Team:Evry/Pill design

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<h1> Capsule design </h1>
<h1> Capsule design </h1>
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<div class="center"><i>or how to transport our bacteria in the intestins</i></div>
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<div class="center"><i>a transport device to deliver <b><span style="color:#bb8900">Iron</span><span style="color:#7B0000"> Coli</span></b> to the intestine</i></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2013/9/9a/Page_capsule_bani%C3%A8re_tristan_ftw.png" alt="Bannière gélule" width="100%"/></div>
<h2>Abstract</h2>
<h2>Abstract</h2>
<p>
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For an optimum efficiency of our iron chelating bacteria, our purpose is to favor its growth in the proximal area of the jejunum. To target this region, we designed a capsule able to deliver our living bacteria in the jejunum and improved its galenic formulation to optimize growth.<br>
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To effectively treat iron over-absorption, we needed a device to deliver our <b><span style="color:#bb8900">Iron</span><span style="color:#7B0000"> Coli</span></b> bacteria to the distal area of the duodenum and the proximal area of the jejunum. Additionally, we want to give <b><span style="color:#bb8900">Iron</span><span style="color:#7B0000"> Coli</span></b> <a href="https://2013.igem.org/Team:Evry/Model3">enough time to produce enterobactins</a>. We thus designed and built a capsule with a methacrylic acid exterior that resists the low pH conditions in the stomach, but will dissolve at intestinal pH to release <b><span style="color:#bb8900">Iron</span><span style="color:#7B0000"> Coli</span></b>. The capsule interior contains colloidal silica and hydroxypropylmethylcellulose (HPMC), which ensures that the bacteria survive in the capsule and have enough time to uptake iron when released in the intestine.<br>
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<img src="https://static.igem.org/mediawiki/2013/5/5f/Capsule_legende.png" alt="capsule_legend" width="40%"/>
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Until now, we managed to design the capsule following the actual norms from the European Pharmacopeoa.<br>
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Our second step is to make our bacteria survive after the complete dissolution of our capsule.
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<div style="margin-left:0.5%;width:80%;float:center;border: 4px ridge black;padding:0.5%;font-size:90%;">
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<p id="norm">
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First, we create a <b>novel capsule following the actual norms from the European Pharmacopeoa</b> concerning gastro-enteric resistant formulations.<br><br>
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Second, we ensure that the bacteria survive following dissolution of the capsule.
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<h2>Capsule design requirements</h2>
<p>
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For a treatment based on bacteria as an medically active entity, we thought of the posology of such a capsule. The patient has to take the capsule on an empty stomach. The reasons are to reduce the exposure to gastric acidity, but also to give the bacteria enough time to grow once delivered in the duodenum/jejunum. In fact, right after the split into the duodenum, the obstruction caused by our galenic formulation has the right viscosity (not to tight, not to loose) to allow the passage of the food. To conclude, the strategy here is to take the capsule before a meal and let the bacteria sufficient time to settle and prepare to chelate the iron.
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We see three main challenges to construction of a device to deliver bacteria to the intestine. First, <b>the capsule must resist the low pH conditions in the stomach</b>, which are lethal to our bacteria. Second, <b>the capsule must rapidly dissolve in the duodenum</b> to deliver its payload to the distal duodenum and the proximal jejunum. Third, <b>the bacteria must have enough time to produce enterobactins to effectively uptake iron</b> before being excreted from the intestine.
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<h2>Barriers to overcome and conditions to fulfill</h2>
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<h2>Capsule design, step by step</h2>
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The challenge here is double. First, we need to overcome the acidity of the stomach which is lethal for most living forms. Secondly, our capsule must dissolve right in the duodenum to deliver the containment at the distal duodenum and the proximal jejunum. Additionnally, we added a component that optimizes the growth for bacteria delivered by a capsule, which will further be explained in more detail.<br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2013/3/37/Capsule.jpg" alt="Capsule" width="7%"/></div>
