Metal-organic frameworks (MOFs) are a uniquely valuable class of materials, in that their structure and composition can be controlled at the atomic level. As the name suggests, MOFs are made of metal ions, or clusters (inorganic), and organic molecular building units, assembling together into crystalline solids [1
]. This construction from building units and the nearly endless pool of different organic and inorganic units to choose from give MOFs their incredible chemical flexibility, and give us control on the atomic level. Each possible building unit contributes its own properties and features, which allows us to make MOFs that have very specific degrees of porosity (pore and window size), surface area, functionality and biocompatibility [5
]. All these attributes make them highly interesting to develop nanostructured smart drug delivery systems capable of bypassing extra- and intracellular barriers [7
In the last 5 years, a number of pioneering studies have been reported that highlight the suitability of MOF nanocarriers as a new type of platform for drug delivery [9
]. So far, these reports have mainly focused on the delivery of single active agents (e.g., one drug), whereas their application to deliver “cocktails” of drugs is still largely unexplored [7
Current chemotherapy faces the challenge that tumours quickly become resistant to a drug during treatment. One possible solution to this problem is the administration of multiple drugs at once to fight resistant cancer strains and prevent formation of new resistances [29
]. This combination therapy has proven to be more effective than single-drug therapies, but faces the challenge that each drug has different physicochemical properties, which leads to heterogeneous pharmacokinetics and tissue distribution.
In this respect, the use of nanocarriers opens up the possibility of co-encapsulating multiple drugs, and thus synchronising their delivery to the cancer cells [29
]. MOF nanoparticle platforms are especially interesting due to their hybrid nature, relying on the synergistic combination of inorganic and organic chemistry [7
]. This allows the creation of chemically diverse internal pore systems able to incorporate drugs with different physicochemical properties. First pioneering studies reporting on such MOF platforms for the delivery of several drugs are very encouraging [32
]. One of them even reports on an enhanced efficiency in tumour reduction due to dual drug delivery with MOF nanoparticles [35
]. While this study shows great promise, the employed nanoparticles were not fully encapsulated. Such an encapsulation would be desirable, though, to prevent the observed drug leakage and to enhance the stability [36
In this paper, we demonstrate that MOF nanoparticles can be simultaneously loaded with multiple drugs. Furthermore, the drug carriers can then be coated with a lipid shell acting as a temporary seal for the encapsulated drugs, and allowing control of interactions with intracellular fluids. The successful synthesis of liposome-coated MOF nanoparticles is based on a simple fusion method. The resulting particles, once loaded, show no premature leakage. As opposed to a previous study [24
], the MOF nanoparticles presented here also show an efficient intracellular release. In our study we focus on iron-based MOF nanoparticles, namely MIL-88A, which are composed of iron(III) and fumaric acid, both naturally occurring in the body [38
]. These particles were loaded with Suberoyl bis-hydroxamic acid (SBHA) alone, or the two drugs irinotecan and floxuridine together. The two latter drugs were chosen because past studies have shown an improved efficacy in preclinical tumour models [39
], making them interesting candidates for the use in combination therapy. Liposomes loaded with both drugs in a 1:1 ratio are currently in an ongoing clinical trial under the name CPX-1 [40
In conclusion, the Lip-MIL-88A platform is a promising tool for use as a platform for combination therapy. It could readily take up a significant amount of biologically active agents (20 wt %) and showed a promising release of its cargo. Due to the liposome coating, no leakage of the cargo could be observed. In cell experiments, Lip-MIL-88A nanoparticles were readily taken up by cells and showed a significant intracellular release after three to four days of incubation. Different drugs (SBHA, irinotecan and floxuridine) were successfully loaded into the particles, and the drug delivery system enables intracellular release profiles. The successful and uncomplicated loading, as well as the effective intracellular release of drugs compared to normal liposome loading procedures, is a promising start for future applications in combination therapy and could as such contribute to improve cancer chemotherapy.
4. Materials and Methods
All chemicals were purchased from Sigma Aldritch (St. Louis, MO, USA) unless noted otherwise.
4.1. UV/Vis Measurements
The UV/Vis measurements were performed on a Lambda 1050 UV/Vis/NIR spectrometer form Perkin Elmer (Waltham, MA, USA). The software used to record the measured spectra was Perkin Elmer UVWinLab (Waltham, MA, USA). For the measurements, the loaded coated particles were dissolved in ALF to facilitate the release of all their cargo. 1 mL of this solution was then diluted with 2 mL H2O yielding a total of 3 mL and measured in a quartz cuvette. For the pure drugs, 1 mL of the aqueous solution was diluted with 1 mL H2O and 1 mL ALF.
