Validation of Therapeutic Agent Conjugation to Polyvinyl Alcohol-Coated Medical Devices

摘要

Here, we present a protocol for derivatizing medical devices with protein therapeutics by conjugating protein therapeutics to a polyvinyl alcohol membrane casted on medical devices. Conjugation of protein-based therapeutics allows for localized and concentrated delivery and is a cost-effective approach that requires vastly lower drug amounts for dosing while minimizing off-target effects.

摘要

Protein-based therapeutics are often limited by their route of administration and inability to confine them to their site of action. One innovative approach we have developed is to covalently bind protein therapeutics to medical devices, allowing more localized and highly concentrated delivery of these agents to their intended site of action. This study aims to evaluate if glucagon-like peptide-2 (GLP-2) can be covalently bound to the vaginal expansion sleeve (VES) and intestinal expansion sleeve (IES) devices in clinically relevant and measurable quantities.

Expansion sleeves were coated with polyvinyl alcohol (PVA) and crosslinked with glutaraldehyde/sulfuric acid vapor to create a chemically active surface capable of binding amine-containing therapeutics such as GLP-2. A standard curve was created by binding 250 µg, 100 µg, 50 µg, 25 µg, and 0 µg of GLP-2 to PVA-coated wells of a 24-well plate. An ELISA standard was created using a rabbit anti-GLP-2 antibody followed by a goat anti-rabbit IgG alkaline phosphatase secondary antibody plus an alkaline phosphatase blue microwell substrate. Colorimetry (yellow to blue at 620 nm) was proportional to the concentration of GLP-2 bound antibodies, enabling calculation of the bound concentration of GLP-2 on the PVA-coated sleeves. The addition of 50 µg of GLP-2 to IES/VES devices bound an average of 22.69 ± 9.32 µg/cm² of GLP-2 with an external IES/VES surface area (9.425 cm²), indicating that 44% of added GLP-2 was immobilized on the PVA coated IES/VES sleeves.

Current human GLP-2 dosing is 50 µg/Kg. Because each sleeve carries 22 µg, this is approximately 44% of a systemic dose in a single device. This methodology makes it possible to add dramatically lower doses of therapeutic agents to get the same effect as systemic administration of the GLP-2 drug while also avoiding systemic effects.

引言

Protein-based therapeutics such as enzymes, antibodies, cytokines, hormones, and growth factors have been shown to be highly effective in the treatment of several diseases¹. If we improve our understanding of how to provide these therapeutic agents, we could significantly advance the biotechnology for applying these substances as medicines/medical treatments¹. The efficacy of protein therapeutics is limited by their biodistribution, cost, and off-target effects. Several approaches have been attempted to improve the pharmacokinetics and pharmacodynamics of protein therapeutics, including PEGylation, glycosylation, lipidat....

研究方案

NOTE: Sections 1 and 2 are completed at the same time. Coating with PVA and crosslinking the PVA membrane should be done at the same time for both the expansion sleeves and the 24-well plate. This allows them to be ready at the same time to create the standard curve with the 24-well plate to use absorbance to calculate the GLP-2 concentration of each sleeve.

1. Creating GLP-2 coated IES and VES devices

1. Make the 5% PVA solution by adding 5 g of PVA into 100 mL of deionized water. To help dissolve, consider stirring on a hotplate. Then, make the 1 mg/mL GLP-2 solution by adding 1 mg of GLP-2 into 1 mL of deionized....

结果

This protocol describes coating and crosslinking IES and VES devices with PVA to allow for the conjugation of GLP-2. Figure 2 shows the standard curve generated to determine the concentration of GLP-2 on each device. Figure 3 shows the GLP-2 concentration of each IES and VES device, and the concentration of the wells used for the standard curve. The sleeves had an average GLP-2 concentration of 22.69 ± 9.32 µg/cm^2, which correspon.......

讨论

We present here a methodology to bind protein-based therapeutics, such as GLP-2, to PVA-coated and crosslinked medical devices. The concentration of the bound GLP-2 was determined by the creation of a standard GLP-2 concentration curve on PVA-coated 24-well plates by the addition of rabbit anti-GLP-2 antibody, goat anti-rabbit IgG alkaline phosphatase, and alkaline phosphatase blue microwell substrate. The secondary antibody conjugated to an alkaline phosphatase enzyme allowed for the addition of an alkaline phosphatase .......

材料

Name	Company	Catalog Number	Comments
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat	Sigma-Aldrich	A0545-1ML
BluePhos Microwell Substrate Kit	Sera Care	5120-0059
GenClone 25-107, 24-Well Cell Culture Plates Flat Bottom Wells, TC Treated, 100 Plates/Unit	Genesee Scientific	25-107
GLP-2 Recombinant Rabbit Monoclonal Antibody (3K3P5)	Invitrogen	MA5-42869
Glucagon-like Peptide-2 (GLP-2) (1-33), rat	Echelon Biosciences	195262-56-7
Glutaraldehyde, 25% Aqueous Solution	Sigma-Aldrich	111-30-8
Nalgene Polypropylene Desiccator with Stopcock	ThermoFisher Scientific	5310-0250
Poly(vinyl alcohol)	Sigma-Aldrich	9002-89-5
Sulfuric Acid (TraceMetal Grade), Fisher Chemical	Fischer Scientific	A510-P212