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<h2>Design of the capsule, step by step</h2>
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<h3>Which galenic formulation is best suited for our goals?</h3>
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<h3>Which galenic formulation suits the best our purpose?</h3>
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<p>
<p>
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The first step in the design of our pill is to determine its galenic formulation. We want a <i>per os</i> administration for our bacteria and had the choice between either a tablet or a capsule. A tablet not only requires a heavy and dry compression but also the bacteria in a lyophilised form. This last step consists of extreme variations of temperature and pression which are not favorable for living beings. However, the capsule is more suited for our purpose because it offers the possibility to contain a non-compressed powder and avoids a lyophilization step, thus representing a softer environment for bacteria transport. Additionnaly, lyophilized bacteria have a delayed recovery before starting any metabolic activity.
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The first step is to determine the galenic formulation of our pill. We want a <i>per os</i> administration for our bacteria and had the choice between either a tablet or a capsule. A tablet requires dry compression of its contents, meaning the bacteria would have to be lyophilized. We decided a capsule is more suited for our purpose because it can contain a non-compressed powder and avoids lyophilization, which would result in a significant delay in metabolic activity of the bacteria after being released.
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<div align="center"><img src="https://static.igem.org/mediawiki/2013/c/c6/IC_in_capsule.jpg" alt="Capsule" width="20%"/></div>
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<h3>How to store the bacteria in the capsule?</h3>
<h3>How to store the bacteria in the capsule?</h3>
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     <b>Figure 1:</b> Testing both moisture absorbents, colloidal silica and maltodextrin.
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     <b>Figure 1:</b> Testing both moisture absorbents.
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The design of a capsule requires every component to be in a powder form. Thus, we tested which one was able to absorb the most LB medium saturated with bacteria. In galenic research, this is called a <i>moisture absorbent</i> and has the right chemical properties to keep our drug (here our bacteria in LB medium) in a dry environment. We experimented <i<maltodextrin</i> and <i>colloidal silica</i> and tried to disperse as much as possible with hand mortar and pestle (figure 1 and 2). As a result, our final choice was <i>colloidal silica</i> for being able to absorb the most medium.<br>
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A pharmaceutical capsule requires every component to be in powder form. We tested which formulation was able to absorb the most LB medium saturated with bacteria. In galenic research, this is called a <i>moisture absorbent</i> and ensures the proper chemical properties to keep our drug (here our bacteria in LB medium) viable in a dry environment. We experimented with several compositions based on <i>maltodextrin</i> and <i>colloidal silica</i> (Figures 1 and 2) and found <i>colloidal silica</i> interacted better with LB medium.</p>
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For the next step, we want to increase the volume to 35 mL which is the sufficient amount to make 50 capsules (standard for one rack, figure 3). Thus, a <i>diluent</i> is required and we opted for HPMC (hydroxypropylmethylcellulose). The entire volume of powder has to be equally distributed in the capsules.
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We produced capsules in batches of 50 (standard for one rack, Figure 3). Here we carefully mix colloidal silica with the HPMC <i>diluent</i> such that the entire volume of powder is equally distributed in the capsules (Figure 4).
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     <b>Figure 5:</b> Testing both moisture absorbents, colloidal silica and maltodextrin.
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     <b>Figure 5:</b> Closing the capsules after a filling the rack.
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   <a title="Iron minion" href="https://static.igem.org/mediawiki/2013/e/ee/Closing-capsules.JPG">
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   <a title="Iron minion" href="https://static.igem.org/mediawiki/2013/6/62/Capsules-finished.jpg">
