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Copper-Gallic acid nanoscale metal-organic framework for combined drug delivery and photodynamic therapy Shalini Sharma, Disha Mittal, Anita K. Verma, and Indrajit Roy ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00116 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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ACS Applied Bio Materials

Gallic acid and photosensitizer co-incorporated nanoscale metal organic frameworks for ROS-mediated and light-activated antitumor therapy 81x44mm (150 x 150 DPI)

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Copper-Gallic acid nanoscale metal-organic framework for combined drug delivery and photodynamic therapy Shalini Sharmaǂ, Disha Mittal║, Anita Kamra Verma║ and Indrajit Roy*ǂ ǂ Department of Chemistry, University of Delhi, Delhi-110007 ║

Department of Zoology, Kirori Mal College, University of Delhi, Delhi-110007 *Email: [email protected]

ABSTRACT: In this paper, we report the synthesis of bioactive copper-gallic acid nanoscale metal-organic framework for the co-delivery of anticancer agent (gallic acid) and photosensitizer (methylene blue) to cancer cells. A supramolecular coordination complex of copper-bioactive frameworks (bio MOFs) were employed as the carrier of two anticancer agents. The first one is the natural phenolic acid (gallic acid), which forms a part of the framework structure (building block). The other one is the photosensitizer methylene blue, loaded as a guest molecule within the amphiphilic pores of the framework. In vitro cytotoxicity and in vivo tumor regression assays revealed enhanced cytotoxicity of dual drug nano-framework when compared with the equivalent dosages of free drugs in the presence of light. Keywords: Copper-gallate metal-organic framework (Cu-GA NMOF), gallic acid (GA), methylene blue (MB), ROS mediated apoptosis, dual drug nano-framework, combination therapy


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A. INTRODUCTION Cancer results due to uncontrolled cell growth that not only affects the infected region, but may also spread to other parts of the body (metastasis).1,2 Currently, natural compounds are of great interest in the management of cancer, which largely avoids the adverse effects associated with traditional chemotherapy agents (e.g. Adriamycin, Cis-platin, etc).3,4,5 Several natural molecules, e.g. Quercetin, Curcumin, Ellagic acid or Gallic acid, are known for their chemo-preventive and therapeutic potential to treat cancer.6,7,8 They induce apoptosis of cancer cells via caspasemediated pathway.9,10 Owing to the presence of free functional groups, they also act as ligands and can be attached to other molecules or macromolecular/nanosized drug carriers. However, such molecules are unlikely to act as efficient monotherapeutic agents in the clinic, unless administered in large amounts, which may promote toxicity. Gallic acid (GA) is a special phytochemical compound among the phenolic family known for its anti-oxidant, anti-inflammatory and anti-carcinogenic behaviour.11,12 It also behaves as a prooxidant in the presence of metals, especially iron or copper, and promotes Fenton-type or autooxidation reaction producing reactive oxygen species (ROS).13,14 Recently, it has been demonstrated that GA is present in a large amount in the herbal formulation Triphala, which exhibits specific protection against prostate cancer.15 It has been used in the treatment of several diseases, such as Type-II diabetes, haemorrhage, albuminuria, Alzheimer’s, Parkinson's, Huntington’s disease, etc.16,17,18 The therapeutic potential of GA has been proven to be better than conventional drugs (e.g. Doxorubicin, 5-fluorouracil) when tested in vitro.19, The nontoxicity of free GA has been demonstrated on rats, showing a high LD50 of 5 g/kg.20 Some studies reported the use of GA conjugated on the surface of ultra-small gold and iron oxide nanoparticles for targeted drug delivery.21



Over the past few years, nanoscale metal-organic frameworks (NMOFs), formed via metalligand coordination bonds, have emerged as excellent drug delivery agents for the treatment of cancer and other diseases.22,23,24 They have several beneficial attributes, such as biocompatibility, controlled porosity, ability to load high amounts of drugs (by physical encapsulation or covalent incorporation), etc. Horcajada et al. reported various non-toxic iron (III) containing NMOFs (e.g. MIL-89, MIL-88, MIL-53), which have been used as efficient carriers of several challenging drugs (e.g. Busulfan, Doxorubicin, Azidothymidine).25 Bioactive ligands, such as nicotinic acid and gallic acid, are also known to form NMOFs in combination with metals such as iron, copper and magnesium.26,27 These are also called bioactive MOFs (bio MOFs), and can be used for drug delivery. Iron nicotinate is an example of bioactive and biodegradable MOF called BioMIL-1, where nicotinic acid is loaded up to 71 % by weight, denoted as vitamin B3 or niacin.28 Iron fumarate (MIL-88A) has been approved as an oral drug for iron supplements and also used for NO storage.29 Anthony Cheetham et al. reported gallate based hybrid materials using Fe(III), Co(II), Mn(II), Ni (II), etc., and studied their different crystalline structures.30 Xueling Mu et al. synthesized ultra-small Fe(III)-gallic acid nanoscale coordination polymer with the help of a protein (BSA) for T1 weighed MR imaging and photothermal therapy in 4T1 tumour bearing mice.31 Photodynamic therapy (PDT) involves the production of cytotoxic ROS, specially singlet oxygen, upon irradiation of photosensitizer (PS) drugs by light of appropriate wavelength. Therefore, photosensitizer-incorporated nanomaterials targeted to tumor sites can cause potent localized toxicity upon photoactivation.32,33,34 Wenbin Lin’s group have demonstrated potent antitumor activity of photosensitizer-entrapped NMOFs.35,36 Sharma et al. reported methylene


