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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.
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.
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, lipidation, and fusion of other proteins2. 'Targetability' is another concern because most of the current protein therapeutics have multiple sites of action, resulting in off-target effects. Specifically, targeting these drugs to desired sites could lower the required therapeutic dose and reduce side effects2. If one could restrict drug delivery to the intended site of action, it might be possible to radically improve treatment while also limiting the undesirable 'off-target' side effects of these drugs.
Multiple medical devices are currently being used that contain therapeutics, such as drug-eluting stents with an antiproliferative drug coating containing clopidogrel, vincristine, and vinblastine to help prevent restenosis and the need for repeat revascularization after cardiac catheterization3. Additionally, biodegradable drug-eluting ureteral stents bearing paclitaxel, doxorubicin, gemcitabine, and other antiproliferative agents have been created to help treat upper tract urothelial carcinoma4. As these stents degrade, the anti-tumor drugs are released locally in a controlled manner, prolonging the drug delivery without the need for additional dosing4. This methodology allows for the long-term effect of the anti-tumor drugs at the intended site of action while also preventing the need for systemic exposure, limiting the drug action on non-tumor cells, and reducing drug toxicity. Also, the biodegradable nature of the stent can also prevent the need for a stent removal procedure4.
Short bowel syndrome (SBS) is another condition where the use of protein-based therapeutics has shown significant improvement in patients5. SBS can occur through various mechanisms but results in inadequate intestinal length, causing malabsorption and malnutrition. Therapies, such as glucagon-like peptide 2 (GLP-2) analogs, can be used before moving to surgical management of this condition5. Human GLP-2, a systemic nutrient peptide hormone, is produced in the distal small intestine and colon by L-type endocrine cells. GLP-2 induces proliferation of intestinal crypt cells, promotes repair of damaged mucosa, inhibits apoptosis of intestinal epithelial cells, and improves intestinal bloody supply5. GLP-2 analogs, such as teduglutide, have been approved for patients 1 year or older for treatment of SBS to help augment the natural intestinal adaptation6. Naturally, intestinal adaptation can take years and results in delayed gastric emptying to increase absorptive time and increased diameter by intestinal dilation to increase the surface area for absorption7. GLP-2 analogs have the same pharmacological activity as the natural GLP-2 while having a more stable structure, allowing a longer half-life and stronger affinity to targets5. Currently, the annual cost of teduglutide is $295,000, and patient dosing is 0.05 mg/kg per day5. Teduglutide side effects include nausea, vomiting, diarrhea, abdominal pain, weight loss, gastrointestinal polyps, and increased growth of existing tumors5.
Previously, our lab has shown that deployment of an intestinal expansion sleeve (IES) (Figure 1) was able to produce an immediate 36.2% elongation of small bowel ex vivo and a 30.2% elongation of small bowel after a one-month deployment in vivo6,8. This device could become a therapy for SBS patients that could shorten parenteral nutrition dependence. The ability to bioconjugate therapeutics to the IES device could further improve the distraction enterogenesis capacity by adding the intestinal mucosal growth agonist GLP-2 to the device. Polyvinyl alcohol (PVA) membranes are hydrophilic polymers that can be prepared by dissolving PVA into water, casting it onto a structural support, and then crosslinking with glutaraldehyde using sulfuric acid as a catalyst9. These hydrogel nanoparticles have been shown to have potential as drug delivery systems through drug diffusion, hydrogel matrix swelling, and chemical reactivity of the drug/matrix10. PVA has been FDA-approved for clinical use in humans due to its excellent biocompatibility and safety11. PVA polymerization results in terminal groups that allow for bioconjugation12.
IES/VES devices are 3 cm in length with an external surface area of 9.425 cm2 that is capable of casting with PVA membranes. These PVA membranes can be bioconjugated with GLP-2 to allow for localized and concentrated delivery of therapeutic agents. Lowering the required dose of the drug and bypassing the need for systemic administration, avoiding negative off-site effects of treatment. This can help mitigate the cost of these expensive drugs by requiring less dosage, and the delayed release of the drug can decrease the frequency of dosing, further lowering costs. Coating the IES device with GLP-2 could lower required dosages, save patients money, avoid systemic administration, prevent negative effects of treatment, and maintain a continuous delivery of the drug to the target site. This report evaluates the extent to which a therapeutic protein, like GLP-2, can be covalently bound to medical devices such as IES/VES devices in physiologically meaningful quantities to provide the local benefit of these drugs while avoiding off-target effects.
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
2. Creating the GLP-2 coated 24-well plate
3. Creation of standard curve and discerning sleeve GLP-2 concentration
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/cm2, which correspon...
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 ...
Jonathan Steven Alexander and Christen J. Boyer hold the patent US10537659B2, 3D-printed polyvinyl alcohol medical devices and methods of activation (2017). The remaining authors have nothing to disclose.
This research was funded by R. Keith White, MD, via the Department of Surgery John C. McDonald Chair Fund, W. Reid Grimes, MD for funding via the Department of Surgery Whitney Boggs Endowed Professorship Fund, and LSU LIFT2 grant, HSCS-2022-LIFT-002.
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 |
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