in that they are non-toxic, biodegradable, have a low production cost, and have pH-dependent solubility and unlike alum can be readily produced as NPs (Lin et al., 2017). Additionally, CaP adjuvants have been shown to provide protection to the antigen cargo from premature enzymatic and proteolytic degradation (Salem, 2015) and prevent elimination by the reticulohistocytic system (RHS) (Posadas et al., 2016). Another advantage of CaP materials is that they can be functionalised with various molecular adjuvants to enhance active cell targeting and vaccine efficacy (Xu et al., 2013; Xu et al., 2014; Zilker et al., 2016). Due to these properties, CaP nanoparticles have high adjuvanticity and show considerable promise as a vaccine adjuvant.

In order for NPs to act as effective vectors for the delivery of antigens to the desired site, a strong interaction between NPs and the antigen is required. Currently, there are four approaches to attach antigen on NPs: adsorption; encapsulation; co-mixtures; and chemical conjugation (Zhao et al., 2014) (Figure 3). The advantages and disadvantages of antigen interaction strategies with CaP NPs are summarized in Table 2. Among these, adsorption and encapsulation are the most widely used strategies for antigen loading on CaP NPs, which can be achieved by charge attraction and hydrophobic interaction (Zhao et al., 2014). A draw back of the adsorption method is that it is a weak interaction effected by salt concentrations, and exogenous lipids and proteins displacing the antigen at the site of injection, which can lead to a burst release of antigen from NPs in vivo or sudden release away from the APC thus reducing immunity (Kamaly et al., 2016). Electrostatic attraction between antigen and CaP NPs plays the most important role in surface adsorption. For example, the adsorption of bovine serum albumin (BSA), ovalbumin (OVA) or lysozyme on HAP arises from electrostatic interaction between Ca²⁺ and PO₄ ³⁻ anions of HAP with the COO⁻ and NH₄ ⁺ cations of the protein antigen (Boonsongrit et al., 2008; Dasgupta et al., 2009; Kandori et al., 2009; Sahdev et al., 2013; Swain and Sarkar, 2013; Ma et al., 2016).

Encapsulation and conjugation can be an appropriate alternative to achieve steady antigen release and delivery of all of the antigen to the APC. In general, encapsulation can be achieved by antigens mixed with chemical reagents and solvents during NP synthesis route, resulting in interaction that is strong, similar to that of chemical conjugation. This method can enhance antigen transfection efficiency as the antigen is not released until the dissolution of NPs, thus delivering the antigen at the desired site. Dasgupta et al. encapsulated BSA protein by first incorporating it with the Ca²⁺ solution prior to mixing with the PO₄ ³⁻ aqueous solution during a precipitation synthesis, resulting in 50–60 nm NPs containing up to 24% by weight BSA protein (Dasgupta et al., 2010). Similarly, Li et al. used the water-in-oil micro-emulsion method to incorporate siRNA in CaP NPs, by adding the siRNA to the PO₄ ³⁻ aqueous solution phase, this resulted in nanoparticles of 60–80 nm in diameter with an 39.8 ± 2.8% encapsulation efficiency of siRNA (Li et al., 2010). Chiu et al. (2012) used a hybrid-encapsulating stabilisation process whereby the antigenic protein was expressed with peptides that bound to and stabilised calcium phosphate. In this method the protein was mixed with the PO₄ ³⁻ aqueous solution phase and upon addition of the Ca²⁺ phase the protein stabilised the calcium-phosphate nucleation to form nanoparticles (60–70 nm) with an amorphous calcium phosphate core with a protein shell.

Chemical conjugation has also been used to incorporate antigenic molecules such as proteins and peptides by covalently linking them to the surface of the NP (Slütter et al., 2010). Ramachandran et al. functionalised the surface of CaP NPs using carbodiimide chemistry to covalently link diamino-PEG and then loaded the PEG-CaP particles with insulin for an oral delivery therapeutic (Ramachandran et al., 2009). In another approach Kozlova et al. synthesised CaP NPs coated with a silica shell that was functionalized by thiol/amino groups or salinization, to covalently attaching antibodies and antigens (Kozlova et al., 2012). The approach of antigen-conjugated NPs has some significant advantages for mucosal immunisation as the conjugated antigen has been found to be protected from proteases in the mucosal tissue (Navarro-Tovar et al., 2016). CaP NPs act as an immune potentiator (adjuvant) as well as a delivery system and conjugation between antigens, adjuvants and NPs has been shown to give better immunity in comparison to a mixed solution of the formulation (Nembrini et al., 2011; Zhao et al., 2017). A Hen Egg Lysozyme (HEL) and TLR9 ligand CaP NP conjugated was found to elicit a significantly higher humoral immune response than non-conjugated NPs (Zilker et al., 2016).

