CN115990275A - A kind of submucosal injection and preparation method thereof - Google Patents

Abstract

The invention provides a submucosal injection, which comprises sodium glycerophosphate, hectorite powder and water; the invention innovatively introduces sodium glycerophosphate, and the multivalent negative charge carried by the sodium glycerophosphate can be electrostatically attracted with the positive charge around hectorite, so that the shear-thinning hydrogel with a stable three-dimensional network structure is assembled. Compared with normal saline, the submucosa injection provided by the invention can be maintained in submucosa tissues for a longer time to form a more stable bulge, so that the submucosa can be better separated from the submucosa, the submucosa can still maintain a complete gel form after complete stripping, and the stripping time is shorter. In addition, la+gp hydrogels significantly promote the generation of blood clots compared to normal saline.

Description

A kind of submucosal injection and preparation method thereof

technical field

The invention specifically relates to a submucosal injection and a preparation method thereof.

Background technique

Digestive endoscopy is widely used in the screening and minimally invasive diagnosis and treatment of early gastrointestinal lesions. At present, typical endoscopic minimally invasive surgery mainly includes endoscopic mucosal resection (Endoscopic mucosal resection, EMR) or endoscopic submucosal dissection (Endoscopic submucosal dissection). dissection, ESD), which can be used for the resection of early intramucosal lesions such as polyps and adenomas. Compared with traditional surgery, endoscopic resection has the advantages of faster recovery, fewer surgical complications, and high economic benefits while achieving the same therapeutic effect. If the lesion is large or associated with cancer, endoscopic submucosal dissection (ESD) is a more ideal endoscopic surgery method, which can not only achieve complete resection but also provide accurate pathological evaluation. Endoscopic submucosal dissection for early gastric cancer (ESD) five-year survival rate of more than 90%. Due to the limitations of the space and field of view of the digestive tract, ESD also has risks such as bleeding and perforation, and the establishment of submucosal space through submucosal injection can reduce the risk of complications and operation time, similar to pneumoperitoneum in laparoscopy of establishment.

The main function of the submucosal preparation is to fully separate the mucosa from the muscular layer to facilitate endoscopic surgery. Existing research includes glucose solution, glycerin, 0.4% sodium hyaluronate (0.4% SH), colloid, etc. However, these submucosal preparations are difficult to prepare or difficult to inject, which may cause tissue damage and thus affect histological evaluation. An ideal submucosal preparation should be able to achieve the desired elevation and maintain it for a sufficient period of time without damaging the tissue. At present, the best choice of submucosal injection in clinical practice has not yet been determined.

Nanoclay is a natural or synthetic layered silicate composed of tetrahedral silicate sheets and octahedral aluminum or magnesium hydroxide sheets in a ratio of 1:1 or 2:1. Among them, artificially synthesized laponite (Laponite, La) is a common shear-thinning material, which is a disc with a thickness of about 1 nm and a diameter of 20–30 nm, with a negatively charged surface and a positively charged edge, and is viscous under shear stress. Fluid, and recovers solid-like properties after stress relief, using this charged property of hectorite, different shear-thinning hydrogels can be synthesized. Previous studies mainly formed gels with chitosan and gelatin, which were widely used in drug delivery, hemostasis and healing, tissue regeneration and other fields.

Contents of the invention

The purpose of the present invention is to provide a more effective and safer submucosal injection, innovatively introducing sodium glycerophosphate, the multivalent negative charges carried by it can be electrostatically attracted to the positive charges around hectorite, and assembled into a stable three-dimensional network shear-thinning hydrogels.

The technical scheme adopted by the present invention is as follows: a submucosal injection comprising sodium glycerophosphate, laponite powder and water.

Preferably, the injection contains 0.28-0.32W/V% sodium glycerophosphate and 2-5W/V% hectorite.

Preferably, a coloring agent is also included.

Preferably, the content of the dyeing agent is 0.04-0.1W/V%.

Preferably, the dyeing agent is indigo carmine or methylene blue.

The present invention also provides a preparation method for the above-mentioned submucosal injection. Mix laponite powder and sodium glycerophosphate powder, add deionized water to fully dissolve, drop in dyeing agent, stir, and the formed hydrogel is the described Submucosal injections.

The beneficial effects of the present invention are as follows: 1, the present invention innovatively introduces sodium glycerophosphate (GP) (the composition of parenteral nutrition commonly used clinically), its multivalent negative charge can be electrostatically attracted with the positive charge around hectorite , a shear-thinning hydrogel that assembles into a stable three-dimensional network structure.

