TIPS GUIDENanotechnology

Lewis, K. Meana, A. Montero, R.Colegio Corazón de María (Gijón)

0. Intro

TIPS stands for Teaching Innovative Practices inSTEM, an Erasmus+ KA219 funded project wherefour 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 onthe project web: http://bit.ly/2rRCpEI This work is done under the terms and conditionsof the Attribution-Noncommercial 3.0 Unported(CC BY-NC 3.0). The aim of this TIPS Guide is to share a bestpractice from a European STEM project amongteachers, empowering them with the means toraise their students interest towards STEM andtherefore supporting the attainment of importantcompetences. On this Guide we will focus onnanotechnology and we will be showcasing oneof the experiments from the nanOpinion projectwhich the Spanish school coordinated in itscountry.

1. Abstract

In this experiment we will illustrate through asimple model how a miniaturized drug deliverysystem is created and how the release of thedrug herein contained can be controlled. Thedrug delivery system is created through a self-assembly encapsulation method, by using anatural polymer (alginate) and a food dye, whichin the model represents the drug. In theexperiment students can learn how the releaseof the “drug” depends from a number ofvariables, like the type of the drug encapsulated(this is tested by changing the food dye) and themedia where the nanocapsules are released in.

Overall the experiment is a simple way ofdiscussing the many variables involved indeveloping nanoscale drug delivery systems.

2. Materials and methods

Materials and stock solutionsThe items below are indicated assumingstudents will work in pairs. Each pair should get:

1 ice cube tray (preferably transparent; anormal 14 or 16 wells ice cube tray is fine)

5 disposable plastic pipettes (or glasspipettes)

6 glass vials with cap with a volume of 2-5mL (or any other small glass or plasticcontainer holding this volume, for example atest tube)

50 mL calcium chloride 0.3 M (from stocksolution)

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 in500 mL in distilled water

Sodium alginate solution: dissolve 6 gr in300 mL hot tap water. Note: add thealginate 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.

form, don’t worry, but don’t use them formaking the beads. Roughly divide theamount in 3 beakers (approx. 100 mL each)and add food dye to two (4 droplets).

TEST 1: Self-assembly a drug delivery systemIn the first part of the experiment students willlearn a simple way of making “capsules” that inthe second part of the experiment are used as“drug carriers”. The capsules form through across-linking process that occurs spontaneouslywhen mixing alginate salt and calcium chloride. Aregular, spherical structure is formed (in thisdocument called “bead”), hence this is a simpleexample of self-assembly.

ProcedureNB: To create the calcium-alginate beads you willneed a container of plastic that can hold about5mL. It is suggested to use an ice cube tray asthis is formed of different “wells” that can beused as reactors.A solution of alginate salt is added to a solutionof CaCl2 using a pipette, drop-by-drop, from adistance of about 15 cm. Students will need onlyfew droplets, so you can give a small beakerwith some solution (with no food dye). As thedrop falls into the CaCl2 solution, an opaque-white bead is form immediately. The beadsform by self-assembly: the calcium ions are“trapped” inside the alginate salt and form astable structure.Students are asked to repeat the test using tapwater instead of CaCl2 and as an additional(optional) activity they can try to make them inmilk.NB. Since milk is white and opaque, it issuggested to try this test using the red alginatesolution. Students will observe that beads do notform, and this can lead to a discussion on whythis happens (milk is not a calcium solution, etc.)

TEST 2: A model of a “drug” release system

In this second part of the experiment, studentswill create a model of a drug delivery system,where the capsule is made of calcium-alginateand the “drug” is simulated by a food dye. In this

part of the experiment you should give eachcouple of students a small beaker with some redalginate solution and another beaker with someblue alginate solution (it is not important howmuch you give them, they will need only fewdroplets, 5 mL in each beaker is more thanenough).As a first thing, students will prepare some redcalcium-alginate beads.Students then compare the effect of immersingthe beads in water and in milk, for differenttimes. In this experiment the milk is used tosimulate a biological fluid (contains proteins,minerals, fat molecules), therefore a much morecomplex media than water.It is suggested to encourage students to thinkabout the possible effects this could have beforerunning the test, what variables could beimportant (time, temperature, pH etc.). Afterwriting their hypothesis, students should runthe test, and write down their observations.Students will observe that the red dye is stableinside the bead, no release is observed afterrelatively short times. However if the beads areleft in water overnight, some release is observed.

Figure 1. Calcium beads do not form in milk

To (visually) estimate the amount of release, it issuggested to remove the surfactant from thevial, place it in a new vial and compare it with acontrol (containing only water).The beads left in milk have also release somedye, and comparing the beads extracted fromwater with those extracted from milk shows aclear difference. After being soaked in milkovernight (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.

