The Progress and Prospect of Zeolitic Imidazolate Frameworks in Cancer Therapy, Antibacterial Activity, and Biomineralization

The progressive development of zeolitic imidazolate frameworks (ZIFs), as a subfamily of metal-organic frameworks (MOFs), and their unique features, including tunable pore size, large surface area, high thermal stability, and biodegradability/biocompatibility, have made them attractive in the field of biomedicine, especially for drug delivery and biomineralization applications. The high porosity of ZIFs gives them the opportunity for encapsulating a high amount of therapeutic drugs, proteins, imaging cargos, or a combination of them to construct advanced multifunctional drug delivery systems (DDSs) with combined therapeutic and imaging capabilities. This review summarizes recent strategies on the design and fabrication of ZIF-based nansystems and their exploration in the biomedical field. First, recent developments for the adjustment of particle size, functionality, and morphology of ZIFs are discussed, which are important for achieving optimized therapeutic/ theranostic nanosystems. Second, recent trends on the application of ZIF nanocarriers for the loading of diverse cargos, including anticancer medicines, antibiotic drugs, enzymes, proteins, photosensitizers, as well as imaging and photothermal agents, are investigated in order to understand how multifunctional DDSs can be designed based on the ZIF nanoparticles to treat different diseases, such as cancer and infection. Finally, prospects on the future research direction and applications of ZIF-based nanomedicines are discussed.


Introduction
Unprecedented advantages of nanomaterials have introduced them in recent decades as a potent platform in biomedicine, DOI: 10.1002/adhm.202000248 which can play a vital role in the therapy and diagnosis of many diseases. [1] For example, biomedical applications of porous materials, such as metal-organic frameworks (MOFs) have extensively been explored with the aim of fabricating novel drug formulations with better biological performance as compared to conventional medicines. [2][3][4] Zeolitic imidazolate frameworks (ZIFs) are a subfamily of MOFs with desirable properties, including high porosity, excellent thermal and mechanical stability, tunable surface properties, and exceptional chemical stability as a result of high resistance to alkaline water and organic solvents. [5] These features have made ZIFs excellent candidates for many applications, such as gas capture, [6] separations, [7] chemical sensors, [8] drug delivery, [9] and catalysis. [10] From a structural point of view, ZIFs are constructed by coordination between M 2+ cations and imidazole (Im) anions, in which Im acts as a linker to create connecting bridges among the metal centers of M(Im) 4 tetrahedral units. [5,11] ZIFs are mainly prepared by solvothermal methods in both organic [5,12] and aqueous solutions. [13] Linker modifications or encapsulation of guest species (e.g., nanoparticles (NPs)) within ZIFs have been commonly employed to control the functionality of ZIFs. In addition, the pore size of ZIFs is simply tunable that, in turn, results in adjustable molecular diffusion/mass transfer and loading of large cargoes. [14,15] This unique property has greatly expanded the application of ZIFs in catalysis science and drug delivery. [16,17] The crystal size and morphology of ZIFs can also be controlled by the type of solvent and metal salt during the synthesis process, the ratio of metal salt to Im linker, the mixing order of the ZIF precursors, and the addition of surfactants. [16] The stability of ZIFs under physiological conditions and their pH-dependent degradability under acidic conditions make this type of MOFs highly attractive for the construction of pH-responsive drug delivery systems (DDSs). [17,18] As a result of the mild acidic condition of the tumor microenvironment (TME), ZIFs, especially ZIF-8, have been extensively investigated as nanocarrier both in vitro and in vivo for cancer ablation. [9,[19][20][21] Furthermore, plenty of research efforts have been devoted to designing novel multifunctional ZIF-based composites both in cancer therapy [22][23][24][25] and also in antimicrobial applications, [26][27][28] bioimaging, [29][30][31] and theranostics (Scheme 1). [24] In the case of ZIF-8, as one of the main subgroups of ZIF nanomaterials, its versatile applications is mainly due to the facile polymerization of Zn 2+ and 2-methylimidazole (2-MeIm) around various objects, including drugs, [19,32,33] NPs, [34][35][36][37][38] and bio-macromolecules [39,40] to render them multifunctionality while preserving structural crystallinity and porosity of ZIF shell. [39,41] It deserves to be pointed out that there are many excellent reviews summarizing the state of the art of the development of MOFs and their applications for therapeutic, imaging, and sensing purposes. [4,17,[42][43][44][45][46][47] However, there is no comprehensive literature review with a focus on the biomedical applications of ZIFs. [11,21,48] In this review, we discuss the synthesis and functionalization of ZIFs and then highlight the most recent progresses on DDSs developed by this type of porous nanostructures for chemotherapy (CT), photothermal therapy (PTT), photodynamic therapy (PDT), antimicrobial applications, development of theranostic nanomedicines, and biomimetic mineralization (Scheme 1). In addition, recent remarkable advances on ZIF-derived nanocomposites with particular emphasis on the multifunctional property of the nanocomposites in the treatment and diagnosis of hard-to-treat diseases are discussed, along with addressing critical challenges and perspectives of these materials. We expect that this review can boost the knowledge of the research community on the potential of ZIFs in biomedicine and its further exploration to introduce novel nanomedicines. Scheme 1. An overview of the ZIF-based DDSs for biomedical applications. ZIF NPs can be used for the loading of the various molecules or the fabrication of nanocomposites. For example, polymers or mesoporous silica NPs (MSNs) can be used for the preparation of core-shell nanosystems.
These porous nanostructures have been prepared by hydrothermal or solvothermal methods in organic solvents, such as dimethylformamide (DMF) [5] and methanol, [12] or even in aqueous solutions [13] with reaction temperatures ranging from room temperature up to 200°C, and reaction times from hours to days. [50] Although hydrothermal/solvothermal methods are commonly used to prepare ZIF-based nanostructures, which are simple and easy, scaling-up of such methods is hard and yield of the products is low. [49] To overcome the shortcomings, sonochemical [54] and mechanochemical [55] methods have been used to increase the yield of ZIF production. Nevertheless, due to a high variety of controllable parameters, including synthesis routes, concentration, and molar ratio of reactants, reaction temperature, solvents, and reaction time, there is a long way to industrialize the production of ZIF-8 and other members of the large family of ZIFs. In addition, the synthesis factors for the scaleup production of ZIFs are so far only partially investigated. [49] Green and sustainable production of ZIFs under mild synthesis conditions and the use of nontoxic solvents [13] and solvent free methods [56,57] are very crucial from environmental protection point of view. For example, in 2017, a fast and scalable method for the synthesis of hierarchical ZIFs, i.e., ZIF-8 and ZIF-67, and one-pot encapsulation of dyes or proteins cargoes using an organic base trimethylamine (TEA) was reported by Zou and co-workers [58] The addition of TEA into the solution of Zn(NO 3 ) 2 ·6H 2 O promoted the formation of ZnO NPs, which rapidly transformed to ZIF-8 NPs after the addition of the 2-MeIm as the linker.
It should be noted that among the family of ZIFs, ZIF-8 is one of the main members that has been intensively used in many applications, such as adsorption, catalysis, electrochemical energy storage, gas separation, drug delivery, sensing, and electronics. [11,21] It has a sodalite-type framework with Zn 2+ linked by 2-MeIm containing cages of 11.6 Å in diameter, which are accessible through a narrow six-ring pore (3.4 Å), creating an intrinsic porosity, which subsequently renders very large surface area of 1630−1700 m 2 g −1 to the ZIF-8. [5] As a result of the ultrahigh thermal stability (stable up to 550°C under an inert atmosphere), ZIF-8 is capable of maintaining its original structure in boiling water/organic solvents for 7 days, and even it is stable in 8 m of NaOH (aq) at 100°C for 24 h. [5] The functionality of ZIF-based nanomaterials can be controlled by different strategies, including the modification of linker or encapsulation of guest species, such as diverse drugs, imagining agents, metal and metal oxide NPs or biomolecules within ZIFs. [11,59] As an example, Lu et al. [38] reported a controlled encapsulation strategy to incorporate various surfactant-capped NPs with different sizes, shapes, and compositions into the ZIF-8 NPs. Interestingly, by adjusting the time of NP addition during the ZIF formation, the spatial distribution of the NPs within ZIF-8 crystals was tunable. Figure 1 shows how adjusting the addition time and sequence (at the beginning (T 0 ) or after a certain time (T) during the growth process of ZIF-8) can affect the spatial distribution of polyvinylpyrrolidone (PVP)-modified Au nanoparticles (AuNPs) inside the ZIF-8 crystals. A similar concept has been applied for the fabrications of various core/shell nanocomposites as nanotheranostic systems to simultaneously diagnose and treat hard-to-treat diseases that have been discussed in the next sections. [60,61] It is also possible to coat the surface of ZIFs with stimuliresponsive gatekeepers to precisely control the release of encapsulated drugs in response to pH, light or reducing agents to improve therapeutic efficiency of ZIFs. [21] Ren et al. developed a pHand redox-responsive DDS with doxorubicin (DOX)-loaded ZIF-8 as core and disulfide-doped organosilica as the shell. [62] The degradation of organosilica shell was confirmed via the breaking down of S-S bond in the presence of tripeptide glutathione (GSH) as a reducing agent, which is at elevated concentrations in the cytoplasm of tumor cells. In vivo studies showed that this DDS exhibited negligible hemolytic toxicity and significantly enhanced therapeutic efficiency against tumor growth compared to the free DOX. This work also stands as a pioneering strategy that opens new opportunities in design and fabrication of multifunctional and stimuli-responsive DDSs based on ZIFs in cancer treatment and diagnosis.
