Team:IISER Bhopal/Implementation Drug delivery

iGEM 2020 || IISER_Bhopal Drug delivery

Drug Delivery

Capsule for delivery - iβeta

Capsule delivery, being a major part of our IHP turned out quite different from what we had envisioned it to be. While the capsule had started as a simple delivery system which would release our drug in the duodenum directly, discussion with Prof. Padma Devarajan helped us ascertain the exact point of drug release. In addition, she also helped us choose the right capsule and helped us improve the efficiency of our drug by advising us to use a muco-penetrative formulation which would help more bacteria cross the mucous layer and reach the crypt cells. She also advised us on prescribing the timing for the patients based on the type of capsule we’ve created.
Here is a Step by Step explanation of the process we intend to follow to create our capsule for our bacterial drug.

Biomanufacturing

MUCO-PENETRATIVE FORMULATION

Microsphere properties include charge and hydrophilicity which have a great influence on their behaviour of penetrating through mucosal barrier. As a consequence, microspheres with a sufficiently hydrophilic and uncharged surface can effectively minimize the adhesive interactions with mucin by reducing hydrophobic or electrostatic interactions, enabling muco-penetration to reach the crypt cells in the duodenum.

Step 1

Preparation of Polymeric Microspheres Polymeric microsphere based drug carriers are spherical particles in the size range of several to hundreds of microns. They can protect unstable drugs during administration. They release the drug over time, providing a prolonged therapeutic effect and reducing the dosing frequency. In addition, microspheres allow for delivery of potent drugs at reduced concentrations, minimizing systemic exposure and adverse side effects.

Microspheres are made from biodegradable polymers such as poly(lactide-co-glycolide) (PLGA). These polymers degrade in vivo by hydrolysis of their ester backbone into non-toxic products, which are excreted by the kidneys or eliminated as CO2 and water through biochemical pathways. PLGA microspheres have been widely used to encapsulate drug molecules and have been used as long-acting, sustained-release pharmaceutical formulations. There are several drug-loaded PLGA microspheres approved by the FDA and marketed for clinical use. When drug-loaded PLGA microspheres are administered, the PLGA polymer starts to degrade in vivo, and as it degrades, the drug molecules are gradually released from the microspheres.

Emulsion solvent evaporation (ESE) Process
ESE can be used to prepare drug-loaded polymeric microspheres. The polymer - PLGA is dissolved in a solvent that is not miscible with water and emulsified using an overhead stirrer, in an aqueous solution containing a surfactant or a polymeric stabilizer, with E. coli Nissle added to the emulsion. After the oil-in-water emulsion is formed, the solvent is removed by evaporation. The particles are washed to remove the surfactant and possible unincorporated drug molecules.

Following is a typical protocol for preparing PLGA microspheres using the ESE process:

  1. Prepare a 5% PLGA solution by dissolving PLGA polymer in methylene chloride. Selected Lactide/Glycolide ratio could vary from 50/50 to 85:15 with different intrinsic viscosities of 0.55–0.75 dL/g.
  2. Prepare a 1% solution of polyvinyl alcohol (PVA) (Mw 85,000–124,000, 87–89% hydrolyzed) solution in DI water.
  3. Mix 5 mL of the 5% PLGA solution prepared in Step 1 with 100 mL of the 1% PVA solution prepared in Step 2 in a 500 mL beaker by continuous mixing using an overhead stirrer.
  4. Keep the emulsion stirred in a fume hood for about 3 h to allow the methylene chloride to evaporate.
  5. Wash the microspheres three times by centrifuginig in a refrigerated centrifugeand discarding the supernatant.
  6. Disperse the pellet in DI water (A).

Step 2

PEGylation of microspheres PEG (Polyethylene Glycol), US FDA - approved polymer is the most prominent noninteracting polymer used for muco-penetrative formulation of drug delivery. It is widely used as coating material in a variety of oral and nanosystems. The PEG‐coating equips the microspheres with an electrically neutral, hydrophilic surface, which prevents hydrophobic and electrostatic interactions with the mucus to facilitate mucopenetration.

PEG 2000

Coating microspheres with low molecular weight (MW) PEG is the most widely studied mucus penetrating strategy. Research has shown that coating with a high density of low MW PEG can reduce the interactions between microspheres and mucus. The experimental results showed that low MW (e.g. 2 kDa) and high-density (e.g. 65–70%) PEG coating can facilitate the NPs to pass through mucus.

