hydrogel composites for

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Injectable maltodextrin based micelle/ hydrogel composites for simvastatin controlled release Shifeng Yan, Jie Ren, Yuhang Jian, Weidong Wang, Wentao Yun, and Jingbo Yin Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01234 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Biomacromolecules

Injectable maltodextrin based micelle/ hydrogel composites for simvastatin controlled release

Shifeng Yan*, Jie Ren, Yuhang Jian, Weidong Wang, Wentao Yun, Jingbo Yin*

Department of Polymer Materials, Shanghai University, 99 Shangda Road, Shanghai 200444, People’s Republic of China

1

ACS Paragon Plus Environment

Biomacromolecules 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

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ABSTRACT: Injectable hydrogels have shown great potential in bone tissue engineering. Simvastatin (SIM), a common hypolipidemic drug, has been suggested as a potential agent to promote bone regeneration. However, due to its hydrophobic nature, the compatibility between SIM and hydrogels is rather poor, thereby greatly affecting the drug release behavior, the mechanical properties and dimensional stability of the hydrogels. Herein, we presented a novel design to entrap SIM in an injectable maltodextrin based micelle/hydrogel composite system. Maltodextrin based micelles were prepared to solubilize and encapsulate SIM. The SIM-loaded aldehyde-modified micelles were anchored to the hydrogel network and served as a crosslinker to realize improved mechanical strength of hydrogel, controlled release and osteogenic capability of SIM. In all, this study demonstrated a strategy to incorporate drug loaded carriers into hydrogels for drug delivery and tissue engineering applications. Keywords:

Injectable

hydrogel;

Controlled

release;

Osteogenicity

2


Micelle;

Simvastatin;

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INTRODUCTION Injectable hydrogels have shown broad application prospects in bone tissue engineering because of minimally invasive surgical procedure, in situ gelation, filling of complex shape defects, simulation of natural extracellular matrix, easy incorporation and localize release of active drugs and soluble factors.1 In order to improve the healing effect of hydrogels on bone defect, the growth factors such as bone morphogenetic proteins (BMPs) have been loaded in hydrogels to promote cell proliferation and differentiation. Bone morphogenetic protein-2 (BMP-2) is currently recognized as an efficient inducer with high osteogenic activity. It can obviously promote bone cell differentiation, bone matrix formation and mineralization.2 However, BMP-2 is suffering from the problems of expensive costing, instability and so on, which limits its clinical application.3 Therefore, it is urgent to obtain simple, inexpensive, safe and effective active drugs. Mundy et al. first reported in Science that statins such as lovastatin and simvastatin can induce expression of BMP-2 gene in bone cells, thus effectively promoting bone regeneration.4 Among various statins, simvastatin (SIM) shows the strongest promoting effect on bone regeneration. Simvastatin is a common used lipid-lowering drug with the advantages of safety, stability, and low costing. It can be used as a substitute for BMP-2. Many subsequent studies have further demonstrated that SIM can reduce the risk of fracture and enhance bone mineral density.5-7 Moreover, simvastatin can provide additional functions, such as promoting the growth of new blood vessels, and anti-inflammatory.8,9 However, as a hydrophobic drug, simvastatin shows poor compatibility with the hydrogel matrix. It tends to aggregate and precipitate from the hydrogel, which makes it difficult to regulate the drug release dose and kinetics. Furthermore, the uneven structure of the drug-loading hydrogel will also affect the mechanical properties and dimensional stability. Therefore, the dispersion and solubilization of SIM within the hydrogel is quite necessary. 3



