Development of novel hydrogels based on Salecan and poly(N-isopropylacrylamide-co-methacrylic acid) for controlled doxorubicin release

Abstract

Salecan is a novel water-soluble extracellular polysaccharide composed of a linear repeating unit of 1-3-linked glucopyranosyl. Salecan is suitable for the preparation of hydrogels for biomedical applications due to its outstanding biological and physicochemical profiles. Here we designed a novel semi-interpenetrating polymer network (semi-IPN) hydrogel that incorporated the hydrophilic polysaccharide Salecan into a stimuli-responsive poly(N-isopropylacrylamide-co-methacrylic acid) (PNM) hydrogel matrix for controlled drug release. In this research, on one hand, Salecan modified the architecture and the pore size of the semi-IPN network. On the other hand, Salecan played a vital role in modulating water content and tuning the water release rate of the developing hydrogel, resulting in an adjustable release rate of the drugs. Doxorubicin (DOX), an anticancer drug, was loaded into the semi-IPN hydrogels as a model drug, and the in vitro release assay exhibited that the release rate was closely related to the content of introduced Salecan, as well as the environmental temperature and pH of the release media. Cytotoxicity experiments demonstrated that all blank semi-IPN hydrogels were non-toxic to HepG2 and A549 cells, while drug released from the DOX-loaded hydrogels could still exert its pharmacological activity and had the ability to kill these two cancer cells. Altogether, this work provides a way to synthesize a new type of hydrogel as general drug delivery vectors with a desired release rate.

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1 Introduction

Hydrogels consist of three-dimensional (3D) networks of hydrophilic polymer chains that can uptake a substantial amount of water or biological fluids without dissolving or losing their structural integrity.^1,2 They have been extensively explored because of their prominent properties like biocompatibility, tunable viscoelasticity and high permeability for water-soluble bioactive agents, resulting in wide-ranging applications including adaptive lenses,³ scaffolds for tissue engineering⁴ and vehicles for drug delivery.² Particularly, smart hydrogels, which exhibit conformational transitions between swollen and collapsed states when experiencing environmental stimuli such as pH,⁵ temperature,⁶ electric/magnetic field,^7,8 light,⁹ etc., have captured a great deal of interest recently. Among these smart hydrogels, pH- and thermo-responsive ones are most appealing, because both parameters are crucial environmental factors in biomedical systems and very easily handled. In many practical applications such as controlled drug delivery, they may provide unique advantages for offering on-demand release of therapeutic biomolecules.²

In the past decades, naturally sourced biopolymers, especially polysaccharides, have commonly been utilized to fabricate intelligent hydrogels as carriers for drug delivery owing to their inherent renewability, biodegradability, biocompatibility and versatility in chemical modification.¹⁰ Nonetheless, there are two major limitations associated with polysaccharide hydrogels when employed as drug delivery devices: one is the weakness of mechanical rigidity; the other is the poor sustained release capability.^11,12 Besides, the drug molecules are typically wrapped in the gel matrices (such as dextran hydrogels) through simple physical forces only, leading to low drug loading. Thus, the development of polysaccharide-based carrier system remains a formidable challenge.⁵ The fusion of polysaccharides and artificial polymers in the form of interpenetrating polymer network (IPN) or semi-interpenetrating polymer network (semi-IPN) hydrogels may contribute to surmount the aforementioned drawbacks and integrate the merits of both components.^13,14 For instance, the polysaccharide parts offer accurate structural control and the synthetic components often provide mechanical strength.^14,15 These biohybrid hydrogels with synergistic combination of functions have a number of fascinating characteristics including facile manipulation, improved mechanical performance, controlled swelling behavior and minimum erosion rate, thereby making them a potential candidate to be utilized in drug delivery.^12,16

Salecan (Cas. no. 1439905-58-4) is a novel soluble extracellular β-glucan obtained as a result of a bacterial fermentation process by a new strain Agrobacterium sp. ZX09. This new microorganism was isolated from soil which was collected from ocean coast of Dongying (China) by our group and its 16S rDNA sequence was deposited in the GenBank database (accession number GU810841).¹⁷ The structure of Salecan comprises of linear repeating units of glucopyranosyl connected together via β-1,3 and α-1,3-glycosidic bonds (Fig. 1).¹⁷ Similar to other β-glucans, Salecan presents attractive biological activities such as nontoxicity (edible safety) and antioxidation, and previous research had demonstrated that it could be applied in the pharmaceutical industries for treating and preventing constipation and in the food industries as a new source of thickening agent.^18–20 In addition, Salecan contains abundant functional hydroxyl groups along its backbone, thereby making it amenable to be chemically tailored in a desired fashion.⁵ Recently, various Salecan-based hydrogels were designed and successfully fabricated by our laboratory.^5,12,21,22 It was discovered that these hydrogels were appropriate materials for biomedical applications.

Fig. 1 Illustration of the preparation and stimuli-responsive drug release of Salecan/PNM semi-IPN hydrogels.

Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel is one of the most vigorously investigated gels, which exhibit an obvious volume phase transition at its lower critical solution temperature of approximately 32 °C, being hydrophobic above this temperature or hydrophilic below it.^6,22 Poly(methacrylic acid) (PMAA) gel is another class of actively studied biomaterials capable of donating or accepting protons upon pH alterations, accompanying reversible conformational changes between the extension or collapse state.^12,23 Attracted by these stimuli-responsive features, a multitude of PNIPAAm/PMAA-based gels are created for a drug delivery purpose.^2,24,25

The aim of the present study is to develop stimulus-responsive (temperature and pH) hydrogels involving Salecan that might be employed as drug carriers. These intelligent semi-IPN hydrogels were fabricated using Salecan as biopolymer, N-isopropylacrylamide and methacrylic acid as monomer, N,N′-methylenebisacrylamide as cross-linker, and ammonium persulfate/N,N,N′,N′-tetramethylethylenediamine as initiator systems. To the best of our knowledge, this is the first report to display the preparation and characterization of the Salecan/PNM semi-IPN hydrogels for controlled and sustained drug release. Their chemical structures, rheological behaviors, swelling profiles, morphologies, release properties of an anticancer drug (doxorubicin) and cytotoxicities were evaluated. The results prove that the produced semi-IPN hydrogels are hopeful devices for drug storage and delivery.

