Engineering zeolitic imidazolate framework-8 nanoparticles for smart drug delivery systems

Zeolitic imidazolate framework-8 nanoparticles (ZIF-8 NPs) are emerging metal–organic framework nanomaterials composed of 2-methylimidazole and zinc ions, which are widely used in biomedical fields due to their distinctive features such as high porosity, bioresponsive degradation, and superior biocompatibility. Especially, the advanced research of ZIF-8 NPs in smart drug delivery systems is providing unique insights into the rational design of versatile nanomedicines for the treatment and diagnosis of serious diseases. This article provides a comprehensive review and outlook on ZIF-8 NPs-based smart drug delivery systems (SDDSs) including the synthesis methods, drug loading strategies, surface modification, and stimuli-responsive release. In particular, we focus on the advantages of ZIF-8 NPs-based drug loading strategies between the metal coordination-based active loading and the physical packaging-based passive loading. Finally, the opportunities and challenges of ZIF-8 NPs as smart drug delivery carriers are discussed.


Introduction
Recently, metal-organic frameworks (MOFs) have attracted much attention as novel inorganic hybrid materials.The term 'metal-organic framework' was first proposed by Yaghi in 1995 [1].They are ligand framework materials with porous structures formed by ligating metal ions/metal clusters with organic ligands [2].Due to their ultra-high porosity, high surface area, tunable structures, easy surface functionalization, and good biocompatibility, MOFs have become one of the most popular materials in biomedical fields, such as MOFsbased drug/cargo delivery and stimuli-responsive systems [3][4][5][6], targeting imaging [7], biocatalysis [8,9] and sensing [10].
Zeolitic imidazolate frameworks (ZIFs), a subclass of MOFs, are novel crystalline porous materials that combine the excellent properties of zeolites and MOFs, such as crystallinity, microporosity, high surface area and exceptional thermochemical stability [11].ZIF-8 is the most widely studied structure among the ZIFs, first synthesized by Park et al using the solvothermal method [12].ZIF-8 NPs are formed by the self-assembly of 2-methylimidazole with the metal atom Zn through ligand bonds [13].Their chemical formula is Zn [MIM] 2 (MIM = 2-methylimidazole) and the sodalite (SOD) topology is shown in figure 1. Owing to their unique advantages, such as good thermochemical stability, large specific surface area, designable structures, easy biodegradability and good biosafety [12,14], ZIF-8 NPs-based smart drug carriers have been widely applied in biomedical and related fields (figure 2).
This article reviews the synthesis methods, drug loading strategies, surface modification, and stimuliresponsive release in smart drug delivery systems, as shown in figure 3. Especially, we elaborate the advantages of ZIF-8 NPs-based drug loading via metal coordination-based active loading strategy over the physical packagingbased passive loading strategy.In addition, the opportunities and challenges of ZIF-8 NPs as intelligent drug delivery carriers are discussed.

Solvothermal synthesis method
Solvothermal method is one of the most popular and efficient methods for the synthesis of ZIF-8 NPs, in which organic solvents are used as reaction media and metal ions are highly crystallized with organic ligands at a specific temperature to form ZIF-8 NPs.To date, ZIF-8 has been synthesized under the following conditions:   methanol, dimethylformamide (DMF) or N,N-diethylbenzamide (DEF) as solvents; temperatures ranging from 80 °C to 150 °C; and reaction times ranging from 24 to 96 h [15].Hayashi et al [16] synthesized ZIF-8 by dissolving zinc nitrate and 2-methylimidazole in DMF and reacting at 140 °C for 24 h.The synthesized ZIF-8 has exceptional porosity, improved thermochemical stability and high crystallinity.However, this method requires longer time and higher temperature, and solvents such as DMF tend to block the pore channels of ZIF-8 particles, affecting particles purity and specific surface area.Given these limitations, a room temperature synthesis method has been proposed by changing the solvent, the ratio of zinc to 2-methylimidazole and adding modulating agents.Among these, methanol works well as a solvent for room temperature synthesis, reducing time and improving the product [15].Cravillon et al [17] first synthesized ZIF-8 NPs with an average particle size of 46 nm at room temperature using methanol as the reaction solvent.So far, solvothermal is still the superior method for the preparation of ZIF-8 NPs and other MOFs.

