Fabrication and Characterization of a Novel ... - ACS Publications

Article pubs.acs.org/journal/abseba

Fabrication and Characterization of a Novel Anticancer Drug Delivery System: Salecan/Poly(methacrylic acid) Semi-interpenetrating Polymer Network Hydrogel Xiaoliang Qi,† Wei Wei,† Junjian Li,† Yucheng Liu,† Xinyu Hu,† Jianfa Zhang,† Lirong Bi,*,‡ and Wei Dong*,† †

Center for Molecular Metabolism, Nanjing University of Science & Technology, Nanjing 210094, China The First Bethune Hospital of Jilin University, ChangChun 130000, China

‡

S Supporting Information *

ABSTRACT: Salecan is a novel linear extracellular polysaccharide with a linear backbone of 1−3-linked glucopyranosyl units. Salecan is suitable for preparing hydrogels for biomedical applications due to its prominent physicochemical and biological profiles. In this contribution, a variety of innovative semi-interpenetrating polymer network (semi-IPN) hydrogels consisting of Salecan and poly(methacrylic acid) (PMAA) were developed via free radical polymerization for controlled drug delivery. The successful fabrication of the semi-IPNs was verified by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and thermogravimetric (TGA) measurements. Scanning electron microscopy (SEM) and rheology analyses demonstrated that the morphological and mechanical behaviors of the resultant hydrogels were strongly affected by the contents of Salecan and crosslinker N,N′-methylenebis(acrylamide) (BIS). Moreover, the swelling properties of these hydrogels were systematically investigated, and the results indicated that they exhibited pH sensitivity. The drug delivery applications of such fabricated hydrogels were further evaluated from which doxorubicin (Dox) was chosen as a model drug for in vitro release and cell viability studies. It was found that the Dox release from the Dox-loaded hydrogels was significantly accelerated when the pH of the release media decreased from 7.4 to 5.0. Toxicity assays confirmed that the blank hydrogels had negligible toxicity to normal cells, whereas the Dox-loaded hydrogels remained high in cytotoxicity for A549 and HepG2 cancer cells. All of these attributes implied that the new proposed semi-IPNs serve as potential drug delivery platforms for cancer therapy. KEYWORDS: Salecan, poly(methacrylic acid), semi-interpenetrating polymer network hydrogels, doxorubicin, drug delivery

1. INTRODUCTION Hydrogels are three-dimensional (3D) cross-linked polymeric networks which possess the ability to imbibe a substantial amount of water from the environment and swell without dissolving.1 Hydrogels have been extensively explored as prospective materials for applications in the drug delivery,2 tissue engineering,3 and biosensor4 fields because of their environmental responsiveness, outstanding biocompatibility, and adjustable mechanical properties simulating native extracellular matrices. Recently, the development of stimuliresponsive hydrogels for drug delivery has drawn much attention. External stimuli to trigger drug release includes, but is not limited to, changes in pH,5,6 temperature,7 and ionic strength7,8 as well as exposure to light,9 electric,10 and magnetic11 fields. Among the family of smart hydrogels, pHsensitive ones have been frequently utilized as self-regulated devices for drug delivery.12 For instance, cancerous tissue (pH 6.8) and endosomes/lysosomes (pH 5.5) present a more acidic circumstance relative to normal tissue (pH 7.4),13 requiring hydrogels that can release the payload drugs in response to a change in pH. © 2015 American Chemical Society

Over the past few decades, numerous polymers have been actively investigated for the design of intelligent hydrogels.14 In particular, natural resourced polysaccharide-constructed hydrogels have gained considerable attention owing to their inherent biocompatibility, renewability, and availability, driving the desire to employ them as drug carriers.12 However, many polysaccharide-based hydrogels lack mechanical strength rigidity and can be vulnerable to rapid erosion and therefore are unstable. This severely hinders the application of polysaccharide hydrogels for drug delivery.15 However, the release of drug from the gel matrices are typically through physical diffusions, which may give rise to premature burst release.16 It is hard to tailor the drug release in an on-demand and predictable manner. To address these issues, the introduction of another polymer (such as a rigid synthetic polymer) to form an interpenetrating or semi-interpenetrating network (semi-IPN) is a feasible approach to reinforce the Received: August 13, 2015 Accepted: October 27, 2015 Published: October 27, 2015 1287

DOI: 10.1021/acsbiomaterials.5b00346 ACS Biomater. Sci. Eng. 2015, 1, 1287−1299

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ACS Biomaterials Science & Engineering

Figure 1. Illustration of the preparation and drug release of Salecan/PMAA semi-IPN hydrogels.

(acrylamide) (BIS). To the best of our knowledge, this is the first report to present the production and characterization of the Salecan/PMAA semi-IPN hydrogels for drug release systems. Their chemical structures, pH-sensitive swelling behaviors, morphologies, rheological properties, the release profiles of an antitumor drug (Dox), and cytotoxicities were assessed. The results demonstrate that the designed semi-IPN hydrogels are envisioned as promising candidates for controlled drug delivery.