Although flexibility, structural diversity, mechanical and thermal stability, as well as control over shape and size of the ZIFs have held great promise in many applications, microporous pores of ZIFs unfavorably restrict fast molecular diffusion and mass transfer, thus limiting their utilities in catalysis, drug delivery, and other applications. [11] Therefore, the fabrication of mesopores or hierarchical porosity (a mixture of micro and mesopores) in ZIFs would markedly expand their applications as hosts to accommodate larger bulky molecules via quick diffusion inside the porous structures. [63][64][65][66][67][68][69] For example, hierarchical ZIF-8 NPs based on surfactant-amino acid co-templating The spatial distribution of the NPs within ZIF-8 crystals can be controlled by their addition sequence (i.e., addition at the beginning (T 0 ) or after a certain time (T) during the ZIF-8 synthesis). Spatial distributions of i) a single NP in the central areas ii) or off the central areas of the ZIF crystals, iii) two types of NPs in the central areas, or iv) one type in the central area but the other type in the transition layers of the ZIF crystals. B) The TEM image of the intermediate product of AuNP/ZIF-8 crystals collected after 3 h of reaction (case (i) in (A)). C) Hybrid crystals obtained when AuNPs were introduced 15 min after the initiation of the reaction (case (ii) in (A)). D) The hybrid crystal that consists of homogeneously distributed 13 nm Au and 34 nm AuNPs in the central area, prepared by simultaneously adding of two types of the NPs at the beginning of the reaction (case (iii) in (A)). E) Hybrid crystals that contain 34 nm AuNP-rich cores, 13 nm AuNP-rich transition layers and NP-free shells obtained by sequentially adding 34 nm AuNPs at the beginning of the reaction and 13 nm AuNPs after 40 min (case (iv) in (A)). Reproduced with permission. [38] Copyright 2012, Springer Nature. strategy were prepared in aqueous solution by Wu et al. [68] They showed that cetyltrimethylammonium bromide (CTAB), as structure-directing agent to form micelles, was not inducing the generation of mesoporous structure, because the hydrated zinc ions were unable to produce strong interactions with the micelles. In contrast, in the presence of histidine aminoacid, the interaction between the ZIF-8 precursors and CTAB micelles was mediated by the amino acid, thus promoting the formation of ZIF-8 network, which had mesopores interconnected by micropores. Encapsulation of NPs within ZIFs and subsequent etching is another strategy to create hierarchical macro-microporous structures. [67,69] Based on this, polystyrene spheres (PSs) as sacrificial NPs, which can dissolve in the methanolic solution, were utilized to synthesize macro-microporous ZIF-8 with cheese-like morphology. The concentration and addition time of the encapsulated PSs were detrimental in controlling the ratio of the www.advancedsciencenews.com www.advhealthmat.de macropores. The introduction of the NPs into the ZIF matrix did not change the well-defined crystal structure of the synthesized ZIF-8. [69] For many biomedical applications, the crystal size and morphology of ZIFs are detrimental factors. [4,11,70] The type of solvent, [53,71] metal salts, [72][73][74] the ratio of metal salt to 2-MeIm, [75] the mixing order of the ZIF precursors, [74] and the addition of surfactants or capping agents [76] have been reported as important factors to control the crystal size of ZIFs. [16] It has been shown that the reactivity of the zinc salt in the growth solution has had a significant impact on the size of the ZIF-8 crystals. [73,74] Small size ZIF-8 crystals (≈50-200 nm) have been harvested by using reactive zinc salt precursors, including zinc(II) acetylacetonate [Zn(acac) 2 ], zinc nitrate (Zn(NO 3 ) 2 ), zinc sulfate (ZnSO 4 ) or zinc perchlorate (Zn(ClO 4 ) 2 ), while nonreactive salts, such as zinc chloride (ZnCl 2 ), zinc acetate (Zn(OAc) 2 ) or zinc iodide (ZnI 2 ) produced ZIF-8 crystals with larger sizes ranging from ≈350 to 650 nm. [72] This was mainly due to the nucleation rate of ZIF crystals, which was controlled by the coordination speed between the metal ions and the 2-MeIm linker. [74] In fact, the solvation of reactive salts (e.g., Zn(NO 3 ) 2 ) in methanolic solution is relatively weak. Therefore, the Zn 2+ ions can be rapidly coordinated by 2-MeIm, leading to fast nucleation and subsequent generation of small-sized NPs. Pan et al. [76] used the surfactant of CTAB as a capping agent to control the size of ZIF-8 nanocrystals. The particle sizes were precisely tuned from ≈100 nm to 4 µm by tuning of the CTAB concentration. This was attributed to the CTAB molecules that were adsorbed on the surface of ZIF-8 crystals in the growth solution and acted as capping agents to prevent further crystal growth.
In order to control the morphology of ZIF NPs, two strategies, i.e., nontemplated and template-mediated methods, have been developed. [11] In the nontemplated methods, different parameters, such as solvent, metal ion to 2-MeIm ratio, and reaction time have been determinant parameters in the morphology of ZIF NPs. [77][78][79][80][81] For example, ordered hierarchical ZIFs with nest-like morphology were prepared using a mixed solvent of methanol (CH 3 OH) and aqueous ammonia (NH 3 ·H 2 O). [77] The architectures with diameters of ≈2-3 µm were formed by self-assembly of numerous nanoplates with a thickness of ≈20-40 nm. Such structures were not produced in the presence of a single solvent. This study demonstrated that the type of solvent had a vital effect on the morphology of ZIFs. In contrast to the template-free approach, the template-mediated methods employ soft (e.g., CTAB [76] ) or hard templates (e.g., PSs [82] ) to control the crystal morphology of ZIF particles. CTAB, as a capping agent, was not only used to control the crystal size of ZIF-8, [76] but also showed that the increase in its concentration changed the crystal morphology from rhombic dodecahedron to truncated rhombic dodecahedron and then to truncated cubes. In fact, the CTAB molecules were adsorbed much more strongly on {100} faces of ZIF-8 than the other two faces, i.e., {110} and {111}, leading to the decrease of growth rate on the {100} face, thereby resulting in the transformation of crystal morphology. [76] Similarly, Hu et al. [83] obtained ZIF-67 nanocubes instead of the commonly observed polyhedrons in the presence of CTAB as a morphological modifying agent. Again, the formation of the new morphology was attributed to the preferential adsorption of CTAB on the hydrophobic surfaces.
Given that hollow metal-organic frameworks (HMOF) have hierarchical porous structures, accessible metal sites, and rapid mass transport property, these types of nano/microstructures have received great attention in many research fields, including catalysis, gas sensors, etc. [84] Generally, three main synthetic strategies have been developed for the construction of HMOFs, including the exterior-templating approach (sacrificial template fabrication technique), the self-templating method, and the twophase interface method. There is a comprehensive review article describing the synthesis of HMOFs and their derivatives using the above-mentioned approaches. [84] Inspired by HMOFs, hollow ZIFs (HZIFs) have also been constructed utilizing the same strategies. For example, different types of HZIFs have been produced by the sacrificial template fabrication technique, in which the ZIF growth on the surface of a core template was followed by a removing step to eliminate the internal template. [82,[85][86][87] In addition, it is imperative to functionalize the surface of the template prior to the ZIF growth process to enhance interaction between the ZIF precursor and the template surface and also to inhibit the competitive homogeneous self-nucleation of ZIFs in the solution. For example, carboxylate-terminated PSs have been utilized as a template for the construction of core-shell PS@ZIF-8 composites, followed by removing the PS cores using DMF as an etchant to obtain HZIF-8 microspheres. [85] This promising strategy will create great opportunities for fabricating novel HZ-IFs with diverse morphologies and unique properties in the near future.
In the self-templating method, which is carried out in the absence of any exterior template, the generated intermediate products during ZIF synthesis can act as a template and direct the formation of HZIFs. [84] In order to have better control over the formation process of HZIFs via the self-templating method, it is necessary to perform a surface protective modification on the intermediate products prior to the subsequent transformation reaction. [84] For example, Hu et al. [88] prepared HZIFs using phenolic acids (PAs), i.e., gallic acid and tannic acid (TA). They demonstrated that PAs not only acted as the surface protecting agents, but also served as an etching agent to create HZIF-8 without destroying the crystallinity of the parent ZIF. In fact, the outer shell of the ZIF-8 was protected by the PAs that blocked the exposed surface of ZIF-8 and the inner part was etched by free H + ions released by the PAs. [89] The HZIFs produced heat upon irradiating to the near-infrared (NIR) light (808 nm), potentially endowing them with photothermal therapy capability. [89] In the two-phase interface method, which is more convenient, the interface of two different phases is employed as a template for growing of ZIF at the interface. This method is classified into three categories: liquid-liquid, solid-liquid, and gas-liquid interface systems, which all have been reported for the synthesis of HZIFs. [66,90,91] Although, there are successful achievements on controlling the pore size, diameter, morphology and surface properties of ZIFs, the reaction parameters have not been systematically optimized and there is no clear guideline for controlling the particle diameter, pore size, and morphology. In addition, most of the current studies have focused only on the ZIF-8. Therefore, further studies are needed on the physicochemical properties of other ZIFs rather than ZIF-8, because the developed techniques are still at an infancy stage. In this context, reproducibil-Adv. Healthcare Mater. 2020, 9,2000248 ity, cost-effective preparation and environmental considerations of the synthesis methods must receive more attention in future studies.

ZIF NPs for pH-Responsive Therapy
The design and construction of novel DDSs have been grown in recent years with central attention devoted to new methods for developing stimuli-responsive DDSs. [92,93] Among different types of stimuli, such as temperature, electric field, light, and magnetic field, [94] pH-sensitive DDSs have been the main core of numerous investigations to develop responsive nanosystems, specifically for cancer therapy goals. [92] This is mainly due to the pH of tumor tissue (pH 5.5-6.0), which is more acidic than the blood and normal tissues (pH 7.4). Such nanosystems have been employed to transport toxic chemotherapeutic drugs to cancer site via blood circulation with minimum undesired drug release before reaching the cancer tissue. [92,95] However, many of the systems suffer from low drug-loading capacity, poor biocompatibility, undesirable biodegradability, and complicated synthesis procedures. As a result of the unique properties of MOFs, this type of porous materials has attracted great attention for achieving a controllable drug release. [4,17] Generally, the encapsulation of drug of interest into MOFs involves several steps, including synthesis of MOF carriers, removal of solvents from their pores, and finally loading of the cargos, which all together make the whole process costly and complicated. [96] Furthermore, the small pore apertures of MOFs limits the entering of large cargoes within the network of MOF. [42,44,97] Recently, ZIFs have successfully been used as drug carriers in the construction of varied DDSs that can load a range of cargos from small molecule drugs to large bio-macromolecules, such as enzymes and proteins for pH-responsive drug delivery ( Table 1). [9,18,21,98] In this section, we provide the most significant signs of progress in the development of simple ZIF-based DDSs in cancer therapy, antibacterial applications, and biomineralization. ZIFs loaded by a cargo, and/or in some cases functionalized by small molecules and polymers, e.g., polyethylene glycol (PEG), are discussed. The components, structures, and properties of ZIFs are highlighted in different examples. In addition, the impact of the ZIF framework, especially the degradation of the network in acidic environments, is discussed along with benefits and critical challenges of each system. In contrast, Section 4 has a focus on the complex multifunctional DDSs derived from ZIF-based composites, integrated/functionalized by some metal NPs, nanorods, mesoporous silicas, quantum dots, polymers, or graphene to form core@shell or hybrid structures for various biomedical applications.

