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Article Cite This: ACS Omega 2019, 4, 1354−1363
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A Self-Healing Metal−Organic Gel (MOG) Exhibiting pH-Responsive Release of a Chemotherapeutic Agent, Doxorubicin: Modulation of Release Kinetics by Partial Dehydration of Matrix Nayuesh Sharma,# Peeyush K. Sharma,# Yashveer Singh,* and C. M. Nagaraja* Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar 140 001, Punjab, India.
ACS Omega 2019.4:1354-1363. Downloaded from pubs.acs.org by 95.85.80.192 on 01/19/19. For personal use only.
S Supporting Information *
ABSTRACT: Use of soft and porous metal−organic gels (MOGs), formed by metal−ligand coordination, has generated immense research interest in fields like conductance, catalysis, chemical sensing, and dye adsorption, but there are only few reports on their use in drug delivery, particularly cancer drug delivery. Consequently, a Cr3+-based metal− organic gel (MOG1) was synthesized from Cr(NO3)3·9H2O and 2,2′bipyridine-4,4′-dicarboxylic acid (BPDA) in DMF, using a solvothermal route. The gel exhibited strong resilience when subjected to a dynamic frequency sweep from 1−100 rad/sec at a constant strain of 0.1% and minimal loss of storage modulus when subjected to repeated cycles of 0.1 and 100% strain for 60 s, and it exhibited self-healing characteristics. The doxorubicin (DOX)-loaded gels were developed to assess the drug release profiles at physiological (7.4) and intratumoral (6.4) pH and found to be pH-dependent. A sustained zero-order release of 54% drug was obtained after 48 h at pH 7.4 and, a faster release, with 87.4% of the drug being released in 12 h following pseudo-Fickian diffusion at pH 6.4. The most unique feature of the gel, not reported earlier, was the transformation of the drug release kinetics from zero order to exponential decay upon dehydration to 80% of its original weight. The drug release slowed down further when the gel was dehydrated to 50% of its original weight. The cell viability assay showed no significant toxicity of MOG1 to mdck (kidney epithelial) cell lines, and more than 75% cells were viable, even after 72 h, with concentrations up to 100 μg/mL, whereas the DOX-loaded MOG1 demonstrated dose-dependent toxicity to cancer cell lines (A549) in the concentration range investigated (0.1−5 μg/mL equivalent DOX). Fluorescence microscopy studies revealed efficient internalization of DOX released from MOG1 into cancer cells. The gel may be employed as an injectable depot for quick and selective delivery of a chemotherapeutic agent to tumoral sites. MOGs for various applications in the field of conductance, catalysis, chemical sensing, and dye adsorption.32−34 The self-healing properties of MOGs are governed by various interactions, such as reversible coulombic interactions, molecular recognitions, hydrogen bonding, hydrophobic interactions, and metal−ligand coordination. Quite a few reports of MOGs exhibiting self-healing property have been reported in literature.35,36 Yan et al. reported a self-healing supramolecular heterometallic MOG based on Co2+ and Ni2+ ions exhibiting the synergistic effect of the constituent metal ́ et al. fabricated a Cu2+-based self-healing MOG, ions.37 Diaz which showed proton conducting properties.38 Yu and coworkers reported a self-healing hybrid gel based on Zn-tpy and polypyrrole, which showed conductivity.39 Nitschke et al. developed a self-healing polymer MOG based on Fe3+ for the release of small molecules in response to competing guests.40 However, the use of MOGs as drug delivery vehicles has
1. INTRODUCTION The self-healing materials have attracted tremendous research interest because of their ability to repair or restore themselves in response to damage.1−5 Materials like polymers, dendrimerclay systems, and nanocomposites have been investigated for their self-healing properties,6−8 but the design of macroscopic self-healing materials by utilizing noncovalent interactions still remains a major challenge. Although design of several supramolecular gels by using molecules containing diverse functional groups has been explored,9−11 the development of metal−organic gels (MOGs) based on metal−ligand coordination bonds has gathered momentum only recently.12−16 A plethora of literature exists on the design of metal−organic frameworks (MOFs) or coordination polymers (CPs)17−19 for stimuli-responsive controlled delivery of small drug molecules20−25 but not as self-healing materials because of their high rigidity.26 MOGs, on the contrary, are soft and can be used to develop self-healing materials by employing the principles of metal−ligand coordination chemistry.27−31 Therefore, current efforts are focused on the development of © 2019 American Chemical Society
Received: October 16, 2018 Accepted: January 3, 2019 Published: January 16, 2019 1354
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Figure 1. (A) FT-IR spectra of freeze-dried MOG1 and BPDA ligand. (B) Plausible structure of MOG1.