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     <img alt="Iron minion" src="https://static.igem.org/mediawiki/2013/e/ee/Closing-capsules.JPG" class="Picture"/>
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     <img alt="Iron minion" src="https://static.igem.org/mediawiki/2013/6/62/Capsules-finished.jpg" class="Picture"/>
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     <b>Figure 6:</b> Hand mortar and pestle, old fashioned but efficient.
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     <b>Figure 6:</b> Final result, 50 capsules uniformally filled with the powder mixture.
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<p>
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The goal of the next step is to increases the volume of powder in a sufficient amount to make 50 capsules (sufficient quantity for 35 mL, which is standard for one rack, figure 3). Thus, a <i>diluent</i> is required and we opted for HPMC (hydroxypropylmethylcellulose). The entire volume of powder has to be equally distributed in the capsules (figure 4, 5 and 6).
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<p style="padding-top:1%;">
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Finally, the 50 capsules contain a total of 11 ml of colloidal silica in which 4 mL of saturated bacteria have been dissolved in 24 mL of HPMC. The last step is sealing the capsules (Figures 5 and 6).
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<div align="center"><img src="https://static.igem.org/mediawiki/2013/3/33/IC_protected_against_acid.jpg" alt="Capsule" width="20%"/></div>
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<h3 id="acidity">How to overcome the acidity of the stomach?</h3>
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  <div class="captionedPicture" style="width:30%;float:left">
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  <a title="Dipping" href="https://static.igem.org/mediawiki/2013/2/24/Alcohol_solution.jpg">
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    <img alt="Dipping" src="https://static.igem.org/mediawiki/2013/2/24/Alcohol_solution.jpg" class="Picture"/>
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    <b>Figure 7:</b> Dipping of the capsule in an alcohol-based methacrylic acid polymer solution.
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  <a title="Hairdryer" href="https://static.igem.org/mediawiki/2013/c/c1/Hairdryer_capsule.jpg">
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    <img alt="Hairdryer" src="https://static.igem.org/mediawiki/2013/c/c1/Hairdryer_capsule.jpg" class="Picture"/>
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    <b>Figure 8:</b> Drying of the capsule by evaporating the alcohol.
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<p style="padding-top:1%;>
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It is a common challenge in pharmaceutical galenical research to bypass the acidity of the stomach and target a medicine to the duodenum/jejunum. To this end, we soaked the capsule in an ethanol-based solution of methacrylic acid and dried it with hot air. This method produced a capsule with a double envelope that resists gastric acidity.
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<div align="center"><img src="https://static.igem.org/mediawiki/2013/f/fd/Dissol_iron_coli.jpg" alt="Capsule" width="20%"/></div>
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<h3>How to overcome the acidity of the stomach?</h3>
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<h3 id="jejunum">How to deliver the bacteria in the intestine?</h3>
<p>
<p>
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It is very common in the pharmaceutical world, and more precisely in galenical research, to overcome the acidity of the stomach and to target the duodenum/jejunum for the delivery of the capsule. By a simple saoking of one side of the capsule in an ethanol-based solution of methacrylic acid and followed by a drying process (hot air), we managed to surround our capsule with a double-enveloppe resistant that confers resistance to gastric acidity. The capsule has to resist as long as possible to the gastric content, but not to much otherwise we miss its dissolution in the duodenum. To conclude, a double-coating is sufficient to fulfill this criteria.
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The gelatine-based composition of the capsule dissolves at neutral pH to deliver its payload to the duodenum. Right after the dissolution of the gelatine capsule, the HPMC swells to form a viscuous obstruction. This process creates an environment in the jejunum where bacteria can statically proliferate. Thus, HPMC, beside its properties as a <i>diluent</i>, is also called a <i>bio-adhesive</i> for its ability to stick to the membranes of the intestins and form an obstruction.
</p>
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<h3>How to deliver the bacteria in the jejunum?</h3>
 
