pH-responsive alginate-based hydrogels for protein delivery

Accepted Manuscript pH-responsive alginate-based hydrogels for protein delivery

Diego S. Lima, Ernandes T. Tenório-Neto, Michele K. LimaTenório, Marcos R. Guilherme, Débora B. Scariot, Celso V. Nakamura, Edvani C. Muniz, Adley F. Rubira PII: DOI: Reference:

S0167-7322(18)31085-7 doi:10.1016/j.molliq.2018.04.002 MOLLIQ 8905

To appear in:

Journal of Molecular Liquids

Received date: Revised date: Accepted date:

2 March 2018 29 March 2018 2 April 2018

Please cite this article as: Diego S. Lima, Ernandes T. Tenório-Neto, Michele K. LimaTenório, Marcos R. Guilherme, Débora B. Scariot, Celso V. Nakamura, Edvani C. Muniz, Adley F. Rubira , pH-responsive alginate-based hydrogels for protein delivery. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Molliq(2017), doi:10.1016/j.molliq.2018.04.002

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pH-responsive alginate-based hydrogels for protein delivery Diego S. Lima1, Ernandes T. Tenório-Neto2‡, Michele K. Lima-Tenório2, Marcos R. Guilherme1, Débora B. Scariot3, Celso V. Nakamura3, Edvani C. Muniz1, and Adley F. Rubira1‡

Department of Chemistry, State University of Maringá, Av. Colombo, 5790, CEP 87020-900,

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Maringá, Paraná, Brazil

Department of Chemistry, State University of Ponta Grossa, Av. Gen. Carlos Cavalcanti,

4748, CEP 84030-900, Ponta Grossa, Paraná, Brazil

Department of Basic Sciences of Health, State University of Maringá, Av. Colombo, 5790,

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CEP 87020-900, Maringá, Paraná, Brazil

Corresponding authors: Tel.: +55 (44) 3011 3686; fax: +55 (44) 3011 5370. E-mail:

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‡

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[email protected] (A.F.Rubira) and [email protected] (E.T.Tenório-Neto)

Abstract

This work describes the synthesis and characterization of pH-responsive hydrogels based on

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alginate for protein delivery. Synthesis approach shown here has included the modification of

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sodium alginate to convert it into a covalently crosslinkable polysaccharide and the subsequent radical polymerization reaction with sodium acrylate and N-vinylpyrrolidone for

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hydrogelation. To evaluate the applicability of the obtained hydrogels as an oral protein delivery system, we studied the cytotoxicity, the drug release profile (using BSA as a protein model), and the swelling performance in the basic and acidic environments. The hydrogels showed a pH-dependent swelling profile with higher value at pH 7.4. The protein release mechanism was demonstrated to be dependent on pH and composition. The proposed materials were shown to be compatible with living cells, indicating great pharmacological potential. These results show that the hydrogels are ideally suited for use as an oral drug delivery device. Keywords: stimuli-responsive hydrogel; glycidyl methacrylate; acrylic acid; biomaterials, alginate.

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1. Introduction Over the past few decades, protein-inspired therapy has emerged as one of the most impactful areas of medicine. The rapid increase in the therapeutic use of proteins is related to complexity of the macromolecules, which enables complex functions with a high degree of specificity unmatched by traditional small molecule drugs, resulting in more effective medicines with fewer side effects [1]. However, the therapy by oral protein administration is

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still a technological challenge to be overcome, because of the acid-catalyzed protein denaturation at the stomach.

There has been a great deal of effort addressed to develop new efficient approaches to

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protect drugs against the acid inactivation. pH-responsive hydrogels (HGs) stand out as an

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important class of biomaterial, owing to their unique physical-chemical properties, biocompatibility and similarity with living tissues. The use of HGs as a polymer carrier appears

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to be a promising drug delivery strategy for oral protein therapy. HGs are three-dimensional (3D) hydrophilic networks of physically or chemically crosslinked polymers. They have been shown to be water absorbers of high performance,

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because of their ability to absorb and retain a large volume of either liquid or biological fluids [2]. Their efficiency to absorb water is related to the porosity, crosslinking density, and

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chemical nature of the polymer chains carrying functional groups, such as, –NH2, –COOH, –

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CONH2, and –SO3H [3]. They may be prepared so that their physical–chemical characteristics (e.g., equilibrium swelling and absorption kinetic) respond to changes in the external environment, such as pH, temperature, ionic strength [4,5]. These are state-of-the-art materials

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or smart HGs.