2. The submucosal injection of the present invention can be successfully injected manually through a 23G endoscopic injection needle through a 5ml syringe. Under the scanning electron microscope, it can be seen that the hydrogel presents a dense, continuous, porous, spongy structure, and the shearing effect of rapid self-healing after stress is eliminated. The recovery property allows the hydrogel to quickly return to the gel state after injection into the submucosal membrane.

3. Compared with normal saline, the submucosal injection of the present invention can last longer in the submucosal tissue, forming a relatively stable bulge, which can better separate the mucosal layer from the submucosal layer and maintain a complete gel form after complete peeling off , and the stripping time is shorter. In addition, compared with saline, La+GP hydrogel can significantly promote the generation of blood clots.

4. Taking normal human gastric mucosal cells GES-1 and mice as the research objects, it has been verified that it has no effect on the proliferation of human gastric mucosal cells GES-1 cells. At the same time, long-term observation of subcutaneous injection in mice also shows that it has no effect on important organs It has an impact, thus indicating the safety of La+GP hydrogel.

Description of drawings

Figure 1. La+GP hydrogel scanning electron micrograph (300×). A-D: SEM microstructures of cross-sections of 2%, 3%, 4%, and 5% La+GP hydrogels, respectively.

Figure 2. Determination of rheological properties and degradation rate of La+GP. A: Oscillatory frequency scan of the hydrogel, scanned at 0.5% stress; B: Oscillatory strain scan of the hydrogel, scanned at 6.3rad/s; C: Oscillatory time scan of the hydrogel, scanned at 0.5% stress and 6.3rad /s scan; D: shear recovery of hydrogel: under 6.3rad/s, 0.5% and 500% stress were applied for 120 seconds; E: viscosity value of hydrogel with shear rate; F: hydraulic coagulation Glue mass change curve. (2%-5% correspond to 2%-5% La+GP hydrogel group respectively).

Figure 3. Determination of La+GP hydrogel injection resistance. Injection resistance of each group of materials injected into the submucosa of the isolated porcine stomach through the injection needle under the endoscope (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 4. Trends in submucosal protrusion height and maintenance rate. A: initial uplift height after submucosal injection (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001); B: uplift height at different time points in each group; C: The maintenance rate of uplift height at different time points in the group; D: The change of submucosal injection area over time. (2%-5% correspond to 2%-5% La+GP hydrogel group respectively)

Figure 5. Submucosal dissection simulating ESD procedure. A-F: The submucosal structure after submucosal injection of normal saline, 0.4% sodium hyaluronate, 2% La+GP, 3% La+GP, 4% La+GP, and 5% La+GP respectively.

Figure 6. Submucosal dissection simulating ESD procedure. Submucosal peeling time in each group (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 7. H&E pathological analysis of the submucosal injection site section of rat stomach (10×). A: Section of injection site on day 0 after injection of normal saline; B: Section of injection site on day 0 after injection of 4% La+GP; C: Section of injection site on day 7 after injection of normal saline; D: Section of injection site on day 0 after injection of normal saline; D: Section of injection site on day 0 after injection of normal saline Profile of the injection site on day 7 after +GP.

Figure 8. Effect of La+GP hydrogel on coagulation time of whole blood. Blood clot formation in each group at different time points.

Figure 9. Effect of La+GP hydrogel on coagulation time of whole blood. Blood coagulation time of each group (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 10. CCK8 method to measure the effect of hydrogel extracts on cell proliferation at different time points (ns: no significant difference).

Figure 11. H&E pathological analysis of major organs of mice after subcutaneous injection for 4 weeks (10×).

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention and corresponding drawings. Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

Preparation of submucosal injection

Weigh 0.03g of sodium glycerophosphate powder and 0.2g of laponite powder respectively, put them into a 10ml glass bottle and vortex to mix, add deionized water to fully dissolve, drop a small amount of 0.04% (W/V, 1g/100ml, 0.04%=0.04g/100ml) indigo carmine is used as a dyeing agent, fully stirred by a magnetic stirrer, and 10mL is constant volume with deionized water to obtain 0.3% sodium glycerophosphate and 2% (W/V, 1g/100ml, 0.2%=0.2 g/100ml) La+GP hydrogel of laponite (invert the glass bottle every 10 seconds, it is judged as gelation if the hydrogel does not flow).

