Question

You are working on an improved polypropylene (–[CH2-CHCH3]n-) hernia correction mesh that releases an anti-inflammatory drug after implantation and is coated with type I collagen to promote bio-recognition to promote tissue integration.

How would you prove the coating is type I collagen?

How would you determine the chemistry and concentration of the drug?

Answer 1

Solution:-

Stain plates after collagen coating with species-specific antibody to type-I collagen. Use a brightfield label (AEC or DAB) to visualize binding. It will show location as well as density of coating.

When we coated our plates and flasks with type-I collagen we noted that a swirling action would deposit the collagen along the periphery and leave the center of the plates and flasks collage-free. We determined that moving the plates/flasks back-and-forth (5x), then side-to-side (5x), then back-and-forth (5x), and then side-to-side (5x), followed by 30 min incubation at rest before removing excess solution gave us the best and most consistent coating as assessed by type-I antibody staining.

At the same time

You can measure the amount of protein that was adsorbed to the tissue culture plastic using a BCA assay. Calculate the difference in total protein in solution pre- vs. post-adsorption.

Use enough (not too much) volume just to cover the surfaces of your wells, to allow even distribution.

The concentration of drug can be determined by volume of distribution(V_D).

V_D of a drug represents the degree to which a drug is distributed in body tissue rather than the plasma. V_D is directly correlated with the amount of drug distributed into tissue; a higher V_D indicates a greater amount of tissue distribution.