Calcium phosphate nanocarriers for drug delivery to tumors: imaging, therapy and theranostics

Dan Huang , Bin He and Peng Mi *
Department of Radiology, Center for Medical Imaging, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Number 17, 3rd Section, Renmin South Road, Chengdu, Sichuan 610041, P.R. China. E-mail: mi@scu.edu.cn

Received 29th May 2019 , Accepted 31st July 2019

First published on 31st July 2019


Calcium phosphate (CaP) was engineered as a drug delivery nanocarrier nearly 50 years ago due to its biocompatibility and biodegradability. In recent years, several approaches have been developed for the preparation of size-controllable, stable and multifunctional CaP nanocarriers, and several targeting moieties have also been decorated on the surface of these nanocarriers for active targeting. The CaP nanocarriers have been utilized for loading probes, nucleic acids, anticancer drugs and photosensitizers for cancer imaging, therapy and theranostics. Herein, we reviewed the recent advances in the preparation strategies of CaP nanocarriers and the applications of these nanocarriers in tumor diagnosis, gene delivery, drug delivery and theranostics and finally provided perspectives.


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Dan Huang

Dan Huang graduated in 2017 from Sichuan University with a B.S. in pharmacy. Upon graduation, she entered the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University as a master's student, where she currently works on theranostic nanoplatforms for cancer imaging and therapy.

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Peng Mi

Peng Mi is a professor at the Department of Radiology, Center for Medical Imaging, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, China. He received his Ph.D. from the University of Tokyo in 2013 under the supervision of Prof. K. Kataoka. After a JSPS postdoctoral fellowship at the Tokyo Institute of Technology until 2015, he joined the Innovation Center of Nanomedicine in Kawasaki as a senior research scientist until 2016. His major research interests relate to biomaterials and nanodevices for precision diagnosis, drug delivery, and targeted therapy.


1. Introduction

Cancer is a leading cause of human death all over the world, accounting for tens of millions of mortalities annually; therefore, it requires effective treatment. Since tumors are complex and heterogeneous, the synergism of diagnosis and therapy enables the pre-selection of patients and facilitates personalized therapy, motivating researchers to find suitable strategies for non-invasively and quantitatively targeting tumors for precision therapy.1,2 Since tumor-tropism imaging and therapy is challenging due to the limited half-life, excretion rate and tumor tissue specificity of probes and bioactive compounds, the development of drug delivery vehicles is expected to circumvent the current limitations in cancer theranostics; in recent decades, nanocarriers have attracted significant attention for drug delivery in the treatment of tumors.3–8 By incorporating cargos, such as imaging probes and bioactive compounds (e.g., anticancer drugs and siRNA), into nanocarriers, the cargos can be specifically delivered to the tumors mainly depending on the enhanced permeability and retention (EPR) effect9 as the nanocarriers can passively extravasate into the tumor tissues from the leaky tumor blood vasculatures and accumulate there due to the impaired lymphatic drainage.10 Moreover, the surface of the nanocarriers can be modified with tumor cell-specific ligands to increase the targeting ability as the ligands can specifically interact with the receptors that are highly expressed on tumor cells;11–13 using nanocarriers, the side effects can be effectively decreased, and the pharmacokinetics properties of the cargos can be enhanced, for instance, decreased toxicity to certain organs,14 extended circulation half-life,15 prevention of drug resistance16 and increased accumulation in tumor;17 this would lead to increased diagnostic sensitivity18 and therapeutic efficacy.19 To date, a number of nanomedicines, including polymeric micelles,20–22 liposomes,23,24 dendrimers,25–27 polymeric nanoparticles28,29 and CaP nanocarriers,30 have been developed for drug delivery, demonstrating high potential for tumor theranostics. For example, CaP nanocarriers have demonstrated high potential for drug delivery31,32 as CaP is a major component of the human bone, demonstrating high biocompatibility and biodegradability that foster the application of these nanocarriers in various biomedical fields, ranging from tissue engineering to drug delivery,33–36 leading to significant progresses in cancer theranostics;20,37–39 in addition, biological evaluations have certified the low toxicity of CaP nanocarriers.40 In general, CaP-based nanocarriers have several potential advantages for drug delivery: (1) biocompatibility without obvious toxicity or immune-response as CaP naturally exists inside the body (e.g., teeth and bones); (2) biodegradability in biological environments; (3) responsiveness to low pH;18,41 (4) easy accessibility at low cost; and (5) stable biochemical properties that do not affect the bioactivity of the payloads.

CaP was first reported in the 1970s as a non-viral gene delivery system to enable gene transfection;42 after this, it has been significantly applied as a drug delivery vehicle for incorporating the imaging probes for cancer diagnosis and bioactive compounds for cancer therapy.43,44 However, the CaP nanocarriers face several challenges including the difficulty of size control, formulation and tumor targeting. In recent years, significant efforts have been devoted towards the development of preparation approaches to achieve CaP nanocarriers with a well-defined structure, size and morphology for efficient drug delivery; the CaP nanocarriers have indeed enhanced the pharmacokinetics and tumor accumulation of payloads, gene transfection efficacy, tumor diagnostic accuracy and therapeutic outcomes. Herein, we summarized various preparation strategies and different applications of CaP-based nanocarriers in cancer imaging, gene (i.e., DNA and siRNA) delivery, as well as cancer theranostics.

