Glycopolymer-Functionalized MOF-808 Nanoparticles as a Cancer-Targeted Dual Drug Delivery System for Carboplatin and Floxuridine

Codelivery of chemotherapeutics via nanomaterials has attracted much attention over the last decades due to improved drug delivery to tumor tissues, decreased systemic effects, and increased therapeutic efficacies. High porosities, large pore volumes and surface areas, and tunable structures have positioned metal–organic frameworks (MOFs) as promising drug delivery systems (DDSs). In particular, nanoscale Zr-linked MOFs such as MOF-808 offer notable advantages for biomedical applications such as high porosity, good stability, and biocompatibility. In this study, we report efficient dual drug delivery of floxuridine (FUDR) and carboplatin (CARB) loaded in MOF-808 nanoparticles to cancer cells. The nanoparticles were further functionalized by a poly(acrylic acid-mannose acrylamide) (PAAMAM) glycopolymer coating to obtain a highly selective DDS in cancer cells and enhance the therapeutic efficacy of chemotherapy. While MOF-808 was found to enhance the individual therapeutic effects of FUDR and CARB toward cancerous cells, combining FUDR and CARB was seen to cause a synergistic effect, further enhancing the cytotoxicity of the free drugs. Enhancement of CARB loading and therefore cytotoxicity of the CARB-loaded MOFs could be induced through a modified activation protocol, while coating of MOF-808 with the PAAMAM glycopolymer increased the uptake of the nanoparticles in cancer cells used in the study and offered a particularly significant selective drug delivery with high cytotoxicity in HepG2 human hepatocellular carcinoma cells. These results show how the enhancement of cytotoxicity is possible through both nanovector delivery and synergistic treatment, and that MOF-808 is a viable candidate for future drug delivery studies.

S3 MOF-808_act: 650 mg of MOF-808 was dispersed in a methanol (260 mL) and water (36.4 mL) mixture and stirred at room temperature for 1 day. The nanoparticles were washed by dispersioncentrifugation cycles with methanol (5 × 40 mL) and dried in a desiccator for two days to yield MOF-808_act.

Synthesis of PAAMAM
PAAMAM was synthesized via RAFT polymerization. Mannose acrylamide (1.0 g, 3.6 mmol) and acrylic acid (0.26 g, 3.6 mmol) were added to a pressure resistant vial and dissolved in mixture of DMF/H2O (70/30 v/v, 1 mL) and purged with N2. Next, [[(butylthio)carbonothioyl]thio]propanoic acid (PABTC, 21.4 mg, 0.09 mmol) and 4,4'-azobis (4-cyanovaleric acid) (ACVA, 2.5 mg, 0.009 mmol) were dissolved in a small amount of DMF (0.3 mL) and were added to the reactor vial. The reaction solution was degassed at room temperature for 15 min. The vial was placed in an oil bath at 70 °C to start the polymerisation. The polymer conversion was monitored by 1 H NMR spectroscopy and the reaction was stopped when full monomer conversion was obtained (12 h). Finally, the polymer was purified by dialysis against distilled water for 2 days and freeze-dried to obtain a white solid.

Drug Loading
For post-synthetic incorporation of FUDR, CARB, and both FUDR and CARB to MOF-808 or MOF-808_act, 120 mg of each MOF was dispersed in methanol (24 mL). In a separate vial 120 mg of FUDR, 120 mg of CARB or both FUDR and CARB (120 mg of each) were dissolved in 24 mL of methanol containing 6.72 mL water and mixed with MOF-808 or MOF-808_act after being sonicated for 15 min at room temperature to obtain FUDR@MOF-808, CARB@MOF-808 and (FUDR+CARB)@MOF-808, or FUDR@MOF-808_act CARB@MOF-808_act and (FUDR+CARB)@MOF-808_act, respectively. The drug solutions were stirred at room temperature for one day and the obtained nanoparticles were washed by dispersion-centrifugation cycles with methanol (5 × 40 mL) and dried in a desiccator for two days.

PAAMAM coating of MOFs
For glycopolymer (PAAMAM) coating of MOF-808, the MOF nanoparticles (75 mg) were dispersed in 15 mL of methanol. In a separate vial, the glycopolymer (7.5 mg) was dissolved in 15 mL of methanol. Then, the solutions were mixed and stirred at room temperature for one day. The coated S4 samples were washed by dispersion-centrifugation cycles with methanol (5 × 30 mL) and dried in a desiccator for two days. The procedure was repeated with MOF-808_act, (FUDR+CARB)@MOF-808 and (FUDR+CARB)@MOF-808_act.
Characterization 1 H Nuclear Magnetic Resonance Spectroscopy (NMR): NMR spectra were collected on either a Bruker AVIII 400 MHz spectrometer, a Bruker AVI 500 MHz spectrometer, or a Bruker DPX-300 spectrometer (glycopolymer) and referenced to residual solvent peaks. Measurements were carried out at room temperature. Thermogravimetric Analysis (TGA): Measurements were conducted using a TA Instruments Q500 Thermogravimetric Analyser. Data were collected from room temperature to 800 °C with a heating rate of 10°C min -1 under an air atmosphere.
Fourier Transform Infrared Spectroscopy (FTIR): IR spectra of solids were collected at a range of 400-4000 cm -1 using an attenuated total reflection (ATR)-FTIR spectrometer (Thermoscientific Nicolet Summit FTIR spectrometer with an Everest ATR) with an accompanying OMNIC software.

