Encapsulation of curcumin nanoparticles with MMP9-responsive and

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Biological and Medical Applications of Materials and Interfaces

Encapsulation of curcumin nanoparticles with MMP9-responsive and thermos-sensitive hydrogel improves diabetic wound healing Juan Liu, Zhiqiang Chen, Jie Wang, Ruihong Li, Tingting Li, Mingyang Chang, Fang Yan, and Yunfang Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03868 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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ACS Applied Materials & Interfaces

Encapsulation of curcumin nanoparticles with MMP9responsive and thermos-sensitive hydrogel improves diabetic wound healing Juan Liu‡, Zhiqiang Chen‡, Jie Wang, , Ruihong Li, Tingting Li, Mingyang Chang, Fang Yan, Yunfang Wang* Tissue Engineering Lab, Institute of Health Service and Transfusion Medicine, Beijing 100850, China. * Address correspondence to: Dr. Y. Wang, Tissue Engineering Lab, Institute of Health Service and Transfusion Medicine. 27, Taiping Road, Haidian District, Beijing, 100850, P.R.China. Tel: +86-10-66931545; Email: [email protected]. ‡ J.L. and Z.C. contributed equally to this work.

KEYWORDS. Curcumin, non-healing wound, MMP9-resposive, drug delivery system, diabetics.

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Abstract Impaired wound-healing in diabetics usually lead to life-threatening complications. In order to develop a system for efficiently and safely fastening skin wound healing in diabetics, thermossensitive hydrogel containing with nanodrug, loaded in form of gelatin microspheres (GMs), was designed to deliver curcumin (Cur) as therapeutic drug. Cur is a naturally existing poly-phenolic compound with a broad range of biological functions useful for potential therapies. Because Cur molecule has weakness in both bioavailability and in vivo stability, delivery of Cur required the assistance from other molecules to act as the carrier vehicles in a sustained manner for therapeutic uses. At first, self-assembly of Cur nanoparticles (CNPs) were prepared to improve bioavailability. The CNPs was further enclosed into GMs for late responding to the matrix metalloproteinases (MMPs) that usually overexpress at diabetic non-healing wound sites. The GMs containing CNPs were loaded into thermos-sensitive hydrogel and finally proved for the capacity of specially induced drug release at wound bed, which promoted the efficacy in healing the standardized skin wounds in streptozotocin-induced diabetic mice. Our results indicated that the successfully developed CNPs delivery system had the capacities to significantly promote skin wound healings, which suggested that it could have potential to become a wound dressing with the properties of anti-oxidants and promotions of cell migration.


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1. Introduction Skin carries the function of repair after injuries because of natural disasters or diseases.1-3 The process of skin wound healing mainly composes with long and sequential events, including clotting cascade, acute and chronic inflammations, re-epithelialization, granulation tissue formation, wound contraction and connective tissue remodeling, etc.4-7 However, skin healing from refractory wound is still one of major challenges for the healthcare presently. For a long while, in order to solve this medical problem completely, causes to induce the refractory wounds are studied. A recent study on diabetic foot ulcers represented one of the endeavors to overcome the obstacle of still impaired or non-healing skin wounds. Through the results of this study, it was encouraging to find that the long-term inflammation accompanied with elevated oxidative stress should be one of the principal reasons for the pathogenesis of diabetic. The results could be helpful to solve the obstacle for how to improve skin wound healings during diabetics. The extensive evidence collectively indicated that curcumin (diferuloylmethane, Cur) had the significant efficacy for wound healings.8-9 Cur is a natural polyphenol existing in the popular Indian spice turmeric and belongs to the ginger family. Particularly, Cur showed some promise as a novel adjuvant treatment due to its antioxidant and anti-inflammatory properties, which acts on various stages of the natural wound healing process to hasten healing.8, 10-13 However, Cur often caused apoptosis found during these actions because of its capability to induce the mitochondrial dysfunctions at high dose. Although the exact mechanism for occurrence of apoptosis is unclear, the function of Cur differed with the cell types in particular.14 Thus, Cur application obviously shows as a double-edged sword. In this regard, using controlled release techniques for Cur delivery was considered by us in order to improve the efficacy. Like many other therapeutic drug of small hydrophobic molecules, Cur has limitations on its efficacy during