To overcome problems associated with protein release into the stomach, a huge amount of strategies to prepare pH-responsive HGs based on polysaccharides and proteins have been reported. Among related natural polymers, the polysaccharides play a relevant role in the biomaterials technology owing to biocompatibility and non-toxicity [6]. An example is alginate (Alg) that has been widely used in the synthesis of HGs for applications in the biomedicine, such as wound healing, tissue engineering, and drug delivery [7–10]. Alginate does not undergo enzyme-catalyze degradation in the human body [11], which makes it attractive for encapsulation (and protection) of enzyme-responsive drugs. Alg is a naturally occurring anionic polymer derived from marine brown algae and from the Pseudomonas and Azotobacter bacteria. Algs are naturally derived polysaccharide block copolymers composed of regions of sequential β-D-mannuronic acid monomers (M-blocks), regions of α-L-guluronic acid (G-blocks), and regions of alternated M and G units. The length

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of the M- and G-blocks and sequential distribution along the polymer chain varies depending on the source of Alg. It is easily gelled in the presence of divalent cations, such as Ca2+, Ba2+, Sr2+, and Zn2+ [11] and the thus formed gel show changeable swelling profile in response to pH. Alg can be combined with other monomers and/or reactants to form advanced systems whose drug release mechanism can be modulated to achieve desired release profile. Efficient

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strategies have been used for engineering such systems, such as grafting copolymerization and chemical modification [12–15].

This work aimed at developing an alginate-based covalent HG that shows variable

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release drug profile in response to pH. The advantage of this system is that the release of drug

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can be controlled by changes in pH. The proposed material was engineered to be a stable vehicle for the delivery of protein. Bovine serum albumin (BSA) was the used as model protein

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(therapeutic agent). As main strategy, Alg was vinyl-modified with glycidyl methacrylate (GMA) in an acidic medium to form a vinylated macromonomer for further copolymerization with sodium acrylate and N-vinyl-pyrrolidone. To evaluate the applicability of the resulting

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material for intended applications, the HG properties, such as, porosity, drug release mechanism, and swelling behavior were studied as function of pH and polymer composition.

2. Experimental 2.1 Materials

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Cell viability studies were performed to demonstrate biocompatibility with living tissues.

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Alginic acid sodium salt from brown algae (Alg), N-vinylpyrrolidone (VP) (99% v/v), glycidyl methacrylate (GMA) (97% v/v), and sodium persulfate (98% m/m) were purchased from Sigma-Aldrich. Acrylic acid (AA) (99.5% v/v) was supplied from Acros Organics. Sodium hydroxide (NaOH) (97% m/m) was obtained from Fmaia. Chlorhydric acid (HCl) (37% v/v) was purchased from Anidrol, N,N,N′,N′-Tetramethylethylenediamine (TEMED) (98% v/v) was supplied from Invitrogen, and acetone (99.5% v/v) was obtained from Synth. All reagents were used as received.

2.2. GMA-based modification of alginate One gram of Alg was dissolved in 30 mL of distilled water while stirring. Then, the pH of the solution was adjusted to 3.5 with addition of aqueous HCl and heated to 60 °C. After that, 0.65 mL of GMA was introduced while stirring and the resultant mixture was left to react

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for 24 h. The thus obtained product (Alg-GMA) was precipitated and washed with acetone. The precipitate was freeze-dried for 24 h.