2. Preparation strategies

The preparation of monodisperse, size controllable and stable calcium phosphate-based nanocarriers is critically important for drug delivery. To date, various preparation strategies, mainly including micro-emulsion, a two-step method, layer-by-layer formulation, and a template-mediated method, have been engineered for the development of CaP nanocarriers (Table 1), and some preparation methods are illustrated in Fig. 1. First, the PEGylated polyanion block copolymers were applied to control the formation of CaP nanoparticles, and the polymer hybrid CaP nanocarriers could be developed. For this method, a Ca2+ solution containing siRNA or DNA was mixed with equal volume of HEPES buffer containing PEG-block-poly(aspartic acid) (PEG-b-PAsp). Then, the mixture was stirred vigorously followed by incubation at 37 °C for 24 h, leading to the self-assembly of the block copolymer hybrid CaP nanocarriers with a narrow size distribution. Bioactive compounds (e.g., siRNA or DNA) could be incorporated inside the core of the CaP nanocarriers, whereas the surface shell of PEG and the internalization of the polyanion with the CaP crystals could control the size and endow the CaP nanocarriers with colloidal stability.45 Second, a two-step method was developed for the preparation of CaP nanocarriers, which could highly improve the stability of the nanoparticles. Typically, the CaP nanocarriers were first prepared by self-assembly using Ca2+, PEGylated polyanion block copolymers and sodium phosphate and subsequently subjected to a hydrothermal synthesis procedure to improve their stability (Fig. 1b). Third, a micro-emulsion method was applied to prepare lipid CaP nanocarriers.46 The amine, carboxylate or polyethylene surface-functionalized CaP nanocarriers were synthesized by employing a reverse microemulsion method (Fig. 1c). Briefly, calcium and phosphate in the water phase were separately dispersed in cyclohexane/surfactants to form two reverse water-in-oil micro-emulsions, and then, the two emulsions were mixed to form nanoprecipitates in the water phase. After this, 3-aminopropyltriethoxysilane or sodium citrate was added to the micro-emulsions to optimize their stability, and the nanocarriers were purified via column chromatography. The obtained nanoparticles could be further conjugated with functional groups, surface-functionalized with PEG47 or coated with lipids.48 Fourth, liposomes were utilized to incorporate the as-synthesized nanoparticles to obtain lipid CaP nanocarriers, which could be purified by column chromatography. Moreover, several lipid materials could be applied such as 1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP), cholesterol, as well as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG-2000)] (DSPE-PEG2000). The method for the preparation of lipid CaP nanocarriers was later improved using phospholipid dioleoylphosphatydic acid (DOPA) to coat the surface of the lipid CaP nanoprecipitates (Fig. 1d); consequently, the purification procedure by column chromatography was not required. The lipid CaP nanocarriers have been developed for the delivery of phosphorylated prodrugs (e.g., gemcitabine phosphates),49 siRNA50 and DNA51 and encapsulating radionuclides (e.g., 111In(III)52 and 177Lu53) for tumor imaging. Although CaP nanocarriers with well-defined particle sizes and morphologies were produced by these microemulsion synthetic routes, the sophisticated removal of surfactant and potential toxic synthetic components could be a potential challenge. The fifth method is the layer-by-layer preparation of CaP nanocarriers with multi-shells.54 The multi-shell CaP nanocarriers were synthesized by first mixing the aqueous solutions of Ca2+ and phosphate to form CaP precipitates and then rapidly mixing a part of this dispersion with a DNA solution to generate single-shell nanoparticles. Then, the Ca2+ and phosphate aqueous solutions were added to the abovementioned nanoparticles to form triple-shell nanomedicines (Fig. 1e). Theoretically, the second process can be repeated to obtain the desired number of layers of the nanoparticles.55
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Fig. 1 Schematic of the different synthetic strategies for the preparation of CaP nanocarriers. The synthetic strategies mainly include PEGylated polyanion hybrid precipitation (a), a two-step method of self-assembly and hydrothermal treatment (b), a micro-emulsion method (c), a lipid coating preparation method (d), and a layer-by-layer preparation method (e).
Table 1 Preparation strategies for obtaining CaP nanocarriers
Method Preparation mechanism Materials Cargos Size (nm) Applications Ref.
PEGylated polyanion hybrid self-assembly ① Interaction between polyanion and Ca2+ to prohibit the mineralization of large CaP blocks; ② PEG shell prevents the further expansion of the CaP core PEG-PAsp, PEG-polymethacrylate siRNA, DNA, pDNA 100 siRNA/DNA delivery 45, 75, 77, 78 and 97
PEG-bisphosphonate GFP-DNA 130–180 DNA delivery 101
PEG-grafted carboxymethyl chitosan hTERT siRNA 102 siRNA delivery 89
PEGylated charge-conversion polymers VEGF siRNA 42 ± 5 siRNA delivery 76 and 86
PEG-ss-siRNA siRNA 90–120 siRNA delivery 79
Triblock polymer (PEG-b-PAsp-b-poly-(L-phenylalanine)) Doxorubicin, chlorin e6 (Ce6) 30–80 pH responsive drug delivery/PDT 110 and 123
Layer-by-layer preparation method Formation of the CaP core, adsorption of bioactive compounds and then formation of an outer layer to improve the stability DNA DNA 152 (single-shell), 415 (double-shell), 236 (triple-shell) DNA delivery 55
Polyanion hybrid precipitation Coordination between the carboxyl groups of PAA and Ca2+ to precipitate the nanocarriers Poly(acrylic acid) (PAA) Doxorubicin 50 pH-Responsive drug delivery 137
The phosphate head group of the template co-precipitates into the CaP nanoprecipitates Alendronate-hyaluronan graft polymer siRNA 170 siRNA delivery 92
Two step method consisting of self-assembly and hydrothermal treatment ① Interaction between polyanion and Ca2+ prohibits the mineralization of large CaP blocks; ② PEG shell prevents the further expansion of the CaP core; ③Hydrothermal treatment improves the mechanical strength and stability of the CaP matrix PEG-P(Glu) Mn2+ 60 pH-Triggered contrast amplification for MR imaging of tumor malignancy 18
Gd-DTPA 80 MR imaging-guided tumor radiotherapy 64
PEG-PAsp Chlorin e6 100 PDT of tumors 122
Water-in-oil micro-emulsion and column chromatography method Amine, carboxylate, or polyethylene surface-functionalized CaP nanoparticles can be conjugated or functionalized with PEG on the surface to increase their stability Citrate ICG, anticancer drug 10–30 NIR imaging, drug delivery, and therapy 30, 46, 47 and 114
Water-in-oil microemulsion and lipid coating preparation method The phosphate head groups of the DOPA co-precipitate into the CaP nanoprecipitate, while positioning the hydrophobic tail in the oil phase Lipid 111In 25 Lymph node metastasis imaging 52
177Lu 36 ± 9 SPECT-guided radiotherapy 53
DNA 40–60 DNA delivery 51
Rapid microwave-assisted solvothermal method The hydrolysis process of phosphorus-containing biomolecules to form phosphate ions to prevent the fast nucleation and disordered growth of the CaP nanoprecipitates Adenosine triphosphate (ATP) as the organic phosphorus source for CaP preparation Eu3+/Gd3+ 53 ± 18 Tumor imaging 71
Docetaxel/Eu3+ 200–300 pH-Sensitive drug release and bioimaging 62


3. Calcium phosphate nanocarriers for cancer imaging

Accurate molecular imaging of cancer is critical for its diagnosis and management, which mainly include (a) diagnosis of the existence and location of the tumors and tumor staging for proper treatment;56 (b) imaging-guided drug delivery and visible therapy;57 (c) tracing of drugs inside the biological systems;58,59 and (d) assessment of their therapeutic effects.60 The development of nanoscale probes can highly promote the diagnostic accuracy. The CaP nanocarriers can be applied as versatile platforms to load several types of imaging probes for tumor molecular imaging, for instance, positron emission tomography (PET), optical imaging, single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). The potential advantages, such as biodegradability, biocompatibility, high loading capacity and pH-responsiveness, of the CaP-based nanoprobes make them precisely probe the tumors with sufficient diagnostic selectivity and sensitivity. Moreover, molecular imaging modalities play a critical role in cancer diagnosis but require promising probes/contrast agents. Using nanoprobes, it is possible to detect cancers at early stages and provide specific biological information of the lesions, which offer an opportunity for cancer treatment.

3.1 Calcium phosphate nanocarriers for the optical imaging of tumors

Optical imaging is a noninvasive imaging technique in oncology that provides insights into the molecular and cellular processes in biological systems. Optical imaging exhibits the advantages of high sensitivity, low cost, real-time imaging ability, radiation safety and convenient operation;61 moreover, the fluorescence imaging technique is the most widely applied imaging tool in preclinical oncological studies. The fluorescent probes possess the advantages of low cytotoxicity and cost, easy accessibility and good optical properties; however, most of them lack tumor-specific imaging ability. Thus, fluorescent probe (e.g., lanthanide and cyanine dye)-loaded CaP nanocarriers have been engineered to improve the optical properties of CaP nanocarriers for tumor imaging in recent years, which can maintain the fluorescence capabilities and improve the in vivo functionalities of the loaded contrast agents; in this regard, a series of studies have been conducted to incorporate cyanine dyes or lanthanides into CaP nanocarriers for the optical imaging of tumors as lanthanide ions, such as europium (Eu3+), can prevent the limitations of high production cost and the dependence of luminescence on the particle size of quantum dots (QDs). For example, europium (Eu3+)-doped amorphous CaP nanocarriers have been engineered for drug delivery and tumor imaging,62 exhibiting pH-responsive drug release and a distinguished fluorescence signal for cancer imaging. However, the excitation wavelength for Eu3+ is 226 nm, which wavelength may be too short for in vivo imaging into deep tissues.