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blue and doxorubicin encapsulated iron-terephthalate metal-organic framework (MIL-88B) for magnetically guided drug delivery and photodynamic therapy.37 These observations prompted us to synthesize photosensitizer-incorporated copper-gallic acid NMOF, and investigate its antitumor activity in vitro and in vivo. We have synthesized gallic acid containing NMOF using copper acetate in the oil-loving core of normal micelles. We have used direct incorporation of antioxidant/anticancer agent (gallic acid) in the framework structure (building block) and investigated their delivery in vitro and in vivo for anticancer applications. The framework was post-synthetically loaded with the photosensitizer methylene blue (MB), for concurrent light-activated photodynamic therapy in vitro and in vivo. B. RESULTS AND DISCUSSION Synthesis and Characterization of Cu-GA NMOF. The reported framework was synthesized by the reaction of polydentate phenolic acid (gallic acid) as the organic ligand and copper acetate as the metallic precursor within the oily core of oil-in-water microemulsion system.The procedure is described in the experimental section and the scheme is given in Scheme 1. TEM image (Figure 1A) showed the framework is needle-like in shape. It constitutes bundles of nanorods of 10 – 15 nm by thickness and 100 – 160 nm by length. This result was matched with FESEM micrograph (Figure 1B), showing nanorod morphology of the framework. The particles were filtered through 0.45 µm syringe filter prior to DLS measurement (Figure 1C). The hydrodynamic radii of aqueous dispersed Cu-GA NMOF is around 168 nm, with a polydispersity index (PDI) of 0.362. The elemental composition of Cu-GA NMOF was analysed using energy dispersive X-ray spectroscopy (EDS), as shown in Figure 2A. The EDS spectrum contains peak of copper



accounting 41 wt%, as a part of MOF structure, whereas C and O contribute 58 wt% collectively, as a contribution from the organic ligand (GA). In this analysis, the sample powder is directly mounted on the sample holder; therefore, there is no additional element present apart from the sample components. The elemental composition was verified by TGA analysis (Figure 2B), which records the change in the weight of the sample with increasing temperature under inert atmosphere. The weight loss below 100 °C is attributed to loss of water molecules, whether coordinated or occupied within the pores of the framework. A prominent weight loss of around 43 wt% was obtained between 100 to 360° C, owing to degradation of GA. This followed the complete breakdown of the frameworks, resulting in a plateau of 52 wt%, corresponding to the formation of copper oxide. The empirical formula and the molecular weight of a probable structure of the as-synthesized Cu-GA NMOF, deduced from TGA thermogram, are Cu3(GA)(H2O) and 377 g/mol, respectively. The crystalline structure and phase characterization of Cu-GA NMOF was obtained using powder XRD (Figure 2C) and selected area electron diffraction (SAED) pattern (Figure 2D). The powder XRD of the as-synthesized Cu-GA NMOF showed polycrystallinity of the material, whose pattern matches well with the simulated pattern of MIL-53 frameworks (Cambridge crystallographic data no. CCDC-220475).38,39 The spectra showed characteristic peaks at 2Ɵ = 7.1°, 10.1°, 13.2°, 20.1°, 28.0°, 31.1°, 41.9° and 42.8°corresponding to (101), (200), (011), (112), (213), (413), (613) and (132) diffraction planes, respectively, which are the typical signatures of MIL-53 structure. The crystallographic signature of the frameworks was further confirmed by SAED pattern, which showed sharp diffraction rings. The calculated interplanar “d” spacing values exactly matched with the powder XRD pattern obtained. MIL-53 frameworks


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are usually composed of 1D chains of MO6, connected by phenolic acid giving a framework with 1D rhombic channels, appearing like the wine rack/diamond structure. The surface functional characterization of Cu-GA NMOF was obtained from Fourier transform infrared (FTIR) spectroscopy (Figure S1, ESI). The characteristic FTIR peak of CuGA NMOF (MIL-53) were observed at 1535 cm-1 and 1395 cm-1, originating from asymmetric and symmetric stretching vibration of C-O of carboxylate group (OCO-), which matches with free GA (1540 cm-1, 1440 cm-1) but with a blue shift because of complex formation. The peak at 1693 cm-1 corresponds to the carbonyl group (C=O) present in free GA, but absent in a complex Cu-GA NMOF, confirming that C=O group of GA is strongly affected on coordination with the copper ion. The bands appearing near ~ 1307 cm-1, 1200 cm-1, 1018 cm-1 correspond to stretching of C-O of phenol (alcohol) present in both free GA and Cu-GA NMOF. The bands appearing between 3500 –3260 cm-1 in free GA are due to O-H symmetry stretching of hydroxyl and carboxyl group in free GA. These bands were absent in Cu-GA nanorods, probably due to the coordination to the copper centre and removal of proton on coordination. This data confirms that both hydroxyl and carboxylate groups of GA participated in coordination with the copper ion. The nitrogen adsorption-desorption isotherm for Cu-GA NMOF resembles with type-IV curve with a hysteresis loop corresponding to the characteristic behaviour of meso- or macroporous material (Figure S2, ESI). The specific surface area of Cu-GA NMOF calculated by using BET method is around 172 m2/g. BJH (Barret-Joyner-Halenda) method was used to calculate the pore volume and average pore diameter, with values of 0.73 cc/g and 2.2 nm, respectively. These data confirmed the mesoporous nature of the as-synthesized Cu-GA NMOF. In contrast, MIL-53 frameworks are reported to be known for their flexible and microporous



nature. The mesoporous behaviour obtained from BET and BJH methods in our case can be justified by the hypothesis that MIL-53 frameworks often shows breathing effect, indicating the reversible transition from small pores (microporous) to large pores (mesoporous) in the presence of small molecules, such as nitrogen, as a guest (Figure S2, ESI).40 The surface characteristics of MIL-53 materials can be different depending upon several factors, such as size, shape, choice of metal ion, chemical functionality of the ligand, synthetic conditions, etc. Anbia et al. reported surface area of 1150 m2/g for MIL-53 (Copper-terephthalate)41 whereas Pu et al. reported the surface area of 80 m2/g for MIL-53 (Iron-terephthalate).42 We then compared the H2O2 producing ability between various concentrations of free GA and its equivalent amounts in Cu-GA NMOF, using o-dianisidine dye assay.The results (Figure S3A, ESI) showed that free GA produces larger amount of H2O2 as compared to bound GA (or CuGA NMOF), because the auto-oxidation of GA is feasible only in its free form. We hypothesize that Cu-GA NMOF produces H2O2 only when it releases GA; therefore, it produces limited H2O2 as compared to free GA. The image depicting the colour change of o-dianisidine detector with increasing concentration of H2O2 has been shown in Figure S3B, ESI. The absorption and fluorescence spectra of MB/Cu-GA NMOF were recorded via UV-Vis spectrophotometry (Figure S4A, ESI) and Spectrofluorometry (Figure S4B, ESI), respectively. The loading efficiency of MB in Cu-GA NMOF matrix in aqueous dispersion reached around 2wt%, which is appreciable enough for current photodynamic therapy. The loading was achieved probably due to the H-bonding interaction between the nitrogen or sulphur groups of methylene blue and the protons of aromatic gallic acid (Figure S4C, ESI). Li et al. reported that the maximum adsorption capacity of MB into MIL-53(Al)-NH2 is 4.5 wt% due to H-bonding