The innate immune system plays a significant role in the induction and development of adaptive immunity to an antigen (Iwasaki and Medzhitov, 2010). It is well established that when the innate immune response is triggered by microbial molecules it can induce and recruit the cellular components of the adaptive immune system and aid in development of immune memory (Medzhitov, 2007). Pattern-recognition receptors (PRRs) on the surface of APCs (particularly DCs) recognise microbial associated molecules, which results in the induction of inflammatory responses and induction of adaptive immune responses (Iwasaki and Medzhitov, 2015). In the last decade, the adjuvant effect of CaP NPs on the innate immune system has been investigated (Kozlova et al., 2014a). Behera et al. demonstrated that Labeo rohita H. S-layer protein (of Aeromonas hydrophila) loaded CaP NPs elicited a robust innate and adaptive immune responses in a fish model following parenteral immunisation (Behera and Swain, 2011). Further, the antigen loaded CaP NPs induced significantly stronger respiratory burst and myeloperoxidase activity compared with negative control groups (Behera and Swain, 2011). Matesanz et al. reported that BSA loaded nano-sized CaP produced a rapid stimulation of the innate immune response in mice (Matesanz et al., 2014). Further, macrophage proliferation and phagocytic activity were greatly decreased, while the generation of inflammatory cytokines such as IL-6, TNF-α was strongly enhanced (Matesanz et al., 2014). Tai et al. found that β-tricalcium phosphate (β-TCP) may also have an immunostimulatory effect on macrophages. Mice injected with β-TCP NPs elicited extensive migration of mouse monocyte/macrophage cells to the injection site, and that it significantly enhanced the production of macrophage inflammatory protein 1 alpha (MIP-1α), suggesting a novel role for β-TCP in vaccine applications (Tai et al., 2014).

As mentioned previously, the innate immune system depends on the recognition of pathogen-associated molecular patterns (PAMPs) by PRRs such as TLRs. TLR activation can elicit inflammatory cytokine production, enhance macrophage phagocytosis activity, and enhance antigen presentation (Krutzik et al., 2005; Moresco et al., 2011). Consequently, TLR agonists in vaccine development have been widely investigated in preclinical studies (Luo et al., 2015). Sokolova et al. evaluated the adjuvant effect of CaP encapsulated with TLR ligands CpG and polyriboinosinic: polyribocytidylic acid (poly (I: C)) in a mouse model. They showed that TLR functionalized NPs elicited high levels of the innate inflammatory cytokines IL-12p70 and TNF-α, which resulted in DC maturation and rapid CD4⁺ T cell activation (Sokolova et al., 2010a). Similarly, other researchers have developed a flagellin-functionalized CaP NPs and studied the immunostimulatory effect on the innate immunity both in vitro and in vivo in mice (Kozlova et al., 2014b). Flagellin is a PAMP that can be recognised by TLR5 on the host cell surface (Hayashi et al., 2001). Compared to free flagellin, flagellin-functionalized CaP NPs produced a high-level of the proinflammatory cytokines IL-1β from bone marrow-derived macrophages (Kozlova et al., 2014a). These findings suggest that CaP NPs could effectively activate the innate immune system and that TLR ligand functionalisation enhances this ability. These key findings should be taken into account in designing CaP NP vaccine formulations, as the addition of TLR ligands will have not only an additional adjuvanticity effect but also a “depot” effect allowing the vaccine to be located in a set area, the ‘injection’ site long enough for dendritic cells and other antigen presenting cells to sequester the vaccine. Furthermore, CaP can be formulated in a number of different ways depending on the Ca to P ratio and this as highlighted above can have significant impact on immunogenicity. Thus although, hydroxyapatite maybe the most stable CaP formulation other CaP formulations such as β-TCP need to be considered.

A potent T cell immune response is indispensable to clearing and recovering from infectious disease. There are several studies that have demonstrated that CaP NPs loaded with viral and bacterial antigens are able to activate protective T cell responses against viral and bacterial infections (Sokolova et al., 2010b; Xu et al., 2013; Zhou et al., 2014; Rojas-Sánchez et al., 2020). A novel HAP vaccine loaded with malaria antigen (merozoite surface protein-119, MSP-119) was found to elicit a vigorous T cell response and secretion of IL-2 and IFN-γ in mice (Goyal et al., 2009). In a separate study hemagglutinin (HA) loaded CaP NPs functionalised with TLR9 ligand (CpG) were found to induce DCs maturation and a strong antigen specific CD4⁺ T cell proliferative response (Sokolova et al., 2010a). In a following study it was found that immunisation of mice with TLR functionalized CaP NPs were effectively taken up by DCs as well as inducing high production of CD4⁺ and CD8⁺ effector T cells (Knuschke et al., 2013). Others have reported that CaP loaded with model antigen ovalbumin (OVA), combined with a fusion protein induced a robust and long-lasting CD8⁺ T cell responses in mice that were characterised by the high production of IFN-γ and TNF-α (Zhou et al., 2014). Interestingly it has been shown that magnesium (Mg) and zinc (Zn) loaded HAP NPs could effectively promote Th1 and Th2 immunity in vitro, and enhance the secretion of type 1 and 2 cytokines from bone marrow dendritic cells (BMDCs), suggesting these mixed ion (Zn, Mg) CaP particles can be used as human adjuvants (Wang et al., 2016). Furthermore, in a recent study, CaP NPs encapsulating the TLR9 ligand, CpG, combined with vial peptides from murine leukaemia Friend retrovirus (FV) were shown strongly enhanced anti-viral immunity (Knuschke et al., 2016). Li et al. developed a CpG-functionalized silica-coated CaP NP (SCP) vaccine and evaluated its effectiveness against human immunodeficiency virus type 1 (HIV-1) infection (Li et al., 2021). When compared to naked DNA vaccination, SCP-based DNA immunisation was found to generate significantly broader humoral and a more robust cellular immune responses in vivo (Li et al., 2021).