2 Experimental section

2.1 Materials

Salecan was fabricated by Center for Molecular Metabolism, Nanjing University of Science & Technology. N-Isopropylacrylamide (NIPAAm, 99%) was purchased directly from Aladdin (Shanghai, China) and purified by recrystallization in toluene/n-hexane before use. Methacrylic acid (MAA) was provided by Sigma-Aldrich (Chemie GmbH, Riedstr., Germany). N,N′-methylenebisacrylamide (MBAAm, 99%), N,N,N′,N′-tetramethylethylenediamine (TEMED, 99.5%) and ammonium persulfate (APS, 99.5%) were all obtained from Sigma-Aldrich (Shanghai, China) and used as received. Doxorubicin hydrochloride (DOX, 98%) was supplied by Dalian Meilun Biology Technology Co., Ltd (Dalian, China). Water used throughout this study was deionized water (Millipore, 18.2 MΩ).

2.2 Hydrogels synthesis

Salecan/poly(N-isopropylacrylamide-co-methacrylic acid) (Salecan/PNM) semi-IPN hydrogels were formulated at 25 °C under nitrogen atmosphere by copolymerizing NIPAAm and MAA in the presence of Salecan using MBAAm as cross-linking agents and APS/TEMED as an initiator system. Briefly, monomers composed of 0.93 g of NIPAAm and 0.3 g of MAA were dissolved in deionized water in a three-neck round bottom flask. Afterword, a certain amount of Salecan solution (2%, w/v), MBAAm (2%, w/v) and TEMED (5%, w/v) were added to the above mixture under agitation at room temperature for 30 min. Argon gas was bubbled through this solution for 15 min to eliminate the dissolved oxygen. With rapid stirring, 1 mL of APS (3.2%, w/v) was pipetted into the pre-gel solution (the total volume of the precursor solution was fixed at 15 mL with deionized water) to initiate polymerization. Subsequently, the polymerization solution was quickly poured out into a Petri dish and allowed to react at 25 °C. After 24 hours curing, the prepared hydrogels were carefully taken out from the dish, cut into small pieces and soaked in deionized water for 7 days by refreshing the water three times a day so as to remove any residual unreacted monomers as well as other impurities. A summary of the Salecan, monomer, and cross-linker are listed in Table 1.

Table 1 Compositions of initial reaction mixtures utilized for the synthesis of Salecan/PNM semi-IPN hydrogels

Ingredient	Designation
	PNM	SPNM1	SPNM2	SPNM3	SPNM4	SPNM5
Salecan (2%, w/v) (mL)	0	3	6	9	9	9
NIPAAm (99%, w/w) (g)	1	1	1	1	1	1
MAA (98%, w/v) (mL)	0.3	0.3	0.3	0.3	0.3	0.3
MBAAm (1%, w/v) (mL)	1	1	1	1	1.5	2
TEMED (5%, w/v) (mL)	1	1	1	1	1	1
APS (3.2%, w/v) (mL)	1	1	1	1	1	1
Deionized water (mL)	10.7	7.7	4.7	1.7	1.2	0.7

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3 Characterization

3.1 Stability of Salecan in semi-IPN hydrogels

To determine the content of Salecan in washing solutions of the semi-IPNs, a colorimetric phenol–sulfuric acid experiment was employed.²⁶ Typically, 2 mL of washing solution was dropped into a test tube with 1 mL of phenol solution (6%, w/w), immediately followed by the incorporation of 5 mL of concentrated sulfuric acid (98%, w/w) and then shaken well. After the mixture was cooled to room temperature, their absorbance was read at 490 nm. Salecan content in the washing solution was obtained using a standard curve as follows: y = 0.017x + 0.0183, R² = 0.9993. All assays were performed in triplicate.

3.2 Fourier-transform infrared spectroscopy (FT-IR)

FT-IR spectra in attenuated total reflectance (ATR) mode of the Salecan and hydrogels were recorded utilizing a Nicolet IS-10 spectrometer (Thermo, USA) over a wide range of 500–4000 cm⁻¹ at an average of 32 scans per sample cycle with a resolution of 4 cm⁻¹.

3.3 X-ray diffraction (XRD) measurements

XRD patterns (the samples were ground through a 200 mesh sieve) were conducted on a DMAX-2200 X-ray diffractometer operating with a Cu Kα radiation at a generator voltage of 30 kV and current of 20 mA. The region of the scanning was chosen from 5° to 60°.

3.4 Thermal gravimetric analysis (TGA)

TGA was performed on a TA Q-600 thermogravimetric apparatus at a heating rate of 10 °C min⁻¹ from 25 to 600 °C in nitrogen atmosphere.

3.5 Scanning electron microscopy (SEM)

SEM images were acquired on a JEOL JSM-6380LV electron microscope. The hydrogels were first immersed in deionized water at 25 °C to attaining equilibrium and subsequently frozen at −80 °C by dipping in liquid nitrogen. Finally, the lyophilized hydrogel disks were fractured and sputtered with gold.

3.6 Rheological properties

Rheological test of hydrogels were carried out by using an Anton Paar MCR101 rheometer implemented with a parallel plate of diameter 50 mm that was attached to a transducer. Gap in the setup for rheological measurements of the swollen hydrogels was 1.0 mm and assays were performed at 25 °C. A strain amplitude sweep was conducted to ensure that experiments were done within the linear viscoelastic regime and a strain percent of 0.01% was selected. The dynamic modulus of the synthetic gel was recorded as a function of frequency (varying from 0.1 to 10 Hz). Results were averaged on three independent runs.

