Mechanochemical approaches towards the in situ confinement of 5-FU anti-cancer drug within MIL-100 (Fe) metal-organic framework

We elucidate two solvent-free mechanochemical methods to achieve one-pot encapsulation of anti-cancer drug 5-Fluorouracil (5-FU) in the iron-based MIL-100 metal-organic framework (MOF). We compare the structural and physicochemical properties of drug@MIL-100 systems derived from in situ manual and vortex grinding, where the former exhibits a slower drug release due to stronger guest-host interactions.

A few studies have reported the use of mechanochemistry for the synthesis of MIL-100 (Fe) and other MOFs, where a milling technique has been used to accomplish neat or liquidassisted grinding to fabricate this porous material. 3 The mechanochemical approach can help to mitigate the harsh conditions (i.e. high-pressure and -temperature paired with the use of nitric (HNO3) and hydrofluoric acids (HF)) conventionally applied in the synthesis MIL-100 (Fe), while greatly reducing the use of toxic and costly organic solvents. 4 The use of these noxious compounds in material synthesis can hinder the prospective bioapplications of this promising host framework.
Although the mechanochemical synthesis of pristine MIL-100 (Fe) material has been demonstrated before, one-pot encapsulation of guest molecules into this Fe-based MOF via mechanochemistry is less understood. Specifically, for MOFs such as MIL-100 (Fe) possessing highly accessible coordinatively unsaturated metal sites (CUS) that act as a strong binding site for guest drug molecules, the solvent-free encapsulation method can eliminate the competition drug molecules might face against the polar solvents for the active binding sites. 5 This is particularly relevant when compared to most commonly applied ex situ encapsulation method, based on the immersion of a pre-synthesized host MOF into a saturated solution of the guest drug for encapsulation by a slow diffusion process. 6 In this work, we report the detailed fabrication of drug@MOF systems using the solvent-free mechanochemical approach. We compare two different mechanochemical methods (manual grinding and automated vortex grinding) to accomplish the in situ encapsulation of the anti-cancer drug: 5-fluorouracil (5-FU), within the pores of the MIL-100 (Fe) host framework, producing the 5-FU@MIL-100 composite systems. We demonstrate its feasibility as a facile solvent-free one-pot strategy. We have selected 5-FU, a commercial anti-cancer drug, as a model guest molecule due to its structural characteristics (i.e. active polar sites) and its hydrophilic character, preventing it from passing through cell membranes without the aid of a drug delivery system. 7 Our manual and vortex grinding strategies were adapted from Stojakovic et al. 8 and Han et al. 9 to enable in situ drug encapsulation, not yet demonstrated in the former studies. Details of the synthetic routes are presented in the ESI.
Figs. 1a-g illustrate the mechanochemical in situ encapsulation process. For the manual grinding approach, all reactants (i.e. organic linker H3BTC, iron nitrate, and 5-FU) were manually ground using a mortar and pestle. We have designed and 3D-printed a customized holder to couple a standard polypropylene container to an automatic vortex mixer (Figs. 1b-e). This setup allowed us to apply a higher number of rotations per minute via the vortex mixer, increasing the number of collisions induced between the solid reactants and the stainless-steel spheres ( Fig. 1f-g).  shows that 5-FU@MIL-100_VG and 5-FU@MIL-100_MG present relatively sharper peaks (higher crystallinity) compared with their pristine counterparts. This might be attributed to the role played by 5-FU molecules in the deprotonation of the H3BTC organic linkers, because the nitrogen atoms of 5-FU are proton acceptor sites (see Fig. S1b).
The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of all the samples are presented in Fig. 1j.