These studies indicated that different kinds of adjuvant used to functionalise or coat CaP NPs can strongly affect T-cell immunity. Knuschke et al. has compared CpG and poly (I:C) functionalized NPs with CaP NPs in vitro, and observed that CpG-CaP NPs elicited a more robust T cell-mediated immunity as compared to the other functionalised and bear CaP NPs (Knuschke et al., 2014c). In another study, CaP NPs encapsulating both CpG and poly (I:C) ligands and the antigen hemagglutinin were found to elicit DC maturation and T cell activation significantly (Sokolova et al., 2010b) (Figure 4). Sokolova et al. demonstrated CaP NPs functionalised with TLR ligand poly (I:C) resulted in a pronounced immunostimulation to liver cells both in vitro and in vivo (Sokolova et al., 2017). From these studies it appears that combining TLR ligands targeting TLR9 and TLR3 singularly or together in a CaP NP formulation enhances CTL immunity by aiding DC targeting and CD4⁺ and CD8⁺ T cell activation. However, these are only two TLR ligands and vaccine formulations, other than CaP NPs have also used ligands for other pattern recognition receptor’s such as TLR2 (Palmitoyl moieties), TLR4 (LPS), TLR5 (flagellin) and NOD1/2 (peptidoglycan) that can be used in future CaP NP studies to enhance targeting, activation and to direct immunity to the desired outcome.

The potential of CaP as a vaccine carrier and adjuvant has been further explored in cancer vaccines (Xu et al., 2014; Zhao et al., 2015; Khalifehzadeh and Arami, 2020a; Scheffel et al., 2020). It is known that the induction of a potent CTL response is crucial for the eradication of cancer cells and prevent cancer recurrence (Rosenberg et al., 2004; Thomas and Prendergast, 2016). Xu et al. studied the effect of NPs on CTL responses in a melanoma mouse model of cancer. Prior to the induction of the disease, mice were immunised with either free melanoma antigen (Tyrosinase-related protein 2, p-Trp2), or free TLR 9 ligand (CpG), or NPs loaded with lipid-calcium-phosphate (LCP) NPs containing p-Trp2 peptide and TLR 9 ligand (Trp2-TLR LCP) (Xu et al., 2013). They showed the mice vaccinated with the Trp2-TLR LCP induced a robust and specific IFN-γ CTL response in vivo which inhibited tumour growth and lung metastasis (Xu et al., 2013). However, the vaccine was found to be less effective in a late-stage melanoma, despite a robust CTL response due to the immune suppressive tumour microenvironment. To overcome this a novel LCP system was trialled that incorporated Transforming Growth Factor-β (TGF-β) siRNA to contribute to tumour immunosuppression. This approach resulted in decreasing levels of TGF-β in the tumour microenvironment and a significant inhibition of tumour growth with increased levels of CD8⁺ T cells compared with vaccine formulations without the TGF-β siRNA (Xu et al., 2014). The anti-tumour effect on advanced stage melanoma has also been improved by a combination of chemotherapeutic agents and LCP (Zhao et al., 2015). Additionally, combinatorial immunotherapy can enhance anticancer efficacy. In a recent study lipid-dendrimer-CaP NPs functionalised with siRNA, plasmid DNA and a hepatocellular carcinoma-targeting peptide were found to increase tumoral infiltration, elicit a strong CD8⁺ T cell response, and suppress hepatocellular carcinoma progression (Huang et al., 2020). Sun et al. formulated mannose-and bisphosphonate (BP)-modified CaP NPs loaded with OVA-encoded plasmid DNA. They found that in C57BL/6J mice, MB-CaP-OVA vaccination greatly suppresses the development of OVA-expressing E.G7 tumour cells (Sun et al., 2021a). Another type of CaP, β-TCP, has also shown anti-tumour activity in vivo and has immune-stimulatory effects on immune cells (Naito et al., 2010). Tai et al. studied the in vivo and in vitro effect of β-TCP in C57bl/6 mice and a macrophage cell lines (J774A.1 cells), respectively. They found that vaccination with β-TCP elicited substantial migration of abundant neutrophils to the injection area in immunocompetent B6 mice. Additionally, β-TCP elicited upregulation of surface marker (CD86) expression and secretion of TNF-α and MIP-1α in mouse monocyte/macrophage cells, indicating the different forms of calcium/phosphate may have significant effects on the immune system and the response induced (Tai et al., 2014).