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the clinical scenarios because of its improper performances including: low hydrophilicity and intrinsic dissolution rate, low physico-chemical stability, rapid metabolization, low bioactive absorption, poor pharmacokinetics and bioavailability, and low penetration and targeting efficacy, etc.15-16 All of these weaknesses significantly affect the efficacy of Cur as a therapeutic molecule. In recent years, drug delivery using nanotechnology extensively improved the delivery efficiency through overcoming solubility, rapid drug metabolism, degradation, and drug stability issues. In addition, drug diffusing or targeting to the certain tissues was also improved through minimizing unintended toxicity to surrounding normal cells/tissues.17-19 The self-carried Cur nanoparticles (CNPs) developed recently have shown with highly effective cancer therapy in vitro and in vivo.20 The prepared CNPs showed the significantly improved dispersibility and outstanding stability in physiological environments with a high drug loading capacity. However, there is still no report on the practical method for releasing of CNPs as the effective molecular into wound bed, constantly and selectively. Therefore, we need to load CNPs in controlled drug release systems.21-23 In the present study, we developed a method efficient and safe for fastening skin wound healing under pathological condition of diabetics. The strategy of this method is summarized in Figure 1. Cur is incorporated to form self-assemble nanoparticles (CNPs) by using a reprecipitation method for improving its solubility and stability. Afterward, CNPs is enclosed in the gelatin microspheres (GMs), which will be able to responsive to the MMP-9, which usually has overexpression and exists at site of non-healing skin wounds of diabetics.24-25 Therefore, the gelatin microspheres loaded with Cur nanoparticles (CNPs@GMs) will release Cur as drug to the wound bed. Finally, CNPs@GMs will effectively cover on wound and mix with thermosresponsive poloxamer hydrogel (CNPs@GMs/hydrogel) to coat on skin wound of diabetics (the


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diabetic mice model here). Preparations of self-carried pure CNPs, CNPs loaded in GMs, and mixed with thermo-sensitive hydrogel for chronic cutaneous wound repair is illustrated in Figure 1. This method was proved in our study for the objectives to evaluate the therapeutic efficacy of CNPs@GMs/hydrogel for dermal wound healing in a diabetic mice model as well as to study the key mechanism on promoting wound healing.

Figure 1. Schematic representations of CNPs@GMs/hydrogel preparation and the process of drug release at wound bed in diabetic mice. (A) Preparation of pure CNPs via a solution exchanged method. (B) CNPs loaded into GMs by emulsion process to get CNPs@GMs. (C) The CNPs@GMs mixed with thermossensitive hydrogel and covered on the wound in diabetic mice. (D) Under the microenvironment of nonhealing wound, GMs were degraded by MMPs, and specifically released the drug.

2. Results and discussion 2.1 Preparation and Characterization of Cur nanoparticles loaded in gelatin microspheres. For a long while, the clinical application of Cur has been largely hampered because of its poor water solubility and low bioavailability. In addition, Cur is also unstable under visible and ultraviolet light. In order to use Cur for skin wound healing under the specially designed situation, at first, the self-assembled CNPs were prepared using a reprecipitation method


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described in our previous report.20 The reprecipitation method has been proved to be very simple but versatile.26 Briefly, free Cur dissolved in tetrahydrofuran (THF) solution was rapidly injected into deionized water under vigorous stirring at a constant speed. The Cur molecules aggregate to form nanoparticles, due to the sudden change of solvent environment. Usually, Cur has a very low aqueous solubility of less than 0.01%, as confirmed by the observation of a ginger slurry.27 However, the Cur concentration of CNPs solution reached to as high as 250 μg/mL with a transparent yellow appearance (Figure 2A). The formed CNPs appeared to be spherical in shape with a well-defined and monodispersed formation state, as determined by scanning electron microscope (SEM) (Figure 2B). The size of nanoparticles was measured by dynamic laser light scattering (DLS). As representative image shown in the inside panel of Figure 2B, the average hydrodynamic diameter of CNPs was 75.4 nm. These results indicated that the obtained CNPs were well dispersed and with narrower granule size distribution, resulting a significant improvement on the water solubility of Cur in aqueous media. The fluorescence properties of free Cur and nanoparticles were further investigated by measuring their fluorescence spectrum. Remarkably, the fluorescence intensity of Cur decreased significantly after formation of CNPs. Figure 2C revealed that the free Cur solution presented strong green fluorescence under UV irradiation, while almost no fluorescence was observed in CNPs solution. Results of fluorescence spectrum also confirmed the fluorescence intensity of Cur decreasing in nanoparticles (Figure 2D), due to that the “π−π stacking” interaction between Cur molecules led to fluorescence quenching. When the Cur molecule released from CNPs, its green fluorescence should be recovered, which enabled the self-monitoring capacity of drug release.