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interaction and - stacking interaction, in contrast to 0.36 wt % in case of MIL-53(Al) where only breathing behaviour contributes to drug loading.43 The photo-induced generation of singlet oxygen by free MB and MB/Cu-GA NMOF was confirmed by using a singlet oxygen detector, a dye called ABMDMA. The plot (Figure 3A) shows steady photoinduced decay in the absorbance curve of ABMDMA treated with free MB or MB/Cu-GA NMOF, whereas the curve of ABMDMA treated with MB/Cu-GA NMOF shows steeper decay. This occurs because of the variation in lifetime of singlet oxygen in different dielectric media. The lifetime of singlet oxygen in aqueous media is in the microsecond regime, during which it can migrate on the order of 100 nm only. Therefore, significant fraction of singlet oxygen is probably lost in reaction in case of free MB, in contrast to MB trapped within the amphiphilic porous framework environment of the NMOF. Therefore, the MB/Cu-GA NMOF formulation appears to be a better photoinduced singlet oxygen generator than free MB in the aqueous system. The release study of both the drugs (GA and MB) from the Cu-GA NMOF was carried out at two different pH (7 and 4) values for 5 days using phosphate buffer at 370C.

It was observed

from the release GA profile (Figure 3B) that a total of 56 % and 69 % of GA were released at neutral (pH 7) and acidic pH (pH 4), respectively. The release of GA can be correlated with the partial and steady degradation of the flexible framework resembling MIL-88A and MIL-89.44 Cu-GA NMOF is a biodegradable matrix not only due to the hydrophilic nature of gallic acid (presence of carboxylate and hydroxyl groups), but also due to the presence of phosphate ions in the biological media, which competes for the metal sites and facilitate its degradation. The relatively higher release of GA at acidic pH is obvious as the protonation of the oxygen group of GA leads to weaker copper-oxygen bond in the framework.



The MB release profile from the MB/Cu-GA NMOF (Figure 3C) shows that at neutral pH (pH 7), MB release is slow and a total of 53% of the drug is released. On the other hand, at acidic pH, a very high release of 94% was observed. This is due to the combined effects of higher degradation of the Cu-GA framework in acidic pH, along with the weakening of the physical interaction between MB and Cu-GA at acidic pH. Overall, the higher release of both these active agents (GA and MB) at acidic pH is beneficial for drug delivery purposes to desired cells. Investigation of in vitro studies using Panc-1 cells. The estimation of intracellular GA was carried out via spectrophotometric analysis of cell lysate after the treatment of cells with different concentration of free GA and Cu-GA NMOF, using Folin’s reagent. The exact concentrations used are mentioned in the experimental section. It was inferred from the data (Figure 4A) that Cu-GA NMOF has better cellular uptake than free GA at all the concentrations studied. This might be due to the hydrophilic nature of free GA, which causes lesser uptake in cells. On the other hand, Cu-GA nanoframework exhibits some lipophilicity, since copper complexation promotes electron delocalisation in the chelating ring system of GA. Increased lipophilicity allows frameworks to interact with the cell membrane more effectively than free GA. The second hypothesis is attributed to the rod-shaped geometry of Cu-GA NMOFs, which enhanced their uptake via endocytosis pathway. It has been reported that geometry influences the uptake profiles due to the different orientation of nanoparticle at the cell surface.45 Similar cellular uptake was quantified via fluorometric analysis of cell lysates after the treatment of cells with free MB and MB/Cu-GA NMOF for 2 hours (Figure 4B). At all the concentrations studied, MB/Cu-GA NMOF has better uptake than free MB, because of the poor penetration of free MB into the cells. There can be two probable reasons for the poor uptake of free MB: (i) MB is known to aggregate at high concentration and pumped out of the cells by MDR mechanism,


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and (ii) MB is known to be reduced to the non-fluorescent Leuco-methylene blue (LMB) in the biological environment. Next, MTT assay was carried out to in order to observe dose-dependent cytotoxicity (nonphotoactivated) between free GA and its equivalent concentration within Cu-GA NMOF. Panc-1 cells were treated with six different concentrations of free GA and equivalent GA as part of CuGA NMOF. The results (Figure 4C) showed that free GA is more cytotoxic than Cu-GA NMOF over the broad range of GA concentration. The IC50 of Cu-GA NMOF in terms of GA concentration is nearly 50 µg/ml, which is two-folds higher than that of free GA (IC50 ~ 25 µg/ml). This result is in agreement with the comparative ROS (H2O2) production between free and NMOF-bound GA, as shown previously in Figure S3, ESI. Overall, free GA autoxidizes more spontaneously/faster and thus produces more ROS as compared to the Cu-GA NMOF, where the GA is released slowly producing less ROS. Thus, Cu-GA NMOFs can be used as slow GA release system with controlled cytotoxicity in vitro. Time-dependent MTT study was also used to investigate the cytotoxic effect of slow release of GA from Cu-GA NMOF in cell culture medium. The data (Fig. 4D) shows that the cytotoxicity increases with time (cell viability of 68 % on the zeroth day to cell viability of 38% on 4 th day). It implies that with more time more GA is released from the Cu-GA NMOF, leading to higher toxicity. Thus, this study proves that GA is released slowly from Cu-GA NMOF in vitro, leading to controlled, time-dependent cytotoxicity. To investigate the effect of ROS quenching on the cytotoxic potential of free and NMOF-bound GA, the interaction of GA and Cu-GA NMOF with the cells was investigated in the presence of the anti-oxidant glutathione (GSH) via MTT assay (Figure S5, ESI). In this experiment, the IC50 concentrations of free GA (25 µg/ml) and Cu-GA NMOF (50 µg/ml GA) were treated with the



