TWI808364B - Adhesive and localizing hydrogel kit and the use thereof - Google Patents

Abstract

This application provides an adhesive and localizing hydrogel kit for localizing the affected area and/or filling up the lesion before surgery, comprising: catechol alginate solution with a concentration of 20 mg/mL and above, oxidant solution, and catalase; wherein the molar ratio of the oxidant to the catechol group of the catechol alginate is 0.1~2.2. In addition, the present application provides a use of catechol alginate hydrogel for preparing a hydrogel for localizing affected area and/or filling up the lesion, and the mixing device thereof.

Description

A water-glue set with both positioning and repairing functions and its application

The invention relates to a hydrogel set with both positioning and repairing functions, in particular, the catechol-alginate hydrogel is used for positioning tumor tissue or malignant nodules before lung cancer surgery, and the hydrogel also has the function of filling pneumothorax.

Lung cancer is one of the most common cancers with a high mortality rate in the world. To reduce the mortality rate of lung cancer, early diagnosis is very important. Lung cancer usually presents small malignant nodules in the early stages, which can be detected by low-dose CT (LDCT). However, during surgical resection, these malignant nodules are small in size and difficult to confirm the location with the naked eye or by touch. Therefore, before surgical resection, thoracic surgeons will first ask radiologists to locate the malignant nodules in the patient's lungs. Methods include implanting a thin hookwire (hookwire) or applying dye (such as patent blue V) to the patient's lungs. stove to assist in positioning. However, in today’s positioning surgery methods, in addition to causing pneumothorax, the metal wires exposed outside the patient’s chest will also cause fear and inconvenience to the patient, and the water-soluble dye will also spread when the patient breathes or moves, resulting in inaccurate positioning or failure. Therefore, we have developed a kind of viscous and colored water glue. After injection into the patient's lungs, it can tightly stick to the lesion without spreading, and block the hole caused by the needle inserted into the lung during positioning surgery, reducing the probability of pneumothorax. It is used to provide a new choice of positioning materials between doctors and patients.

In recent years, studies have found that the tentacles of mussels can still adhere to the surface of various materials in a water-containing environment. The reason is that its tentacles can secrete mucus containing a large amount of catechol. The structure of catechol can form covalent and non-covalent bonds with the surface of inorganic and organic substances, thereby producing stickiness.

Since 2012, Catechol-conjugated alginate has been synthesized and applied in research in several medical fields. CJ Kastrup et al. used catechol-alginate (Catechol alginate) as an adhesive in the paper "Painting blood vessels and atherosclerotic plaques with an adhesive drug depot" to treat vascular diseases. In the paper "Mussel-inspired alginate gel promoting the osteogenic differentiation of mesenchymal stem cells and anti-infection", S. Zhang et al. used the same material to successfully promote the osteogenic differentiation of mesenchymal stem cells. F. Scognamiglio in the paper "Enhanced bioadhesivity of dopamine-functionalized polysaccharides membranes for general surgery applications" made a film by mixing catechol-alginate and hyaluronic acid, and wrapped the film around the anastomosis to try to reduce Bowel anastomotic leakage. The idea of using catechol-alginate as a binder has also been proposed. In the paper "Enzymatically crosslinked alginate hydrogels with improved adhesion properties", Junxia Hou et al. used horseradish peroxidase (HRP) and _H2O2 to oxidize catechol- _alginate to prepare in situ adhesives, which showed that catechol-functionalized alginate gels were more effective than phenol-functionalized alginate and alginate-tyramine The hydrogel (Alg-TA) exhibited significantly improved adhesive properties, however, its time to reach the gel point was too long to be applicable to the drugs used, and the enzyme was too expensive to be suitable for clinical applications.

In view of the problems existing in the prior art, this application provides a water glue set with both positioning and repairing functions, which is used for positioning and/or repairing the affected part before surgery, including: Catechol-alginate solution, concentration above 20 mg/mL (mg/mL); oxidant solution; and Catalase; Wherein, the molar ratio of the oxidant to the catechol group in the catechol-alginate is 0.1 to 2.2.

In one embodiment, the oxidizing agent is selected from the group consisting of periodate, iodate, fibrinogen, tyrosinase, horseradish peroxidase and hydrogen peroxide. Preferably, the oxidizing agent is sodium periodate.

In one embodiment, the concentration of the catechol-alginate solution is above 35 mg/mL; the molar ratio of the oxidant to the catechol group in the catechol-alginate is 0.2-0.8; the concentration of the catalase is 600 IU/mL-1000 IU/mL.