For fluorescence imaging, the potential limitations include auto fluorescence from the biological systems themselves (e.g., the tissues) and limited light penetration depth in the tissues (i.e., 2–3 mm). To address these limitations, near-infrared fluorescent (NIRF) dyes have been incorporated into the CaP nanocarriers for tumor imaging, which can minimize the background fluorescence as tissue chromophores significantly reduce light absorption in the near-infrared range. There are several types of near-infrared fluorescence imaging probes and dyes, for instance, cypate, indocyanine green (ICG), nanocrystals (i.e., quantum dots) and fluorescent biological nanoparticles. The bio-safe indocyanine green (ICG) dye, approved for clinical applications by FDA, as a near-infrared fluorescent dye was incorporated into the CaP nanocarriers, which could increase the diagnostic sensitivity and selectivity of these nanocarriers for in vivo molecular imaging. For instance, Adair et al. have encapsulated ICG into CaP to obtain PEGylated ICG-CaP nanocarriers (ICG-CPNPs) with the average particle size of 16 nm; this has improved the loading efficiency and quantum efficiency of ICG (Fig. 2a). The PEG shell of the ICG-CPNPs could improve the circulation time of ICP in blood circulation as well as its accumulation in the tumors, and ex situ tissue imaging revealed that the ICG-CPNPs exhibited deep-tissue imaging ability and early detection of solid tumors (Fig. 2b–d).47 The CPNPs were further modified with human holotransferrin and anti-CD71 antibody through a coupling reaction between avidin–biotin and PEG and gastrin peptide and PEG-maleimide. The in vivo NIRF imaging demonstrated that the CPNPs could selectively and effectively target human breast and pancreatic tumors for the NIRF imaging of tumor. The results demonstrated that the CaP nanocarriers could be applied for the incorporation of fluorescent dyes for in vivo tumor optical imaging, demonstrating high potential in biomedical imaging and tumor diagnosis.46


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Fig. 2 The ICG-incorporated CaP nanocarriers (CPNPs) for the near-infrared fluorescence imaging of cancer. (a) A TEM image showing the morphology of ICG-CPNPs; (b) the half-life of ICG-CPNPs and ICG with continuous illumination; (c) ligand-conjugated ICG-CPNPs target transferrin receptors for the imaging of subcutaneous tumor model of breast cancer. (i–iv) represent the free ICG, ICG-incorporated PEG-CPNPs, ICG-incorporated anti-CD71-avidin-CPNPs, and ICG-incorporated human holotransferrin-avidin-CPNPs; (d) ligand-conjugated ICG-CPNPs target gastrin receptors for the imaging of orthotropic tumor model of pancreatic cancer. (i–iii) represent ICG-incorporated PEG-CPNPs, ICG-incorporated Gastrin-10-PEG-CPNPs, and ICG-incorporated pentagastrin-avidin-CPNPs. The figures were reproduced with permission from ref. 47, Copyright©2008, American Chemical Society.

3.2 Calcium phosphate nanocarriers for the MR imaging of cancer

MRI is a noninvasive imaging modality with high spatial resolution and deep tissue penetration, which has attracted significant attention for the molecular imaging and diagnosis of cancer. However, MRI has limited sensitivity and requires contrast agents (CAs) to modulate the water proton relaxation times in the tissues to achieve contrast enhancement for diagnosis. Thus, a series of magnetic materials, including Gd- or Mn-based materials for T1-weighted MR imaging and FeCo, Fe3Pt, and Fe3O4 nanoparticles for T2-weighted MR imaging, have been applied as CAs for MRI. Although MRI can provide physiological and anatomical information, CAs that specifically probe the tumors for distinguishing the malignant regions are still lacking.63 Considering that the nanocarriers can specifically deliver CAs to the tumors, CaP-based nanocarriers have been applied for loading CAs (e.g., Gd- or Mn-based contrast agents) for tumor imaging by MRI. For example, diethylenetriaminepentaacetic acid gadolinium (Gd-DTPA) was prepared through a two-step method using PEG-block-poly(glutamic acid) (PEG-b-P(Glu)) for self-assembly with Ca2+ and HPO42−. The Gd-DTPA-loaded CaP nanocarriers (Gd-DTPA/CaP) were around 80 nm in size and presented the molecular relaxivity of 22.19 mM−1 s−1, a value much higher than that of the clinically applied contrast agent Gd-DTPA (i.e., 3.48 mM−1 s−1). Gd-DTPA/CaP exhibited long circulation in blood and high accumulation in the tumors, which could increase contrast enhancement in the tumors for cancer diagnosis by MRI. The Gd-DTPA/CaP could enhance the contrast in tumors mainly depending on the accumulation of the contrast agent Gd-DTPA in the tumors.64 However, in some cases, it was difficult to distinguish some tumors and tumor microenvironments, for instance, metastatic tumors and hypoxia in tumor microenvironments, by the accumulated CAs. Therefore, MRI nanoparticle-CAs that can elicit high contrast enhancement in tumor microenvironments would highly increase the diagnostic sensitivity. For this purpose, Mn2+-doped PEGylated CaP nanocarriers (PEGMnCaP) were engineered using PEG-b-P(Glu) for self-assembly with Ca2+ and HPO42− through the two-step synthesis procedure (Fig. 3a). The loading efficacy of Mn2+ in PEGMnCaP could reach up to 4% by weight. The PEGMnCaPs were approximately 60 nm in diameter and highly monodisperse, demonstrating reduced toxicity, slightly enhanced distribution in the tumor tissue and prolonged circulation half-life. The PEGMnCaPs were stable in normal biological environments but could respond to low pH in tumor microenvironments and liberated Mn2+, which could bind to proteins in the surrounding tumor microenvironments to non-linearly increase the relaxivity for contrast amplification. This could quickly enhance the contrast in solid tumors selectively and enable the probing of hypoxic areas inside the tumors (Fig. 3b and c) and the detection of millimeter-sized micro-metastasis of colon tumors in the liver (Fig. 3d), whereas there was almost no contrast enhancement in normal tissues. The signal amplification strategy of PEGMnCaPs by responding to tumor-specific pathophysiological parameters, such as pH, to rapidly amplify the diagnostic signals makes PEGMnCaPs potentially applicable for the diagnosis of ultra-small tumors (e.g., 1 mm liver metastasis), monitoring of biological processes in tumors (i.e., hypoxia imaging) and improvement of the cancer diagnostic accuracy. The results highlighted that the MRI CA-incorporated CaP nanocarriers could be well-formulated, showing high potential for tumor malignancy imaging by MRI with high sensitivity and selectivity as well as future potential applications in clinical diagnosis.18
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Fig. 3 The pH-responsive CaP nanocarriers with contrast amplification ability for the MR imaging of malignant tumors. (a) Mn2+-Doped polymeric CaP micelles (PEGMnCaPs) present a monodisperse distribution and an active release Mn2+ of at different pathological pH values. (b) PEGMnCaPs enhance the contrast in C26 tumors for MR imaging and indicate higher contrast of hypoxic regions in the tumors (c). (d) PEGMnCaPs allowed precise imaging of 1–2 mm micrometastasis in liver when the pathological study of liver metastasis was conducted (e). The figures were reproduced with permission from ref. 18, Copyright©2016, Springer Nature Limited.

