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Nanogel-integrated pH Responsive Composite Hydrogels for Controlled Drug Delivery Gunce E. Cinay, Pelin Erkoc, Mohammad Alipour, Yoshihide Hashimoto, Yoshihiro Sasaki, Kazunari Akiyoshi, and Seda Kizilel ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00670 • Publication Date (Web): 16 Jan 2017 Downloaded from http://pubs.acs.org on January 17, 2017

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ACS Biomaterials Science & Engineering

Nanogel-integrated pH-Responsive Composite Hydrogels for Controlled Drug Delivery

Gunce. E. Cinay, Pelin Erkoc, Mohammad Alipour, Yoshihide Hashimoto, Yoshihiro Sasaki, Kazunari Akiyoshi, Seda Kizilel* ––––––––– G.E. Cinay, M. Alipour, Assoc. Prof. S. Kizilel Chemical and Biological Engineering, College of Engineering, Koc University, Sariyer, Istanbul, 34450, Turkey P. Erkoc, Assoc. Prof. S. Kizilel Biomedical Science and Engineering, Koc University, Sariyer, Istanbul, 34450, Turkey Dr. Y. Hashimoto, Assoc Prof. Y. Sasaki, Prof. K. Akiyoshi Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan E-mail: [email protected]

Abstract A novel pH-sensitive hydrogel system consisting of poly(methacrylic acid-g-ethylene glycol) (P(MAA-g-EG)) and acryloyl group modified-cholesterol-bearing pullulan (CHPOA) nanogels was developed for the controlled delivery of an anticonvulsant drug, pregabalin (PGB). Here, the hydrophilic hydrogel network provides the pH-sensitive swelling behavior, whereas nanogel components form separate reservoirs for the delivery of drugs with different hydrophobicities. These nanocarrier-integrated hybrid gels were synthesized through both surface-initiated and bulk photopolymerization approaches. The swelling and drug release behavior of these pH-responsive hydrogels synthesized by different photopolymerization approaches at visible and UV light wavelenghts were studied at acidic and basic pH values. Nanogel-integrated hydrogels exhibited higher swelling behaviour compared to plain hydrogels in reversible swelling experiments. Similarly, the presence of nanogels in hydrogel network enhanced the loading and release percentages of PGB and the release was analyzed to describe the mode of transport through the network. In vitro cytotoxicity assay suggests that hydrogels in altered groups are nontoxic. This is the first report about the visible lightinduced synthesis of a pH-responsive network incorporated CHPOA nanogels. Responsive 1 - Environment ACS Paragon -Plus


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and multifunctional properties of this system could be used for pH-triggered release of therapeutic molecules for clinical applications. Keywords: CHPOA, nanogel, pH sensitive, hybrid hydrogel, drug delivery, pregabalin

1. Introduction One of the foremost needs in drug delivery is the development of novel technologies for controlled release of therapeutic molecules. Significant challenges exist for targeted and controlled delivery of easily degradable molecules and small molecular weight drugs through responsive systems.1 Drug delivery via oral route remains the most preferred site for delivery as opposed to intravenous and subcutaneous injections, and has patient compliance and lower production cost.2 Peptide and protein drugs have high therapeutic efficacy with high activity, specificity and low toxicity. However, large molecular weight peptides and proteins have poor oral bioavailability due to their enzymatic degradation in gastrointestinal (GI) tract and low absorption through intestinal membrane.3 Development of a stimuli-responsive drug delivery system is promising for controlled release of therapeutic molecules with determined physicochemical properties, such as hydrophobicity, molecular weight, and pH stability at certain environmental pH or proteolytic enzyme concentration.4-5 Hydrogels are threedimensional and cross-linked polymeric networks, and are especially attractive due to their high water content and biocompatibility.6 Their application extends from drug delivery to diagnostics and tissue engineering.7-9 In particular, stimuli-responsive hydrogel networks can alter their physicochemical properties depending on changes in environmental temperature, light, electric field, ionic strength and pH.10-17 Notably, pH-responsive hydrogels has shown great promise for the delivery of antitumor drugs into a tumor site, utilizing the pH difference between healthy and acidic tumor tissue environment, and for oral drug administration, where drastic pH alteration occurs through GI tract.8-9, 18-19