3.7 Swelling behaviors

To determine the water uptake ability of the Salecan/PNM semi-IPN hydrogels, the confirmed weight of lyophilized hydrogel was immersed in various buffer solutions at given temperature and pH (the ionic strength of each buffer solution was selected at 0.1 M, which was obtained by adding an appropriate amount of NaCl). Over a specified period of time, the hydrogel was taken out, and the mass was measured after sweeping surplus water on the surface of the hydrogel. The equilibrium swelling ratio (ESR) of the hydrogels was calculated using the following expression:

ESR = W_e − W_d/W_d (1)

where, W_e and W_d are the weight of the equilibrium swollen hydrogels and the initial dried sample, respectively. For each gel sample, the assays were repeated three times, and the ultimate results were calculated by averaging the replicates.

3.8 Water retention tests

The kinetics of deswelling of the semi-IPN hydrogels was measured gravimetrically at 45 °C and 25 °C. The hydrogels were allowed to swell to equilibrium in deionized water at 25 °C, and then the swollen hydrogels were transferred into an oven at 45 °C (or 25 °C). At certain time intervals, the hydrogel was gently taken out. Thereafter, the gel sample was weighed and put into the oven again to continue the water loss process. Water retention (WR) is defined as:

WR (%) = (W_t/W_eq) × 100 (2)

in the equation, W_t and W_eq correspond to the weights of the deswelling hydrogel at time t and the fully swollen hydrogels, respectively. All measurements were performed in triplicate.

3.9 Drug loading and release

3.9.1 Drug loading. DOX was selected as a model drug to study the drug loading and controlled release behavior of semi-IPNs. The DOX-loaded hydrogels were fabricated using the swelling-diffusion method. Typically, powder of DOX was dissolved in a 0.01 M phosphate-buffered saline (PBS, pH 7.4) to acquire the DOX solution at a concentration of 50 μg mL⁻¹. Then, the preweighed dried hydrogels (80 mg) were placed in 30 mL of the drug solution under shaking for 1 day at 25 °C. After swelling equilibrium was reached, the hydrogels were gently taken out from the DOX solution and rinsed thoroughly with PBS twice to remove the superficial adsorbed drug. The remanent DOX solution and the PBS used to wash the DOX-loaded hydrogels were collected together. The amount of DOX left in the immersing solution was measured by an UV-vis spectrophotometer (TU-1900, China) at a wavelength of 481 nm.²⁷ The drug loading efficiency (DLE) was defined as:

DLE (wt, %) = (W₀ − W_e)/W₀ × 100 (3)

where, W₀ and W_e are the total weight of the DOX in soaking medium before and after loading of the gel specimens, respectively.

3.9.2 Drug release. For in vitro drug release assays, the above DOX-loaded hydrogels were immersed in 40 mL of PBS (pH = 5.0 and 7.4, I = 0.1 M). Afterward, these hydrogels were incubated at 25 °C or 37 °C with constant shaking speed at 100 rpm. At pre-determined time intervals, 2 mL of the release media was withdrawn and 2 mL of fresh buffer was replenished. The amounts of released DOX were quantified by UV-vis spectrophotometry at 481 nm and the cumulative percent drug release was obtained by formula (4):where, m_DOX indicates the amount of DOX in the hydrogel, V₀ means the volume of the release medium (V₀ = 40 mL), and C_n denotes the concentration of DOX in the nth sample. All the release experiments were performed in triplicate, and the average results were presented.

3.10 Cellular experiments

3.10.1 Cell culture. Human lung adenocarcinoma cells (A549) and hepatocellular carcinoma cells (HepG2) were obtained from Center for Molecular Metabolism (Nanjing University of Science & Technology, China). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 1% penicillin/streptomycin solution. Cells were cultured in a humidified incubator at 37 °C with 5% CO₂. The medium was changed every three days.

3.10.2 Cell viability. Cell viability of the fabricated hydrogels against three cell lines including HepG2 and A549 cells was monitored using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay by an indirect extraction method according to ISO 10993-5 standard, as depicted previously.²⁸ Prior to the cytotoxicity experiments, the drug-free hydrogels were sterilized with 70% ethanol followed by washing with sterile PBS (pH 7.4). After that, the sterile hydrogels were added into 5 mL of DMEM at 37 °C for 48 h. Next, the hydrogels were carefully removed and the obtained extract solutions were filtered through 0.22 mm syringe filters.

Meanwhile, the drug-loaded hydrogels were soaked in fresh PBS (pH 7.4) for 48 h and then gently taken out from the buffer solutions. After that, free DOX solutions and the extract liquids were sterilized by a 0.22 μm filtration and diluted with DMEM medium (the concentration of DOX in the extract liquid was identical with that of the free DOX, ultimate concentration: 0.5, 1, 2, 4 and 8 μg mL⁻¹).

Considering the MTT testing, all cells (5000 cells per well) were separately seeded in a 96-well plate and cultured overnight to allow cell attachment. Subsequently, the culture medium in each well was aspirated and replaced with 200 μL of drug-free hydrogel extracts, free DOX or drug-loaded hydrogel extract solution. 24 h later, the medium was substituted with fresh DMEM containing 20 μL of MTT, and the cells were incubated for another 4 h. Upon the removal of MTT solution, the purple formazan crystal produced by viable cells was dissolved with dimethyl sulfoxide (DMSO). The relative cell viability was measured by comparison control well containing only cell culture medium. All data were averaged over three calculations.

