Metal-Organic Framework Nanoparticle-Based Biomineralization: A New Strategy toward Cancer Treatment.

Cancer treatment using functional proteins, DNA/RNA, or complex bio-entities is important in both preclinical and clinical studies. With the help of nano-delivery systems, these biomacromolecules can enrich cancer tissues to match the clinical requirements. Biomineralization via a self-assembly process has been widely applied to provide biomacromolecules exoskeletal-like protection for immune shielding and preservation of bioactivity. Advanced metal-organic framework nanoparticles (MOFs) are excellent supporting matrices due to the low toxicity of polycarboxylic acids and metals, high encapsulation efficiency, and moderate synthetic conditions. In this review, we study MOFs-based biomineralization for cancer treatment and summarize the unique properties of MOF hybrids. We also evaluate the outlook of potential cancer treatment applications for MOFs-based biomineralization. This strategy likely opens new research orientations for cancer theranostics.

1. Introduction

Cancer is an urgent global public problem and the focus of significant worldwide research 1-3. Cancer treatments include low-molecular-weight drugs, gene therapy via DNA/RNA 4, immunotherapy via antibodies and antigens 5, and biotherapy via proteins and oncolytic viruses 6. However, these biological approaches are limited by the complicated tumor microenvironment, so the delivery systems were studied for the application of the biomacromolecules. These traditional delivery systems were designed to load the biomacromolecules on the surface of the nanomaterials, or coated inner the nanomaterials while the self-assembly of the polymer or amphipathic molecule. And, with help of the target modification, the biomacromolecules could gather in the function tissues/cells.

Recently, many researchers have focused on metal-organic frameworks (MOFs), which are novel porous materials 7-11. MOFs are constructed by the coordination of metal cations or clusters and organic bridging ligands. They offer a tunable design and a network structure with controlled chemical functionality, high crystallinity, and good porosity. Due to their unique structures and properties, MOFs have been applied in gas storage and separations 12, catalysis 13, 14, energy 15, 16, and sensing 17, 18.

Nanomedicines were designed to overcome biological barriers and selectively target tissues. They are efficient therapeutics 19. MOFs can be tailored for specific biomedical applications 20-23. MOFs are promising platforms for molecular imaging or drug delivery because of their porosity, tunable design, and low toxicity 24-28. Furthermore, MOFs were also good delivery platforms for the biomacromolecules and the loading strategies of biomacromolecules can be categorized in four ways: (i) the biomacromolecule was adsorbed on the surface of MOFs due to the physical absorption 29-30; (ii) the biomacromolecule was conjugated on the surface through the chemical coupling with the organic bridging ligands or the chelation reactions with the chelation reactions 31-33; (iii) the biomacromolecule was infiltrated into the pore taking advantage of the mesoporous nanostructure 34-37; (iv) the biomacromolecule was encapsulated within the MOFs networks during the self-assembly reaction of the mixed solution containing metal cations, organic bridging ligands and the biomacromolecules 38-40.

These MOF biocomposites obtained by surface coating or bio-conjugation enabled the biomacromolecule in the outermost surface, which the MOFs mostly serve as the carrier but leads to insufficient protection of biomacromolecules. While the biomacromolecules loaded on the pore networks of MOFs could protect the biomacromolecules from the external environment, and yet the infiltration strategy was dependent on pore size of the MOFs, in which the pore size must exceed the biomacromolecule.

During the self-assembly (biomineralization), the activity of the biomolecules or living organisms are retained and even improved in some cases 41-44. The diversity of the metal-connecting points and organic-bridging ligands as well as straightforward self-assembly makes MOFs excellent supporting matrices for the immobilization of DNA, enzymes, peptides, proteins and living organisms (e.g., viruses and cells) (Figure ) 22. Furthermore, these surface-coated matrices of MOFs help the biomolecules or living organisms with prolonged bio-activity to enhance their stability under physiological conditions. MOFs-based biomineralization has been applied for storage, transport, treatment, and sensing. Cargo includes biological catalysts, biomolecules, or living organisms 41.

In this review, we summarize the application of MOFs-based biomineralization in cancer treatment (Figure ). We discuss potential obstacles for practical use including toxicity arising from unintended interactions of MOFs with healthy organisms and the relevant implications for rational design. We also detail relevant insights into future applications of MOFs and how nano-bio interactions will be key to the safe design of MOFs as a platform for cancer treatment.