Example 2

Same as Example 1, except that the amount of hectorite powder added is 0.3g, and what is obtained is lithium soap containing 0.3% sodium glycerophosphate and 3% (W/V, 1g/100ml, 0.3%=0.3g/100ml) Stone La+GP hydrogel.

Example 3

Same as Example 1, except that the amount of hectorite powder added is 0.4g, and what is obtained is lithium soap containing 0.3% sodium glycerophosphate and 4% (W/V, 1g/100ml, 0.4%=0.4g/100ml) Stone La+GP hydrogel.

Example 4

Same as Example 1, except that the amount of hectorite powder added is 0.5g, and what is obtained is lithium soap containing 0.3% sodium glycerophosphate and 5% (W/V, 1g/100ml, 0.5%=0.5g/100ml) Stone La+GP hydrogel.

results and analysis

1. The hydrogel prepared in the above-mentioned examples 1-4 is subjected to characterization analysis, and the specific analysis methods and results are as follows:

1.1 Gelation time and injectability evaluation of hydrogel

Invert the glass bottle every 10 seconds. If the hydrogel does not flow, it is judged as gelation. Record the corresponding gelation time of the hydrogels in Examples 1-4.

The hydrogel was filled in a 5ml syringe and assessed for injectability using a 23G endoscopic needle.

Results: Mix 2%-5% laponite and 0.3% sodium glycerophosphate powder, then add deionized water and magnetically stir evenly, a colorless and transparent hydrogel can be formed after standing still, and with the increase of laponite content Increasing, the gelation time of the system gradually shortened (as shown in Table 1). When the hectorite content reaches 4% concentration, it can be gelled by standing for about 180 seconds.

The 4% La+GP hydrogel loaded in a 5ml syringe can be successfully injected manually through a 23G endoscopic injection needle, and immediately re-forms a gel with a fixed shape after injection.

Table 1. Gelation time of hydrogels with different concentrations

1.2 SEM observation

Take 3ml of 4% La+GP hydrogel in a 6cm-diameter petri dish, and place it in a -80°C refrigerator for 12 hours;

Put the sample into the freeze dryer after preheating for 30 minutes, and take it out after 24 hours of freeze drying.

The freeze-dried hydrogel was cut open with a blade, and the cross-section was sprayed with gold and observed under a scanning electron microscope (S3400 20kv10.3mm x100 SE).

Results: The cross-section of the hydrogel was observed under a scanning electron microscope, and the interior of the La+GP hydrogel showed a dense and continuous sponge-like porous structure. Among them, the structure of 2%-3% La+GP hydrogel is relatively loose (as shown in Figure 1A and B). When the concentration of hectorite increases, the structure becomes more and more regular, and the interior of 4% and 5% La+GP is uniform The shingled arrangement (shown in Figure 1C, D).

1.3 Degradation rate of hydrogel

Put 2ml (W ₀ ) of different concentrations of La+GP hydrogels into petri dishes with a diameter of 6cm, add PBS (pH7.4) solution to immerse the hydrogels, and place them on a shaker (rotating speed 100r/min);

Take it out every 30 minutes within 0-120 minutes, and weigh (Wt) after wiping off excess water on the surface with absorbent paper. (n=5). Calculation formula: degradation rate=(W ₀ −W _t )/W ₀ ×100%.

Results: The submucosal injection gel used as an in vivo injection can indirectly reflect the maintenance capacity of the hydrogel by immersing in PBS that is in line with the in vivo environment, and the 2% La+GP hydrogel is reduced by half after 30 minutes. quality, while the maintenance rate of the hydrogel in PBS within 2 hours was also more stable with the increase of hectorite concentration. When the hectorite concentration reached 5%, the hydrogel not only did not degrade, but absorbed water to 107% of its original mass after 2 hours. (Figure 2F)

1.4 Determination of rheological properties

A 2%-5% La+GP hydrogel was prepared and distributed into 5ml syringes; evenly placed between the parallel plates of the rotational rheometer. Oscillatory time sweep was performed at a strain of 0.5% and a shear rate of 6.3rad/s to measure the storage modulus (G') and loss modulus (G") of hydrogels with different concentrations;

Oscillation frequency sweep was carried out under the condition of fixed strain of 0.5%, and the storage modulus (G') and loss modulus (G") of hydrogels with different concentrations were measured;

The shear rate was fixed at 6.3rad/s for oscillatory strain scanning, and the storage modulus (G') and loss modulus (G") of hydrogels with different concentrations were measured;

The change of viscosity with strain force was determined.