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pH-responsive hydrogels possess pendant acidic (-COOH), amino (R-NH2 or R2-NH) or other functional groups within the polymer network that ionize at a pH above the pKa of the polymeric network.20,21 Due to the electrostatic repulsion in the presence of ionized groups, chain extension in hydrogel structure occurs, followed by increased water uptake and swelling.22 P(MAA-g-EG) hydrogels consisting of methacrylic acid (MAA) backbones with poly(ethylene glycol) (PEG) tethers have been investigated for oral drug delivery applications in previous studies.23,24 In acidic environments, ionizable carboxyl groups on MAA form hydrogen bonds with etheric oxygens on PEG chains. At pH values above pKa of 4.8, carboxyl groups are deprotonated, and hydrogel network expands by electrostatic repulsion.22 The drastic pH alterations along GI tract have been utilized for the oral delivery of proteins with a broad molecular weight range (3.4-57 kDa) through P(MAA-g-EG) network.23, 25 Nanoparticles have been easily incorporated into polymer structures and provide unique physical and chemical properties to the existing polymers.26 In the last few decades, the significant developments in nanoparticle field lead to the discovery of nano-polymers with different hydrophobicities and allowed researchers to develop amphiphilic hybrid polymers with desirable properties, for various biomedical applications.27-28 Previously, Akiyoshi et al. reported the synthesis of physically cross-linked cholesterolbearing pullulan (CHP) nanogels with 30 nm diameter size, where cholesteryl and pullulan groups contributed to hydrophobicity and hydrophilicity, respectively.29 CHP nanogels have various advantageous properties, such as their ability to entrap bioactive molecules including DNA, RNA, and proteins, preserve their stability by molecular chaperone-like function, and release proteins in the native form.30-31 Sekine et al. further engineered CHP nanogels to obtain polymerizable acryloyl group modified-cholesterol-bearing pullulan (CHPOA) nanogels.29 CHP nanogels and the derivatives have been tested in clinical studies and were proposed as candidate carriers for vaccine formulations and cancer therapeutics.32 To overcome the problems associated with low drug loading capacity and poor physicochemical 3 - Environment ACS Paragon -Plus


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stability, CHP nanogels can be combined with other hydrophilic delivery vehicles, such as hydrogels, and chemically crosslinked with PEG derivatives.29, 33-34 The functionalization of a stimuli-responsive hydrogel network with hydrophobic nano-sized domains would lead to the development of a hybrid system which has potential to provide sustained and site-specific multiple drug delivery with altered hydrophobicities. In our previous study, we have developed a pH-sensitive hybrid hydrogel system consisting of a hydrophilic P(MAA-g-EG) hydrogel network and hydrophobic styrene-butadiene-styrene (SBS) structures based on visible light-induced photopolymerization.24 Here, we improved the pH-responsive hydrogels further to achieve a nanostructured network through CHPOA nanogel integration. Hybrid hydrogels were synthesized through both surface-initiated and bulk photopolymerization mechanisms for controlled crosslink density. Surface-initiated polymerization approach used here can serve as a control parameter on the microstructure of hydrogels, by altering the number of pores and pore sizes. These alterations can result in different swelling and drug release profiles, which may be useful for specific needs in clinical applications. As we compare bulk and surface-initiated polymerization approaches here, utilization of surface-initiated polymerization approach for hydrogel synthesis might be advantageous for the development of complex and biocompatible delivery systems. Due to low energy requirements of visible light photopolymerization and clinically acceptable curing times, utilizing both surface-initiated mechanism and visible light for photopolymerization for the synthesis of hydrogels can present a promising approach for controlled drug delivery.35

2. Experimental Section 2.1. Materials Poly (ethylene glycol) monomethyl ether monomethacrylate (PEGMMA; 1000 g/mole), methacrylic acid (MAA) and tetraethylene glycol dimethacrylate (TEGDMA) were received from Polysciences Inc. (Warrington, PA). Eosin Y, L-glutamine (200 mM), penicillin4 - Environment ACS Paragon -Plus

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streptomycin, (10,000-unit penicillin and 10 mg streptomycin/mL), trypsin-EDTA (25 %), potassium phosphate and ethanol (99.8 %) were obtained from Sigma-Aldrich. Triethanolamine (TEA) and sodium chloride were obtained from Merck (Darmstadt, Germany) and 3,3-dimethylglutaric acid (DMGA) was from Fisher Scientific (New Jersey, USA). Dulbecco Modified Eagle’s Medium (DMEM), 10 % fetal bovine serum (FBS, heat inactivated), and phosphate-buffered saline (PBS) tablets were purchased from Life Technologies (Paisley, UK), Biological Industry, and Amresco (Solon, Ohio), respectively. Hydrochloric acid (HCl, 37 % (wt)) and sodium hydroxide (NaOH) were obtained from Riedel-de Haen. Powder form Pregabalin was kindly supplied by Abdi Đbrahim (Pharmaceutical Company, Istanbul, Turkey). All chemicals were used as received except for MAA, which was passed through a hydroquinone remover column (Sigma-Aldrich) prior to use. Deionized water was used in all studies. 2.2. Preparation of Buffers The preparation of artificial gastric fluid (AGF, pH 2.0) and artificial intestinal fluid (AIF, pH 7.4) were done as follows. Hydrochloric acid (7 mL, 37 % (wt)) and 2.0 g of sodium chloride were dissolved in 1000 mL of deionized water for the preparation of AGF. To prepare AIF, potassium dihydrogen phosphate (6.8 g) was dissolved in 500 mL of deionized water, and then pH was adjusted to 6.8. The solution was then diluted to 1000 mL by the addition of deionized water. Necessary pH adjustments were performed before use. 0.1 M DMGA buffer solution prepared in distilled water was utilized in drug release experiments, as was consistent with the previous study of our group.24 In order to maintain the ionic strength of the buffer, 0.1 M sodium chloride was added and the solution was kept at room temperature at dark. pH 2.0 and pH 7.4 DMGA buffers were prepared before use. 2.3. Synthesis of Plain and Nanogel-integrated Hydrogels CHPOA nanogels were prepared as was detailed previously.29 The method used to prepare hybrid pH-responsive hydrogels is shown in Figure 1 and Figure 2. 5 - Environment ACS Paragon -Plus