3.10.3 Cell uptake. For the cellular uptake experiments, A549 and HepG2 cells were seeded at density of 1 × 10⁴ cells per well in a 6-well plate and incubated for 24 h. 200 μL of the above mentioned free DOX (4 μg mL⁻¹) or the extract liquid of DOX-loaded hydrogel (4 μg mL⁻¹ equivalent DOX concentration) was then added into the cell culture medium to initiate cell uptake. After exposure to culture medium for 4 h, the cells were rinsed three times with pre-warmed PBS to eliminate the excess superficial adsorbed DOX. The fluorescent intensity was captured by fluorescence microscopy (Olympus, Japan).

4 Results and discussion

4.1 Fabrication of Salecan/PNM semi-IPN hydrogels

In this contribution, a new type of Salecan/PNM semi-IPN was prepared with Salecan, NIPAAm, MAA and the cross-linker MBAAm in radical polymerization. APS was applied as initiator, and TEMED acted as activator to accelerate production of sulfate free radicals.² The general synthesis route for Salecan/PNM hydrogel is presented in Fig. 1. Briefly, an aqueous solution of MAA and NIPAAm was first blended with an aqueous solution of Salecan in the presence of TEMED as catalyst. Subsequently, the pre-gel solution was degassed under argon bubbling at 25 °C, followed by the introduction of APS to initiate the reaction. After purification and lyophilization, Salecan/PNM semi-IPNs were acquired. This fabrication process allowed for Salecan to be easily loaded in the pure PNM hydrogel, which can uniformly diffuse and physically entangle within PNM network through hydrogen-bonding interaction between the hydroxyl groups of the Salecan and carboxyl groups of the MAA, ensuring the retention of integrity of the Salecan structure after semi-IPN formation.

4.2 Stability of incorporated Salecan in semi-IPNs

To confirm the stability of Salecan within the semi-IPNs, we determined the concentration of Salecan in the soaking solution of Salecan/PNM semi-IPNs adopting phenol–sulfuric acid method.²⁶ The associated data are displayed in Table S1 (ESI†), corroborating that the Salecan remained stable and its chains well interlocked within the PNM network.

4.3 FT-IR spectroscopy

The structures of Salecan, PNM and Salecan/PNM hydrogels were characterized by FT-IR as clearly illustrated in Fig. 2A. For the FT-IR curve of pristine Salecan, a broad peak centered at 3297 cm⁻¹ was likely due to the –OH moieties in the backbone of the polysaccharide.²¹ Main characteristic peaks of Salecan appeared in the range of 1100–800 cm⁻¹.¹² More specifically, the peak of 1040 cm⁻¹ was associated with C–OH stretching in the glucopyranose ring, a small absorption band at 894 cm⁻¹ indicated that D-glucopyranose possessed a β-configuration and a weak peak located at 813 cm⁻¹ corresponded to the existence of a little α-glucopyranose.^12,17

Fig. 2 FTIR (A), XRD (B), TGA (C) and DTG (D) curves of Salecan, semi-IPN and PNM hydrogels.

Regarding the spectrum of PNM, the PNIPAAm segment in the PNM hydrogel was identified by both the appearance of amide I peak (C=O stretching vibration) at 1640 cm⁻¹ and amide II peak (N–H bending vibration and C–N stretching vibration) at 1533 cm⁻¹.^29,30 Meanwhile, a twin band at 1387–1368 cm⁻¹ with almost equal intensity (corresponding to deformation mode of the isopropyl group of NIPAAm),³¹ was also found in the FT-IR spectra of PNM. The PMAA component in the PNM hydrogel was appraised by a prominent absorption band at 1705 cm⁻¹, which was ascribed to C=O stretching modes of MAA.³² Furthermore, the presence of cross-linker units in the PNM hydrogel was confirmed by a band at approximately 1170 cm⁻¹ arising from the C–N stretching (secondary amine) in the MBAAm structure.³³

In case of the absorption line of Salecan/PNM semi-IPN hydrogel, there was no significant discrimination between the peaks of pure PNM and Salecan/PNM, in addition to the appearance of a new peak at 1040 cm⁻¹ (attributed to C–OH stretching of Salecan) and the slightly weaker band (particularly for the peak at 1705 cm⁻¹ which was assigned to C=O stretching vibration from carboxylic groups of MAA) of the latter, resulting from the formulation of hydrogen bonding between Salecan and the PNM hydrogel matrix.²³ Similar results were obtained from other publications.^34,35 Moreover, the interaction (intermolecular and intramolecular hydrogen bonds) between the Salecan and hydrogel directed the hydroxyl peak of PNM to shift to higher wavenumber region (from 3297 to 3311 cm⁻¹) with Salecan addition. These observations provided straightforward testimony to the successful incorporation of chains of Salecan into neat PNM network.

4.4 XRD studies

To get more detailed insight into the structural information of the different samples, XRD experiments were performed on these materials. As sketched in Fig. 2B, Salecan displayed a single broad halo centred at 2θ = 21°, which was associated with the crystalline regions of the polysaccharide structure. The origin of those crystalline regions may be ascribed to the inter-chain correlations between the polysaccharide backbone and the intra-chain correlations involving the –OH side groups of Salecan.^5,22 However, for the XRD patterns of Salecan/PNM, the intensity of the characteristic peak of Salecan decreased drastically to almost nil. This suggested that Salecan was either molecularly dispersed or homogeneously distributed in an amorphous state in the PNM matrix without aggregation, leading to the destruction of the crystallization.⁵

