Doxorubicin Loaded-UiO-66-NH2 Coated With Poly(N-Vinylcaprolactam) For Controlled Release of Doxorubicin Against A549 Lung Cancer


 The use of nano metal-organic frameworks (NMOFs) has been developed for drug delivery systems due to their high porosity and large specific surface area. In this work, UiO-66-NH2 NMOFs were synthesized via the microwave heating method and doxorubicin (DOX) molecules were incorporated into the UiO-66-NH2 NMOFs. Then, poly(N-Vinylcaprolactam) (PNVCL) synthesized by the free radical polymerization was coated on the NMOFs surface to fabricate the pH/temperature-sensitive carrier against A549 lung cancer cells death in vitro. The synthesized nanocarriers were characterized using FTIR, XRD, SEM, FESEM, TGA, and BET analysis. The average particle sizes of UiO-66-NH2 MOF and PNVCL coated-UiO-66-NH2 /DOX MOF nanoparticles were found to be 175 nm and 235 nm, respectively. TGA analysis showed that the PNVCL percentage coated on the UiO-66-NH2 NMOFs surface was about 17.5 %, and 27.3% for NMOFs incubated in 1% and 2% PNVCL solutions, respectively. The BET surface area of UiO-66-NH2 NMOFs, UiO-66-NH2 NMOFs/DOX 100 μg mL-1, and PNVCL 1% coated-NMOFs/DOX was found to be 1052, 121, and 87 m2g-1, respectively. The DOX release data of UiO-66-NH2 and PNVCL coated- UiO-66-NH2/DOX were evaluated under pH values of 5.5, 7.4, and temperatures of 25 °C, 37°C. The anticancer activity of synthesized NMOFs was investigated against lung cancer cells (A549) in vitro. The maximum cytotoxicity of A549 cancer cells was found to be 76% using PNVCL 1% coated-UiO-66-NH2/DOX 100 μg mL-1 NMOFs.