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The skin regeneration requires a balance between the degradation of collagens and the synthesis of new extracellular matrix (ECM) components,28 which is determined by matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). However, in diabetic conditions this balance is disturbed resulting in wounds. There is considerable conflicting information in the literature concerning the overexpression of specific MMPs in nonhealing wounds, such as gelatinases, also referred to as MMP9.29 Gelatin, a natural protein derived from the hydrolysis of collagen, is the definite substrate of MMP9, which can be degraded and specifically release the pharmaceutical molecule at wound site with excellent biocompatibility.30 Thus, gelatin formed microspheres are supposed to be a suitable biomaterial for delivering drugs and releasing dugs specifically at non-healing wound.31-32 To encapsulate CNPs into GMs, GMs and CNPs@GMs were prepared by using emulsion chemical cross-linking method with gelatin as wall material, which could cracked into smaller molecules by MMPs and released their contents, glutaraldehyde, as the commonly cross-linking agent, was chosen to use to solidify and precipitate the microspheres from water, and to improve the thermal and mechanical stability of the microspheres under physiological conditions. In addition, glutaraldehyde could make GMs solidify in neutral conditions, in which Cur was very stable. For analyses, the GMs and CNPs@GMs suspending liquid was dropped on glass slides, respectively, and observed under microscope. The representative images in panels of Figure 2E and F showed that the CNPs@GMs and GMs exhibited the uniform stable spheric morphology with the similar size. The diameter for majority of these particles distributed from 4 to 8 μm, indicating their homogeneous size. Quantification studies were performed in the case of the CNPs@GMs sample, in which the Cur loading efficiency and encapsulation rate here were determined to be 24.75 ± 3.18% and


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94.82 ± 0.52%, respectively, using a standard absorbance technique (Figure. S1). This loading capacity was considerably higher than those achieved with other carrier-based drug delivery system (typically 98%) was purchased from Nanjing Goren bio-technology company (China), gelatin (>98%), tetrahydrofuran (THF), paraffin liquid, 50% glutaraldehyde solution, and isopropanol were purchased from sinopharm chemical reagent company(China), span-80,pluronic F127, pluronic F68, and streptozotocin were purchased from sigma-aldrich (USA). Human normal skin fibroblast BJ cells and immortalized human epidermal cells HaCat cell lines were purchased from Chinese academy of medical science & Peking union medical college (China). The cell culture medium and fetal bovine serum were from Gibco (USA), 0.25% Trypsin-EDTA, antibiotic solutions (penicillin and streptomycin) were purchased from was purchased from sigma-aldrich (USA). BJ cells were maintained in DMEM medium and HaCat cells were maintained in α-MEM with 10% fetal bovine serum and 1% antibiotic solutions. These cells were cultured under a humidified atmosphere containing 5% CO2 at 37 °C. Preparation and characterization of self-assembled CNPs In a typical run, 1 mg of Cur was firstly dissolved in 1 mL tetrahydrofuran (THF). 200 µL of the solution was then quickly injected into 5 mL of high-purity water under vigorous stirring in 1400-1600 rpm at 20-25 oC for 30 min. Then the obtained CNPs were lyophilized, by a freeze dryer, and was observed by the scanning electron microscopy (SEM, HITACHI S-4300). The SEM samples were prepared by drying the nanoparticles onto a Si substrate followed by a 2 nm layer of Au coating. Hydrodynamic sizes of the CNPs were measured in aqueous solutions using a DLS instrument (Malvern Zetasizer Nano ZS). Fluorescence spectra was recorded with a microplate reader (Ensight, PerkinElmer).