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Figure 1. Schematics of (A) the components and parallel crosslinked chemical structure of P(MAA-g-EG) hydrogel, (B) criss-crossed CHPOA nanogel structure and CHPOA nanogels in pH-responsive hydrogel, and (C) chemical structure of pregabalin (PGB). 6 - Environment ACS Paragon -Plus

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Figure 2. Schematics for the preparation of (a) bulk and (b) surface-initiated polymerization of hybrid pH-responsive P(MAA-g-EG) hydrogels through UV and visible light photopolymerization. Briefly, monomers MAA (3.6 g, 0.033 mol) and PEGMMA (2.0 g, 0.020 mol) were first dissolved in 1:1 (w/w) ethanol and water solution. Cross-linker TEGDMA was added in the amount of 0.5 % mol of the total monomer (Figure 1). For bulk synthesis of P(MAA-g-EG) hydrogels 225 mM TEA and 0.030 mM eosin Y were added into the prepolymer solution (Figure 1 and 2). For surface-initiated synthesis of hydrogels, 225 mM TEA was added into the prepolymer solution, where 1 mM eosin Y solution, which is prepared in distilled water, was applied to the surface of the molds in which the polymer solution will be poured into, and the molds were air dried at dark. Same prepolymer solutions were used for both UV and visible light-induced synthesis of hydrogels. Separately, 10 mg/mL CHPOA nanogel solution was prepared in 1X PBS and added to the prepolymer mixture at different final concentrations (0, 1 and 5 % (v/v)). Next, 50 µL of plain prepolymer solution and prepolymer solutions with altered nanogel ratios were placed into the molds separately and exposed to either UV light 7 - Environment ACS Paragon -Plus


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(300 nm, 44 mW) or green light (argon ion laser, Coherent Inc., Santa Clara, CA, 514 nm, 7.5 mW/cm2) for 20 min. All resulting hydrogels were rinsed in distilled water at room temperature to remove unreacted chemical residues and excess eosin Y. Finally, hydrogels were freeze-dried (Labconco, FreeZone 2.5 L Benchtop Freeze Dry System) and dry weights were recorded. Plain hydrogels and hybrid hydrogels integrated with 1 or 5 % (v/v) CHPOA nanogels were identified as (B-0, B-1, and B-5) and (SI-0, SI-1, and SI-5) for bulk and surface-initiated photopolymerization, respectively. 2.4. Gel Fraction Hydrogel samples were placed in eppendorf tubes and dried until constant weight was measured at 60 °C. Then, sol was extracted in 1:1 (w/w) ethanol and water solution at 60 °C for 1 day and washed in distilled water for three times. The remaining gel was dried to constant weight at 60 °C. Gel fraction was determined gravimetrically and by using Equation (1), 36-37

% = ⁄ × 100

(1)

where Wo is the initial weight of dry gel and Wd is the weight of dry gel after extraction. 2.5. Surface Characterization by Field Emission Scanning Electron Microscopy (FESEM) FESEM images were used to investigate the final microstructure and uniformity of pore size for the gels that were synthesized with UV or visible light. For FESEM imaging, all samples were dried under vacuum and fixed on carbon tape attached specimen stubs. A thin layer of gold (10 nm thickness) was coated on samples using a Cressington Sputter Coater (108 Auto with Cressington Thickness Monitor MTM-10). All samples were observed with Zeiss Ultra Plus field emission scanning electron microscope. Next, images were analyzed using an automatic image capture software. To capture the pore size differences in the bottom and top sections of the hydrogels synthesized via surface-initiated polymerization, FESEM images were taken from the bottom and top part of the side view. 8 - Environment ACS Paragon -Plus