2. MOFs-based biomineralization of proteins

Protein-based pharmaceuticals (Pps), a kind of high-molecular weight therapeutic substance (>1kDa), have emerged as dominant tools (including cytokines, therapeutic antibody, protease and protein vaccines) in the treatment of various cancer 45. Compared with traditional small molecule chemotherapeutics, Pps have tunable properties, increased therapeutic efficacy and reduced systemic toxicity 46-50. Since rituximab was approval for lymphoma target therapy in 1997, therapeutic proteins have become a fast-growing category among cancer therapeutic drugs 51-62. Data from various La Merie financial reports indicate that total sales of Pps in 2017 reached $188 billion, and the rapid growth will continue over the next several years.

The predominance of protein-based biopharmaceuticals is likely to remain among the anticancer drugs in the foreseeable future. For example, monoclonal antibody is one of the most successful anticancer biopharmaceuticals, which has been developed and applied to treat various cancers (e.g., breast cancer, colorectal cancer, lung cancer, et al.). As the fastest growing segment of the biopharmaceutical market, protein-based biopharmaceuticals have significantly extended the lives of many cancer patients. However, proteins are xenobiotics that can elicit adverse immune reactions 6, and may lose their biological activity or even be degraded at low pH and near proteolytic enzymes in the tumor microenvironment 63-65. With the help of advanced delivery nanosystems, bioactive proteins can be efficiently delivered to the tumor tissues/cells for targeted anticancer theranostics.

The encapsulation of protein in MOF architectures provides a novel strategy for its efficient loading without concerning the size of the biomolecules 41, 66-67. The co-precipitation during the self-assembly procedure of the MOFs precursor solution containing the protein is a one-pot embedding. Furthermore, the addition of the protein also promotes the rapid growth of MOFs. To date, the study of the MOFs biomineralized proteins has attracted the researchers a wide range of interests, and the applicability of this strategy has demonstrated toward different proteins and MOFs (Table ).

Recently, Liang et al. reported a simple approach to encapsulate bovine serum albumin (BSA) and horseradish peroxidase (HRP) into MOFs by de novo assembly 11. This is especially useful for biomineralization through the crystal growth of the zeolitic imidazolate framework-8 (ZIF-8) using Zn(II) ion and 2-methylimidazole (2-MIM). The controlled crystal formation enabled each BSA to be encapsulated with ~22 Zn(II) ion and ~31 2-MIM; the protein encapsulation efficiencies were 82% to even near 100% for different proteins. More importantly, the encapsulated proteins retained bioactivity at elevated temperatures, low pH, and organic solvents, leading to improved outcomes.

In another example, Feng et al reported the preparation of Antibody@MOFs by a facile biomineralization procedure 68. The in vitro self-assembly reaction could protect the antibodies in the framework, and the antibodies could release within 10 s. After encapsulated by the ZIF-90 or ZIF-8, the Human immunoglobulin G (IgG) polyclonal antibody (H-IgG) and Goat anti BSA IgG polyclonal antibody (G-IgG) could show few aggregations in the harsh condition including high temperature (75 °C), methanol or acetone, freeze-thawing cycles (-80 °C~37 °C) and high pressure (20 Mpa). Furthermore, the adalimumab (Ada) was biomineralized by ZIF-90 or ZIF-8 with high encapsulation efficiencies, and high recover efficiencies. After treated with 5 °C heat or 5 days storage plus 10 freeze-thawing cycles, the protected Ada could recover high bioactivities.

The good protection effect was quite an advantage for the protein@MOFs to adapt the complex tumor environment, and showed potential application to the cancer treatment. The protein@MOFs must possess good biocompatibility and low immunological risks 69-73. In addition, most of the early MOFs-based biomineralization of protein used micron-sized carriers display limitations in vivo applications (Table ) 22, 74. With the development of the MOF preparation, the size, Zeta potential, and biocompatibility of some protein-encapsulated MOFs could match the needs of nanomedicine. In 2018, Chen et al. synthesized BSA@ZIF-8 NPs with a size of 92 ± 7.9 nm. The encapsulation efficiency was 93%, and the BSA loading capacity was ∼52.2 μg in 1 mg BSA@ZIF-8 NPs—these were useful for intracellular studies 75. The BSA delivery system was also extended to other proteins and even multiple proteins in one single ZIF-8 NPs for co-delivery with high loading capacities; only the size changed. This might be due to the different pre-nucleated clusters surrounding the proteins.