cells, which were pre-treated with GSH (20 mM) for 2 hours. The results showed that in both the cases, the cell viability increased significantly (around 100% viability) in the presence of the non-enzymatic antioxidant (GSH). GSH pre-treatment protects the cells against ROS induction by free GA and Cu-GA NMOF. This result again proves that GA induces cytotoxicity via ROS generation as a result of spontaneous auto-oxidation. Next, we probed the effect of photosensitizer incorporation within the Cu-GA NMOFs. In this in vitro experiment, the cytotoxicity of free MB and MB/Cu-GA NMOF was evaluated, without (dark) and with (light) laser-light irradiation, in Panc-1 cells (Figure 5A). The light-activated cytotoxicity for MB/Cu-GA NMOF is significantly better than that of free MB over all three concentrations tested. The maximum effect was observed at high concentration of MB/Cu-GA NMOF (50µg/ml GA, 3µM MB), where the cell viability decreased from 56 % (dark) to only 2 % (light). The reason is attributed to the combined effects of GA-induced ROS generation and MB-light induced singlet oxygen production (photodynamic therapy) on the cells. The combined GA and MB/light (PDT) mediated cytotoxic effects in vitro was repeated with EAC cells (Figure 5B). The samples (Cu-GA NMOF, MB/Cu-GA NMOF, free GA and free MB) were treated with EAC cancer cells, without (dark) and with light irradiation. The results revealed the IC50 of Cu-GA NMOF in terms of GA is nearly 50 µg/ml, while the same for MB/Cu-GA (dark) is about 25 µg/ml. Overall, at all the concentrations tested for MB/Cu-GA NMOF, there is enhanced cytotoxicity on light irradiation, although the effect is somewhat subdued in comparison to that obtained using Panc-1 cells. Interestingly, free MB was found to be completely non-toxic without light irradiation (dark), and showed only marginal light-induced cytotoxicity (cell viability about 85%) at its highest concentration tested (3 µM). Overall, the results validate the combined GA and MB/light (PDT) mediated cytotoxic effects in vitro.


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Finally, in vivo anticancer studies were performed in mice bearing subcutaneous EAC tumors, using the various samples and controls. The therapeutic potential of MB/CuGA NMOF, free MB and placebo CuGA NMOF were assessed by tumour regression analysis. Intravenous injections of the samples and controls were given once a week for two weeks. Half of the animals receiving the injections were irradiated with laser light at the tumor (light), whereas the other half did not receive light treatment (dark). Significant tumor growth inhibition was observed in mice receiving combined treatments of MB/Cu-GA and light-irradiation of the tumor (Figure 6A). On the other hand, poor tumor growth inhibition was observed in mice treated with free MB or placebo CuGA (both light and dark), as well as MB/Cu-GA (dark). Not surprisingly, maximum tumor growth was observed in saline treated control. Clearly, the superior antitumor effect of MB/Cu-GA NMOF with light irradiation is due to the synergistic effects of Cu-GA and PDT. The tumor uptake of MB/Cu-GA after intravenous injection is probably due to the EPR effect. Localized irradiation with a 635 nm laser (38 W/cm2, 10 min) significantly regressed the tumor, without any noticeable toxicity. Thus, this photosensitizer-loaded ROS-producing nanoformulation is an extremely promising PDT drug carrier for anticancer studies. The pictures of the excised tumours from the various treatment groups are given in Figure 6B. Tumour weight analysis was carried out from the sacrificed mice, which confirmed maximum tumour regression in case of MB/Cu-GA NMOF and light treatment (Fig. S6, ESI).The comparative tumour volumes on the respective days in different groups are also provided (Table S1, ESI).

C. CONCLUSION Cu-GA based nano-framework (Cu-GA NMOF) was synthesized, which were rod-shaped and a biodegradable matrix, since 69 % of GA (drug) was released over a period of 5 days.These



crystalline nanomaterials were efficiently uptaken by cells (Panc-1 cells), in comparison to free GA. The cell viability assay (using Panc-1 cells) has confirmed Cu-GA induced cytotoxicity via ROS generation with a IC50 of 50 µg/ml, which is two folds higher than that of free GA (25 µg/ml). The photosensitizer MB loaded Cu-GA NMOFs (MB/Cu-GA NMOFs) have been successfully used for synergistic of anticancer drug delivery and photodynamic therapy in cells in vitro (using Panc-1 and EAC cells). Next, the tumour regression curve in vivo showed that intravenously administered MB/Cu-GA NMOFs, in combination with photoirradiation of the tumor, induces significant reduction of the tumour burden, owing to combined effects of controlled ROS generation and photodynamic therapy. Therefore, CuGA NMOFs are promising new nanomaterials for in vivo pre-clinical cancer phototherapy applications.

D. EXPERIMENTAL SECTION Materials used. Copper acetate, a blue coloured powder used as a source of copper ion (Cu2+), was procured from SRL whereas the linker Gallic acid was purchased from Spectrochem. Methylene blue purchased from SRL and Aerosol OT obtained from Alfa Aesar. Horseradish peroxidase and o-dianisidine hydrochloride obtained from sigma Aldrich. Human pancreatic carcinoma epithelial cells (Panc-1) were bought from National Centre for Cell Science (NCCS), Pune, India. It was cultured according to the standard protocols. The cell culture biochemicals such as phosphate buffer saline (PBS, 1X), fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM, w/o phenol red) were obtained from High Media. The other chemicals such as amphotericin-B (antifungal), trypsin (detachment of cells from flask), and penicillin streptomycin (PS, anti-microbial) were purchased from Thermo Fisher scientific. The cells were cultured in high glucose DMEM medium supplemented with 10 % of FBS and 1 % of PS, along


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with amphotericin-B. The cells were placed in a humidified incubator operating at 37 °C and 5 % CO2. MTT reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide) was acquired from Sigma Aldrich. Synthesis of Cu-GA NMOF and MB/Cu-GA NMOF. In brief, 0.22 g of surfactant AOT was solubilized in double distilled water at room temperature, followed by the addition of 400 µl of nbutanol forming a transparent micellar solution. To this clear micellar solution, 200 µl each of 0.1 M copper acetate and gallic acid prepared in DMF, were added after maintaining a constant temperature of 70-80 °C. The reaction was continued for 12 hours, followed by addition of ethanol to precipitate the particles. The addition of ethanol removes the AOT and excess of non-coordinating solvent DMF from the frameworks. The as-synthesized blank framework was referred as Cu-GA NMOF. Then, we added 200 µl of methylene blue (15.7 mM in water) into the 10 ml aqueous solution of purified Cu-GANMOF (4 mg) under stirring for 1 hour at room temperature. We have called this resulting MB encapsulated copper-gallate NMOF as MB/Cu-GA NMOF. The scheme is shown in Scheme 1. Characterization. The as-synthesized framework (Cu-GA NMOF) was characterized with dynamic light scattering (DLS, MALVERN ZETASIZER), transmission electron microscope (TEM, TECNAI G2 T-30) and field emission scanning electron microscope (FESEM, MIRA3 TESCAN) for size and shape analysis. Then, energy dispersive X-ray spectroscopy (EDS, JEOL-JSM 6610LV) and thermogravimetric analysis (TGA, Perkin Elmer SII) were carried out for elemental/ compositional analysis. Powder X-ray diffraction (PXRD, Bruker D8 Discover) and selective area electron diffraction (SAED, TECHNAI G2 T-30) helped in determining the crystalline structure and phase characterization. Fourier transform infrared spectroscopy (FTIR, Perkin Elmer RXI) was carried out for identifying surface functionality of the frameworks. BET (AUTOSORB-1) technique was carried out for evaluation of surface area and porosity measurement, an important parameter for