2.3. Synthesis of alginate-based HG Before gelation, acrylic acid was neutralized with NaOH to form acrylate salt (sodium acrylate, SA) [3],[16]. For that purpose, acrylic acid (AA) was added dropwise to acetone and

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the mixture was kept under mild stirring until a clear solution was formed. Then, NaOH was slowly added while stirring ([acid/base] molar ratio of 1:1). After few hours, the mixture became whitish which was filtered and washed with acetone. The residual acetone was

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withdrawn from solid phase (SA) by solvent evaporation at 35 °C using a ventilated oven.

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For HG synthesis, known amounts of Alg-GMA, VP and SA were added to 10 mL of distilled water while stirring. After complete solubilization, 100 mg of sodium persulfate and

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2 drops of TEMED were added, which was kept under stirring at room temperature until a transparent, stiff material (gelation) be formed. Table 1 summarizes the HG compositions. The samples are labeled using the following notation: AlgxVyAz, where Alg, V and A are the

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modified alginate, VP, and SA, respectively; x, y, and z is related to the amount of each monomer: the numbers “1” and “2” are associated to the smallest and the biggest amount used

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of each component.

Table 1. Contents of Alg-GMA, VP, and SA used in the hydrogel-forming suspensions. Alg-GMA(g)

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Samples

VP(mL)

SA(g)

Alg1V1A1

0.25

0.5

0.5

Alg1V1A2

0.25

0.5

1.0

Alg1V2A1

0.25

1.0

0.5

Alg1V2A2

0.25

1.0

1.0

Alg2V1A1

0.50

0.5

0.5

Alg2V1A2

0.50

0.5

1.0

Alg2V2A1

0.50

1.0

0.5

Alg2V2A2

0.50

1.0

1.0

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2.4. Measures of swelling degree (capacity of water absorption) in dependence on the pH The dependence of swelling degree on the pH was investigated using buffer solutions of pH 1.2 (simulated gastric fluid – SGF) and pH 7.4 (simulated intestinal fluid – SIF) at 37 °C. The sample was brought to a Becker containing 100 mL of distilled water and 100 mL of buffer solution. The final concentration of the buffer solutions was 0.1M and the Ionic

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strength was kept constant. At a certain time, the HGs were withdrawn from the solution buffers; the excess water droplets on the surface were wiped off carefully, the samples were weighed at each new time step. This procedure was done until to achieve the swelling

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equilibrium.

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The swelling degree (SW) was determined from Eq. (1) that correlates the mass of the sample swollen at a specific time (Mt) to the initial mass of sample (M0). Herein, the swelling

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measurements were performed immediately after HG synthesis. To check the reproducibility, for each sample, the swelling experiments were performed in duplicate. 𝑀𝑡 𝑀0

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(1)

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2.5. Release of bovine serum albumin from HGs

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Prior to gelation, BSA was introduced to the HG-forming solution to be loaded during the HG synthesis. The amount of BSA corresponded to 10% (w/w) of the reactants used in the

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feed solutions. The approach to prepare BSA-loaded HGs was the same of Section 2.3. A known weight of BSA-loaded HG was added to a glass reactor containing 200 mL of either simulated gastric fluid (pH 1.2) or simulated intestinal fluid (pH 7.4) at 37 °C. In order to prevent gradient concentration, these solutions were homogenized under mild stirring. Aliquots of 5 mL were collected at specific times, and then the absorption readings were done at 277 nm, which is the wavelength for the maximum absorption of BSA, by means of an UVvis spectrophotometer (Thermo Scientific Genesys™ 10s). After that, the aliquots were brought back into the reactor to prevent volume loss. The concentration of BSA released from the HGs were determined from analytical curves (see supplementary material) correlating the absorption to the concentration of BSA.

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2.6. Characterizations 2.6.1. Fourier transform infrared spectroscopy (FTIR) FTIR spectra of GMA, Alg-GMA, and Alg were recorded on a Bruker FTIR spectrometer (VERTEX-70V). The samples were analyzed as dry powders prepared into pellets with KBr. A total of 128 scans were run for each spectrum to reach the resolution of 4 cm-1.