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2.6. Characterization of Swelling Behavior of Plain and Nanogel-integrated Hydrogels Reversible swelling experiments were carried out to examine the response of plain and nanogel-integrating P(MAA-g-EG) hydrogels to cyclic pH changes and understand the mechanical stability hydrogels. Reversible swelling/deswelling behavior was investigated by immersing the cylindrical gels at different pH solutions (pH 2.0 (AGF) to mimic the stomach environment, and pH 7.4 (AIF) to mimic the intestine environment) at 25 oC.38 In every 20 min throughout the experiment, hydrogels were taken out from the solutions and blotted with tissue paper to remove excess water at the gel surface, and wet weight of swollen hydrogels were measured. First, hydrogels were placed in pH 2.0 (AGF) solution. Swelling data in acidic condition was recorded for 120 min, next hydrogels were transferred into pH 7.4 (AIF) solution, and wet weight of hydrogels were recorded for 100 min. Next, hydrogels were transferred into pH 2.0 and pH 7.4 solutions in turn, and weight measurements were recorded for 240-340 min and 360-460 min, respectively. Equilibrium weight swelling behavior was calculated using equation (2) as follows: 20

q=

Ws Wd

(2)

where q is the equilibrium weight swelling ratio, Ws is the weight of swollen hydrogel, and Wd is initial dry weight of the hydrogel. The minimum and maximum values of the equilibrium weight swelling ratio obtained from each experiment group used to create bar graphs shown in Figure 7. 2.7. Drug Loading and Release Studies PGB was used as a model drug for release studies. The solubility of PGB at room temperature, in aqueous media is greater than 30 mg/mL within pH range of 1-13. PGB is classified as highly soluble and highly permeable under the Biopharmaceutical Classification System, and the drug product is shown to dissolve almost completely.39 In our system, hydrogels were incubated in 20 mL PGB

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solution (0.25 mg/mL) at pH 7.4 1X PBS for 16 hours at 25 oC with constant shaking.38 Next, PGB loaded hydrogels were collapsed by adding 2 µL of 6 N HCl, bringing pH of the solution to pH 2.0, to entrap PGB within the network. Then, hydrogels were rinsed with 20 mL distilled water once to eliminate any drug adsorbed on the surface. The absorbance of the remaining solution was measured by UV-Vis spectrophotometer (Shimadzu UV-3600 – UVVIS-NIR Spectrophotometer) at 210 nm to determine the amount of PGB loaded into hydrogels.30 Standard calibration curve for the absorption of PGB in aqueous solution was prepared to determine PGB concentration in unknown samples. PGB release studies were performed in 20 mL 0.1 M DMGA buffer at pH 2.2 and pH 7.4, for 8 hours at 37 ˚C. PGB loaded hydrogels were placed in pH 2.2 DMGA buffer for the first 2 h and then hydrogels were transferred into pH 7.4 DMGA buffer for the following 6 h. Samples (800 µL) were taken every 30 minutes and replaced with the same amount of fresh 0.1 M DMGA buffer at the same pH to sustain sink conditions. PGB concentration at each time point was calculated utilizing respective absorbance values and used to calculate the mass released at time t (Mt) using the following equation:40

M t = C t * V + ∑ C t −1 * V s

(3)

where Ct is PGB concentration at time t, V is the total volume of release solution (20 mL) and

Vs is the sample volume (800 µL). Using the Mt values, the % release of PGB was determined with the following equation:41

M  % Mass Re lease =  t  x100  M∞ 

(4)

where M∞ is the total weight of PGB released during the experiment. Pregabalin release behavior was modeled using the following power-law correlation:42

Mt = kt n M∞

(5)

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where k is the proportionality constant and n is the diffusional exponent and describes the mode of drug transport from the network. Cumulative release data was fitted to a sigmoidal curve obtained from Hill equation. ‘Sigmoid Emax model’ was modified as can be seen bellow:43

∝

=

!

∝

(6)

" ∝

R is the predicted drug release percent, Rmax is the maximum release, C is the drug concentration at time t, T50 is the time for which 50% of maximum release is obtained and α is the Hill coefficient of sigmoidicity. 2.8. In vitro Cell Viability Test Cytotoxicity of hybrid hydrogels was evaluated by CellTiter-Glo Luminescent Cell Viability Assay (Promega). Human BJ fibroblast cells (Passage #: 8-12) were cultured with DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% Penicillin/Streptomycin in an incubator with 5% CO2 at 37 oC. The plain and CHPOA-integrated P(MAA-g-EG) hydrogels were immersed in culture medium for 24 hours at 37 °C before cell seeding. Hydrogel samples were placed in 24-well plate and fibroblasts were seeded onto the gel surface (12000 cells/0.2 cm2). After 15 minutes, 1 mL culture medium was added to each well and placed into the incubator. Prior to measurement, ATP standard curve was prepared in cell culture medium. Next, gels were transferred into 1:1 (v/v) mixture of Cell Titer-Glo reagent and cell culture medium. Both samples and ATP solutions were incubated at 25 oC, 100 rpm for 15 min. Luminescence was measured using a plate reader (Biotek, Synergy H1).

