**3. Applications of BioMOFs: selected examples**

process favours a rapid and uniform nucleation process, which results into a more homoge‐ neous particle size distribution. Regarding ultrasounds, it has shown to be a highly efficient

Sonochemical synthesis or sonocrystallization method not only promotes the nucleation process but also stimulates the homogeneity of the nucleation, what represents an advantage over the traditional solvothermal methods. This approach is prone for industrial applications

Mechanochemistry is a green, solvent-free and efficient strategy to build MOFs. It is based on the direct grinding of the linkers and the metal salts either in a mortar or in a ball mill, without recurring to solvent (neat grinding, NG) or recurring only to catalytic amounts of solvent to activate the process (liquid-assisted grinding, LAG). Alternatively, also catalytic amounts of ionic salts can be used to trigger the process (ion- and liquid-assisted grinding, ILAG). This is a simple method and the absence of solvent makes it very appealing to biomedical applications

The loading of relevant molecules, such as imaging and therapeutic agents, into MOFs can be

The direct incorporation implies using those molecules directly to assemble the framework. This strategy also encloses the networks in which paramagnetic metal ions, such as Gd3+, Fe3+ and Mn2+, do not act only as the metal sites to connect the ligand but act also as magnetic resonance imaging contrast agents. High loadings of the relevant compounds can be achieved by this strategy; however, it is necessary to tune the morphology and physicochemical properties of these MOFs for each case and it is important to guarantee that there is no

The postsynthesis strategy requires high porosity and the active compound is incorporated within the MOF by noncovalent or covalent interactions. In the case of noncovalent loading, the process is reversible and therefore the drug release can be premature. On the other hand, the covalent loading creates a prodrug in which the drug release happens at the same time as

The improved biomedical properties of MOFs also depend on the rational design of the surface. However, the task of changing the outer surface of the MOF without changing its character‐ istics is still very difficult. Ideally, MOFs should have a coating shell to confer stability to the material under the different physiological media, but it must be non-toxic and must not interfere with the pores [86]. There are two approaches to achieve the surface modifications: covalent and noncovalent attachments. The choice of the best method relies on the parameters and nature of the MOF, as well as on the nature of the molecule to be grafted [88]. To date only a few successful examples have been reported of which we highlight the following three.

the MOF degradation and thus it may be considered a more robust approach [87].

**2.3. Loading of drugs and other biomedically relevant compounds into MOFs**

done directly during the MOF synthesis or in the postsynthesis.

degradation of the compound during the synthesis [21].

method [86].

1406 Metal-Organic Frameworks

[63, 82, 83, 86].

due to its easy scale-up [63].

**2.4. Surface modifications**

The first biomedical applications of nanoscale MOFs were as delivery vehicles for imaging contrast agents and molecular therapeutics. However, the large amount of paramagnetic metal ions in these systems further allows their exploration for magnetic resonance imaging (MRI). [21]. Furthermore, BioMOFs are also being studied as materials for drug storage as well as controlled drug delivery and release. A few examples of such applications are briefly discussed and the details of the mentioned BioMOFs are presented in **Table 1**. For a matter of clarification, examples of different bio-inspired applications of ZIFs are given in the next section.

#### **3.1. Exploring synergetic effects between metal and drug within a BioMOF**

As previously mentioned, one of the best approaches to construct BioMOFs is the direct incorporation of therapeutically active molecules containing multiple complexing groups with biocompatible metal cations (Ca2+, Ag2+, Zn2+, Fe2/3+), and thus the delivery of the active compounds is accomplished via framework degradation [25, 91, 94–97]. Tamames-Tabar et al. recently discussed the possibility of directly introducing azelaic acid as linker and an endog‐ enous low-toxicity transition metal cation (Zn2+) [98]. Both linker and metal exhibit interesting antibacterial and dermatological properties for the dermatological treatment of several skin disorders and their combination results into a novel biocompatible and bioactive MOF, named BioMIL-5. It was synthesized by hydrothermal methods and its stability was assessed through tests in water and in bacteria broth at 37°C; also antibacterial activity studies against two Grampositive bacteria *Staphylococcus aureus* and *Staphylococcus epidermidis* were conducted [91].