The protein-encapsulated ZIF-8 NPs was further coated with a layer of polyvinylpyrrolidone (PVP) to enhance its stability in cell media (Figure ). The BSA@MOF hybrid could decompose in acidic buffer (pH = 5.5), and the BSA was released within 0.5 h and completed after 2 h. Next, the fluorescein (FITC)-labeled BSA was incubated in the ZIF-8 NPs, and the resulting MOF hybrid offered much higher FITC fluorescence than the pure BSA in the HeLa cells, implying that the PVP-coated MOF hybrid improved the delivery efficiency of protein. In addition, the cell fluorescence study of the early endosomes and lysosomes indicated that the endocytic pathway accounted for the MOF hybrid. Notably, the ZIF-8 NPs could be decomposed in the weakly acid environment of early endosomes and lysosomes. The encapsulated protein was then released into the cytoplasm. The development of the MOF hybrid could enable the proteins delivered to the live cell and escaped from the endo-lysosomes to avoid degradation/denaturation. Thus, the biomineralized MOF designed with this rationale holds great potential for cancer theranostics.

(1) Therapeutic proteins

Nowadays, many therapeutic proteins were applied in the cancer therapy. However, the systemic delivery of therapeutic proteins to target cancer sites is impeded by many factors including rapid degradation and systemic elimination of 'naked' proteins in biological systems. A variety of delivery nanosystems have been invented to deliver therapeutic proteins to target cancer tissues 76-79. For example, the tumor necrosis factor-related apoptosis inducing ligand (TRAIL) could induce the apoptosis of cancer cells sparing normal tissue 80-83. To effective delivery the TRAIL ligands, Lin et al applied a micellar hybrid nanoparticle to carry TRAIL (IPN@TRAIL) 6. The IPN@TRAIL could localize to the tumor tissues, and the TRAIL therapeutic efficiency enhanced by TRAIL nano-vectorization.

The MOFs-based biomineralization delivery system was also investigated for cancer-associated theranostic proteins. Caspase 3 could induce the death of cells 84-86, and prior work co-encapsulated caspase 3/HSA in ZIF-8 NPs with a further PVP coating 75, 87. When the HeLa cells were treated with caspase 3/HSA@MOF hybrid, the caspase 3 could be delivered to the cancer cells, and the cell plasma membrane was damaged. Further cell viability studies suggested that the caspase 3/HSA@MOF hybrid had obvious cytotoxicity that varied with concentration; no cytotoxicity was found for the HSA@MOF hybrid.

In another example, Cheng et al. also encapsulated protein using the biomineralization of ZIF-8 (Figure ) 88. With the modified procedure, the encapsulation efficiency of the MOF-protein (MP) was 94%, and the protein loading capacity was as high as 41%. This was 3 to 50 times that of surface conjunction or adsorption loading. The biomineralized MOFs retained their nanostructure in PBS (pH = 7.4) but degraded in acidic buffer (pH = 5.0) after 1 h with the cargo releasing. Previous studies have demonstrated that extracellular vesicle (EV) could enhance blood circulation time, reduce phagocyte uptake, and preferentially gather in the homotypic tumor area 89-93. These authors coated EVs on the surface of MP nanoparticles to increase the physiological solution stability and then used them for cancer therapy. In MDA-MB-231 cells, the pure gelonin showed low protein transduction efficiency (∼1.27%), but the MP nanoparticles obviously promoted the transduction efficiency (∼43.7%). Furthermore, after coated with EV, the prepared EV-MOF-protein (EMP) enabled the transduction efficiency increased to ∼77.5%, far higher than the liposome-enveloped MP nanoparticles. An in vivo study with MDA-MB-231 tumor-bearing mice demonstrated that the multifunctional MOFs could accumulate in the tumor area within 24 h post-injection (iv). The mice treated with EMP nanoparticles could effectively inhibit the tumor growth than the other groups and the collected tumors were less than the other groups, implying the good protein delivery effect of the prepared biomineralization system.