structure elucidation. Optical studies (UV-visible, Shimadzu UV 1601 and fluorescence spectrometry, Cary Eclipse, Varian Polo) were carried out for drug loading and release at acidic and neutral medium. Release profile of GA from Cu-GA NMOF. The time-dependent release of GA from Cu-GA NMOF was investigated in phosphate buffer at two different pH (4 and 7) via Folin-Ciocalteau assay. Folin-Ciocalteau phenol reagent is a mixture of polyphosphomolybdic and polyphosphotungstic acid, which in alkaline medium gets reduced by polyphenols (e.g. GA) forming blue coloured molybdenum oxide/tungstic oxide absorbing at 740 nm.46 To estimate the release of GA from Cu-GA NMOFs, we prepared six aliquots containing a fixed concentration of Cu-GA NMOF (100 µg/ml) in 1 ml of phosphate buffer (0.1 M) and stored at 4 °C. Each aliquot was taken out every day from 4°C to room temperature for 5 days. At the fifth day, all the aliquots were centrifuged. The supernatant collected contained free GA, which has been released from the frameworks. To the supernatant, we added 25 µl of 7 % Na2CO3 along with 20 µl of Folin’s reagent and incubated for 1-hour prior to recording ODs at 740 nm. H2O2 Determination. The auto-oxidation of gallic acid (GA) produces ROS (especially H2O2) which is estimated by o-dianisidine reagent via absorption spectroscopy. For the in situ determination of H2O2, we carried out o-dianisidine assay which uses a colorimetric probe odianisidine dihydrochloride, a colourless detector. The analysis is based on the transition of reduced o-dianisidine (colourless) to oxidised o-dianisidine (yellow/orange) by H2O2.47 The detailed procedure is mentioned in the ESI. Photo-excited singlet oxygen generation assay. We studied the photo-induced singlet oxygen generation by Cu-GA NMOF, MB/Cu-GA NMOF and free MB in aqueous medium via measurement of photobleaching of the dye called ABMDMA. We studied the qualitative estimation


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of singlet oxygen by addition of 15 µM solution of ABMDMA with Cu-GA NMOF (25 µg/ml GA), MB/Cu-GA NMOF (25 µg/ml GA; 1.5µM MB) and free MB (1.5 µM) into 3 ml of water, followed by photoirradiation by 635 nm laser (power output 38 mW/cm2) for different periods of time (e.g. 5, 10 and 15 min). The absorbance decay of ABMDMA due the production of singlet oxygen at 380 nm is plotted as a function of irradiation time. In vitro studies (using Panc-1 cells) Cellular uptake of free GA and Cu-GA NMOF. The cellular uptake of free GA and Cu-GA NMOF, which contained equivalent amounts of GA, was investigated via intracellular Folin’s assay. When the cell confluency reaches to 70-80 %, we treated the cells with three different concentrations of GA (12, 25 and 50 µg/ml) as free and Cu-GA NMOF for 2 hours. The treated cells were washed using ice cold PBS, followed by lysis for supernatant extraction. The cell lysis reagent TX-100 (1 % prepared in PBS buffer) was added to the treated cell wells for 30 min. The mixture (cells + nanoparticles + TX-100) were scratched and centrifuged at 2000 rpm for 2 min just to remove the cell debris from the supernatant. The supernatant (cell lysate) was collected for spectrophotometric analysis. The estimation of intracellular GA was monitored when cell lysate was mixed with 500 µl of Folin Ciocalteu reagent and 1 ml of Na2CO3 (7 % aqueous solution). The resultant solution turned blue (indicating the presence of GA) and the ODs was recorded at 740 nm after 12 hours of incubation. Cellular uptake of MB/Cu-GA NMOF. We analysed the comparative cellular uptake of free MB and MB/Cu-GA NMOF via fluorometric analysis of cell lysates. The cells were treated with three different concentrations of MB/Cu-GA NMOF (50 µg/ml GA: 3 µM MB, 25 µg/ml GA: 1.5 µM MB, 12.5 µg/ml GA: 0.75 µM MB) and equivalent free MB for two hours.The cell lysates



was collected for fluorescence analysis under excitation wavelength of 650 nm. This study determined the comparative passive uptake between free MB and MB/Cu-GA NMOF. Concentration dependent GA-induced cytotoxicity. When the cell confluency in each of the well reaches to 40 – 50 %, the cells were treated with different concentration of GA (3, 6, 12, 25, 50 and 100 µg/ml) from free and Cu-GA NMOF for a duration of 24 hours. Afterwards, we washed all the wells with ice cold PBS, followed by replacement with fresh medium and incubation for 24 hours. Finally, 100 µl of MTT reagent (5 mg/ml in PBS) was added to the wells and further incubated for 2 hours in dark condition. Finally, the media was aspirated and 1 ml of DMSO were added to dissolve the formazan crystals formed (kept for 2 hours). The optical density of purple coloured solution was monitored at 540 nm using UV-vis spectrometer. Time dependent GA-induced cytotoxicity. The time-dependent cytotoxicity of GA, released from Cu-GANMOF w as carried out in vitro. A fixed concentration of Cu-GA NMOF (25 µg/ml) was dispersed into the high glucose DMEM media for different intervals of time/days (up to 4 days). These prepared aliquots of Cu-GA NMOF in cell culture media was used to treat the Panc-1 cells for 24 hours. Photoactivated toxicity. This study evaluated the light-activated cytotoxicity of three different concentrations of free MB and MB/Cu-GA NMOF, namely high, medium and low, as listed below: High: MB/Cu-GA NMOF (50 µg/ml GA; 3 µM MB), Medium: MB/Cu-GA NMOF (25µg/ml GA; 1.5 µM MB); Low: MB/Cu-GA NMOF (12.5 µg/ml GA; 0.75 µM MB) vs their equivalent free MB (3, 1.5 and 0.75 µM). In brief, the cells were treated with above listed concentrations of free MB and MB/Cu-GA NMOF for 2 hours. Afterwards, the cell wells were washed, replenished with fresh media, followed by further incubation of 2 hours. Next, each cell