3.3 Calcium phosphate nanocarriers for SPECT/PET tumor imaging

The PET imaging modality works by labeling biological molecules with positron-emitting isotopes such as 15O, 13N, 11C, 18F, 14O, 64Cu and 68Ga, which can generate two γ rays, detected by the scintigraphic equipment. PET demonstrates high molecular sensitivity and offers quantitative information for the detection and staging of a wide range of cancers. The single-photon emission computed tomography (SPECT) technique is less sensitive than PET, and it mainly uses positron-emitting isotopes, such as 99mTc, 111In, 123I, and 131I, with different γ cameras. Both PET and SPECT have relatively poor spatial resolution; thus, both are generally combined with CT to overcome these limitations.65 Small molecular diagnostic tracers for detection equipment have been established; however, some critical issues, including fast elimination, non-specific distribution, and longitudinal imaging, of the small molecular theranostic tracers are required to be mitigated. The nanoparticle-based imaging tracers have shown various advantages as the nanoparticles can extend the circulation time, bind to specific cancer cells by attaching with the targeted ligands, and serve as a platform where different imaging modalities can be functionalized to overcome the imaging limitations. The radiolabeling approaches can be summarized as follows: (1) chelating radio-metal ions with complexing ligands through coordination chemistry;66 (2) direct bombardment of nanoparticles through hadronic projectiles;67 (3) preparation of nanoparticles with radioactive ingredients;68 and (4) post-synthetic radiolabeling without a chelator.69,70

The complexation of a radionuclide and nanoparticles through chelators can form successful nanoparticle-radiotracers. The unique properties of CaP nanocarriers allow the efficient encapsulation of radiometals without the use of chelators. Moreover, versatile lipid-calcium-phosphate (LCP) nanocarriers have been developed with tumor-targeting function and SPECT/PET imaging ability for cancer theranostic applications. The LCP nanocarriers were developed by directly doping 111In3+ into the CaP core through the microemulsion method and decorating the outer leaflet coating with lipid and DSPE-PEG2000. The 25 nm LCP nanocarriers were able to penetrate into tumor tissues and enter the lymphatic system and could accumulate in the lymph nodes because of their small size, well PEGylated lipid surface and slightly negative surface charge. The 111In3+-doped LCP nanocarriers could specifically accumulate in the lymph nodes through lymphatic drainage; this suggested that the 111In3+-doped LCP nanocarriers could be applied in the SPECT/CT imaging of systemic lymph node metastasis. The imaging results showed that the 111In3+ signals were observed in the heart, vena and lymph nodes, demonstrating high potential of these nanocarriers for imaging lymph node metastasis based on the 4T1 breast tumor model.52 Moreover, when incorporated with other trivalent radiometals, such as 90Y or 192Ir, these nanocarriers can have potential applications, which can be effective therapeutic radionuclide candidates for future studies. The radioisotope-loaded nanoparticles did not exhibit nanoparticle-mediated toxicity, difficulty in scale-up, and high expenses because of the high encapsulation efficiency of radionuclides.

3.4 Calcium phosphate nanocarriers for the multimodal imaging of tumors

Each molecular imaging modality has intrinsic benefits and limitations; for instance, PET has high intensity but low spatial resolution, and ultrasound cannot provide whole body anatomical imaging. Thus, the combination of multiple imaging modalities has been applied for precise tumor imaging. The development of imaging agents for multiple imaging modalities in combination can overcome their respective limitations, providing complementary information of tissues or diseases. The CaP nanocarriers can be employed for incorporating different types of contrast agents into multimodal imaging systems to increase the imaging accuracy.

The CaP nanocarriers enable the co-delivery of multiple imaging molecules for tumor multimodal molecular imaging. For tumor multimodal imaging, Eu3+ and Gd3+ have been doped inside the calcium phosphate nanospheres and hydroxyapatite nanorods for combined imaging. Recently, a rapid microwave-assisted solvothermal method for obtaining Eu3+/Gd3+-doped fluorescent hydroxyapatite nanorods has been reported using adenosine triphosphate (ATP) as the organic phosphorus source for size control.71 The Eu3+/Gd3+-doped nanorods were 53.0 ± 18.2 nm in diameter with narrower size distribution and exhibited high drug loading efficacy in the range from 653.5 to 841.4 mg g−1 by weight ratio. The Eu3+/Gd3+-doped nanorods indicated high potential for multimodal tumor molecular imaging by MRI, photoluminescence imaging and computed tomography (CT) imaging. In a subsequent study, the versatile Eu3+/Gd3+-CaP vesicle-like nanospheres were developed for the combined functions of photoluminescence, magnetism and drug delivery by adding amphiphilic monomethoxy-PEG-block-polylactide (mPEG-PLA) at room temperature to improve the colloidal stability (Fig. 4a).72 The Eu3+/Gd3+-CaP vesicle-like nanospheres with diameter around 100 nm exhibited high drug loading capacity and essentially inappreciable cytotoxicity to the cells. The administration of Eu3+/Gd3+-CaP vesicle-like nanospheres in mice led to significant NIR fluorescence intensity as compared to the case of the control group with no obvious NIR fluorescence; this indicated that these nanospheres were effective for NIR fluorescence imaging in vivo (Fig. 4c). This was due to their high drug loading capacity and physiological stability for efficient drug delivery to the tumors. As shown in Fig. 4e and f, an X-ray image of the mice whole-body was obtained after the administration of Eu3+/Gd3+-CaP vesicle-like nanospheres, and obvious signals appeared under X-ray irradiation. The non-invasive visualization of mice indicates that the nanospheres are promising for fluorescence and X-ray imaging.


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Fig. 4 (a) Formation of a polymer hybrid, vesicle-like Eu3+/Gd3+-CaP nanospheres; (b) optical and near-infrared fluorescence images; (c) X-ray and fluorescence overlay image (d), as well as the X-ray image (e) of the tumor-bearing mice with the subcutaneous injection of Eu3+/Gd3+-CaP vesicle-like nanospheres to the right side, where Eu3+[thin space (1/6-em)]:[thin space (1/6-em)]Gd3+ = 1[thin space (1/6-em)]:[thin space (1/6-em)]2; (f) signal intensity measured from the X-ray images, whereas 1–4 means the subcutaneous injection position, viz., abdomen, chest, as well as the spine. The figures were reproduced with permission from ref. 72, Copyright©2012, Elsevier.

In addition, the CaP nanocarriers employed for multimodal imaging systems are expected to overcome the drawbacks, including limited tissue penetration and low spatial resolution, of fluorescence imaging. The co-delivery of other types of imaging agents in addition to fluorescent agents inside the CaP nanocarriers can be achieved. The CaP nanocarriers with ICG/Gd3+ doped inside the CaP core and polyethyleneimine (PEI) cap tagged with 99mTc-methylene diphosphonate (99mTc-MDP) were developed for tri-modal molecular imaging, demonstrating 0.15% loading capacity of ICG, 0.002 ng mg−1 of 99mTc by weight, and 3.38% of Gd3+ by mole ratio. The PEGylation of the CaP nanoparticles prolonged the blood circulation time and periodic redistribution. The toxicity assessment revealed that multimodal contrast agents based on CaP nanocarriers exhibited biocompatibility and low toxicity. The near-infrared, MRI and nuclear images were acquired for mice models with a clearer visualization of small blood vessels in the liver tissues, demonstrating high potential of these nanocarriers for liver angio-imaging.44

4. Calcium phosphate nanocarriers for gene delivery

Neoplastic diseases are associated with changes in the genomic phenotype. RNA interference (RNAi) and gene therapy are aimed at treating genetic diseases including cancer, neurodegenerative disorders, peripheral vascular diseases, and acquired immunodeficiency syndrome (AIDS). The realization of efficient gene delivery is a major challenge in gene therapy and RNAi.73 The key factor towards successful gene delivery is the achievement of effective gene transfection. Although current gene delivery vehicles are mostly based on cationic polymers (polyplexes) or cationic lipids (lipoplexes), their potential immune-responses can be a potential hurdle for clinical translation.74 The CaP nanocarriers have attracted significant attention for the delivery of a variety of genes, including DNA, small interfering RNA (siRNA), as well as messenger RNA (mRNA), due to their efficient gene transfection ability, biocompatibility and biodegradability. To date, several studies have applied nucleic acid-incorporated CaP nanocarriers for treating diseases by RNA interference, gene regulation and gene therapy. Various materials, such as polyanions,75,76 bisphosphonate and even DNA, have been applied to yield well-dispersed CaP nanocarriers for efficient gene transfection, whose surface can be coated with lipids, PEG or mucopolysaccharide to extend their half-life time in blood circulation and increase their stability.