3. Results and Discussion 3.1. Material Synthesis and Characterization The plain and hybrid P(MAA-g-EG) hydrogels with different CHPOA nanogel ratios were prepared via visible light-induced photopolymerization (Figure 2). Hybrid hydrogel systems synthesized with bulk polymerization included photoinitiator eosin Y in the prepolymer. Also,



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CHPOA nanogels were suspended in the prepolymer solution of hybrid hydrogels, where altered ratios of CHPOA nanogels (1, 5 and 10% (v/v)) were used. For the synthesis of hydrogels via surface-initiated photopolymerization, eosin Y was physically adsorbed on polystyrene substrate surfaces. Visual examination of visible-light induced hydrogels in altered groups demonstrated that gel morphology was influenced both by the presence of nanogels and polymerization mechanism. Disk-shaped hydrogels in all groups remained opaque at pH 2.0, while they appeared soft and transparent at pH 7.4 (Figure 3).

Figure 3. Visible light photopolymerized pH-responsive P(MAA-g-EG) hydrogels in collapsed state (pH 2.0) and in swollen state (pH 7.4). Plain, 1 % and 5 % CHPOA nanogelintegrated hydrogels synthesized via (a) bulk polymerization and (b) surface-initiated polymerization. The distance between digits is 1 cm. Incorporation of nanogels into the network did not compromise swelling behavior and pHresponsive properties, and that hydrogels remained in collapsed state at low pH, and retained swollen structure at high pH medium. Also, the sizes of the hydrogels prepared with 5 % nanogel concentration at swollen state were larger compared to gels prepared with 1 % nanogels. Surface morphology of plain and hybrid hydrogels were characterized by Field Emission Scanning Electron Microscope (FESEM). Surface morphology of plain, 1% and 5% CHPOA


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nanogel-incorporated hydrogels synthesized via bulk and surface-initiated polymerization methods were demonstrated in Figure 4.

Figure 4. FESEM images of plain and CHPOA nanogel-integrated P(MAA-g-EG) hydrogels obtained via bulk or surface-initiated visible light photopolymerization (Scale bar: 10 µm).

FESEM images of hydrogels synthesized with bulk and surface-initiated visible light photopolymerization resulted in the formation of openings on the surface of all hydrogel structures that serve for the loading and release of biomolecules (Figure 4). Micrographs of plain hydrogels obtained via bulk and surface-initiated polymerization demonstrated morphological

differences.

In

surface-initiated

polymerization,

the

adsorption

of

photoinitiator on the surface leads to initiation of polymerization reactions and growth of hydrogels from substrate surface, which results in the formation of gradients in crosslink density.44-47 However, in bulk polymerization photoinitiator is homogeneously dispersed in prepolymer solution which is the most possible reason for the varieties in pore morphologies. Integration of nanogels into the prepolymer solution increased the number of pores in two polymerization types, however pore size was affected differently. Pore sizes were increased with nanogel addition in bulk polymerized gels but the trend was reverse in surface initiated samples. The effect of nanogel ratio on pore size was not differentiable from FESEM images.



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Figure 5. FESEM images of P(MAA-g-EG) hydrogels synthesized via visible light induced surface-initiated polymerization. Gel-air interface of 1 % nanogel-integrated hydrogel (a) scale bar: 10 µm, and (b) scale bar: 1 µm. Substrate-gel interface of 1 % nanogel-integrated hydrogel (c) scale bar: 10 µm, and (d) scale bar: 1 µm.

Figure 5a demonstrates FESEM images of the samples at gel-air interface of hydrogels, where Figure 5c shows gel-substrate interface. Examination of hydrogels synthesized with surface-initiated polymerization showed variations in the surface microstructure, both at surface-gel and gel-air interfaces. The surface of hydrogels appeared flat and stiff at substrategel interface, whereas porous structure was observed with large pore sizes at gel-air interface, which can also be explained by gradients in crosslinking density. Also, spherical nanogel structures dispersed in hydrogel network were imaged by FESEM (Figure 5b and 5d). Nanogel distribution appeared different at the top and bottom surfaces of gels. While nanogels


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stand as single, isolated particles in the gel-air interface of 1 % nanogel-integrated hydrogel hydrogels synthesized via surface-initiated polymerization, they existed as small aggregates in the substrate-gel interface. The presence of nanogel aggregates can be explained by limited dispersion of nanogels in aqueous media before hydrogel polymerization, due to the formation of hydrogen bonds in nanogel drying process.48 Hybrid hydrogels were also synthesized via UV light-induced polymerization. FESEM images of hydrogels obtained via surface-initiated polymerization were illustrated in Figure S1 indicating that gel-air interfaces of gels were more porous than surface-gel interface. Porosity in gel-air interfaces of these gels were increased with the addition of nanogels in a concentration dependent manner. Differences between the micrographs of UV- and visible light-induced photopolymerized hydrogels demonstrated morphological differences indicating the influence of the wavelength of light on the microstructure of hydrogels, as were observed in our previous study.24 Visual observation of these gels demonstrated better integrities for hydrogels synthesized with visible light compared to the ones synthesized with UV light exposure. This observation could be explained by efficient photoinitiation mechanism and crosslinking reactions taking place via visible light exposure. 3.2. Gel Fraction and Swelling Behavior To examine the effects of two different polymerization mechanisms and altered concentrations of nanogel incorporation on the formation of P(MAA-g-EG) hydrogels, we conducted a gel fraction experiment. Neither the polymerization mechanisms nor the addition of nanogels caused significant alterations in the values of gel fraction. For all sample sets including bulk and surface-initiated polymerized hydrogels with different nanogel ratios (0, 1 and 5%), gel fraction values altered between 50-70 % (Figure 6), where higher nanogel concentration in the prepolymer solution lead to higher gel fraction for bulk photopolymerization.