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well was irradiated for 5 min by using a laser of 635 nm light (output power 38 mW/cm2). After 48 hours of incubation, the cells were analysed by MTT assay. In vitro and in vivo studies, using Ehrlich-Lettre ascites carcinoma (EAC) cell line. In vitro. The interaction of Cu-GA NMOF, MB/Cu-GA NMOF, free GA and free MB with EAC cells, which are obtained from mice, were investigated in vitro. The procedure is similar to that used for the treatment of Panc-1 cells. In vivo. The in vivo experiment was carried out in accordance with the Animal Ethical Committee of University of Delhi, India, under registration number 1666/GO/Re/S/12/CPCSEA (Protocol no. DU/KR/IAEC/2018/07). EAC cells were introduced in vivo in female Balb/c mice (20 to 25 g) by serial intraperitoneal passage at 7-10 days intervals, as published by Verma et al.48 Treatment schedule. The Balb/c mice were divided into six groups, with five animals in each group. Group I was control (tumor mice, untreated), receiving intravenous injection (post 7 and 14 days of inoculums) of only saline; Group II, Cu-GA per se, were given iv once a week for 2 weeks; Group III was free MB (Light-activated); Group IV was free MB (non-light-activated, or dark); Group V was MB/Cu-GA NMOF (light-activated); and Group VI was MB/Cu-GA NMOF (dark). A dose of 150μg/ml body weight was given intravenously, and 18 hours post injection MB/Cu-GA (light) group and MB (light) group mice were anesthetized using Sodium Pentabarbital at a concentration of 90 mg/kg of body weight and the tumours were irradiated by using laser of 635nm light (output power 38 mW/cm2). The tumour volume was estimated on alternate days.



ASSOCIATED CONTENT Supporting Information The characterization analysis using FTIR, BET and the optical studies including UV-Vis Spectra and fluorescence spectra has been included. The cell death mechanism is demonstrated using MTT data. Tumor Weight data is also included. AUTHOR INFORMATION Corresponding Author *Email: [email protected] ACKNOWLEDGMENT This study was mainly supported by Nanomission grant (sanction no. SR/NM/NS-1076/2015(G) received from the Department of Science and Technology, Government of India). SS and DM want to acknowledge fellowship support from University Grant Commission (UGC), Government of India. ABBREVIATIONS NMOF, nanoscale metal-organic framework; GA, gallic acid; Cu-GA NMOF, copper-gallate NMOF; MB/Cu-GA NMOF, Methylene blue encapsulated NMOF. REFERENCES 1

Vermeulen, L.; Sprick, M. R.; Kemper, K.; Stassi, G.; Medema, J. P. Cancer stem cells-old concepts, new insights. Cell Death Differ. 2008, 15(6), 947-958.

2

Popper, H. H. Progression and metastasis of lung cancer. Cancer Metastasis Rev. 2016, 35, 75-91.


Page 20 of 33

Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

Abd-Rabou, A. A.; Zoheir, K. M.; Ahmed, H. H. Potential impact of curcumin and taurine on human hepatoma cells using Huh-7 cell line. Clinical Biochem. 2012, 45, 1519-21.

4

Harlev, E.; Nevo, E.; Ephraim, P.; Lansky, E. P.; Lansky, S.; Bishayee, A. Anticancer attributes of desert plants: a review. Anticancer Drugs 2012, 23, 255-71.

5

Aggarwal, B. B.; Shishodia, S. Molecular targets of dietary agents for prevention and therapy of cancer. Biochem. Pharmacol. 2006, 71, 1397-421.

6

Surh, Y. J.; Chun, K. S. Cancer Chemopreventive effects of curcumin. Adv. Exp. Med. Biol. 2007, 595, 149-72.

7

Paivarinta, E.; Pajari, A. M.; Torronen, R.; Mutanen, M. Ellagic acid and natural sources of Ellagitannins as possible chemopreventive agents against intestinal tumorigenesis in the Min mouse. Nutr. Cancer 2006, 54(1), 79-83

8

Giftson, J. S.; Jayanthi, J. S.; Nalini, N. Chemopreventive efficacy of gallic acid, an antioxidant and anticarcinogenic polyphenol, against 1,2 dimethyl hydrazine induced rat colon carcinogenesis. Invest. New Drugs 2010, 28(3), 251-9.

9

Liang, C. Z.; Zhang, X.; Li, H.; Tao Y. Q.; Tao, L. J.; Yang, Z. R.; Zhou, X. P.; Shi, Z. L.; Tao, H. M. Gallic acid induces the Apoptosis of Human Osteosarcoma cells In Vitro and In Vivo via the Regulation of Mitogen-Activated Protein Kinase Pathways. Cancer Biother. Radiopharm. 2012, 27(10), 701-710.

10

Ji, B. C.; Hsu, W. H.; Yang, J. S.; Hsia, T. C.; Lu, C. C.; Chiang, J. H.; Yang, J. L.; Lin, C. H.; Lin, J. J.; Suen, L.; Wood, W. G.; Chung. J. G. Gallic acid induces Apoptosis via Caspase-3 and Mitochondrion-Dependent Pathways in Vitro and Supresses Lung Xenograft Tumor Growth In Vivo. J. Agric. Food Chem. 2009, 57(16), 7596-7604.



11

Ulasli, S. S.; Celik, S.; Gunay, E.; Ozdemir, M., Hazman, O.; Ozyurek, A.; Koyuncu, T.; Uniu, M. Anticancer effects of thymoquinone, caffeic acid phenethyl ester and resveratrol on A549 non- small cell lung cancer cells exposed to benzo(a) pyrene. Asian Pac. J. Cancer Prev. 2013, 14, 6159-64.

12

Kroes, B. H.; van den Berg, A. J.; van Ufford, H. C.; van Dijk, H.; Labadie, R. P. Antiinflammatory activity of gallic acid. Planta Med. 1992, 58, 499-504.