Figure 2. (A) MOG1 in an inverted vial after 24 h. (B,C) SEM images of a freeze-dried sample of MOG1.
drug distribution profile of chemotherapeutic agents by attaching it to polymers or nanoparticles;49 and local targeting, where the chemotherapeutic agent is directly administered to locally accessible tumors.50 Also, the chemotherapeutic agent must be selectively released in tumors,46,47 which can be achieved by loading the anticancer drug into the drug delivery systems employing interactions sensitive to enzymes overexpressed in tumors, acidic pH, or hypoxia. A pH-responsive system capable of controlled release of chemotherapeutic agents to tumor sites is highly desirable as it can be directly administered to solid tumors in a minimally invasive manner, where it can act as a drug depot for prolonged periods.51,52 A viscoelastic gel system with self-healing capability can be a suitable candidate for such applications.53 Based on these considerations, we fabricated a Cr3+-based metal−organic gel, MOG1, in DMF using the solvothermal route and subjected it to amplitude, frequency, and temperature sweeps along with alternating cycles of strains to investigate its viscoelastic and self-healing characteristics. DOX, a chemotherapeutic agent, was loaded into the MOG1, drug release kinetics was studied in aqueous buffers of physiological and tumoral pH, and modulation of drug release kinetics by partial dehydration of matrix was also investigated. The cell viabilities of kidney epithelial (mdck) cell lines against MOG1 and antitumor activities of DOX-loaded MOG1 against A549 cell lines were assessed using the MTT assay. Finally, the intracellular distribution of DOX released from MOG1 was observed under a fluorescence microscope.
received only limited attention. For instance, Zhao et al. demonstrated the use of Fe3+-BDC-based MOG for DOX loading and release, but the self-healing characteristics of the gel was not explored and the drug release kinetics was too fast to be effective for a chemotherapeutic agent.41 Recently, non-osmotic gels have generated interest because of their unique ability to retain intrinsic water like hydrogels but resist the inevitable swelling under physiological conditions, which is undesirable in the semiconfined in vivo spaces, as swelling can affect their mechanical robustness as well as other intrinsic properties.42,43 Viscoelastic gels derived from the metal coordinated with organic linkers are an example of such nonswelling systems, and it has been shown that the intrinsic water content of such systems significantly influence their physicochemical characteristics.44 Hence, a viscoelastic system with high water retention and swelling resistance characteristics could have immense potential as an implant material for intratumoral delivery of chemotherapeutic agents, offering the possibility of modulating drug release properties by controlling the intrinsic water content of the system.45 Efficient cancer drug delivery has manifold challenges.46,47 Since chemotherapeutic agents are associated with significant side effects, cancer drug delivery must incorporate drug targeting strategies. A chemotherapeutic agent can be targeted to tumors by employing one of the following targeting approaches: active targeting, achieved by attaching the chemotherapeutic agent to ligands with high affinity for cancer cell receptors;48 passive targeting, achieved by changing the 1355
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Figure 3. Viscoelastic characteristics of MOG1: (A) amplitude sweep for LVE determination, (B) frequency sweep at a constant strain of 0.1%, (C) temperature sweep from 25−120 °C at a constant strain of 0.1%, and (D) self-healing properties when subjected to alternating cycles of 0.1 and 100% strain for 60 s.