Protein-MOFs possess the following advantages: i) The biomineralized MOFs have an extremely high protein loading capacity relative to the surface modification; ii) The MOFs protect therapeutic protein from enzyme degradation in the circulation and tumor microenvironment, and maintained the bioactivity in the cancer cells; iii) The MOFs can degrade in the weakly acidic solution, leading to the encapsulated proteins being released from the MOFs structure in the acidic endo-lysosomes. The biomimetic MOF system needed minimum dosage of the therapeutic proteins and showed good cancer therapy. This was a significant platform for other therapeutic proteins leading to clinic applications of the protein-based nanomaterial.

(2) Antigen and Antibody

Cancer immunotherapy-based antibodies and/or antigens are important in cancer treatment 94-100. Nano-carrier gave vaccines more effective immune response than pure protein antibody/antigen 101, 102. The antigen-presenting dendritic cells (DCs) can present the danger signal to T-lymphocyte and generating the CD4+ and CD8+ T cells, resulting of the cancer immunotherapy and induce the “immune memory” effect 103, 104. The nano-carrier can “programming” the activation state of DCs and induce the cellular and humoral immunity 105. In studies of antigen-induced immunotherapy, ovalbumin (OVA) is a common model antigen 106, 107. In 2012, Irvine's group used lipid-coated poly(lactic-co-glycolic acid) (PLGA) NPs as efficient carrier of OVA, and combined this with the adjuvant of the nano-vaccine to elicit three orders of magnitude higher serum anti-OVA IgG titers versus pure soluble OVA 108.

The biomineralized MOF system is also a good potential carrier of the protein antigen as first described by Qu et al 109. The OVA was encapsulated into the ZIF-8 NPs with a loading capacity of 7%, and then the cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) was adsorbed on the surface of OVA@ZIF-8. Afterwards, in the group of RAW264.7 cells treated with OVA@ZIF-8-CPG, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) had much higher expression versus the other groups, implying the enhanced immunostimulatory activity of the nanocomposites. Later, an in vivo immune response of OVA@ZIF-8-CPG was studied after the nanocomposites subcutaneously injected into Kunming mice. The anti-OVA IgG titers were much higher than the OVA/CPG mixture. In addition, the CD4+ T and CD8+ T cells after OVA@ZIF-8-CPG treatment were also higher than the OVA/CPG mixture group. These results implied that the antigen-biomineralized MOFs possessed superior loading capacity, efficient delivery, and pH-responsive release for antigen-presenting cells. This led to a strong humoral immune response. A good cancer immunotherapeutic effect was achieved by antigen-biomineralized MOFs.

Moreover, tumors may evade immune destruction by endogenous “immune checkpoints” and terminate the antigen-activated immune responses, the presence of immune inhibitory receptors in T cells 99. So, the immune-checkpoint-pathway inhibitors such as anti-PD-L1 antibody, anti-PD1 antibody or anti-CTLA-4 antibody can recover the immune responses and develop cancer immunotherapeutic approaches 110-113. The delivery of the antibody with an efficient strategy and controllable release of antibodies can spare the essential dose of the antibody and decrease the cost of treatment 114. The antibody biomineralized MOFs could protect the protein against various severe environment, we assume that the antibody biomineralized MOFs can act as prominent delivery strategy of the immune-checkpoint-pathway inhibitors and feature powerful clinical potency in cancer immunotherapy.

(3) Enzyme

For enzyme-induced cancer treatment, glucose oxidase (GOx) could catalyze glucose resulting in the production of gluconic acid and hydrogen peroxide (H2O2) 115-117. The rapid growth of the tumor was dependent on the glucose supply in the cancer cells, and the starvation therapy could eliminate tumors by the reduction of glucose via GOx 118-121. In addition, as the hypoxia enhanced, the therapeutic effect of hypoxia-activated prodrugs could increase, and the synergy effect was achieved 122, 123. Also, the generated H2O2 could produce the hydroxyl radical through the Fenton reaction 124, 125, or oxidize L-Arginine (L-Arg) with a production of NO, resulting of the starvation synergistic cancer therapy 126. In recent years, the biomineralization of enzyme has been extensively investigated to enhance the enzymatic activity and extend the enzyme's application environment 42, 74, 127.