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the target delivery in the duodenum/jejunum is possible thanks to the gelatine-based composition of the capsule. By a simple dissolution in water, it delivers its containment.<br>
 
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Additionnally, beside the moisture absorbent, we need to have a sufficient volume of powder to be able to make our capsules (in our case, 50 at a time). This is called a <i>diluent</i>. In galenic formulations, we have the choice between a lot of different diluents, but one particularly has some interesting properties: HPMC (HydroxyPropylMethylCellulose). It is able to swell in the presence of water and, depending on its density, is more or less viscuous. This process creates an environment where bacteria can statically proliferate. So, when the capsule splits into the duodenum, the swelling of the HPMC occurs in the jejunum and creates a temporary obstruction. In this situations, our bacteria will be able to proliferate in the obstruction area, thus optimizing growth. We had to choose the right viscosity to have a sufficient stability in the jejunum and to have the right amount for an optimal penetrance of the water. Thus, HPMC, beside its properties as a <i>diluent</i>, is also a <i>bio-adhesive</i> for its ability to stick to the membranes of the intestin and form an obstruction.
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<div align="center"><img src="https://static.igem.org/mediawiki/2013/6/68/Capsule_ironcoli_relargage.png" alt="Capsule" width="20%"/></div>
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<h2>Norms of the European Pharmacopeoa</h2>
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<h3 id="Norms">How to prove the real efficiency of our capsule?</h3>
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  <div class="captionedPicture" style="width:30%;float:left">
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  <a title="Hairdryer" href="https://static.igem.org/mediawiki/2013/1/1e/Dissolution_machine.jpg">
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    <img alt="Hairdryer" src="https://static.igem.org/mediawiki/2013/1/1e/Dissolution_machine.jpg" class="Picture"/>
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    <b>Figure 9:</b> The dissolution machine.
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  <a title="Hairdryer" href="https://static.igem.org/mediawiki/2013/4/4d/Dissolution_basket.jpg">
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    <img alt="Hairdryer" src="https://static.igem.org/mediawiki/2013/4/4d/Dissolution_basket.jpg" class="Picture"/>
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    <b>Figure 10:</b> Basket that dips the capsules into a liquid solution.
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Figure 1 and 2 show the material required to test the resistance of our capsule to the acidity. At the left side of the machine, a lever repeatedly moves the basket with the capsules inside the solution (figure 1). In the basket, 
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The dissolution machine lifts raises and lowers a basket into a 800mL beaker (Figure 9). In each experiment, we were able to test 6 capsules spread into 6 separate columns (Figure 10). These columns can be sealed on top with a lid to increase the dissolution and mimic different segments of the intestines.
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  <a title="Capsule integrity 1 hour" href="https://static.igem.org/mediawiki/2013/b/b7/081_25pc.jpg">
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    <b>Figure 11:</b> The capsules do not dissolve after 1 hour of exposure to gastric acid.
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    <img alt="Turbidity 1 hour" src="https://static.igem.org/mediawiki/2013/d/d1/082_25pc.jpg" class="Picture"/>
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    <b>Figure 12:</b> The acid solution is clear after containing the capsules for 1 hour.
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We observed that the capsules maintained their integrity after one hour of exposure to gastric acidity (Figure 11) and didn't release their contents into solution (Figure 12).
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  <a title="Capsule integrity 2 hours" href="https://static.igem.org/mediawiki/2013/e/e6/084_25pc.jpg">
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    <img alt="Capsule integrity 2 hours" src="https://static.igem.org/mediawiki/2013/e/e6/084_25pc.jpg" class="Picture"/>
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    <b>Figure 13:</b> Capsules resist dissolving after 2 hours of exposure to gastric acid.