In one embodiment, the concentration of the catechol-alginate solution is above 40 mg/mL, the oxidant is sodium periodate, the molar ratio of the oxidant to the catechol group in the catechol-alginate is 0.25-0.6, and the concentration of the catalase is 800 IU/mL.

In one embodiment, the hydrogel kit with both positioning and repairing functions further includes dyes, fluorescent dyes and/or developers.

In one embodiment, the water glue set with both positioning and repairing functions further includes: a first syringe containing the catechol-alginate solution; a second syringe containing the oxidizing agent solution; a multi-way valve; Wherein, the first syringe contains the catalase.

In one embodiment, the multi-way valve is a two-way valve or a three-way valve.

In addition, the present application further provides a combination of catechol-alginate, oxidizing agent and catalase for preparing water gel for positioning and repairing affected parts. Alternatively, a catechol-alginate hydrogel is used for preparing the affected part positioning and repairing hydrogel.

In one embodiment, the affected part is tumor tissue or malignant nodules in the lung.

To sum up, the water glue set or application provided by this application with both positioning and repairing functions is to use catechol functional group dopamine (Dopamine) through 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (N-hy Droxysuccinimide (NHS) was grafted onto highly biocompatible sodium alginate to form catechol-alginate. In operation, catechol-alginate, oxidizing agent (sodium periodate, NaIO ₄ ) and catalase are mixed to form an injectable hydrogel. Through the tests of gelation time, mechanical properties, burst pressure and positioning effect, it is confirmed that the water gel developed by us has appropriate viscosity and obvious color, so as to facilitate the application in clinical operations such as tissue positioning and lung repair.

The advantages and features of the present invention and methods for achieving them will be more easily understood by describing in more detail with reference to exemplary embodiments and accompanying drawings. However, the invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, for those skilled in the art, these embodiments are provided to make this disclosure more thorough and comprehensive and fully convey the scope of the present invention, and the present invention will only be defined by the appended claims. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be more understandable that terms such as those defined in commonly used dictionaries should be understood as having meanings consistent with the contents of the relevant fields, and will not be understood in an overly idealized or overly formal meaning unless clearly defined herein. As stated herein, range values are intended to be shorthand for describing each and every value within the range, and any value within the range can be selected as an endpoint of the range.

This application provides a water gel set with both positioning and repairing functions, which is used for positioning and repairing the affected part before surgery, including: Catechol-alginate solution, concentration above 20 mg/mL (mg/mL); oxidant solution; and Catalase; Wherein, the molar ratio of the oxidant to the catechol group in the catechol-alginate is 0.1 to 2.2.

In one embodiment, the oxidizing agent is selected from the group consisting of periodate, iodate, fibrinogen, tyrosinase, horseradish peroxidase and hydrogen peroxide. Preferably, the oxidizing agent is sodium periodate.

In one embodiment, the concentration of the catechol-alginate solution is above 35 mg/mL; the molar ratio of the oxidant to the catechol group in the catechol-alginate is 0.2-0.8; the concentration of the catalase is 600 IU/mL-1000 IU/mL.

In one embodiment, the concentration of the catechol-alginate solution is above 40 mg/mL, the oxidant is sodium periodate, the molar ratio of the oxidant to the catechol group in the catechol-alginate is 0.25-0.6, and the concentration of the catalase is 800 IU/mL.

In one embodiment, the hydrogel kit with both positioning and repairing functions further includes dyes, fluorescent dyes and/or developers.

In one embodiment, the water glue set with both positioning and repairing functions further includes: a first syringe containing the catechol-alginate solution; a second syringe containing the oxidizing agent solution; a multi-way valve; Wherein, the first syringe contains the catalase.

In one embodiment, the multi-way valve is a two-way valve or a three-way valve.

In addition, the present application further provides a combination of catechol-alginate, oxidizing agent and catalase for preparing water gel for positioning and repairing affected parts. Alternatively, a catechol-alginate hydrogel is used for preparing the affected part positioning and repairing hydrogel.

In one embodiment, the affected part is tumor tissue or malignant nodules in the lung.

Several examples are listed below as examples to illustrate the implementation of this application; those skilled in the art can easily understand the advantages and effects of this creation through the contents of this specification, and make various modifications and changes without departing from the spirit of this creation to implement or apply the content of this creation.