In order to achieve better bone regeneration, it is necessary to maintain sufficient time and proper dose of SIM in bone defect area. The scaffolds with long-term SIM release were found to be more desirable in repairing critical size bone defects.10 Bae et al. directly mixed SIM into photo-cured hyaluronic acid-based hydrogels.11 The SIM-loaded hydrogels exhibited a concentration-dependent release behavior and obvious initial burst release. Jia et al. utilized the SIM-mPEG micelles as SIM carriers to improve the water solubility and bioavailability of SIM.12 However, frequent injection of the drug-loaded micelles was still needed to ensure the effective concentration of SIM in bone defect. The encapsulation of SIM-loaded micelles into injectable hydrogels is expected to combine the advantages of micelles and injectable hydrogels. The loading of SIM into the micelles can improve the drug solubility and dispersion in the hydrogel, and the injectable hydrogel serves as a carrier for the active drug-loaded micelles and a scaffold for the cells, facilitating tissue growth. In this study, we presented a novel hydrophobic drug entrapment strategy in an injectable maltodextrin based micelle/ hydrogel composite system to improve the water solubility, dispersion ability, controlled release, as well as the osteogenic capability of SIM. SIM was loaded in micelles and the SIM-loaded aldehyde-modified micelles were anchored to the hydrogel network by Schiff base linkage (SIM@micelle-CHO/hydrogel). For comparison, the injectable SIM-loaded micelle/ hydrogel system (SIM@micelle/hydrogel) without chemical linkage and the SIM/hydrogel mixture system (SIM@hydrogel) were also studied. The preparation of micelles and the micelle/ hydrogel composites, drug loading and release were investigated. The mouse osteogenic precursor cells (MC3T3-E1) were encapsulated within the composite hydrogels to evaluate their cytocompatibility and osteogenic capability.

EXPERIMENTAL SECTION 4


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Materials. Maltodextrin (MDex, Mw = 15,000) was purchased from Shandong Xiwang Sugar Co., Ltd. (Shandong, China). Carboxymethyl chitosan (CMCS, Mw = 200 kDa) and simvastatin (SIM) were purchased from Dalian Meilun Biotech Co., Ltd. (Dalian,

China).

Palmitic

acid

(PA),

Sodium

periodate

(NaIO4),

N,N'-Carbonyldiimidazole (CDI), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), 4-dimethylaminopyridine (DMAP) and Rhodamine B were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Synthesis and Rhodamine B Labelling of MDex-C16 and MDex-C16-CHO. Maltodextrin-graft-palmitic acid (MDex-C16) amphiphilic polymers were prepared by using CDI as a condensing agent. MDex-C16 with different grafting degrees was prepared by changing the molar ratio of PA to MDex (PA/MDex = 0.08, 0.10, 0.12, and 0.14). The mole ratio of CDI to PA was set at 1.1. Taking PA/MDex = 0.10 as an example, 0.2202 g of CDI and 0.3166 g of PA were dissolved in 15 mL of anhydrous dimethyl sulfoxide (DMSO). After stirring for 2 h at 50 oC under N2 flow, 2 g of pre-dried MDex was added into the solution. The reaction was performed at 80 oC for another 5 h. After being precipitated and washed in anhydrous ethanol, the product was dissolved in a proper amount of deionized water and dialyzed against deionized water for 2 days. Finally, the solution was lyophilized to obtain MDex-C16. The 1H NMR spectra of MDex and MDex-C16 were determined using the BRUKER AV 500 MHz instrument, and D2O was used as solvent. Aldehyded-functionalized MDex-C16 (MDex-C16-CHO) with different oxidation degrees was prepared by using NaIO4 as an oxidizing agent (NaIO4/MDex-C16 molar ratio: 0.10, 0.15 and 0.20), according to the procedure previously reported.13 Taking NaIO4/MDex-C16 = 0.15 as an example, 0.5 g of MDex-C16 (degree of grafting, DG = 5.6 %) was dissolved in 50 mL of deionized water and then 0.086 g of NaIO4 was added. After stirring for 2 h at room temperature in the dark, equivalent molar amounts of ethylene glycol was added and stirred for another 0.5 h to stop the reaction. The resulted solution was dialyzed against deionized water for 2 days and 5