Hydrothermal synthesis method
The major difference between hydrothermal synthesis and solvothermal synthesis is that the solvent used is water rather than an organic solvent.Organic solvents are pricy, hazardous, flammable and environmentally unfriendly.In recent years, water has been explored as a green solvent to replace organic solvents in the preparation of ZIF-8 NPs.Pan et al [18] successfully synthesized ZIF-8 NPs with an average particle size of about 85 nm at room temperature using water as the solvent for the first time.However, the synthesis of ZIF-8 NPs in aqueous systems requires a substantial number of ligands to be involved in the reaction.Studies have shown that organic amines such as n-butylamine [19], triethylamine (TEA) [20], polyamines [21], and pyridine [22] can be introduced into the reaction system as deprotonating agents, effectively reducing the need for organic ligands.It has also been demonstrated that ammonia can deprotonate imidazole ligands and coordinate the release of metal ions [23].Chen et al [24] synthesized ZIF-8 by rapidly pouring an aqueous Zn(NO 3 ) 2 6H 2 O into an aqueous ammonia solution of 2-methylimidazole and stirring at room temperature for 24 h.This approach is straightforward, cost-effective and efficient, and has great potential for practical applications such as the largescale manufacture of ZIF-8.

Sonochemical synthesis method
Sonochemistry is a facile and environmentally friendly route for the rapid synthesis of MOFs.It primarily employs ultrasound (US) to trigger high-energy chemical reactions, and US has been proven to be superior to other conventional energy sources in terms of slashing reaction times and increasing energy efficiency [25].Seoane et al [26] synthesized ZIFs such as ZIF-7, ZIF-8, ZIF-11 and ZIF-20 by acoustic crystallization, where ZIF-8 was synthesized under 47 kHz and 110 W sonication for 4 h.The resulting crystals had a small and narrow size distribution but low yields.Cho et al [27] carried out a large-scale synthesis of ZIF-8 with 85% yield in 2 h continuously using a sonochemical method by adding the deprotonator TEA, adjusting the pH and increasing the substrate concentration (figure 4).Thus, using a sonochemical method, a rapid, cost-effective, high-yield and large-scale synthesis of ZIF-8 is feasible.

ZIF-8 NPs based drug loading
Given the large internal pore structure, ZIF-8 NPs are widely used as nanocarriers for drug loading.According to different interaction mechanisms, the ZIF-8 NPs-based drug loading strategies can be divided into two main parts: non-covalent passive loading (physical packaging) and covalent active loading (metal coordination) (figure 5).For the former, there are two types of passive loading, including one-pot method and impregnation method.Essentially, these drug molecules are not directly involved in the composition of ZIF-8 NPs.For the latter, these drug molecules contain active groups, such as carboxyl, which directly coordinate with metal-bound ligands.In this section, we focus on the mechanisms and applications of these different loading modes.

One-pot method
The one-pot method, in which functional molecules are introduced for in situ encapsulation during the growth of MOFs to construct molecular/MOF composites [40].The one-pot method is one of the easiest, most costeffective and efficient methods for loading drugs into MOFs.So far, many drugs have been reported to be successfully encapsulated in ZIF-8 NPs by this method, such as DOX [41], hinokiflavone (HF) [42], camptothecin [43], curcumin [44], 3-methyladenine (3-MA) [40], etc Su et al [45] successfully synthesized ICG&Cur@ZIF-8 NPs by encapsulating both anticancer drug curcumin (Cur) and photothermal agent indocyanine green (ICG) in ZIF-8 NPs by one-pot method, and thus the synthesized ICG&Cur@ZIF-8 NPs have pH-responsive drug delivery and good photothermal properties.In addition, some enzymes have also been successfully packaged into ZIF-8 NPs.Lyu et al [46] directly embedded cytochrome c (Cyt c) in ZIF-8 NPs by a co-precipitation method.The resulting Cyt c in ZIF-8 NPs showed a 10-fold increase in peroxidase activity compared to its native counterpart in solution.This enabled rapid, highly sensitive, visible detection of trace amounts of highly explosive organic peroxides in solution (figure 6).Wu et al [47] synthesized GOX&HRP/ZIF-8 NPs by encapsulating glucose oxidase (GOX) and horseradish peroxidase (HRP) simultaneously in ZIF-8 NPs using a one-pot method at 25 °C in aqueous solution, and this ZIF-8 NPs-based multi-enzyme system showed high catalytic efficiency, high selectivity and higher stability.