In 2018, Qu's group reported an enzyme@MOF hybrid for the starvation-activated therapy 128. Through a self-assembly procedure (Figure ), the GOx and prodrug tirapazamine (TPZ) were encapsulated in the ZIF-8 MOF, and the erythrocyte membrane was further coated on the surface of the MOF hybrid to enhance the tumor accumulation and blood circulation 129-136. The loading amounts of GOx and TGZ were ~10 wt % and 13.2 wt% respectively, which were high enough for the further study. The GOx and TGZ were released into cytoplasm due to the acidity of the lysosome/endosome environment. The GOx could consume the endogenous glucose and O2 to starve the cancer cells. Concurrently, a hypoxic microenvironment was enhanced and thus the TPZ was transformed into highly cytotoxic radical leading to synergistic therapy.

The TGZ@eM showed high cytotoxicity against CT26 cells than the other groups implying the combined treatment effect. The hypoxia-inducible factor-1α (HIF-1α) staining assays of CT26 multicellular tumor spheroids (MCTS) after incubation with different nanostructures implied the O2 reduction effect of GOx. A LIVE/DEAD kit was used after the TGZ@eM treatment to study cell cytotoxicity, and this was confirmed in the MCTS. The GOx in the TGZ@eM nanoreactor deprived the glucose and O2 in the cancer cells resulting in starvation therapy and the hypoxia-induced chemotherapy. When studied in vivo of CT26 tumor-bearing mice, the TGZ@eM group showed satisfactory therapeutic outcomes, implying the synergistic effect of the GOx-based starvation therapy and the activated TPZ therapy.

Willner's group co-immobilized insulin/GOx or anti-vascular endothelial growth factor aptamer (VEGF aptamer)/GOx in ZIF-8 NPs (Figure ) 137. When the biomineralized MOFs were immersed in the glucose solution, the glucose was transformed to gluconic acid and the acidity of the solution increased. Afterwards, the ZIF-8 nano-mixture was decomposed, and the co-encapsulated insulin or VEGF aptamer was released. And, in the PBS (pH = 7.4), the ZIF-8 nano-mixture stained nanostructure without any change. In a further study, the release of insulin or VEGF was accelerated with the increasing of glucose concentrations. Glucose selectivity studies showed that only glucose could induce the release of insulin or VEGF. The glucose in angio-epithelial cells is higher than the normal cells 138-139, and the VEGF aptamer could inhibit the proliferation and migration of the epithelial cells 140-142. Thus, the VEGF aptamer/GOx ZIF-8 NPs could respond in the epithelial cells, and the glucose was reduced along with release of the VEGF aptamer. The VEGF aptamer/GOx ZIF-8 NPs showed potential applications in cancer therapy. In the tumor tissues, the reduction of glucose by GOx might damage the cancer cells and epithelial cells via starvation; the released VEGF aptamer could inhibit angiogenesis.

Willner's group also demonstrated that two- or three enzyme cascades possessed better catalytic activity than the enzyme mixture in solution because the MOF nanoreactors provided the enzymes with intercommunication on the nanoscale 143. The GOx and horseradish peroxidase (HRP) were encapsulated into ZIF-8 NMOFs, and the enzyme cascade was activated with the addition of glucose. The biocatalytic activity of the obtained two-enzyme cascade was 7.5-fold enhanced than the GOx/HRP homogeneous mixture. The β-galactosidase (β-Gal)/GOx/HRP three-enzyme cascade was also evaluated, and the biocatalytic activity 5.3-fold enhanced than the β-Gal/GOx/HRP homogeneous mixture.

MOFs that possessed stable nanostructure and enhanced enzyme catalytic activity in the endo-lysosome or cytoplasm might be another approach to the cancer treatment 144-148. We assume that enzyme biomineralized MOFs can be applied to cancer therapy in two ways: (i) the enzyme-MOFs decompose in the tumor microenvironment and thus the enzyme show catalytic effects combine with therapy of the other released drug (the MOFs decomposed due to the catalytic effect and the co-encapsulated drug was released); (ii) the enzyme-MOFs show enhanced catalytic effect in tumor tissues and influence the endo-biochemical system leading to the therapeutic effect.