mechanical properties of hydrogels and their resistance to corrosion.17 Semi-IPN and IPN hydrogels are an intriguing class of systems comprising the fusion of one or two polymeric networks that are physically interlocked and entangled on the molecular scale without chemical-covalent bonds between polymer chains of different type.17−19 The superior performances of IPNs, like enhanced mechanical strength,20,21 improved thermal stability,15 and adjustable responsive properties,22 have drawn great interest for delivering bioactive molecules.23,24 Especially, semi-IPN delivery systems, which consist of stimuli-responsive macromolecules (such as PMAA) and natural polymers (polysaccharides), are an exciting development, as they confer the components with responsive and biological characteristics.25,26 Salecan is a novel water-soluble extracellular polysaccharide (CAS No. 1439905-58-4) produced by fermentation from a new strain Agrobacterium sp. ZX09. This new strain was isolated from soil collected from the ocean coast of Shandong (China) by our laboratory, and its 16S rDNA sequence was deposited in the GenBank database (accession number GU810841).27 Structurally, Salecan consists of a linear backbone of β-Dglucopyranosyl (Glcp) units linked by α-1,3 and β-1,3 glycosidic bonds (Figure 1).27 Like other β-glucans, Salecan exhibits fascinating biological activities like antioxidation and nontoxicity (edible safety), which are often required in pharmaceutical and food industries.28,29 Moreover, Salecan possesses a high number of hydroxyl groups, located on the main chain, thereby making it possible to be chemically modulated in a controlled and specific fashion. Recently, a range of Salecan-based hydrogels materials were prepared by our group.30,31 It was found that these hydrogels were suitable materials for biomedical applications. Poly(methacrylic acid) (PMAA) is a synthetic polyelectrolyte capable of accepting or donating protons upon pH changes, accompanying reversible conformational alterations between the collapse and extension states.8,32 Furthermore, it is nontoxic and has a high mechanical strength.33 Attracted by these features, biomedical engineers are creating a multitude of PMAA-based hydrogels with diverse functions for drug delivery purposes.33,34 The aim of this article is to develop hydrogels containing Salecan that might be useful for loading and delivering drugs. These pH-sensitive intelligent semi-IPN hydrogels were synthesized by radical polymerization of methacrylic acid in the presence of Salecan and cross-linker N,N′-methylenebis-

2. EXPERIMENTAL SECTION 2.1. Materials. Salecan was prepared by Center for Molecular Metabolism, Nanjing University of Science & Technology. Methacrylic acid (MAA) was obtained from Sigma-Aldrich (Chemie GmbH, Riedstr., Germany). BIS and ammonium persulfate (APS) were supplied by Aladdin Industrial Corporation (Shanghai, China). Doxorubicin hydrochloride (Dox) was purchased from Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). The cell proliferation and cytotoxicity detection kit was provided by Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). 2.2. Fabrication of Hydrogels. Semi-IPN hydrogels of Salecan and PMAA were synthesized by free-radical cross-linking copolymerization using BIS as a cross-linker and APS as an initiator, in an aqueous solution at 70 °C for 6 h. In a typical procedure, 1 mL of MAA (98%, w/v), a certain content of BIS (1%, w/v), and Salecan (2%, w/v) were mixed according to the desired blending ratios in a tube and shaken for 10 min in an ultrasonic bath. Then, the solution was bubbled with nitrogen for 30 min to eliminate the dissolved oxygen. After that, 1 mL of APS (0.8%, w/v) was added, and the resulting mixture (the final volume of the solution was fixed at 10 mL with deionized water) was immediately transferred to a circular glass mold, which served as a polymerization reactor. The polymerization was conducted for 6 h at 70 °C. After reaction, the hydrogels were carefully taken out from the mold and cut into small pieces. Finally, the hydrogels in the form of discs were soaked in deionized water for 7 days (the deionized water was refreshed three times every day to remove the impurities). The feed ratios of the reaction mixtures are summarized in Table 1.

3. CHARACTERIZATION 3.1. Stability of Salecan in Semi-IPNs. The concentration of Salecan in the washing solutions of Salecan/PMAA semiIPNs was calculated using the phenol-sulfuric acid method.34 Briefly, 1 mL of phenol solution (6%, w/w) was dropped into a test tube with 2 mL of washing solutions, followed by the addition of 5 mL of concentrated sulfuric acid and then shaken well. The mixture was cooled at ambient temperature, and the absorbance was read at 490 nm. The concentration of Salecan 1288


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by adding an appropriate amount of NaCl). At each predetermined time interval, the hydrogels were taken out from the swelling medium and weighed after removing the surface adsorption of buffer with wet filter paper. The measurement was continued until constant weights of the swollen hydrogels were obtained. The swelling ratio (SR) and equilibrium swelling ratio (ESR) were calculated using eqs 1 and 2, respectively:

Table 1. Composition of the Initial Reaction Mixtures Utilized for the Preparation of Hydrogels Based on Salecan and PMAA designation ingredient

PMAA

Salecan (2%, w/v) (mL) MAA (98%, w/v) (mL) BIS (1%, w/v) (mL) APS (0.8%, w/v ) (mL) deionized water (mL)

0

SPM1 SPM2 2

4

SPM3 SPM4 SPM5 6

4

4

SPM6 4

1

1

1

1

1

1

1

1

1

1

1

2

1.5

0.5

1

1

1

1

1

1

1

7

5

3

1

2

2.5

3.5

SR = Wt /Wd

(1)

ESR = We/Wd

(2)

where, Wt is the weight of hydrated hydrogels after a certain time, We is the weight of equilibrium swollen hydrogels, and Wd is the weight of the initial dried hydrogels. The swelling kinetics of the hydrogels in various salt solutions (KCl, CaCl2, and FeCl3) was measured in the same way. All experiments were conducted in triplicate. 3.8. Water Retention Tests. The dried semi-IPN hydrogels were first soaked in deionized water at 25 °C to achieve equilibrium. A certain quantity of swollen gels was heated at 37 °C in an air oven for different intervals (t, min). Then, the quality of the remaining gels was weighed. Water retention (WR) of the hydrogels was defined as follows:

in the washing solution was determined using a standard curve (y = 0.017x + 0.0183, R2 = 0.9993). The measurements were performed in duplicate. 3.2. Fourier Transform Infrared (FT-IR) Spectra. The FTIR spectra of the Salecan and the dried hydrogels were recorded on a Nicolet IS-10 spectrometer working in attenuated total reflectance mode, in the range of the scanning wave numbers 4000−500 cm−1 with a 32 scans per sample cycle and a resolution of 4 cm−1. 3.3. X-ray Diffraction (XRD) Measurements. X-ray diffraction (XRD) patterns of the samples were obtained with a DMAX-2200 X-ray diffractometer using Cu Kα radiation with a wavelength of 0.154 nm at a voltage of 30 kV and a current of 20 mA over the range of 5−50°. 3.4. Thermogravimetric Analysis (TGA). Thermogravimetric analysis (TGA) was carried out using a TA Model Q600 thermal gravimetric analyzer at a heating rate of 10 K/min between 25 and 600 °C under a nitrogen atmosphere. The typical sample weight was between 3 and 5 mg. 3.5. Scanning Electron Microscopy (SEM). SEM images were taken with a JEOLJSM-6380LV electron microscope. The Salecan/PMAA semi-IPNs were first swollen to equilibrium in deionized water at room temperature. Then, the semi-IPNs were cut into thin slices and lyophilized using liquid nitrogen as the cryogen. Finally, these dried samples were coated with gold to enhance the conductivity. 3.6. Mechanical Properties. The rheological properties of the Salecan/PMAA semi-IPNs were determined using an Anton Paar MCR101 rheometer. A 50 mm parallel-plate geometry and a gap of 1 mm were used for all assays which were done at 25 °C. To ensure the measurements were performed in the linear region of viscoelasticity, an amplitude sweep was carried out, and the results displayed no variation in elastic modulus up to a strain of 0.01%. The dynamic modulus of the semi-IPN hydrogels were recorded as a frequency function, where the frequency sweeps were measured between 0.1 and 10 Hz. The tests were repeated three times, and the results were averaged. 3.7. Swelling Studies. The swelling properties of the synthesized semi-IPN hydrogels were investigated by gravimetric analysis. Dried hydrogel samples were precisely weighed and immersed in buffer solutions with various pH values at room temperature (pH 1.2, HCl/NaCl buffer; pH 5.0, potassium hydrogen phthalate buffer; pH 7.4, disodium hydrogen phosphate/potassium dihydrogen phosphate buffer; pH 9.18, sodium tetraborate decahydrate buffer; the ionic strength of each buffer solution was 0.1 M, which was acquired

WR (%) = (Wt /Weq) × 100

(3)

where, Wt is the weight of unswollen hydrogel at time t, and Weq is the weight of the swollen hydrogel at equilibrium in deionized water. Results were averaged on three independent runs. 3.9. In Vitro Drug Loading and Release Behaviors of the Hydrogels. 3.9.1. Drug Loading. Dox was chosen as a model drug and loaded to the as-prepared hydrogels using the swelling diffusion method. Specifically, the preweighed dried hydrogels (0.1 g) were soaked in 20 mL of Dox solution (50 mg/mL) at room temperature. After swelling in the Dox solution for 24 h, the hydrogel was swollen to equilibrium. Subsequently, the swollen hydrogels were gently removed from the Dox solution and washed with deionized water to remove the superficial adsorbed Dox. The concentration of Dox remaining in the soaking solution was determined spectrophotometrically at 481 nm using an UV−vis spectrophotometer (TU-1900, China). The drug loading efficiency (DLE) of drugloaded hydrogel was calculated according to eq 4: DLE (wt, %) = (W0−We)/W0 × 100

(4)

where, W0 and We are the total amounts of the Dox in immersing solution before and after loading of the hydrogels, respectively. 3.9.2. Drug Release. The in vitro drug release performances were investigated in PBS at pH 5.0 (endosomal pH in cancer cells) and pH 7.4 (physiological pH). The above-mentioned drug-loaded hydrogels were first placed into a flask containing 40 mL of PBS buffer solution (pH 5.0 and 7.4). Then, these flasks were put into a constant temperature shaking incubator at 37 °C at 100 rpm in the dark. At specific time intervals, 2 mL of the release media was taken out; at the same time, 2 mL of fresh PBS solution was supplemented. The cumulative release ratio of Dox from hydrogels was measured by UV−visible spectrophotometer at the absorbency of 481 nm. The accumulative percent drug release (Er) was obtained using the following equation: 1289


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ACS Biomaterials Science & Engineering n−1

Er (%) =

Ve ∑1

Ci + V0Cn

mDox

× 100

4.2. Stability of Salecan in Semi-IPN Hydrogels. The amount of Salecan remaining in the washing solution of Salecan/PMAA semi-IPNs was determined using the phenolsulfuric acid method.35 The results are presented in Table S1 (Supporting Information), suggesting that the Salecan was stable and that its chains were successfully incorporated into the PMAA hydrogel. 4.3. FT-IR Spectral Analysis. Figure 2a shows the FT-IR spectra for the Salecan, PMAA, and Salecan/PMAA semi-IPN

(5)