Impregnation method
The impregnation method, in which functional organic molecules are loaded into the pores of MOFs by capillary forces, electrostatic interactions and ligand reactions [48].Zhao et al [49] successfully synthesized Pd/ ZIF-8 NPs using ZIF-8 NPs as the carriers and Pd(NO 3 ) 2 •2H 2 O as the metallic salt by the impregnation method.Compared with conventional inorganic carriers (silica and alumina) and organic carriers, Pd nanoparticles on ZIF-8 carriers exhibited higher catalytic activity and selectivity for hydrocinnamic aldehyde (HCAL) (up to 91.9%).The prepared Pd/ZIF-8 NPs catalysts could be reused at least four times without significant loss of activity or selectivity.Soltani et al [50] used the impregnation method to load gentamicin (GEN) into nanoscale zeolite imidazole framework-8 (NZIF-8) and obtained GEN@NZIF-8 NPs with uniform size and morphology and an average diameter of 200 nm.The antibacterial activity results demonstrated that the antibacterial activity of GEN was enhanced by NZIF-8 encapsulation.
Despite these advances, low loading efficiency is an inherent defect that plagues all passive drug loading techniques based on ZIF-8 NPs.For example, Soltani et al [50] prepared ZIF-8 NPs to achieve a loading efficiency of only 19% for GEN, and Yin et al [51] designed polydopamine-modified ZIF-8 NPs to achieve a drug loading of only 16.45% for MTX.

Active drug loading
Based on the chemical structures of the loading drugs, a novel active loading strategy is presented to promote the loading efficiency.Our previous research demonstrated that the metal coordination-based active drug loading strategy of ZIF-8 NPs exhibited excellent superiority in enhancing the drug loading efficiency.It has been reported that curcumin (an antitumor drug) can chelate transition metal ions via its monoanionic enol form, which can protect curcumin against degradation (the attack of a hydroxide ion at the diketo/enol moiety), thus achieving higher solubility and stability (figure 7) [52][53][54].As depicted in figure 8, curcumin and 2-methylimidazole (organic ligand) were simultaneously coordinated by zinc ions to form Cur-ZIF hybrid complex according to active loading approach.This route had a higher loading capacity and efficiency of 41.4 ± 2.9% and 69.6 ± 2.1%, respectively [55].Unlike passive loading, this active loading has a higher drug loading capacity via a reversible chemical reaction, in which the therapeutic activity of the drug is not affected.Nevertheless, the presence of active groups in drug molecules is essential for active loading.

Surface modification of ZIF-8 NPs
Exogenous nanoparticles suffer from rapid clearance mediated by endocytic cells and the reticuloendothelial system in vivo.Numerous studies have shown that surface modification of ZIF-8 NPs can significantly enhance their encapsulation ability, confer biocompatibility and brand new features and functions.

Biomembrane camouflaging
In addition to these conventional materials, biomembranes can also be employed to functionalize ZIF-8 NPs.These biomembrane-mimetic-based ZIF-8 NPs inherit the membrane composition and complex antigenic spectrum of source cells, and thus represent many excellent properties, such as homotypic targeting, prolonged circulation time and mitigated immunogenicity [63].Therefore, increasing attention has been paid to this biomembrane-mimetic strategy.For example, erythrocyte membrane-coated nanoparticles were capable of immune escape and prolonged blood circulation [64]; cancer cell membrane-camouflaged nanomaterials were designed to target homotypic cancer cells [65]; neutrophil membrane-wrapped nanodrugs were exploited to be recruited to inflammatory sites [66,67]; the biotropism of platelets to vasculature trauma could be utilized to target the residual microtumors after tumor surgical resection [68,69]; macrophage membranes can be  employed to endow nanoparticles tumor-homing abilities [70].Zhang et al [71] developed a MOF-based biomimetic nanoreactor in which glucose oxidase (GOX) and the prodrug tirapazamine (TPZ) were encapsulated in an erythrocyte membrane-camouflaged ZIF-8 NPs to obtain TPZ-GOX-ZIF-8 NPs@erythrocyte membrane (TGZ@eM).This biomimetic strategy reduced immune clearance and promoted nanoparticles to accumulate at tumor tissue through the enhanced permeability and retention (EPR) effect [72].Thus, TGZ@eM has immune escape and prolonged blood circulation properties and can effectively accumulate at tumor tissues for starvation-activated colon cancer therapy (figure 10).

Application of ZIF-8 NPs in smart drug delivery systems
ZIF-8 NPs possess reversible metal coordination bonds that endow them with responsive degradation properties for drug delivery.Moreover, ZIF-8 NPs-based drug loading can attenuate the toxic side effects of drugs.Doxorubicin (DOX), an anthracycline antineoplastic medication, is widely used in various diseases.However, it suffers from some significant flaws that severely limit its clinical use, including a short half-life, poor absorption and, in particular, severe cumulative dose-dependent cardiotoxicity.Furthermore, it has been demonstrated that Zn 2+ can competitively inhibit the ability of anthracyclines to bind to cardiac myosin, lessening the adverse effects of drugs and acting in a cardioprotective manner [73].Vasconcelos et al [74] synthesized DOX-ZIF-8 by integrating doxorubicin into ZIF-8.Compared with pure DOX, DOX-ZIF-8 can compensate for the deficiency of DOX alone and achieve a continuous, slow release of DOX for 30 days with a drug release of 66%.In addition, DOX-ZIF-8 has higher antitumor potential and lower cytotoxicity in HL-60 and MCF-7 cell lines.In view of the above, ZIF-8 NPs are widely used as ideal nanocarriers for drug controlled release based on various strategies.

