Team:Evry/Pill design

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Iron coli project

Capsule design

a transport device to deliver our bacteria to the intestine



Bannière gélule

Abstract

To effectively treat iron over-absorption, our engineered bacteria need to be delivered to the distal area of the duodenum and the proximal area of the jejunum. We thus designed a capsule with a methacrylic acid exterior that resists the low pH conditions in the stomach, but will dissolve at intestinal pH. 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.

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. 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 effectively uptake iron

Capsule design, step by step


Capsule

Which galenic formulation is best suited for our goals?

The first step in the design of our pill is to determine its galenic formulation. We want a per os administration for our bacteria and had the choice between either a tablet or a capsule. A tablet requires a heavy and dry compression and the bacteria in a lyophilized form, entailling high temperatures and pressures that would result in significant bacterial mortality. We decided a a 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 more favorable environment for bacteria storage. In addition, a capsule avoids lyophilizing the bacteria, which would delay their metabolic activity 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 and colloidal silica (figure 1 and 2). Our final choice was colloidal silica.



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

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 diluent is required and we opted for HPMC (hydroxypropylmethylcellulose). The entire volume of powder has to be 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 all together 11 ml of colloidal silica (where 4 mL of saturated bacteria have been dissolved in) and 24 mL of HPMC. The last step is the closing of the capsules (figure 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.

It is very common in pharmaceutical galenical research to overcome the acidity of the stomach and to target the duodenum/jejunum for medical delivery. 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.


Capsule

How to deliver the bacteria in the jejunum?

The delivery in the duodenum/jejunum is already possible due to the gelatine-based composition of the capsule. In contact to water, the capsule dissolves and delivers its containment in the duodenum. This is only feasable when the capsule has already resisted to the gastric acidity. Moreover, we added a HPMC as a diulent. In the presence of water, right after the dissolution of the gelatine capsule, it is able to swell. Depending on its density, the polymere forms a more or less 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: The basket that dips into the solution.

The dissolution machine is able to lift a basket up and down into a 800mL beaker (figure 9). For every experience, we were able to test 6 capsules spread into 6 apart columns (figure 10). Keep in mind that these columns have the possibility to be closed by a lid on the top to increase the dissolution and mimic different segments of the intestins.




Capsule integrity 1 hour
Figure 11: Capsule dissolvement stage after 1 hour of exposure to gastric acid.
Turbidity 1 hour
Figure 12: Water turbidity after 1 hour of exposure to gastric acid.

We observe that after one hour of exposure to gastric acidity, the capsules are not dissolved. They kept their integrity (figure 11) and didn't deliver its contanment (figure 12).

Capsule integrity 2 hours
Figure 13: Capsule dissolvement stage after 2 hours of exposure to gastric acid.
Turbidity 2 hours
Figure 14: Water turbidity after 2 hours of exposure to gastric acid.

We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).

Final solution color
Figure 15: Final solution color after 2 hours of exposure to gastric acidity.

We observe that the water is very clear (figure 15), thus meaning that the first condition of the European Pharmacopeoa to create a gastro-enteric resistant capsule is 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: Water turbidity after 0 minute of exposure to PBS buffer (pH = 7,2).

We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).

Capsule integrity 15 minutes
Figure 18: Capsule dissolvement stage after 15 minutes of exposure to PBS buffer (pH = 7,2).
Turbidity integrity 15 minutes
Figure 19: Water turbidity after 15 minutes of exposure to PBS buffer (pH = 7,2).

We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).

Capsule integrity 30 minutes
Figure 20: Capsule dissolvement stage after 30 minutes of exposure to PBS buffer (pH = 7,2).
Turbidity integrity 30 minutes
Figure 21: Water turbidity after 30 minutes of exposure to PBS buffer (pH = 7,2).

We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).

Capsule integrity 40 minutes
Figure 22: Capsule dissolvement stage after 40 minutes of exposure to PBS buffer (pH = 7,2).
Turbidity integrity 40 minutes
Figure 23: Water turbidity after 40 minutes of exposure to PBS buffer (pH = 7,2).

We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).

Capsule integrity 50 minutes
Figure 16: Capsule dissolvement stage after 50 minutes of exposure to PBS buffer (pH = 7,2).
Turbidity integrity 50 minutes
Figure 17: Water turbidity after 50 minutes of exposure to PBS buffer (pH = 7,2).

We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).

Capsule integrity 60 minutes
Figure 16: Capsule dissolvement stage after 60 minutes of exposure to PBS buffer (pH = 7,2).
Turbidity integrity 60 minutes
Figure 17: Water turbidity after 60 minutes of exposure to PBS buffer (pH = 7,2).

We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).