13

Benherlal, P. S.; Arumughan, C. Studies on modulation of DNA integrity in Fenton’s system by Phytochemicals. Mutat. Res. 2008, 648, 1-8.

14

Babich, H.; Schuck, A. G.; Weisber, J. H.; Zuckerbraun, H. L. Research strategies in the study of the pro-oxidant nature of polyphenol nutraceuticals. J. Toxicol. 2011, 1-12.

15

Russell Jr, L. H.; Mazzio, E.; Badisa, R. B.; Zhu, Z. P.; Agharahimi, M.; Oriaku, E. T.; Goodman, C. B. Auto-oxidation of Gallic acid induces ROS-dependent death in Human Prostate cancer LNCaP cells. Anticancer Res. 2012, 32(5),1595–1602.

16

Patel, S. S.; Goyal, R. K. Cardioprotective effects of gallic acid in diabetes-induced myocardial dysfunction in rats. Pharmacognosy Res. 2011, 3(4), 239-45.

17

Sameri, M. J.; Sarkaki, A.; Farbood, Y.; Mansouri, S. M. Motor disorders and impaired electrical power of Pallidal EEG improved by gallic acid in animal model of Parkinson’s disease. Pak. J. Biol. Sci. 2011, 14(24), 1109-16.

18

Hsieh, C. L.; Lin, C. H.; Wang, H. E.; Peng, C. C.; Peng, R. Y. Gallic acid exhibits risk of inducing muscular hemorrhagicliposis and cerebral hemorrhage - its action mechanism and preventive strategy. Phytother. Res. 2015, 29(2), 267-80.


Page 22 of 33

Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19

Dorniani, D.; Hussein, M. Z.; Kura, A. U.; Fakurazi, S.; Halim Shaari, A.; Ahmad, Z. Preparation of Fe3O4 magnetic nanoparticles coated with gallic acid for drug delivery. Int. J. Nanomed. 2012, 7, 5745–5756.

20

Shahrzad, S.; Aoyagi, K.; Winter, A.; Koyama, A.; Bitsch, I. Pharmacokinetics of gallic acid and its relative bioavailability from tea in healthy humans. J. Nutr. 2001, 131, 1207– 1210.

21

Daduang, J.; Palasap, A.; Daduang, S.; Boonsiri, P.; Suwannalert, P.; Limpaiboon, T. Gallic acid enhancement of gold nanoparticle anticancer activity in cervical cancer cells. Asian Pac. J. Cancer Prev. 2015, 16(1), 169-74.

22

Cai, W.; Chu, C. C.; Liu, G.; Wáng, Y. Metal-organic Framework-based Nanomedicine Platforms for Drug Delivery and Molecular Imaging, Nano, Small Micro 2015, 11(37), 4806-4822.

23

He, C.; Liu, D.; Lin, W. Nanomedicine Application of Hybrid Nanomaterials Built from Metal-Ligand coordination bonds: Nanoscale Metal-Organic Frameworks and Nanoscale Coordination Polymers. ACS Chem. Rev. 2015, 115, 11079−11108.

24

Zhuang, J.; Kuo, C. H.; Chou, L.Y.; Liu, D.Y.; Weerapana, E.; Tsung, C. K. Optimized metal-organic framework nanospheres for drug delivery: evaluation of small molecule encapsulation. ACS Nano 2014, 8, 2812- 2819.

25

Horcajada, P.; Chalati, T.; Serre, C.; Gillet, B.; Sebrie, C.; Baati, T.; Eubank, J. F.; Heurtaux, D.; Clayette, P.; Kreuz, C.; Chang, J. S.; Hwang, Y. K.; Marsaud, V.; Bories, P. N.; Cynober, L.; Gil, S.; Ferey, G.; Couvreur, P.; Gref, R., Porous metal-organic framework nanoscale carriers as potential platform for drug delivery and imaging. Nat. Mater. 2010, 9, 172.

26

Barcelo, J. M.; Guieb, M.; Ventura, A.; Nacino, A.; Pinasen, H.; Viernes, L.; Yodong, T.;



Estrada, B.; Valdez, D.; Binwag, T., Antibacterial, Prooxidative and Genotoxic Activities of Gallic acid and its Copper and Iron Complexes against Escherichia coli. Asia Pac. J. Multidiscip. Res. 2014, 2(6), 45-56. 27

Cooper, L.; Hidalgo, T.; Gorman, M.; Farnandez, T. L.; Vazquez, R. S.; Olivier, C.; Guillou, N.; Serre, C.; Martineau, C.; Taulelle, F.; Borges, D. D.; Maurin G.; Fernandez A. G.; Horcajada, P.; Devic, T. A biocompatible porous Mg-gallate metal-organic framework as an anti-oxidant carrier, Chem. Commun. 2015, 51, 5848-5851.

28

Mantion, A.; Massuger, L., Rabu, P.; Palivan, C.; McCusker, L. B.; Taubert, A. MetalPeptide Frameworks (MPFs): “Bioinspired” Metal Organic Frameworks. J. Am. Chem. Soc. 2008, 130, 2517.

29

McKinlay, A. C.; Xiao, B.; Wragg, D. S.; Wheatley, P. S.; Megson, I. L.; Morris, R. E. Exceptional behaviour over the whole adsorption-storage delivery cycle for NO in porous metal-organic frameworks. J. Am. Chem. Soc. 2008, 130, 10440.

30

Feller, R. K.; Cheetham, A. K. Fe(III), Mn(II), Co(II) and Ni(II) 3,4,5-trihydroxybenzoate (gallate) dehydrates; a new family of hybrid framework materials. Solid State Sciences 2006, 8, 1121–1125.

31

Mu, X.; Yan, C.; Tian, Q.; Lin J.; Yang, S. BSA-assisted synthesis of ultrasmall gallic acidFe(III) coordination polymer nanoparticles for theranostics. Int. J. Nanomed. 2017, 12, 7207–7223.

32

Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; Piette, J.; Wilson, B. C.; Golab, J. Photodynamic therapy of cancer: an update. CA Cancer J. Clin. 2011, 61, 250−281.

33

Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B. W.; Hasan, T. Imaging and Photodynamic therapy: mechanism, monitoring, and optimization. Chem. Rev. 2010, 110, 2795−2838.


Page 24 of 33

Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34

Roy, I.; Ohulchanskyy, T. Y.; Pudavar, H. E.; Bergey E. J.; Oseroff A. R.; Morgan, J.; Dougherty T. J.; Prasad P. N. Ceramic-based nanoparticles entrapping water insoluble photosensitising anticancer drugs: a novel drug carrier system for photodynamic therapy. J. Am. Chem. Soc. 2003, 125, 7860.