The vibrational data allow us to investigate in detail the chemical structure of the samples in the mid-infrared region. The characteristic bands of MIL-100 (Fe) (i.e. (C-H) in the organic linker at 707 cm -1 and 760 cm -1 , ν(O-C-O) and δ(O-H) at 1371 cm -1 and 1441 cm -1 , and ν(C=O) in the carboxylic group at 1623 cm -1 ) 11 were detected, confirming retainment of the chemical bond integrity after 5-FU loading via both encapsulation techniques. The spectrum of unencapsulated 5-FU enables the identification of the drug bands in the drug@MOF systems from experiments and theory (density functional theory calculations) ( Figure S3). The spectra of 5-FU@MIL-100_MG and 5-FU@MIL-100_VG display changes in the regions of 800-1300 cm -1 and 1680-1800 cm -1 , in comparison to the spectrum of pristine MIL-100 (Fe), confirming the presence of drug molecules in the drug@MOF assemblies. However, there are salient differences between the spectra of the two different drug-loaded systems, indicating distinct guest-host interactions resulting from the two encapsulation techniques. In 5-FU@MIL-100_VG spectrum the modes at 945 cm -1 , 995 cm -1 , and 1140 cm -1 associated to δ(N-H) and ν(C-N-H), respectively, are evident. In contrast, in the 5-FU@MIL-100_MG spectrum these modes are more supressed while the vibrations at 810 cm -1 , 1210 cm -1 , and 1247cm -1 , associated with δ-ring and ν(C-F), respectively, are apparent (see Figs. S2 and S3 in the ESI for details and schematic representation of the vibrational modes). These observations suggest the higher efficacy of the manual grinding encapsulation process towards the successful confinement of 5-FU drug molecules within the MIL-100 (Fe) pores.
The level of guest encapsulation was evaluated by Brunauer-Emmett-Teller (BET) surface area from N2 adsorption isotherms (Fig. 2a), and further estimated by thermogravimetric analysis (TGA) (Fig. 2b). The surface area of the pristine MIL-100 (Fe)_MG (793 m 2 g -1 ) and MIL-100 (Fe)_VG (753 m 2 g -1 ) are in good agreement with the values of other reported mechanochemically synthesized MIL-100 (Fe) samples (Table S2, ESI). We have determined the drug loading in 5-FU@MIL-100_MG and 5-FU@MIL-100_VG to be 20.2 wt.% (0.25 g/g MOF) and 18.3 wt.% (0.22 g/g MOF), respectively. The BET surface area of the drug@MOF samples was found to be greatly reduced to 333 m 2 g -1 (reduction of ~58 %) and 697 m 2 g -1 (reduction of ~7.5 %), respectively. These results further indicate that although the vortex grinding is very effective for the synthesis of pristine MIL-100 (Fe) MG, the manual grinding process appears to be more efficient for the encapsulation of 5-FU within the MOF pores. As indicated by the FTIR vibrational data, we hypothesize that the C=O⋯Fe coordination strongly limits the displacement of nitrogen atoms, thus suppressing the δ(N-H) and δ(C-N-H) modes in 5-FU@MIL-100_MG (Fig. 1j). Conversely, the identification of drug molecule "free" motions in the 5-FU@MIL-100_VG spectrum suggests that 5-FU molecules could be adsorbed onto the external surfaces of the MIL-100 crystals by weaker intermolecular interactions (e.g. π-π interactions between 5-FU and BTC linker).
An increase in the thermal stability of 5-FU@MIL-100_MG and 5-FU@MIL-100_VG was observed when compared to MIL-100 (Fe) MG and MIL-100 (Fe) VG samples (see Table S3 in the ESI for further detail). This can be attributed to the improved crystallinity presented by the drug@MOF systems in comparison to their pristine counterparts (see Fig 1i).