2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of MOG1. In the current work, we prepared a new Cr3+-BPDA-based metal− organic gel (MOG1) solvothermally from the reaction of Cr3+ nitrate and 2,2′-bipyridine-4,4′-dicarboxylic acid (BPDA) ligand in DMF at 100 °C. Cr3+ metal was chosen as it is oxophilic, and in the presence of DMF as the solvent, BPDA2− ion coordinates to Cr3+ ion through carboxylate groups, resulting in a stable gel formation. This was further supported by the observation that under similar synthesis conditions, the use of relatively soft metal ions, such as Fe2+, Co2+, Ni2+, Cu2+, and Zn2+, did not result in a stable gel formation. FT-IR spectra of freeze-dried MOG1 clearly showed a shift in the carbonyl stretching frequency to 1634 cm−1 compared to that of free ligand (1725 cm−1), indicating the coordination of carboxylate groups of BPDA to Cr3+ (Figure 1A,B).54 The gel formation was further confirmed by inversion test in which the vial containing “as-synthesized” gel was kept in an inverted position and was found to be stable even after 24 h, suggesting stable gelation (Figure 2A). SEM images of the gel revealed its porous nature with leaf-like morphology (Figure 2B,C). Based on the above analyses, we propose that the structure of MOG1 is constituted by the interaction of BPDA2− with Cr3+ ions through carboxylate oxygens (Figure 1B), resulting in the formation of a porous metal−organic gel with the general formula [Cr2(BPDA)3]·XDMF, similar to the MOFs constructed from BPDA ligand and M3+ ions.55 The solvent DMF molecules are trapped in the pores to provide soft and wet
nature to the Cr-BPDA network, thus providing gel characteristics. Thermogravimetric analysis of freeze-dried MOG1 showed an initial weight loss of 12.12% in the temperature range of 25−110 °C, corresponding to the loss of about five guest water molecules (Figure S1, Supporting Information). The second weight loss of 30.38% in the temperature range of 110−467 °C corresponded to the loss of one BPDA2− anion (calcd. 29.15% based on the formula [Cr2(BPDA)3]), and above 612 °C, the compound decomposed with the loss of other two BPDA2− moieties. 2.2. Viscoelastic Characterization of MOG1. The gel was subjected to amplitude, frequency, and temperature sweeps along with alternating cycles of strains to investigate its viscoelastic and self-healing characteristics. The small amplitude dynamic oscillatory rheology studies provided an insight into the viscoelastic characteristics of the gel. Application of a dynamic amplitude sweep from 0.01 to 200% provided a maximum storage modulus value of 1939.5 Pa, while the loss modulus values drifted around the 120 mark throughout the LVE range, extending up to 10% strain, and the crossover point was found to be around 79.45%, after which the gel transformed into a liquid-like entity (Figure 3A). This crossover strain is one of the highest values reported for MOGs. Crossover at higher amplitudes indicates efficient deformation of the gel structure under strains while resisting failure or yielding. A comparable crossover value of 300 Pa was reported by Sahoo et al.30 albeit in terms of stress amplitude 1356
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Figure 4. (A) Self-healing of two discrete pieces of MOG1 and the diffusion of fluorescein dye from loaded to unloaded regions. (B−E) Injectability of MOG1 from a syringe needle and instant self-healing into a gel.
