TIPS GUIDE Nanotechnology EN
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This document provides instructions for a classroom experiment on nanotechnology and drug delivery systems. The experiment uses calcium alginate beads to model encapsulated drug delivery. Students create calcium-alginate beads by dropping an alginate-dye solution into a calcium chloride solution, causing the beads to self-assemble. They then test how quickly different dye-filled beads release their contents in water versus milk, modeling drug release in different fluids. Comparing a red and blue dye, students observe the blue dye diffuses more quickly, demonstrating how a drug's chemical structure influences its release rate. The experiment teaches variables involved in developing controlled drug delivery at the nanoscale.
TIPS GUIDE Nanotechnology EN
- 1. TIPS GUIDE Nanotechnology Lewis, K. Meana, A. Montero, R. Colegio Corazón de María (Gijón) 0. Intro TIPS stands for Teaching Innovative Practices in STEM, an Erasmus+ KA219 funded project where four European schools from Belgium, France, Italy and Spain, have worked together for two years (2015-2017) sharing their best practices on STEM (Science, Technology, Engineering and Maths) subjects. More info on the project can be found on the project web: http://bit.ly/2rRCpEI This work is done under the terms and conditions of the Attribution-Noncommercial 3.0 Unported (CC BY-NC 3.0). The aim of this TIPS Guide is to share a best practice from a European STEM project among teachers, empowering them with the means to raise their students interest towards STEM and therefore supporting the attainment of important competences. On this Guide we will focus on nanotechnology and we will be showcasing one of the experiments from the nanOpinion project which the Spanish school coordinated in its country. 1. Abstract In this experiment we will illustrate through a simple model how a miniaturized drug delivery system is created and how the release of the drug herein contained can be controlled. The drug delivery system is created through a self- assembly encapsulation method, by using a natural polymer (alginate) and a food dye, which in the model represents the drug. In the experiment students can learn how the release of the “drug” depends from a number of variables, like the type of the drug encapsulated (this is tested by changing the food dye) and the media where the nanocapsules are released in. Overall the experiment is a simple way of discussing the many variables involved in developing nanoscale drug delivery systems. 2. Materials and methods Materials and stock solutions The items below are indicated assuming students will work in pairs. Each pair should get: 1 ice cube tray (preferably transparent; a normal 14 or 16 wells ice cube tray is fine) 5 disposable plastic pipettes (or glass pipettes) 6 glass vials with cap with a volume of 2-5 mL (or any other small glass or plastic container holding this volume, for example a test tube) 50 mL calcium chloride 0.3 M (from stock solution) 10 mL sodium alginate (with no food dye) 10 mL sodium alginate with red food dye 10 mL sodium alginate with blue food dye 50 mL distilled water 50 mL full fat milk 1 tweezers 1 empty medium size beaker (for washing) 1 glass rod for mixing (or a teaspoon) Paper towel Gloves Eye protection Before starting the experiment in class, teachers should prepare the stock solutions: Calcium chloride solution (0.3 M): 22.05 gr in 500 mL in distilled water Sodium alginate solution: dissolve 6 gr in 300 mL hot tap water. Note: add the alginate slowly as some lumps could form, stir the solution while preparing it. If lumps The European Commission support for the production of this publication does not constitute an endorsement of the contents which reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.
- 2. form, don’t worry, but don’t use them for making the beads. Roughly divide the amount in 3 beakers (approx. 100 mL each) and add food dye to two (4 droplets). TEST 1: Self-assembly a drug delivery system In the first part of the experiment students will learn a simple way of making “capsules” that in the second part of the experiment are used as “drug carriers”. The capsules form through a cross-linking process that occurs spontaneously when mixing alginate salt and calcium chloride. A regular, spherical structure is formed (in this document called “bead”), hence this is a simple example of self-assembly. Procedure NB: To create the calcium-alginate beads you will need a container of plastic that can hold about 5mL. It is suggested to use an ice cube tray as this is formed of different “wells” that can be used as reactors. A solution of alginate salt is added to a solution of CaCl2 using a pipette, drop-by-drop, from a distance of about 15 cm. Students will need only few droplets, so you can give a small beaker with some solution (with no food dye). As the drop falls into the CaCl2 solution, an opaque- white bead is form immediately. The beads form by self-assembly: the calcium ions are “trapped” inside the alginate salt and form a stable structure. Students are asked to repeat the test using tap water instead of CaCl2 and as an additional (optional) activity they can try to make them in milk. NB. Since milk is white and opaque, it is suggested to try this test using the red alginate solution. Students will observe that beads do not form, and this can lead to a discussion on why this happens (milk is not a calcium solution, etc.) TEST 2: A model of a “drug” release system In this second part of the experiment, students will create a model of a drug delivery system, where the capsule is made of calcium-alginate and the “drug” is simulated by a food dye. In this part of the experiment you should give each couple of students a small beaker with some red alginate solution and another beaker with some blue alginate solution (it is not important how much you give them, they will need only few droplets, 5 mL in each beaker is more than enough). As a first thing, students will prepare some red calcium-alginate beads. Students then compare the effect of immersing the beads in water and in milk, for different times. In this experiment the milk is used to simulate a biological fluid (contains proteins, minerals, fat molecules), therefore a much more complex media than water. It is suggested to encourage students to think about the possible effects this could have before running the test, what variables could be important (time, temperature, pH etc.). After writing their hypothesis, students should run the test, and write down their observations. Students will observe that the red dye is stable inside the bead, no release is observed after relatively short times. However if the beads are left in water overnight, some release is observed. Figure 1. Calcium beads do not form in milk To (visually) estimate the amount of release, it is suggested to remove the surfactant from the vial, place it in a new vial and compare it with a control (containing only water). The beads left in milk have also release some dye, and comparing the beads extracted from water with those extracted from milk shows a clear difference. After being soaked in milk overnight (Figure 2): The European Commission support for the production of this publication does not constitute an endorsement of the contents which reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.