Applications of ZIF NPs in Cancer Therapy
For the first time, Sun et al. [9] demonstrated that ZIF-8 could be used for the delivery of anticancer drugs in vitro. Remarkable loading of 660 mg of 5-FU in 1 g of ZIF-8, and pH-triggered controlled release of the drug, which was much faster in acidic condition (pH 5) than neutral one (pH 7.4), were observed, suggesting ZIF-8 as an excellent pH-sensitive DDS. [9] However, as the pore window of ZIF-8 is 3.4 Å, this structural feature limits the entrance of large molecules inside the pores, thus leading to low cargo loading within the pores and burst release, as a result of drug adsorption on the surface of the particles. [37,119] In order to tackle this hurdle, a simple process that combined ZIF synthesis and cargo encapsulation in a one-pot manner was reported by Zheng et al. [100] In this process, the anticancer drug DOX and three organic dyes, were successfully encapsulated within ZIF-8 and ZIF-67 with high cargo loading (14-20 wt%). Firstly, the metal ion and dye/drug molecules self-assembled to form coordination polymers. After the addition of the organic linkers, the coordination polymers disassembled, and thus, the subsequent generation of ZIF network caused to encapsulating of target molecules within the ZIF hosts. The drug/dye-loaded crystals possessed hierarchical pore structures containing ordered micropores and homogeneously distributed mesopores filled by the guests. Interestingly, hierarchical micro and mesoporous ZIF-based structures were harvested by the removal of the organic drug/dyes from the pores. DOX-loaded-ZIF-8 showed pH-responsive release behavior, in which the drug was not released under physiological condition (pH 7.4), while the release of the drug occurred in a controlled manner at lower pH values of 5.0−6.5. Cytotoxicity assays on breast cancer cell lines showed that the DOX@ZIF-8 had higher toxicity than that of free DOX. [100] In a different study on the controlled release behavior of DOX-loaded into ZIF-8 and ZIF-7, it was shown that ZIF-8 released the drug in a more controlled manner than ZIF-7 under acidic condition, demonstrating that ZIF-7 was more stable at acidic pH than the ZIF-8 carrier, highlighting the effect of ZIF type in the design of controlled DDSs. [20] Despite excellent advantages of ZIFs, low dispersity, cytotoxicity, and aggregation of the MOFs under physiological condition, nonactive targeting capability, and concerns related to its favorable biocompatibility have seriously limited their in vivo applications. [102,120,121] In order to overcome these limitations, several studies have been demonstrated the surface functionalization of the ZIFs by folic acid (FA)-PEG, [108,110] hyaluronic acid (HA), [105] phenolic lipid, [120] peptide, [122] and phospholipid bilayer. [102] HA, a targeting ligand of the CD44 receptor, is overexpressed in many growing tumor cells. This ligand was attached on the surface of curcumin (CCM)-loaded ZIF-8 through coordinative interaction to promote the cellular uptake of NPs. The surface modification endowed the nanocomposite (CCM@ZIF-8/HA) with active targeting ability and enhanced its biocompatibility. The CCM@ZIF-8/HA showed enhanced dispersity in phosphate buffer saline (PBS) and a longterm pH-responsive drug release behavior and successfully delivered CCM into cancer cells. [105] Efficient autophagy inhibition was observed by chloroquine (CQ), as autophagy inhibitor, -loaded ZIF-8 (CQ@ZIF-8) after functionalization by methoxy FA-PEG. Compared to healthy cells (HEK293 cells), the cancer cells (HeLa cells) showed lower viabilities when treated with FA-PEG/CQ@ZIF-8 NPs, demonstrating targeting ability of FA and inhibiting the process of autophagy flux in the cancer cells, as well as the formation of autophagosome more effectively than the free CQ and CQ@ZIF-8. [108] www.advancedsciencenews.com www.advhealthmat.de  Adv. Healthcare Mater. 2020, 9,2000248 www.advancedsciencenews.com

www.advhealthmat.de
Surface chemical functionalization of ZIFS with safe materials can increase their biocompatibility; however, it might reduce pore sizes or even block the pores. Zhao and co-workers, used a mechanical ball-milling method to develop a surface defection strategy on the external surface of ZIF-8 to tune the hydrophobichydrophilic balance of ZIF-8, resulting in significantly higher cell viability without decreasing its cargo loading and release capacity. [123] In the mechanical ball-milling method, unsaturated Zn-sites and N-sites were created on the external surface of ZIF-8, leading to the binding of H 2 O molecules on the surface and generating a hydrophilic surface that significantly improved cell viability of ZIF-8.
Horcajada and co-workers evaluated the cytotoxicity of a series of MOF NPs on two cell lines (J774 and HeLa) by the MTT assay and indicated that the cytotoxicity was strongly depended on the MOF composition since Fe-based MOFs exhibited less toxicity than the Zn-or Zr-MOF NPs. [121] Recent toxicological investigations suggested that toxicity of Zn was related to the high solubility of Zn 2+ cations. [124] Therefore, the research group proposed that ZIF-8 NPs were progressively degraded into Zn 2+ and 2-MeIm in cell culture media containing phosphates ions and in the acidic endosomal environment. The high toxicity of ZIF-8 was related to the competition of the released/dissolved Zn 2+ with Fe 2+ and Ca 2+ ions through ion channels and/or DNA damage. [121] These observations highlighted the importance of surface modification of the ZIFs to overcome the toxicological concerns.
A series of pharmacokinetics and biodistribution studies of ZIF-8 NPs were conducted by Zhang and co-workers to understand biofate of the NPs within the body. [70] After intravenous administration to rats (32 mg kg −1 ), the serum zinc concentration steadily declined over time and elimination half-life was 3.6 h with a clearance of 0.187 L h −1 g −1 . Low accumulation of Zn was detected in tissues, including the kidney, heart, brain, and testis (up to ≈25 µg g −1 ). In contrast, ZIF-8 NPs were captured much more by reticuloendothelial system (RES) organs (up to ≈210, ≈35, and ≈32 µg g −1 in the lung, liver, and spleen, respectively). Unexpected accumulation of Zn in the lung was probably due to the particle size of the NPs. However, after 7 days, the concentration of Zn in the RES tissues decreased dramatically, suggesting the fast degradation and elimination of the NPs. In addition, around 65% and 12% of the NPs were eliminated by feces and urine within 7 days, respectively. Interestingly, due to the high pulmonary accumulation and fast elimination of the NPs, the 5-Fu-loaded ZIF-8 NPs had a significant therapeutic effect on a tumor lung metastasis bearing mouse model. Significant accumulation of ZIF-8@DOX in the lung was also reported by Cheng et al., which can be used for the chemotherapy of lung cancer. [125] This phenomenon was related to the aggregation of the NPs in the blood circulation, leading to the entrapment of the nanosystem in abundant capillaries of the lung. Interestingly, after coating of the NPs with 4T1 cancer cell membrane, accumulation in the lung was remarkably decreased, highlighting importance of the biomimetic coating in enhanced biocompatibility of the ZIFbased DDSs.
Although there are extensive studies on the applications of ZIFs in cancer diagnosis and treatment, the most attention has mainly focused on ZIF-8. In order to exploit other members of the ZIF family, Jiang et al. [126] synthesized nanoscale ZIF-90 with a negative zeta-potential, which exhibited better cell biocompatibility, mitochondria targetability, and in vivo survival rate compared to the ZIF-8 NPs with positive zeta-potential. In addition, in vivo toxicity studies confirmed the excellent biocompatibility of ZIF-90 and minimal side effects on the renal and liver functions. The surface of DOX-loaded ZIF-90 NPs was surface-modified by Y1 receptor ligand [Asn 6 , Pro 34 ]-NPY (AP) to realize targeted adenosine triphosphate (ATP) and induce in vivo pH-responsive triple-negative breast cancer treatment. ATP-responsive property of the nanosystem was due to the stronger coordination between ATP and Zn 2+ than the Im and Zn 2+ , enhancing the therapeutic efficacy of the nanosystem in the killing of cancer cells. Naturally, the concentration of ATP in the tumor cells is ≈100 times higher than the normal cells, allowing the targeting of ATP for cancer therapy. [127,128] Multidrug resistance (MDR) is one of the important hurdles inhibiting effective cancer chemotherapy. [129] This phenomenon mainly originates from either an acquired resistance in cancer cells by the stimulus of anticancer drugs to overexpress ATP-binding cassette (ABC) transporter (e.g., p-glycoprotein (pgp)) or intrinsic high expression of ABC transporter proteins. This overexpressed transporter can efflux the chemotherapeutic drugs from the cytoplasm of cancer cells to reduce their accumulation within the cells, resulting in an extremely low therapeutic outcome. [130,131] To circumvent MDR, the combination of small molecule anticancer drugs or chemosensitizers with macromolecular therapeutic genes, in a single DDS is an efficient way to treat cancer. [130] As a result of the inherent biocompatibility and pH-degradability of ZIF-8, this porous material was employed to construct a co-delivery nanosystem containing DOX and verapamil (VER) to overcome MDR in tumor cells for achieving efficient in vivo cancer therapy. [33] The (DOX+VER)@ZIF-8 was further functionalized by methoxy poly FA-PEG to avoid aggregation of (DOX+VER)@ZIF-8 particles, and thus, prolonging its blood circulation as an active DDS (Figure 2A). VER, as a p-glycoprotein inhibitor, could reverse the drug resistance associated with p-glycoprotein, resulting in increased local concentrations of DOX in MDR tumor cells and improving the efficiency of the formulation. Transmission electron microscope (TEM) images showed that PEG-FA/(DOX+VER)@ZIF-8 particles had uniform spherical-like morphology and the diameter of the NPs was determined to be 185 nm with narrow size distribution ( Figure 2B). For both DOX and VER, faster drug release in acidic condition (pH ≈ 5.0) compared to the neutral release medium (pH ≈ 7.4) was observed from the (DOX+VER)@ZIF-8 NPs, confirming the degradation of ZIF-8 in the acidic environment and drugs release acceleration. Cytotoxicity assay on B16F10 and MCF-7/A cells showed that the inhibition rates of PEG-FA/(DOX+VER)@ZIF-8 were higher than that of free drug of DOX and DOX@ZIF-8 NPs ( Figure 2C). This fact was attributed to the targeting property of the folate ligand and the role of VER in overcoming the drug efflux mediated through the overexpressed p-gp in MDR cancer cells, i.e., MCF-7/A cells. Fluorescence inverted microscopy showed that PEG-FA/(DOX+VER)@ZIF-8 NPs have a higher intensity of red fluorescence than (DOX+VER)@ZIF-8 NPs in MCF-7 cells, confirming the specific binding between folate ligand and overexpressed FA receptors on the cancer cells. In agreement with fluorescence inverted microscopy, flow cytometry analy- sis proved that the cellular uptake of (DOX+VER)@ZIF-8 NPs into p-gp-overexpressed MCF-7 cells was higher than free DOX and DOX@ZIF-8, demonstrating the inhibitory effect of VER on drug efflux induced by p-gp. As shown in Figure 2D, different formulations were studied in vivo. Among all the formulations, the mice treated by PEG-FA/(DOX+VER)@ZIF-8 NPs exhibited the best tumor inhibition effect. This behavior was also confirmed by the tumors dissected and photographed after the last injection ( Figure 2E), ascribed to the pH-responsive property of the NPs, active targeting capability via FA and the MDR reversal mediated by VER. Altogether, this DDS broadened the applications of ZIFs in the biomedical field and was an efficient formulation in reversing the MDR for targeted cancer therapy goals. www.advancedsciencenews.com www.advhealthmat.de

Antibacterial Applications of ZIFs
MOFs have extensively been used as bactericidal agents through the release of antimicrobial metal ions (e.g., Ag + , Zn 2+ , and Co 2+ ) or antimicrobial agents from their framework. [43] It has been shown that particle size, shape, zeta-potential and chemical properties of NPs have a significant impact on their antibacterial activity. [132,133] As particle size, morphology, and composition of the MOFs are tunable, there is a great opportunity to treat various microbial infections by adjusting their physicochemical properties, a unique benefit that is difficult to achieve by conventional antibiotics. [27,43] Although MOFs have huge surface area and high porosity, their small pore size limits the loading of large molecules via post-synthetic methods. The access to MOFs containing large pore apertures is challenging yet and needs complicated synthesis procedures. [15,64,[134][135][136] As mentioned above, ZIFs can encapsulate many organic/inorganic cargos via growing around the guests, while maintaining the functionality of the cargos. [100] ZIFs have been successfully employed to encapsulate antibiotics, such as ciprofloxacin, [137] gentamicin, [138] physcion, [112] ceftazidime, [111] and vancomycin (Van) [114] for antimicrobial therapy. For example, Song et al. [26] prepared 2-nitrobenzaldehyde (o-NBA) and rifampicin (RFP)loaded ZIF-8 (o-NBA-RFP@ZIF-8) through a one-pot method for light-controlled antibacterial therapy. Under UV-light treatment (365 nm), o-NBA was converted to 2-nitrosobenzoic acid, thus serving as pH-jump reagent to generate an acidic environment and degrade the ZIF-8 network, leading to burst release of RFP antibiotic and Zn 2+ and, ultimately, resulting in synergistic antibacterial therapy to inhibit bacteria-induced wound infection and promoting wound healing ( Figure 3A). In the presence UVlight, TEM images proved the pH-dependent degradation of o-NBA-RFP@ZIF-8 NPs ( Figure 3B). Light-triggered antibiotic release studies revealed no drug release from RFP@ZIF-8 NPs in the absence of o-NBA ( Figure 3C). In contrast, a maximum of 80 wt% of RFP was released within 150 min upon UV-light irradiation of the o-NBA-RFP@ZIF-8 NPs, confirming pH dependency of the drug release by in situ light-mediated acid generation ( Figure 3C) and subsequent ZIF network dissolvation and, thus, pH decreasing and production of Zn 2+ ions in the release medium ( Figure 3D,E).