In the antibacterial activity studies, the MIC/MBC (MIC = minimal inhibitory concentration; MBC = minimal bactericidal concentration) values in *S. aureus* and *S. epidermidis* demonstrate that the antimicrobial activity of azelaic acid and Zn2+ is maintained after the synthesis [91]. Regarding the stability tests, BioMIL-5 has shown to be stable in water and in bacterial culture medium, but especially in water, leading to the progressive release of both Zn2+ and azelaic acid. Indeed, this progressive and slow release of the active Zn2+ and azelaic acid in both media led to interesting and time-maintained antibacterial properties when used for 7 days against *S. epidermidis* [91]. The high stability demonstrated and the maintenance of its antibacterial properties (**Figure 1**) turn BioMIL-5 into a promising candidate for future applications in the treatment of several skin disorders and in the cosmetic industry [91].


**Table 1.** Details on the presented BioMOFs.

**Figure 1. (Left)** Delivery profile of azelaic acid (AzA) and zinc (Zn) in Mueller Hinton cation adjusted broth or MHCA (M) and in water (W): AzA(M) (pink), AzA(W) (blue), Zn(M) (orange) and Zn(W) (green); **(right)** Bacterial growth curves comparing the control group (orange) with BioMIL-5 at different concentrations (mg mL−1): 0.9 (green), 1.7 (blue), and 4.3 mg mL−1 (pink) after 1 week in *S. epidermidis* (image from Tamames-Tabar et al. [91]—Copyright © 2014, Royal Society of Chemistry).

#### **3.2. BioMOFs for the controlled drug delivery of ibuprofen**

properties (**Figure 1**) turn BioMIL-5 into a promising candidate for future applications in the

**BioMOF Metal Ligand Application Ref**  BioMIL-5 Zn2+ Treatment of skin disorders [93]

MIL-100 Fe3+ Drug delivery of ibuprofen [22, 23]

MIL-101 Fe3+ Drug delivery of ibuprofen [22, 23]

Bio-MOF-1 Zn2+ Drug delivery of procainamide HCl [30]

MIL-53 Fe3+ Drug delivery of ibuprofen – "breathing

CaZol nMOF Ca2+ Drug delivery of Zol – targeted anticancer

IRMOF-3 Zn2+ Drug delivery of Paclitaxel – targeted

**Table 1.** Details on the presented BioMOFs.

effects"

agent

anticancer agent and MRI applications

[22]

[94]

[95]

treatment of several skin disorders and in the cosmetic industry [91].

1428 Metal-Organic Frameworks

Bearing in mind that BioMOFs are envisaged as new tools for the controlled drug delivery [22, 19-23, 25, 26], Horcajada et al. prepared the first examples of MOFs for the delivery of ibuprofen in the gastrointestinal tract: MIL-100 (with trimesic acid) and MIL-101 (with terephthalic acid) [22, 56]. Ibuprofen was chosen as a model drug because it is a worldwide used pharmaceutical compound with analgesic and antipyretic features [56]. Both MOFs have large pores: MIL-100 contains pore diameters of 25–29 Å with pentagonal window openings of 4.8 Å, and hexagonal windows of 8.6 Å; MIL-101 contains 29–34 Å pore diameter with a large window opening of 12 Å for the pentagonal and 16 Å for the hexagonal windows. They exhibit a very high drug storage capacity: up to 0.35 g of ibuprofen per gram of porous solid for MIL-100 and 1.4 g of ibuprofen per gram of porous solid for MIL-101 [22, 23, 25, 56]. MIL-101 displays a higher loading capacity due to the fact that ibuprofen can fit in both pentagonal and hexagonal windows of MIL-101, but not into the smaller pentagonal window of MIL-100 [22, 23, 25]. This demonstrates the real importance of material's pore size in drug loading [25, 50]. The kinetics of ibuprofen delivery to stimulated body fluid at 37°C was also studied, revealing a complete drug controlled release from 3 to 6 days [22, 23, 25].