Introduction
Metal-organic frameworks (MOFs) as crystalline porous materials have been widely used for targeted delivery of anticancer drugs due to their high speci c surface area, large porosity, good biocompatibility, and ne pore sizes [1][2][3][4][5][6][7]. The nanosized-MOFs (NMOFs) prepared by the microwave heating method exhibited unique physic-chemical properties in comparison to the micrometer scale of MOFs [1,8]. The advantages of MOFs compared with other inorganic drug delivery systems (DDS) are the high encapsulation e ciency and their easier functionalization for targeted delivery [9][10][11][12][13][14]. UiO-66 MOFs ([Zr6O4(OH)4]) with high stability and high biocompatibility as well as good biodegradability and high speci c surface area had a high potential for delivery of anticancer drugs [15][16][17]. Furthermore,  MOFs with Zr-O clusters, and metal sites, as well as octahedral and tetrahedral cavities, could be considered for the controlled release of Doxorubicin (DOX) due to having a coordination interaction between the Zr (IV) clusters of UiO-66 and hydroxyl groups of DOX [4,18]. Moreover, the presence of open cavities and clusters in the UiO-66 MOFs matrix causes the incorporation of the high content of drug molecules, and following its release could occur in a controlled manner. UiO-66 MOF is also a good candidate for the controlled release of anticancer agents into the cancer tissues as a pH-sensitive carrier [18]. Thus, DOX could be released from UiO-66 MOFs into the acidic tumor sites due to the protonation of phosphate and weaknesses of the interaction between DOX and Zr-O clusters of UiO-66 MOF under acidic condition [18,19].
The accumulation of NMOFs in the bloodstream could be decreased by the coating of NMOFs surface with polymers which improve the stability of NMOFs and provide the effective application of NMOFs in DDSs of anticancer drugs during the in vitro and in vivo therapy of various cancers through the targeted delivery of anticancer agents into the tumor tissues [20][21][22]. Furthermore, the speci c distribution of anticancer drugs on the cancerous cells could decrease the adverse side effects of anticancer drugs via decreasing the non-selective distribution of drugs on the healthy cells. The coating of stimuli-responsive polymers on the MOFs surface could result in the enhancement of the permeability of the anticancer drug on the cancerous cells [23][24][25][26].
The lower critical solubility temperature (LCST) thermoresponsive polymers such as poly (N-isopropyl acrylamide) (PNIPAAm), and poly (N-vinyl caprolactam) (PNVCL) have been developed for DDSs of anticancer drugs [27][28][29][30]. Among LCST polymers, PNVCL as a hydrophilic biocompatible temperaturesensitive polymer is used for various biomedical applications [30][31][32][33][34]. Furthermore, the biocompatibility of PNVCL is higher than PNIPAAm, due to the hydrolysis of acrylamide groups of PNIPAAm and the production of small compounds which is not suitable for biomedical applications [35]. The LCST of PNVCL is ranging from ~32-34° which is close to physiological temperature. Thus the release rate could be controlled by the coating of PNVCL on the MOF surface at body temperature through the delay in the release of anticancer agents.
2.2 Synthesis of UiO-66-NH 2 and UiO-66-NH 2 /DOX UiO-66-NH 2 NMOFs were synthesized via the microwave method as described in reference [5]. Brie y, ZrCl 4 (125mg) and BDC-NH 2 (134mg) were dispersed into the 15 mL DMF and 1 mL HCl under sonication for 1 h. Then, the mixture was transferred into the microwave and the heating proceeded at 130°C for 1h.
Then, the product was washed with DMF and methanol three times and was dried at 60 °C for 6 h.
To load DOX molecules into the UiO-66-NH 2 MOF, 5 mg of UiO-66-NH 2 NMOFs were dispersed into DOX solutions (10, 50, and 100 μg mL -1 ) for 24 h under stirring. Then, the prepared UiO-66-NH 2 /DOX was washed with ethanol and distilled water three times. Then, the prepared products were centrifuged at 12000 rpm for 20 min. The nal content of DOX in NMOFs samples was determined according to the initial content of DOX, the nal content of DOX in solution, and DOX content in the supernatant after centrifugation of NMOFs using UV-Vis spectrophotometer at a wavelength of 481 nm. The drug encapsulation DEE (%) is evaluated as follows:

Synthesis of PNVCL and PNVCL coated-UiO-66-NH 2 /DOX
The carboxylated PNVCL is synthesized by free radical polymerization of NVCL, MPA, and AIBN in isopropanol at 70 °C for 8 h. Then, diethyl ether was added to precipitate the synthesized product. Later, the precipitated product was dried under vacuum. The prepared sample was dispersed in deionized water and was dialyzed for 4 days (MWCO of 5000) to remove the unreacted materials. Finally, the obtained product was lyophilized.
To coat the UiO-66-NH 2 /DOX surface with PNVCL, the predetermined amount of MOF nanoparticles was dispersed in 1% and 2% PNVCL (dissolved in distilled water) under stirring for 24 h and then, centrifuged at 12000 rpm for 39 min to remove the non-attached PNVCL on the MOFs surface. The prepared UiO-66-NH 2 /DOX/PNVCL samples were washed three times with deionized water and dialyzed for 3 days (MWCO of 12000) and then dried at 30°C for 24 h. Finally, the obtained product was lyophilized for further investigations. The schematic of the PNVCL and PNVCL/UiO-66-NH 2 /DOX synthesis is illustrated in Fig.1.

