TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

Summary

We describe the simplest protocol to prepare biodegradable medical glue that has an effective hemostatic ability. TAPE is a water-immiscible supramolecular aggregate prepared by mixing of tannic acid, a ubiquitous compound found in plants, and poly(ethylene) glycol, yielding a 2.5 times greater water-resistant adhesion compared with commercial fibrin glue.

Abstract

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant adhesion strength than commercial tissue adhesives. Medical adhesives have attracted great attention as potential alternative tools to sutures and staples due to their convenience in usage with minimal invasiveness. Although there are several protocols for developing tissue adhesives including those commercially available such as fibrin glues and cyanoacrylate-based materials, mostly they require a series of chemical syntheses of organic molecules, or complicated protein-purification methods, in the case of bio-driven materials (i.e., fibrin glue). Also, the development of surgical glues exhibiting high adhesive properties while maintaining biodegradability is still a challenge due to difficulties in achieving good performance in the wet environment of the body. We illustrate a new method to prepare a medical glue, known as TAPE, by the weight-based separation of a water-immiscible supramolecular aggregate formed after a physical mixing of a plant-derived, wet-resistant adhesive molecule, Tannic Acid (TA), and a well-known biopolymer, Poly(Ethylene) glycol (PEG). With our approach, TAPE shows high adhesion strength, which is 2.5-fold more than commercial fibrin glue in the presence of water. Furthermore, TAPE is biodegradable in physiological conditions and can be used as a potent hemostatic glue against tissue bleeding. We expect the widespread use of TAPE in a variety of medical settings and drug delivery applications, such as polymers for muco-adhesion, drug depots, and others.

Introduction

In a past decade, efforts have been made to replace current surgical sutures and staples to close wounds with biodegradable/bioabsorbable adhesives due to their convenience in usage and low tissue invasiveness during surgical treatments. Commercially available tissue-adhesives are classified into four types: (1) cyanoacrylate derivatives¹, (2) fibrin glues formed by enzymatic conversion from fibrinogen to fibrin polymers by thrombin^2,3, (3) protein-based materials such as chemically or physically cross-linked albumin and/or gelatin^4,5, and (4) synthetic polymer-based ones⁶. Although they have been used in many clinical applications, all adhesives have their own intrinsic disadvantages and drawbacks that can be obstacles to their widespread usage. Cyanoacrylate-based glues show high adhesion strength to tissues, but their toxic by-products such as cyanoacetate and formaldehyde formed during degradation, often cause significant degrees of inflammatory responses⁷. Fibrin glues and albumin or gelatin-based materials have safety issues regarding the transmission of infectious components, such as viruses from animal sources: human blood plasma for fibrin glues and animals including cattle, chicken, pigs, and fish for gelatin-based glues⁸. Although a few synthetic polymer-based adhesives have been approved by the Federal Drug Administration (FDA), most adhesives made of synthetic polymers continue to have difficulties in minimizing the manufacturing process steps and achieving biocompatibility⁹. Most importantly, all glues suffer from poor mechanical and adhesion strength to wet tissues¹⁰. Recently, biomimetic tissue adhesives inspired by marine mussels^11-13, geckos¹⁴, gecko with mussel¹⁵, and endoparasitic worms¹⁶ have been emerging as promising alternatives to current medical glues due to their tunable mechanical and adhesive properties with biocompatibility. However, to this day, there are still issues to be addressed before they become commercial products¹⁷.

Here, we report an entirely new type of medical glue called TAPE that is prepared by the intermolecular hydrogen bonding between a plant-derived adhesive molecule, Tannic acid (TA), and a bio-inert polymer Poly(ethylene glycol) (PEG), as its name indicates. TA is a representative hydrolysable tannin ubiquitously found during the secondary metabolism of plants. It has attracted much attention due to its anti-oxidant, anti-mutagenic, and anti-carcinogenic properties and has been shown to participate in supramolecular interactions with many polymers, such as poly(N-isopropylacrylamide) (PNIPAM) and poly(N-vinylpyrrolidone) (PVPON), to form layer-by-layer (LbL) films^18-20 and drug-releasing microcapsules^21-23. In this study, we discover that TA can act as an efficient water-resistant adhesive functional moiety to form a medical adhesive, TAPE. By simple mixing with TA, a non-fouling polymer PEG becomes a supramolecular glue with 2.5-fold increased adhesion strength compared with commercial fibrin glue, and this adhesion was maintained throughout up to 20 cycles of attachment and detachment, even in the presence of water. Its hemostatic ability was tested on a liver bleeding model in vivo and showed good hemostatic ability to stop bleeding within a few seconds. TAPE has its significant meaning in a related field as the first plant-derived adhesive that can reveal new insight into solving the drawbacks of current problems with bio-inspired approaches. We also expect the widespread use of TAPE in a variety of medical and pharmaceutical applications such as muco-adhesives, drug-releasing patches, wound-care dressings, and others due to its simple preparation method, scalability, tunable biodegradation rate, as well as highly wet-resistant adhesion properties.