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  <a title="Turbidity 2 hours" href="https://static.igem.org/mediawiki/2013/a/a1/083_25pc.jpg">
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    <img alt="Turbidity 2 hours" src="https://static.igem.org/mediawiki/2013/a/a1/083_25pc.jpg" class="Picture"/>
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    <b>Figure 14:</b> The acid solution remains clear after containing the capsules for 2 hours.
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We continued the experiment for an additional hour to confirm that even after two hours of exposure to a pH=2 solutes, the capsules were still not dissolved (Figure 13) and hadn't released their contents (Figure 14).
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  <a title="Final solution color" href="https://static.igem.org/mediawiki/2013/6/6b/085_25pc.jpg">
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    <img alt="Final solution color" src="https://static.igem.org/mediawiki/2013/6/6b/085_25pc.jpg" class="Picture"/>
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    <b>Figure 15:</b> Final acid solution color after containing the capsules for 2 hours.
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The acid solution was clear after removing the capsules (Figure 15), showing that the first condition of the European Pharmacopeoa to create a gastro-enteric resistant capsule has been fullfilled.
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  <a title="Capsule integrity 0 minute" href="https://static.igem.org/mediawiki/2013/c/ce/086_25pc.jpg">
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    <b>Figure 16:</b> Capsule dissolvement stage after 0 minute of exposure to PBS buffer (pH = 7,2).
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  <a title="Turbidity integrity 0 minute" href="https://static.igem.org/mediawiki/2013/a/ac/088_25pc.jpg">
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    <b>Figure 17:</b> PBS (pH = 7,2) turbidity at the beginning of the experiment.
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The capsules were fully intact when they were transferred to a PBS (Figure 16) and none of the contents were immediately released (Figure 17).
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  <a title="Capsule integrity 15 minutes" href="https://static.igem.org/mediawiki/2013/5/58/091_25pc.jpg">
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    <img alt="Capsule integrity 15 minutes" src="https://static.igem.org/mediawiki/2013/5/58/091_25pc.jpg" class="Picture"/>
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    <b>Figure 18:</b> Capsules begin dissolving after 15 minutes in PBS buffer (pH = 7,2).
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  <a title="Turbidity integrity 15 minutes" href="https://static.igem.org/mediawiki/2013/e/e9/090_25pc.jpg">
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    <img alt="Turbidity integrity 15 minutes" src="https://static.igem.org/mediawiki/2013/e/e9/090_25pc.jpg" class="Picture"/>
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    <b>Figure 19:</b> PBS buffer turbidity after containing capsules for 15 minutes.
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The capsules began to dissolve after 15 minutes in PBS buffer (Figure 18) and the dye solution began to be released (Figure 19).
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  <a title="Capsule integrity 30 minutes" href="https://static.igem.org/mediawiki/2013/0/02/096_25pc.jpg">
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    <img alt="Capsule integrity 30 minutes" src="https://static.igem.org/mediawiki/2013/0/02/096_25pc.jpg" class="Picture"/>
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    <b>Figure 20:</b> Capsules after 30 minutes in PBS.
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  <a title="Turbidity integrity 30 minutes" href="https://static.igem.org/mediawiki/2013/a/a6/095_25pc.jpg">
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    <img alt="Turbidity integrity 30 minutes" src="https://static.igem.org/mediawiki/2013/a/a6/095_25pc.jpg" class="Picture"/>
 +
  </a>
 +
  <div class="caption">
 +
    <b>Figure 21:</b> PBS turbidity after containing capsules for 30 minutes.
 +
  </div>
 +
  </div>
 +
 +
<p style="padding-top:1%;">
 +
The capsules continue to dissolve after 30 minutes in PBS buffer (Figure 20) and released their contents (Figure 21).
 +
<div style="clear: both;"></div>
 +
 +
  <div class="captionedPicture" style="width:30%;float:left">
 +
  <a title="Capsule integrity 40 minutes" href="https://static.igem.org/mediawiki/2013/b/bf/098_25pc.jpg">