where mDox is the amount of Dox in the hydrogel, V0 represents the volume of the release medium (V0 = 40 mL), and Cn is the concentration of Dox in the nth sample. The experiment for in vitro Dox release was performed in triplicate. 3.10. In Vitro Cell Assay. 3.10.1. Cell Viability. The cytotoxicity of the hydrogels against COS7, A549, and HepG2 cells was assessed using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) assay by an indirect extraction method on the basis of the ISO 10993-5 standard as described previously.31 These three cells were cultured in a DMEM medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL of penicillin, 2 mM L-glutamine, and 100 mg/mL of streptomycin at 37 °C and 5% CO2. Meanwhile, the Dox-free hydrogels were sterilized with ethanol (70%) followed by rinsing with sterile pH 7.4 PBS. Then, the sterile hydrogels were added into 5 mL of DMEM at 37 °C for 48 h. Subsequently, the hydrogels were carefully taken out, and the resulting extract solutions were filtrated through a 0.22 μm syringe filter. Additionally, the Dox-loaded hydrogels were immersed in fresh pH 7.4 PBS for 48 h and then taken out from the buffer. After that, free Dox solution and the extract liquid were sterilized by filtration (0.22 μm) and diluted with DMEM culture media (the concentration of Dox in extract solution was the same as that of free Dox; ultimate concentration 0.25, 0.5, 1.5, 3, 6, and 12 μg/mL). For the MTT assay, these cells were separately seeded into 96-well plates at a density of 5000 viable cells per well and incubated overnight to allow cell attachment. Then, the culture media in each well was replaced with 200 μL of DOX-free hydrogel extracts, free Dox, or Dox-loaded hydrogel extract solution. After 24 h, the medium was replaced with fresh DMEM containing 50 μL of MTT, and the cells were cultured for an additional 4 h. Upon the withdrawal of MTT solution, the purple formazan crystals generated by viable cells were dissolved with DMSO. The relative cell viability was calculated by comparing the absorbance at 570 nm with control wells containing only cell culture medium. The obtained data were expressed as mean values of three measurements. 3.10.2. Fluorescence Microscopy. A549 and HepG2 cells were seeded in 6-well plates at a density of 1 × 104 cells per well in 2 mL of DMEM and incubated for 24 h. The cells were then treated with free Dox (6 μg/mL) or the above-mentioned extract solution of Dox-loaded hydrogel (6 μg/mL equivalent Dox concentration) for 4 h. After washing three times with PBS, the cells were observed by fluorescence microscopy (Olympus, Japan).

Figure 2. FTIR (a), XRD (b), and TGA (d) curves of Salecan, semiIPN, and PMAA hydrogels.

4. RESULTS AND DISCUSSION 4.1. Preparation of Salecan/PMAA Semi-IPN Hydrogels. The Salecan/PMAA semi-IPN hydrogel synthesis scheme is shown in Figure. 1. In brief, the initiator APS was first decomposed at 70 °C to generate free radicals. Subsequently, these radicals initiated the copolymerization of MAA and BIS in the presence of Salecan. Finally, a three-dimensional network of PMAA was formed, and Salecan chains interdiffused and became physically entangled within this PMAA hydrogel network through hydrogen-bonding interactions.

hydrogels, respectively. With regard to the spectrum of the Salecan, a broad absorption band entered at 3291 cm−1 was assigned to the O−H stretching vibration, as well as the intermolecular hydrogen bonding of the polysaccharide moieties.30 Characteristic peaks of Salecan emerged in the range of 1100−800 cm−1. Specifically, the band at 1040 cm−1 was related to C−OH stretching in the glucopyranose ring, a small peak at 893 cm−1 suggested that D-glucopyranose had a βconfiguration, and a weak peak at 814 cm−1 belonged to the presence of a little α-glucopyranose.27,31 For the spectrum of 1290


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Figure 3. SEM photos of Salecan/PMAA semi-IPN hydrogels: (a) SPM1, (b) SPM2, (c) SPM3, (d) SPM4, (e) SPM5, and (f) SPM6. Scale bars represent 100 μm.

neously dispersed in an amorphous state in the PMAA matrix, resulting in the deformation of the lattice structure.42 4.5. Thermal Gravimetric Analysis (TGA). Figure 2c shows the thermal properties of the Salecan, PMAA, and Salecan/PMAA semi-IPN hydrogels. Salecan exhibited a small mass loss of approximately 12% upon heating even below 150 °C, ascribed to the vaporization of absorbed and bonded water. Subsequently, about 48.0% mass loss occurred in the 200−400 °C temperature range, and further a 20.0% mass loss took place between 400 and 600 °C, which may be due to the splitting of Salecan structure and chain scission.30,31 Similar results were obtained for other polysaccharides.43−45 In the case of the PMAA thermogram, three stages were noticed. In the first stage, a moderate decrease in weight (8.5%) before 150 °C could be attributed to the release of moisture. The second stage started from around 160 °C and finished at 300 °C with a weight loss of 14.0%. It belonged to the dehydration and decarboxylation of the polymer.37 After 300 °C, PMAA lost its major mass (77.0%), mostly because of the intensive pyrolysis of the poly(methacrylic anhydride) backbone chains.37,44 Here, the semi-IPN hydrogel also underwent three steps of thermal decomposition. An initial 8.0% of weight loss in the temperature range 30−160 °C was attributed to the elimination of imbibed water from the hydrogel network. Then, almost 9.0% weight loss was observed in the 160−300 °C temperature range. At the third step, a 64.0% mass loss was experienced. The latter two steps belonged to a sophisticated process where fragmentation of the Salecan skeleton, depolymerization of the PMAA chain, and breakage of the cross-linked network was dominant. Typically, after Salecan was loaded into the gel, the maximum mass loss rate appeared at a higher temperature (414 °C) than that of the PMAA (406 °C), indicating a better thermal stability of the semi-IPN.31 4.6. SEM Observations. Figure 3 depicts representative SEM photos of the cross-section for lyophilized Salecan/PMAA semi-IPN hydrogels. Cryo-SEM was used here to make sure that the microstructure and morphology of all hydrogel samples were well-retained and that the observations discussed herein are in accordance with reality. All hydrogels demonstrated homogeneous and porous architectures consisting of polyhedral pores. The average diameter of pores calculated by the available software (ImageJ) was around 50.2 ± 6.3, 80.1 ± 8.6, 104.6 ±