4.5 Thermal gravimetric analysis (TGA)

Thermal degradation properties of the Salecan, neat PNM and Salecan/PNM semi-IPN hydrogels were investigated by TGA technique. As described in Fig. 2C, the Salecan exhibited a minor weight loss of about 12.5% from 30 °C to 200 °C, belonged to the evaporation of free and bound water. Subsequently, approximately 44.0% mass loss occurred between 200 and 370 °C, and further a 24.0% weight loss took place in the temperature range 400–600 °C, which was most likely due to the pyrolysis of side groups and splitting of polysaccharide rings.^5,21,36 In terms of the pristine PNM thermogram, the destruction could be divided into two stages. In the first stage, nearly 7.5% weight loss was noticed before 150 °C, corresponding to the elimination of absorbed water from the cross-linked network. The second stage commenced at a temperature of 160 °C and completed at 600 °C with a weight loss of around 78.0%. It attributed to the deformation of the polymeric chains and backbone decomposition.³⁷ Similarly, the Salecan/PNM hydrogel also underwent two steps of weight loss. An initial 7.7% of mass loss was appeared in the 30–200 °C temperature range, manifesting the removal of imbibed water. Following this period, a further decrease in weight (77.9%) was experienced. This step was ascribed to a series of sophisticated processes where fragmentation of Salecan, breakage of PNM chain and scission of hydrogel skeleton was dominant. From the TGA data, it can be concluded that the incorporation of Salecan caused notable changes in the thermal properties of the obtained hydrogels. Typically, after Salecan was introduced into hydrogel, the maximum mass loss rate presented at a higher temperature (386 °C, Fig. 2D) than that of the pure PNM (380 °C, Fig. 2D), evidencing a better thermal stability of the Salecan/PNM. Besides that, the thermal data was highly consistent with the FT-IR results discussed earlier in the paper, which implied considerable interaction of the Salecan chains with the neat hydrogel that was attributed to the entanglements and hydrogen bonding among Salecan and the PNM network.

4.6 Mechanical behaviors

Desirable mechanical properties, particularly mechanical strength and elasticity, are crucial for hydrogel materials to be employed in practical devices.³⁸ Fig. 3A and B illustrate the storage modulus (G′) and loss modulus (G′′) of the synthetic hydrogels with variable loading of Salecan and cross-linker MBAAm. The G′ (solid symbols) representing the elastic part of the hydrogel exhibits the energy storage of the active network after perturbation, whereas the G′′ (hollow symbols) corresponding to the viscous part reflects the energy loss of hydrogel matrices through dissipated heat or relaxation.³⁸ The solid-like behaviors of these hydrogels were clearly identified by the fact that the G′ and G′′ curves were linear and parallel with G′ was dominant across the entire range of frequencies investigated and further verified as the value of storage modulus G′ was always greater than that of the loss modulus G′′ in each case.^38,39 On the other hand, it was noted that their stiffness were easily manipulated by varying the Salecan and cross-linker concentration. As reported in Fig. 3A, G′ decreased from 527 Pa to 195 Pa when the Salecan concentration elevated from PNM to SPNM2. This trend was likely that the incorporation of Salecan helped bring about an enhanced swelling ratio (as will be revealed in later), which caused a decline in the mechanical strength of the gels, and in turn, a reduction in the G′. Additionally, the G′ obviously increased from 70 to 120 and to 277 Pa as the concentration of the cross-linker MBAAm increased from 1 to 1.5 and to 2 mL, respectively. An explanation was that the higher content of MBAAm induced a higher intermolecular crosslinking within the hydrogels and created a more compact network structure, thus leading to a greater stiffness.⁴⁰

Fig. 3 Frequency dependence of (A) dynamic storage modulus (G′) and (B) dynamic loss modulus (G′′) of PNM and Salecan/PNM semi-IPN hydrogels.

4.7 Swelling test

The ability of the synthesized Salecan/PNM semi-IPNs to uptake and retain water would be decisive for its later application as drug carriers.⁴¹ For that purpose, their swelling parameter was comprehensively evaluated. At first, the swelling behavior of the semi-IPNs responding to external temperature stimuli was investigated. Subsequently, pH dependence, swelling and deswelling kinetics were studied, respectively.

4.7.1 Effect of temperature on swelling behaviors. Fig. 4A depicts the typical temperature-dependent equilibrium swelling ratios (ESRs) of the Salecan/PNM hydrogels over the temperature range of 25–45 °C in deionized water (pH 7.0). Although the obtained swelling ratios for the semi-IPN hydrogels were not the same, it was observed that the results were similar for all hydrogel samples and that the dependences on the temperature followed the same trend. Markedly, all semi-IPNs were characterized by a fast dehydration that took place at nearly 32 °C. Further, the loss of absorption liquids was basically stable up to 45 °C. Taking SPNM3 as an example, ESRs were 40.0 at 25 °C, 32.6 at 32 °C, and 3.1 at 45 °C. In general, the main reason for this unique characteristic of the semi-IPNs can be assigned to the thermo-responsive component, PNIPAAm, which exhibited a lower critical solution temperature (LCST) of around 32 °C and could undergo distinctive and rapid transition from the swollen to the de-swollen states.^42,43 At temperatures below the LCST, the PNIPAAm component of the semi-IPN hydrogel was swellable and formed hydrogen bonds with water molecules. These bonds acted cooperatively to construct a stable shell of swelling around the hydrophobic groups, leading to greater water absorption and producing larger swelling ratios.⁴³ As the external temperature enhanced, the fragile hydrophilic/hydrophobic balance in the semi-IPNs was broken thus, raising the aggregation of the PNIPAAm segments and, consequently, causing the subsequent shrinkage of the hydrogel matrix.^11,43,44

Fig. 4 Swelling behavior of the Salecan/PNM hydrogels: equilibrium swelling ratio values in deionized water at 25–45 °C (A) and different buffer solutions at 25 °C (B); swelling kinetic (C) and water retention (D) curves.