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then lyophilized to obtain MDex-C16-CHO. Aldehyde content was determined by potentiometric titration and the oxidation degree (OD) was defined as the percentage of MDex saccharide structural units that were oxidized.14,15 The Rhodamine B-labelled MDex-C16 and Rhodamine B-labelled MDex-C16-CHO were prepared by esterification of the carboxyl groups of rhodamine B with the hydroxyl groups of MDex-C16 or MDex-C16-CHO. Briefly, 100 mg of MDex-C16 or MDex-C16-CHO (DG = 5.6%) was dissolved in 5 mL DMSO, then 13.65 mg of rhodamine B, 6.3 mg of EDC·HCl and 2.199 mg of DMAP were added. The mixture solution was stirred at room temperature for 24 h followed by dialysis against deionized water for 5 days. Finally, the Rhodamine B-labelled MDex-C16 or Rhodamine B-labelled MDex-C16-CHO was obtained by freeze-drying. Preparation of SIM-loaded MDex-C16 and MDex-C16-CHO Micelles. SIM-loaded micelles were prepared by thin-film hydration method.16 Briefly, 20 mg of SIM and 100 mg of MDex-C16 or MDex-C16-CHO were dissolved in 15 mL of N, N-Dimethylformamide (DMF) in a flask. Then, DMF was removed by using a rotatory evaporator at 50 oC for 1.5 h. After that, a SIM/polymer mixture film was obtained on the inner wall of the flask. Then, 40 mL of deionized water was added into the flask and stirred at 40 °C for 1 h to hydrate the film. The unloaded SIM was separated by centrifugation at 10000 rpm for 3 min and the supernatant was lyophilized to obtain the

SIM-loaded

MDex-C16

(SIM@micelle)

or

MDex-C16-CHO

micelles

(SIM@micelle-CHO) powder. The drug loading content (DLC) and drug loading efficiency (DLE) of micelles were determined by the common formulas reported in the literature.17 Formation of Injectable SIM-loaded Micelle/ Hydrogel Composites. SIM-loaded micelle/hydrogel composites were prepared by mixing CMCS/ phosphate buffer (PBS) solution and

OMDex/PBS solution

containing the

SIM-loaded

micelles

(SIM@micelle/hydrogel or SIM@micelle-CHO/hydrogel). As presented in Supporting information, the oxidation degree (OD) of OMDex was 41.1% (Figure S1a) and the 6


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amino content of CMCS was 3.38 mmol/g (Figure S1b). The solid content of precursor polymer solution and the -CHO/-NH2 molar ratio were set at 2 wt% and 0.5, respectively. The SIM loading concentration in the resultant composites was set at 1 mg/ml and 2 mg/ml by incorporating appropriate amount of SIM-loaded micelles. A centrifugal tube inversion method was adopted to measure the gelation time.18 The precursor solutions were mixed evenly in a 5 mL centrifugal tube, and the hydrogel was formed when the solution no longer flowed. Characterization of Micelles. The critical micelle concentration (CMC) of the polymeric micelles was determined using the surface tension method.19 Briefly, a series of MDex-C16 aqueous solutions at various concentrations ranging from 0.01 mg/ml to 4 mg/ml were prepared. Then the surface tension values were measured and analyzed as a function of polymer concentration. The CMC value was estimated from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentrations.20 The particle size distribution of MDex-C16 and MDex-C16-CHO micelles were determined by dynamic light scattering (DLS; Malvern Zetasizer Nano ZA90, UK). The measurement was performed at 25 °C and an incident angle of 90°. X-ray diffraction (XRD) patterns were determined by XRD (D/MAX2550, Rigaku) using Cu Kα radiation. The samples including SIM powder, freeze-dried MDex-C16-CHO micelle, SIM/MDex-C16-CHO micelle mixture, and SIM loaded MDex-C16-CHO micelle were scanned between 2θ = 5-60° with a scanning rate of 2 °/min at a voltage of 40 kV and 30 mA. The morphologies of micelles before and after SIM loading were investigated using transmission electron microscopy (TEM; JEM-200cx, Japan). The samples were prepared by dropping 20 μL of micelles aqueous solution (0.1 mg/mL) onto the copper meshes. Characterization of Micelle/ Hydrogel Composites. The morphologies of the micelle/ hydrogel composites were investigated by scanning electron microscopy 7