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Figure 6. Percent gel fraction of P(MAA-g-EG) hydrogels with CHPOA nanogels synthesized via bulk and surface-initiated visible light photopolymerization. Reversible swelling experiments were performed to investigate pH-responsive behavior and cyclic swelling/deswelling response of plain and hybrid hydrogels in different groups. All hydrogels from each experiment set remained intact throughout two cycles of the swelling/deswelling experiment. Figure 7 represents the comparison of swelling behavior of hydrogels formed by bulk and surface-initiated photopolymerization, where nanogel concentrations of 0, 1, and 5 % were included in the prepolymer.


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Figure 7. Comparison of swelling ratios for pH-responsive hydrogels synthesized by (a) bulk, and (b) surface-initiated visible light induced photopolymerization for 0, 1, and 5 % nanogel concentrations at pH 2.0 and pH 7.4. At high pH values, carboxyl groups in MAA monomer become negatively charged, and electrostatic repulsion occurs between neighboring PEG chains. PEG chains within the network act as both hydrogen bonding points with water and also coil extension suppliers. At low pH condition, carboxyl groups in MAA structure become protonated. In the absence of repulsive forces, hydrogen bonds form between PEG chains and MAA. For the case of anionic P(MAA-g-EG) hydrogels, a collapsed state is retained at low pH conditions.2, 49 As shown in Figure 7, low swelling ratios were observed in acidic condition (pH 2.0), where swelling ratios were increased for all hydrogels observed at pH 7.4. We observed higher swelling at pH 7.4 for hydrogels formed with visible light-induced photopolymerization compared to the gels prepared with UV light (Figure S2). This can be attributed to the formation of different pore sizes in hydrogel structures polymerized with two different polymerization mechanism. Equilibrium weight swelling ratios decreased with the addition of 1% CHPOA nanogel in P(MAA-g-EG) hydrogel synthesized with both polymerization mechanisms at pH 7.4 (Figure 7). This decrease was explained in a previous study where swelling and release profiles of P(MAA-g-EG) hydrogels combined with hydrophobic PMMA nanoparticles were investigated. Reduced swelling was attributed to reduced ionic repulsion between carboxyl groups in MAA, and decreased water uptake due to increased hydrophobicity introduced by PMMA particles.2 Similarly, the increased hydrophobicity coming from CHPOA nanogels could be associated with the reduced swelling ratios in our study. Surprisingly, with further increase in the nanogel ratio to 5%, swelling increased above the values shown for nanogel-free hydrogels (Figure 7). We believe that increased amount of nanogels lead to the formation of nanogel aggregates (Figure 6d) and contributed to high pore size. These pores could allow the transport of more water into the hydogel network which results in higher swelling.50 17 - Environment ACS Paragon-Plus


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3.3. Controlled Release of PGB PBG release experiments were carried out in DMGA buffer solution at pH 2.2 during the first 2 hours, followed by 6 hours in the same buffer at pH 7.4 to mimic the physiological condition in digestive system. Percent loading of PGB into the gels and percent release of PGB were calculated for visible light-induced crosslinking, where bulk or surface-initiated photopolymerization was used with altered nanogel concentrations (Table 1). Table 1. Percent PGB loading and percent PGB release from hybrid gels formed via bulk or surface-initiated visible light-induced polymerization with altered nanogel-incorporation. Formulation of the hydrogel B-0 % B-5 % SI-0 % SI-5 %

PGB loading (%)

PGB release (%)

83.03±0.37 86.54±1.97 78.77±2.86 83.88±0.63

87.91±0.79 99.86±2.18 90.64±1.22 94.36±4.41

Differences in the network structure and different ratios of nanogels present in the hydrogel caused different loading efficiencies. As shown in Table 1, for plain hydrogels percent PGB loading and release were measured as 83.03 and 87.91%, respectively. When 5% nanogel integrated gels were used, percent PGB loading and release were measured as 86.54% and 99.86%, respectively. Similarly, amount of PGB loaded into and released from 5% nanogel integrating gels were slightly higher than that of plain gels formed via surface-initiated photopolymerization. These results suggest that it would be possible to improve drug loading and drug release capacity of a pH-responsive network slightly through nanogel conjugation into the network. This observation is also consistent with our previous study investigating the delivery of PGB from pH sensitive P(MAA-g-EG) hydrogels at acidic pH as well as other studies in the literature about the delivery of various drugs from pH-responsive hydrogels.24, 51-52

In addition, the amount of PGB loaded in the hydrogels produced by bulk polymerization