pure PMAA hydrogel, a broad peak in the region between 3600 and 3000 cm−1 corresponded to the O−H stretching vibration.36 The band at 2992 cm−1 can be ascribed to C−H stretching in the −CH2 group.37 Two characteristic bands of PMAA emerged at 1693 and 1252 cm−1, which were due to the stretching vibration of CO and C−O of the carboxylic group.38 Moreover, the peaks at 1480 and 1388 cm−1 were assigned to the CH2 and CH3 deformation, while a band at around 1158 cm−1 was attributed to the C−N stretching (secondary amine) from BIS.36,38,39 Considering the FTIR spectra of semi-IPN hydrogels, it could be clearly observed that some absorption bands were altered compared to Salecan and PMAA. For instance, a strong transmittance peak located at 1694 cm−1 was associated with stretching vibration of CO of the PMAA segments, whereas the occurrence of bands at 1043 and 893 cm−1 was characteristic of the polysaccharide skeleton of the Salecan molecule. Following the literature, similar results were obtained.36,40,41 Furthermore, a broad band appeared at 3360 cm−1, corresponding to the formation of hydrogen bonds between the −COOH groups of MAA and the −OH groups of Salecan.36 In addition, other FT-IR features of SPM3, such as C−N stretching at 1161 cm−1, C−O stretching at 1256 cm−1, −CH3 deformation at 1388 cm−1 and −CH2 deformation at 1447 cm−1, can be considered as proof of PMAA hydrogel formation. These together proved that Salecan chains had been entrapped within the pure PMAA network successfully. 4.4. XRD Studies. XRD experiments were employed to characterize the Salecan, pure PMAA, and Salecan/PMAA semi-IPN hydrogels (Figure 2b). According to Figure 2b, a broad and diffuse diffraction hole centered at 2θ = 21° was observed in the XRD patterns of Salecan. These accorded with the data from the literature and inferred that the Salecan had an amorphous structure.30 In contrast, from the spectrum of pure PMAA hydrogel, it could be seen that the PMAA showed a prominent diffraction peak positioned around 2θ = 15°, suggesting high crystallinity. With the addition of Salecan, the diffraction peak of Salecan/PMAA semi-IPN hydrogel was similar to that of PMAA; the relative peak intensity decreased, and the amorphous halo of Salecan was not found. This implied that Salecan was either molecularly distributed or homoge1291


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Figure 4. Frequency dependence of (a) dynamic storage modulus (G′) and (b) dynamic loss modulus (G″) of PMAA and Salecan/PMAA semi-IPN hydrogels.

Figure 5. Swelling ratio values in deionized water (a), swelling kinetics in different buffer solutions (b), salt solutions (c), and water retention graphs (d) of Salecan/PMAA semi-IPN hydrogels.

20.1, 57.7 ± 9.4, 72.3 ± 15.7, and 95.9 ± 18.4 μm for SPM1, SPM2, SPM3, SPM4, SPM5, and SPM6, respectively. Interestingly, the addition of Salecan and BIS appeared to impact the diameter of pore size in a concentration dependent manner. For example, in Figure 3a−c, an increment in the Salecan dose from 2 to 6 mL resulted in an increase in the average pore size from 50.2 ± 6.3 to 104.6 ± 20.1 μm. The cause may be that introduction of Salecan enhanced the hydrophilic ability of the network. As a result, water molecules could easily diffuse into the gel matrix. Therefore, larger pores were created in the composite hydrogels by water sublimation resulting from the lyophilization process.46,47 On the contrary, the average pore size of the hybrid gels clearly decreased as the BIS content increased, ranging from 95.9 ± 18.4 to 57.7 ± 9.4 μm (Figure 3d−f). The reason was that, with the incorporation

of BIS, the cross-linking density of the network was enhanced and the water content of semi-IPNs declined, which was prone to the formation of a smaller ice crystal during the freeze-drying process and concomitant smaller pores.48 Variation in morphologic properties may involve changes in the physical−chemical behaviors of the hydrogels. As we shall describe below, the mechanical, swelling, and drug release characteristics were also significantly influenced by this semiIPN structure. 4.7. Rheological Analysis. Rheological studies of the hydrogels were undertaken to obtain further insight on their characteristics (Figure 4). For each hydrogel, the storage modulus (G′) exceeded the loss modulus (G″) over the entire angular frequency range, and both moduli were independent of frequency, demonstrating an elastic solid behavior.49,50 1292