2.3. Doxorubicin Loading/Release Studies and Effect of Dehydration on the Release Kinetics. We investigated the loading and release of DOX from MOG1 at pH 7.4 (physiological) and 6.4 (tumoral).57 DOX is a chemotherapeutic agent exhibiting broad-spectrum antitumor activity against various cancers, including breast, ovarian, and gastric cancers as well as the acute lymphoblastic leukemia, either alone or in combination with other agents.58 An equilibrium DOX loading was achieved after the complete removal of DMF from the bulk of the gel. The gel was initially immersed in DI water to achieve the solvent exchange. This solvent exchange was conducted to provide the gel a more biocompatible aqueous environment, thus rendering it suitable for biomaterial applications. The absence of DMF was confirmed by a UV− Vis spectrophotometer (200−225 nm). Once fully hydrated, the gel was suspended in 15 mL DOX solution (2.5 mg/mL) and incubated for 24 h, with constant shaking to facilitate diffusion of drug molecules into the gel. The loading of DOX was found to be 8.12 μg per mg of fully hydrated gel, as estimated from absorption measurements (Figure S3, Supporting Information). We speculate that the significant loading of DOX into MOG1 could be ascribed to its hydrogen bonding interactions, involving the O−H groups in DOX and pyridine N atoms in MOG1 (Figure S4, Supporting Information).59,60 The hydrogen bonding interactions are supported by the fact that FT-IR spectroscopy showed shifting of bands at 1939 and 1000 cm−1, corresponding to O−H and C−O bonds of DOX loaded in MOG1, respectively, compared to those of free DOX (Figures S5 and S6, Supporting Information). The drug release and impact of inherent water content of the gel and pH of the dissolution media on the release kinetics was investigated. An experimental design comprising two variables, pH and partial dehydration of gel, were selected to investigate the DOX release behavior. The gel was dehydrated up to 80 and 50% of its original hydrated state (100%) to obtain three different samples with a variable degree of hydration. All three gel samples were loaded with the same amount of DOX. To achieve this uniformity, 40 mg of the gel was taken for 100% hydrated sample, while 32 and 20 mg of gels were taken for samples dehydrated to 80 and 50% of its original weight,
rather than percent strain, and a lower crossover value of only 7% was reported by Foster et al.56 Dynamic frequency sweep measurements carried out at a constant strain of 0.1% revealed virtually unaffected gels across the frequency variation of 1− 100 rad/sec (Figure 3B). Overall, these results suggest the resilient characteristics of the gel against strains of varying frequency and amplitude. To assess the thermoresponsive behavior of the gel, a temperature sweep from 25 to 120 °C was performed, keeping the amplitude constant at 0.1%. The results showed a linear improvement in the values of viscoelastic parameters, which could be attributed to partial escape of solvent molecules from the gel matrix, resulting in a denser structure with improved overall gel strength (Figure 3C). The gel exhibited self-healing property with minimal loss in the values of storage modulus when subjected to repeated cycles of 0.1−100% strain for 60 s each. This instantaneous healing ability is resulting from coordination-based interactions between metal and ligands, which can easily realign themselves once the strain is removed. As can be seen in Figure 3D, the rheological parameter (storage modulus) of MOG1 recovered instantly after every cycle of a higher strain of 100% (relaxation) with no overall reduction. The results are comparable to previous reports on similar materials where the gels were shown to recover instantly when the strain was relaxed.30,56 No significant deviation in viscoelastic and self-healing characteristics of the gel was observed after exchanging the solvent from DMF to water, indicating that the structural integrity of gel was not affected by the solvent exchange process (Figure S2, Supporting Information). The self-healing characteristics of MOG1 were evident visually too. Two separate pieces of the gel, one as-synthesized and another fluorescein dye-loaded, fused with each other when brought together, and there was a diffusion of fluorescein dye throughout the gel network, indicating efficient self-healing (Figure 4A). The gel could be passed through a syringe needle, and it immediately self-healed into a gel, which indicates that gels possess thixotropic characteristics (Figure 4B−E; see also Video S1). 1357
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Figure 5. Percentage DOX released from MOG1 against time in buffer: (A) 7.4 (physiological) and (B) 6.4 (tumoral). The DOX-loaded MOG1 was dehydrated up to 80 and 50% of their original weights.