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Preparation and characterization of CNPs encapsulated in gelatin microspheres (CNPs@GMs) Gelatin microspheres (GMs) were prepared through glutaraldehyde crosslinking of a gelatin aqueous solution as reported previously. Briefly, 100 mg gelatin was added to 900 μL highpurity water under vigorous stirring in 800-1000 rpm at 55℃ for 30 min to form aqueous phase. 45 μL Span-80 was added to 4.5 mL paraffin liquid, and stirred in 800-1000 rpm at 55 ℃ for 30 min, to form oleic phase. Aqueous phase was dropped into oleic phase with injection syringe gently and uniformly, while oleic phase was stirred in 1100 rpm at 55 ℃ for 30 min sequentially. And then, the mixture was transferred to ice-water bath, with adding 25% glutaraldehyde solution for stirring another 30 min. Isopropyl alcohol was added in the mixture for 20 min in order to dehydration. Diethyl ether and isopropyl alcohol was used for washing for the compound alternately for 3 times. Then organic solvent was removed by rotary evaporator. GMs were obtained by applying these above steps. 20 μL gelatin microsphere suspending liquid was drop on glass slide, and observed by inverted microscope. Then the particle diameter was measured by nano measurer 1.2 software, and analyzed by Origin 2017. To prepare CNPs@GMs, 30 mg CNPs and 100 mg gelatin was added to 900 μL high-purity water under vigorous stirring in 800-1000 rpm at 55 oC to form aqueous phase. The following steps were the same as the preparation of GMs. The resultant CNPs@GM samples were lyophilized and stored at 4 °C until for further evaluation. The amount of Cur in CNPs@GMs was further defined. Cur was dissolved in THF with a series concentration from 0.01 to 50 μg/mL, and their absorption photometry was measured by Ensight at 425 nm to obtain the standard curve. Freeze-dried CNPs@GMs were also dissolved


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THF and measured its concentration through the standard curve. The encapsulation efficiency and the drug loading efficiency were calculated from the following equations: Encapsulation efficiency = (weight of Cur in CNPs@GMs/weight of Cur fed initially) × 100% Drug loading efficiency = (weight of Cur in CNPs@GMs/weight of CNPs@GMs) × 100% Preparation and characterization of thermos-sensitive hydrogel To study the thermos-sensitive property of pluronic F127, 16%, 18%, 20%, and 22% (w/w) pluronic F127 in saline were placed in the refrigerator at 4 ℃ till it was fully dissolved. Phase transition temperature was detected with changing the temperature of water bath kettle. Further, the mixture of pluronic F127 and F68 with different proportions (F127: 18%, 20%, 22%; F68: 3%, 6%, 9%) in saline were prepared, and the following steps were the same as above. For preparation of CNPs@GMs loaded into hydrogel, 20% pluronic F127, 6% F68, and CNPs@GMs containing 1 mg Cur were mixed in saline, stored at 4 oC until to use. Drug release measurement First, the release profile of CNPs@GMs with MMP9 treated at a serial concentration from 0.1 to 50 nM was detected by analysis their fluorescence spectrum. Further in order to reduce the disturbance, the absolute quantification of drug release was detected by dialysis using minidialysis tubes with a molecular mass cut off of 6 kDa to 8 kDa (Millipore, USA). A suspension of CNPs@GMs in water was divided equally (100 μL) into multiple minidialysis tubes. All these minidialysis tubes were soaked in a same beaker containing 10 mL PBS with or without 10 nM MMP9 and stirred at 37 oC. After 0 min, 10 min, 30 min, 1 h, 2 h, 4 h, 10 h, 24 h,