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2.6.2. 1H NMR measurements

H NMR spectra were recorded on a Varian spectrometer (model Mercury Plus BB) by

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applying frequencies of 300.0583 MHz for 1H nucleus. To record the 1H NMR spectra, 20 mg

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of powdered samples were added to 0.7 mL of CDCl3, as the internal standard (0 ppm), and dissolved with the help of an ultrasonic bath 1440 A (Ondontobrás) by applying a frequency

2.6.3. Scanning electron microscopy (SEM)

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of 44 kHz. The angle pulse and the relaxation time were fixed in 45° and 1 s, respectively.

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Prior to SEM imaging, the HGs were swollen into buffer solutions of pH 1.2 and pH 7.4 for 24 h. The swollen HGs were withdrawn from the solutions and immediately frozen by immersion in liquid nitrogen before being lyophilized for 48 h. Under these conditions, it is

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supposed that the morphology of the swollen HGs is maintained. The samples were fractured

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and sputter-coated with a thin layer of gold. The SEM images were obtained on a scanning electron microscope (Shimadzu, model SS550 Superscan) by applying acceleration voltage of

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15 kV and current intensity of 30 µA.

2.6.4. Cytotoxicity assay

Epithelial colorectal adenocarcinoma cells obtained from Homo sapiens (HT-29) were maintained in DMEM (Dulbecco´s Modified Eagle’s Medium), supplemented with fetal bovine serum 10% (FBS) and incubated at 37 °C and 5% CO2 tension, during 72 h. HT-29 cell suspension containing 2.5x105 were placed in 96-wells microplate after trypsinization. Cells adhered during 24 h and 500 µg mL-1 of HGs were dispensed over the cells monolayer and the microplate was incubated at the same conditions described above. Cellular viability was determined after 48 h by using MTT method (3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide - Amresco®). Briefly, MTT in phosphate buffer solution (PBS) was prepared at 2 mg mL-1 and 50 μL were placed in each well. Microplates were incubated during 4 h, in the absence of the light and, next, formazan crystals were soluble with DMSO.

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Purple color generated from mitochondrial enzymatic metabolism of viable cells was measured on spectrophotometer microplate reader, at 570 nm wavelength. Cell viability percentage was showed considering as 100% the viability of negative control.

2.6.5. Gel permeation chromatography (GPC) of Alg-GMA GPC of Alg-GMA was conducted for better understanding the HG degradation during

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the swelling. For that purpose, approximately 1.0 g of Alg-GMA was dissolved into a buffer solution of pH 7.4 at 37 ºC. After complete solubilization of Alg-GMA, an aliquot of 30 mL was taken from recently prepared solution (Alg-T0) and after 150 h (Alg-T150). Both samples

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were lyophilized and the average molecular weight of Alg-GMA was measured by GPC

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(Viscoteck GPCmax VE2001, Malvern Instruments Ltd, UK), equipped with Viscotek VE3580 RI detector and a Shodex SB-806M-OH column using 0.3M/0.2 M CH3COOH/CH3COONa as

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an eluent at room temperature with a flow rate of 1.0 mL/min. The aliquots of Alg-T0 and AlgT150 were diluted with 0.3M/0.2 M CH3COOH/CH3COONa for 24 h to final concentration of 3.0 mg/mL. Then, 200 µL of each one of them were injected into GPC equipment to make the

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readings. The average molecular weight of Alg-GMA was estimated using the calibration curve

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of the standard poly(ethylene oxide) (PEO).