4.1 Calcium phosphate nanocarriers for siRNA delivery

Double-stranded RNA with short sequences of nucleotides can be applied for inhibiting genetic diseases including cancer; however, its delivery to specific target cells in vivo is a challenge. Thus, a series of gene delivery vectors, including CaP-based nanocarriers, have been applied for delivering siRNA to the cancer cells as the phosphate groups in siRNA can readily interact with Ca2+ to be stably doped inside the CaP nanocarriers. For instance, the incorporation of the block copolymer PEG-b-PAsp into calcium phosphate cores was exploited for developing safe and efficient gene delivery CaP nanocarriers.77 The organic–inorganic hybrid CaP nanocarriers demonstrated small size by adjusting the concentrations of PEG-b-PAsp block copolymers and phosphate anions, high loading efficiency and colloidal stability. The in vitro experiments revealed that siRNA could be released in the intracellular compartment with appreciably lower Ca2+ concentrations as compared to that in the extracellular fluid. Moreover, another polyanion, poly(methacrylic acid) (PMA), was constructed into a block polymer structure to facilitate the endosomal-escape function of the CaP nanocarriers.78 Moreover, considering the discordance of regulated crystal growth with PEG palisades and efficient siRNA entrapment, the later studies have reported the use of redox-responsive disulfide bonds to link PEG and siRNA to solve this problem.79 By this strategy, the hybrid micelles could encage a larger amount of siRNA inside the CaP core, and the encapsulated siRNA was five-fold greater (i.e., 400 mg mL−1) than that in the previous two systems. The PEG-siRNA-incorporated CaP nanocarriers were stable under physiological conditions, exhibiting high cellular uptake and effective gene silencing of luciferase genes in Huh7 hepatocarcinoma cells. Recently, another polymer hybrid CaP nanocarriers consisting of CaP and amphiphilic polyethylene glycol-L-α-phosphatidylethanolamine (mPEG-PE) copolymer have been developed for efficient siRNA delivery.80 The mPEG-PE/CaP hybrid nanoparticles with the average size of 53.2 nm and surface negative charge of approximately −16.7 mV could enable the efficient encapsulation of siRNA (about 80%) and protect the siRNA from ribonuclease (RNase) degradation. In addition, metalloproteinase -responsive PEG/lipids/CaP hybrid nanocarriers were developed for siRNA delivery, and efficient siRNA delivery to the tumors was achieved using them without obvious toxicity.81

The LCP nanocarriers further modified with PEG conjugated with the sigma-1 receptor-specific ligand anisamide were developed, combining the merits of liposome and CaP nanocarriers to acquire prolonged circulation half-life in the blood stream and promoted endosome escape function for achieving the efficient systemic delivery of siRNA to the cancer cells;82 the LCP nanocarriers of around 40 nm size with a positive charge exhibited the siRNA encapsulation efficiency of 91%. Moreover, the LCP nanocarriers decorated with anisamide-installed PEG could specifically interact with the Sigma receptors expressed on the B16F10 cancer cells. The ligand-installed LCP nanocarriers formulated with luciferase siRNA led to a significant in vivo gene silencing activity (i.e., 78%) in the C57BL/6 mice bearing B16F10 lung metastatic models. The LCP nanocarriers could also incorporate multiple siRNAs, including vascular endothelial growth factor (VEGF), c-myc, and mouse double minute 2 homolog (MDM2),83 for the cocktail treatment of metastatic tumors, resulting in the effective silencing of related oncogenes in metastatic nodules by intravenous injection. They could significantly inhibit lung metastasis with the efficiency of approximately 70–80% at a low dose (0.36 mg kg−1), and their survival rate was significantly enhanced.

Moreover, combined delivery of a gene and a drug was carried out using the LCP nanocarriers for the improved intracellular delivery of siRNA and promotion of the gene transfection efficiency. The folic acid (FA) and EGFR-specific single chain fragment antibody (ABX-EGF scFv)-installed LCP nanocarriers (LCP-FA-scFv) exhibited significantly enhanced cellular uptake and tumor accumulation, indicating their valuable targeting ability for the diagnosis and therapy of human breast cancer.48 The LCP-FA-scFv further significantly improved the siRNA delivery to MDA-MB-468 breast cancer cells as well as enhanced its tumor accumulation.84 Moreover, the polycation liposome-encapsulated CaP nanocarriers (PLCP) were developed for VEGF siRNA delivery, which could reduce 60–80% VEGF gene expression in vitro. Moreover, when these nanocarriers were treated with PLCP/VEGF siRNA or combined with doxorubicin, significant inhibition of tumor growth and angiogenesis was observed in MCF-7 xenograft tumor-bearing mice.85

In addition, another strategy for promoting the endosomal escape was applied using the block copolymer of PEGylated charge-conversion polymers to prepare the polymeric CaP nanocarriers, where the polymer charge could change from anionic (negative charge) to cationic (positive charge) inside the acidic cellular organelle (e.g., endosome and lysosome). The charge-conversion block copolymer hybrid CaP nanocarriers exhibited a high siRNA-loading efficiency (i.e., around 80% of the dose), whereas the converted positively charged polymers could disrupt the endosomes/lysosomes to allow for efficient intracellular siRNA delivery. By incorporating the VEGF siRNA, these nanocarriers could knockdown approximately 80% of the mRNA expressed in the pancreatic cancer cells (BxPC3).76 In the following study, the VEGF siRNA-loaded PEG-CCP/CaP nanocarriers were investigated for cancer treatment, demonstrating significant prohibition of subcutaneous BxPC3 tumor growth and gene silencing (i.e., 68%).86 The siRNA-loaded PEG-CCP/CaP nanocarriers were further investigated for spontaneous bioluminescent pancreatic tumors in transgenic mice. The systemic administration of siRNA-loaded PEG-CCP/CaP nanocarriers demonstrated promising in vivo gene silencing results, as confirmed by bioluminescence imaging.87 In another study, a pH-triggered charge reversible conjugate was synthesized to form siRNA (siBcl-2) loaded CaP nanocarriers. The tumor microenvironment pH could trigger the charge conversion and size change of the siBcl-2-incorporated lipid CaP nanocarriers, leading to high accumulation in the tumor tissues and significant inhibition of tumor growth.88 In another study, carboxymethyl chitosan (CMCS) with enhanced solubility, non-cytotoxicity and pH-sensitivity was conjugated with PEG to form siRNA-loaded polymer hybrid siRNA/CaP nanocarriers.89 The PEG-CMCS hybrid siRNA/CaP nanocarriers exhibited efficient systemic delivery of siRNA. They also demonstrated pH-responsive release of siRNA and better gene silencing effect than Lipofectamine 2000 while demonstrating significant suppression of tumor growth through the specific gene silencing effect and induction of cell apoptosis in nude mice xenografted with A549 tumors. The siRNA and PEGylated CaP nanocarriers incorporated with ultra-low levels of doxorubicin were developed,90 showing enhanced caveolin-mediated endocytosis and internalization by the cancer cells. They could effectively deliver siRNA to cause the marked down-regulation of the X-linked inhibitor of the apoptosis protein (XIAP) and tumor growth inhibition.