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48 h, and 72 h, one minidialysis tube was withdrawn. The unicellular suspension remained in each minidialysis tube was transferred and the Cur content was quantified as described above. The percentage of CNPs@GMs released at different time (Cx) is calculated as follows: Cx = (C0Cx)/C0 × 100%, C0 is the initial platinum content added in the tube, and Cx is the platinum content remained in the tube at different time. All experiments were carried out in triplicate. Cell cytotoxicity assay The cytotoxicity of the Cur different formations to BJ and HaCat cells was determined by MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide) assay. Briefly, 100 μL culture medium containing cells at a suitable density (BJ, 5000 cells per well; HaCat, 8000 cells per well) were plated in 96-well plates. After incubation for 24 h, the cells were treated with free Cur, CNPs, CNPs@GMs, CNPs@GMs pretreated with 10 nM MMP9 for 72 h at equal Cur concentration from 0.01 to 100 μg/mL for 24 h. Then the medium was replaced with 100 μL MTT at 0.5 mg/mL and further cultured for another 4 h. Subsequently, the supernatant was discarded, and 100 μL DMSO was added to each well. Finally, the absorbance at 570 nm of each well was measured using a microplate reader (Ensight, PerkinElmer). Similarly, the biocompatibility of pluronic F127 and F68 was assessed using the same method. For assessment the protective effect of antioxidation, firstly, BJ cell and HaCat cell was treated with hydrogen peroxide (H2O2) at a series concentrations from 1 to 3000 μg/mL for 24 h. The following steps were the same as above. Further, BJ cell and HaCat cell were treated with 255 or 336 μg/mL H2O2, respectively, and co-treated with different Cur formations at 0.01, 0.1, or 1 μg/mL for 24 h. The following test steps were the same as above.


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Flow cytometry assays Cellular uptake of free Cur and CNPs by BJ and HaCat cells were investigated by flow cytometry. For this experiment, 5 × 105 cells per well were seeded into 6-well plates. After 24 h incubation at 37 oC, the cells were incubated with medium containing free Cur and CNPs at 10 μg/mL of the Cur equivalent concentration for 1 h, washed twice with ice-cold PBS, trypsinized and suspended in PBS. Subsequently, the uptake of free Cur and CNPs by cells was monitored using flow cytometry (Attune acoustic focusing cytometer, Applied Biosystems, Life Technologies, Carlsbad, CA, USA). For cell apoptosis assay, BJ and HaCat cells were cultured in DMEM or α-MEM containing free Cur, CNPs, CNPs@GMs, or CNPs@GMs pretreated with MMP9 at Cur concentration of 10 μg/mL for 24 h. And then cells were harvested and collected. Cell apoptosis was assessed using a FITC-annexin V and propidium iodide (PI) kit (Invitrogen, Molecular Probes). The collected cells were suspended in 100 μL staining buffer, incubated for 15 min, and kept on ice until analysis through flow cytometry. Cells, which were negative for both PI and FITC-annexin V stainings were considered to be living cells, while cells that were PI-negative and FITC-annexin V-positive were considered to be early apoptotic cells, and that showed both PI-positive and annexin V-positive were those in the later stages of apoptosis. For antioxidant effect assay, BJ and HaCat cells were treated with H2O2 or co-treated H2O2 with free Cur, CNPs, CNPs@GMs, or CNPs@GMs pretreated with MMP9 at Cur concentration of 1 μg/mL for 24 h. And then cells were harvested, collected, and stained with 5 μM CellROX™ Deep Red Reagent and 100 μM monochlorobimane (mBCl) for 15 min. The fluorescence of the staining cells was detected by flow cytometry. CellROX is the reagent


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localized in the cytoplasm and become brightly fluorescent by reacting with ROS. mBCl is essentially nonfluorescent until conjugated, readily reacts with GSH. Migration-promoting effects assay The ability of the different formulations to improve cell proliferation and migration to remodeling tissue was evaluated by monitoring cell migration in real time and using the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument equipped with a CIM-plate 16 (Roche, Indianapolis, IN). The CIM-plate 16 is a 16-well system in which each well is composed of upper and lower chambers separated by an 8-μm microporous membrane. Migration/invasion was measured as the relative impedance change (cell index) across microelectronic sensors integrated into the bottom side of the membrane. 7.5 × 104 BJ and HaCat cells were added in duplicates to the upper chambers, and medium containing Cur different formations at 1 μg/mL was added in the lower chambers. Migration was monitored every 15 min for 72 h. For quantification, the cell index at indicated time points was averaged from at least three independent measurements. Further, the migration of fibroblast was evaluated by measuring the cell expansion on wound surface using a scratch assay method. Cells were cultured in 6 well plates until complete monolayer confluence. Then a linear wound was generated using a sterile plastic pipette tip. The scattered fragments of cells were removed by washing with fresh medium. Cells were treated with Cur different formulations containing 1 μg/mL Cur and incubated for 12 h, 24 h, and 36 h. At the end of experiments, cells were fixed with 4% paraformaldehyde and stained with Hoechst 33258 to visualize the cell nucleus. Cells were captured under the microscope with Vectra® 3 (PerkinElmer).