#### **3.3. Cation-triggered release of procainamide HCl from BioMOF-1**

Another example of a BioMOF constructed by the direct incorporation of simple biomolecules and biocompatible metal cations in their structures is Bio-MOF-1 proposed by An et al. [30] Bio-MOF-1 is based on (i) adenine, a purine nucleobase, as a biomolecular ligand, (ii) a second ligand, biphenyldicarboxylic acid, which was used to promote the formation of larger accessible pores, and (iii) Zn2+ as a biocompatible metal cation [30]. Bio-MOF-1 has shown to be stable and maintains its crystallinity for several weeks in biological buffers. Due to the intrinsic anionic nature of Bio-MOF-1, An et al. explored its potential use as a system for the storage and release of cationic drug molecules [30], more specifically the storage and release of procainamide HCl, an effective antiarrhythmic agent used to treat a variety of atrial and ventricular dysrhythmias with a short half-life *in vivo* making necessary its administration every 3–4 h [29, 30, 99]. Procainamide HCl was successfully encapsulated into the pores of Bio-MOF-1 through a cation exchange process and the complete loading (0.22 g/g material) was achieved after 15 days corresponding to approximately 2.5 procainamide molecules per formula unit residing in the pores and 1 procainamide molecule at the exterior surface [29, 30]. Due to the ionic interaction between procainamide and Bio-MOF-1, cationic drugs are triggered by cations and then released from the framework. Steady procainamide release was observed within 20 h and a complete release was observed after 72 h (**Figure 2**) [30].

**Figure 2. (Left)** Scheme depicting cation-triggered procainamide release from Bio-MOF-1; **(right)** procainamide release profiles from Bio-MOF-1 (blue—PBS buffer; red—deionised nanopure water) (image from An et al. [30]—Copyright © 2009, American Chemical Society).

#### **3.4. Exploring the potentialities of the breathing effects on BioMOFs**

Some MOFs can present structural flexibility or "breathing effects," which allows them to modulate their pore size upon adsorption of organic molecules into the pores, while their crystallinity is maintained [22, 50, 54, 59]. One example of BioMOFs presenting a "breathing effect" is MIL-53 [22, 54, 100]. The structure of MIL-53 consists on terephatalate anions and trans-chains of metal (III) octahedra sharing OH groups and thus creating a 3D framework with one-dimensional pore channel systems [22, 100]. The capacity to expand its structure upon heating explains the ``breathing effect'' observed in MIL-53 (**Figure 3**) [22]. In this study, Horcajada et al. also observed that aluminium and chromium MIL-53lt (lt is low temperature) present a reversible pore opening involving atomic displacements by 5.2 Å upon dehydration, whereas the iron analogue only open its pores during the adsorption molecules [101, 102]. This can be explained by the formation of hydrogen bonds between the water molecules and the inorganic hydrophilic parts of the pore. After approximately 3 weeks, a complete release of ibuprofen is observed, where 20 wt% of ibuprofen loading was achieved at high temperature (**Figure 3**) [22].

#### **3.5. pH-responsive BioMOFs**

An interesting example that shows the potential use of BioMOFs in biomedical applications is the recently disclosed work of Au et al. which is based on the reformulation of zoledronate (Zol) exploring nanotechnology to develop a new nanoscale MOF (nMOFs) formulation of Zol, turning a bone antiresorptive agent into an anticancer agent [92].

