Green and Smart: Hydrogels To Facilitate Independent Practical

Laboratory Experiment pubs.acs.org/jchemeduc

Green and Smart: Hydrogels To Facilitate Independent Practical Learning Glenn A. Hurst* Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom S Supporting Information *

ABSTRACT: A laboratory experiment was developed to enable students to investigate the use of smart hydrogels for potential application in targeted drug delivery. This is challenging for students to explore practically because of the extremely high risks of handling cross-linking agents such as glutaraldehyde. Genipin is a safe and green alternative that has been combined with chitosan and poly(vinyl alcohol) to form a pH-sensitive hydrogel. In this problem-based learning mini-project, students can study the swelling behaviors, oscillatory functionality, gelation, fluorescence, optical, and release properties of this system. Students in the third year of an undergraduate chemistry degree program have successfully conducted this laboratory experiment as part of a problem-based learning mini-project. KEYWORDS: Upper-Division Undergraduate, Polymer Chemistry, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, Colloids, Green Chemistry, Materials Science

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INTRODUCTION One methodology to prepare upper-division undergraduate students for exploratory research projects is to utilize problembased learning (PBL) mini-projects to reflect real-life problem solving scenarios.1 Such open-ended laboratory experiments aid in the development of independent practical learning through the acquisition and enhancement of research skills and criticalthinking abilities.2 Furthermore, as such experiments can be designed to be research-led, it is likely that students can also benefit from a deeper understanding of the subject matter through engagement via such an active and problem-based approach.3 An area where there is little practical support available to instructors yet is exciting for undergraduate students to explore is the development of vehicles for controlled drug delivery. Smart polymeric hydrogels have been explored as “intelligent” delivery systems that can release entrapped therapeutic moieties at the appropriate time and site of action in response to specific physiological triggers.4 A hydrogel is a network of hydrophilic polymer chains that is typically composed of over 90% water. Smart polymeric hydrogels are macromolecular networks that can exhibit reversible conformational rearrangements upon variation in the local environment. Indeed, pH-sensitive hydrogels have been utilized to selectively deliver antibiotics to the gastrointestinal (GI) tract to treat Helicobacter pylori infections as well as to release cancer therapeutics in tumor cells.5,6 Cationic hydrogels such as chitosan are suited to this purpose because of their ability to swell at acidic pH. Chitosan is produced from the thermochemical alkaline deacetylation of chitin, a natural and abundant polymer found in the exoskeletons of invertebrates. However, to enhance its stability, chitosan is routinely cross-linked with agents such as glutaraldehyde, © XXXX American Chemical Society and Division of Chemical Education, Inc.

formaldehyde, or epoxy compounds, all of which pose serious hazards and are not suitable for undergraduate use.7−10 Furthermore, as an alternative, temperature-sensitive poly(Nisopropylacrylamide)-based hydrogels have been utilized for drug delivery, although these also require the use of ammonium persulfate as an initiator, which poses systemic health hazards.11 To this end, a Green Reagents and Sustainable Processes (GRASP) approach was employed to substitute the aforementioned cross-linking agents with a safe and green alternative, and the resultant gel was implemented for use with undergraduate students as part of a PBL mini-project.12 Genipin is a green cross-linking agent obtained from geniposide in gardenia fruits, and it reacts with primary amines to form a fluorescent structure with blue pigmentation.13 The fluorescent attributes provide a basis for students to investigate fluorescence as a function of gelation time. Second, poly(vinyl alcohol) (PVA) can be incorporated into the network by means of hydrogen bonding to chitosan moieties in order to enhance the elasticity of the bulk material. This study outlines the possible evaluative methodologies students can utilize in order to determine the suitability of a “green” genipin-cross-linked chitosan−PVA (G-Ch-PVA) hydrogel for use in delivery to the GI tract. The chemical structures of the gel constituents are shown in Figure 1. This laboratory experiment builds on work conducted by Chen et al.,14 in which an anionic hydrogel in the form of a Special Issue: Polymer Concepts across the Curriculum Received: March 29, 2017 Revised: July 12, 2017