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In vivo wound healing and tissue collections Female BALB/c mice of 18–20 g were purchased from Vital River Company (Beijing, China) and raised under standard pathogen-free conditions. All animal experiments were performed in accordance with the principles of care and use of laboratory animals. Following 10 days of acclimatization, a single injection of STZ (75 mg/kg) in citrate buffer solution (0.1 M, pH 4.5) was intraperitoneally administered to initiate the induction of diabetes. Blood glucose concentrations were monitored with glucometer by tail blood. When the blood glucose level reached 19.2 mM, the mice were ready for next experiment. Mouse backs were shaved then covered in a thin layer of Nair hair removal cream. A wound measuring 1×1 cm2 (≈100 mm2) was created on the dorsal thoraco-lumber region of the diabetic mice under ketamine anesthesia. The prepared 200 µl Hydrogel, Cur/Hydrogel, and CNPs@GMs/Hydrogel were applied directly to wounds. Following surgery, the mice were wrapped with tegaderm (3M) to cover and protect the wound area. Subsequent measurement of wound area was taken every day post-wounding. The results of wound measurements on various days were expressed as percentage of wound contraction. The values were expressed as percentage values of the day 0 measurements and were calculated by Wilson's formula as follows: Wound contraction (%) = (wound area on day 0 – wound area on a particular day) / wound area on day 0 ×100%. Animals were sacrificed on postoperative day 10 and 20 and wound tissue was excised and fixed for immunohistochemical and histological evaluation. Tissue histology


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Individual wounds were excised, imaged and fixed by immersion in a 4% paraformaldehyde solution for 24 h. The fixed skin grafts were bisected and paraffin embedded. Serial 5 µm sections were obtained from each of the paraffin embedded wounds using the microtome (Leica). Sections were collected and stained with hematoxylin and eosin stain (Sigma) according to the protocol. Images of the stained samples were captured by a light microscope (Vectra® 3, PerkinElmer). Immunohistochemistry Briefly, paraffin sections were rehydrated, incubated in antigen retrieval solution and blocking serum, and stained using primary antibodies to GPx, α-SMA, CD31, Ki67, and MMP9 (Abcam), respectively. Subsequently, the sections were incubated with the universal secondary antibody (VECTOR) and VECTASTAIN Elite ABC reagent, washed in PBS, reacted with ImmPACT DAB enzyme substrate. Finally, the sections were counterstained with hematoxylin and mounted using antifade mounting medium. Images of the stained samples were also captured by a light microscope (Vectra® 3, PerkinElmer). Image J software was used to analysis the positive marker percentage. Statistical analysis All experiments were carried out in triplicate unless otherwise indicated. Error bars represent standard deviations. Data are presented as mean value ± SD from three independent measurements. Graphs were plotted using origin 9.0 software. ASSOCIATED CONTENT Supporting Information.


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Standard curve of free Cur in 80% EtOH; Molecular formula of pluronic F127 or F68 and phase-transition temperature of pluronic F127 at different concentrations; Oxidative damage of BJ and HaCat cell by H2O2; Wound closure levels in scratch assay; Cell viability assays of BJ and HaCat cells with treatment of pluronic F127 and F68; Phase-transition temperatures of the mixture solution containing F127 and F68 at different proportions in PBS. This material is available free of charge via the Internet at http:// pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Address correspondence to: [email protected]. Author Contributions ‡These authors contributed equally. Conflict of Interest: The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Dr. Jinfeng Zhang for her support on preparation of curcumin nanoparticles. This work was supported by the National Key Research and Development Program of China (No. 2016YFC1101305), and the National Natural Science Foundations of China (No. 31370990, 31700878, and 81170388).


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Encapsulation of curcumin nanoparticles with MMP9-responsive and

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