(83.03% for B-0 and 86.54% for B-5) was higher than that of hydrogels synthesized by surface-initiated polymerization (78.77% for SI-0 and 83.88% for SI-5). 18 - Environment ACS Paragon-Plus

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Figure 8 demonstrates PGB release from 0 % and 5 % nanogel-integrated gels formed with bulk and surface-initiated crosslinking with visible light exposure at low and high pH. As can be seen in Figure 8, P(MAA-g-EG) hydrogels released nearly half of their drug load in the first 30 min of pH shift from low to neutral. PGB release reached maximum in 5 h for all plain and nanogel-incorporating polymers synthesized via bulk and surface-initiated polymerization. This result is also consistent with the observation that P(MMA-g-EG) hydrogels demonstrate higher swelling at pH values higher than their pKa of 5.4 which promotes high drug release.53 Since the P(MAA-g-EG) hydrogel structure collapse at low pH and swells as the buffer pH becomes more than the hydrogel pKa, this behavior of release from the hydrogel structure was expected. This fast release can be explained by the presence of PGB close to the surface of the hydrogel, due to the already initiated diffusion in pH 2.2. On the other hand, nanogel-incorporating pH responsive hydrogels exhibited slower release which is more desirable for controlled drug release purposes. In previous literature, it has been shown that the typical rapid swelling response of pH-responsive hydrogels can be restricted in the presence of highly cross-linked interpenetrating networks.54 Thus, linear swelling profiles achieved by higher crosslinking can be useful to minimize burst release of biomolecules in oral delivery applications. Similarly, here we suggest that the increased amount of polymerizable CHPOA nanogels incorporated in the pH-responsive hydrogel have contributed to the linear swelling behavior.54



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Figure 8. Release behavior of PGB at pH 2.2 and pH 7.4 from (a) plain and (b) 5 % nanogelintegrated P(MAA-g-EG) hydrogel formed with bulk polymerization synthesized with visible light, (c) plain and (d) 5 % nanogel-integrated P(MAA-g-EG) hydrogel formed with surfaceinitiated polymerization synthesized with visible light. Furthermore, nanogel-incorporating hydrogels released less PGB in low pH environment, compared to plain P(MAA-g-EG) hydrogels. This reduction is advantageous since it prevents the degradation of the drug in stomach, reduces possible toxic effects of drug to the stomach, and increases the possible maximum amount of drug to be released in the neural pH of intestines.50 To evaluate the kinetics of release, drug release data was fitted to a modified Hill Equation. (Equation 6), where an s-shaped (sigmoidal) drug release curve was obtained (Figure 9). The release profile included a lag-time of 150 min, followed by a continuous drug release for the


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remaining 350 min. A pH dependent release was taken into consideration, since drug release started together with change of buffer after 150 min.55

Figure 9. Fitting of cumulative PGB release data to Hill equation at pH 2.0 (first 150 min) and pH 7.4 (from 150 min until the end of the experiment). PGB release profile from plain and 5 % nanogel-integrated P(MAA-g-EG) hydrogel formed through (a, b) bulk and (c, d) surface-initiated polymerization, respectively. Red circle: experimental data, blue line: fitting curve. T50 indicates the time at which 50% of maximum drug release is obtained and α is the Hill coefficient of sigmoidicity. Both parameters were calculated for plain and 5 % nanogelincorporated hydrogels prepared with bulk and surface-initiated polymerizations in low and neutral pH, and results are summarized in Table 2.



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Table 2. T50 and Hill coefficient of sigmoidicity values calculated for hybrid gels formed via bulk or surface-initiated visible light-induced polymerization with altered nanogelincorporation.

T50 (min) 217 184 218 256

Formulation of the hydrogel B-0% SI-0% B-5% SI-5%

∝ 2.62 3.15 2.99 2.65

T50 values demonstrated that the gels synthesized via surface-initiated polymerization and in the absence of nanogels released 50% of the drug around 184 minutes, whereas with 5% nanogel incorporation into hydrogel T50 was calculated as 256 minutes. This suggests that the addition of nanogels into the hydrogel network for surface-initiated polymerization extended the time needed for drug release. This could be explained by differences in the porosity of hydrogels with nanogel integration as was illustrated in Figure 4. 3.4. Cytocompatibility Test Cytocompatibility of plain and SBS-incorporated P(MAA-g-EG) hydrogels were confirmed by both in vitro Cell-Titer Glo assay on fibroblasts and in vivo irritation test on a rabbit model, in our previous study.24 Here, we investigated the effect of CHPOA nanogel integration into P(MAA-g-EG) hydrogels on cellular viability. Biocompatibility of pH-responsive hybrid hydrogels was investigated on human BJ fibroblast cells in vitro. Fibroblast cells were seeded onto the surface of hydrogels and viability was characterized after 6 h and 24 h using Cell-Titer Glo viability assay. The results of in vitro cell viability assay for plain, 1 % and 5 % nanogel-incorporated P(MAA-g-EG) hydrogels synthesized under visible light, both via bulk and surface-initiated polymerization, are shown in Figure 10.