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ACS Biomaterials Science & Engineering Furthermore, their G′ and G″ values were easily tuned within a given range (from 0.1 to 10 Hz) by modulating the amounts of Salecan and BIS utilized to produce the hydrogel. More specifically, as illustrated in Figure 4a, before the introduction of Salecan, the PMAA hydrogel possessed the strongest storage modulus (G′) value of approximately 7177 Pa. By increasing the amount of Salecan solution in the initial mixture to 2, 4, and 6 mL, the G′ value gradually declined to around 5289, 2719, and 1250 Pa, respectively. These data were likely that more Salecan would cause a higher swelling ratio (as demonstrated in swelling analysis), which led to a decline in the stiffness of the gels and, as a result, a reduction in the storage modulus. Besides that, it can be observed that a higher initial BIS concentration led to a higher G′ of the hydrogels (Figure 4a). Incorporation of BIS in hydrogels raised the density of polymer chains and created a more dense structure, thus attaining higher storage moduli.49 4.8. Swelling Characteristics. 4.8.1. Swelling Kinetics. Swelling kinetics and time-dependent swelling profiles of the semi-IPN hydrogels in deionized water at 37 °C were plotted in Figure. 5a. It was found that all of the lyophilized hydrogels revealed a rapid swelling rate in the first 7 h of immersion and achieved water saturation by 15 h of incubation. The equilibrium swelling ratio of SPMA1, SPMA2, SPMA3, SPMA4, SPMA4, SPMA5, and SPMA6 was 10.3, 14.4, 19.1, 16.6, 13.2, and 11.9, respectively. Furthermore, the value of the ESR varied with the concentration of Salecan and BIS in the hydrogels. For instance, as is vividly apparent in Figure. 5a, SPM1 fabricated with less hydrophilic Salecan revealed a lower ESR (10.3) than SPM3 (19.1) containing the more hydrophilic Salecan. One possible explanation for this phenomenon can be assigned to the appearance of −OH groups on the Salecan skeleton that attract water from the surrounding environment, facilitating the penetration of the water molecules into the gel matrix.51 Consequently, the hydrogel enhanced its ability to take up water. Also, the ESR changed with BIS constituents in the hydrogel network; it gradually reduced from approximately 16.6 to 11.9 when the BIS content increased from 0.5 to 2 mL (Figure 5a). This was because the increment in the number of cross-linker BIS hindered the mobility of the hydrogel chains and, ultimately, the water absorbency. Others have published similar results.31,52 Overall, dynamic swelling experiments denoted that the swelling behaviors of the as-prepared hydrogels were found to be tunable by adjusting the hydrophilicity and cross-linking density of the network, which can simply be regulated by controlling the Salecan and BIS contents in the hydrogels composition. 4.8.2. Effect of pH on Swelling. To better imitate the in vivo conditions, equilibrium swelling experiments were conducted in simulated physiological fluid with different pH values (pH 1.2, 5.0, 7.4, and 9.18). The ionic strength in various buffer media was adjusted to be 0.1 M with NaCl. As revealed in Figure. 5b, the general feeling was that all of the semi-IPNs exhibited the same trend, namely, the ESR of the samples enhanced continuously from pH 1.2 to 7.4, but reduced abruptly at pH 9.18. Taking SPM3 as an example, the ESRs were 3.4 for pH 1.2, 8.1 for pH 5.0, 19.1 for pH 7.4, and 17.4 for pH 9.18. The remarkable differences found in these values may be assigned to the protonation and ionization balance of the carboxyl acid groups appearing in the hydrogel chains, whose pKa value was approximately 5.5.53 At pH 1.2 and 5.0 (pKa of the PMAA), the −COOH groups of PMAA chains dissociated, which can not only undermine the H-bonds between the Salecan and PMAA chains but also induce the electrostatic repulsion among the ionized −COO− groups, causing the increment of the hydrogel volume. Further increasing the pH to 9.18, the excess counterions (such as potassium ion) shielded the charge of the carboxylate ion and decreased the effective repulsion force, leading to reduced swelling.55 On the whole, equilibrium swelling studies clearly revealed that these semiIPN hydrogels were capable of responding to pH of the external media, making them quite attractive to be utilized as a controlled drug delivery vehicle. 4.8.3. Influence of Salt on Swelling. Figure 5c presents the swelling behavior of the semi-IPN hydrogel (SPM3) in different salt solutions with various concentrations. As seen from Figure 5c, water absorbency of the SPM3 was significantly affected by salt concentrations of the surrounding medium. Regardless of salt species, the ESRs of SPM3 dropped drastically as the salinity ratios increased. Specifically, the ESRs of the gel were 11.3, 10.4, 9.4, 8.4, and 8.3 for CaCl2 concentration (%, w/w) = 0.3, 0.6, 0.9, 1.2, and 1.5, respectively. The mechanisms of these phenomena can be understood as follows. On the one hand, the osmotic pressure between the external incubation medium and the interior hydrogel was strengthened with the increment of saline solution concentration. In this case, water in the hydrogel network tended to diffuse out to the swelling solution, which had a high salt concentration, leading to acceleration of water molecules release and reduction of swelling capacity.56 On the other hand, the counterions (such as K+) penetrated into the inside structure of the hydrogel and combined with carboxylic groups (−COO−) of PMAA units, causing the shielding effect and weakening the anion−anion electrostatic repulsion.53,57 Hence, the moistures were squeezed out from the gel. It is also exhibited in this graph that the ESRs strongly depended on ionic valence of the introducing salt. Apparently, at a given salt concentration, the swelling of the hydrogel was trivalent Fe3+ < divalent Ca2+ < monovalent K+. It could be explained that the complexing capability of the acrylate anions (−COO−) can induce the formation of intra- and intermolecular complexes with multivalent cations. In other words, the high charge cations (e.g., Ca2+ and Fe3+) could cross-link PMAA via electrostatic interaction with anionic groups (COO−) on PMAA polymer chains, which augmented the cross-linking density of the hydrogel, giving rise to a dense structure. The resulting compact architecture limited the environmental solution entry into the network of the hydrogels, thus leading to a remarkable decrement of ESR with increase of the metal cationic valence of the salt solution.58,59 4.8.4. Deswelling Kinetics. Figure 5d displays the deswelling kinetics of the semi-IPN hydrogels containing different Salecan and BIS contents under identical experimental conditions. The semi-IPN hydrogels were characterized by a rapid dehydration that occurred during the first 6 h. Further, the loss of absorption water was basically stable up to 15 h. An explanation was that, initially, the water molecules in the hydrogel interior were easy to diffuse out, and with the passage of the drying time, the relative proportion of strong absorbed water was 1293


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Figure 6. In vitro Dox release behaviors from the semi-IPN sample (a) at the two different pH values of 5.0 and 7.4 and (b) at pH 7.4, followed by pH 5.0; (c) SPM1−SPM3 and (d) SPM4−SPM6 hydrogels in pH 5.0 buffers.