ATP/pH-responsive release
There are two mechanisms for the inherent responsive degradation of ZIF-8 NPs.One is based on ATP-triggered degradation, which is based on the principle that the coordination between ATP and Zn 2+ is much more muscular than between 2-methylimidazole and Zn 2+ .Thus, ATP and Zn 2+ can promote ZIF-8 NPs disassembly by competitive chelation [75].Taking advantage of this property, Wang et al [76] synthesized multifunctional RhB@ZIF-8 NPs by encapsulating the fluorescent indicator rhodamine B (RhB) in ZIF-8 NPs by a one-step method.RhB@ZIF-8 NPs exhibited good sensitivity and selectivity to ATP and respond rapidly to ATP through competitive ligand interactions, with ZIF-8 NPs decomposition and fluorescence shutdown.Coupled with their good biocompatibility and high cell permeability, RhB@ZIF-8 NPs have been successfully used for real-time monitoring of mitochondrial ATP fluctuations in living cells during photodynamic therapy.
Another type is based on pH-triggered disintegration.ZIF-8 NPs have good stability in neutral pH environments (e.g.blood and normal tissues), however, they can be auto-decomposed into harmless components in acidic environments (tumor tissue conditions) [77].Organic ligands can be protonated under acidic conditions, which lead to the breakdown of ZIF-8 NPs by cleavage of the Zn 2+ -imidazole ion coordination bonds [78,79].Based on this property, Zhang et al [80] constructed a novel delivery system for CpG ODNs by encapsulating CpG oligodeoxynucleotides (CpG ODNs) into ZIF-8 NPs for the first time.The ZIF-8/CpG ODNs complex exhibited good biocompatibility and high stability in a physiological environment but degraded in the acidic environment of endolysosomes to release CpG ODNs.CpG ODNs could activate the immune system and induce Th1 responses by stimulating Toll-like receptor 9 (TLR9) in the acidic endolysosomal compartment, thus enhancing their immunostimulatory activity (figure 11) [81,82].

Redox-responsive release
In normal tissues, intracellular glutathione (GSH) concentration (2∼10 mmol l −1 ) is substantially higher than the extracellular concentration (2∼10 μmol l −1 ), and it has been validated that the concentration of GSH in tumor tissues is at least 4 times higher than that in normal tissues [83][84][85].Therefore, medication release regulated by redox processes also holds enormous promise [86].Ma et al [87] synthesized redox-responsive ZIF-8 NPs/CA@PTX using ZIF-8 NPs as a drug delivery vehicle, cysteamine (CA) as a linker and redox-sensitive material, and paclitaxel (PTX) as an antitumor drug.The cysteamine coated onto the surface of ZIF-8 NPs played a dual role as a linker and responded to redox stimulation due to the amine functional group (-NH2) at the end of its molecular chain and the disulfide bond (S-S) in the middle of the chain.It connected the antitumor drug to the surface of the drug delivery vehicle, while glutathione (GSH) can cleave the disulfide bond, allowing the release of the drug.It was shown that higher GSH concentration and lower pH facilitated the release of PTX.Thus, this redox-responsive paclitaxel delivery system based on metal-organic framework had a remarkable tumoricidal effect.

Ligand receptor targeting release
Targeted ligand modification on the surface of ZIF-8 NPs allows the construction of active targeting drug delivery systems, where specific binding of ligand to target receptors overexpressed on the surface of tumor cells mediates endocytosis and increases drug accumulation in the cells.Hyaluronic acid (HA) is a negatively charged hydrophilic natural polysaccharide abundant in the extracellular matrix and has good biocompatibility and biodegradability.In addition, HA can actively target specific tumor cells and bind to CD44 receptors overexpressed on the surface of tumor cells [88,89].Yu et al [44] prepared Cur@ZIF-8 NPs and Cur@ZIF-8@HA NPs in aqueous solutions.Compared with Cur@ZIF-8 NPs, Cur@ZIF-8@HA NPs could effectively reduce the leakage of Cur in ZIF-8 NPs, minimize the side effects on normal tissues and enhance the sustained release of Cur, contributing to the prolonged drug circulation time in the body and the enhanced chemotherapeutic efficacy.In addition, through the active targeting ability of HA, Cur@ZIF-8@HA NPs could induce more tumor cells uptake and effectively inhibit tumor growth and metastasis.This targeted accumulation and pH-responsive slow release effect endowed Cur@ZIF-8@HA NPs with excellent potential in the treatment of breast cancer.