Embodiment 1─catechol-preparation of alginate

Dissolve 0.5 g of medium-viscosity sodium alginate (Sodium Alginate, ALG) powder and 1.0 g of low-viscosity ALG powder in 30 ml of water, then pass through argon gas at room temperature to deoxygenate and stir for 2-3 hours. Next, EDC was slowly added to the ALG solution (molar ratio 3:1), and stirred for 30 minutes at room temperature and pH 5-5.5 to allow EDC to fully activate the carboxyl groups on ALG. Next, slowly add deoxygenated dopamine hydrochloride (DOPA) aqueous solution into the EDC-activated ALG solution (molar ratio 5:1), and stir for about 5-10 minutes. Finally, the NHS aqueous solution was slowly added to the ALG mixed solution (the molar ratio of EDC to NHS was 1:1) to form a catechol-alginate (Catechol alginate, C-ALG) solution. Its response is as follows:

Adjust the pH of the C-ALG solution to 9 with 1 N aqueous sodium hydroxide (Sodium Hydroxide, NaOH) solution, at this time the C-ALG solution should appear brownish red. The argon gas was continuously passed through to react until the next day, so that the grafting reaction was complete. After the reaction was completed, the product was poured into 99.5% methanol to precipitate it, and this step was repeated 3 times to wash away unreacted DOPA. Finally, the product was dried under reduced pressure for long-term storage.

Example 2 - Formation of catechol-alginate hydrogel

The dried C-ALG was redissolved with pH 7.4 phosphate buffer saline (PBS), and 800 IU of catalase was added to each 1 mL solution. Finally, equal volumes (100 uL) of different concentrations of NaIO ₄ and C-ALG solutions were dropped into a 2 mL vial and mixed evenly, and allowed to cross-link at 37°C to form a hydrogel. Crosslink formation was tested by vial tilting assay, and crosslink time was defined as the time for the solution to lose fluidity when the vial was tilted. Each sample was tested for crosslinking time 3 times and was considered a failure if the crosslinking time took more than 20 minutes. Table 1 below: Table 1: Ratio of material composition and crosslinking time C-ALG concentration (mg/mL) NaIO ₄ concentration (mg/mL) Molar ratio (NaIO ₄ /catechol base) Cross-linking time (seconds) Test No. 15 0.72 0.25 Failed (>20 minutes) -- 1.44 0.5 Failed (>20 minutes) -- 2.88 1 Failed (>20 minutes) -- 5.76 2 Failed (>20 minutes) -- 30 0.72 0.125 548.67 8.62 -- 1.44 0.25 178.7 6.0 CA-1 2.88 0.5 565 13.5 -- 5.76 1 Failed (>20 minutes) -- 11.52 2 Failed (>20 minutes) -- 40 0.96 0.125 313.67 9.7 -- 1.92 0.25 140.3 10.5 CA-2 3.84 0.5 122.7 7.5 CA-3 7.68 1 66.67 7.51 -- 15.36 2 701 19.3 --

The color of the hydrocolloid changes during the crosslinking process, and the gelled hydrocolloid is dark brown, as shown in Figure 1A. A high concentration of C-ALG will lead to faster cross-linking, but when the concentration of the oxidant is too high, the cross-linking time will be longer. For example, the sample with a C-ALG concentration of 40 mg/mL and a NaIO ₄ /catechol molar ratio of 2 has a longer cross-linking time. When a high concentration of oxidizing agent is used, the crosslinking reaction of the C-ALG solution with the oxidizing agent may also be uneven. For example, in the sample with a C-ALG concentration of 40 mg/mL and a _NaIO4 /catechol molar ratio of 1, although its crosslinking time is short, in the case of a high _NaIO4 /catechol molar ratio, due to the high reaction rate, its crosslinking forms an inhomogeneous hydrogel with particles.

C-ALG hydrogels with a crosslinking time within 2-5 minutes are suitable for topical surgery. If the cross-linking time is too long or cannot be cross-linked after being injected into the lung, the C-ALG solution will still flow in the lung when the patient breathes, resulting in inaccurate tumor localization. And the cross-linking time should not be too short, otherwise the C-ALG solution will be difficult to push and inject through the syringe when it is mixed in the syringe. Therefore, according to the advice of thoracic surgeons, materials that cross-link within 2-5 minutes were selected for follow-up tests, so CA-1, CA-2 and CA-3 were selected for follow-up tests.

Embodiment 3─viscoelasticity test

In order to determine the mechanical properties of the C-ALG hydrogel, this example determined the viscoelasticity by means of a rheometer equipped with a parallel plate with a diameter of 20 mm, and controlled the temperature at 37°C through the bottom plate, measured the changes in the storage modulus (G') and loss modulus (G'') in frequency sweep mode, and recorded the final conditions and stability of each test group, in which fully cross-linked hydrogels (incubated overnight at 37°C in advance) were prepared as samples (CA-1, CA-2 and CA-3) before testing, and Operate at a frequency of 0.1 to 1 Hz, with a depth setting of 10% and a strain setting of 1%.