As shown in Figure 3F, in vitro antibacterial assays showed that o-NBA-RFP@ZIF-8 had higher antibacterial activity against both the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative ampicillin-resistant Escherichia coli than the other samples under UV-light treatment. This was attributed to the synergistic effect between the antibiotic and Zn 2+ ion released from the o-NBA-RFP@ZIF-8. In vivo antibacterial efficacy was evaluated by MRSA and ampicillin-resistant E. Coli-induced wound infection model on the back of BALBc mice. As shown in Figure 3G, the wound size using o-NBA-RFP@ZIF-8 as a treatment agent under UV-light irradiation decreased much faster (80%) than the other groups. In the group treated with the o-NBA-RFP@ZIF-8 + light, well-established collagen fibers and dermal layer formation was confirmed by Masson's trichrome staining, demonstrating high efficacy of the synergistic treatment to eradicate the infection, promote derm generation, and modulate the collagen alignment, thus leading to accelerated wound healing. [26]

ZIFs for Biomimetic Mineralization
Biomimetic mineralization (bio-min) with MOFs is an emerging and robust strategy to shield sensitive biological materials toward enhanced thermal stability, protection against denaturing proteolytic agents/mechanical stress/UV radiation and extended shelf-life time. [41,[139][140][141][142][143] Using this strategy, many encapsulated bio-macromolecules including enzymes, [39,41,[144][145][146][147][148][149][150][151][152] proteins, [40,[153][154][155][156][157][158][159] vaccines, [160][161][162] antibodies, [163] or even more complex living systems [139,[164][165][166][167] (e.g., bacteria, viruses, and eukaryotic cells) have been made with enhanced bioactivity and stability. For the first time, a rapid and low-cost bio-min method was introduced by Liang et al. [143] as a protective approach for the coating of bio-macromolecules. They showed that a wide variety of bio-macromolecules, including enzymes, proteins, and DNA could successfully be encapsulated within MOFs framework. The resulting biocomposites were stable and kept their activity even after exposure to harsh conditions. The change of pH was used to control the release of the cargos from the MOF-coated biomacromolecules. For the bio-min process, ZIF NPs, especially ZIF-8, have been shown to be a superior candidate to protect biomacromolecules under the mild condition of the biomineralization process. [41] For example, the bio-min of ZIF-8 can be performed within a few minutes in aqueous solutions and under ambient temperature without using toxic organic solvents. [158] However, despite many examples available on bio-min of diverse biomacromolecules, the mechanism of such bio-min has not fully been investigated. [143] Enzymes are efficient and environmentally benign catalysts that can selectively catalyze different biological processes to produce various chemicals and pharmaceutically active medicines. [168] However, their unfavorable stability against heat and organic solvents and undesirable recyclability have limited the use of enzymes toward practical applications. [41] Therefore, the immobilization of enzymes within a porous matrix is a straightforward and efficient method to solve the above-mentioned problems and improve the performance of enzymatic reactions. [41,141] ZIFs have been able to encapsulate various enzymes, such as lipase, [144,169] horseradish peroxidase, [145,147] catalase, [38,148,149,170] glucose oxidase, [39,[145][146][147] chloroperoxidase, [146] lysozyme, [143] cytochrome c, [156] andgalactosidase, [147] to render them high stability. In this condition, the porous shell allowed substrates to diffuse through the pores and undergo catalysis by the caged enzyme, while keeping the inlaid enzyme from denaturation by various chemicals. It should be added that two or even three enzymes have been simultaneously embedded in ZIF NPs, acting as nanoreactors in biocatalytic cascades transformations. [145][146][147] In 2018, Chen et al. [39] fabricated two smart glucose-responsive DDSs based on ZIF-8 NPs. In the first system, glucose oxidase (GOx) enzyme and insulin protein were loaded within ZIF-8 NPs in a one-pot method, while the second nanosystem was composed of anti-vascular endothelial growth factor (VEGF) aptamer and GOx co-encapsulated into the ZIF-8 NPs. In the presence of glucose, GOx-mediated aerobic oxidation of glucose to H 2 O 2 /gluconic acid and subsequent creation of an acidic microenvironment occurred. As a result, insulin and VEGF were released from the particles, known as acid-degradable sense-andtreat systems. Both of these DDSs were potentially suggested as  "intelligent" carriers for the therapy of diabetes and macular disease, respectively. In the case of controlled release of insulin from the insulin and GOx-coloaded ZIF-8 (IG@ZIF-8) NPs, loading of the two cargos was performed in a one-pot method, as shown in Figure 4A. Glucose acted as a key player to control the local pH at the nanoenvironment of the IG@ZIF-8 NPs. In fact, the local acidic nanoenvironment created by the conversion of glucose to gluconic acid via the biocatalytic effect of GOx led to the degradation of the IG@ZIF-8 and, in turn, induced the release of insulin. In the confocal laser scanning microscopy images of the ZIF-8 NPs loaded with FITC-labeled insulin (green) and the coumarinfunctionalized GOx (blue) ( Figure 4B, panel I and panel II, respectively), the overlapped turquoise color was related to the internal integration of the two cargos in the ZIF-8 NPs ( Figure 4B, panel IV), confirming the successful co-immobilization of the proteins within the porous ZIF. Scanning electron microscope (SEM) image of the IG@ZIF-8 NPs with rhombic dodecahedral shape and particle size of ≈300−350 nm, are shown in Figure 4C panel (I). This morphology was retained when the IG@ZIF-8 NPs were treated with the buffer solution without glucose (Figure 4C, panel III). Interestingly, after treatment with glucose solution (50 × 10 −3 m), the corrosion of the protein-loaded ZIF-8 occurred, proving the degradation of the IG@ZIF-8 due to local acidic nanoenvironment, generated by the GOx-mediated oxidation of glucose ( Figure 4C, panel II). Release study of FITClabeled insulin from the IG@ZIF-8 showed that when the concentration of glucose increased, the release of the insulin was accelerated for a fixed time of 1 h, again confirming the glucosedependent degradation of the IG@ZIF-8 to control the insulin release ( Figure 4D). In addition, among different saccharides, only glucose triggered the release of insulin from the IG@ZIF-8 ( Figure 4E). A switchable ON/OFF release of insulin was also observed when an increase/decrease of the glucose concentration was applied ( Figure 4F). All these observations clearly proved that the intelligent controlled DDS of insulin could potentially be developed for the treatment of diabetes. The same results were ob-www.advancedsciencenews.com www.advhealthmat.de tained when VEGF aptamer and GOx were encapsulated within ZIF-8 (VG-ZIF-8). The authors showed that the release of VEGF aptamer was glucose-dependent in the VG-ZIF-8 NPs, which was consistent with the increased local acidity at the aptamer-loaded ZIF-8 due to the GOx-catalyzed conversion of glucose to gluconic acid, and thus, degradation of ZIF-8 matrix and accelerated release of the VEGF aptamer. These results hold a great promise for the potential application of the VG-ZIF-8 as an "intelligent" system for the inhibition of VEGF-induced angiogenesis in macular disease. [39] Recently, GOx and hemoglobin encapsulated-ZIF-8 NPs were prepared in which GOx and hemoglobin acted as nutrient starvation and radical generation agents respectively, endowing the nanosystem anti-cancer property in vitro. [171] Although great results were obtained in the in vitro phase of this study, the in vivo studies in animal models were missing and seems to be imperative in future studies with similar concept.
Cell membrane coating nanotechnology has emerged as a promising approach to facilitate the delivery of therapeutic agents through the camouflagation of NPs within a layer of a specific cell membrane. Utilizing such bio-inspired technology could enhance the colloidal stability of the NPs and minimize their nonspecific tissue accumulation. [172,173] Using distinctive advantages of neutrophils, including inflammation targeting ability and antitumor/antibacterial properties, artificial super neutrophils were constructed by encapsulating two enzymes, i.e., GOx and chloroperoxidase (CPO) into ZIF-8 NPs, and further coverage of neutrophil membrane (NM) on the surface of GOx/CPO-caged ZIF-8 NPs. It has been shown that the neutrophil-mimicked nanosystem produced seven-fold higher reactive hypochlorous acid (HClO) than the natural neutrophils through GOx/CPOmediated cascades reactions for eliminating tumors and infections. [146] The encapsulation of vaccines into MOFs, i.e., ovalbumin and attaching the cytosine-phosphate-guanine oligodeoxynucleotides (CPGO) in/on ZIF-8 (OVA/CPGO-ZIF-8), was carried out by Quʼs research group. [161] Their results showed that the OVA/CPGO-ZIF-8 had pH-responsive property, efficiently enabling the system to release the antigen and CPGO in the same antigen presenting cells, inducing potent humoral and cellular immune responses. This study sheds light on producing novel/effective MOF-based vaccines against a range of ailments.
In addition to the embedding of a wide variety of drugs, enzymes, proteins, and vaccines, living cells [165] and viruses [164,167] were used to encapsulate/cage within the ZIF frameworks. [139] For the first time, biomimetic mineralization of tobacco mosaic virus (TMV) using ZIF-8 precursors as a robust MOF for encasing and protecting the virus against foreign denaturing environmental stressors was investigated by Gassensmithʼs research group. [167] They showed that discrete rod-shaped TMVencapsulated ZIF-8 with good uniformity could be obtained. They also demonstrated that the metal concentration and ligand to metal molar ratio affected distinct morphologies of the core−shell composites and the size of ZIF-8 NPs, which greatly influenced the stability of the core-shell hybrid particles. [164] On the encapsulation of living cells, i.e., yeast, it has been shown that mechanical constraints imposed by the ZIF-8 coating prohibited the living cells from reproducing, while cell metabolic processes were maintained due to the microporous shell of ZIF-8 that allowed the transfer of small molecules like oxygen and glucose to the cell. Interestingly, this biomimetic mineralization, i.e., the ZIF-shell, was easily be removed by mildly acidic pH or ethylenediaminetetraacetic acid (EDTA), leading to a "switching-OFF" effect and the recovery of the full functionality of the encapsulated cells or bio-macromolecules. [143,165]

Biomedical Applications of Multifunctional ZIF-Based Composites
As mentioned in the introduction section, ZIF-based composites, especially the ones prepared by ZIF-8, have extensively been applied in many biomedical applications, including cancer therapy [22][23][24][25] and antimicrobial purposes. [26][27][28] Nevertheless, the poor water disparity of ZIF-8 has limited its advanced applications. [174] On the other hand, the integration of functional materials (e.g., metal NPs/nanorods, mesoporous silicas, photosensitizers (PS), light-absorbing dyes, quantum dots, polymers, graphene, proteins, and enzymes) with ZIFs is a necessity to combine the merits of both the components, including the flexibility and high porosity of ZIFs with unique imaging, targeting properties and dispersiblity of functional materials (Scheme 1). Thus, the unique features of the composites, resulting from the synergistic combination of both ZIFs and other active components, have made them as highly attractive hybrid materials for many biomedical applications, which are not attainable with an individual component. Table 2 summarizes the recent biomedical applications developed by ZIF-based composites. In this section, we have classified the applications of ZIF-8 into three categories of monotherapy, combined therapy, and theranostics applications, and discuss them in detail.