Figure 2. : Red calcium alginate beads after overnightimmersion in surfactant (left); water (middle) and milk(right).

TEST 3: Influence of the drugThe importance of the type of “drug” entrappedin the capsule is investigated in TEST 3, wherestudents compare what happens when adifferent food dye is used (blue food dye). Aswith TEST 2, it is encouraged that students thinkwhat the effect of using a food dye with adifferent “colour” might be.Then they prepare some blue calcium-alginatebeads and then repeat the same diffusion test inwater and in milk.

Figure 3. Calcium alginate beads with a blue food dye.

It should be noted that the blue dye diffuses veryquickly out of the calcium alginate beads, after15 minutes a clear difference is noted comparedwith the red calcium alginate beads. Studentsare encouraged to reflect upon why this happensand should come to the conclusion that thechemical structure of the dye and itsinteraction (or lack of) with the calciumalginate polymer must be different (variables arecharge, pKa of functional groups, conformation,hydrogen bridges...).

Figure 4. Blue calcium alginate beads after overnightimmersion in milk (left); water (middle) and surfactant(right).

3. Results and evaluationSodium alginate is a polymer obtained fromseaweed. It has a linear structure with manycarboxyl groups sticking out (a “carboxyl group”is a combination of carbon atoms and two oxygenatoms carrying a single negative charge). When asolution of sodium alginate is combined with asolution of calcium chloride, the calcium ions(Ca2+, with a double positive charge) are able“bridge” two different alginate strands. The resultis a cross-linked polymer which has a gel-likeconsistency (Figure 5).

Figure 5. (Above) Sodium alginate structure (repeatunit); (Below) schematic representation of sodiumalginate crosslinked polymer (through calcium atoms).

Beads form immediately in CaCl2; the shape ofthe bead is spherical unless the droplet isreleased too closely/low above the CaCl2

solution.To appreciate this process, students can trymaking 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.

ions, for instance tap water and milk. In bothcases, beads don’t form. Milk is suggestedbecause students will have the generalknowledge that milk “contains calcium”.However, milk is not a solution of calcium ions,rather a colloid, where calcium ions are trappedinside between an ionic solution and a colloid).The different diffusion behaviour of the red andblue dye is most likely due to the difference inchemical structure and charge between the twomaterials. Figure 6 provides the chemicalstructure of the dyes contained in the food dyessolutions:

Figure 6. The chemical structures of red and blue fooddye used in this experiment.

As it can be seen, the two dyes have a verydifferent chemical structure. It is not theintention of the author to investigate in detailsthis matter, which requires an advancedunderstanding of chemistry, not suitable for theage group at which this experiment is targeted.The suggestion to teachers is to raise the issue ofchemical difference between the dyes focusingon how this might influence the way the dye is“trapped” inside the bead and its “willingness” todetach from it. The ultimate aim is to getstudents to realize that, when designing a drugdelivery system, scientists need to considernumerous variables, among which the type ofdrug that needs to be encapsulated and carried:the capsule should carry the drug but theneventually release it!

Figure 7. The beads of red and blue food dye used inthis experiment.

4. AcknowledgementsThe authors would like to thank the Erasmus+programme of the European Union for fundingthe TIPS project under grant 2015-1-ES01-KA219-015719_1.

5. References1. B. Criswell «Connecting acids and bases

with encapsulation...and chemistry andnanotechnology», Journal of ChemicalEducation (2007), 84:7, 1136-1139

2. H. G. Bagaria et al., «Self-assembly andnanotechnology: real-time, hands-on, andsafe experiments for K-12 students», Journalof Chemical Education (2011), 88, 609-614

3. L. Filipponi, D. Sutherland,"Nanotechnologies, Principles, Applications,Implications and Hands-On Activities, Acompendium for educators", 2013, free todownload at:http://ec.europa.eu/research/industrial_technologies/publications-reports_en.html . Seein particular pages: 166-171

4. ”A step toward minute factories that producemedicine inside the body”:htt p:/ /port al . acs .org /port al / acs / corg/ conte n t ? _nf pb =t rue&_page Label =PP _ARTI CLE MAIN &n od e _ i d = 223 &c o n t e n t _ i d = C N B P _ 0 3 0456 &u s e _ s e c= t r u e & se c _ u r l _ v ar = r e g i on 1 &_ uu i d=d 9 b f e4 e d - 47 b 3 - 427 c - 8a 8 e - a938384166 4f

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.