As shown in Figure 2, the values of (G') and loss modulus (G'') of porcine lung were higher than those of CA-1, CA-2 and CA-3, and in addition, the values of G' and G'' of CA-1, CA-2 and CA-3 were independent of the set frequency range, which was similar to the normal breathing frequency. This indicates that the cross-linked networks of CA-1, CA-2, and CA-3 hydrogels are stable and uniform when they are implanted into lung tissue in vivo. Compared with CA-1 and CA-2, CA-3 with higher C-ALG and _NaIO4 concentrations had higher G' value and more cross-linking network, and was closer to G' of porcine lung. Therefore, CA-3 hydrogel has similar mechanical properties to porcine lungs and is softer than lungs, which can minimize the adverse effects of CA-3 hydrogel on surrounding tissues.

Example 4 - Burst Pressure Test

In this example, fresh lungs of rats were used to compare the pressure before/after the material supplemented lung air leakage. Connect one end of the air gauge to a high-pressure air cylinder, and the other end to the main trachea of the dissected rat lung. When gas was poured into the mouse lung to make it inflated, a 32-gauge needle was used to randomly create a hole with a depth of 1 mm on the surface of a lung lobe. At this time, the damaged area was immersed in normal saline, and the air bubbles were observed to confirm the lung leak. After filling the damaged part of the lung with water glue, infuse air again, observe whether there are still air bubbles, and measure the pressure value (bursting pressure) after filling the lung at this time.

As shown in Figure 3, before using C-ALG hydrogel, all the pressures in the rat lung defect model were 190 mm-H ₂ O, while C-ALG hydrogel could greatly increase the burst pressure to 266 15 ~ 385 13mm- _H2O . CA-3 and CA-2 were able to withstand higher stress compared with CA-1 (p < 0.05), but there was no significant difference between CA-3 and CA-2. This example shows that the hydrogel provided by the present application can prevent pneumothorax by sealing the injection hole of the lung after local surgery.

Example 5─Positioning effect test

In this example, pig lungs purchased in the traditional market were used for testing, and the localization effects of C-ALG hydrogel and the clinically used dye Patent Blue V were compared. 0.3 mL of C-ALG hydrogel and Patent Blue V were dropped onto the surface of pig lungs of fixed size (length 5 cm × width 3 cm × height 0.5 cm), and soaked in PBS at 37 °C to simulate the physiological environment in vivo. Afterwards, the effects of 0 h/1 h/2 h/4 h/8 h/24 h of water gel fixation on pig lungs were observed respectively, and the diffusion area of water gel material and dye was calculated by Image J.

Please refer to Figure 4A, C-ALG hydrogel on the left and Patent Blue V on the right. After crosslinking, the spreading area of C-ALG hydrogel slightly increases from 0.575 ± 0.092 cm ² to 0.728 ± 0.058 cm ² , 0.8 ± 0.047 cm ² and 0.818 ± 0.051 cm ² in 2 hours, 8 hours and 24 hours respectively . On the contrary, after 2, 8 and 24 hours, the average area of the dark blue color of the dye Patent Blue V increased widely from 1.594±0.878 cm ² to 4.014±0.206 cm ² , 6.401±1.274 cm ² and 7.946±1.461 cm ² , respectively, please refer to Figure 4B.

In addition, C-ALG hydrogel firmly adhered to the lung surface without displacement in localization experiments. On the contrary, the dye Patent Blue V spread almost 1/2 of the whole lung specimen, and the blue color on the surface of the lung also faded and became blurred. It shows that the C-ALG hydrogel provided by this application can effectively and accurately locate the affected part of the operation.

This example shows that the C-ALG water gel provided by this application can directly have the positioning effect due to its original dark brown color, but in practice, different general dyes, fluorescent dyes or developers can be added to enhance its positioning effect according to actual needs.