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3. Results and Discussion

3.1. Spectroscopic analysis of chemically modified sodium alginate (Alg-GMA) The modification of polysaccharides with GMA has been shown to occur by

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transesterification and/ or epoxide ring-opening reaction mechanisms [3],[17]. Both mechanisms depend on pH and chemical nature of the solvent. In this work, the modification of the polysaccharide with GMA was processed at pH 3.5. Under this condition, GMA reacts with carboxylic and hydroxyl groups in Alg by epoxide ring-opening mechanism. Figure 1 shows the FTIR spectra of GMA, Alg-GMA, and Alg in the spectral range of 2000 to 800 cm-1. The signals observed for alginate at 1612 cm-1 and 1416 cm-1 are attributed to asymmetric and symmetric stretching vibrations of carbonyl groups (νC=O), respectively. Both signals can be also observed in the Alg-GMA spectra. Furthermore, in the Alg-GMA spectra, the band of νC=O from GMA appears as a shoulder-type peak (at nearly 1700 cm-1, indicated as *), probably, due to overlapping bands of C=O stretching from both Alg and GMA. The stretching of C-O bonds (from ester groups) was observed at 1100 cm-1 and 1200 cm-1. On the other hand, the bands of νasC–C and νC–O of the epoxy ring from GMA (at 910 and 860

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cm−1, respectively) were not observed suggesting that alginate reacted with GMA by an

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epoxide ring-opening mechanism [18].

Figure 1. FTIR spectra of GMA, Alg-GMA, and Alg in the spectral range of 2000 to 800 cm.

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NMR-1H analysis was performed (Figure 2) for a more detailed analysis of modified

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polysaccharide. A scheme of the product resulting from coupling reaction of Alg with GMA is

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shown in inset. The signals at δ 2.67, δ 2.84, and δ 3.23 in the spectrum of GMA were attributed to hydrogen from epoxide ring (6 and 7). These signals were not observed in the spectrum of

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Alg-GMA, which confirm the alginate modification have occurred by the epoxide ring-opening mechanism [17]. The signals at δ 6.17 and δ 5.74 in the spectrum of Alg-GMA were assigned to vinyl carbon-linked hydrogen from both isomers (inset). In the same spectrum, the signal corresponding to methyl carbon-linked hydrogen from vinyl carbons (c) could be observed at δ 1.94. Other signals of the Alg-GMA structure were overlapped by hydrogen signals from Alg.

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Figure 2. 1H NMR spectra of GMA, Alg-GMA, and modified Alg.

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3.2. HG morphology

Figure 3 shows the SEM micrographs of the HGs. As described in the experimental section, the SEM images were made from the lyophilized HGs after being swelled up to

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equilibrium in water. The samples were shown to be porous, indicating typical structure of

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HGs. Porous structure serves as a means for diffusion of macromolecules, like proteins [6]. HGs showed relevant changes in the pore sizes in response to pH. HGs swollen at pH 7.4 were

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found to be more porous than those swollen at pH 1.2. Morphology differences in porosity are related to ionizable groups present in the HG network. For example, in the solutions with pH 1.2, the carboxylic groups (pKa of ca. 4.5) from acrylate and Alg-GMA are in non-ionized form (COOH), increasing the attractive forces that pull swollen network polymer back. As a result, HG shrinks forming a denser polymer network that affect the water absorption. At pH above the pKa of the acid, the COOH groups ionize to form fixed-charge carboxylate groups (COO−), which generate electrostatic anion-anion repulsion forces that make easier the polymer networks to expand in water. The structural changes in the HGs influenced by the environmental variations are responsible for the alterations on the morphological properties [16].

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Figure 3. Micrographs of hydrogels with different amounts of GMA-Alg, N-vinyl-pyrrolidone, and sodium acrylate taken from samples freeze-dried after being swollen to equilibrium in water. Scale bars of images are 20 µm.

3.3. Swelling performance

Figure 4 shows the time-dependent swelling curves for HGs swollen at different pH. To have a comprehensive understanding on swelling performance of the HGs, this approach was performed at the pH 1.2 for simulating the gastric fluid and at the pH 7.4 for simulating the intestinal fluid. HG showed changes in volume in response to pH. As discussed in section 3.2 this effect is related to ionizable groups present in the HG network. The higher the pH of swelling, the greater the water absorption capacity of HG. At pH 1.2, HGs quickly reached a pseudoequilibrium state (see Figure S2 - supplementary material), which was followed by continuous

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loss of water. This behavior was believed to be the direct result of an increase in the crosslinking density caused by the hydrogen bonds, which contribute to increase the attractive forces. In consequence, absorbed water is expelled into external solution, decreasing the weight

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of HG [16],[18].