- 3. Figure 2. : Red calcium alginate beads after overnight immersion in surfactant (left); water (middle) and milk (right). TEST 3: Influence of the drug The importance of the type of “drug” entrapped in the capsule is investigated in TEST 3, where students compare what happens when a different food dye is used (blue food dye). As with TEST 2, it is encouraged that students think what the effect of using a food dye with a different “colour” might be. Then they prepare some blue calcium-alginate beads and then repeat the same diffusion test in water and in milk. Figure 3. Calcium alginate beads with a blue food dye. It should be noted that the blue dye diffuses very quickly out of the calcium alginate beads, after 15 minutes a clear difference is noted compared with the red calcium alginate beads. Students are encouraged to reflect upon why this happens and should come to the conclusion that the chemical structure of the dye and its interaction (or lack of) with the calcium alginate polymer must be different (variables are charge, pKa of functional groups, conformation, hydrogen bridges...). Figure 4. Blue calcium alginate beads after overnight immersion in milk (left); water (middle) and surfactant (right). 3. Results and evaluation Sodium alginate is a polymer obtained from seaweed. It has a linear structure with many carboxyl groups sticking out (a “carboxyl group” is a combination of carbon atoms and two oxygen atoms carrying a single negative charge). When a solution of sodium alginate is combined with a solution of calcium chloride, the calcium ions (Ca2+ , with a double positive charge) are able “bridge” two different alginate strands. The result is a cross-linked polymer which has a gel-like consistency (Figure 5). Figure 5. (Above) Sodium alginate structure (repeat unit); (Below) schematic representation of sodium alginate crosslinked polymer (through calcium atoms). Beads form immediately in CaCl2; the shape of the bead is spherical unless the droplet is released too closely/low above the CaCl2 solution. To appreciate this process, students can try making the beads in solutions alternative to CaCl2 that they probably expect to contain some Ca2+ The European Commission support for the production of this publication does not constitute an endorsement of the contents which reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.
- 4. ions, for instance tap water and milk. In both cases, beads don’t form. Milk is suggested because students will have the general knowledge that milk “contains calcium”. However, milk is not a solution of calcium ions, rather a colloid, where calcium ions are trapped inside between an ionic solution and a colloid). The different diffusion behaviour of the red and blue dye is most likely due to the difference in chemical structure and charge between the two materials. Figure 6 provides the chemical structure of the dyes contained in the food dyes solutions: Figure 6. The chemical structures of red and blue food dye used in this experiment. As it can be seen, the two dyes have a very different chemical structure. It is not the intention of the author to investigate in details this matter, which requires an advanced understanding of chemistry, not suitable for the age group at which this experiment is targeted. The suggestion to teachers is to raise the issue of chemical difference between the dyes focusing on how this might influence the way the dye is “trapped” inside the bead and its “willingness” to detach from it. The ultimate aim is to get students to realize that, when designing a drug delivery system, scientists need to consider numerous variables, among which the type of drug that needs to be encapsulated and carried: the capsule should carry the drug but then eventually release it! Figure 7. The beads of red and blue food dye used in this experiment. 4. Acknowledgements The authors would like to thank the Erasmus+ programme of the European Union for funding the TIPS project under grant 2015-1-ES01-KA219- 015719_1. 5. References 1. B. Criswell «Connecting acids and bases with encapsulation...and chemistry and nanotechnology», Journal of Chemical Education (2007), 84:7, 1136-1139 2. H. G. Bagaria et al., «Self-assembly and nanotechnology: real-time, hands-on, and safe experiments for K-12 students», Journal of Chemical Education (2011), 88, 609-614 3. L. Filipponi, D. Sutherland, "Nanotechnologies, Principles, Applications, Implications and Hands-On Activities, A compendium for educators", 2013, free to download at: http://ec.europa.eu/research/industrial_tech nologies/publications-reports_en.html . See in particular pages: 166-171 4. ”A step toward minute factories that produce medicine inside the body”: http://portal.acs.org/portal/acs/corg/conten t? _nfpb=true&_pageLabel=PP_ARTICLEMAIN& node_id=223&content_id=CNBP_030456&use _sec=true&sec_url_var=region1&_uuid=d9bfe 4ed-47b3-427c-8a8e-a9383841664f The European Commission support for the production of this publication does not constitute an endorsement of the contents which reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.