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DOI: 10.1021/acs.jchemed.7b00235 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

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Laboratory Experiment

MATERIALS Chitosan (medium molecular weight, 428,180 g mol−1), PVA (molecular weight 146,000−186,000 g mol−1), glacial acetic acid, paracetamol, and genipin were obtained from SigmaAldrich in the United Kingdom. Glycine (pH 2), phthalate, (pH 4), and phosphate (pH 7) buffers were obtained from Fisher Scientific in the United Kingdom. Chitosan was dissolved at room temperature in a 1% aqueous acetic acid solution (pH 2.6) with continuous mechanical stirring for 24 h to obtain a 1.5% (w/v) pale-yellow, viscous solution. PVA was dissolved in distilled water at 85 °C with continuous mechanical stirring for 1 h to yield a 5% (w/v), transparent, homogeneous solution. A 0.5% (w/v) genipin solution was produced by dissolving genipin in distilled water for 15 min using a sonication bath at room temperature. Enzyme-free simulated gastric fluid (pH 2.0) was prepared by dissolving 1.03 g of sodium chloride in 50 mL of distilled water. Following this, 870 μL of 3 M hydrochloric acid was added to the sodium chloride solution. This was then diluted to 200 mL with distilled water to form the GI fluid.16

Figure 1. Chemical structures of chitosan, PVA, and genipin.

contact lens based on poly(2-hydroxyethyl methacrylate-comethacrylic acid) was used to demonstrate reversible pHresponsive behavior, and work by Neeves et al.,15 in which gelatin-based hydrogels were used to deliver food dye upon network cleavage with bromelin, a protease enzyme.15 Both of the prior laboratory experiments are for use at the high school level. This work utilizes a pH-responsive cationic hydrogel to explore the pH-sensitive swelling and reversibility behavior, the gel color and fluorescence intensity as functions of gelation time, the porous architecture, and the release of paracetamol (or p-acetylaminophenol, also known as acetaminophen) in simulated GI fluid and is hence appropriate for a team of upperdivision undergraduate students. Following hydrogel preparation, students may use UV−vis spectroscopy in conjunction with fluorescence spectroscopy to monitor gelation as a function of time. Students can conduct gravimetric swelling experiments in order to investigate how the network swells as a function of pH. Upon establishing the pH-dependent swelling behavior of the hydrogel, students can study how the network responds in the environment in which it will be applied (simulated gastric fluid solution). This not only allows students to determine how the hydrogel will respond in the target environment but also enables them to consider the effect of ionic strength on network swelling. Students also have the opportunity to confirm that the hydrogel exhibits reversible pH-sensitive swelling behavior by conducing oscillatory experiments. This not only confirms the utility of the hydrogel as a switchable material but also demonstrates its potential applicability in coupling the network with a reaction that oscillates in pH, exemplifying prospective implementation to facilitate pulsatile release of imbibed therapeutics. Students can also use UV−vis spectroscopy to monitor the release of a drug (in this case paracetamol) from the gel network while it is immersed in simulated gastric fluid solution. Finally, the porous structure of the hydrogel can be examined using optical microscopy. This is convenient, as in most cases scanning electron microscopy is routinely utilized in order to study the gel architecture, requiring sample pretreatment (hence leading to a change in network architecture) and expensive instrumentation unavailable for use with undergraduate students. This experiment allows students to explore the behavior of a smart hydrogel via numerous techniques in the context of developing a material suitable for application as a drug delivery vehicle. It is advantageous that the students are introduced to the system they will be exploring with respect to the application together with discussing potential characterization techniques that they may wish to explore in order to understand the associated structure−property relationships. The experiment is particularly relevant for a team of third-year undergraduate students studying materials science or polymer chemistry modules.