PEGylation of the E.coli loaded microspheres is achieved by adding PEG to the dispersion of the pellet in DI water (A) prepared in step 1 and incubating in a refrigerator for about 6-8 hours to allow PEG to adsorb on the microspheres (Dispersion B) as shown in the figure below.

PEGylated microspheres penetrate the mucus layer to reach epithelial cells

FREEZE DRYING/Lyophilisation

This is a dehydration process. The process is performed by freezing probiotics in the presence of carrier material at low temperatures, followed by sublimation of the water to water vapour under vacuum. One of the most important advantages is the water phase transition, and oxidation are avoided. To improve the probiotic activity upon freeze-drying and also stabilize them during storage, there is the addition of cryoprotectants. Freeze-drying is a costly technology; it is probably most often used to dry probiotics.

Procedure

To Dispersion B trehalose is added as a cryoprotectant and the sample freeze dried by freezing to -20oC and then drying at pressures well below the triple point of water. The steps are shown in the flowsheet below:

HPMC CAPSULE

Capsule Name - ACGCAPSTM HD

  • Delays release of formulation by up to 60 minutes or until the capsules is in the intestine (i.e. pH > 5.5)
  • Eliminates the need for additional protective coating of formulation or capsule
  • Made from natural plant sourced cellulose
  • Ideal choice for products like probiotics and enzymes to prevent degradation
  • Easier and more convenient way to develop a modified release product
  • Meets the functional, cultural and dietary preferences & compliances

CAPSULE SIZE CHART

After our experimental trials interpret the amount of dosage required for a patient, we will use the capsule to incorporate the microspheres as per the standard size chart given below.

Size 000 00 0 1 2 3 4 5
Density
0.6 g/ml		 822 mg 570 mg 408 mg 300 mg 222 mg 180 mg 126 mg 78 mg
Density
0.8 g/ml 		1096 mg 760 mg 544 mg 400 mg 296 mg 240 mg 168 mg 104 mg
Density
1 g/ml 		1370 mg 950 mg 680 mg 500 mg 370 mg 300 mg 210 mg 130 mg
Density
1.2 g/ml 		1644 mg 1140 mg 816 mg 600 mg 444 mg 360 mg 252 mg 156 mg

References

  1. L.E.Garcia-Amezquita, J.Welti-Chanes, F.T.Vergara-Balderas, D.Bermúdez-Aguirre. Freeze-drying: The Basic Process. https://doi.org/10.1016/B978-0-12-384947-2.00328-7
  2. Essi Taipaleenmäki Brigitte Städler (2020) Recent Advancements in Using Polymers for Intestinal Mucoadhesion and Mucopenetration. https://doi.org/10.1002/mabi.201900342
  3. Lengyel, M.; Kállai-Szabó, N.; Antal, V.; Laki, A.J.; Antal, I. Microparticles, Microspheres, and Microcapsules for Advanced Drug Delivery. Sci. Pharm. 2019, 87, 20. http://dx.doi.org/10.3390/scipharm87030020
  4. Min Liu, Jian Zhang, Wei Shan, Yuan Huang (2015) Developments of mucus penetrating nanoparticles. Asian Journal of Pharmaceutical Sciences. https://doi.org/10.1016/j.ajps.2014.12.007
  5. Y.Y. Wang, S.K. Lai, J.S. Suk, et al. Addressing the PEG mucoadhesivity paradox to engineer nanoparticles that “slip” through the human mucus barrier Angew Chem Int Ed Engl, 47 (2008), pp. 9726-9729. https://doi.org/10.1002/anie.200803526
  6. Polyethylene glycol selection guide.
  7. http://informahealthcare.com/doi/abs/10.3109/03639045.2010.546801
  8. https://doi.org/10.3389/fmicb.2016.00494
  9. Empty capsule size chart
  10. ACG-World.
  11. Evonik Industries.
  12. Different types of pill guide
  13. Five different types of Pills
  14. Technology to protect probiotic bacteria.

THE BIGPIE

A project by the iGEM Team of the Indian Institute of Science Education & Research Bhopal.

Funded by the Department of Biotechnology, Government Of India.

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iGEM IISER Bhopal 2020