3. MOFs-based biomineralization of DNA/RNA

The cancer gene therapy based on the RNA or DNA has attracted significant attention in the past two decades 149-152.The RNA interference (RNAi) has been applied to the sequence specific silencing of target messenger RNA (mRNA), and thus decrease the suppression of gene and protein, resulting of the gene therapy 153-154. And, the synthetic short interfering RNAs (siRNAs) and plasmid DNA (pDNA) are the most widely studied therapeutic drugs for the RNAi 155. However, these gene drugs are large biomacromolecules, cannot efficiently delivered to the tumor tissues and cross the cell membrane to cytoplasm, and in the serum can be degraded by the nuclease. The current major strategies to delivery RNA or DNA are delivery nano-vehicles, including various cationic materials such as liposomal, polymeric formulations and inorganic nanoparticles 156. There are some problems for these common delivery systems, such as suboptimal transfection effect and the potential hemolytic effect 157.

MOFs also can be applied in gene delivery system 158-159. Mirkin and the other researchers recently prepared DNA-MOFs using click chemistry to conjugate the dibenzocyclooctyne-functioned DNA (DNA-N3), azide-functioned MOFs (MOFs-N3) 160,161, or coordination chemistry between the phosphate of DNA and the external metal nodes (Zr, Hf, Fe) of MOFs 162-164. Lin and other researchers loaded the RNA on the surface of MOFs via metal ion connections or physical absorption 33, 164.

However, the complicated modification of DNA/RNA and the lack of release stimuli-environmental limit its application to gene therapy. Coating the DNA/RNA with non-toxic inorganic shells can leave out the DNA/RNA modification and extend the delivery system while keeping the length of the DNA/RNA unconstrained. In addition, the biomineralization can prolong the shelf life time and offer possibilities for clinical applications of genomic drugs 11.

Recently, calcium phosphate (CaP) has been used to coat living agents due to its good biocompatibility, stability, non-immunogenicity, and pH-responsive decomposition in the tumor tissues 165. And, the CaP can be used as the biomineral shells of the DNA/RNA via the interfacial interactions between calcium and phosphate ions, and the DNA/RNA can release due to the pH in the tumor cells 166, 167. But the traditional biomineralization coating was critical to their surface, and direct biomineralization of the genetic macromolecules was difficult to realize. However, Liang et al. encapsulated DNA using ZIF-8 with a high encapsulation efficiency of 75% implying that the MOFs could be potentially applied as an effective bioactive DNA carrier 11.

Khashab et al. first reported RNA-biomineralized MOFs 168. The site-specific gene editing CRISPR/Cas9 platform was incorporated with a Cas9 protein and single guide RNA (sgRNA) for cancer treatment 169-171. The co-delivery of the Cas9 protein or mRNA and sgRNA was necessary to translate this into the internal environment. To co-encapsulate the Cas9 protein and sgRNA, these authors encapsulated the CRISPR/Cas9 platform with ZIF-8 (Figure ) and the loading capacity was 1.2 wt% with an encapsulation efficiency of 17%. The pH-responsive nature of the ZIF-8 enabled the endosomal escape of the CRISPR/Cas9@ZIF-8 and the release of the gene editing platform. When the enhanced green fluorescent protein (eGFP) Chinese hamster ovary cells were treated with the eFGP editing CRISPR/Cas9@ZIF-8 nano-composites, the eGFP was reduced after 2 or 4 days, which was far more efficient than the free CRISPR/Cas9. The CRISPR/Cas9@ZIF-8 could also be potentially applied to cancer immunotherapy; this protein and RNA co-biomineralization strategy showed guidance for further RNA or protein-RNA delivery via MOFs.

Aside from the in situ biomineralization method, Zhou et al. presented a precise DNA loading method inner the isoreticular MOFs (NiIRMOF-74) 172. By changing the length of the organic linker, the authors prepared NiIRMOF-74-II to -V with 2.2 to 4.2 nm pore sizes. The pore of the NiIRMOF-74 provided a host-guest interaction to the NiIRMOF-74 and single-stranded DNA (ssDNA). This enabled precise loading of the ssDNA inside the pores. Upon addition of the complementary DNA (cDNA), the ssDNA was released due to the interaction between the ssDNA and cDNA (Figure ). The loading capacity was up to 6.9 wt%, which is far higher than other MOFs. The ssDNA loaded in NiIRMOF-74 could maintain 95% survival after incubation in FBS for 24 h, while the other porous nanostructures had low ssDNA survival, implying the good protection of NiIRMOF-74 vectors. When the MCF-7 cells were treated with 33-nucleotide ssDNA (DNAzyme)-loaded Ni-IRMOF-74-II, it inhibited 76% EGR-1 gene expression. The precise DNA loading rate and excellent transfection efficiency showed good potential in cancer gene therapy.