the Dox loading content in the semi-IPN (SPM4) reached 84.5% (Table S2, Supporting Information), indicating that the prepared semi-IPNs were indeed efficient vehicles for Dox loading. 4.9.2. Drug Releasing. For the design of efficient drug vehicles, achieving a controllable drug-release property is the main challenge.62 In this article, Dox was loaded into semi-IPN hydrogels through electrostatic interactions between the negatively charged PMAA chains and positively charged Dox molecules.63 It should be noticed that because the segment of PMAA is a weak acid with a pKa of 5.5,53 it can be easily protonated under acidic conditions, which disrupted the electrostatic interaction between Dox and hydrogel (pH < 5.5), favoring Dox release. Considering this factor, the pH value of simulated cancer environments was selected as 5.0. Figure 6a presents the Dox release profiles of the Dox-loaded semi-IPNs (SPM3) in phosphate buffered saline (PBS) with a pH of 7.4 (mimicking the normal physiological environment) and a pH of 5.0 (corresponding to the pH in endosomes of tumor cells) at 37 °C, respectively. As illustrated in Figure 6a, the release of Dox from SPM3 displayed distinct pHdependence and was markedly accelerated with decreasing pH. At pH 7.4, only around 23.0% of the Dox was released after 5 h, implying that the designed vehicles were stable under physiological conditions. However, approximately 62.8% of Dox was released within the same period when pH was lowered to 5.0, and in the end, total Dox release quantity at pH 5.0 was 2.4-fold that at pH 7.4. Besides that, successive exposure of SPM3 to a different release medium at pH 7.4 and 5.0 brought about a pH-dependent “off−on” switching of drug release. By adjusting the pH value from 7.4 to 5.0, Dox release from the hydrogels was activated, and the release speed was augmented drastically (Figure 6b). These differences in release rates might

enhanced, resulting in a relatively slow deswelling. The same effect was obtained by Sacco and co-workers.60 However, as shown in Figure 5d, the semi-IPN hydrogels containing more Salecan exhibited a faster dehydration rate and released more water within the identical time. For instance, within 500 min, SPM1, SPM2, and SPM3 shrank and expelled about 83.5%, 96.7%, and 98.6% solvent, respectively. The reasons for this phenomenon were that the incorporation of the hydrophilic chains, Salecan, could disrupt the formation of the dense layer on the surfaces of the hydrogels and that the hydrophilic chains acted as water-releasing channels when the collapse took place. Thus, the water contained in the gel matrix was more easily broken free.51 Conversely, it can be observed from Figure 5d that the water retention capacity of semi-IPN hydrogels decreased with BIS dosage. The water retention value of the SPM4 with 2 mL of BIS was 88% at time 600 min; however, water retention reached 93% when 1.5 mL of BIS was applied and was 97% with the addition of 0.5 mL of BIS. Such improvement of water-retention capacity might be attributed to the fact that higher content of BIS produced a highly crosslinked hydrogel, which in turn lowered the conformational freedom of polymeric chains, restricting the outflow of water from the network.39 4.9. Triggered Release of Dox in Vitro. 4.9.1. Drug Loading. Doxorubicin (Dox), a DNA interacting drug, is widely used in the treatment of several pediatric and adult cancers.61 To assess the potential application of semi-IPNs as drug carriers, Dox was selected as a model anticancer drug to explore the loading and controlled release behavior of the hydrogels. In the current research, Dox was incorporated into the aforementioned hydrogels through electrostatic interactions between the negatively charged hydrogels and positively charged Dox molecules. According to eq 4, it was found that 1294


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Figure 7. (a) Fluorescent microscopy images of A549 and HepG2 cells after 4 h of incubation with 6 μg/mL free Dox solutions and the extract liquid of Dox-loaded hydrogel; (b) cell viability of COS7, A549, and HepG2 cells after treatment with blank hydrogel extracts; (c) cell viability of A549 and HepG2 cells after treatment with Dox loaded hydrogel extracts and free Dox solutions.

network was gradually collapsed due to the hydrogen bonding interactions between the protonated carboxylic groups, thereby retarding and impeding the release rate of Dox.69−71 Figure 6c and d demonstrates the cumulative releases of Dox from semi-IPN hydrogels prepared using various concentrations of Salecan and containing different cross-linker contents at 37 °C in PBS solutions with a pH of 5.0. It was found that the Dox release was progressively enhanced by either the addition of Salecan or lowering the cross-linker BIS. Especially, Dox was slowly released from the SPM1 with no more than 42.9% release being achieved at the end of the release. In contrast, the release rate of SPM3 was much faster, with approximately 71.6% of Dox being released in the same time scale. A probable explanation for this increment in Dox release lies with the interior structure of the hydrogels.68 Typically, hydrogels with higher Salecan contents exhibited a larger average pore size (as verified by SEM results), which facilitated Dox release. Moreover, the Dox-releasing capacities increased significantly with decreasing the feeding ratios of the cross-linker. In detail, the release rate of the SPM4 was always lower than SPM6 during the whole incubation period and the final release percent of the hydrogels reached 46.9% and 64.7% for SPM4 and SPM6, respectively. This phenomenon could be explained by considering the formation of a denser polymeric network in the case of a higher cross-linking reagent, which restricted the release of encapsulated Dox from the semi-IPN hydrogels. Others have published similar results.72,73 Taking the data in section 4.9 together, the Dox-containing semi-IPN hydrogels were highly sensitive to pH: it greatly minimized the side effects to normal cells under physiological conditions by reducing premature drug loss and undergoing accelerated release in an acidic tumor environment, both of which could be definitely beneficial to the overall therapeutic efficacy of cancer treatment.74 These properties make the