Example 6 - Operation of the water glue set with both positioning and repairing functions

The practical operation method of the hydrogel set with both positioning and repairing functions provided by this application is to quickly and fully mix the catechol-alginate solution, the oxidizing agent solution, and catalase before applying it to the patient, so that it undergoes a cross-linking reaction to form a hydrogel, and before it is completely gelled and still has fluidity, it is injected into the patient's tumor to be surgically resected for positioning. Since the catechol-alginate solution and the oxidizing agent solution must be quickly and uniformly mixed to form a homogeneous particle-free water gel, as shown in Figure 5A, the catechol-alginate solution (111) can be placed in a first syringe (110), and sodium periodate (121) can be placed in a second syringe (120), and then the first syringe (110) and the second syringe (120) are connected to any two ends of a three-way valve (130). And close the remaining end, press alternately through the first syringe (110) and the second syringe (120) several times, use physical and mechanical force to make the catechol-alginate solution and the oxidant solution mix conveniently, quickly and uniformly, and finally form the catechol-alginate hydrogel (240), and push it all into the first syringe, as shown in Figure 5B. Next, as shown in Figure 5C, when the mixed catechol-alginate hydrogel is ready to be injected into the affected part of the patient's tumor or malignant nodule to be surgically removed, the three-way valve can be switched to close the empty end of the second syringe, and the needle can be directly connected to the remaining end of the three-way valve to be administered to the patient.

The first syringe and the second syringe can be connected to different ends of the three-way valve arbitrarily, and at the same time, the mixed catechol-alginate hydrogel can also be pushed to the first syringe or the second syringe. In addition, when preparing to inject the mixed catechol-alginate hydrogel into the tumor site of the patient to be surgically removed, the syringe containing the catechol-alginate hydrogel can also be separated from the three-way valve, and the needle can be directly put on the syringe to be administered to the patient. Furthermore, multi-way valves such as two-way valves and four-way valves can be arbitrarily selected to mix the catechol-alginate solution and the oxidizing agent solution.

100:Water glue set with both positioning and repair functions 110: The first syringe 111: Catechol – Alginate Solution 120: second syringe 121: oxidant solution 130: multi-way valve 200: Water glue set with positioning and repair functions 210: The first syringe 220:Second Syringe 230: multi-way valve 240: Catechol – Alginate Hydrogel 300: Water glue set with positioning and repair functions 310: first syringe 320: second syringe 330: multi-way valve 340: Catechol – Alginate Hydrogel 350: Needle

Figure 1 shows the color change of C-ALG solution during the crosslinking process.

Figure 2 shows the results of the viscoelasticity tests.

Figure 3 shows the results of the burst pressure test.

Figure 4A shows the results of the localization effect test.

FIG. 4B shows the calculated result of the diffusion area of the material in FIG. 4A .

FIG. 5A shows a schematic diagram of the operation of the hydrogel kit with positioning and repairing functions of the present application.

FIG. 5B shows a schematic diagram of the operation of the hydrogel kit with positioning and repairing functions of the present application.

FIG. 5C shows a schematic diagram of the operation of the hydrogel kit with positioning and repairing functions of the present application.

100:Water glue set with both positioning and repair functions

110: The first syringe

111: Catechol-alginate solution

120: second syringe

121: oxidant solution

130: multi-way valve

Claims (9)

A hydrogel kit with both positioning and repairing functions, used for positioning and repairing affected parts before surgery, comprising: catechol-alginate solution with a concentration of 35 mg/ml (mg/mL) or more; an oxidizing agent solution, the oxidizing agent being periodate; and catalase; wherein the molar ratio of the oxidizing agent to the catechol group in the catechol-alginate is 0.1-2.2. The set of claim 1, wherein the concentration of the catechol-alginate solution is above 35 mg/mL, the molar ratio of the oxidant to the catechol group in the catechol-alginate is 0.2-0.8, and the concentration of the catalase is 600IU/mL-1000IU/mL. The set of claim 1, wherein the concentration of the catechol-alginate solution is above 40 mg/ml (mg/mL), the oxidizing agent is sodium periodate, the molar ratio of the oxidizing agent to the catechol group in the catechol-alginate is 0.25-0.6, and the concentration of the catalase is 800 IU/mL. The set according to claim 1, further comprising a dye, a fluorescent dye and/or a developer. The kit according to claim 1, further comprising: a first syringe containing the catechol-alginate solution; a second syringe containing the oxidant solution; a multi-way valve; wherein the first syringe contains the catalase. The set of claim item 5, wherein the multi-way valve is a two-way valve or a three-way valve. A use of the set as claimed in claim 1, which is used to prepare water gel for positioning and repairing the affected part. A use of the catechol-alginate hydrogel prepared by the set of claim 1 for preparing the affected part positioning and repairing hydrogel. According to the set according to any one of claims 1 to 6, the affected part is tumor tissue or malignant nodule in the lung.