Monotherapy by ZIF Nanocomposites
Chemotherapy using nanoscale DDSs is one of the most effective monotherapeutic approaches in cancer ablation in vivo. [208,209] As discussed in the previous section, ZIF-8 intrinsically possess the properties of large surface areas, high porosities, and well-defined structures, making this material capable of loading and releasing different chemotherapeutic agents. [21] However, its small pore size (3.4 Å) and poor dispersity in aqueous solution have limited its wide application in chemotherapy. Therefore, many attempts have been made on the design and fabrication of hybrid structures to overcome these problems. [21] Thorough a facile and simple method, polyacrylic acid@ZIF-8 (PAA@ZIF-8) NPs were fabricated by Ren et al. [37] with ultrahigh DOX loading capability (1.9 g of DOX g −1 composite), successfully employed as a pH-dependent DDS in cancer treatment in vitro. As shown in Figure 5A, PAA@ZIF-8 composite was prepared by ionexchange between Zn 2+ and Na + on the surface of poly(acrylic acid sodium salt) (PAAS) NPs followed by dropping the resultant NPs into a methanol solution of 2-MeIm to form the hybrid PAA@ZIF-8 nanostructure, showing an particle size of 128 nm measured by SEM ( Figure 5B). The NPs were highly dispersed in serum and water with no obvious aggregation, confirming their good stability and dispersity. As a result of the pHdependent degradability of the nanosystem, a faster drug release rate was observed in acidic medium (pH 5.5) rather than www.advancedsciencenews.com www.advhealthmat.de  [199] (Continued) Adv. Healthcare Mater. 2020, 9,2000248 www.advancedsciencenews.com www.advhealthmat.de at the neutral pH of 7.4 after 60 h ( Figure 5C). The 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) cell assay on MCF-7 cells showed that DOX-loaded PAA@ZIF-8 composite had similar toxicity with the free drug at different loadings. The results also confirmed that the PAA@ZIF-8 NPs were nearly nontoxic to live cells, proving the biocompatibility of the NPs ( Figure 5D). Although excellent results were obtained in vitro, the assessment of the developed NPs in vivo was not performed to identify its translational potential.
www.advancedsciencenews.com www.advhealthmat.de In 2017, the same nanocomposite was prepared by Yan et al. [34] and successfully applied as vectors to deliver DOX for anticancer therapy in mice model, greatly enhancing drug therapeutic efficacy. Toxicity assessments, including hematoxylin and eosin staining analysis on tumor and major organs, blood chemistry tests, and hematology analysis, confirmed that the nanocomposites were highly biocompatible.
As mentioned in the sections above, the MDR is one of the major challenges in the treatment of cancer chemotherapy. Codelivering chemotherapeutic drugs and MDR gene silencing siRNAs paves a rational and effective way to circumvent the MDR shortcomings in conventional cancer chemotherapy. [210][211][212] In 2018, Pan et al. [35] in situ synthesized an ultrathin ZIF-8 film on carboxylated mesoporous silica (MSN-COOH) surface and prepared a novel DDS for dual delivery of siRNAs and DOX. The mesoporous channels of MSN-COOH acted as a reservoir to load DOX drug and the positively charged ZIF-8 shell was exploit for efficient loading of siRNA via electrostatic interactions and, in turn, protected siRNA from nuclease degradation. The microporous shell also acted as pore blocker to overcome premature release of DOX from the mesoporous silica channels. In vitro studies demonstrated that the positively charged ZIF-8 film promoted the uptake of the dual drug-loaded NPs in MDR cancer cells of MCF-7/ADR and SKOV-3/ADR. In fact, decomposition of the ZIF thin layer in the acidic endo-lysosome, and subsequently, the intracellular release of siRNAs and chemotherapeutic DOX, significantly enhanced chemotherapeutic efficacy. Although in vivo studies were not investigated, this study highlighted the power of ZIF-8 as a pore blocker of porous materials and an excellent platform to adsorb/control the delivery of siRNAs and its protection against nuclease degradation. [35] As a result of the dissolution of ZIF-8 in acidic media and EDTA, various hollow structures, including hollow TiO 2 , [213] polyphenol-metal networks, [214,215] ZnS nanocages, [216] and hierarchical-pore MOFs [136] have been successfully synthesized. Size and morphology-controllable hollow MSNs (HMSN), including cubic and dodecahedral morphologies ranging from ≈80 to ≈3000 nm in particle size, were fabricated by acid-degradable ZIF-8 as a self-sacrificial template (Figure 6A). [192] After encapsulating the HMSNs with DOX (HMSN@DOX) and subsequent coating of HMSN@DOX with ZIF-8 layer as pore blocker, the resulting pH-responsive DDS showed remarkable efficiency in killing SMMC-7721 cancer cells (human liver cancer cell lines) ( Figure 6B,C).
As above-mentioned, the pore diameter of ZIF-8 is very small (3.4 Å), which is even smaller than most of the anticancer drugs. [14] Such small pores impede the high drug loading potential of NPs, and subsequently hinder highly efficient chemotherapy. [100,217] In 2018, Zhao and co-workers [181] constructed ZnO@ZIF-8 nanocomposites containing mesoporous  [192] Copyright 2017, The Royal Society of Chemistry.
ZnO core and ZIF-8 shell. In the mesoporous/microporous composite, the ZnO core acted as drug carrier to easily load anticancer drug, DOX, at the mesoporous channels of ZnO core and the microporous shell served as a cap to prevent premature release of chemotherapeutic DOX at physiological environment. In addition, the ZnO core not only functioned as a drug carrier, but also used as a Zn ion source to create the ZIF-8 shell. The authors showed that more than 80% of the anticancer drugs were released at the pH value of 5.5. This was mainly attributed to the dissolution of ZnO core and ZIF-8 shell in acidic conditions, endowing the composite with good pH-responsive drug release behavior. The MTT assay in HeLa cells showed that the toxicity of DOX-encapsulated ZnO@ZIF-8 was significantly higher than that of the composite alone and free DOX, which was mainly due to the synergistic toxic effect of released anticancer DOX and ZnO@ZIF-8 decomposition under acidic TME. [181] A similar approach was reported by Zhou et al. [186] in which diselenide-containing triblock copolymer (PEG-PUSeSe-PEG) was employed as a drug carrier and ZIF-8 acted as a gatekeeper to delay drug release from the copolymer micelles. The selenium-containing polymers not only served as DOX storage agent, but also it was disassembled in the presence of redox agents (H 2 O 2 /GSH), which are overproduced in cancer cells. Although their cancer therapy studies were conducted in vitro, the design/fabrication of such redox/pH-sensitive nanocomposite with good loading capacity, controllable drug release property and excellent biocompatibility paved a rational way to fabricate multiresponsive DDSs in cancer treatment.
As above-mentioned, another limitation of ZIF-8 is the poor water dispersibility and inaccessibility of its free surface functional groups, preventing its further applications toward advanced biomedical goals. Therefore, the construction of waterdispersible targeted ZIF-8 based DDSs is of particular importance, especially in chemotherapy applications. [102] Macrocyclic water-soluble carboxylated pillar [6]arene (WP6) was used to functionalize ZIF-8@DOX via the coordination between metal nodes of the drug-loaded ZIF-8 and the carboxyl group of WP6 to improve water dispersibility of the DDS. [174] In addition, through host-guest complexation between the WP6 and a galactose derivative as targeting ligand, a highly dispersed/targeted DDS was prepared to increase the anticancer efficiency of ZIF-8@DOX NPs toward galectin overexpressing hepatoma cancer cells. Confocal laser scanning microscope studies and flow cytometry showed that the pH-responsive DDS took up by HepG2 cells and DOX were released efficiently within the cells. This study was a pioneering work on the fabrication of rational targeted pHresponsive water dispersible drug carriers using the combination of supramolecular hybrid structures and ZIF-8 for potential cancer therapy.
Chemodynamic therapy (CDT) as an emerging and unique therapeutic strategy has also been recently developed for cancer therapy through reactive oxygen species (ROS) generation www.advancedsciencenews.com www.advhealthmat.de mediated by Fenton reaction. [218] In this process, • OH radicals can directly be generated by the Fenton reaction of overproduced endogenous H 2 O 2 and metal ions catalysts, e.g. Fe(II) or Cu(I), in tumor sites, leading to apoptosis induction and tumor growth inhibition. [218][219][220] However, one of the main obstacles of cancer CDT is the insufficient concentration of H 2 O 2 in the TME that causes poor anticancer performance of this technique. [221] In 2019, the proof-of-concept of singlet oxygen-based CDT for selective tumor eradication using hypochlorous ion (ClO − )-loaded ZIF-8 was proposed by Zhao co-workers ( Figure 7A). [184] ClO − could react with H 2 O 2 in TME to stoichiometrically generate singlet oxygen and kill cancer cells. [222] On the other hand, the enrichment of H 2 O 2 in TME was achieved by simultaneous intraperitoneal (i.p.) administration of the ascorbate (Asc) to enhance the efficiency of CDT. It was shown that Asc could promote H 2 O 2 accumulation in the extracellular fluids of solid tumors at its pharmacological concentration. [223,224] They showed combinational ClO − and Asc delivery via intravenous (i.v.) and i.p. administration, respectively, is a potent CDT system for selective cancer therapy in vivo ( Figure 7A). The ClO − -loaded ZIF-8 (ClO@MOF) was prepared in a one-pot reaction and further coated with poloxamer 188 (Pluronic F68) to increase the stability of the nanosystem. The hydrodynamic size of the F68-modified ClO@MOF (ClO@MOF/F68) NPs was 177.9 ± 13.2 nm (Figure 7B). As shown in Figure 7C, the prepared NPs exhibited a slightly positive surface charge. Significant amounts of ROS even at the absence of Asc was observed in murine mammary carcinoma 4T1 cells treated by ClO@MOF/F68 NPs ( Figure 7D), attributed to the reaction of ClO − with intracellular H 2 O 2 . Interestingly, the cells co-treated by ClO@MOF/F68 NPs plus free Asc generated the highest level of ROS, confirming Asc-mediated elevation of intracellular H 2 O 2 level to boost the ROS concentration. Compared to ClO@MOF/F68 alone, the co-supplement of ClO@MOF/F68 with Asc remarkably reduced the cell viability of 4T1 cells due to the enhanced ROS production ( Figure 7E). In addition, significant GSH reduction was observed using the combination of ClO@MOF/F68 and Asc in vitro ( Figure 7F). GSH is a major antioxidant inside the tumor cells, which hinders the performance of ROS-based nanomedicines to treat cancer. In agreement with the in vitro cytotoxicity experiments, in vivo antitumor studies on 4T1 tumor-bearing xenograft mice showed that the combinational formulation, i.e., ClO@MOF/F68 plus Asc, exhibited significant anti-tumor activity than the other groups (Figure 7G). Altogether, this study paves new ways to overcome the side effects of traditional PDT and sonodynamic therapy (SDT), including the limited tissue penetration of light in PDT, low quantum yield of singlet oxygen in SDT, and the toxicity of photosensitizers/sonosensitizers, to develop effective ROS-based antitumor therapy with lower side-effects.

Combined Therapy by ZIF Nanocomposites
Although monotherapy of cancer by novel DDSs enhances therapeutic efficiency and decreases side effects of toxic chemotherapeutic drugs, still the MDR of cancer cells in chemotherapy is a big hurdle, remaining as an important challenge in effective cancer treatment. [131,225] Therefore, the construction of multifunctional cancer treatment systems, combining multiple therapeu-tic modalities, including chemotherapy, PTT and PDT in a single system for more effective cancer treatment has been an imperative goal. [3,17,21,[226][227][228][229] Herein, we will discuss about the ZIFbased multifunctional nanocomposites developed very recently for combined cancer therapy.