4.7.2 Influence of pH on water uptake capacity. To assess the effect of pH on swelling ratios, the semi-IPN hydrogels were swollen in buffered solutions at different pH (1.2, 5.0, 7.4 and 9.18). The ionic strength of each buffer media was regulated to be 0.1 M with sodium chloride. As illustrated in Fig. 4B, all of the semi-IPNs displayed a similar trend with different extend of hydration. Overall, the ESR of the hydrogels increased consecutively from pH 1.2 to 7.4, but dropped abruptly at pH 9.18. For instance, the ESRs of SPNM2 were 7.5 for pH 1.2, 17.9 for pH 5.0, 29.3 for pH 6.86 and 26.3 for pH 9.18. This phenomenon may be related to the protonation and ionization balance of the carboxyl moieties existing in the semi-IPN hydrogel, whose pK_a value was around 5.5.^23,42,45 At pH below the pK_a of the PMAA (pH 1.2 and 5.0), most of the carboxylate groups on the PMAA units were protonated and transformed into –COOH.²³ The H-bonding among the –OH groups in Salecan, –COOH groups in PMAA and –CONH– groups in PNIPAAm restricted the swelling of the polymeric network.^5,22 Nonetheless, the carboxyl group in PMAA changed into –COO⁻ groups when the external pH was increased to 7.4 (beyond the pK_a of the PMAA). On the one hand, the electrostatic repulsion operating between the carboxylic anions of methacrylic acid was strengthened, which benefited the flexibility of the hydrogel chains.^44,46 On the other hand, the aforementioned hydrogen bonds were dissociated and overwhelmed by the hydrophobic interactions, yielding a higher swelling ratio.⁴⁷ With further augmenting the pH to 9.18, the surplus counter ions (such as potassium ion) in the incubation media shielded the charged carboxyl groups, thereby weakening the electrostatic repulsion force and the swelling ability.

4.7.3 Dynamic swelling properties. The pH- and temperature-dependence of the swelling ratio only described the equilibrium hydration states of semi-IPN hydrogels at different pH and temperatures. Actually, the dynamic swelling properties of the hydrogels were more important in drug delivery applications.⁴⁸ The dynamic swelling behaviors were determined in various temperatures and pHs (ESI†). Taking the semi-IPN hydrogels in pH 7.4 PBS at 25 °C as an example (Fig. 4C), on the whole, the swelling ratio values positively correlated with the incubation time of all gels which revealed a similar absorption profile during the initial 4 h of cultivation. After 10 h of immersion, the semi-IPNs achieved water saturation, and hydration induced a maximum mass increment of 22.9, 29.3, 39.9, 33.7 and 26.2 with respect to the SPNM1, SPNM2, SPNM3, SPNM4 and SPNM5, respectively. Interestingly, it can be concluded from these data that the hydration degree of the semi-IPNs was affected apparently by the concentration of Salecan and cross-linker MBAAm. As vividly exhibited in Fig. 4C, an enhancement in the Salecan dose from 3 to 9 mL brought about an increase in ESR from 22.9 to 39.9. The incorporation of Salecan improved remarkably the affinity of the semi-IPN hydrogels for water, due to the appearance of hydroxyl groups on the Salecan skeleton, facilitating the penetration of water molecules into the hydrogel matrix and, ultimately, elevating its water absorbability.^44,49 Meanwhile, the value of ESR corresponded to the MBAAm content in the hydrogel network. Namely, the level of ESR changed from 39.9 for the hydrogels crosslinked with 1 mL of MBAAm, to 26.2 for those hydrogels prepared with 2 mL of MBAAm. One reasonable explanation was that increasing the number of cross-linker MBAAm accelerated the formation of covalent bonding in the gel structure, hindering the mobility of the hydrogel chains.^50,51 Accordingly, the hydrogel enhanced its capability to take up water.

4.7.4 Water retention tests. In the next stage, deswelling kinetics research of the semi-IPN hydrogels was conducted in deionized water at 45 °C (Fig. 4D) and 25 °C (ESI†). As evidenced in Fig. 4D, the water retention capacity of Salecan/PNM hydrogels decreased with Salecan dosage. The water retention of the SPNM1 with 3 mL of Salecan was 80% at time 90 min; however, this value reached 90% when 6 mL of Salecan was added and was 96% with the incorporation of 9 mL of Salecan. An explanation was that the introduction of the hydrophilic Salecan chains disturbed the formation of dense layer on the surface of the gels and that the hydrophilic Salecan segments could acted as water-releasing channels when the collapse happened, facilitating the expulsion of water.^5,50 Additionally, the cross-link density also influenced the water retention ability of the hydrogels. The semi-IPNs containing more cross-linker MBAAm preserved more water and manifested a slower dehydration rate within equal time intervals. Compared to sample SPNM5, the gels SPNM3 and SPNM4 displayed higher deswelling kinetics. SPNM5 released over 80% of its water content after 90 min whereas, during the identical time period, the semi-IPN hydrogels, SPNM4 and SPNM3, lost about 86% and 91% of their water content, respectively. Such improvement of water-retention might be ascribed to the fact that higher content of cross-linker produced a dense network, which in turn reduced the conformational freedom of polymer chains, limiting the outflow of water from the hydrogel.^12,50

4.8 Morphology of semi-IPN hydrogels

The microstructure of hydrogels directly affects their ultimate properties as drug delivery vehicles, whereas the kinetics will be influenced by the surface area and pore density of the hydrogel.⁴⁸ Accordingly, the hydrogel structure was analyzed by SEM. Prior to the imaging; each formulation was submerged in deionized water to reach equilibrium, shock-frozen with liquid nitrogen, and then lyophilized. This procedure had minimal effect on the architecture of the hydrogel.^12,13 As reported in Fig. 5, all hydrogels displayed an interconnected porous structure with homogeneously distributed pores, resembling other freeze-dried hydrogel systems.^2,6 The incorporation of Salecan led to a relatively loose network, with significantly bigger pores ranging from 28.3 ± 7.5 to 78.2 ± 15.6 μm for PNM and SPNM2, respectively. The reason was that, as the amount of Salecan increased, the water content of semi-IPNs was enhanced (as verified in Section 4.7), which was prone to the emergence of a larger ice crystal during the cryogenic freezing process and concomitant greater pores.^5,52 It should also be noted that the sample with higher MBAAm concentration possessed much smaller internal holes, compared with that of lower MBAAm content. In particular, the pore size of the hydrogel reduced in the order of SPNM3 (111.4 ± 25.7 μm), SPNM4 (94.7 ± 19.1 μm), and SPNM5 (53.3 ± 11.5 μm) with the increased cross-links MBAAm applied during gel synthesis. The cause may be that addition of MBAAm enhanced the cross-linking density of the hydrogel network, curtailing the free space available for reservation of water. As a result, water molecules were hardly diffused into the hydrogel matrix. Therefore, smaller pores were created in response to the formation of smaller ice crystal during the lyophilization process.^2,53