The CaP nanocarriers can also be decorated with cancer cell targeting moieties to increase the targeting ability and therapeutic efficacy. For instance, hyaluronan is a type of negatively charged polysaccharide that enables the stabilization of nanoparticles and CD44-mediated tumor targeting as it can specifically interact with the CD44 receptors that are highly expressed on the cancer cells. The hyaluronic acid and DOPA-stabilized CaP/siRNA nanocarriers were formulated, exhibiting improved accumulation of siRNA in the solid tumors and targeted gene silencing effect through i.v. injection.91 Effective and safe systemic siRNA delivery could also be achieved by calcium phosphate and siRNA co-precipitates with a shell of alendronate-hyaluronan graft polymer (AHA).92 Alendronate and hyaluronan were covalently conjugated to be anchored around the CaP nanocarriers to control the size of these nanocarriers, enhance their cellular internalization and tumor accumulation through CD44 receptor-mediated tumor targeting. The CaP-AHA/siRNA nanocarriers exhibited the diameter of 170 nm, a negative charge and improved physical stability owing to the surface coating of hydrophilic HA (Fig. 5a). The in vitro test indicated that the CaP-AHA/siRNA nanocarriers exhibited gene silencing efficacy in A549 cancer cells by CD44 receptor-mediated endocytosis to significantly down-regulate the expression of EGFR (Fig. 5c and d) while demonstrating significant inhibition of tumor growth by silencing the EGFR expression in the A549 tumor xenograft. In another recent study, the polysaccharide derivative of thiolated hyaluronic acid (HA-SH) was applied to stabilize the CaP nanocarriers for achieving extended duration of cycling in blood and enhanced accumulation in the tumors.93 It could suppress 80% of gene expression and the growth rate of the B16F10 xenograft tumors. In another study, the HA-based RNAi nanoplatform based on CaP was developed using the phosphate receptor Zn(II)-dipicolylamine (DPA/Zn) to bind RNA for siRNA delivery.94


image file: c9bm00831d-f5.tif
Fig. 5 The hyaluronan-decorated CaP nanoparticles for siRNA delivery. (a) The size distribution of CaP nanoparticles; (b) the schematic of pH-triggered siRNA release from CaP nanoparticles; (c) cellular uptake of CaP-AHA10/siEGFR nanoparticles; (d) lysosomal escape of CaP-AHA10/siEGFR nanoparticles as tested in the A549 cells. The figures were reproduced with permission from ref. 84, Copyright©2016, The Royal Society of Chemistry.

To date, the CaP-based nanocarriers have demonstrated high potential for siRNA delivery including efficient intracellular siRNA delivery with endosome escape function, RNA interference, as well as tumor therapy. Although there have been some progresses, new strategies/optimization are required for the further optimization of the CaP nanocarriers in siRNA delivery, including the optimization of the colloidal stability, tumor targeting ability, and therapeutic efficacy. In addition, further studies on the immunogenicity and toxicity of the CaP-based vector materials are required for the translation of these materials from bench to bedside.

4.2 Calcium phosphate nanocarriers for DNA delivery

The application of gene therapy with plasmid DNA has attracted significant attention for the treatment of genetic diseases (e.g., cancer), and the development of gene carriers for the effective delivery of genes to cells is critically important to achieve a successful treatment. The CaP precipitates were found to increase the transfection effect of human adenovirus 5 DNA to the cells in 1973,42 and now, calcium phosphate-DNA co-precipitation has become one of the most widely used transfection methods. However, the heterogeneous size distribution of DNA-loaded CaP nanocarriers resulted in a large deviation in the transfection efficiency.95 The size of the DNA-loaded CaP nanocarriers is important for the development of DNA delivery systems.77 Many efforts have been made to regulate the size as well as improve the stability and transfection efficiency of these nanocarriers.96

The first organic–inorganic CaP nanocarriers were exploited for DNA delivery, which laid the foundation for the further development of calcium phosphate as a drug carrier. As abovementioned, the PEG-PAA block polymer was initially utilized to form stable and uniform CaP/DNA composites. The self-assembled nanoparticles had the small size of around 100 nm and good colloidal stability with the capability of incorporating DNA into the core with reduced cytotoxicity.97 Intriguingly, in a subsequent study, the CaP core was coated with DNA for improving the colloidal stability.98 The DNA-coated CaP nanocarriers could be coated with another DNA/calcium phosphate shell to form multi-shell calcium phosphate/DNA nanomedicines for achieving higher transfection efficiency. It was further revealed that the cells could deal with the associated calcium uptake. Later, this method was optimized with several kinds of small molecules with similar functional groups and applied for the transfection of pDNA and siRNA to different cells.99,100 Moreover, the bisphosphonate-stabilized CaP nanocarriers of around 200 nm size were developed for DNA delivery.101 The nanoparticles revealed the DNA incorporation efficiency of 70% and enhanced the in vitro gene transfection efficiency with low cytotoxicity. Moreover, a reproducible, one-step procedure was described for preparing aminosilane-modified CaP nanocarriers.102 It yielded CaP nanocarriers with stability, narrow size distribution (i.e., diameter of 140 nm), high pDNA condensation capability and high transfection of pDNA in A549 cells with low toxicity. Recently, magnetic CaP nanocarriers with the average diameter of 185 nm have been used for DNA transfection, exhibiting enhanced transfection efficiency in the A549 and HepG2 cancer cells under an external magnetic field.103 The magnetic nanocarriers could enhance the transfection efficiency by about 30% when applied under an external magnetic field. Furthermore, the polyethyleneimine (PEI)-modified pDNA-loaded CaP nanocarriers were constructed as a durable gene transfection system, which featured slow degradation and release of pDNA with highly durable gene expression.104 A single injection of the PEI-modified pDNA-loaded CaP nanocarriers led to effective tumor suppression. In addition, it was reported that short CaP nanorods with the length of 40–60 nm incorporating pEGFP-N1 pDNA could transfer EGFP-N1 to the SGC-7901 cells, and the uptake efficiency of the nanocarriers was 80% of the uptake achieved by Lipofectmine-2000.105

5. Calcium phosphate nanocarriers for cancer chemotherapy

Generally, the chemotherapeutic loading efficiency of calcium phosphate nanocarriers is far from satisfactory to achieve cancer chemotherapy. Extensive efforts have been made to enhance the loading efficiency of anticancer bioactive compounds. Instead of encapsulating the therapeutic drugs into CaP, the chemotherapeutics are complexed with biomolecules such as cyclodextrin (CD) and human serum albumin (HSA). Shi et al. reported that the Pt-IV prodrug was complexed with HSA and further coated with CaP to form the Pt-HSA/CaP nanoparticles, in which about 0.27% of Pt was loaded.106 The Pt-HSA/CaP nanoparticles were stable in systemic circulation, and CaP was dissolved upon cellular internalization for intracellular drug delivery, resulting in the efficient inhibition of tumor cell growth. In another study, β-CD was utilized to form complexes with the hydrophobic anticancer drug docetaxel through non-covalent interactions in the hydrophobic cavities, exhibiting the docetaxel loading efficiency of 6.24%.107 Owing to its remarkable internalization with the A549 cancer cells, it enhanced the cytotoxicity towards the cancer cells as tested in vitro. Moreover, Choi et al. developed versatile RNAi nanoformulas (NFs) based on hyaluronan, conjugated them with the artificial RNA receptor Zn(II)-dipicolylamine (DPA/Zn) for RNA loading, and stabilized with the CaP layer (CaP-HDz/RNA-NF) (Fig. 6a and b).94 Doxorubicin could be encapsulated within the hydrophobic inner core of the nanocarriers, exhibiting pH-sensitivity and targeted drug delivery (Fig. 6c). The co-delivery of doxorubicin and MDR 1 gene target siRNA by this system could overcome the MDR of OVCAR8/ADR cancer cells towards doxorubicin and improve the antitumor efficiency in vitro and in vivo (Fig. 6d). Moreover, mesoporous silica nanoparticles (MSNs) have attracted significant attention in drug loading and release with a CaP nanocoating. Lee et al. reported CaP covered mesoporous silica nanocontainers for the controlled release of doxorubicin guest molecules.108 Doxorubicin was loaded into the pores of MSNs, and then, doxorubicin-loaded MSNs were capped by CaP coatings through urease-mediated surface mineralization. The doxorubicin-loading content inside the MSNs was 4.2% by weight. Similarly, Zhu et al. designed doxorubicin-loaded with MSNs as the fundamental cores and then coated them with CaP, followed by liposome encapsulation with zinc phthalocyanine (ZnPc). Doxorubicin was loaded into MSNs@CaP and MSNs@CaP@PEGylated liposomes at 41% and 20% by weight, indicating that the nanoplatform could effectively load doxorubicin for chemotherapy.109
image file: c9bm00831d-f6.tif
Fig. 6 The development of CaP nanocarriers for tumor chemotherapy. (a) Preparation of CaP-HDz/RNA-NF; (b) mean size and zeta-potential of HDz-NP, HDz/siRNA-NF, Ca-HDz/siRNA-NF, and CaP-HDz/siRNA NF; (c) RNA and drug release from NFs incorporated with both paclitaxel (PTX) and siRNAs; (d) in vivo bioluminescence imaging (BLI) of firefly luciferase (fLuc) gene expression in fLuc-expressing HCT116 tumor-bearing mice intravenously injected with PBS, free siLuc, or CaP-HDz/siLuc-NFs; (e) antitumor efficacy of CaP-HDz/DOX/siMDR-NFs in drug-resistant OVCAR8/ADR tumor-bearing mice (n = 4) after the systemic administration of NFs. The figures were reproduced with permission from ref. 94, Copyright©2014, American Chemical Society.