been investigated earlier. It provides a unique capability to modulate the drug release in such networks by controlling the hydration. Another feature of MOG1 is its ability to retain its structural integrity even after the drug release, which is advantageous over previously reported materials, where the small molecule release was due to temporal degradation of the gel architecture.31 2.4. Cell Viability of MOG1 and Cr3+ Release from the Matrix. The safety and toxicity profiles of MOG1 were assessed. All in vitro cell studies were conducted utilizing indirect extraction method on the basis of ISO Standard 10993-5.65 Initially, MTT assay-based cell viability studies were conducted using mdck (kidney epithelial) cell lines to assess the in vitro toxicity of MOG1 at various concentrations, ranging from 20 to 100 μg/mL (Figure 6). The results showed
respectively. All samples were then subjected to limited sink conditions of pH 7.4 and 6.4. As can be seen from Figure 5, the DOX-loaded gel with 100% hydration showed a sustained zero-order release of 54% DOX after 48 h at pH 7.4. The DOX release data gave a regression value of 0.994 when fitted for zero-order release kinetics model.61 This particular observation highlights the significance of MOG1 for sustained delivery of drugs for longer durations of time. At pH 6.4, the release kinetics was faster, supposedly following a pseudo-Fickian diffusion model,62,63 with 87.4% of the drug released within 12 h. This significant acceleration in the drug release at a lower pH can be attributed to reduction in the hydrogen bonding interactions between the drug and MOG1.64 The data reported here offers a significant improvement in comparison to earlier reports. The release properties of metallohydrogels developed by Banerjee and co-workers were assessed using a model drug, caffeine.31 Most of the drug was released in less than 100 min, and the release was sustained only for 5 h. The MOG developed by Zhao and co-workers was shown to release DOX, but the release was sustained only for 90 min.41 Both release profiles are too quick to be effective. It is also to be noted that the MOG1 developed in the present work was found to be intact at the end of release experiments, which implies a diffusion-driven drug release behavior for this gel as opposed to abovementioned materials, where the release behavior was predominantly degradation-driven.31,41 Therefore, it can be concluded that MOG1 is pharmacokinetically more suitable as an implantable/injectable depot for the DOX release. The MOG1 samples dehydrated up to 80% of its original weight showed a release of 50.9% of DOX after 48 h at pH 7.4 following an exponential decay model (Figure 5 and Table S1, Supporting Information). At pH 6.4, a maximum release of 82% DOX was observed after 24 h following a pseudo-Fickian diffusion model, albeit with a low regression value of 0.925 when fitted for a Korsmeyer−Peppas model. The lower regression and a slower release as compared to 100% hydrated samples can be attributed to reduced diffusion of the release medium into the gel matrix due to partial dehydration of the gel sample. Following a similar pattern, the samples dehydrated to 50% of their original weights showed an even slower and lower release of DOX although following a non-Fickian diffusion model (Figure 5 and Table S1, Supporting Information). The transition of drug release kinetics from zero order to an exponential decay upon partial dehydration of MOG1 is a characteristic feature of this gel, which has not
Figure 6. Cell viability of mdck cells incubated with different concentrations of MOG1 for 24 and 72 h (n = 3).
no significant toxicity, and majority of cells were viable under the conditions studied. Even upon exposure to high concentrations of MOG1 (100 μg/mL) for 72 h, more than 75% of cells were found viable, which can be compared with a similar material based on a silver-coordinated pyridyl system with an IC50 value of 50 μg/mL66 and tartaric acid-based aluminum-coordinated gels with 80% viability at 7.81 μg/ mL.67 The cell viabilities were further ascertained using brightfield microscopy images, which indicated excellent compatibility of gel toward cells (Figure S7, Supporting Information). In addition, the MP-AES analyses of the release media were carried out and showed no significant leaching of Cr3+ ions from the gel network for up to 72 h (Table S2, Supporting 1358
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Figure 7. Cell viability of A549 cells incubated with different concentrations of free DOX and extracts of DOX-loaded MOG1 (MOG1-DOX): (A) 0−24 h. (B) 24−48 h. (C) IC50 values. Asterisk (*) denotes p < 0.05 (significant difference).
consistent cytotoxicity was observed with IC50 values of 1.861 and 2.965 μg/mL after 24 and 48 h, respectively (Figure 7C). The dose-dependent cellular toxicity was further confirmed with bright-field microscopy images (Figure S8, Supporting Information), which showed the healthy cells at minimal DOX/equivalent DOX concentrations but cellular debris at higher concentrations, with minimal adherent cells visible at the highest concentration (5 μg/mL) investigated. The extracts from free MOG1 exhibited minimal toxicity at all concentrations against A549 cells, similar to normal mdck cell lines, but the cellular toxicities of MOG1 extract against A459 cells were much lower than mdck. About 87.54 ± 6.15% of mdck cells were viable when treated with 100 μg/mL of MOG1 extract, whereas 85.32 ± 5.54% of A549 cells were viable when treated with 616.5 μg/mL of extract. The intracellular distribution of DOX released from MOG1 was observed using a fluorescence microscope and compared with that of free DOX (Figure 8).70 The studies attain significance as it ascertains the stability of DOX during the release process and its ability to enter the cells so as to exert the cytotoxic effect. The DIC and fluorescence images of cells treated with extracts from DOX-loaded MOG1 (5 μg/mL equivalent DOX) exhibited co-localization of DOX (red emission) with nuclei-staining DAPI (blue emission) within the cytoplasm and nuclei, thus indicating efficient intracellular distribution of DOX released from MOG1, similar to the free DOX, but a lower intensity was observed for the released DOX as compared to the free DOX (equivalent concentrations), indicating incomplete release of DOX from MOG1-DOX even after 48 h of extraction. As expected, no such co-localization was observed in cells treated with extracts of free MOG1.
