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Copper Metal-Organic Framework Nanoparticles Stabilized with Folic Acid Improve Wound Healing in Diabetes Jisheng Xiao, Yunxiao Zhu, Samantha Huddleston, Peng Li, Baixue Xiao, Omar K. Farha, and Guillermo A. Ameer ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b01850 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018
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Copper Metal-Organic Framework Nanoparticles Stabilized with Folic Acid Improve Wound Healing in Diabetes Jisheng Xiao1,6, Yunxiao Zhu1, Samantha Huddleston1, Peng Li2,6, Baixue Xiao1, Omar K. Farha2,6 and Guillermo A. Ameer1, 3, 4, 5, 6*
1
Biomedical Engineering Department, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA
2
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA
3
Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
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Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA
5
Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL, 60611, USA
6
International Institute for Nanotechnology, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208,
USA *Corresponding Author: Guillermo A. Ameer, e-mail:
[email protected] ABSTRACT The successful treatment of chronic non-healing wounds requires strategies that promote angiogenesis, collagen deposition, and re-epithelialization of the wound. Copper ions have been reported to stimulate angiogenesis; however, several applications of copper salts or oxides to the wound bed are required, leading to variable outcomes and raising toxicity concerns. We hypothesized that copper based metal-organic framework nanoparticles (Cu-MOF NPs) referred to as HKUST-1, which are rapidly degraded in protein solutions, can be modified to slowly release Cu2+ resulting in reduced toxicity and improved wound healing rates. Folic acid was added during HKUST-1 synthesis to generate folic acid-modified HKUST-1 (F-HKUST-1). The effect of folic acid incorporation on NP stability, size, hydrophobicity, surface area, and copper ion release 1 ACS Paragon Plus Environment
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profile was measured. In addition, cytotoxicity and in vitro cell migration processes due to FHKUST-1 and HKUST-1 were evaluated. Wound closure rates were assessed using the splinted excisional dermal wound model in diabetic mice. The incorporation of folic acid into HKUST-1 enabled the slow release of copper ions, which reduced cytotoxicity and enhanced cell migration in vitro. In vivo, F-HKUST-1 induced angiogenesis, promoted collagen deposition and reepithelialization, and increased wound closure rates. These results demonstrate that folic acid incorporation into HKUST-1 NPs is a simple, safe, and promising approach to control Cu2+ release, thus enabling the direct application of Cu-MOF NPs to wounds. KEYWORDS: metal-organic framework, copper, folic acid, wound healing, diabetic ulcer
Chronic non-healing wounds are a significant problem for people with diabetes and contribute to increased healthcare costs.1-3 Specifically, diabetic foot ulcers (DFUs) result in 73,000 non-traumatic lower limb amputations in the U.S.A. and impose a cost burden of at least $9 billion to insurance companies, healthcare providers, and patients each year.4 Despite these expenditures, there are no consistently effective therapies to treat chronic non-healing DFUs.5 When developing strategies to address this problem, technologies that avoid the use pharmaceuticals or biologics are attractive to lower product development costs and reduce time to market. Of particular interest is the use of copper ion (Cu2+) as it has been shown to stimulate angiogenesis and collagen deposition processes that lead to improved wound healing.6-9 However, multiple applications of copper salts and copper oxide are necessary, increasing the risk of copper-induced toxicity to the patient.10-14 However, the risk of copper ion toxicity can be reduced if the ion is slowly released.15-18
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Metal-organic frameworks (MOFs) are a class of crystalline porous coordination polymers consisting of inorganic metal ions and organic ligands that interact to form clusters with short- and long-range order.19-21 MOFs traditionally have been used for gas adsorption and separation, catalysis, luminescence, sensing and proton conduction.22-34 Because the chemical and physical properties of MOFs are tunable based on the choice of metal nodes and organic linkers, they may be suitable for the storage and release of Cu2+.35 However, copper based-MOFs (CuMOFs) tend to be not stable in physiological protein-containing solutions, hindering their direct use in wound healing.36 The instability of Cu-MOFs in protein solutions can potentially be addressed with surface modification techniques.36 For example, hydrophobic fluoropolymer coatings have been reported to enhance HKUST-1 stability by preventing water molecules from clustering in the pores and subsequently breaking the metal–carboxylate linkages.37 However, fluoropolymers are considered to be toxic in vivo. Therefore, use of these molecules would pose a major regulatory challenge for clinical adoption. We hypothesized that HKUST-1 modification with a small carboxyl containing biological molecule that is sparingly soluble in water would safely increase HKUST-1 stability in protein solutions by increasing the MOF’s hydrophobicity. Folic acid, also known as vitamin B9, is biocompatible, present in foods, and consumed as a dietary supplement. The folate form has been used to target compounds to cells overexpressing the folate receptor in order to image and/or kill tumors.38-39 The chemical groups that comprise folic acid, p-aminobenzoic acid, pteridine, and glutamic acid, render it slightly soluble in water at neutral pH and compatible with the synthesis reaction conditions of HKUST-1.40-42 Herein, we report the use of folic acid as a stabilizer for Cu-MOFs in protein solutions, reducing their degradation and Cu2+ release rates. HKUST-1 was chosen as the first model Cu3 ACS Paragon Plus Environment