be ascribed to the following two factors. (i) In an acidic environment, the hydrophilicity of Dox increased due to the protonation of amino groups native to its structure (Dox is a weak base and has a pKa of 8.264). Thus, the driving force for entrapped Dox to escape from the hydrogels profoundly increased. As a result, the soluble Dox could be freely distributed out from the networks. (ii) When the pH of the culture media was reduced to 5.0, the protonation of PMAA chains could break up the electrostatic interactions between −COO− of MAA moieties and −NH3+ of Dox,65 which might be helpful for diffusion of the Dox. More attractively, at pH 5.0, the release behavior of Dox displayed apparently a biphasic pattern characteristic. That was, a fast release stage (the initial 5 h) followed by a slow release stage (from 5 to 18 h). As noted in Figure 6a, the hydrogel released 62.8% of Dox rapidly within 5 h (stage I), but only another 8.8% of Dox was squeezed out during the next 13 h (stage II). This was similar to the findings of Yu et al.66 With regard to this phenomenon, the present authors inferred that Dox release from the hydrogel was mainly related to diffusion and the internal structure change of the hydrogels.67 Prior to the incubation, the electrostatic attraction between the negatively charged carboxyl groups from PMAA and the positively charged Dox molecules promoted most of the Dox molecules to stay inside the swollen equilibrium hydrogels. However, when these hydrogels were suddenly subjected to a lowered pH, the carboxyl groups of PMAA chains were highly protonated, which disrupted the electrostatic interaction between Dox and the hydrogel, leading to a fast dissociation of Dox from the hydrogel.68 Consequently, Dox diffused out through the pores of the gel matrices in a short time (stage I). Obviously, in stage I, the Dox release rate was dominated by environmental pH. After that, the release proceeded to stage II, and the release profile maybe governed by the swelling− shrinking behavior of the hydrogel. In stage II, the polymer 1295


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IPN hydrogels with different pore sizes ranging from 50.2 to 104.6 μm were prepared. Therefore, the properties of the fabricated hydrogels were precisely controlled, including swelling properties, mechanical behaviors, and drug release kinetics. Additionally, the sustained Dox release from the semiIPN hydrogel could also be modulated by varying the pH of the release media. The in vitro release of the entrapped Dox from the hydrogels was greatly inhibited under neutral environment (pH 7.4) mimicking normal tissue, whereas a rapid release was achieved in acidic conditions (pH 5.0) such as in the endosome or lysosome of cancer cells. Moreover, the in vitro cellular uptake assay and cytotoxicity studies verified that the released Dox could be sufficiently taken up by HepG2 and A549 cells, then efficiently restrain the proliferation of these two cancer cells, and finally induce cell death. With these excellent features, we can foresee that this Salecan-based pH-responsive semi-IPN hydrogel holds great promise to be utilized as a vehicle for anticancer drug delivery.

prepared semi-IPN hydrogels attractive platforms for effective drug carriers in tumor therapy. 4.10. In Vitro Cell Uptake. The cell uptake study was conducted in the A549 and HepG2 cell lines. Since Dox is a fluorescent molecule, its internalization by tumor cells can be monitored using fluorescence microscopy.75 Figure 7a illustrates bright field and fluorescence microscope images of these two cells after 4 h of treatment with free Dox and the extract liquid of Dox-loaded hydrogels with an equivalent amount of Dox (6 μg/mL). Notably, the intense red fluorescence emitted by Dox was visibly observed inside both A549 and HepG2 cells, verifying that the released Dox had been effectively taken up by these two cells. 4.11. Cytotoxicity Assay. Following pH triggered release and cellular internalization, another key point was to evaluate the potential toxicity of proposed hydrogels for drug delivery applications. To act as a safe and efficient drug carrier, the vector itself was required to be nontoxic to the normal cells, and the drug loaded vehicle should cause high cytotoxicity to tumor cells. Herein, the cytotoxicity of Dox-free hydrogels to normal cell lines COS7 was first investigated by an indirect MTT assay.76 As presented in Figure 7b, the viabilities of COS7 cells cultured with different extracts of empty hydrogel samples were all above 90% after 24 h of incubation. This demonstrated that these hydrogels were biocompatible with negligible cytotoxicity and could be utilized as a delivery platform. Further, we screened the cytotoxic effect of the blank hydrogel in HepG2 and A549 cancer cell lines. It also can be seen from Figure 7b that the hydrogel alone did not exhibit any appreciable cytotoxicity to both HepG2 and A549 cells. Ultimately, the antitumor activity of Dox-loaded hydrogels (SPM3) against two tumor cells (HepG2 and A549 cells) was also estimated via the MTT method, and the free Dox was employed as a control. After a 24 h of exposure, Dox-loaded hydrogels showed a dose-dependent cell proliferation inhibition behavior for either HepG2 cells or A549 cells (Figure 7c), which was similar to that of free Dox, revealing that the cytotoxicity was caused by released Dox and not by the hydrogel system itself.77 The result also displayed that Dox released from the hydrogels can still exert its activity (as evidenced by the cellular uptake analysis) and effectively induce cellular apoptosis.67 However, as shown in Figure. 7c, Doxloaded hydrogels exhibited lower cell growth inhibition efficiency (regardless of whether HepG2 or A549 cells were used) as compared with pure Dox at the equivalent drug concentration. For instance, the cell viability of free Dox (6 μg/ mL) and Dox-loaded hydrogels (at an equivalent DOX concentration of 6 μg/mL) was 37.4% and 28.1% for A549 cells and 30.8% and 20.5% for HepG2 cells, respectively. This fact might be ascribed to the delayed but sustained release of Dox from the Dox-loaded hydrogels and the decline in cellular uptake of Dox within 24 h.78,79 The cell viability data, coupled with the above-mentioned Dox release results, rendered the obtained semi-IPN hydrogels as intriguing vehicles for application in the field of targeted drug delivery.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.5b00346. Drug loading efficiency of the semi-IPN hydrogels and the content of Salecan in hydrogel and washing medium (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(L.B.) E-mail: [email protected]. *(W.D.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China under the Grant 30870625 and the Fundamental Research Funds for the Central Universities, NUST2011XQTR07.

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REFERENCES

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