PTT, a light-induced method, is a minimally invasive treatment that employs the photothermal agents located in tumors to convert NIR energy into heat, leading to irreversible cellular damage and subsequent tumor killing. [230] Various photothermal agents including gold NPs, [231] MXenes, [232,233] bismuthcontaining nanomaterials, [234][235][236] organic NPs, [237] liquid metal nanodroplets, [238] and 2D noncarbonaceous materials [239] have been developed for PTT. Simultaneous chemo-photothermal therapy (CPTT) can be realized by combining photothermal agents and chemotherapeutic drugs within ZIFs. Tian et al. [185,195] developed DOX-encapsulated ZIF-8/graphene quantum dot (GQD) nanocomposites, utilizing a one-pot method with a particle size of 50-100 nm, to achieve synergistic photothermal therapy and controlled drug delivery ( Figure 8A). Since ZIF-8 is stable under neutral conditions (pH 7.4) and can disintegrate under acidic environment, DOX release was much faster at acidic condition than in neutral environment from the DOX@ZIF-8/GQD ( Figure 8B). The drug-loaded NPs efficiently converted NIR irradiation into heat, which was attributed to the high photothermal conversion efficiency and excellent thermal conductivity of GQD as a photothermal agent ( Figure 8C). In vitro studies on the viability of 4T1 breast cancer cell line showed a synergistic effect to kill the cancer cells, owning to the photothermal effect of GQDs and the pH-dependent DOX release from the multifunctional DDS, holding great promise for CPTT of cancer ( Figure 8D).
Wang et al., [176] for the first time, demonstrated that copper sulfide (CuS)-incorporated ZIF-8 (CuS@ZIF-8) NPs decomposed under physiological conditions, i.e., pH 7.4, while exposed to NIR laser irradiation, which was related to the uneven thermal expansion of ZIF-8. The CuS@ZIF-8 nanosystem was prepared by PVP-mediated in situ growth of ZIF-8 around the CuS NPs. The incorporation not only did not alter the low pH-degradability of ZIF-8 NPs, but also boosted a synergistic chemo-photothermal therapeutic effect on tumor cells killing after DOX loading into the CuS@ZIF-8 nanocomposite.
Recently, noble bimetallic nanomaterials have gained great interest as promising photothermal agents owing to the advantages of facile synthesis, chemical inertness, and high photothermal conversion efficiency. [240,241] However, their unstable structures and shapes, and potential biotoxicity have limited their biomedical applications. [242] As a result of the low toxicity of palladium (Pd) at concentrations required for photothermal therapy, DOXloaded Au-Pd bimetallic nanocomposites encapsulated within ZIF-8 (Au-Pd/DOX@ZIF-8) were used to fabricate a multifunctional DDS for synergistic in vitro CPTT of cancer cells (Figure 9A). [36] Pd NPs were first prepared and then covered by Au nanosheets. To obtain Au-Pd/DOX@ZIF-8 nanocomposites, the Au-Pd NPs and DOX were synchronously encapsulated by ZIF-8 in a single step. The Pd-Au was able to convert NIR light into heat and accelerate the release of chemotherapeutic DOX through local hyperthermia. Furthermore, the ZIF-8 shell was degraded at acidic tumor microenvironment, leading to the release of encapsulated DOX. Such a synergistic scenario introduced a cytotoxic nanosystem against SMMC-7721 cells, which was not attainable  via a single modality. Cancer cell viability reached 11% after pretreating with Au-Pd/DOX@ZIF-8 (80 µg mL −1 ) for 24 h under irradiation of NIR laser for 10 min, which the viability was lower when no pretreatment was used, confirming the accelerated release of the encapsulated DOX under laser irradiation.
Although metal-based NIR adsorbing agents have extensively been utilized for PTT, they are expensive and suffer from low photostability under long-term laser irradiation. [36,233,[243][244][245] Organic polymers, such as polyaniline (PANI) [243,[246][247][248] and polypyrrole [249][250][251][252] have good biocompatibility, low cost, high mechanical flexibility and strong NIR absorption. [243,249] These features endow them with excellent capacities to be exploited as alternative photothermal agents in PTT. [253] In 2018, Silva et al. [183] synthesized PANI-decorated ZIF-8 NPs (PANI@ZIF-8) and after 5-FU drug loading, this multifunctional DDS was used as an efficient CPTT platform for in vitro cancer therapy ( Figure 9B). The authors showed that the release of 5-FU from the polymer-coated ZIF-8 reached to 68% at pH 5.2, while at the same condition and upon NIR laser irradiation (980 nm, 0.8 W cm −2 ), the drug release was ≈80%. Therefore, the PANI not only accelerated the drug release, but also served as NIR-light to heat conversion agent to kill cancer cells.
It has been reported that unmodified ZIF-8 has high toxicity, which limits its in vivo applications in the design/fabrication of advanced DDSs. [22,100] Therefore surface modification of ZIF-8 is an imperative task to tune/improve the biocompatibility of this carrier. Using cytotoxicity and in vivo acute toxicity assessments, Wu et al. [22] demonstrated that the biosafety of ZIF-8 NPs was significantly improved by PDA coating. The PDA also acted as photothermal agent to achieve PTT. To precisely control the rate of drug release from the DOX-loaded ZIF-8/PDA NPs, the authors used a phase-change material (PCM), i.e., tetradecanol, for NIR-mediated drug release. As the melting point of tetradecanol is 38-40°C and it does not dissolve under physiological conditions, a more control on DOX release was obtained upon NIR irradiation via tetradecanol dissolvation by increasing local temperature through light-to-heat conversion effect of PDA. Therefore, a controlled CPTT was successfully achieved by the biocompatible DOX-loaded PDA-PCM@ZIF-8 in vivo. The cytotoxicity studies in HepG2 cells indicated that the ZIF-8 itself has high cytotoxicity, while the PDA@ZIF-8 and PDA-PCM@ZIF-8 had favorable biocompatibility. When 15 mg kg −1 of ZIF-8 was injected to the mouse through the i.v. route, the survival rate was only 60% and an abnormality was seen in the liver of the mouse after dissection. In contrast, in the case of PDA@ZIF-8, the survival rate was 80% for the administration dose of 75 mg kg −1 , again proving increased biocompatibility after surface modification.  [36] Copyright 2017, The Royal Society of Chemistry. B) Schematic application of 5-FU-loaded PANI@ZIF-8 in CPTT. Reproduced with permission. [183] Copyright 2018, American Chemical Society.
The same group used ZrO 2 coating onto the surface of ZIF-8 NPs to improve the biocompatibility of the NPs. [205] The ZrO 2 shell not only boosted biocompatibility of ZIF-8, but also served as an excellent CT contrast agent, owning to the high atomic number of Zr element. In addition to DOX loading, ionic liquid was also loaded on the surface of ZIF-8@ ZrO 2 for synergistic chemo/microwave thermal therapy of cancer.
Successful in vitro and in vivo synergistic CPTT was reported by loading DOX into AuNR coated ZIF-8 (AuNR@ZIF-8-DOX) NPs, and controlled release under NIR irradiation ( Figure 10A). In this study by Li et al., [60] the authors showed NIR and pH dual stimuli-responsive DOX release from AuNR@ZIF-8-DOX ( Figure 10B,C). The core-shell nanostructures significantly induced photothermal anticancer effect both in vitro and in vivo ( Figure 10D,E). As a result of the high light-to-heat conversion capability of AuNR, the AuNR@ZIF-8-DOX NPs possessed the highest toxicity at cellular and animal models as compared to the single therapeutic treatment approaches, i.e., separate chemotherapy and PTT. This study introduced the AuNR@ZIF-8-DOX NPs as a powerful multifunctional DDS for combined CPTT of cancer. [60] The combination of CDT and PDT (CPDT) has emerged as a new strategy for cancer treatment. [200,[254][255][256] However, overexpression of GSH and hypoxia in TME are two main obstacles that severely affect the successful treatment of cancer through the combined strategies. [257] Recently, O 2 -Cu/ZIF-8@Ce6/ZIF-8@F127 nanocomposite was prepared to simultaneously decrease hypoxia and GSH level for enhanced CPDT. [258] It is noteworthy that one of the important features of ZIFs is its gas storage capability. [6] ZIF-90 as a member of ZIFs has been reported as an O 2 carrier to overcome tumor hypoxia for efficient PDT. [200] However, in the O 2 -Cu/ZIF-8@Ce6/ZIF-8@F127 nanocomposite, ZIF-8 acted as O 2 carrier and the doped Cu 2+ in the ZIF network doubled the O 2 loading capacity of ZIF-8 to increase the amount of ROS generated by PDT using Ce6 photosensitizer under 650 nm laser irradiation. Moreover, the released Cu 2+ reduced GSH through oxidation reaction and the produced byproduct, i.e., Cu + acted as CDT agent and generates cytotoxic •OH via Fenton-like reaction by reacting with H 2 O 2, which was overexpressed in tumor cells. [259,260] In addition to cancer therapy, ZIF-mediated combined therapy has been proposed for other diseases. [26,261,262] For example, endophthalmitis is a disease that occurs in many intraocular surgeries, particularly cataract surgery due to the localization of the pathogenic microorganisms in the damaged tissue. This infection leads commonly to intraocular inflammation, uveitis, and damage to the vitreous cavity within the eyeball. [263,264] Recently, the combination of chemotherapy and PDT was used for successful inhibition of infectious endophthalmitis by ZIFs. [262] To this end, ammonium methylbenzene blue (MB)-loaded ZIF-8-PAA (ZPM) was first prepared, followed by AgNPs incorporation, using dopamine as reducing agent and then further modification with Van/NH 2 -PEG to give NIR and pH-responsive ZIF-8-PAA-MB@AgNPs@Van-PEG (ZPMAVP) nanocomposite for the treatment of bacterial endophthalmitis (Figure 11A). The MB acted as photosensitizer antibacterial agent and PAA endowed the nanocomposite with higher pH-responsive properties and better drug loading capacity. The bactericidal and antiadhesive activities of the NPs were investigated by agar plate counting method and SEM imaging. In the case of ZPM NPs, treating with three kinds of bacteria, i.e., S. aureus, E. coli, and MRSA, resulted in no apparent decrease of bacterial growth when laser treatment was not applied. In contrast, an obvious reduction of bacteria was observed under laser irradiation at 650 nm, confirming the high efficiency of the PDT. As a result of the antibacterial property of AgNPs, the Ag-incorporated-ZPM (ZPMA) showed higher bactericidal effects. It has been reported that Van, which is a highly efficient antibiotic can act as a targeting molecule, especially towards Gram-positive bacteria. [265] As shown in Figure 11B-D, the grafting of the targeting molecule and hydrophilic PEG chain on ZPMA further increased the bacteria-killing efficiency in all three kinds of bacteria. SEM imaging of the remaining/corpse bacteria on silicon wafers confirmed the positive impact of laser treatment, i.e., PDT, on biofilm eradication function of ZPM, ZPMA, and ZPMAVP to decrease the number of adhered bacteria on the surface. The antibacterial mechanism of the ZPMAVP NPs was attributed to the generation of ROS by Ag + released from the NPs and also through the PDT. In vivo antibacterial tests were carried out on bacterial endophthalmitis model constructed in the eyes of New Zealand White Rabbit. Compared to the eyes treated by Van, the symptoms of endophthalmitis in the groups treated by ZPMAVP NPs under laser irradiation were mild and conjunctival congestion and anterior chamber empyema were not obviously observed ( Figure 11E,F). The number of bacteria after 7 days in the vitreous humor of the eyeball of the rabbit cultured on an agar plate and administrated with Van and ZPMAVP/laser groups was less than 1 × 10 5 and 1 × 10 3 CFU mL −1 , respectively, while this number in the control group was more than 1 × 10 10 CFU mL −1 ( Figure 11G). Collectively, the in vitro and in vivo antibacterial experiments demonstrated the effect of the PDT on endophthalmitis therapy, opening new ways in the PDT-mediated treatment of eye diseases due to naturally good light transmission of the eyes. This nanosystem not only introduced ZIF-8 as a biocompatible carrier, but also microporous structure of ZIF-8 was reported as a molecular cage to prevent the self-aggregation of MB as photosensitizer. Recently, this property of ZIF-8 has been used in encapsulating hydrophobic PS squaraine (SQ) to enhance PDT efficacy of SQ against drug-resistant planktonic bacteria and its biofilm. [116]

Theranostics Applications of ZIF Nanocomposites
Nanotheranostic is the integration of therapeutic and diagnostic modalities into a single nanosystem to achieve imagingguided therapy. [266] Such nanoplatforms have received a great Figure 11. A) Schematic preparation of ZPMAVP nanosystem and its application in combined CPDT of endophthalmitis. Bacterial viability of B) S. aureus, C) E. coli, and D) MRSA, using plate counting methods ( # p < 0.05, ## p < 0.01, ### p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001) after treatments with ZPM NPs, ZPMA NPs, and ZPMAVP NPs. Photographs and slit lamp micrographs of endophthalmitis caused by E) S. aureus and F) MRSA at days 1, 3, and 7 after treatment with PBS, Van, and ZPMAVP+laser. G) Agar plate culturing of S. aureus and MRSA at day 7 available in the vitreous fluid after treatment with PBS, Van, and ZPMAVP+laser NPs. The power of the laser in all the experiments was 202 mW cm −2 . Reproduced with permission. [262] Copyright 2019, Wiley-VCH.  [194] Copyright 2018, The Royal Society of Chemistry.