Information). The evident cytocompatibility can be attributed to negligible leaching of Cr3+. Prior literature indicates that Cr3+ is a trace micronutrient, with a normal-to-modest intake regarded as safe.68 The in vitro toxic doses range up to 2000 μg/mL and are known to cause minimal in vivo toxicity. 2.5. Cell Viability of DOX-Loaded MOG1 and Uptake Studies. The cytotoxic efficiency of the DOX released from the DOX-loaded MOG1 was assessed against A549 cell lines using MTT assay69 and compared to the free DOX (positive control) and equivalent concentrations of DOX released from MOG1-DOX at pH 7.4 and 6.4 for two distinct time periods: up to 24 h and 24−48 h. The MOG1-DOX (50 mg) was immersed into 2 mL of phosphate buffer (PB) of pH 6.4 and 7.4 at 37 °C for 48 h, with media being replaced after 24 h. The release media was collected and sterilized by passing through a 0.22 μm filter and appropriately diluted to get equivalent DOX concentrations. The cell viabilities when treated with free DOX (0.1−5 μg/mL) and MOG1-DOX extract (0.1−5 μg/mL equivalent DOX) were measured using MTT assay. The in vitro toxicity data suggested that the DOX was released from the MOG1-DOX in a pH-dependent manner, with a faster release at pH 6.4, yielding a lower cell viability as compared to that at pH 7.4 (Figure 7A). Owing to the faster release at pH 6.4, the release media from 24−48 h showed significantly higher cell viabilities, while the release media at pH 7.4 showed a sustained cell-killing efficiency because of the zero-order release profile of DOX from MOG1DOX at pH 7.4 (Figure 7B). The IC50 of free DOX was found to be 1.079 μg/mL, whereas for up to 24 h release media at pH 6.4, IC50 values were found to be 1.377 μg/mL, which drastically increased to 8.370 μg/mL for 24−48 h release media, indicating maximum release within the initial 24 h. For release media at pH 7.4, a 1359
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DMF (8 mL) was stirred for 30 min in a 30 mL glass vial. The vial was capped tightly, sealed with Teflon tape, and kept in a preheated oven at 100 °C for 7 days. After 7 days, the vial was cooled to room temperature, and the excess solvent was removed to yield a dark green-colored gel. The reactions were done in triplicate. 4.3. Rheological Characterization. The viscoelastic properties of MOG1 gel were investigated by dynamic oscillatory rheology, using an Anton Paar MCR 102 rheometer equipped with a dynamic Peltier heating system. The measurements were performed by parallel plate geometry using a 25 mm PP25-type parallel plate probe with a gap of 300 μm throughout the experiment. The experiments were performed by scooping out a sufficient amount of preformed gel onto the Peltier plate stabilized at 25 °C. The gels were allowed to stabilize for 10 min at the temperature provided prior to each experiment. The gels were initially subjected to a dynamic amplitude sweep from 0.1 to 100% to verify the viscoelastic nature of the gel as well as to estimate the linear viscoelastic (LVE) range. The gels were further subjected to a frequency sweep of 100 to 0.1 rad/sec and a temperature sweep of 25 to 100 °C, with amplitude kept constant within the LVE range. The self-healing or thixotropic nature of gels was investigated by exposing it to alternate cycles of high and low amplitude of strain (0.1 and 200%, 60 s) to assess the deformation and recovery upon application and removal of high strain. Dynamic amplitude sweep and self-healing studies, as described above, were repeated after exchanging the bulk solvent in MOG1 from DMF to water. 4.4. Doxorubicin-Loaded MOG1. In order to conduct loading and release studies, the bulk solvent in MOG1 was exchanged from DMF to water (hydrated MOG1) by immersing the gel (500 mg) into water (5 mL) in a glass vial and incubated at 37 °C for 24 h. The samples were collected periodically to estimate the amount of DMF released from the gel bulk. The hydrated MOG1 gel (500 mg) was immersed into a centrifuge tube containing 15 mL of aqueous DOX solution (2.5 mg/mL) and incubated at 37 °C for 48 h, with continuous shaking at 100 rpm. It was taken out of the vial, and contents were centrifuged for 5 min at 4000 rpm to ensure no dispersed gel in the supernatant. The supernatant was quantified for DOX using UV−Vis spectrophotometry. The amount of DOX loaded onto the gel was estimated by taking the difference in amounts of DOX initially present in solution and the supernatant. All experiments were done in triplicate. 