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EXPERIMENTAL METHODS

Hydrogel Preparation

The 1.5% (w/v) chitosan solution (1 mL), 5% (w/v) PVA solution (200 μL), and 0.5% (w/v) genipin solution (100 μL) were combined in cylindrical polyethylene vials (13 mm internal diameter and 5 mL volume). The mixtures were mechanically stirred at room temperature for 30 min and placed in an oven at 37 °C for various time periods. The gels were removed from the vials by inserting a pencil through the container after the base and lid were cut off. The cylindrical gels were cut into discs (12 mm diameter and approximately 3 mm thickness) using a ruler and a Stanley knife. Gels were stored at 2 °C before use. UV−Vis Spectroscopy of the Hydrogels

Hydrogels were prepared in polystyrene cuvettes (as opposed to polyethylene vials) and allowed to undergo gelation at 37 °C. At predetermined time intervals, scanning UV−vis spectra were recorded at 300−800 nm using a Shimadzu UV-1800 spectrometer. Distilled water was used as a reference in all cases. Fluorescence Spectroscopy of the Hydrogels

Fluorescence spectroscopy was utilized to record the excitation and emission spectra of hydrogels placed in polystyrene cuvettes using a Shimadzu RF-5301 fluorescence spectrometer. The excitation spectra were recorded using 462 nm as the emission wavelength while scanning the excitation wavelength from 250 to 800 nm. The emission spectra were recorded utilizing 360 nm as the excitation wavelength while scanning the emission wavelength from 250 to 900 nm. Both the excitation and emission spectra were recorded using a slit width of 5 nm. Optical Microscopy of the Hydrogels

Hydrogels were characterized using a Leica ICC50 HD optical microscope with a 10× magnification. Samples that were equilibrated in water (or an appropriate buffer solution) were placed on glass microscope slides, and the porous structures were analyzed. B



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Swelling Measurements on the Hydrogels

RESULTS This experiment has been completed by groups of six students as part of a third-year PBL mini-project in undergraduate chemistry. The groups completed the experiment within six lab days (totaling approximately 36 h in the laboratory). Representative data (averages of three tests) for all parts of the experiment are presented as follows. After an outline of the project was presented to students, they completed a risk assessment (available in the Supporting Information). Figure 2 shows the effect of varying the gelation

Swelling measurements were determined gravimetrically after the hydrogels had been immersed in various solutions to include glycine, phthalate, phosphate, and simulated gastric fluid. Preweighed hydrogels were immersed in one of the aforementioned solutions at a constant temperature (20 °C). At predetermined time intervals, swollen hydrogels were removed, and excess solution was blotted from the sample surfaces. To facilitate transfer of a sample from solution to the weighing scale, the hydrogel was enclosed in a weighing boat. The change in mass was then recorded, and the gravimetric swelling ratio was calculated according to eq 1:

W Ø = s Ø* Wd


(1)

where the swelling ratio (Ø/Ø*) is dimensionless, Ws is the weight of a swollen hydrogel (wet weight − dry weight), and Wd is the dry weight of the hydrogel. Both Ws and Wd are measured in grams. Oscillatory Swelling Measurements on the Hydrogels

The oscillatory response of the hydrogels was evaluated by manually alternating the pH of the solution in which samples were immersed between 2 (glycine buffer) and 7 (phosphate buffer) at a constant temperature (20 °C). Hydrogels were immersed in one of the solutions for 15 min, followed by determination of the swelling ratio as discussed previously, and then placed in the other buffer solution. This process was repeated to investigate the reversibility of swelling.

Figure 2. Variation in absorbance of a G-Ch-PVA mixture at 610 nm as a function of gelation time studied via UV−vis spectroscopy.