35

Lan, G.; Ni, K.; Xu, Z.; Veroneau, S. S.; Song, Y.; Lin, W. Nanoscale Metal-Organic Framework overcomes Hypoxia for Photodynamic Therapy Primed Cancer Immunotherapy. J. Am. Chem. Soc. 2018, 140(17), 5670-5673.

36

Lu, K.; He, C.; Lin, W. Nanoscale Metal-Organic Framework for Highly Effective Photodynamic therapy of Resistant Head and Neck cancer. J. Am. Chem. Soc. 2014, 136(48), 16712-15.

37

Sharma, S.; Sethi, K.; Roy, I. Magnetic nanoscale metal-organic frameworks for magnetically aided drug-delivery and photodynamic therapy, New J. Chem., 2017, 41, 11860-11866.

38

Sanchez, M. S.; Diaz, K.; Getachew, N.; García, M. D.; Diaz, I. Synthesis of metal-organic frameworks in water at room temperature: salts as linker sources. Green Chem., 2015, 17, 15001509.

39

Loiseau, T.; Serre, C.; Huguenard, C.; Fink, G.; Taulelle, F.; Henry, M.; Bataille, T.; Ferey, G. A rationale for the large breathing of the porous aluminium-terephthalate (MIL53) upon hydration. Chem. Eur. J. 2014, 10, 1373–1382.

40

Ling, S.; Walton, I. R.; Slatera, B. Theoretical study of conformational disorder and selective adsorption of small organic molecules in the flexible metal-organic framework material MIL-53 Fe. Molecular Simulation 2015, 41, 1348–1356.

41

Anbia, M.; Sheykhi, S. Synthesis of nanoporous Copper-terephthalate [MIL-53(Cu)] as a



novel methane storage adsorbent. J. Natural Gas chemistry 2012, 21(6), 680-68. 42

Pu, M.; Ma, Y.; Wan, J.; Wang, Y.; Wang, J.; Brusseau, M. L. Activation performance and mechanism of a novel heterogeneous persulfate catalyst: Metal Organic Framework MIL53(Fe) with FeII/FeIII mixed-valence coordinative unsaturated iron center. Catal. Sci. Technol. 2017, 7, 1129-1140

43

Li, C.; Xiong, Z.; Zhang, J.; Wu, C.; The strengthening role of amino group in metalorganic framework MIL-53(Al) for methylene blue and malachite green dye adsorption. J. Chem. Eng. Data 2015, 11, 3414-22.

44

He, C.; Liu, D.; Lin, W. Nanomedicines Applications of Hybrid Nanomaterials Built from Metal-Ligand Coordination Bonds: Nanoscale Metal-Organic Frameworks and Nanoscale Coordination Polymers. Chem. Rev. 2015, 115 (19),11079–11108.

45

Zhang, S.; Li, J.; Lykotrafitis, G.; Bao G.; Suresh, S. Size-Dependent Endocytosis of Nanoparticles. Adv. Mater. 2009, 21,419-424.

46

Arogba, S. S. Phenolics, Antiradical Assay and Cytotoxicity of Processed Mango (Mangiferaindica) and Bush Mango (Irvingiagabonensis) Kernels. Nigerian Food Journal 2014, 32 (1), 62-72.

47

Fernandes, F. H.; Salgado, H. R. Gallic Acid: Review of the Methods of Determination and Quantification. Crit. Rev. Anal. Chem. 2016, 46(3), 257-65.

48

Verma, A. K.; Leekha, A.; Kumar, V.; Moin, I.; Kumar, S. Biodistribution and In-vivo Efficacy of Doxorubicin Loaded Chitosan Nanoparticles in Ehrlich Ascites Carcinoma (EAC) Bearing Balb/c Mice. J. Nanomed. Nanotechnol. 2018, 9, 510-515.


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Scheme 1. Schematic representation of synthesis of MB/Cu-GA NMOF in micellar medium



Figure 1. (A) Transmission electron microscopic (TEM) image having a scale bar of 200 nm; (B) Field emission scanning electron microscope (FESEM) image having a scale bar of 200 nm; (C) Dynamic light scattering (DLS) data of Cu-GA NMOF


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Figure 2. (A) Energy dispersive spectrum (EDS) and the inset containing tabular data containing weight percentage of elements present within Cu-GA NMOF; (B) thermogravimetric analysis (TGA) of Cu-GA NMOF; (C) Powder high resolution X-ray diffraction, and (D) selected area electron diffraction (SAED) of Cu-GA NMOF



Figure 3. (A) A comparative plot between log (A/Ao) versus time showing decrease in the optical density at 380 nm; (B) Represents the time-dependent release profile of GA from Cu-GA NMOF detected using Folin’s assay; (C) Release profile of MB from MB/Cu-GA NMOF using fluorescence emission intensity measured at 680 nm for MB


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Figure 4. The bar graph showing the concentration dependent cellular uptake of (A) Free GA and Cu-GA NMOF using Folin’s reagent absorbing at 760 nm; (B) Free MB and MB/Cu-GA NMOF using fluorescence emission at 680 nm for MB; (C) MTT assay showing the concentration-dependent cytotoxicity of free GA and its equivalent concentrations within Cu-GA NMOF, treated with Panc-1 cells for 24 hours; (D) MTT assay displaying time dependent- cytotoxicity of Cu-GA NMOF at 25 µg/ml dosage treated with Panc-1 cells for 24 hours



Figure 5. (A) MTT assay of free MB and MB/Cu-GA NMOF treated Panc-1 cells for 48 hours at three different dosages of MB and Gallic acid; For MB/Cu-GA NMOF: High (50 µg/ml GA, 3 µM MB), Medium (25 µg/ml GA, 1.5 µM MB) and Low (12.5 µg/ml, 0.75 µM MB) and Free MB: High (3 µM), Medium (1.5 µM), Low (0.75 µM), without (dark) and with light irradiation for 5 min.; (B) MTT assay of free MB, MB/Cu-GA NMOF and CuGA NMOF treated EAC cells for 48 hours at three different dosages mentioned above., without (dark) and with light irradiation for 5 min


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Figure 6. (A) Tumour regression curve post- laser therapy; (B) Photographs of excised tumours on Day 21.


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