Fig. 5 SEM images of the prepared hydrogels: (A) PNM, (B) SPNM1, (C) SPNM2, (D) SPNM3, (E) SPNM4, (F) SPNM5. Scale bars represent 100 μm.

4.9 Loading and in vitro release of DOX

4.9.1 Drug loading. In view of the foregoing discussions, the extended investigation was necessary to estimate whether the elaborated pH-thermo dual responsive semi-IPN hydrogel had a regulation effect on drug release. Here, doxorubicin hydrochloride (DOX), one of the most widely used potent chemotherapeutic agent in the treatment of various types of tumors via intercalating with DNA,^54,55 was chosen as a model drug and incorporated into polymeric networks by a swelling-diffusion approach. Relying on eqn (4), a satisfied drug entrapped efficiency of 82.3% (SPNM3) was achieved (Table S2, ESI†) thanks to the electrostatic interactions between the negatively charged carboxylic acid groups on PMAA units and positively charged DOX molecules,⁵⁶ as well as the interconnected porous structure of the designed hydrogels.^57,58

4.9.2 Triggered release of DOX. The stimuli-responsive semi-IPN hydrogels provide an opportunity to fine-manipulate the release properties of DOX. Fig. 6A presents the cumulative release profiles of DOX-loaded gel specimen (SPNM3) in pH 7.4 PBS at predetermined temperatures (25 °C and 37 °C). As expected, the release of DOX from DOX-loaded semi-IPN hydrogel was temperature-dependent, and the release rate at 37 °C was much quick than that at 25 °C. At 25 °C, the extent of drug release was suppressed and only 22.2% of DOX could be released when incubated for 4 h. While at 37 °C, the DOX release from semi-IPNs was distinctly boosted. The accumulated release amount of DOX was more than 58.3% within the same period. The rapid escape of encapsulated drug from the semi-IPN at elevated temperatures was also found in other hydrogel vesicles containing PNIPAAM segments.^11,59 This behavior was probably ascribed to the hydrophilic-to-hydrophobic conversion of the PNIPAAM components above the LCST (37 °C), which squeezed the semi-IPN hydrogels, leading to shrinkage of the networks along with the expulsion of DOX.⁶⁰ Moreover, the hydrogen bonds between the semi-IPN hydrogels and drug molecule DOX were weakened at 37 °C, benefiting for the diffusion of entrapped DOX from the interior of the hydrogel to the release medium.¹¹

Fig. 6 In vitro DOX release profiles from the semi-IPN sample (A) at temperature 25 °C and 37 °C and (B) at the two different pH values of 5.0 and 7.4; (C) SPNM1-SPM5 hydrogels in pH 5.0 buffers. (D) UV-vis spectrum of free and released DOX.

Next, cumulative release profiles were investigated under different PBS buffers of pH 5.0 and 7.4, which mimicked the environments of tumor tissues and healthy tissues, respectively. As depicted in Fig. 6B, cumulative drug release from the DOX-loaded SPNM3 hydrogel was increased in response to changes in the pH value of the culture medium from 7.4 to 5.0. Over the course of 21 h, 74.8% of DOX were released at pH 5.0, whereas only 27.5% of DOX were released at pH 7.4. This distinctive pH-responsive behavior could be explained by the following two factors. (1) It is well known that DOX is a weak base, whose pK_a value is approximately 8.2.⁶¹ When the pH of the incubation medium was reduced to 5.0, the hydrophilicity of DOX enhanced due to the protonation of NH₂ on DOX, thereby increasing the solubility of DOX in buffer solutions and as a result, facilitating the escape of DOX.^5,12 (2) In an acidic environment, the deionization of PMAA chains broke up the electrostatic interactions between the –NH³⁺ of DOX and –COO⁻ groups of MAA,⁶² which induced the increased electrostatic forces, enabling a fast drug release. The pH-sensitive release behavior of the semi-IPN hydrogel would be desirable for cancer treatment, since it can maintain a low DOX leakage in the normal physiological conditions and ensure accelerated release stimulated by the weakly acidic pH in the lysosomes (pH 4–5) and endosomes (pH 5–6) of cancer cells.

Furthermore, we explored the release kinetics of DOX from five different hydrogel formulations synthesized containing diverse concentrations of Salecan and using various cross-linker contents at room temperature in PBS buffer with a pH of 5.0. In all cases, the DOX release was profoundly augmented by either the incorporation of Salecan or lessening the cross-linker MBAAm. In detail, less than 43.0% DOX was released from SPNM1 in 21 hours when the Salecan content was 3 mL. Nonetheless, nearly 53.9% DOX was released when the SPNM2 containing 6 mL of Salecan, and there was more than 74.8% DOX release when the Salecan content reached 9 mL. Specimens consisting of more hydrophilic Salecan were thought to produce higher swelling of hydrogels (as confirmed in Section 4.7), which would enhanced the driving forces for the free diffusion of DOX molecules, thereby being responsible for the increment of DOX release.^5,11 Also, it was found from Fig. 6C that the lower DOX release was accompanied by a higher feeding ratio of cross-linker in the fabricated hydrogel. Specially, within 12 h, SPNM3, SPMN4, and SPNM5 shrank and expelled approximately 73.8%, 60.3%, and 49.4% DOX, respectively. This phenomenon could be understood by considering the higher extent of gelation and the formation of denser structures in the case of a greater MBAAm amount, which hampered the escape of encapsulated drugs from the semi-IPN hydrogel. These results are consistent with the findings of Nagahama et al.⁶³ On this basis, the release rate of the DOX can be tuned by simply adjusting the Salecan and crosslink contents in the hydrogel compositions.