Prior to gelation, 10 mg of paracetamol was added to a mixture of chitosan, PVA, and genipin, which was placed in an oven at 37 °C for 24 h. A Shimadzu UV-1800 spectrometer was utilized to determine the wavelength at which the absorbance of paracetamol was greatest in aqueous solution (243 nm). A calibration curve of absorbance against paracetamol concentration was constructed, and the paracetamol-loaded hydrogel was immersed in 20 mL of simulated gastric fluid. The solution was maintained at 37 °C, and 500 μL aliquots were removed, diluted, and transferred to a polystyrene cuvette, where the absorbance at 243 nm was determined. The absorbance was monitored as a function of time, and the volume of the simulated gastric fluid solution was maintained throughout by addition of 500 μL of the simulated gastric fluid following each withdrawal.

time at 37 °C on the color of the G-Ch-PVA hydrogel as determined using UV−vis spectroscopy. On the basis of an initial scanning spectrum together with the fact that the hydrogel is blue, the absorbance at 610 nm was monitored as a function of gelation time. With increasing gelation time, the sample develops a dark-blue pigmentation, indicative of oxygenradical-induced polymerization of genipin and its reaction with amine functional groups (in chitosan) upon exposure to air.17,18 Figure 2 shows that the rate of cross-linking decreases after 12 h, as most of the amine groups have reacted to form cross-links. Subsequently, students investigated how the fluorescence intensity of the G-Ch-PVA hydrogel changes with gelation time (Figure 3) using fluorescence spectroscopy. Upon combination of chitosan and genipin, the network is known to fluoresce,19 and hence, the degree of cross-linking can be determined as a function of fluorescence intensity. From Figure 3 it is clear that the fluorescence intensity increases with gelation time up to a point where a plateau is likely to be reached, as all of the amine

HAZARDS Chitosan, PVA, sodium chloride, and the glycine, phthalate, and phosphate buffer solutions are not classified as hazardous substances, although normal lab practice should be followed. Genipin causes serious eye irritation and is also toxic if swallowed. Paracetamol is harmful if swallowed, causes skin irritation and serious eye irritation, and may cause respiratory irritation. Acetic acid is a flammable liquid and vapor, and it causess severe skin burns and irritation. Hydrochloric acid may be corrosive to metals, causes severe skin burns and eye damage, and may cause respiratory irritation. However, students are not required to handle acetic acid or hydrochloric acid, as the chitosan and simulated gastric fluid solutions can be prepared in advance. Students should wear safety goggles and a long-sleeved lab coat throughout the investigation. For safe disposal, the gels should be placed in a solid-waste container.

Figure 3. Variation in the fluorescence intensity of a G-Ch-PVA mixture followed by fluorescence emission spectroscopy at 440 nm as a function of gelation time.

UV−Vis Spectroscopy To Monitor Paracetamol Release

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Figure 4. Mechanism of the reaction between chitosan and genipin.

Figure 5. Proposed reaction mechanism for the formation of a conjugated genipin derivative.

groups have reacted to form cross-links. This behavior is in accordance with the variation in absorbance at 610 nm, indicating that the color of the network and the degree of fluorescence are linked. If students wish to study the mechanism of the reaction between chitosan and genipin, this is shown in Figure 4, and Figure 5 presents a proposed mechanism detailing how the autofluorescence arises from the formation of a conjugated genipin derivative.19 Upon formation of a hydrogel, changes in the swelling ratio with time can be determined upon varying the pH of the solution in which the sample was immersed (Figure 6). All of Figure 7. Oscillatory gravimetric swelling measurements on G-ChPVA hydrogels equilibrated in a pH 2 glycine buffer and then a pH 7 phosphate buffer in an alternating fashion with a time period of 15 min.

Figure 6. Gravimetric swelling measurements of G-Ch-PVA hydrogels in pH 2 (glycine), pH 4 (phthalate), and pH 7 (phosphate) buffer solutions.