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Figure 10. Percent survival of human BJ fibroblasts on (a) bulk, and (b) surface-initiated visible light polymerized sets of pH-responsive P(MAA-g-EG) hydrogels with altered nanogel concentrations. ATP calibration curves were generated and ATP amounts were calculated for each sample from luminescence values. We normalized viability results on nanogel integrated P(MAA-g-EG) hydrogels with the viability on plain gels without nanogels. This is consistent with a previous

study, where researchers did not include cells seeded on cell culture dishes as controls in cytotoxicity experiments of P(IA-g-EG) microparticles, P(MAA-g-EG) microparticles, and P(MAA-g-EG) hydrogels containing nanoparticles.2, 56-57 Since CHP nanogels and their derivatives were proposed as biocompatible in previous studies, it was expected that CHPOA nanogel incorporation into P(MAA-g-EG) hydrogels would not compromise with biocompatibility.29,

32

According to Figure 10, cell viability was slightly

decreased after 24 h for cells seeded on all hydrogels. Peppas et al. also demonstrated that cell viability on PMMA nanoparticle containing P(MAA-g-EG) gels at 24 h was lower compared to cells exposed to the same environment for 6 h or 12 h.58 In our previous study, slight decrease was also observed on viability of fibroblast cells seeded onto P(MAA-g-EG) hydrogels on day 1; however, long term cytocompatibility of P(MAA-g-EG) hydrogels was confirmed on a rabbit model in vivo.24 This also suggest that interaction of individual cells in 23 - Environment ACS Paragon-Plus


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cell culture plate may compromise cell viability, whereas in vivo interaction of the gels with the natural extracellular matrix may be more tolerable by the cells and the tissue. Contrary to hydrogels synthesized by bulk polymerization, significant increase in cell viability was observed with the addition of nanogels in 6 h for the hydrogels synthesized by surface-initiated polymerization. Also, cell viability was higher on surface-initiated hydrogels compared to gels in the other group. As shown in Figure 4, increasing nanogel ratio resulted in larger pore sizes on the surface of bulk polymerized hydrogels, in contrast to the opposite trend shown in surface-initiated polymerized hydrogels. Obviously, cells cared about the pore size differences of the underlying P(MAA-g-EG) hydrogels, and hence responded differently which resulted in altered cytotoxicity of hydrogels. Differences between cross-linking of hybrid polymers synthesized via bulk and surface-initiated polymerization mechanisms influences stiffness of the gels, which affect the cytocompatibility of the substrates. As shown in previous studies, the surface stiffness influences the attachment and growth of cells.59 The lower metabolic activity observed on hydrogels synthesized via bulk polymerization on day 1 could be associated with this phenomenon. In addition, the addition of polymerizable CHPOA nanogels into prepolymer solution affect polymerization kinetics and crosslinking density, which is an additional parameter that affects cell viability, as was explained in earlier sections. To investigate the effect of nanogel integration on mechanical properties of gels, we conducted a rheology experiment (Figure S3). Integration of nanogels lead to the formation of looser networks (lower Gmax′), in a concentration independent manner. Gelation was started at 945 s for the gel without nanogel, gelation times were 1052 s and 1017 s for gels prepared with 1% and 5% nanogel concentrations. The addition of CHPOA nanogels into hydrogels synthesized by surface-initiated polymerization improved cell survival. Previously, we showed that the addition of SBS particles into P(MAA-g-EG) hydrogels improved the percent fibroblast survival results, which 24 - Environment ACS Paragon-Plus

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is attributed to higher stiffness of hybrid hydrogels and better adhesion of fibroblasts on SBS domains.24 Similarly, improved cell survival with the addition of CHPOA nanogels could be explained through better adhesion of cells on nanogel domains. Here, we showed that the addition of CHPOA nanogels at these ratios are not toxic to cells. Conclusions In this study, we developed pH-sensitive hybrid hydrogel systems consisting of pH-sensitive P(MAA-g-EG), and CHPOA nanogels for controlled delivery of PGB. The P(MAA-g-EG) hydrogels were synthesized with bulk and surface-initiated photopolymerization, where both UV and visible light-induced crosslinking were used. In this study, we proposed a nanostructured, hybrid hydrogel system consisting of hydrophilic pH-responsive P(MAA-gEG) polymer and amphiphilic CHPOA nanogel components. This approach would allow for stimuli responsive drug delivery from hybrid networks with altered hydrophobicities and croslinking gradients. Acknowledgements: This study was funded by FP7-IRG-239471 and Koc University seed grant to SK. FESEM characterizations were performed at Koc University, Surface Science Center (KUYTAM). Supporting Information: FESEM and swelling experiment data for UV light polymerized pH responsive hydrogels were shown in Supporting Information which is available from the ACS Publications Online Library or from the author.



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