Altogether, the temperature- and pH-dependent DOX release rendered the semi-IPN hydrogels with an attractive platform in terms of controlling drug release rate. That is to say, the drug is adequately stable in the designed hydrogel vehicles with minimal premature drug loss during the blood circulation, whereas a rapid release of drug can be triggered by the tumor microenvironment with slightly acidic pH and body temperature.⁶⁴

4.9.3 Activity of released DOX from semi-IPN hydrogels. It is important that the designed DOX release vehicles not only deliver the DOX at an on-demand manner but also maintain the activity of the DOX. Drug denaturation may take place during the preparation, storage and release periods.⁶⁵ Fig. 6D displays the UV-vis absorption spectra of pristine and released DOX. It can be noticed that the maximum absorption wavelength remained unchanged, and no new peaks appeared. Consequently, the DOX was liberated in its original form, as well as no detectable impurities were produced by this system, implying that our strategy to incorporate DOX was effective and DOX was not damaged during the encapsulation and release periods.^27,65

4.10 In vitro cytotoxicity

A further study on the activity of released DOX was evaluated whether the released DOX could effectively kill cancer cells. Here, the inhibition effects of blank hydrogels (drug-free), drug-loaded hydrogels and free DOX on the growth of HepG2 and A549 cell lines were quantitatively investigated via the MTT assay. It was indicated in Fig. 7A that the leachates from empty semi-IPN hydrogels possessed negligible cytotoxicity, as the eventual average viabilities of the both cells exceeded 90% at all tested concentrations of hydrogel extracts. This observation demonstrated the biocompatibility of the drug delivery devices themselves.⁶⁶ Contrarily, pure DOX and DOX-loaded hydrogels induced significant cytotoxicity to cells. As presented in Fig. 7B, a dose-dependent cytotoxicity was found for either DOX-loaded hydrogels or free DOX. When the DOX concentration was raised, cell viability was reduced simultaneously. For instance, DOX-loaded hydrogels can kill about 51.4%, 66.6% and 84.6% of HepG2 cells and 51.8%, 72.2% and 84.5% of A549 cells at a concentration of 2, 4 and 8 μg mL⁻¹, respectively. This result also proved that DOX released from the semi-IPNs remained biologically active. Besides that, free DOX was more cytotoxic than DOX-loaded hydrogels. At an equivalent DOX concentration, DOX-loaded hydrogels exhibited a slightly lower inhibition of the cell proliferation as compared to pure DOX in view of the slow but sustained release of the payload drug from the hydrogels.⁶⁷

Fig. 7 Cell viability of HepG2 and A549 cells after treatment with blank hydrogel extracts (A) and free DOX and DOX-loaded hydrogel extracts solutions (B). (C) Fluorescent microscopy photos of HepG2 and A549 cells after 4 h incubation with 4 μg mL⁻¹ the extract liquid of DOX-loaded hydrogel and free DOX solutions.

4.11 In vitro cell uptake

Facilitated by the inherent red fluorescence of DOX, the imaging tracing of cell uptake was monitored via fluorescence microscopy.^68,69 Fluorescent photos were acquired 4 h post-incubation of HepG2 and A549 cells with either free DOX or the extract liquid of DOX-loaded semi-IPN hydrogels (4 μg mL⁻¹). Fig. 7C summarizes the DOX uptake results. Significantly, a bright red fluorescence was observed and distributed uniformly in both HepG2 and A549 cells, demonstrating that the released DOX had been taken up by these two cancer cells successfully.⁶⁰ The cell uptake results, coupled with the aforementioned cell viability and DOX release data, indicated that the created hydrogel is envisioned as a potential candidate for controlled drug delivery.

5 Conclusions

Collectively, a new class of semi-IPN hydrogels comprised of Salecan and P(NIPAAm-co-MAA) was designed and formulated via free radical polymerization. The resulting semi-IPN hydrogels were thoroughly characterized and employed as drug reservoirs to investigate DOX release profiles for cancer therapy. Their chemical architecture and molecular characteristic were visualized by UV-vis, FT-IR, XRD and TGA analysis, which proved the successful incorporation of Salecan into the PNM matrix. It was found that the physicochemical properties of the created hydrogels, such as mechanical strength, swelling behavior, morphology and drug release kinetic, could be precisely tailored by varying the amount of Salecan and MBAAm in the hydrogel formulation. Besides that, the in vitro release of the entrapped drug DOX from the semi-IPN hydrogels was also controllable by adjusting the temperature and pH of the release medium. Namely, DOX release was suppressed in neutral conditions mimicking normal tissue, while it was markedly enhanced under acidic environment at body temperature typically appeared at tumor cites. Moreover, cell assays identified that all empty hydrogels were non-toxic to HepG2 and A549 cells, whereas DOX released from the semi-IPNs could still exert its pharmacological activity and had the ability to induce cell death. All these results hint that the developed semi-IPN hydrogels are suitable carriers for the drug storage/release.

Acknowledgements

This work was supported by National Natural Science Foundation of China under the Grant 51573078 and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Footnote

† Electronic supplementary information (ESI) available: Drug loading efficiency of the Salecan/PNM hydrogels and the concentration of Salecan in washing medium and hydrogel. See DOI: 10.1039/c6ra10716h

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