promise in real-time monitoring and treating cancer and other hard-to-treat diseases, while playing a pivotal role in the development of personalized medicines. [266] Recently, nanoscaled MOFs (NMOFs), including ZIFs, have emerged as one of the most promising nanotheranostic systems due to their huge porosity, large surface area, tunable functionality, and good biocompatibility/biodegradability. [4,21,45,200,[267][268][269][270] Single or even multimodal imaging by ZIF-based theranostic systems has been recently studied. [24,61,194] Herein, we discuss how these multifunctional nanosystems work to simultaneously monitor and treat cancer. Fluorescent carbon nanodots (C-dots) as safe imaging agents, which had strong fluorescence intensity were encapsulated within ZIF-8 and were used as theranostic after 5-FU loading for simultaneous pH-responsive DDS and fluorescence imaging of cancer cells in vitro. [61] In the study, it was also shown that, using a facile two-step method, the size of the ZIF-8 NPs was tuned by changing the initial concentrations of 2-MeIm and Zn 2+ ions and it was confirmed that fluorescence intensity C-dots encapsulated ZIF-8 was adjustable by varying the amount of C-dots.
Although there are many examples on the construction of core−shell structures using ZIFs for theranostic applications, these systems might suffer from complexity and high cost of the methods. Surface modification of ZIFs is an alternative approach to incorporate imaging agents on ZIFs to make multifunctional theranostic nanosystems. [21] Similar to the report of Zheng et al. [100] discussed in the previous sections, Shu et al. [194] loaded DOX into ZIF-8 through a one-pot process and then the resultant DOX@ZIF-8 was subsequently coated with PDA. In order to add a magnetic resonance imaging (MRI) agent, the PDA coated DDS was successively chelated with Fe 3+ and further conjugated with HA (DOX@ZIF-8-HA) (Figure 12A). The HA acted as targeting ligand toward the PC-3 prostate cancer cell line, proved by confocal laser scanning microscopy and flow cytometry. Innate acid degradability of ZIF-8 caused pH-responsive release of DOX from the multifunctional DDS ( Figure 12B). Furthermore, the Fe 3+ functioned as a contrast agent for MRI (Figure 12C). Therapeutic activity of DOX@ZIF-8-HA was assessed using the cell counting kit-8 (CCK-8), demonstrating targeting www.advancedsciencenews.com www.advhealthmat.de ability of the nanosystem toward CD44 overexpressed PC-3 cells and improving its intracellular uptake and enhancing its in vitro chemotherapeutic efficacy, as compared to the free DOX. (Figure 12D). In addition, free HA had a negligible impact on the therapeutic efficacy of DOX@ZIF-HA.
As a result of the high effectiveness, noninvasiveness, and concurrent fluorescence imaging property of PDT, this method has gained great attention to be part of theranostic systems for cancer therapy. A PS, oxygen, and light are key components for successful PDT. [271] However, one of the main challenges in PDT is the self-aggregation of PSs in aqueous environments because of its hydrophobic nature, leading to quick quenching of PDT and poor efficiency of the systems. [272] Xu et al. [177] utilized ZIF-8 NPs as a stabilizer to encapsulate and separate water-insoluble photosensitizer zinc(II) phthalocyanine (ZnPc) molecules inside nanoscale molecular cages of ZIF-8 to maintain the PS as monomeric in aqueous solution for efficient PDT. The ZnPc@ZIF-8 nanosystem showed highly efficient cytotoxic singlet oxygen generation capability and excellent photodynamic activity towards HepG-2 cancer cells. This system also exhibited red fluorescent emission after endocytosing by the cancer cells, paving a way to overcome self-aggregation/bioavailability problems of PSs for imaging-guided PDT. It should be noted that the self-aggregation of PSs and hypoxia at the tumor sites are the main limitations in the development of cancer PDT methods. As a result of the degradation of manganese oxides (MnO 2 ) into Mn 2+ ions in an acid solution of H 2 O 2 , such chemistry can boost the concentration of O 2 in TME, therefore increasing the efficiency of PDT process. [273][274][275] In addition, released Mn 2+ ions were exploited as contrast agent for MRI. Utilizing the nanoenzyme-like/O 2 -generating behavior of MnO 2 , BSA-MnO 2 NPs were loaded onto the surface of Ce6, a hydrophobic photosensitizer, encapsulated-ZIF-8 (BSA-MnO 2 /Ce6@ZIF-8) NPs for MRI-guided PDT of cancer ( Figure 13A). [276] The successful synthesis of BSA-MnO 2 /Ce6@ZIF-8 was confirmed by TEM, dynamic light scattering (DLS) and energy-dispersive X-ray spectroscopy (EDS) (Figure 13B-D). For example, EDS revealed the presence of O, N, Zn, S, and Mn, proving the existence of BSA-MnO 2 NPs on the surface of Ce6@ZIF-8 NPs ( Figure 13C). As shown in Figure 13E, BSA-MnO 2 /Ce6@ZIF-8 NPs were able to produce more oxygen at pH 5.0 than the pH 7.4. Such behavior was attributed to the simultaneous presence of H + and H 2 O 2 that triggered the catalase-like activity of nanosystem, confirming the O 2 -generating capability of the BSA-MnO 2 /Ce6@ZIF-8 NPs to relieve hypoxia in cancer therapy using PDT methods. Both cell viability and in vivo tumor growth inhibition studies confirmed the outstanding killing effect derived from the PDT under 650 nm NIR laser irradiation ( Figure 13F-H). Moreover, MRI after injection of the NPs proved that the BSAMnO 2 /Ce6@ZIF-8 could effectively accumulate in the tumor sites, holding great promise in MRI-guided cancer therapy ( Figure 13I). [276] Visible-light photosensitizer graphitic carbon nitride (g-C 3 N 4 ) nanosheets, as a photodynamic therapeutic agent, were encapsulated within ZIF-8 by growing ZIF-8 components in the presence of g-C 3 N 4 nanosheets. [180] Chemo-photo combination therapy was conducted by DOX-loaded g-C 3 N 4 @ZIF-8 NPs, generating singlet oxygen for PDT and delivering DOX with a pH-sensitive manner for chemotherapy. Blue fluorescence of g-C 3 N 4 nanosheets and red fluorescence of DOX rendered nanotheranostic property to the particles due to the combined imagingguided chemo/phototherapy of cancer cells.
It is important to consider that sufficient interaction between ZIF-8 and drugs is essential to achieve high drug encapsulation through ship-in-bottle approaches, i.e., concurrent drug loading and ZIF construction. However, only drugs containing polar functional groups (PFG), e.g. carboxylic acid, carbonyl, and sulfonic groups, have shown satisfactory results in the loading step. Therefore, drug molecules without these functional groups need to be functionalized with appropriate PFGs to reach acceptable drug loading. Zhang et al. [187] developed a versatile prodrug strategy, using cytarabine as a model drug that had insufficient loading in ZIF-8, and was covalently attached with new indocyanine green (IR820), containing sulfonic groups to produce a prodrug via an amide linkage. Sulfonic groups of prodrugs coordinated with the zinc ions in ZIF-8-IR820, strengthening the interaction of prodrug with ZIF-8, thereby resulting in a high drug loading content of 39.8%. Importantly, due to light to heat conversion capability and fluorescence emission capability of IR820, the developed DDS, after functionalizing with HA as targeting ligand, was successfully applied in fluorescence imaging-guided CPTT in vitro breast cancer.
Although single-modality therapies, i.e., chemotherapy, PTT, or PDT, have attracted much attention in cancer therapy, each modality has its own disadvantages, which force the usage of multi-therapies to induce a synergistic effect. [1] The combination of chemotherapy, PDT, PTT, and immunotherapy by ZIF-8 as a nanomedicine platform was developed by Yang et al. [24] (Figure 14). DOX, CuS NPs, and protoporphyrin IX (PpIX) used as chemotherapy, PTT, and PDT agent, respectively, were loaded within ZIF-8. The CuS NPs not only served as PTT agent to convert light (808 nm) to heat, but also accelerated DOX release under NIR irradiation. The resulting DOX-CuS-PpIX@ZIF-8 was coated by unmethylated cytosine phosphateguanine (CpG) oligonucleotide as immune adjuvant through electrostatic interaction for immunotherapy with the aim of preventing cancer metastasis/recurrence. To realize MR imaging, MnO 2 nanosheets were grown on the CpG coated NPs after surface modification of the NPs by the PDA layer. The PDA acted as a gatekeeper to prevent the drug leakage before reaching to cancer sites and enhanced the PTT effect. Therefore, an "all-inone" nanotheranostic was successfully utilized for MRI-guided chemo/photothermal/photodynamic/immunotherapy of cancer with anti-metastasis/recurrence property. This example clearly demonstrated how the interior and exterior surfaces of ZIF-8 can truly be exploited to construct multifunctional nanocomposites intended for concurrent diagnosis and multi-modal therapy.