Zol is a third-generation nitrogen heterocycle containing bisphosphonate that is widely used as an antiresorptive agent for bone cancer metastasis. In the preclinical data, it was observed that bisphosphonates such as Zol have direct cytotoxic effects on cancer cells. However, such effect has not been firmly established in the clinical settings, what led Au et al. to develop a new bioresorbable sub-100 nm diameter pH-responsive calcium zoledronate (CaZol) nMOF as a potential cytotoxic anticancer agent. Folate receptor (FR) is known to be overexpressed in tumours, and therefore folate (Fol) was incorporated as a target ligand into the CaZol nMOFs to facilitate tumour uptake. This study successfully demonstrated that the active-targeted CaZol nMOF possesses excellent chemical and colloidal stability on physiological conditions, encapsulating more Zol than other existing drug delivery systems. It further shows higher efficiency than small molecule Zol in inhibiting cell proliferation and inducing apoptosis in FR-overexpressing H460 non-small cell lung and PC3 prostate cancer cells *in vitro*. Au et al. also validated these results *in vivo* and observed that Fol-targeted CaZol nMOF proved to be an effective anticancer agent, increasing the direct antitumour activity of Zol by 80–85% [92].

ventricular dysrhythmias with a short half-life *in vivo* making necessary its administration every 3–4 h [29, 30, 99]. Procainamide HCl was successfully encapsulated into the pores of Bio-MOF-1 through a cation exchange process and the complete loading (0.22 g/g material) was achieved after 15 days corresponding to approximately 2.5 procainamide molecules per formula unit residing in the pores and 1 procainamide molecule at the exterior surface [29, 30]. Due to the ionic interaction between procainamide and Bio-MOF-1, cationic drugs are triggered by cations and then released from the framework. Steady procainamide release was

**Figure 2. (Left)** Scheme depicting cation-triggered procainamide release from Bio-MOF-1; **(right)** procainamide release profiles from Bio-MOF-1 (blue—PBS buffer; red—deionised nanopure water) (image from An et al. [30]—Copyright ©

Some MOFs can present structural flexibility or "breathing effects," which allows them to modulate their pore size upon adsorption of organic molecules into the pores, while their crystallinity is maintained [22, 50, 54, 59]. One example of BioMOFs presenting a "breathing effect" is MIL-53 [22, 54, 100]. The structure of MIL-53 consists on terephatalate anions and trans-chains of metal (III) octahedra sharing OH groups and thus creating a 3D framework with one-dimensional pore channel systems [22, 100]. The capacity to expand its structure upon heating explains the ``breathing effect'' observed in MIL-53 (**Figure 3**) [22]. In this study, Horcajada et al. also observed that aluminium and chromium MIL-53lt (lt is low temperature) present a reversible pore opening involving atomic displacements by 5.2 Å upon dehydration, whereas the iron analogue only open its pores during the adsorption molecules [101, 102]. This can be explained by the formation of hydrogen bonds between the water molecules and the inorganic hydrophilic parts of the pore. After approximately 3 weeks, a complete release of ibuprofen is observed, where 20 wt% of ibuprofen loading was achieved at high temperature

An interesting example that shows the potential use of BioMOFs in biomedical applications is the recently disclosed work of Au et al. which is based on the reformulation of zoledronate (Zol) exploring nanotechnology to develop a new nanoscale MOF (nMOFs) formulation of Zol,

turning a bone antiresorptive agent into an anticancer agent [92].

**3.4. Exploring the potentialities of the breathing effects on BioMOFs**

2009, American Chemical Society).

14410 Metal-Organic Frameworks

(**Figure 3**) [22].

**3.5. pH-responsive BioMOFs**

observed within 20 h and a complete release was observed after 72 h (**Figure 2**) [30].

#### **3.6. Magnetic nanoscale MOF as potential anticancer drug delivery system, and imaging and MRI contrast agent**

The combination of both imaging and therapeutic agents in the same MOF greatly facilitates the efficacy studies of theranostic nanoparticles. Having this in mind, Chowdhuri et al. developed a new magnetic nanoscale MOF (IRMOF-3) consisting of a MOF with encapsulated

**Figure 3. (Top)** Schematic 3D representation of the breathing effect of MIL-53(Cr) hybrid solid upon dehydration-hy‐ dration; **(bottom)** ibuprofen delivery **(left)** from MIL-53(Cr) and MIL-53(Fe) materials and **(right)** from MIL-53 in com‐ parison with MIL-101, MCM-41 and MCM-4 (images from Horcajada et al. [22]—Copyright © 2008, American Chemical Society).