Characterization tests
The X-ray diffraction (XRD) patterns were recorded using Philips X'pert diffractometer in the range of 10- to compare the morphology and structure of UiO-66-NH 2 /DOX and PNVCL coated-UiO-66-NH 2 /DOX MOF nanoparticles. The thermal analysis of UiO-66-NH 2 /DOX and PNVCL coated-UiO-66-NH 2 /DOX MOF nanoparticles was carried out using thermogravimetric analysis (TGA Q500) by the heating rate of 10 °C min −1 ranging from 50-700°C. The Brunauer-Emmett-Teller (BET) method was used to measure the speci c BET surface area of MOFs (Micromeritics ASAP 2010, Micromeritics, Norcross, GA, USA) by the N2 adsorption-desorption isotherm. The UV-Vis spectrophotometer (JAS.CO V-530, Japan) was used to measure the nal concentration of DOX at a wavelength of 481 nm.

Drug release and kinetic studies
To obtain the DOX release behavior from synthesized MOFs, the drug loaded-MOFs were incubated in 50 mL of 0.1 M phosphate buffer solution at temperatures of 25, 37 °C and pH values of 5.5, 7.4. At predetermined time intervals, 2 mL of incubation solution was collected from the solution medium. While 2 mL of fresh PBS was simultaneously added into the dissolution medium. The DOX release was determined according to its concentration at a certain time and actual drug content in MOFs. The release experiments were carried out three times and the average values were reported. The kinetic data of DOX release were analyzed by the zero-order, Higuchi [36], and Korsmeyer-Peppas [37] pharmacokinetic models to obtain the drug release mechanism from synthesized MOFs.

Cell viability
To investigate the biocompatibility of MOFs samples, the synthesized nanoparticles were incubated in the L929 normal broblast cells (Institute Pasteur of Iran, IPI, Tehran, Iran) cultured in RPMI with 10% fetal calf serum and 1% penicillin-streptomycin at 37°C in a humidi ed atmosphere of 5% CO2. The cell viability of synthesized DOX loaded-MOF nanoparticles (100 µg/mL) against A549 lung cancer cell lines (IPI, Tehran, Iran) was evaluated after 24, 48, and 72 h incubation time as described previously [38]. The ELISA microplate reader (Multiskan MK3, Thermo Electron Corporation, USA) at a wavelength of 570 nm was used to determine the cell viability of A549 cells treated with MOF nanoparticles. The CyFlow ow cytometer (Partec, Germany) by staining of cells with Annexin V-uorescein isothiocyanate (VFITC) /propidium iodide (PI) was used to compare the apoptosis of A549 cancer cells in the presence of various MOFs samples [39].

Characterization of MOF nanoparticles
The XRD patterns of UiO-66-NH 2 MOF and PNVCL coated UiO-66-NH 2 /DOX MOF nanoparticles are illustrated in Fig.2. The presence of sharp diffraction peaks at 2θ=7.5˚ and 8.5˚ corresponding to (111), and (002) planes indicated the formation of pure UiO-66-NH 2 MOF nanoparticles [5]. The SEM images and FESEM images of UiO-66-NH 2 MOF and PNVCL coated-UiO-66-NH 2 /DOX MOF nanoparticles are illustrated in Fig.3. As shown, the uniform nanoparticles ranging from 100-200 nm were obtained for UiO-66-NH 2 MOF particles. The FESEM image of synthesized UiO-66-NH 2 MOF nanoparticles revealed the presence of irregular shapes (spherical and polyhedral) of UiO-66-NH 2 MOF nanoparticles with an average particle size of 175 nm. The SEM image of PNVCL coated-UiO-66-NH 2 /DOX MOF indicated the increase in the particle sizes of particles. The particle sizes were obtained ranging from 100-300 nm with an average particle size of 235 nm. The increase in particle sizes was further con rmed by the dynamic light scattering (DLS) measurements. As shown, the average hydrodynamic sizes of UiO-66-NH 2 NMOFs and DOX loaded-UiO-66-NH 2 NMOFs were about 230 nm and 275 nm, respectively. The adsorption/desorption isotherms for UiO-66-NH 2 , UiO-66-NH 2 /DOX and PNVCL coated-UiO-66-NH 2 /DOX are illustrated in Fig.6. For UiO-66-NH 2 , the BET surface area and pore volume were found to be 1052 m 2 g -1 , and 0.58 cm 2 g -1 , respectively. After loading DOX into the NMOFs, the BET surface area and pore volume were decreased to 121 m 2 g -1 and 0.12 cm 2 g -1 , respectively which demonstrated the high loading of DOX molecules into the pores of nano bers. The blockage of NMOFs pores with DOX molecules resulted in a signi cant decrease in speci c BET surface area after loading of DOX into the NMOFs. After the coating of PNVCL, the speci c BET surface area and pore volume were decreased to 87 m 2 g -1 and 0.08 cm 2 g -1 , respectively.

