Purdue University Graduate School
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DEVELOPING COLLAGEN AND HYALURONAN BASED HIGH-FIDELITY, HIGH-THROUGHPUT IN VITRO PLATFORMS FOR BIOTHERAPEUTIC SCREENING

thesis
posted on 2024-06-27, 17:52 authored by Paulina M BabiakPaulina M Babiak

Biopharmaceuticals, such as insulin, monoclonal antibodies, growth hormones, and vaccines, have emerged as a major class of therapeutic molecules. Subcutaneous administration of biotherapeutics is a convenient drug delivery method that is less invasive, requires shorter clinic times, improves patient compliance, and reduces cost to the healthcare system compared to intravenous administration. The mass transport of a therapeutic injected into the subcutaneous tissue is dictated by physiochemical properties of the molecule such as size and electrostatic charge. The bioavailability and efficacy of the therapeutic formulation depend on efficient transport of the molecule from the injection site to lymphatic or blood vessels. The injected biotherapeutic needs to traverse complex structures of the subcutis and the extracellular matrix (ECM) before it arrives at the uptake site. In vitro transport screening platforms provide insights into the effects of tissue and therapeutic properties on macromolecular transport through biological barriers.

In this work, we develop an in vitro Transwell macromolecular recovery platform, an economical and high-throughput method that can be used to systematically evaluate effects of ECM components on mass transport properties of macromolecules. In Chapters 2-3, we engineer subcutaneous tissue models based on collagen type I ((Col I), the most abundant fibrillar protein in the subcutaneous ECM) and hyaluronic acid ((HA), an anionic and highly viscous polysaccharide). In Chapter 2, we optimize protocols to reproducibly fabricate Col I and combined Col I and HA (ColHA) hydrogels. In Chapter 3, we establish a workflow to characterize collagen material from different sources (animal sources, different vendors, and between batches of identical material) since inherent variabilities can occur.

Next, we develop and optimize a high throughput Transwell platform, and we screen the transport of macromolecules, which are representative of current therapeutics used in subcutaneous injections. We demonstrate that macromolecular transport within Col type I (Col I), blended collagen I and II (Col I/II), blended Col I and III (Col I/III), and combined Col I and HA hydrogels (ColHA) hydrogels is inversely related to the hydrodynamic radius of the diffusing macromolecules. Blending col I/II and I/III gels results in altered fibril morphologies (smaller fibrils), which decrease mass recovery rates. Increasing HA concentration within the Col I hydrogels decreases macromolecular recovery. This decrease is mainly a consequence of increased viscosity within the matrix. Recovery rates of large molecules such as immunoglobulin G (IgG), a molecule similar in size to therapeutic antibodies, were highly sensitive to HA concentration in col hydrogels. Smaller molecules, such as myoglobin and lysozyme, that are similar in size to insulin experience electrostatic effects as HA concentration increases within col gels. Recovery of macromolecules in an HA solution was a function of both electrostatic and steric interactions. The results from these studies were highly reproducible and highlighted the robustness of the optimized assay.

Our results thus demonstrate that the Transwell platform can be utilized for systematic evaluation of therapeutic transport as a function of molecular characteristics. The results presented can inform desirable physiochemical properties for efficient biotherapeutic transport within the subcutaneous tissue.

In the last main portion of the thesis, we work with elastin, another biologically derived material. In this portion, we developed an optimized method for expression and purification of elastin-like polypeptide proteins. We then present a method to chemically alter the material to introduce underwater adhesive properties to the material.

Funding

Graduate Research Fellowship Program(GRFP)

Directorate for Education & Human Resources

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Eli Lilly

Frederick N. Andrews Fellowship

History

Degree Type

  • Doctor of Philosophy

Department

  • Chemical Engineering

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Julie C. Liu

Additional Committee Member 2

Luis Solorio

Additional Committee Member 3

Sangtae Kim

Additional Committee Member 4

You-Yeon Won