Protocol

All animal care and experiments are performed in accordance with the ethical protocol provided by the KAIST (Korea Advanced Institute of Science and Technology) IRB (Institutional Review Board).

1. TAPE Formation

1. For preparing a TA solution, place a 4 ml-sized glass vial on a magnetic stirrer, and add 1 ml of distilled water with a stirring bar. Add 1 g of tannic acid to the vial, and dissolve it in the water by gentle stirring at 200 rpm for more than 1 hr. When the TA is completely dissolved, the mixture becomes transparent with a brown color.
2. Prepare a PEG solution by adding 1 g of PEG powder (4-arms, 10 kDa, and linear, 4.6 kDa) to 1 ml of distilled water followed by mixing them by vortexing for few seconds to make a white slurry. Keep this slurry in the incubator at 60 °C for 10 min. until the white one becomes completely clear.
   NOTE: The melting point of PEG with 10 kDa molecular weight is around 55 - 60 °C, and the 4 kDa one is 53 - 58 °C. Melted PEG becomes water-miscible so that a high concentration of PEG in water up to 1 g/ml can be achieved as a clear solution. Once a clear PEG solution is formed at a high temperature, the solution is still stable at room temperature after cooling.
3. Add 329 µl of the PEG (4-arms, 10 kDa) solution prepared in step 1.2 to 671 µl of the TA solution prepared in step 1.1 (In the case of a linear PEG with 4.6 kDa, add 311 µl of a PEG solution to 689 µl of a TA solution) in a micro-centrifuge tube. Gently blend the two viscous and honey-like solutions with a narrow spatula to mix them homogeneously.
   CAUTION: Both solutions are quite viscous, so the scientist must slowly but sufficiently pull up and transfer the solutions with a micropipette.
4. Spin the mixture prepared in step 1.3 at 12,300 x g for 3 min in a centrifuge equipped with a fixed-angle rotor.
5. Carefully remove as much of the supernatant as possible using a micropipette, and collect the product that has settled down: This is the fully formed TAPE. After TAPE formation, store it in the refrigerator (4 - 8 °C) for up to several weeks. NOTE: TAPE can be sterilized by gamma radiation or electron beam treatment prior to use in surgical applications.

2. Measurement of the Adhesion Strength of TAPE 

1. Prepare two pieces of porcine skin tissue with a diameter of 6 mm by cutting with a biopsy punch after removing all fat on the skin tissue.
   NOTE: The porcine skin tissue was obtained from healthy porcine flank skin and was purchased from a local meat market located in Daejeon in South Korea.
2. Apply commercial cyanoacrylate glue to the outer side of each tissue, and attach the tissue to the metallic rod.
   NOTE: The metallic rod is used as a supplementary handle so tissues are not directly gripped by the machine. Accordingly, it can be replaced with other materials following the configuration of the tensile machine.
3. Apply a drop of TAPE (a drop of TAPE is approximately 3 - 6 mg) to one side of the tissue. Then, spread the TAPE uniformly using another tissue between the two tissues on their inner sides so they are attached as shown in Figure 2A.
4. Then, manually attach and detach the two sides of tissues several times to homogenously mix and maximize the interface between each tissue and TAPE.
5. With the UTM, carefully grip each side of the rod. The adhesion strength will be determined by the force needed to detach two tissues attached with TAPE. First, apply a force of 20 N for 1 min. Next, with the machine, pull each rod in an opposite direction at a rate of 1 mm/min. until the tissues are completely detached.
   NOTE: Data will be given as a force-distance (F-D) curve detected by the motion of each rod.
6. Calculate the adhesion strength of TAPE by dividing the maximum force (kN) shown at the F-D curve achieved in step 2.5 by the specimen surface area, that is, 3.14 x (0.003 m)².
7. For monitoring the adhesion strength in the presence of water, add 20 µl of water on the detached area between two tissues, and attach them immediately. With the machine, perform the detachment test again.