Fe3O4 nanoparticles for targeted anticancer drug delivery with cell imaging and magnetic resonance imaging (MRI). More specifically, authors conjugated the magnetic nanoscale MOF with folic acid and labelled it with the fluorescent molecule rhodamine B isothiocyanate due to its fluorescent properties. These systems were then successfully loaded with the hydropho‐ bic anticancer drug paclitaxel. The efficiency of this nMOF towards targeted drug delivery was evaluated using an *in vitro* cytotoxicity 5-diphenyltretrazolium bromide (MTT) assay and fluorescence microscopy, revealing that the loaded nMOF targeted and killed the cancer cells in a highly effective manner. Furthermore, they had also tested the effectiveness of MRI of this nMOF *in vitro* and observed a stronger T2-weighted MRI contrast towards the cancer cells, which proved the possible use of this system in imaging (**Figure 4**) [93].

**Figure 4. (Left)** *In vitro* T2-weighted spin-echo MR phantom images of magnetic nanoscale Fe3O4@IRMOF-3 and mag‐ netic nanoscale Fe3O4@IRMOF-3/FA at different concentrations incubated in HeLa cells; **(right)** *in vitro* paclitaxel re‐ lease from magnetic nanoscale Fe3O4@IRMOF-3/FA at different time intervals (images from Chowdhuri et al. [93]— Copyright © 2015, Royal Society of Chemistry).

### **4. Bio-inspired applications of ZIFs: selected examples**

There are many applications for ZIFs, specifically ZIF-8 (**Figure 5**). However, this type of materials has largely been explored as a way to deliver anticancer drugs and other chemo‐ therapeutics. Only a few relevant examples are mentioned herein.

**Figure 5.** (a) Synthesis of ZIF-8; (b) fragment of the crystal structure of ZIF-8 (images adapted from Katsenis et al. [103] —Copyright © 2015, Rights Managed by Nature Publishing Group); and (c) image generated for ZIF-8 in http:// www.chemtube3d.com (University of Liverpool).

#### **4.1. Slow release of the anti-cancer drug doxorubicin from ZIF-8**

Fe3O4 nanoparticles for targeted anticancer drug delivery with cell imaging and magnetic resonance imaging (MRI). More specifically, authors conjugated the magnetic nanoscale MOF with folic acid and labelled it with the fluorescent molecule rhodamine B isothiocyanate due to its fluorescent properties. These systems were then successfully loaded with the hydropho‐ bic anticancer drug paclitaxel. The efficiency of this nMOF towards targeted drug delivery was evaluated using an *in vitro* cytotoxicity 5-diphenyltretrazolium bromide (MTT) assay and fluorescence microscopy, revealing that the loaded nMOF targeted and killed the cancer cells in a highly effective manner. Furthermore, they had also tested the effectiveness of MRI of this nMOF *in vitro* and observed a stronger T2-weighted MRI contrast towards the cancer cells,

**Figure 4. (Left)** *In vitro* T2-weighted spin-echo MR phantom images of magnetic nanoscale Fe3O4@IRMOF-3 and mag‐ netic nanoscale Fe3O4@IRMOF-3/FA at different concentrations incubated in HeLa cells; **(right)** *in vitro* paclitaxel re‐ lease from magnetic nanoscale Fe3O4@IRMOF-3/FA at different time intervals (images from Chowdhuri et al. [93]—

There are many applications for ZIFs, specifically ZIF-8 (**Figure 5**). However, this type of materials has largely been explored as a way to deliver anticancer drugs and other chemo‐

**Figure 5.** (a) Synthesis of ZIF-8; (b) fragment of the crystal structure of ZIF-8 (images adapted from Katsenis et al. [103] —Copyright © 2015, Rights Managed by Nature Publishing Group); and (c) image generated for ZIF-8 in http://

which proved the possible use of this system in imaging (**Figure 4**) [93].

