A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Summary

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

Abstract

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 °C, -20 °C, -80 °C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

Introduction

Recently, hydrogels have attracted great interest in the biomedical field because they are three-dimensional networks with high water content and are soft and flexible, so they can mimic natural tissues easily¹. Also, they do not dissolve in aqueous medium at physiological temperature and pH but present a large swelling². Hydrogels can act as tissue engineering scaffolds, hygiene products, contact lenses, and wound dressings; because they can trap and release active compounds and drugs, they are used as drug delivery systems³. Depending on their application, hydrogels can be made from natural or synthetic polymers, or a combination of both, in order to obtain the best characteristics⁴.

The properties of hydrogels are a consequence of many physical and chemical factors. At the physical level, their structure and morphology depend on their porosity, pore size and pore distribution⁵. At the chemical and molecular level, the polymer type, the hydrophilic group content in the polymer chain, the crosslinking point type, and the cross-linking density are the factors that determine the swelling capacity and the mechanical properties^6,7.

According to the type of crosslinking agent used to form the network, the hydrogels are classified as chemical hydrogels or physical hydrogels. Chemical hydrogels are joined by covalent interactions between their chains, which are formed through UV and gamma irradiation or using a crosslinking agent^7,8. Chemical hydrogels usually are strong and resistant but, generally, the crosslinking agent is toxic to the cells and its removal is difficult, so its application is limited. On the other hand, physical hydrogels form by the connection of the polymer chains through non-covalent interactions, avoiding the use of crosslinking agents^4,9. The main non-covalent interactions in the network are hydrophobic interactions, electrostatic forces, complementary and hydrogen bounds⁷.

Poly(vinyl alcohol) (PVA, Figure 1a) is a synthetic and water-soluble polymer with excellent mechanical performance and biocompatibility that can from crosslink agent-free hydrogels through the freeze-thawing method^10,11. This polymer has the capacity to form concentrated zones of hydrogen bonds between -OH groups of their chains (crystalline zones) when they are freezing¹². These crystalline zones act as crosslinking points in the network, and they are promoted by two events: the approaching of the polymer chains when the crystal water expands and the PVA conformational changes from isotactic to syndiotactic PVA during freeze¹³. Because of the freeze-drying, the water crystals are sublimated, leaving void spaces that are the pores in the hydrogel¹⁴. To obtain hydrogels with better properties, PVA can been easily combined with other polymers.

In that sense, chitosan constitutes an option as it is the only biopolymer from natural sources with positive charges. It is obtained by the deacetylation of chitin and it is composed of random combinations of β-1,4 linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)^15,16 (Figure 1b). Chitosan is biodegradable by human enzymes and it is biocompatible. Also, by its cationic nature, it can interact with the negative charge of the cell surface, and this property has been associated with its antimicrobial activity¹⁷. This polymer is easy to process; however, their mechanical properties are not sufficient and some materials have been added to form complexes with better characteristics.

Considering specific characteristics of chitosan and PVA, the successful manufacture of hydrogels has been reached by the freeze-thawing method^2,18 to avoid the use of toxic crosslinking agents. In chitosan-PVA hydrogels, the crystalline zones of PVA are also formed, and chitosan chains are interpenetrated and form simple hydrogen bonds with -NH₂ groups and -OH groups in PVA. The final chitosan-PVA hydrogel is mechanically stable, with high rates of swelling and low toxicity, and with antibacterial effect¹⁸. However, depending on the freezing conditions used in the preparation (temperature, time and number of cycles), the final characteristics may change. Some studies report that increasing the number of freezing cycles decreases the swelling degree and increases the tensile strength^19,20. In order to strengthen the network, other agents such as gamma and UV radiation and chemical crosslinkers have been used additionally after the freeze-thawed preparation^21,22,23. Hydrogels with a higher chitosan proportion have a more porous network and high swelling capacity but less strength and thermal stability. In this context, it is important to consider the preparation conditions to obtain suitable hydrogels for their target application.

The purpose of this work is to present in detail how the freezing conditions (temperature of freezing and number of cycles) affect the final characteristics of CS-PVA hydrogels. FT-IR spectra, morphological and porosity characteristics and swelling capacity were evaluated, as well as drug loading and release capacity. In the release studies, diflunisal (Figure 1c) was used as model drug, due to its size suitable to the hydrogel structure.

Protocol

1. Preparation of chitosan-PVA hydrogels

1. Prepare 2% (w/w) chitosan and 10% (w/w) PVA solutions. Dissolve 0.2 g of chitosan in 10 mL of 0.1 M CH₃COOH solution (previously filtered) at room temperature and maintain continuous mechanical stirring overnight. Dissolve 1 g of PVA in 10 mL of distilled water and stir at 80 °C for 1 h.
2. Mix both solutions 1:1 using a magnetic stirrer until they are homogeneous at room temperature, and pour the mixtures on Petri dishes. Leave the samples for 2 h at atmospheric pressure to degas.
3. Freeze the hydrogels at -4 °C, -20 °C or -80 °C for 20 h and 4 cycles (samples CP4-4, CP4-20 and CP4-80, respectively). Freeze another hydrogel at -80 °C for 20 h using 5 or 6 freezing cycles (samples CP5-80 and CP6-80). After the third freezing cycle, wash the hydrogels with deionized water. At the end, freeze-dry the hydrogels at -46 °C for 48 h and store for further characterization (methodology adapted from²).