3. In Vitro Degradation Test

1. Cut a cap (d = 8 mm) of micro-centrifuge tube, and weigh the cap to define it as W_c.
2. Fill the cap with 150 mg of TAPE, and weigh all together again to set it as a total initial weight W₀.
   CAUTION: Do not overload TAPE in the cap. The height of TAPE should be lower than the top of the cap, as it can be a physical barrier to a stream of PBS buffer generated by the stirring process during the incubation in step 3.4.
3. Put the cap containing TAPE into a cell culture flask (75 cm²), and add 50 ml of PBS buffer (1x, pH 7.4) to the cell culture flask so the TAPE in the cap is completely immersed in the PBS buffer, as shown in Figure 3A (n = 5).
4. Incubate the cell culture flask prepared in step 3.3 in an orbital shaking incubator at 37 °C, similar to physiological conditions, with gentle stirring (50 rpm).
   CAUTION: Keep the stirring condition at 50 rpm. Higher rpm might cause a collapse of TAPE.
5. At each time point, take the cap with TAPE from the cell culture flask, and then dry them by blowing nitrogen gas. Weigh the cap containing remaining TAPE. Set the weight at each time point to W_t. Replace the fresh PBS again, and shake it again after measuring W_t at each time point.
6. Calculate the relative remaining weight (%) the following equation.
   Relative remaining weight (%) = (W_t - W_c)/(W₀ - W_c) x 100%

4. Hemostatic Ability of TAPE

       NOTE: All animal experiments should be performed in accordance with the guidelines and ethical protocol provided by the Korean Ministry of Health and Welfare.

1. To evaluate the in vivo hemostatic ability, review the hemorrhaging mouse liver model as described in ref ²⁴.
2. Anesthetize fifteen mice (normal ICR mouse, 6 weeks, 30 - 35 g, male) with an intraperitoneal injection of tiletamine-zolazepam (33.333 mg/kg) and xylazine (7.773 mg/kg) (n = 5 per group). To confirm proper anesthetization, pinch the animal's paw gently and observe movements such as withdrawing the paw, etc. No movement indicates that the animal is sufficiently anesthetized to do surgery.
3. To prevent dryness of animal's eyes, apply vet ointment to eyes sufficiently while under anesthesia. Expose the liver via a midline abdominal incision, and prick the liver with an 18 G needle to induce bleeding.
4. Remove the flowing blood with sterile gauze, and put 100 µl of TAPE or fibrin glue (positive control) immediately on the bleeding site.
   NOTE: No further suturing is needed after applying TAPE due to its highly blood-resistant adhesive properties on wound tissues. For the negative control, no treatment occurs at the site of bleeding.
5. In each case, put a filter paper with known mass underneath the liver to collect the blood from the damage site. Replace the paper with a fresh one every 30 sec for 4 times (i.e., 2 min).
6. Measure the mass of absorbed blood on each filter paper collected every 30 sec. After the animal experiment, sacrifice the mice through CO₂ asphyxiation euthanasia.

Results

TAPE is a supramolecular aggregate that settles down after centrifuging the mixture of two aqueous solutions containing TA (1 g/ml in distilled water) and PEG (1 g/ml in distilled water) with 2:1 volume ratio (Figure 1A). The mixing ratio is the key factor in achieving high adhesion strength; when TAPE is formed by a 2:1 mixing ratio, 20 units of the hydroxyl group (-OH) in 25 units of TA interact with each ether group (-O-) in PEG, resulting in the highest intermolecular...

Discussion

We developed an entirely new class of hemostatic adhesive named TAPE inspired by the water-resistant molecular interaction of a plant-derived polyphenolic compound, TA. TA is a representative hydrolysable tannin that has significantly attracted attention due to its anti-oxidant, anti-bacterial, anti-mutagenic, and anti-carcinogenic properties.

The process of making TAPE is extremely simple, scalable, and environmentally friendly, as it is just the one-step mixing of two aqueous solutions follo...

Materials

Name	Company	Catalog Number	Comments
Tannic acid	Sigma-aldrich	403040
Poly(ethylene oxide), 4-arm, hydroxy terminated	Aldrich	565709	Averge Mn ~10,000
Poly(ethylene glycol)	Aldrich	373001	Average Mn 4,600
Biopsy punch	Miltex	33-36	Diameter = 6 mm
Aron Alpha®	Toagosei Co., Ltd.		Instant glue
Universal testing machine (UTM)	Instron	5583
Microcentrifuge tubes	SPL life science	60015	1.5 mL
Petri dish	SPL life science	10090	90 x 15 mm
Sodium phosphate monobasic	Sigma	S5011	1x PBS ingredient
Sodium phosphate dibasic	Sigma	S5136	1x PBS ingredient
Sodium chloride	Duchefa biochemie	S0520.5000	1x PBS ingredient
Incubating shaker	Lab companion	SIF6000R
ICR mice	Orient bio	Normal ICR mouse	6 weeks, 30-35 g, male
Tiletamine-zolazepam (Zoletil 50)	Virbac
Zylazine (Rompun)	Bayer
PrecisionGlideTM needle (18 G)	BD	302032	18 G
Filter paper	Whatman	1001 125	Diameter = 125 mm
Parafilm	Bemis Flexible Pakaging	PM996