One of the main advantages of multifunctional NPs is multimodal imaging, enabling them to combine two or more imaging modalities for simultaneous imaging and therapy. As each imaging modality has its own intrinsic limitations, therefore Reproduced with permission. [276] Copyright 2019, American Chemical Society.
the integration of several imaging agents within a single system may allow more accurate diagnosis of diseases through. [1] For the first time, the intrinsic two-photon fluorescence imaging property of ZIF-8 NPs derived from 2-MeIm was reported by Zhao et al. [182] Such an intrinsic property holds great potential for the fabrication of novel theranostic platforms. In-spired by the excellent advantages of ZIF-8 in the constriction of multifunctional NPs, Bian et al. [23] prepared an "all-in-one" imaging system, i.e., Fe 3 O 4 @PAA/AuNCs/ZIF-8, integrating trimodal imaging of MRI, CT, and optical imaging (OI), where Fe 3 O 4 and AuNCs acted as MRI and CT/OI agents, respectively. To prepare the nanocomposite, oleic acid (OA) capped Fe 3 O 4 Figure 14. A) Schematic depiction of preparing "all-in-one" nanocomposite of DOX-CuS-PpIX@ZIF-8-PDA-MnO 2 . B) Theranostic property of the nanocomposite for MRI-guided chemo/photothermal/photodynamic/immunotherapy to eliminate the primary solid tumor. Reproduced with permission. [24] Copyright 2018, The Royal Society of Chemistry.
NPs were firstly modified by CTAB surfactant. After the coating of the CTAB-functionalized NPs by PAA molecules, the Fe 3 O 4 @PAA core-shell NPs were obtained. Subsequently, the resultant Fe 3 O 4 @PAA NPs were incorporated by glutathione capped AuNCs to form Fe 3 O 4 @PAA/AuNC NPs. Finally, the Fe 3 O 4 @PAA/AuNCs/ZIF-8 nanocomposite was prepared by the addition of Zn 2+ and 2-MeIm. The nanocomposite showed ultrahigh DOX loading (1.54 mg DOX per 1 mg of NPs) and high magnetic property. In addition, the NPs showed low systematic toxicity in vivo and suppressed the tumor growth effectively by DOX delivery into the tumor tissue of Balb/c mice through intravenous injection. DOX-loaded Fe 3 O 4 @carbon@ZIF-8 multifunctional nanotheranostics were also fabricated by He et al. [202] for pH-triggered release of DOX in vitro. The superparamagnetic iron oxide nanocrystals and carbon dots acted as MR and fluorescence imaging contrast agents, respectively. In addition, the remarkable inhibition of tumor growth without side effects was obtained in vivo in the A549 lung cancer mice model after intravenous injection.
As mentioned in the previous sections, one of the main obstacles of cancer CDT is the insufficient concentration of H 2 O 2 in TME, leading to the poor performance of this technique. [221] Artificial in situ generation of H 2 O 2 in TME, for example, through the conversion of -d-glucose to gluconic acid and H 2 O 2 by GOx, is a reliable way to overcome the shortcomings of CDT. [221] In 2018, Zhang et al. [277] developed an ATP-responsive autocatalytic Fenton nanosystem, called GOx@ZIF@MPN, by the incorporating of GOx in ZIF-8, and then, coating the whole system with Fe(III)/TA polyphenol network (MPN) for suppressing tumor growth mediated by enhanced chemodynamic-starvation therapy (Figure 15A,B). As a result of the ATP upregulation in tumor cells, as compared to normal cells, and potential degradation of Fe(III)/TA MPNs in overproduced ATP environments, the MPN shell of the designated nanosystem was degraded into Fe(III) and TA in tumor cells while the internal GOx was exposed.
Afterwards, GOx was reacted with the endogenous glucose to generate sufficient H 2 O 2 for CDT, while TA as reducing agent converted Fe(III) to Fe(II) to further push forward the Fenton reaction to generate highly toxic hydroxyl radical ( • OH) by Fe(II) as a catalyst. It should be noted that TA played a crucial role in accelerating Fe(III)/Fe(II) conversion to guarantee highly efficient Fenton reaction mediated CDT. ATP-responsive degradation of GOx@ZIF@MPN was shown by the release of iron at different ATP concentrations ( Figure 15C). The iron release was increased when ATP concentration and treating time increased, demonstrating the ATP-responsive property of the nanosystem, due to the strong binding affinity of ATP to Fe(III). After intravenous injection of Cy5.5-loaded GOx@ZIF@MPN NPs to 4T1 tumor-bearing mice, the fluorescence intensity was gradually enhanced over time and then weakened, but still retained in a desirable level after 24 h, confirming long time blood circulation of the NPs ( Figure 15D). In addition, the MRI signal was detected after 10 min of the post-injection due to the T1-weighted contrast effect of GOx@ZIF@MPN after intratumoral injection. The MRI signal was gradually enhanced over time till 30 min, demonstrating rapid degradation of the MPN shell in response to ATP at the tumor site and generation of excellent T1-weighted imaging signal for tumor diagnosis ( Figure 15E). As shown in Figure 15F,G, studies on 4T1 cells treated with GOx@ZIF@MPN showed that the nanosystem had very high toxicity against the cancer cells both in vitro and in vivo, when compared to control groups of GOx@ZIF and ZIF@MPN, confirming a synergistic effect of CDT and starvation therapy on the cancer ablation. Such an enhanced therapeutic effect was also observed by the photographic imaging of harvested tumors from each treatment group after sacrificing mice ( Figure 15G). Very recently, a biomimetic cascade nanoreactor based on ZIFs, i.e., DOX-loaded GOx@ZIF camouflaged by the tumor cell membrane, was also developed for synergistic chemotherapy and starvation therapy. [125] Indeed, the biomimetic membrane endowed the NPs with superior immune  [277] Copyright 2018, American Chemical Society. Figure 16. A) Schematic illustration of the Au@MOF-DOX synthesis and its imaging and synergistic anticancer property. B) TEM images Au@MOF and C) yolk-shell structural Au@MOF. D) The heating and cooling curves of Au@MOF (200 ppm of Au) irradiated at 1064 nm NIR laser. E) Temperature change profile of Au@MOF with different concentrations of Au and F) the profile at various power densities for 10 min with 50 ppm of Au concentration exposed to 1064 nm NIR laser. G) In vivo ITI images of H22 tumor-bearing mice. The mice were intravenously injected with PBS or Au@MOF NPs irradiated with 0.8 W cm −2 1064 nm laser for 5 min. H) In vivo PAI images of Balb/c nude mice. The mice were intravenously injected with Au@MOF NPs. I) Time-dependent tumor growth curves of tumor-bearing mice in different groups. Reproduced with permission. [282] Copyright 2019, American Chemical Society. evasion and homologous targeting capacities, thereby introducing a novel and efficient method for precise tumor therapy.
Yolk-shell nanocomposites (YSCs), integrating different compositions and functionalities in a single system have attracted wide attention in biological research. [278,279] In fact, the hollow cavity of the YSCs can act as a container for loading more car-gos and the shell can serve as a protective layer for the yolk, which can be made of a wide variety of NPs. [280,281] Recently, an interesting multifunctional YSC was constructed by integrating star-shaped gold (Au star) NPs and ZIF-8 as the shell [282] (Figure 16A). As a result of the acid-degradability of ZIF-8 NPs, the part of the ZIF-shell was selectively removed by TA as an etchant www.advancedsciencenews.com www.advhealthmat.de Scheme 2. Schematic illustration of the facing challenges and future progresses of ZIF-based nanostructures for biomedical applications.
to obtain the yolk-shell structures (Au@MOF) ( Figure 16B,C). The yolk-shell Au@MOF nanocomposite not only worked as a photothermal agent at NIR-I (750-1000 nm), but also showed very high photothermal transformation efficiency at NIR-II region (1000-1700 nm; Figure 16D-F), which is of particular importance in deeper tissue penetration and higher spatiotemporal resolution than NIR-I region. Moreover, due to the strong NIR light absorbance of the Au yolk, the nanosystem was able to be used for ITI and PAI of cancer tissue ( Figure 16G,H). After DOX encapsulation and upon the laser irradiation at NIR-II window (1064 nm), the Au@MOF exhibited outstanding synergistic tumor ablation effect based on the light to heat conversion property of the Au yolks and anti-cancer property of DOX ( Figure 16I), which caused a remarkable synergistic anticancer effect through combined chemo-phototherapy.

Conclusions
Recently, the scientific community has witnessed the considerable achievements of ZIFs for versatile biomedical applications, especially in the stimuli-responsive DDSs and biomineralization of elegant bio-macromolecules due to their unique features, including tunable pore size, large surface area, high thermal stability, and favorable biodegradability/biocompatibility. Herein, we have comprehensively discussed the very recent developments for the controlling of the physicochemical properties of ZIFs and their impact on diverse cargo loading and the release trend of various molecules, including drugs, bio-macromolecules, photosensitizers, and photothermal agents, in order to produce optimized DDSs for the treatment of different diseases, such as cancer and infection. Although the study of biomedical applications of ZIFs is in its infancy phase, it has opened promising and intriguing prospects based on their high performance in many applications, which was discussed in this review paper (Tables 1  and 2). However, despite the growing interest and impressive advancements in this field, some critical issues and foreseeable challenges are crucial to be considered and addressed for the ra-tional design of ZIF-based nanosystems for versatile biomedical applications (Scheme 2). First, broadening the biomedical applications of ZIFs and improving the therapeutic efficacy of its derived nanomedicines should be further investigated. The intrinsic characteristics of ZIFs endow them with superior capability in the loading of different cargos in a single particle to exploit them as multifunctional carriers. For example, in the case of cancer therapy or bacterial infection, efficient boosting of the therapeutic efficacy might be achieved by the integration of immunotherapeutic and starvation agents with chemo and phototherapeutic cargos to increase the efficacy of the ZIF nanosystems. While the current advancements on the of the ZIFs have focused on the abovementioned diseases, other biomedical applications, including biosensing, brain disease therapy, tissue engineering, inflammation alleviation, etc., are yet to be investigated. In addition, the synthesis of other novel and multifunctional derivatives of ZIF NPs rather than ZIF-8 might expand the applications of the porous ZIF-based nanomaterials in nanomedicine. In this context, the physicochemical properties of ZIFs, including particle size, pore size, surface modification/targeting, compositional and morphological features must be carefully tuned to meet standard requirements for in vivo studies.
The second issue is related to the large scale preparation of ZIFs with adjustable particle diameter and pore size and the exploration of new targeting ligands for surface modification of the ZIFs. This issue can affect the drug loading into the ZIFs, alter drug accumulation at a specific site, and markedly reduce/increase the uptake of the NPs by RES organs. At present, ZIFs are mainly prepared in a lab-scale and researchers must work on the development of synthesis approaches for the rapid and reproducible production of these nanomaterials in a large scale. Since the toxicity and poor loading are the main concerns for the bench to bedside movement, preparation of HZIFs can be suggested as an alternative to enhance the drug loading efficiency and reduce the frequency of administration, thus overcoming the toxicity concerns of ZIF carriers.
At present, due to the novelty of ZIFs and limited understanding of their biological effects, most of the research is towards www.advancedsciencenews.com www.advhealthmat.de developing "proof-of-concept" studies that are not clinically relevant. For future clinical translation, short and long-term biosafety on different organs, biodegradability, biodistribution, blood halflife, and clearance of the ZIFs must be studied comprehensively. Such studies should be conducted not only in small animals, such as rabbit and mouse, but also in big animals, including pigs and dogs to produce clinically applicable ZIF-based nanoscale formulations. To this end, we do believe that the physicochemical properties of ZIFs are essential factors that should be optimized to push forward these nanosystems for clinical application. Moreover, since the interaction of ZIFs with the immune system has remained largely unknown, the influence of these NPs with immune cells should be clarified before developing new techniques for their large scale production.
Taken all together, the engineering of ZIFs for biomedical applications is still in an infancy stage and there are plenty of opportunities to design and fabricate advanced DDSs in this burgeoning field of nanomedicine. To achieve this goal, closer and strengthened collaboration among experts from diverse fields will highly be imperative if we expect novel clinical innovations and personalized medicine in the near future through revealed therapeutic and diagnostic mechanisms of ZIF-based "magic bullet."