2. FT-IR characterization

1. Place a little piece (1 mm x 2 mm) of hydrogel in the FT-IR spectrometer in ATR mode. Take the FT-IR spectra from 4000 to 600 cm^-1 (2 cm^-1 of resolution and average of 32 scans).

3. Swelling assays

1. Cut out discs (13 mm in diameter and 10 mm in height) from the hydrogel and weigh them. Incubate the discs in 50 mL of deionized water with shaking at 25 °C. Repeat three times.
2. Every 30 min remove the sample from the medium, blotter to eliminate the excess of water, and weigh. Calculate the swelling degree using the equation 1 and calculate the equilibrium state of swelling, , at 24 h using the equation 2.
   )
   Where is the weight of the dry hydrogel and is the weight of the wet hydrogel.
   

4. Electronic Microscopy

1. Cover a little piece of hydrogel with a thin gold layer (30 s and 10 mA) in a sputter coater.
2. Put the sample in a scanning electron microscope (SEM). Analyze the samples under vacuum at 20 kV and take the images with a 500x and 1500x magnification.

5. Porosimetry

1. Place discs 15 mm in diameter weighing around 0.26 g into the penetrometer (a solid penetrometer, having a bulk volume of 0.3660 mL and 5.7831 mL of stem volume). Analyze the porosity and pore size by Mercury Intrusion Porosimetry (MIP).
2. Conduct the experiment in the hysteresis mode (intrusion-extrusion). Measure the total intrusion volume (mL/g), total pore area (m²/g), pore diameter (µm), porosity (%), permeability (mDarcy) and tortuosity. Repeat twice.

6. Drug loading and release

1. Before loading, prepare 4 L of 15 mg/L diflunisal solution and stir overnight. Confirm the concentration of the solution by UV-Vis spectroscopy (initial concentration). Indeed, swell 400 mg of freeze-dried samples of hydrogel in 6 mL of distilled water for 24 h.
2. For loading, fill a flask with 50 mL of diflunisal solution and maintain at 25 °C with constant stirring. Submerge each swelled hydrogel in the flask.
   1. Take aliquots of remaining diflunisal solution (2 mL) at different times in order to determine the plateau region of the curve, for example: 3, 6, 24, 27, 30 and 48 h. After 24 h replace the solution with a fresh one.
3. Measure the absorbance at 252 nm of each aliquot, and determine the concentration of diflunisal present in the solution, using a calibration curve of diflunisal. Calculate the amount of diflunisal retained in the hydrogel at 24 and 48 h, as the difference of initial and final concentrations, taking into account the total volume (56 mL).
   1. Determine the encapsulation efficiency (EE) using the equation 3.
      
   2. Freeze the loaded hydrogels at -80 °C and lyophilize them at -50 °C.
4. For drug release, submerge 300 mg of freeze-dried diflunisal loaded hydrogels in 50 mL of phosphate buffer (pH 7.4) at 25 °C. Maintain constant stirring. Withdraw aliquots of 2 mL at different times and replace with fresh medium to keep a constant volume.
   1. Determinate the diflunisal released spectrophotometrically at 252 nm, according to a calibration curve.
5. Deduce the predominant drug release mechanism in the hydrogels adjusting the drug release data corresponding to the first 60%, to the Korsmeyer-Peppas model (Equation 4), to obtain the kinetic (k) and the diffusion (n) constants. The n values indicate the mechanism of drug release^24,25. Then, n values close to 0.5 are related to Fickian diffusion, meanwhile values of 0.5-1.0 for anomalous transport, where are involved diffusion and relaxation chains, and finally, values of 1.0 are related to case II transport.
   
   1. To confirm the results, use the Higuchi, First order, and Zero order mathematical models (Equations 5 to 7) and select the better fit.
   2. 
      
      
      where t represents the release time, Mt the amount of drug delivered at a given time, and M_∞ the total amount of drug delivered at the end of the process.

Results

Hydrogels preparation
Chitosan-PVA hydrogels were obtained at -4 °C, -20 °C and -80 °C with 4 freezing cycles and at -80 °C with 5 and 6 freezing cycles by the previously reported freeze-thawing method². All hydrogels were homogeneous, semi-transparent, flexible and resistant against manipulation.

FT-IR characterization
The FT-IR spectra are shown in

Discussion

The freeze-thawing method is a suitable process to prepare biocompatible hydrogels focused in biomedical, pharmaceutical or cosmetical applications^34,35,36. The most important advantage of this method, compared with other well-known methods to prepare hydrogels, is that crosslinking agent use is avoided, which could cause an inflammatory response or adverse effects in the human body³⁴. This is a versatile...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials:
Chitosan medium molecular weight	Sigma-Aldrich	448877	Mw determined by capillary viscometry (637,000 Da) and deacetylation degree of 70%
Diflunisal (2'-4'-difluoro-4-hydroxy-3-biphenyl-carboxylicacid)	Merck
Glacial acetic acid	Sigma-Aldrich	1005706
Poly(vinyl alcohol)	Sigma-Aldrich	341584	Mw 89,000-98,000, 99+% hydrolyzed
Equipment:
Cressington Sputter Coater 108 auto	TED PELLA INC
Cryodos Lyophilizator	Telstar
Falcon tubes	Thermo Fisher Company
FT-IR spectroscopy	Nicolet iS50	in ATR mode
Lyophilizator	LABCONCO
Micromeritics Autopore IV 9500	Micromeritics
Scanning electron microscope	Pemtron SS-300LV
UV-visible spectrophotometer	Agilent 8453