the buffer solutions had the same ionic strength (0.36 mol kg−1). The pH-dependent behavior can be observed whereby the swelling ratio increases as the hydrogels are immersed in increasingly acidic media. The pH-dependent swelling is likely to arise from protonation of the amine functional groups within the network, which induces further electrostatic repulsion between such moieties, leading to hydrogel swelling. Following on from the study of pH-dependent swelling behavior, the swelling reversibility of the network can be demonstrated (Figure 7). After initial gel swelling, oscillatory behavior is observed when the acidic pH 2 buffer solution is exchanged between the gel matrix and the surrounding pH 7 phosphate buffer solution. Students investigated how the swelling ratio of the G-ChPVA hydrogel changes when the ionic strength of the solution in which it is immersed is varied by contrasting glycine at pH 2 with simulated gastric fluid at pH 2 (Figure 8). It is clear that when the hydrogel is immersed in the solution with low ionic

Figure 8. Gravimetric swelling measurements of G-Ch-PVA hydrogels in pH 2 glycine buffer solution and pH 2 simulated gastric fluid after immersion for 180 min.

strength, the swelling ratio after 180 min increases (for reference, when G-Ch-PVA hydrogels are immersed in distilled water, an average swelling ratio of 750% is recorded). Solutions with a high ionic strength result in an enhanced formation of ionic atmospheres, increasing the amount of charge screening, which leads to a decrease in ionic Coulomb potential (and hence charge distribution within the network). In turn, this lowers the Donnan potential (defined as the electrical potential between the gel and the surrounding solution), decreasing the osmotic pressure exerted by the gel and resulting in a lower swelling ratio. The potential of G-Ch-PVA hydrogels to release paracetamol in simulated gastric fluid as determined by UV−vis spectrosD



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copy is shown in Figure 9. This allows students to recognize that imbibed moieties can be released from the gel in its target environment where the pH is sufficiently low to induce network swelling.


ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00235. Student documentation (PDF, DOCX) Instructor presentation (PDF) Student risk assessment form (PDF, DOCX) Technician notes (PDF, DOCX) Student pro-forma (PDF, DOCX) Instructor pro-forma (PDF, DOCX) Mini-project marksheet (PDF, DOC) Poster marksheet (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Figure 9. Paracetamol release from G-Ch-PVA hydrogels in simulated gastric fluid determined via UV−vis spectroscopy and the simultaneous gravimetric swelling data recorded.

ORCID

Finally, optical microscopy can be used to examine the porous architecture of G-Ch-PVA hydrogels (Figure 10). This

The author declares no competing financial interest.

Glenn A. Hurst: 0000-0002-0786-312X Notes

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ACKNOWLEDGMENTS G.A.H. thanks the students who participated in this experiment and Dr. Nigel Lowe for producing the marking criteria. REFERENCES

(1) Mc Donnell, C.; O’Connor, C.; Seery, M. K. Developing Practical Chemistry Skills by Means of Student-Driven Problem-Based Learning Mini-Projects. Chem. Educ. Res. Pract. 2007, 8 (2), 130−139. (2) Flynn, A. B.; Biggs, R. The Development and Implementation of a Problem-Based Learning Format in a Fourth-Year Undergraduate Synthetic Organic and Medicinal Chemistry Laboratory Course. J. Chem. Educ. 2012, 89 (1), 52−57. (3) Healey, M. Linking Research and Teaching to Benefit Learning. Journal of Geography in Higher Education. 2005, 29 (2), 183−201. (4) Priya James, H.; John, R.; Alex, A.; Anoop, K. R. Smart Polymers for the Controlled Delivery of Drugs. Acta Pharm. Sin. B 2014, 4 (2), 120−127. (5) Vashist, A.; Vashist, A.; Gupta, Y. K.; Ahmad, S. Recent Advances in Hydrogel Based Drug Delivery Systems for the Human Body. J. Mater. Chem. B 2014, 2, 147−166. (6) Zhang, H.; Mardyani, S.; Chan, W. C. W.; Kumacheva, E. Design of Biocompatible Chitosan Microgels for Targeted pH-Mediated Intracellular Release of Cancer Therapeutics. Biomacromolecules 2006, 7 (5), 1568−1572. (7) Monteiro, O. A. C.; Airoldi, C. Some Studies of Crosslinking Chitosan-Glutaraldehyde Interaction in a Homogeneous System. Int. J. Biol. Macromol. 1999, 26 (2−3), 119−128. (8) Singh, A.; Narvi, S. S.; Dutta, P. K.; Pandey, N. D. External Stimuli Response on a Novel Chitosan Hydrogel Crosslinked with Formaldehyde. Bull. Mater. Sci. 2006, 29 (3), 233−238. (9) Wan Ngah, W. S.; Endud, C. S.; Mayanar, R. Removal of Copper(II) Ions from Aqueous Solution onto Chitosan and CrossLinked Chitosan Beads. React. Funct. Polym. 2002, 50 (2), 181−190. (10) Paddock, J. R.; Maghasi, A. T.; Heineman, W. R.; Seliskar, C. J. Making and Using a Sensing Polymeric Material for Cu2+. J. Chem. Educ. 2005, 82 (9), 1370−1371. (11) Schueneman, S. M.; Chen, W. Environmentally Responsive Hydrogels. J. Chem. Educ. 2002, 79 (7), 860−862. (12) Garrett, B.; Matharu, A. S.; Hurst, G. A. Using Greener Gels to Explore Rheology. J. Chem. Educ. 2017, 94 (4), 500−504. (13) Muzzarelli, R. A. A.; El Mehtedi, M.; Bottegoni, C.; Aquili, A.; Gigante, A. Genipin-Crosslinked Chitosan Gels for Tissue Engineering