 +
    <img alt="Capsule integrity 40 minutes" src="https://static.igem.org/mediawiki/2013/b/bf/098_25pc.jpg" class="Picture"/>
 +
  </a>
 +
  <div class="caption">
 +
    <b>Figure 22:</b> Capsules after 40 minutes exposure to PBS buffer (pH = 7,2).
 +
  </div>
 +
  </div>
 +
 +
  <div class="captionedPicture" style="width:30%;float:left">
 +
  <a title="Turbidity integrity 40 minutes" href="https://static.igem.org/mediawiki/2013/e/e7/097_25pc.jpg">
 +
    <img alt="Turbidity integrity 40 minutes" src="https://static.igem.org/mediawiki/2013/e/e7/097_25pc.jpg" class="Picture"/>
 +
  </a>
 +
  <div class="caption">
 +
    <b>Figure 23:</b> PBS turbidity after containing capsules for 40 minutes.
 +
  </div>
 +
  </div>
 +
 +
<p style="padding-top:1%;">
 +
The capsules continue to dissolve after 40 minutes in PBS buffer (Figure 22) and release their contents (Figure 23).
 +
 +
<div style="clear: both;"></div>
 +
 +
  <div class="captionedPicture" style="width:30%;float:left">
 +
  <a title="Capsule integrity 50 minutes" href="https://static.igem.org/mediawiki/2013/6/62/104_25pc.jpg">
 +
    <img alt="Capsule integrity 50 minutes" src="https://static.igem.org/mediawiki/2013/6/62/104_25pc.jpg" class="Picture"/>
 +
  </a>
 +
  <div class="caption">
 +
    <b>Figure 24:</b> Capsule dissolution after 50 minutes in PBS buffer (pH = 7,2).
 +
  </div>
 +
  </div>
 +
 +
  <div class="captionedPicture" style="width:30%;float:left">
 +
  <a title="Turbidity integrity 50 minutes" href="https://static.igem.org/mediawiki/2013/2/22/102_25pc.jpg">
 +
    <img alt="Turbidity integrity 50 minutes" src="https://static.igem.org/mediawiki/2013/2/22/102_25pc.jpg" class="Picture"/>
 +
  </a>
 +
  <div class="caption">
 +
    <b>Figure 25:</b> PBS turbidity after containing capsules for 50 minutes.
 +
  </div>
 +
  </div>
 +
 +
<p style="padding-top:1%;">
 +
The capsules continue to dissolve after 50 minutes in PBS buffer (Figure 24) and release their contents (Figure 25).
 +
<div style="clear: both;"></div>
 +
 +
  <div class="captionedPicture" style="width:30%;float:left">
 +
  <a title="Capsule integrity 60 minutes" href="https://static.igem.org/mediawiki/2013/2/2a/106_25pc.jpg">
 +
    <img alt="Capsule integrity 60 minutes" src="https://static.igem.org/mediawiki/2013/2/2a/106_25pc.jpg" class="Picture"/>
 +
  </a>
 +
  <div class="caption">
 +
    <b>Figure 26:</b> Capsules were fully dissolved after 60 minutes in PBS buffer (pH = 7,2).
 +
  </div>
 +
  </div>
 +
 +
  <div class="captionedPicture" style="width:30%;float:left">
 +
  <a title="Turbidity integrity 60 minutes" href="https://static.igem.org/mediawiki/2013/f/f8/105_25pc.jpg">
 +
    <img alt="Turbidity integrity 60 minutes" src="https://static.igem.org/mediawiki/2013/f/f8/105_25pc.jpg" class="Picture"/>
 +
  </a>
 +
  <div class="caption">
 +
    <b>Figure 27:</b> The PBS turbidity after 60 minutes of exposure to PBS buffer (pH = 7,2).
 +
  </div>
 +
  </div>
 +
 +
<p style="padding-top:1%;">
 +
The capsules were fully dissolved after 60 minutes in PBS (Figure 26) and had released their contents into the PBS solution (Figure 27).
 +
<div style="clear: both;"></div>
 +
</br>
 +
<h2 id="conclusion">Conclusion and perspectives</h2>
 +
<p>
 +
Our polymeric capsule successfully bypassed stomach acidity (pH=2) and rapidly dissolved to release its contents at neutral pH as in the duodenum and jejunum. The capsule thus fulfills the requirements of the European Pharmacopeia.
 +
</p>
 +
 +
<p>
 +
The purpose of this capsule is to <a href="https://2013.igem.org/Team:Evry/Model3>slow down the flush of bacteria</a> right after it is delivered in the duodenal region. Bacteria don't have enough time to produce their iron chelators. The addition of HPMC will create an obstruction in the area of iron absorption to allow enterobactin production.
 +
</p>
 +
 +
<p>
 +
Also, we calculated its cost and evaluated the <a href="https://2013.igem.org/Team:Evry/Economy"> impact</a> such a treatment could have on the health system. Blood-lettings are expensive and this treatment can be seen as an alternative or a complement to blood-lettings.
 +
</p>
 +
 +
<p>
 +
The next step for the capsule project is to mix the colloidal silica and hydroxypropylmethylcellulose with a concentrated culture of our siderophore-overexpressing <b><span style="color:#bb8900">Iron</span><span style="color:#7B0000"> Coli</span></b>. After encapsulating them in gelatine and methacrylic acid, we must establish that our bacteria can be released from the capsule and survive. If these tests are conclusive it could be possible to test the capsule in hemochromatosis mice.
 +
</p>