4.5. Doxorubicin Release from MOG1 and Effect of Dehydration. The MOG1 gels were partially dehydrated under vacuum for different durations to obtain gels with 80 and 50% of their original weights. Two samples each of DOXloaded MOG1 (fully hydrated and partially dehydrated to 80 and 50%) were immersed in two separate vials containing 1.5 mL of PB of pH 7.4 and 6.4. The vials were shaken at a rate of 100 rpm in darkness at 37 °C, and aliquots (150 μL) were withdrawn at different time points. The release medium was replenished with an equal amount of PB. The amount of DOX released was determined by measuring the absorbance at 485 nm, and the data presented are mean ± standard deviation of three experiments. 4.6. Cell Viability Assay of Unloaded MOG1. MDCK cells (Madin−Darby canine kidney epithelial cells) were seeded in 96-well plates at an initial density of 5 × 104 cells/ well in a DMEM medium supplemented with 10% FBS.
Figure 8. Fluorescence microscopy images of A549 cells, after 4 h of incubation with free DOX (5 μg/mL), extracts of DOX-loaded MOG1 (MOG1-DOX) with equivalent DOX concentrations, and MOG1, showing intracellular uptake of DOX in cells treated with free DOX and DOX released from MOG1-DOX but not in cells treated with extracts of unloaded MOG1.
3. CONCLUSIONS In conclusion, we report the first example of a Cr3+-based MOG1 exhibiting strong resilience and self-healing characteristics. Upon loading with DOX, a chemotherapeutic agent, MOG1, demonstrated a pH-responsive drug release: a quicker release following pseudo-Fickian diffusion at intratumoral pH and sustained zero-order release at physiological pH. The most remarkable feature of this gel is its capability to transform the drug release kinetics from zero order to exponential decay upon partial dehydration. This unique capability could be further explored in a hybrid system, where an appropriately functionalized polymer can be incorporated into the gel to endow it with swelling−deswelling capabilities for a tunable drug release. The unloaded gels were significantly nontoxic to normal epithelial and A549 cell lines, but the DOX-loaded MOG1 demonstrated dose-dependent cytotoxicity against A549 cell lines and efficient intracellular distribution. On account of its self-healing and injectable characteristics, MOG1 loaded with a chemotherapeutic agent can be directly administered to locally accessible tumoral sites, without employing active or passive targeting strategies. Once in the tumor, the gel will provide a pH-responsive, quick, and selective release of chemotherapeutic agents. 4. EXPERIMENTAL SECTION 4.1. Materials and Methods. All reagents employed were commercially available and used as provided, without further purification. The Cr(NO3)3·9H2O (98%) and 2,2′-bipyridine4,4′-dicarboxylic acid (98%, BPDA) were obtained from Sigma-Aldrich, and doxorubicin hydrochloride (98%) was obtained from Dalian Meilun Biotech, China. FT-IR spectra were recorded in the 4000−600 cm−1 region on dry KBr pellets using a Bruker IFS 66v/S spectrometer. Scanning electron microscopy (SEM) images were recorded using JEOL JSM-6610LV. Thermogravimetric analyses (TGA) were carried out in temperature range of 25−900 °C (heating rate: 10 °C min−1) using a Mettler Toledo thermogravimetric analyzer under a nitrogen atmosphere (flow rate: 50 mL min−1). UV−Vis spectra were measured on Shimadzu spectrophotometer. 4.2. Synthesis of MOG1. A mixture of Cr(NO3)3·9H2O (40.4 mg, 0.1 mM), BPDA ligand (24.9 mg, 0.1 mM), and 1360
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*E-mail:
[email protected] (C.M.N.).