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MOFs due to its ease of synthesis and large scale production and the low toxicity of its organic ligand 1,3,5-benzenetricarboxylic acid (H3BTC) (LD50: 8.4 g/kg in rats).17, 43 We demonstrate that F-HKUST-1 is less toxic than HKUST-1, can slowly release copper ions, can promote cell migration in vitro, and significantly accelerates wound healing in diabetic mice. RESULTS AND DISSCUSSION Folic Acid can be incorporated into HKUST-1. Folic acid-modified HKUST-1 (F-HKUST-1) was successfully synthesized by dropwise addition of 1 mL of copper acetate monohydrate (75 mg/mL) aqueous solution to a mixture of H3BTC and folic acid, followed by stirring at room temperature for 40 min to form a gel-like green suspension. The detailed synthesis and characterization of HKUST-1 and F-HKUST-1 are described in the Methods section (Figure S1-S4, Table S1). The chemical features of F-HKUST-1 were examined using 1H nuclear magnetic resonance (1H NMR) spectroscopy and X-ray photoelectron spectroscopy (XPS) (Figure S1, S3). 1H NMR spectroscopy on an acid-digested sample revealed the presence of both 1,3,5benezenetricarboxylic acid with a resonance at 8.52 (s, 3H) ppm and folic acid with resonances at 8.69 (s, 1H), 8.06 (s, 1H), 7.55 (s, 2H), 7.38 (s, 1H), 6.63 (s, 2H), 4.52 (s, 2H), 4.20 (s, 1H), 3.32 (s, 1H), 3.25 (s, 1H) 2.18 (s, 2H), 1.72-1.84 (d, 2H) ppm (Figure S1). XPS further confirmed the successful synthesis of F-HKUST-1, as the nitrogen characteristic peak from folic acid was observed in the spectra of F-HKUST-1 but not in HKUST-1 (Figure S3). The elemental analysis results revealed the F-HKUST-1 formula to be Cu3(H3BTC)1.92(folic acid)0.12. The X-ray powder diffraction (XRD) data confirmed that the crystal structure of F-HKUST-1 was similar to that of HKUST-1 (Figure 1A, B). The stability of F-HKUST-1 in 5% FBS at 37°C was evaluated using XRD and transmission electron microscopy (TEM). After exposure to 5% FBS for 24 hrs, the signature XRD peaks 4 ACS Paragon Plus Environment
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from HKUST-1 spectra disappeared while three main peaks remain for F-HKUST-1 (Figure 1A, B). TEM images confirmed the XRD results (Figure 1C). However, F-HKUST-1 NPs were observed after incubation in 5% FBS, confirming that folic acid protected F-HKUST-1 from FBSmediated degradation (Figure 1D). These results demonstrate that folic acid modification improves HKUST-1 stability, possibly due to an increase in MOF hydrophobicity and/or a reduction in NP internal surface area. To confirm the above hypotheses, water-in-air contact angles and N2 isotherms were measured to calculate surface tensions and surface areas, respectively, for HKUST-1 and FHKUST-1. The contact angle for HKUST-1 and F-HKUST-1 was 34.7 ± 0.6° and 70.7 ± 9.3°, respectively. The corresponding surface tension for HKUST-1 and F-HKUST-1 was 63.3 ± 4.7 dyn/cm and 42.2 ± 4.1 dyn/cm, respectively (Figure 1E). These results demonstrate that folic acid incorporation into the MOF increases the hydrophobicity of HKUST-1, which may prevent protein and water molecules from crowding in the pores and subsequently breaking the Cu– carboxylate linkages.37 The N2 isotherm of HKUST-1 and F-HKUST-1 measured at 77̊ K revealed a Brunauer-Emmett-Teller (BET) surface area of 1500 m2/g and 300 m2/g, respectively. These results suggest that folic acid blocked a significant percentage of pores within HKUST-1, which would limit the diffusion of water or protein molecules to Cu2+ sites in HKUST-1 and further enhance NP stability in protein solutions (Figure 1F). The modification of HKUST-1 with folic acid resulted in the sustained release of Cu2+ from F-HKUST-1 at 37°C in 5% FBS for approximately 96 hrs. F-HKUST-1 released 56% and 69% of Cu2+ within 24 hrs and 48 hrs, respectively (Figure 1G). In contrast, HKUST-1 released 94% and 98% of Cu2+ within 24 hrs and 48 hrs, respectively (Figure 1G). The lower amount of
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Cu2+ released from F-HKUST-1 is due to the higher stability of F-HKUST-1 in proteincontaining media as Cu2+ content in both NPs was the same.
Figure 1. Characterization of F-HKUST-1 and HKUST-1 stability in 5% FBS, hydrophobicity, surface area, and Cu2+ release. (A) XRD patterns of HKUST-1 (B) XRD patterns of F-HKUST-1. Spectra
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were obtained before and after incubation in 5% FBS for three time intervals. TEM digital images of (C) HKUST-1 and (D) F-HKUST-1 before (C1, D1) and after incubation in 5% FBS for 24 hrs (C2, D2) and 96 hrs (C3, D3). (Scale bars: 100 nm). (E) Digital images of the water-in-air contact angles of water droplets placed on powders of (E1) HKUST-1 and (E2) F-HKUST-1; Quantitative analysis of (E3) contact angle and (E4) surface tension of HKUST-1 and F-HKUST-1. (F) N2 adsorption - desorption isotherms of HKUST-1 and F-HKUST-1. (G) Cu2+ release from HKUST-1 and F-HKUST-1 in 5% FBS. (n = 3, **P