The samples morphology was examined via scanning electron microscopy (SEM) as presented in Figs. S4-S5. SEM images show no significant changes upon 5-FU loading. The nature of the grinding process appears to favour the formation of fragmented aggregates of MIL-100 (Fe) crystals, which have a non-uniform distribution (ca. 2-30 µm). Fig. 3 shows results of the drug release study of 5-FU from MIL-100 (Fe), conducted in a phosphate buffered saline (PBS) of pH 7.4 used to simulate physiological conditions. We track the evolution of the 266 nm absorption band of 5-FU using UV-Vis to construct a calibration curve (Figs. 3a-b) to determine the cumulative release of the drug molecules. Drug release profiles obtained for the drug-loaded samples are presented in Fig. 3c. Interestingly, 5-FU@MIL-100_MG shows a significantly slower showing material decomposition as a function of increasing temperature. Insets show the derivative weight change with respect to temperature. Note that the decrease of the temperature in the TGA plots over 300 ºC is an due to highly exothermic process related to the departure of the organic linker, exceeding the set program temperature. The furnace temperature thus decreases in order to follow the initial program temperature. 6 release of 5-FU. During the first release stage (initial 5 hours), we detected a drug release of ~60% from the vortex grinding sample in comparison to ~26% released from the manual grinding counterpart. The percentages were calculated by considering the total loading of the drug in the drug@MOF systems, determined by TGA. The second release stage (i.e. up to 72 hours) happened in a considerably slower fashion, which stabilizes after 48 hours. 62% and 34% of the 5-FU loaded into 5-FU@MIL-100_VG and 5-FU@MIL-100_MG were released, respectively. Fitted curves of the release profiles revealed that the release process follows a generalized hyperbolic curve for both samples (see Table S4 in ESI for further details). We assessed the stability of the host framework during the release experiments ( Fig. S6-S7) to ensure the ongoing stability of the host material during the release of the guest molecules. UV-Vis spectra of MIL-100 (Fe) (Fig. S6) show no changes in the absorbance bands of the MIL-100 host during the drug release measurement. Likewise, PXRD patterns of the host collected after the release experiment (Fig. S7a) display no signs of MIL-100 (Fe) degradation. The FWHM measured before and after the release process (Fig. S7b) shows a slight broadening of the (022) peak. However, no evidence of amorphization was found.
The data from the 5-FU release study indicate that the manual grinding technique provides a more effective nanoconfinement of 5-FU within the pores of MIL-100 (Fe), while the vortex grinding approach appears to yield weaker guest-host interactions due to adsorption of guest molecules outside the cages of the host framework. Specifically, this explains the large difference in the amount released in the first hour (doubling that of the vortex ground sample). A slower diffusion at the molecular level throughout the pores is usually associated with strong guest-host interactions. 6 Consequently, the slower release kinetics of 5-FU from 5-FU@MIL-100_MG indicates stronger guest-host interactions, further supporting the notion that the 5-FU molecules are bound to the CUS through C=O⋯Fe coordination. The conditions for the establishment of such strong coordination seem to be favoured by the continuous and homogenous grinding provided by the manual grinding approach, but disfavoured by the localized collisions that occur during the vortex grinding process.
In summary, we have demonstrated that mechanochemistry offers new opportunities for the fabrication of complex MOFs such as MIL-100, and more specifically it is a promising strategy for the nanoconfinement of guest drug molecules within the MOF porosity. By leveraging two mechanochemical based techniques (i.e. manual and vortex grinding) for the encapsulation of 5-FU anti-cancer drug in MIL-100 (Fe), we show modifications in the vibrational behaviour of each drug@MOF assembly, revealing the underpinning intermolecular interactions arising from each encapsulation technique. The outcomes are reflected in the release kinetics of 5-FU from the MIL-100 host, in which the stronger guest-host interactions of the manually ground system led to a slower release of the 5-FU drug. Finally, we found that while the vortex grinding approach we used is very effective for the fabrication of pristine MIL-100 (Fe), a continuous grinding/milling process (i.e. manual grinding) allows the enhanced entrapment of the guest molecules. We hope the findings will instigate further development of 'green' solventfree approaches for controlled binding and release of drug molecules from promising drug@MOF composite systems.
B.E.S. thanks the Minas Gerais Research Foundation (FAPEMIG CNPJ n21.949.888/0001-83) for a DPhil scholarship award. J.C.T. thanks the ERC Consolidator Grant under the grant agreement 771575 (PROMOFS) for research funding. We thank Professor Ronald Roy and Dr Jason Raymond in the Department of Engineering Science, University of Oxford, for access to the UV-Vis spectrometer. We thank the Research Complex at Harwell (RCaH) for access to the TGA-Q50 and the Nicolet iS10 FTIR spectrometer.

Conflicts of interest
There are no conflicts to declare.