MOG1 (20 mg/mL) was suspended separately in DMEM with 10% FBS and incubated for 24 and 72 h to completely extract out the MOG1 components. After 24 h, MOG1 extracts at concentrations of 20, 40, 60, 80, and 100 μg/mL were added to the cells followed by the fresh DMEM medium, and PB was taken as the control. The cells were further incubated for 24 h, and the culture medium was removed and replaced with MTT solution and further incubated for 4 h. The MTT solutions were discarded, and DMSO was added to each well to dissolve formazan crystals. Absorbance of the dissolved formazan crystals was recorded using a microplate reader (Tecan Infinite Pro) at 570 nm. The relative cell viability was estimated by comparing the absorbance in control wells with the culture medium. The data presented here are mean ± standard deviation of six experiments. 4.7. Cell Viability Assay of DOX-Loaded MOG1. The A549 (adenocarcinomic alveolar basal epithelial) cells were cultured in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin, in a humidified environment at 37 °C and 5% CO2. A stock solution of DOX (1 mg/mL) was prepared in DMEM, and 100% hydrated DOX-containing MOG1 was subjected to release in phosphate buffer of pH 6.4 and 7.4 at 37 °C for 48 h, with release media being replaced after 24 h. The release media were filtered through 0.22 μm membrane filters and diluted with DMEM to obtain the equivalent DOX concentrations of 0.1, 0.5, 1, 2.5, and 5 μg/ mL (the amount of extract taken from DOX-loaded MOG1 and unloaded MOG1 was as per the DOX loading efficiency). The antitumor activity was estimated using MTT assay, as described above, but instead of MOG1 extracts, free DOX and release media of DOX-loaded MOG1 were added to cells and PB was taken as the negative control. Studies were done in triplicate. 4.8. Cell Uptake Studies of DOX, DOX-Loaded MOG1, and Unloaded MOG1. The cells (A549) were seeded in a 24-well plate at a density of 2 × 104 cells/well and incubated for 48 h prior to treating with free DOX (1 mL) and equivalent extract solutions (5 μg/mL) followed by incubation of 4 h. The cells were subsequently washed with DPBS (×2), fixed with 3.7% glutaraldehyde, and counterstained with DAPI for 30 min. Finally, cells were washed again with DPBS (×2) and observed under a fluorescence microscope. 4.9. Statistical Analysis. Statistical differences (*p < 0.05) were determined using one-way ANOVA followed by Bonferroni post hoc test in GraphPad Prism 7. Differences with p < 0.05 were considered significant.
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ORCID
Yashveer Singh: 0000-0003-1625-8156 C. M. Nagaraja: 0000-0002-4271-6424 Author Contributions #
N.S. and P.K.S. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Funding
Grant #02(0245)/15/EMR-II (CSIR India). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Authors thank Dr. Vishwajeet Mehandia, Assistant Professor, Department of Mechanical Engineering, IIT Ropar for giving access to the cell culture laboratory. Y.S. acknowledges financial assistance from CSIR, New Delhi [grant #02(0245)/15/EMR-II]. N.S. is grateful to CSIR for the SRF, and P.K.S. is grateful to IIT Ropar for the institute fellowship.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02833. TGA analysis of freeze-dried MOG1; viscoelastic and drug loading characteristics of MOG1; FT-IR spectra of MOG1, DOX-loaded MOG1, and DOX; bright-field images of mdck and A549 cells; DOX release parameters; and MP-AES analyses (PDF) Video of thixotropic characteristics of MOG1 (AVI)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
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[email protected] (Y.S.). 1361
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