Glucose-responsive release
Yin et al [90] successfully synthesized GOX-Au@ZIF-8 NPs by wrapping glucose oxidase (GOX), gold nanoparticles (AuNPs) and the hypoglycemic agent metformin in ZIF-8 NPs by a co-precipitation method (i.e.one-pot method).In this system, glucose was first oxidized by oxygen to produce gluconic acid and H 2 O 2 , and then catalyzed by GOX, thereby lowering the surrounding pH, and ZIF-8 NPs thus were degraded to release the loading drug.In addition, the generated H 2 O 2 was converted to H 2 O and O 2 catalyzed by AuNPs, which not only reduced the hazard of reactive oxygen species but also regenerated and recycled oxygen in the oxidation reaction of glucose (figure 12).This glucose-responsive closed-loop drug delivery system had a positive correlation between drug release and glucose concentration, opening avenues for the treatment of diabetes under hypoxic conditions.
In addition to the above responsive releases, light-responsive release [91,92], thermal-responsive release [93], and magnetic-responsive release [94] can be achieved by surface functionalization of ZIF-8 NPs or  introduction of specific functional molecules.In practical applications, two or even three responses can be combined simultaneously to control the precise targeting of drug release, thereby maximizing therapeutic efficacy.

Conclusion and outlook
In this work, the synthesis methods, drug loading strategies and surface modifications of ZIF-8 NPs were systematically reviewed.Especially, we demonstrated the superiority of the metal coordination-based active loading strategy over the physical packaging-based passive loading strategy.We then focused on the applications of ZIF-8 NPs in smart drug delivery systems.Due to their intrinsic ATP/pH-responsive properties, drug-loaded ZIF-8 NPs could release drug with high concentrations in the acidic, ATP-rich tumor environment.In addition, ZIF-8 NPs could be designed as other stimuli-responsive drug delivery systems (e.g.light, heat, magnetic, targeted localization, GSH and hydrogen peroxide stimulation) to control drug release at specific sites, minimize toxic side effects at normal tissue sites and tremendously improve the precision and efficacy of treatment.Although intelligent drug delivery based on ZIF-8 NPs has made significant progress, most studies are still at the proof-of-concept stage, and there are still some thorny issues that we should consider and solve, such as metabolites of ZIF-8 NPs, pharmacokinetic studies and in vivo safety assessment, which need further advanced research.Therefore, there may be three directions to develop the safer and smarter ZIF-8 drug delivery system in the future research: (1) innovate the active drug loading modes via distinct physical and chemical ways to replace traditional passive loading, (2) explore the surface functionalization strategies via biomembrane camouflage or targeted ligand modification to reduce nonspecific distribution in vivo, (3) establish the diversified self-assembly forms via metal chelation to integrate multiple therapeutic blocks.

Figure 2 .
Figure 2. Timeline of key historical events related to ZIF-8 NPs and their applications in biomedicine.

Figure 3 .
Figure 3.The synthesis methods of ZIF-8 NPs and their stimuli-responsive release.

Figure 5 .
Figure 5. Two kinds of drug loading strategies based on ZIF-8 NPs.

Figure 6 .
Figure 6.Preparation of the cytochrome c-embedded ZIF-8.A schematic showing the synthesis of the Cyt c/ZIF-8 composite.TEM images of the Cyt c/ZIF-8 composite obtained after a reaction time of 5 min (a) and 24 h (b) [46].Reproduced with permission from Ref. 46.Copyright © 2014, American Chemical Society.

Figure 8 .
Figure 8. Schematic illustration of the synthetic process of Cur-ZIF NPs and CM/Cur-ZIF@IQ NPs, and the following anti-tumor application[55].Reproduced with permission from[55].Copyright © 2022, The Royal Society of Chemistry.

Figure 10 .
Figure 10.Schematic illustration of the Preparation of the (A) TGZ@eM Nanoreactor and (B) Erythrocyte Membrane Cloaked MOF Biomimetic Nanoreactor for Starvation-Activated Colon Cancer Therapy [71].Reproduced with permission from Ref. 71.Copyright © 2018, American Chemical Society.