**4. Bio-inspired applications of ZIFs: selected examples**

therapeutics. Only a few relevant examples are mentioned herein.

Copyright © 2015, Royal Society of Chemistry).

14612 Metal-Organic Frameworks

www.chemtube3d.com (University of Liverpool).

Zheng et al. successfully developed a simple one-pot synthesis of ZIFs that contain encapsu‐ lated organic molecules. One-pot synthesis is a new approach that combines MOF synthesis and molecule encapsulation in a one-pot process and that has been extremely used to overcome the drawbacks observed when using the two processes separately [104].

In this study, the doxorubicin: ZIF-8 complex, which aims to treat mucoepidermoid carcinoma of human lung, human colorectal adenocarcinoma (HT-29) and human promyelocytic leukaemia (HL-60) cell lines, exhibits lower toxicity than pure doxorubicin, probably due to the slow release of the drug that is achieved with this complex (**Figure 6**) [69, 104]. Furthermore, ZIF-8 crystals loaded with doxorubicin proved to be efficient pH-responsive drug delivery systems, in which the drug is released in a controlled manner at low pH (5.0–6.5). With this work, Zheng et al. opened a new opportunity to develop multifunctional materials for biomedical applications using this simple, scalable, and environment-friendly one-pot synthesis [104].

**Figure 6. (Top)** Schematic representation of the pH-induced one-pot synthesis of MOFs with encapsulated target mole‐ cules; **(bottom left)** The pH-responsive release of doxorubicin from doxorubicin@ZIF-8 particles determined by UV-vis spectrophotometry; **(bottom right)** TEM image of an MDA-MB-468 cell; and the inset is an enlarged image of the area marked by the square showing individual ZIF-8 particles (blue arrows) and their aggregates (red arrows) (image from Zheng et al. [104]—Copyright © 2016, American Chemical Society).

#### **4.2. ZIF-8 as efficient pH-sensitive drug delivery**

"Smart" drug delivery of anticancer drugs is being explored making use of pH-sensitive systems [65–68]. The interest in the use of a pH-responsive drug vehicle is due to the fact that they can reduce undesired drug release during transportation in blood circulation and improve the effective release of the drug in the tumour tissue or within tumour cells [105, 106].

Sun et al. evaluated the possibility to use ZIF-8 as a pH-responsive drug vehicle and they have demonstrated that ZIF-8 exhibits a remarkable loading capacity for the anticancer drug 5 fluorouracil (around 600 mg of 5 FU g−1 of desolvated ZIF-8) (**Figure 7**) [66]. Ren et al. further developed polyacrylic acid@ZIF-8 (PAA@ZIF-8) nanoparticles that exhibit ultrahigh doxoru‐ bicin loading capability (1.9 g doxorubicin/g nanoparticles) and that thus can be used as pHdependent drug delivery vehicles [65].

**Figure 7. (Left)** Schematic illustration showing two approaches of the encapsulated 5-Fu released from ZIF-8 (C = grey, N = blue, O = red, F = light blue, Zn = green); **(right)** 5-Fu delivery (% 5-FU vs. t) from ZIF-8; the inset shows the release process from 0 to 24 h (images from Sun et al. [66]—Copyright © 2012, Royal Society of Chemistry).

**Figure 8.** Schematic representation of the synthetic route of the C-dots@ZIF-8 for simultaneous anticancer drug deliv‐ ery and fluorescence imaging of cancer cell (image from He et al. [67]—Copyright © 2014, Royal Society of Chemistry).

Zhuang et al. successfully encapsulated small molecules, such as fluorescein and the anticancer drug camptothecin, in ZIF-8 nanospheres for drug delivery. In this study, the evaluation of fluorescein-encapsulated ZIF-8 in the MCF-7 breast cancer line demonstrated cell internaliza‐ tion and a minimal cytotoxicity. Furthermore, the pH-responsive dissociation of the ZIF-8 framework likely results in endosomal release of the small-molecule cargo proved that ZIF-8 can be an ideal drug delivery vehicle [68].