Shear recovery: At 6.3 rad/s, 500% stress was applied for 120 s to induce shear thinning, after which the stress was reduced to 0.5% stress for 120 s to allow recovery of the gel. Five cycles were performed, and the storage modulus (G') and loss modulus (G") were measured as a function of time.

Results: When La+GP hydrogels with different concentrations were subjected to frequency changes in the range of 0.1-100rad/s, the storage modulus G' was always greater than the loss modulus G", and G' was about 17 times that of G", indicating that The tested sample is a stable hydrogel system (solution: G'<G", solid: G'>G") (Fig. 2A). And it was observed in the oscillation time scan that as the concentration of laponite in the gel increased, the values of the storage modulus and loss modulus both increased (Fig. 2B).

When a gradually increasing strain is applied, the storage modulus of the gel decreases gradually, while the loss modulus increases until the two intersect to a critical point, that is, the gel-sol transition occurs. When the strain force of the hydrogel continues to increase, the storage modulus is continuously smaller than the loss modulus, and the hydrogel system becomes liquid. The process of hydrogel changing from solid to liquid state with increasing strain force indicated the shear-thinning property of La+GP hydrogel (Fig. 2C).

Next, under 6.3rad/s, the strain force was changed every 120 seconds, and the storage modulus (G') and loss modulus (G") were recorded over time, and the La+GP hydrogel was studied. Gel-sol transition ability (Figure 2D). When a small strain force of 0.5% was applied in the first 120s, the G′ value of each gel was significantly higher than the G″ value, indicating that the hydrogel structure was stable. When the strain force increases to 500%, the G' value drops rapidly, below the G" value, the system behaves as a liquid, that is, a gel-sol transition has occurred. When the first cycle ends, the strain force returns to 0.5% When , G' and G" return to their initial values almost instantaneously, that is, they return from the thinned and flowing liquid state to the solid gel state instantaneously.

During the three cycles of strain stress change, when the stress changed from large to small, all the gels could quickly self-repair to the initial gel state, and the storage modulus (G') and loss modulus (G") were the same as the initial gel state. Consistent. These data demonstrate the strong reversibility of the La+GP hydrogel.

As the hectorite concentration increased, the viscosity of the hydrogel also increased, and the initial viscosity of 5% La+GP was 7 times that of 2% La+GP without shear force (Fig. 2E). It shows that the higher the hectorite concentration is, the stronger the shaping ability of the hydrogel is, and the weaker the fluidity is. When subjected to shear force, the viscosity of hydrogels with various concentrations decreased rapidly and greatly, and the fluidity was enhanced. The viscosity characteristic curve indicated that La+GP hydrogel was a non-Newtonian fluid.

This part verifies the gelation time of La+GP composite nano hydrogels with different concentrations synthesized with 2%-5% nanometer mineral hectorite (La) and 0.3% sodium glycerophosphate (GP). As the La concentration increases, the gelation time gradually shortens. 2% and 3% La require 5-10 minutes of gelation time due to the low concentration. When the La concentration increases to 5%, although the gelation time is the shortest, the The higher concentration of La is difficult to mix completely within 90 seconds and the injection resistance is greater, making it difficult to pass through the slender endoscopic injection tube. Therefore, the 4% La+GP hydrogel with a gelation time of 180 seconds is the most suitable for application, and can be manually injected through a 23G endoscopic injection needle through a 5ml syringe. Under the scanning electron microscope, it can be seen that the hydrogel presents a dense and continuous porous sponge-like structure.

Shear thinning refers to the property that the viscosity of the fluid decreases with the increase of the shear rate. It can be seen from the oscillatory strain scan that the gel-sol transition occurs when the applied strain force is greater than the critical value of shear thinning. The modulus G' (reflecting the elasticity of the material) is smaller than the elastic loss modulus G" (reflecting the degree of viscosity of the material), that is, the hydrogel changes from a semi-solid to a flowing liquid. When the strain force disappears, it becomes a flow The dynamic gel can quickly return to the initial semi-solid gel state. The La+GP hydrogel with shear-thinning properties is easy to pass through a 23G submucosal injection needle, and the shear recovery is rapid and self-healing after stress relief The properties make the hydrogel quickly return to the gel state after being injected into the submucosal membrane. These rheological properties provide the possibility for the La+GP hydrogel to become a potential formulation for submucosal injection.