Another strategy is loading the drug inside the CaP shell or hollow CaP nanospheres. Lee et al. designed a triblock copolymer by grafting hydrophobic poly(L-phenylalanine) on PEG-polyanion to form a hydrophobic inner core for loading doxorubicin.110 CaP was mineralized with PAsp to form doxorubicin-loaded core–shell-corona micelles. The average hydrodynamic diameter and the drug loading capacity of the micelles were 62.8 nm and 7.8% by weight, respectively, and controlled drug release was observed from the hybrid nanocarriers. The micelles exhibited enhanced tumor accumulation and antitumor therapeutic efficacy.111 In addition, phosphorylated prodrugs are a prospective strategy for loading drugs into the CaP nanocarriers. Overall, the delivery of chemotherapeutics by CaP nanocarriers could be improved in further studies, and more CaP-based nanocarriers for anticancer drug delivery will appear in future.

6. Calcium phosphate nanocarriers for cancer theranostics

Considering that molecular imaging can provide the anatomical information of tumors, it can be combined with tumor therapy, which is called cancer theranostics. The theranostic approaches integrate imaging function into therapy, offering a visible and quantitative way to trace the drug delivery/release, cellular targeting and uptake, image the pathological characteristics of tumors, as well as monitor the drug therapeutic efficiency and side effects. Supported by these concrete evidences from precise cancer diagnosis, it is possible to offer personalized medicine and dose adjustment, which can optimize and evaluate the therapeutic strategies.112 During the past few decades, the combination of therapy and imaging methods has been achieved with various nanoplatforms, including CaP nanocarriers,113 as it enable the incorporation of multiple payloads for simultaneous tumor-targeting, imaging and therapy.

The CaP nanocarriers have been applied for imaging-guided cancer chemotherapy as CaP enables the pH-triggered drug release of the desired dose at the tumor sites to enhance the therapeutic efficacy with reduced side effects. For instance, Morgan et al. encapsulated both Cy3 and therapeutic agents into CaP nanocarriers for tumor fluorescence imaging and therapy.114 The nanocarriers were approximately 20–30 nm in diameter, as characterized by a transmission electron microscope (TEM), exhibiting colloidal stability and nearly 5-fold enhancement in the fluorescence quantum yield over Cy3. In another study, CaP-based metallic nanocarriers based on Fe3O4@CaP-capped gold nanocages (AuNCs) were reported, which demonstrated high potential for tumor magnetic targeting, photothermal therapy and chemotherapy.115 The Fe3O4@CaP-capped AuNCs could achieve dual pH/NIR-triggered drug release, making it possible to achieve synergistic effect in dealing with tumors.

In addition, the trivalent cation lutetium (177Lu3+) was doped into the LCP nanocarriers for cancer therapy and imaging.53177Lu3+ is an attractive option in cancer theranostics as a radionuclide to provide simultaneous imaging and therapeutic functions. Only a low dosage of 177Lu3+ is required to achieve therapeutic effects as trivalent cations are preferentially encapsulated into the 177Lu3+-loaded LCP nanocarriers, minimizing the heavy metal-induced toxicity. The encapsulation efficiency of 177Lu could reach up to approximately 70% inside the 36 ± 9 nm nanoparticles. Both SPECT and Cerenkov imaging modalities have shown that the 177Lu3+-loaded LCP nanocarriers can accumulate in the tumors. The therapeutic results demonstrated that the 177Lu3+-loaded LCP nanocarriers could significantly inhibit the tumor growth mainly by inducing the apoptosis of cancer cells by breaking the double-stranded DNAs, remodeling of the tumor microenvironment to be less malignant, as well as creating more disordered conditions.

Recently, CaP nanocarriers with the average diameter of 67 nm have been developed using polyacrylic acid (PAA)/CaP, acting as a shell packing the aggregation of Au nanoclusters (Au NCs) to enhance the fluorescence property as well as load the chemotherapeutic agent doxorubicin with the high drug storage capacity of 92%. Moreover, nearly 100% of doxorubicin could be released after 7 h incubation at pH 5.1. The doxorubicin and Au NC-incorporated PAA/CaP nanocarriers can be applied for simultaneous dual-modality tumor imaging as the aggregation of Au nanoclusters (AuNCs) can be applied for CT imaging at high spatial resolution as well as fluorescence imaging with high sensitivity, which is very helpful in tumor diagnosis, guiding and monitoring of the chemotherapeutic effect by the incorporated chemotherapy drug doxorubicin. The AuNCs-A@PAA/CaP nanocarriers have been applied for dealing with liver tumor, demonstrating high potential for visualizing and monitoring the chemotherapeutic process using dual-modal imaging modalities.116 Moreover, the multifunctional theranostic carbon/CaP/Fe3O4 nanocarriers of 140 ± 20 nm were engineered for MRI and pH/NIR-activatable drug delivery.117 The nanocarriers exhibit the efficient drug encapsulating capability of 67%, pH/NIR-responsive drug release and theranostic functions due to the unique structure of mesoporous carbon matrix incorporated with CaP and Fe3O4 components. Further, yolk–shell nanoparticles of average length and width of around 100 and 65 nm composed with gold nanorod (AuNR) core/DOX-loaded CaP shell were fabricated for photoacoustic (PA) and CT imaging as well as chemotherapy and photothermal therapy.118 The photothermal effects could induce the release of doxorubicin with enhanced therapeutic effect against the cancer cells. Moreover, the hollow mesoporous fluorescent Gd2O3:Eu/CaP nanospheres were developed using PAA-CaP as a template, exhibiting the high drug loading capability of 73.2%, low cytotoxicity, pH-sensitivity and sustained drug release for bioluminescence imaging, MR imaging and therapy.119 Furthermore, a theranostic drug delivery system based on AuNP and CaP was developed for CT imaging and cancer therapy.120 The gold nanoparticles were modified with PEG-Asp-Cys polymers and coated with the CaP shell containing doxorubicin to prepare PEGylated Dox-AuNP@CaP with the average diameter of about 79.8 ± 18.7 nm. Because of the relatively high atomic mass of gold, the loading efficiency of doxorubicin was 27.9%, which was a little lower than that for the aforementioned nanocarriers. The PEGylated doxorubicin-AuNP@CaP with enhanced stability and pH-triggered drug release properties showed high potential for cancer CT imaging and treatment. In addition, another theranostic system was developed by encapsulating iron oxide crystals with self-assembled gelatin (AGIO) and simultaneous co-deposition of the doxorubicin-CaP layer on the AGIO.121 The AGIO@CaP-doxorubicin nanocarriers showed an average size of 120 nm, CaP shell thickness of about 20 nm, and loading efficiency of 38%, while the in vitro characterization also demonstrated efficient MRI contrast enhancement and efficient cellular internalization with the HeLa cells.