4. Other biomineralization

Other than the biomacromolecule, various living bacteria and virus also have been applied in the cancer treatment. Even though the living bacteria and virus are promising in cancer treatment, sometimes the biomedical applications are limited by the host immune antiviral effect and inefficient tumor accumulation 173. Artificial biomineralization of the living bacteria or virus agent can solve the problem of living agent-based cancer treatment 174. By coating the living agent surface with a mineral layer or encapsulating several living agents into one mineral nanostructure, the living agent can accumulate in the tumor.

Biomineralization using CaP can be realized through the interactions between calcium ions and amino acids on the surface of the bacteria or virus, followed by the addition of phosphate ions 174, 175. However, the certain amino acids on the surface of the living agents limited the universality for the strategy, and the morphology of the mineral hybrids need further study to meet the clinical research standard. As the core-shell nanostructure with MOF shell was studied, and combine with the study of the MOFs-based biomineralization, the various MOFs can also be applied in the biomineralization of the living agents.

(1) Bacteria

Nowadays, the bacteria especially the salmonella has been applied as antitumor agents due to its tumor preferentially amplifying and even 1000 times than the normal tissues 176, 177. The salmonella can express the prodrug-converting enzymes, such as herpes simplex thymidine kinase. In addition, the salmonella induces the production of the Tumor Necrosis Factor-α (TNF-α) from immune cells, and thus lead to the immune killing of cancer cell. With the development of the bacterial adjuvants, the applications of bacteria in the cancer treatment have gained much research interest 178-181.

The Yang's group attempted the biomineralization of anaerobic bacteria using MOF based on the interaction between the Zr4+ and the phosphate units on surface of the bacteria 182. As shown in Figure , the Zr6O4(OH)4(BTB)2(OH)6(H2O)6 clusters (BTB = 1,3,5-benzenetribenzoate) was coated on the surface of Moorella thermoacetica (M. thermoacetica) in all its life cycle. And, when treated with the ROS, the viability of M. thermoacetica-MOF was obvious higher than the pure the M. thermoacetica, implying the good protection effect of the coated MOF shell. The efficient coating of the bacteria using MOFs and the protection effect could enable the application of the bacteria@MOFs in cancer treatment. In addition, with the rational design of the organic ligands, the bacteria@MOFs could be prepared to release in the tumor microenvironment or under the external excitation, and thus the bacteria showed therapeutic effect.

(2) Virus

In addition, oncolytic viruses (OVs) including poxvirus, paramyxovirus, reovirus, and picornavirus are the most widely studied among virus immunotherapy 183, 184. OVs are genetically modified to selectively infect the cancer cells and replicate in the cells regardless of the normal cells. The preclinical OVs have effective antitumor effect 185. Viral gene delivery systems include adenoviruses (Ads) and lentiviruses. The Gassensmith's group used tobacco mosaic virus (TMV) as a template-fabricated TMV@MOF using ZIF-8 (Figure ) 44, 186-187. The prepared TMV@MOF had a tuned nanostructure via changes in the synthetic conditions. Meanwhile, the surface of the TMV@MOF could also be conjugated with organic molecules or biomolecule via chemical reactions. Later, this group demonstrated that a high zinc concentration near the TMV could catalyze the growth of ZIF-8 on the surface of the TMV. This would benefit the other virus-MOF core-shell systems.

It was supposed that the OVs can be encapsulated into MOFs during direct self-assembly (Figure 68. And, the surface modification enhances the targeting effect and biocompatibility of the OVs@MOF. Unlike protein or DNA/RNA, the OVs are more difficult to store and transport. MOFs-based biomineralization gives the OVs@MOF a longer lifetime after assembly versus free OVs. In addition, the targeting modified OVs@MOF hybrid (OVs@MOF-T) is more suitable for cancer treatment. The OVs@MOF-T can specifically accumulate in the tumor tissues more efficiently regardless of the immunogenic effect of OVs. Subsequently, OVs are released from the endosomes due to the pH-response MOF disassembly, achieving significant therapeutic efficacy in cancer treatment.