Figure 4. Time-dependent swelling curves at the indicated pH at 37°C.

In the basic environment, the swelling equilibrium was not reached. The cause of this effect is related to long time period of swelling (150 h). The same behavior was found when the tests were repeated, suggesting that this is an intrinsic property of these HGs. For a better understanding of this result, two hypotheses were raised: i) HG networks were crumbled by a high osmotic pressure and/or ii) the Alg chains were cleaved by hydrolysis. The first hypothesis is less relevant considering the fact that HGs did not loss its original shape when swollen. If the second hypothesis is true, the Alg chains are broken down by the basic medium, but this occurs at a molecular level. To address this issue, additional experiments were performed as follows: known amount of Alg-GMA was dissolved in a buffer solution with pH 7.4 at 37 °C. Then, two aliquots of 30 mL were collected from this solution at initial time (Alg-

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T0) and at 150 h (Alg-T150). Both samples were lyophilized and the average molecular weight of Alg-GMA was measured by Gel Permeation Chromatography (GPC) (see Figure S3 supplementary material). The average molar mass (MW) of Alg-T0 and Alg-T150 were found to be 99,872 Da and 8,992 Da, respectively. This means that the Alg-GMA is cleaved, to some extent, during the swelling.

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3.4. Cell viability To evaluate the pharmaceutical potential of HG, the cell viability studies were performed (Figure 5). This approach is excellent to evaluate the toxicity of new materials prior

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to in vivo tests.

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The cell viability was shown to be higher than 70%, indicating that none of HGs significantly inhibited the cell growth. This result indicates that the proposed materials are

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biocompatible with living cells. The effect of sample composition on the cell growth was observed. Comparing Alg1V1A1 and Alg2V1A1 samples, no important effect on the cell viability was observed with the increase in the amount of modified-alginate. On the other hand,

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the cell viability reduced when the amount of N-vinyl-pyrrolidone increased.

Figure 5. In vitro cytotoxicity results of alginate-based hydrogels at concentration of 500 µg mL-1 using MTT approach. Data presented are the mean ± SD (n=5).

The effect of sodium acrylate seems to be related to the amount of Alg-GMA. For example, at low concentrations of Alg-GMA (Alg1), the cell viability decreased with an

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increase in SA. However, for Alg-GMA-richer sample, the cell viability showed a positive response influenced by the high amount of SA. 3.5. BSA release Figure 5 shows the time-dependent release curves of BSA from HGs. The t50 and t90 release times represent, respectively, the time at which 50% and 90% of initial BSA load was released. Ct and Ceq are the concentrations of BSA released at any time and at the equilibrium,

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respectively. Alg1V1A1, Alg2V1A2, and Alg2V2A2 were selected to release tests considering

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their similar values of cytotoxicity and swelling.

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Figure 5. Time-dependent release curves of BSA at the indicated pH at f 37°C: (A-B) Alg1V1A1, (C-D) Alg2V1A2, (E-F) Alg2V2A2.

Comparing the t50 values of Alg1V1A1 and Al2V1A2 at pH 1.2 (Figures 5A and 5C), the release rate of BSA decreased approximately two times. The low t50 values were associated with tighter polymer structure of these HGs, which makes protein release rate into surrounding

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liquid slower. However, contrary to expectations, this effect is not observed for Alg2V1A2 (Figure 5C) and Alg2V2A2 (Figure 5E), for which the release rate was more rapid, independently of the polymer network density. The only difference in the composition of these

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samples is the amount of VP; Alg2V2A2 has double of Alg2V1A2. It is reasonable to suggest

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that VP somewhat assists in the BSA release. This idea is strengthened considering that the fact that both VP and BSA are negatively charged in the acidic environment. The positive charges

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on BSA are generated at pH lower than 4.7 (isoelectric point of the protein) [18] and ones on VP results of the resonance from a -N-C=O to a -N+=C-O- group [19]. At pH 7.4, the t90 values were found to be higher for denser polymer HGs (see Figures 5B, D, and F). This indicates