Another example of a pH-responsive drug vehicle using ZIF-8 is the work of Liu et al., who fabricate green fluorescent carbon nanodots@ZIF-8 (c-dots@ZIF-8 NPs). In this work, the authors observed that the nanoparticles synthesized exhibit green fluorescence and micro‐ porosity, characteristics that unveil its ability as potential platforms for simultaneous pHresponsive anticancer drug vehicle and fluorescence imaging in cancer cells (**Figure 8**). Moreover, the fluorescence intensity and size of c-dots@ZIF-8 NPs can be tuned by varying the amount of C-dots and the concentration of the precursors [67].

#### **4.3. ZIFs as potential carriers to brain capillary endothelial cells**

Sun et al. evaluated the possibility to use ZIF-8 as a pH-responsive drug vehicle and they have demonstrated that ZIF-8 exhibits a remarkable loading capacity for the anticancer drug 5 fluorouracil (around 600 mg of 5 FU g−1 of desolvated ZIF-8) (**Figure 7**) [66]. Ren et al. further developed polyacrylic acid@ZIF-8 (PAA@ZIF-8) nanoparticles that exhibit ultrahigh doxoru‐ bicin loading capability (1.9 g doxorubicin/g nanoparticles) and that thus can be used as pH-

**Figure 7. (Left)** Schematic illustration showing two approaches of the encapsulated 5-Fu released from ZIF-8 (C = grey, N = blue, O = red, F = light blue, Zn = green); **(right)** 5-Fu delivery (% 5-FU vs. t) from ZIF-8; the inset shows the release

**Figure 8.** Schematic representation of the synthetic route of the C-dots@ZIF-8 for simultaneous anticancer drug deliv‐ ery and fluorescence imaging of cancer cell (image from He et al. [67]—Copyright © 2014, Royal Society of Chemistry).

Zhuang et al. successfully encapsulated small molecules, such as fluorescein and the anticancer drug camptothecin, in ZIF-8 nanospheres for drug delivery. In this study, the evaluation of fluorescein-encapsulated ZIF-8 in the MCF-7 breast cancer line demonstrated cell internaliza‐ tion and a minimal cytotoxicity. Furthermore, the pH-responsive dissociation of the ZIF-8

process from 0 to 24 h (images from Sun et al. [66]—Copyright © 2012, Royal Society of Chemistry).

dependent drug delivery vehicles [65].

14814 Metal-Organic Frameworks

One extraordinary example of the biomedical applications of ZIFs is the recent work from Chiacchia et al. who synthesized and characterized nanospheres of biodegradable zincimidazolate polymers (ZIPs) as a delivery system into human brain endothelial cells, the main component of the blood-brain barrier (BBB) [107].

**Figure 9.** Synthesis and assembly of loaded ZIP particles and their uptake into human brain endothelial cells: (I) encap‐ sulation process of cargo species into the ZIP matrices at the point of synthesis; (II) cross-section of the human cerebral microvasculature and cell-uptake of loaded ZIP particles by the isolated and immortalized human brain endothelial cell line (image from Chiacchia et al. [107]—Published by The Royal Society of Chemistry).

In this work, both biodegradable particles synthesized, RhB@ZIP and AuNP@ZIP, have shown to be able to encapsulate fluorophores and inorganic nanoparticles at the point of synthesis with extremely high loading efficiencies. Furthermore, these ZIP particles are non-cytotoxic, stable in cell culture medium and able to penetrate the hCME\D3 human cerebral microvas‐ cular endothelial cell line. This cell line is a well-established *in vitro* functional model for the human BBB, which expresses the same levels of transporters, cell-specific receptors and tight junction proteins found in healthy human brain microvessels [108, 109], to release their cargos within the cell cytoplasm (**Figure 9**) [107].

Nevertheless this work needs more studies related to the exact cellular uptake mechanism, clearance rate and blood-stream stability of the ZIPs, but this is a promising result in the use of ZIPs as a novel platform for brain-targeting treatments [107].