Besides, the polymer hybrid CaP nanocarriers were applied for the incorporation of photosensitizer chlorin e6 (Ce6) for pH-sensitive tumor photodynamic therapy (PDT) (Fig. 7a and b).122 The Ce6-incorporated CaP nanocarriers with a diameter of about 100 nm could significantly increase the intracellular delivery of Ce6 with enhanced phototoxicity than that of free Ce6 (Fig. 7c). The Ce6-incorporated CaP nanocarriers exhibited long circulation in blood vessels, which could target tumor tissues through the EPR effect, and the therapeutic results have shown that it could suppress the tumor progression (Fig. 7d–f). Lee also reported Ce6-loaded core–shell-corona polymer hybrid CaP micelles of 74.6 nm size with loading content of 10% by weight and loading efficiency of 95%.123 The optical imaging results after the intravenous injection of Ce6-loaded CaP micelles showed improved cancer specificity and superior phototoxicity compared to that of free Ce6 due to the enhanced stability and high tumor accumulation. In another study, the photosensitizer temoporfin, tumor targeting peptide RGDfK, and a fluorescent dye were incorporated in CaP nanocarriers for multifunctional NIRF imaging and PDT of tumors.124 The multifunctional CaP nanoparticles with a diameter of about 200 nm and a zeta potential of approximately +22 mV could be preferentially accumulated in the tumors for achieving enhanced therapeutic efficacy as well as reduced side effects such as skin photosensitivity. The CaP nanocarriers incorporated RGD peptide could target tumors, reduce the side effects such as skin photosensitivity, and achieve high therapeutic efficacy by inducing apoptosis and destroying the tumor vascularization. Moreover, aptamers can also be used for tumor targeted imaging, for example, lactoferrin-treated Fe3O4 (Fe3O4-bLf) loaded CaP nanoparticles modified with locked nucleic acid (LNA) have been exploited,125 which exhibit a diameter of 205 ± 102 nm. The LNA-aptamer decorated CaP nanocarriers were found to completely regress the xenograft tumors in 90% of the triple positive colon cancer (EpCAM, CD133, and CD44), while the multimodal imaging efficacy was also determined by NIR, MRI, and CT.


image file: c9bm00831d-f7.tif
Fig. 7 Polymer hybrid CaP nanocarriers for tumor theranostics. (a and b) Strategy for developing Ce6-incorporated CaP nanocarriers (CaPCe6) for tumor PDT; (c) average diameter of CaPCe6; (d) the fluorescence intensity of CellROX inside the cancer cells; (e) the cell viability of CaPCe6 with/without laser irradiation; (f) tumor therapeutic results of CaPCe6 as tested on subcutaneous A549 tumor models. The dose was 200 μg based on Ce6. The figures were reproduced with permission from ref. 123, Copyright©2016, The Royal Society of Chemistry.

Moreover, the imaging could be applied for imaging-guided pinpoint local radiotherapy, which can prevent hamper normal tissues.126 Gd-DTPA/CaP could also be applied for MR imaging-guided gadolinium neutron capture therapy (GdNCT). Gd-DTPA is a clinically applied MRI contrast agent and Gd can capture thermal neutrons to generate γ rays that kill cancer cells.5,43,127 The loading efficacy of Gd-DTPA inside Gd-DTPA/CaP was estimated to be 6.85 ± 0.22%. Gd-DTPA/CaP could delivery Gd-DTPA to the tumors with high selectivity, increasing the contrast in the tumor tissues for precise positioning. Thereafter, the thermal neutron irradiation was conducted on the detected tumor regions for gadolinium neutron capture therapy, which demonstrated effective tumor ablation effect. All the results indicated that Gd-DTPA/CaP was an efficient vehicle for imaging-guided tumor radiotherapy.127,128

In addition, Janus nanoparticles have been engineered, which could provide asymmetry compartments and encapsulate different payloads within a single particle for a wide set of technological and biomedical applications,129,130 such as drug delivery,131,132 catalysis,133 or solid surfactants.134,135 The Janus nanoparticles render them truly multifunctional platforms for cancer theranostics since their asymmetric structure allows multifunctionality within a single nanoparticle.136 The CaP-based multifunctional Janus nanoparticles (JNPs) have been engineered with FA-PEG-SH and PAA-Ca on the surface of the Au nanoparticles, while the pre-formed nanoparticles further reacted with sodium hydrogen phosphate (Na2HPO4) to form the CaP shell on the PAA-Ca side.137 The “exposed” Au domains with FA-PEG-SH achieve high contrast enhancement in the tumors for CT imaging by targeting the cancer cells, while the PAA/CaP sides with a mesoporous structure could incorporate doxorubicin with a drug loading efficiency of 95%. The FA-Au@PAA/CaP JNPs with size of about 139 nm possessed good biocompatibility and pH-responsive properties, and provided multiple functions for tumor CT imaging and tumor-targeted chemotherapy. Besides, spherical polydopamine hybrid mesoporous calcium phosphate hollow JNPs (PDA/CaP H-JNPs) were developed later in a facile synthetic approach.138 The PAA nanoparticles were applied as the template, which then interacted with ICG and PEG-SH on the PDA domains, while the mesoporous CaP with a hollow cavity served for incorporating the chemotherapeutic agent doxorubicin.137 The hollow CaP structure was formed because PAA coordinated to calcium ions can be washed away due to its solubility in water. The loading efficiency of doxorubicin inside the CaP hollow cavities was approximately 92%. The PEG-ICG-PDA/CaP H-JNPs demonstrated high drug encapsulating capability, high photothermal conversion ratio and NIR intensity, as well as pH- and NIR-sensitivities for PA imaging, imaging-guided chemotherapy, and photothermal therapy.

7. Conclusions and perspectives

Recent progresses in the development of CaP nanocarriers have demonstrated high potential of these nanocarriers for drug delivery; there has been significant improvement in the preparation of CaP nanocarriers by enhancing the size, drug loading efficacy, and stability. The CaP nanocarriers have been demonstrated as a versatile platform to load different cargoes, such as imaging probes and therapeutic compounds, for enhanced tumor diagnosis and targeted therapy. Although there have been several scientific advances over the past few decades, the translation of CaP nanocarriers towards clinical applications for cancer diagnosis and therapy is required. Therefore, future efforts may focus on the optimization of CaP nanocarriers for clinical applications, including large scale preparation, systemic toxicity evaluation, pharmacokinetic and pharmacodynamic studies, as well as study of the efficacy. The imaging-functionalized CaP nanocarriers may facilitate the prediction and monitoring of the outcomes of the therapeutic CaP nanocarriers.139 We believe that more and more attempts will pave the way for CaP nanocarriers towards clinical applications in the near future for both bio-imaging and drug delivery.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was funded by the National Key R&D Program of China (2017YFA0207900), the Recruitment Program of Global Experts (D1424002A) and the Sichuan Science and Technology Program (2018RZ0134).

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