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that, in the basic medium, the release rate dependents on polymer composition. The experimental data were also adjusted by applying the more general version of the

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power law equation, described in eq (2). This is the most comprehensive mathematical model

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used to determine the release performance of drug from a polymer matrix [20],[21]: 𝐶𝑡 = 𝑘𝑡 𝑛 𝐶∞

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where Ct and C∞ are the concentrations of BSA released from HG at a specified time at the equilibrium, respectively, k is a constant, and n is a parameter used to interpret the release mechanism. The conceptual meanings of n is dependent of the sample shape (i.e. cylinder, thin film, and sphere). For thin film, which is the geometry of the alginate HGs, when n=0.5 the swelling mechanism is termed as Fickian transport. This mechanism is characterized when the solvent diffusion rate is slower than relaxation rate. If n=1 (case II transport), the mechanism is driven by macromolecular relaxation of the polymer chains. When 0.5 < n < 1.0 the mechanism is termed anomalous transport (which is contribution of both Fickian diffusional and relaxational mechanisms. Finally, when n > 1, it indicates supercase II mechanism which is the contribution of diffusion, macromolecular relaxation, and erosion of the polymer chains [22]. The values of n and k were obtained from slopes of the logarithmical curves of Ct /C∞ as

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a function of time (Table 2). Moreover, it is important to mention that the eq (1) is restricted to the first 60% of the released drug (linear part) [23].

Table 2. Fitting parameters (n and k) obtained from the power law equation. pH

n

k

Alg1V1A1

1.2

0.21 ± 0.01

0.470 ± 0.013

7.4

0.77 ± 0.02

0.161 ± 0.004

1.2

1.04 ± 0.07

0.099 ± 0.010

7.4

0.97 ± 0.11

1.2

0.40 ± 0.10

7.4

0.57 ± 0.03

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0.054 ± 0.013 0.423 ± 0.201 0.257 ± 0.01

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Alg2V2A2

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Alg2V1A2

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The parameter k is a constant characteristic of HG and may be correlated with the diffusion coefficient (D) [24]. Moreover, k may be correlated to t50 since the eq (1) is restricted

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to the first 60% of the BSA released. As a general trend, the k value is higher at acidic pH,

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indicating that the release rate was faster at pH 1.2. This result matches with those of Figure 5. The release mechanisms were found to be supercase II for Alg2V1A2 and Fickian diffusion

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for Alg2V2A2, suggesting a dependence on the sample composition. In both cases, the release mechanisms were nonresponsive-pH. On the other hand, the mechanism of Alg1V1A1 was driven by pseudo-Fickian diffusion (when n < 0.5) at pH 1.2 and anomalous transport at pH 7.4.

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Conclusion Alg-based HGs as polymer carrier for protein release were successfully prepared. HGs showed a pH-dependent swelling profile with higher value at pH 7.4. At pH 1.2, HGs quickly reached a pseudo-equilibrium state followed by continuous loss of water. This behavior related to an increase in the crosslinking density caused by the HG bonds. The release mechanisms were found to be supercase II for Alg2V1A2 and Fickian diffusion for Alg2V2A2, suggesting

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a dependence on the sample composition. The mechanism of Alg1V1A1 was driven by pseudoFickian diffusion at pH 1.2 and anomalous transport at pH 7.4. The proposed materials were

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shown to be compatible with living cells, indicating great pharmacological potential.

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Acknowledgements

This work was supported by Conselho Nacional de Desenvolvimento Científico e

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Tecnológico (CNPq) (processes n° 150268/2016-5, 152109/2016-1, and 118454/2017-0).

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Graphical Abstract

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Highlights 

The hydrogels were completely degraded after six days in simulated intestinal fluid.

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Drug release mechanisms were controlled by adjusting the pH.

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The hydrogels have great pharmacological potential for drug delivery.

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The proposed materials are biocompatible and suitable for use in biological

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environments.