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(SEM; JXA-840, JEOL, Japan). The cross-section of the samples were gold-coated before observation. Rheological experiments were carried out on a rheometer (AR2000, TA Instruments, USA) using parallel plates configuration at 37 °C in an oscillatory mode. For the frequency sweep test, scanning ranged from 0.1 to 100 rad/s at 0.1% strain.21 In Vitro Micelles and Drug Release from Micelle/ Hydrogel Composites. In order to research on the release behavior of micelles, Rhodamine B-labelled micelle/ hydrogel composites were prepared with a SIM loading of 2 mg/mL. The Rhodamine B content was determined by UV-vis spectroscopy at a wavelength of 554 nm.22 In order to investigate the release behavior of SIM, 0.5 mg of the hydrogel sample (blank

CMCS/OMDex

hydrogel,

SIM@hydrogel,

SIM@micelle/hydrogel,

or

SIM@micelle-CHO/hydrogel) was immersed in 10 mL of PBS solution at pH7.4. A 3 ml of PBS solution from release system was replaced with 3 mL fresh PBS solution at every predetermined time points. The concentration of SIM released was measured at 238 nm using an ultraviolet-visible (UV) spectroscopy (KENREAL, USA).11 Cytotoxicity Test. The cytocompatibility of the blank hydrogel, SIM@hydrogel, and SIM@micelle-CHO/hydrogel was evaluated by live/dead assay. The samples with a SIM loading concentration of 2 mg/mL were prepared by mixing two precursor solutions into a 48-well tissue culture plate (200 μL/well) through a double-barrel syringe. CMCS/OMDex blank hydrogel without SIM loading was served as a control group. Mouse osteoblastic pre-osteoblasts (MC3T3-E1) were seeded onto the sample surface at a density of 2×105 cells/cm2 with 500 μL of cell culture medium (DMEM containing 10% FBS and 1% penicillin-streptomycin). The seeded cells were incubated for 1 day, 4 days and 7 days at 37 °C in a humidified atmosphere of 5% CO2. Fresh medium was changed once a day. Cells were stained using a Calcein-AM/PI Double stain kit and observed under an inverted fluorescence microscope. Live cells were stained into green and dead cells were red.21 In Vitro Osteogenesis Evaluation. Inserted Porous Tissue Culture Plates 8


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(Transwell®) were used to investigate the effect of the various hydrogel samples (blank

CMCS/OMDex

hydrogel,

SIM@hydrogel,

SIM@micelle/hydrogel,

or

SIM@micelle-CHO/hydrogel) on osteogenic differentiation of MC3T3-E1 cells. MC3T3-E1 cells (obtained from Wuhan Seville Technology Co. Ltd., Hubei, China) were seeded onto 24-well plates at a density of 2×105 cells/cm2. A 200 μL of sample with a SIM loading concentration of 2 mg/mL was placed on the chamber. SIM could diffuse into the culture medium through the microporous membrane in the lower part of the chamber. Cells were cultured for 7 days and 14 days in osteogenic medium (DMEM medium with 10% FBS, 1% penicillin-streptomycin 10 nM dexamethasone, 10 mM β-glycerophosphate, 25 μg / mL ascorbic acid). The culture medium were changed every 2 days. Alizarin Red S staining was used to characterize the osteogenic mineralization of MC3T3-E1 cells. After removing the medium from the 24-well plate, the cells were fixed with 95% ethanol for 15 minutes, and then stained with 150 μL alizarin red S for 30 minutes. The stain was rinsed off with PBS. For quantitative analysis, the dye was extracted with 10% cetylpyridinium chloride and the absorbance was measured at 560 nm. The osteogentic activity of MC3T3-E1 cells was detected by an alkaline phosphatase (ALP) assay reagent kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), as described previously.23 After 1 day, 7 days, and 14 days of culture, cells were lysed, mixed with ALP assay working solution. The absorbance of reaction product was measured at 420 nm. Statistical Analysis. Statistical analysis was conducted using ANOVA. All the data were expressed as means ± standard deviations (SD). Statistically significant differences was set at p

hydrogel composites for

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