Scanning Electron Microscopy (SEM):
The powder samples were coated with Au/Pd for 50 seconds using Polaron SC7640 sputter coater and imaged using a Carl Zeiss Sigma Variable Pressure Analytical SEM with Oxford Microanalysis. Particle size distribution was measured manually using ImageJ software version 1.52a (National Institutes of Health, Bethesda, MD, USA) by randomly selecting 100 particles from an SEM image.
Dynamic Light Scattering (DLS)/Zeta potential: The hydrodynamic size, polydispersity index (PDI) and zeta potential of MOFs in water were determined using a Malvern Dynamic Light Scattering (DLS) S5 with a Zetasizer Nano ZS potential analyser equipped with Non-Invasive Backscatter optics (NIBS) and a 50 mW laser at λ = 633 nm. The samples were prepared with a concentration of 0.25 mg mL -1 by sonication for 20 min prior to measurement. The pH of the solutions was adjusted to about 7.4 to eliminate pH effect on zeta potential of the nanoparticles. All measurements were carried out in triplicate.
Gas Uptake and Pore-Size Distribution: N2 adsorption isotherms were recorded on a Quantachrome Autosorb iQ gas sorption analyser at 77 K. Samples were degassed under vacuum at 120 °C for 20 h by using an internal turbo pump. BET surface areas were calculated from the isotherms using the Micropore BET Assistant in the Quantachrome ASiQwin operating software. Pore size distributions were calculated using N2 at 77 K on carbon, slit/cylinder/sphere pore QSDFT, adsorption model within the same software (version 3.01).
UV/Vis Spectroscopy: UV/Vis absorbance spectra were recorded on a Shimadzu UV-1800 and analyses of data were performed using the software UVProbe (version 2.51).
FUDR Determination: 2 mg of the prepared formulations were dispersed in 10 mL of PBS (1x, pH 7.4) and stirred for 4 days at room temperature. The degradation solutions were lyophilized on a Christ Alpha 2-4 LO plus freeze dryer to evaporate PBS and then, the degradation product was dissolved in methanol (4 mL) and centrifuged. The supernatant was filtered through 0.2 µm PTFE filters prior to analysis by HPLC and was analysed by a Shimadzu reverse-phase HPLC (RP-HPLC) system equipped with Shimadzu LC-20AT pumps, a Shimadzu SIL-20A autosampler and a Shimadzu SPD-20A UV-Vis detector (monitoring at λ = 268 nm for FUDR and λ = 205 nm for benzene-1,3,5-tricarboxylic acid) using a C18 Kinetex® reverse-phase LC column (250 × 4.6 mm, 5 µm). Collection of the chromatograms was completed using a mobile phase of water:acetonitrile (95:5% with 0.1% trifluoroacetic acid) for 20 min at a flow rate of 1 mL min -1 . The drug concentration was calculated according to a calibration curve of FUDR prepared in methanol. FUDR loading content was reported (v/v) FBS, 1% (v/v) penicillin (100 U/mL) streptomycin (100 μg/mL), 2 mM L-glutamine and 1% (v/v) non-essential amino acid (NEAA) solution. All cells were grown in a humidified incubator at 37°C in 5% CO2 and passaged every 2-3 days.

MCF-7 and PANC-1 cells at a density of 5000 cells/well and HepG2 cells at a density of 8000 cells/well
were seeded into 96-well plates separately and incubated at 37°C in a 5% CO2 atmosphere. On the second day, they were subjected to the samples at different concentrations with fresh culture medium and incubated for additional 24 h or 72 h. Cells without any treatment were employed as positive controls. At the end of incubation, Alamar Blue reagent was introduced to each well according to the manufacturer's instructions (10% v/v) and the cells were further incubated at 37°C in 5% CO2 for 4 h. Fluorescence reading was performed under 557/10 nm excitation and 593/10 nm emission using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany). The relative cell viability was calculated in reference to the untreated control cells using the following equation: (2) The average cell viability was calculated using three different passages of cells with 3 repeats with each (n = 3x3).