Drug encapsulation e ciency, drug release, and kinetic studies
The DOX encapsulation e ciency for NMOFs incubated at 10, 50 and 100 μgmL -1 DOX is presented in Table 1. As shown in this table, the maximum drug encapsulation e ciency (DEE%) was found to be 55.5% from 1% PNVCL coated-NMOFs containing 10 μg/mL DOX. By increasing DOX concentration, the DEE was gradually decreased. Furthermore, a coating of 2% PNVCL on the NMOFs surface resulted in a decrease of DEE in comparison to DEE of PNVCL 1% coated-NMOFs in the same condition.

Cytotoxicity of NMOFs
The cytotoxicity of UiO-66-NH 2 , UiO-66-NH 2 /PNVCL 1%, and UiO-66-NH 2 /PNVCL 2% against normal broblast cells are illustrated in Fig. 8a. The gradual decrease in the cell viability of pure UiO-66-NH2 by time could be attributed to the Zr-O clusters release into the medium which increased the cytotoxicity of cells treated with broblast cells treated with UiO-66-NH 2 NMOFs. Whereas, there was no signi cant cytotoxicity toward broblast normal cells treated with PNVCL coated-NMOFs.
The DAPI staining images of untreated A549 cells and A549 cells treated UiO-66-NH 2 /DOX 100 μg mL -1 NMOFs and NMOFs coated with 1% and 2% PNVCL after 72 h incubation time are illustrated in Fig. 9. As shown, the nuclear fragmentation in their chromatin of cells was detected in the presence of 100 μg mL -1 DOX loaded-NMOFs and NMOFs/DOX coated with 1% and 2% PNVCL. with PNVCL. The comparison of SEM and DLS results indicated that the average particle sizes of synthesized NMOFs reported by SEM were lower than that of DLS. The hydrodynamic radius of MOFs was found to be higher than that of particle sizes of dried nanoparticles. Similar trends were reported by other researchers [40,41]. The comparison of FTIR spectra of pure PNVCL and PNVCL 1% coated-UiO-66-NH 2 /DOX indicated that the carboxyl peak intensity in the FTIR spectrum of UiO-66-NH 2 /DOX/PNVCL was decreased in comparison to the carboxyl peak intensity of pure PNVCL which could be attributed to the interaction of carboxylic groups of PNVCL with amine groups of UiO-66-NH 2 [42].

Conclusion
The PNVCL coated-UiO-66-NH 2 /DOX NMOFs were successfully synthesized and its application was investigated for controlled release of DOX against A549 lung cancer cells. The XRD and FESEM image of UiO-66-NH 2 NMOFs demonstrated the formation of crystalline nanoparticles with an average particle size of 175 nm. The FTIR spectra of PNVCL coated NMOFs revealed the interaction of carboxylic groups of PNVCL with amine groups of UiO-66-NH2. Based on TGA results, the PNVCL polymer percentages coated on the UiO-66-NH 2 NMOFs surface were found to be 17.5 %, and 27.3% for NMOFs incubated in 1% and 2% PNVCL solutions. The BET surface area and pore volume of UiO-66-NH 2 NMOFs were found to be 1052 m 2 g -1 , and 0.58 cm 2 g -1 , respectively. The maximum drug encapsulation e ciency (DEE %) was found to be 55.5% from NMOFs coated with 1% PNVCL and 10 μg mL -1 DOX.