Figure 10. Optical microscopy image of a G-Ch-PVA hydrogel.

is of particular importance to aid students in visualizing the conformation of the network at the microscale. As an extension, students can record images of how the architecture changes as a function of the pH of the solution in which the gel was immersed prior to observation. Following completion of the practical component of the project, students produce a group research poster and an individual report as part of the assessment. The marking criteria for the poster and the report are provided in the Supporting Information.

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CONCLUSIONS This experiment enables students to investigate a smart hydrogel for potential application as a targeted drug delivery vehicle via completion of a PBL mini-project. The research-led activity allows students to characterize the material through a multitude of experimental methodologies, helping students to develop an integrated understanding of the system. A GRASP approach was employed to identify and substitute harmful cross-linking agents for genipin, a green alternative, making this area of chemistry suitable for undergraduate students to practically explore. E




and Regeneration of Cartilage and Bone. Mar. Drugs 2015, 13 (12), 7314−7338. (14) Chen, Y.-H.; He, Y.-C.; Yaung, J.-F. Exploring pH-Sensitive Hydrogels Using an Ionic Soft Contact Lens: An Activity Using Common Household Materials. J. Chem. Educ. 2014, 91 (10), 1671− 1674. (15) Sylman, J. L.; Neeves, K. B. An Inquiry-Based Investigation of Controlled Drug-Delivery from Hydrogels: An Experiment for High School Chemistry and Biology. J. Chem. Educ. 2013, 90 (7), 918−921. (16) Bergstrand, M.; Soderlind, E.; Eriksson, U. G.; Weitschies, W.; Karlsson, M. O. A Semi-Mechanistic Modeling Strategy to Link In Vitro and In Vivo Drug Release for Modified Release Formulations. Pharm. Res. 2012, 29, 695−706. (17) Muzzarelli, R. A. A. Genipin-Crosslinked Chitosan Hydrogels as Biomedical and Pharmaceutical Aids. Carbohydr. Polym. 2009, 77, 1−9. (18) Mi, F. L.; Shyu, S. S.; Peng, C. K. Characterization of RingOpening Polymerisation of Genipin and pH Dependent Cross-Linking Reactions between Chitosan and Genipin. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 1985−2000. (19) Chen, H.; Ouyang, W.; Lawuyi, B.; Martoni, C.; Prakash, S. Reaction of Chitosan with Genipin and its Fluorogenic Attributes for Potential Microcapsule Membrane Characterization. J. Biomed. Mater. Res., Part A 2005, 75A, 917−927.

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Green and Smart: Hydrogels To Facilitate Independent Practical

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