Latest revision as of 03:07, 29 October 2013

Iron coli project

Capsule design

a transport device to deliver Iron Coli to the intestine



Bannière gélule

Abstract

To effectively treat iron over-absorption, we needed a device to deliver our Iron Coli bacteria to the distal area of the duodenum and the proximal area of the jejunum. Additionally, we want to give Iron Coli enough time to produce enterobactins. We thus designed and built a capsule with a methacrylic acid exterior that resists the low pH conditions in the stomach, but will dissolve at intestinal pH to release Iron Coli. The capsule interior contains colloidal silica and hydroxypropylmethylcellulose (HPMC), which ensures that the bacteria survive in the capsule and have enough time to uptake iron when released in the intestine.

capsule_legend

First, we create a novel capsule following the actual norms from the European Pharmacopeoa concerning gastro-enteric resistant formulations.

Second, we ensure that the bacteria survive following dissolution of the capsule.


Capsule design requirements

We see three main challenges to construction of a device to deliver bacteria to the intestine. First, the capsule must resist the low pH conditions in the stomach, which are lethal to our bacteria. Second, the capsule must rapidly dissolve in the duodenum to deliver its payload to the distal duodenum and the proximal jejunum. Third, the bacteria must have enough time to produce enterobactins to effectively uptake iron before being excreted from the intestine.

Capsule design, step by step


Capsule

Which galenic formulation is best suited for our goals?

The first step is to determine the galenic formulation of our pill. We want a per os administration for our bacteria and had the choice between either a tablet or a capsule. A tablet requires dry compression of its contents, meaning the bacteria would have to be lyophilized. We decided a capsule is more suited for our purpose because it can contain a non-compressed powder and avoids lyophilization, which would result in a significant delay in metabolic activity of the bacteria after being released.


Capsule

How to store the bacteria in the capsule?

Iron minion
Figure 1: Testing both moisture absorbents.
Iron minion
Figure 2: Hand mortar and pestle, old fashioned but efficient.

A pharmaceutical capsule requires every component to be in powder form. We tested which formulation was able to absorb the most LB medium saturated with bacteria. In galenic research, this is called a moisture absorbent and ensures the proper chemical properties to keep our drug (here our bacteria in LB medium) viable in a dry environment. We experimented with several compositions based on maltodextrin and colloidal silica (Figures 1 and 2) and found colloidal silica interacted better with LB medium.



Iron minion
Figure 3: Preparing the rack with 50 empty capsules.
Iron minion
Figure 4: Filling the capsule with the total amount of powder.

We produced capsules in batches of 50 (standard for one rack, Figure 3). Here we carefully mix colloidal silica with the HPMC diluent such that the entire volume of powder is equally distributed in the capsules (Figure 4).



Iron minion
Figure 5: Closing the capsules after a filling the rack.
Iron minion
Figure 6: Final result, 50 capsules uniformally filled with the powder mixture.

Finally, the 50 capsules contain a total of 11 ml of colloidal silica in which 4 mL of saturated bacteria have been dissolved in 24 mL of HPMC. The last step is sealing the capsules (Figures 5 and 6).



Capsule

How to overcome the acidity of the stomach?

Dipping
Figure 7: Dipping of the capsule in an alcohol-based methacrylic acid polymer solution.
Hairdryer
Figure 8: Drying of the capsule by evaporating the alcohol.


Capsule

How to deliver the bacteria in the intestine?

The gelatine-based composition of the capsule dissolves at neutral pH to deliver its payload to the duodenum. Right after the dissolution of the gelatine capsule, the HPMC swells to form a viscuous obstruction. This process creates an environment in the jejunum where bacteria can statically proliferate. Thus, HPMC, beside its properties as a diluent, is also called a bio-adhesive for its ability to stick to the membranes of the intestins and form an obstruction.


Capsule

How to prove the real efficiency of our capsule?

As a proof of concept, we fulfilled the two basic requirements of the European Pharmacopeia to make a gastro-enteric resistant capsule which are as follows:

  1. No dissolution of the capsule after 2 hours of exposure to gastric acid (solution at pH = 2)
  2. Dissolution of the capsule within 1 hour of exposure to water (Phosphate Buffer solution, pH = 7) right afterwards

Hairdryer
Figure 9: The dissolution machine.
Hairdryer
Figure 10: Basket that dips the capsules into a liquid solution.