5. Conclusions and outlook

MOFs-based biomineralization is useful in biological catalysis or nanomedicine. The biomineralization of protein, DNA/RNA, or viruses can be done with MOF encapsulation. Biotherapy for cancer with this approach has shown progress—the MOFs can be loaded with cancer-associated biomacromolecules. The biomacromolecules maintain their biological activity regardless of the extreme tumor microenvironment and delivery via MOF protection.

For application of the biomineralization towards cancer therapy, the MOF should possess the following properties: (i) the MOF synthetic conditions should be moderately facile, and the MOF should interact with the biomacromolecules and protect them from the surroundings; (ii) the size, biological stability, and biosecurity of the MOF should meet the requirements for cancer treatment nanomedicine; (iii) the MOF should be responsive to the tumor microenvironment and thus the encapsulated biomacromolecules or living agents released. Currently, the preparation method for the MOFs-based biomineralization is mostly limited to the ZIF-8 or ZIF-90, and the organic bridging ligands still have in vivo toxicity. However, better understanding of nano-bio interactions to exploit MOF-architectures for cancer treatment can be adapted or modified to improve their performance and dose efficiency—this reduces patient exposure to the therapeutics.

In summary, the application of a MOF-biomineralized therapeutic protein, antigen/antibody, enzyme, and DNA/RNA leads to improved cancer treatment efficacy. Their properties allow for unique opportunities in modulating key components of the healthy organism system such as the inherent activation of immune cells and functional delivery of biomacromolecules as well as biosensing and monitoring of treatment response. The field of MOF-based cancer treatment combines researchers from multiple subdisciplines of material science, nano-chemistry, and nano-biological interactions, immunology, and medicinal chemistry. This leads to new opportunities to synergistically complement and improve MOF-based cancer nanomedicine.

Table 1

Summary of preparations and applications of the protein-biomineralized MOFs.

MOF	Protein	Size	Application	Ligand	Metal ion	Temperature	Time	Ref.
ZIF-8	BSA, HSA, OVA, HRP, insulin, hemoglobin, lysozyme	1-5 μm	Bioprocessing or delivery	2-methylimidazole (BSA)	Zn2+ (BSA)	Rt (BSA)	12 h (BSA)	22
ZIF-90	catalase	~1.5 μm	Biocatalysis	Dithiodiglycolic acid ( disulfide )	Mn2+	150 ℃	24 h	74
ZIF-8	GOx	>1 μm	Biocatalysis	2-methylimidazole	Zn2+	4 ℃	12 h	127
NU-1003	organophosphorus acid anhydrolase (OPAA)	300 nm (minimum size)	Biocatalysis	benzoicacid	Zr4+	80 ℃	1 h	145
ZIF-8	urease	120-500 nm	Bioprocessing	2-methylimidazole	Zn2+	Rt	Over-night	146
NPCN-333	tyrosinase	100 nm	Biocatalysis and cancer therapy	4,4',4''-s-triazine-2,4,6-triyl-tribenzoic acid (TATB)	Al3+	95 ℃	24 h	147
ZIF-8	insulin/GOx, GOx/HRP or β-Gal/GOx/HRP	~ 500 nm	Biocatalysis	2-methylimid azole (insulin)	Zn2+ (insulin)	/	Over-night	137, 143
ZIF-8	BSA (DOX)	70-110 nm	Cancer drug delivery	2-methylimidazole	Zn2+	/	10 min	148
ZIF-8	OVA	~ 200 nm	Cancer immunotherapy	2-methylimidazole	Zn2+	Rt	10 min	109
ZIF-8	BSA	53-153 nm	Bioprocessing for cancer treatment	2-methylimidazole	Zn2+	30 ℃	20 min	75
ZIF-8	BSA, cytochrome c or gelonin	< 100 nm	Bioprocessing for cancer treatment	2-methylimidazole (BSA)	Zn2+ (BSA)	/	50 min(BSA)	88