4.2. Fluorescence Microscopy
The fluorescence microscope images were recorded with a Zeiss Observer SD (Jena, Germany) spinning disk confocal microscope using a Yokogawa (Musashino, Japan) CSU-X1 spinning disc unit and an oil objective with 63× magnification and BP 525/50 and LP 690/50 filters. The setup was heated to 37 °C and a CO2 source was provided to keep the atmosphere at 5% CO2. For both excitation of the calcein and the cell marker a laser with a wavelength λ = 488 nm was used. The images were processed with the Zen software by Zeiss to optimize contrast.
4.3. Fluorescence Spectroscopy
The fluorescence spectroscopy experiments were recorded with a MD-5020 setup from PTI Photon Technology International (Birmingham, UK). The software Felix32 was used for recording and evaluating the measured data. For the experiments hollow caps were filled with 50 µL of a 1 mg/mL particle stock solution. Depending on the experiment, 100 µL of water or ALF or 90 µL water and 10 µL 20% triton X-100 solution were added, before the caps were sealed with a dialysis membrane and placed into cuvettes filled with water together with a stirring rod. The measurement temperature was 37 °C, with an excitation wavelength of 495 nm and an emission wavelength of 512 nm.
4.4. Cell Culture
All cell experiments were prepared in a Hera-Safe cell culture unit from Heraeus (Hanau, Germany). The cells were incubated at 37 °C/5% CO2 in Hera Cell incubators also from Heraeus. Cells were grown in Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% PenStrep. These chemicals were purchased from Thermofisher Scientific (Waltham, MA, USA).
4.5. MTT Assays
The MTT-assays were performed with a Spectra Fluor Plus from Tecan (Männedorf, Switzerland) and were then evaluated with Excel 2010 and Origin. 5000 cells were seeded per well and incubated together with 100 µL DMEM (10%FBS, 1% PenStrep) for 1 day before the particles were added. Each concentration was tested on three different days and on each day in triplicate. After the allotted incubation time the plates were washed with Hank’s Balanced Salt solution (HBSS) to remove dead cells, before the MTT reagent diluted in DMEM (0.5 mg/mL) was added. After two hours of incubation the MTT reagent solution was removed from the cells, and the cells were frozen at −80 °C for 0.5 h. Before the measurement 100 µL dimethyl sulfoxide (DMSO) were added to each well.
4.6. Synthesis of the Uncoated and Coated MIL-88A Nanoparticles
4.6.1. Synthesis of MIL-88A NPs
MIL-88A nanoparticles were synthesized in a microwave assisted approach based on the results of Chalati et al. [38
]. In this synthesis route, an aqueous solution of FeCl3
O (1.084 g, 4.01 mmol) and fumaric acid (485 mg, 4.18 mmol) are given to water (20 mL, Milli-Q). The reaction mixture was stirred until the metal salt was completely dissolved. The reaction mixture was then given into a Teflon tube (80 mL) and placed into a microwave oven (Synthos 3000, Anton-Paar, (Graz, Austria)) along with 3 additional vessels. Two of these vessels are filled with water (20 mL, Milli-Q), the third vessel is filled with an aqueous FeCl3
(20 mL, 1.084 g, 4.01 mmol) and is used to monitor the reaction progress. The vessels were heated under stirring with the sequence shown in Table 2
To remove residual reactants, the sample was subsequently washed via centrifugation (7840 rpm, 20 min) and redispersion of the pellet in ethanol (20 mL). This washing cycle was repeated 3 additional times. To also remove bulk material formed during the reaction, the dispersion was then centrifuged 3 times (3 min, 3000 rpm) and the pellet fraction of the product discarded.
4.6.2. Preparation of the Liposome Coating Solution
A 1 mg/mL PBS solution of DOPC was prepared and extruded through an extruder with a 100 nm pore sized membrane 11 times for cleaning.
4.6.3. Preparation of the Loaded and Coated Particles
1 mg of MIL-88A NPs were suspended in 1 mL of a 1 mM solution of calcein, suberohydroxamic acid (SBHA), irinotecan or floxuridine and incubated overnight for loading. Next, they were centrifuged for 5 min at 14,000 rpm, to discard the supernatant and the pellet was dissolved in 0.2 mL of the liposome coating solution and 0.2 mL water and incubated for 2 h. The particles were then centrifuged (5 min at 14,000 rpm) and redispersed in 1 mL PBS after washing several times.
For the preparation of the particles loaded with both irinotecan and floxuridine the MIL-88A particles were immersed in mixtures of 1 mM irinotecan and floxuridine solutions in the desired ratios, before following the same coating and washing procedure as outlined above.