The dissolution machine lifts raises and lowers a basket into a 800mL beaker (Figure 9). In each experiment, we were able to test 6 capsules spread into 6 separate columns (Figure 10). These columns can be sealed on top with a lid to increase the dissolution and mimic different segments of the intestines.




Capsule integrity 1 hour
Figure 11: The capsules do not dissolve after 1 hour of exposure to gastric acid.
Turbidity 1 hour
Figure 12: The acid solution is clear after containing the capsules for 1 hour.

We observed that the capsules maintained their integrity after one hour of exposure to gastric acidity (Figure 11) and didn't release their contents into solution (Figure 12).

Capsule integrity 2 hours
Figure 13: Capsules resist dissolving after 2 hours of exposure to gastric acid.
Turbidity 2 hours
Figure 14: The acid solution remains clear after containing the capsules for 2 hours.

We continued the experiment for an additional hour to confirm that even after two hours of exposure to a pH=2 solutes, the capsules were still not dissolved (Figure 13) and hadn't released their contents (Figure 14).

Final solution color
Figure 15: Final acid solution color after containing the capsules for 2 hours.

The acid solution was clear after removing the capsules (Figure 15), showing that the first condition of the European Pharmacopeoa to create a gastro-enteric resistant capsule has been fullfilled.




Capsule integrity 0 minute
Figure 16: Capsule dissolvement stage after 0 minute of exposure to PBS buffer (pH = 7,2).
Turbidity integrity 0 minute
Figure 17: PBS (pH = 7,2) turbidity at the beginning of the experiment.

The capsules were fully intact when they were transferred to a PBS (Figure 16) and none of the contents were immediately released (Figure 17).

Capsule integrity 15 minutes
Figure 18: Capsules begin dissolving after 15 minutes in PBS buffer (pH = 7,2).
Turbidity integrity 15 minutes
Figure 19: PBS buffer turbidity after containing capsules for 15 minutes.

The capsules began to dissolve after 15 minutes in PBS buffer (Figure 18) and the dye solution began to be released (Figure 19).

Capsule integrity 30 minutes
Figure 20: Capsules after 30 minutes in PBS.
Turbidity integrity 30 minutes
Figure 21: PBS turbidity after containing capsules for 30 minutes.

The capsules continue to dissolve after 30 minutes in PBS buffer (Figure 20) and released their contents (Figure 21).

Capsule integrity 40 minutes
Figure 22: Capsules after 40 minutes exposure to PBS buffer (pH = 7,2).
Turbidity integrity 40 minutes
Figure 23: PBS turbidity after containing capsules for 40 minutes.

The capsules continue to dissolve after 40 minutes in PBS buffer (Figure 22) and release their contents (Figure 23).

Capsule integrity 50 minutes
Figure 24: Capsule dissolution after 50 minutes in PBS buffer (pH = 7,2).
Turbidity integrity 50 minutes
Figure 25: PBS turbidity after containing capsules for 50 minutes.

The capsules continue to dissolve after 50 minutes in PBS buffer (Figure 24) and release their contents (Figure 25).

Capsule integrity 60 minutes
Figure 26: Capsules were fully dissolved after 60 minutes in PBS buffer (pH = 7,2).
Turbidity integrity 60 minutes
Figure 27: The PBS turbidity after 60 minutes of exposure to PBS buffer (pH = 7,2).

The capsules were fully dissolved after 60 minutes in PBS (Figure 26) and had released their contents into the PBS solution (Figure 27).


Conclusion and perspectives

Our polymeric capsule successfully bypassed stomach acidity (pH=2) and rapidly dissolved to release its contents at neutral pH as in the duodenum and jejunum. The capsule thus fulfills the requirements of the European Pharmacopeia.

The purpose of this capsule is to impact such a treatment could have on the health system. Blood-lettings are expensive and this treatment can be seen as an alternative or a complement to blood-lettings.

The next step for the capsule project is to mix the colloidal silica and hydroxypropylmethylcellulose with a concentrated culture of our siderophore-overexpressing Iron Coli. After encapsulating them in gelatine and methacrylic acid, we must establish that our bacteria can be released from the capsule and survive. If these tests are conclusive it could be possible to test the capsule in hemochromatosis mice.