2. Application of hydrogel: submucosal injection and hemostatic effect in vitro and in vivo

2.1 Determination of injection resistance

Using a syringe pump and a manometric system to evaluate the pressure of different concentrations of hydrogel injected through the endoscopic injection needle in the submucosa of the isolated porcine stomach, and compared with the control group of normal saline and 0.4% sodium hyaluronate. Among them, the endoscopic injection needle (22 gauge, 1.8m long, NET2422-C4, Endo-Flex, Germany), the digital pressure gauge (HT-1895, Xinsite, China) and the syringe were connected to the three-way cock, and then fixed with the syringe pump Syringe to set injection speed. Use a 5mL syringe to measure the injection pressure at an injection speed of 0.1mL/s, and record the stabilized pressure value.

Results: Compared with normal saline, the injection resistance of 2%-4% La+GP hydrogel was slightly larger, but it was significantly lower than that of 0.4% sodium hyaluronate commonly used in clinical practice (Figure 3), indicating that the hydrogel has good reliability. Injectable, and La+GP hydrogel can maintain a stable and long-lasting submucosal bulge.

2.2 In vitro experiment of submucosal injection

Choose a suitable injection point at the bottom of the freshly isolated pig stomach, inject 2 mL of La+GP hydrogel, 0.4% sodium hyaluronate, and normal saline into the submucosa with a syringe with a 23G needle, and repeat 5 times in each group. Time, compare the change of uplift height with time in each group. ;

Record the initial height of the mucosal bulge, measure the height of the submucosal bulge with a ruler and a needle at 0, 15, 30, 60, 90, and 120 minutes after submucosal injection, and calculate the maintenance rate of the mucosal bulge;

Maintenance rate = measured height / initial uplift height × 100%

All experimental operations were completed by the same person, and the assistant was responsible for recording the experimental data.

Results: Compared with the initial height (6.50±0.50mm) of the normal saline group commonly used in clinical practice, the initial uplift heights of hydrogels of various concentrations after injection were significantly higher, and the differences were statistically significant (P<0.01). 0.4% sodium hyaluronate (SH) is a submucosal preparation commonly used to replace normal saline in clinical practice. mm) was significantly higher than that of the SH group (P<0.01), and the initial height of the 2% and 3% La+GP groups (10.67±0.58mm, 11.33±1.53mm) was not significantly different from that of the SH group ( P>0.5). Comparing the 2%-5% La+GP hydrogel groups with each other, only the 2% and 4% La+GP groups had differences in initial bulge height (P<0.05) (Figure 4).

After submucosal injection, the bump heights of each group at different time points are shown in Table 2. Compared with the normal saline group, the height of the submucosal bulge in the 2%-5% gel group was statistically different at each time point (P<0.05). Compared with 0.4% sodium hyaluronate (SH), only the higher concentrations of 4% and 5% La+GP groups had statistical differences in height at each time point (P<0.05).

Table 2. Changes in the height of mucosal bumps with different submucosal injections over time

P<0.05。Note: Compared with isotonic saline treatment, *P<0.05; compared with 0.4% sodium hyaluronate, #P<0.05; compared with 2% concentration of laponite, △P<0.05; compared with 3% concentration of laponite,

P<0.05.

In the gel groups with different concentrations, there was no obvious difference in the heights of the 2% and 3% groups at each time point. At 30 minutes, the bulge heights of the 2% and 3% groups were 9.00±0.50mm, 8.67±0.57mm, respectively. Both were lower than the 4% and 5% groups, and the difference was statistically significant (P<0.05). At 90 minutes, the height of the bulge in the saline group dropped to 1.71±0.2mm, which was only about 1/4 of the initial height, and the heights of the 2% and 3% gel groups were 7.33±0.57mm and 7.83±0.76mm, respectively. The maintenance rates were 68.79±4.67% and 69.60±7.88%, which were significantly higher than those of the normal saline group. The bulge heights of the 4% and 5% groups were 10.50±0.50mm and 11.83±1.04mm, and the maintenance rates were 78.78±1.12% and 90.99±2.27%, there were differences compared with the 2% and 3% groups respectively (P<0.05). At 120 minutes, the bulge height of the 4% and 5% groups can still be well maintained, which is statistically significant compared with the 2% and 3% groups (Table 2).

With the time of submucosal injection, the injection gradually diffused to the surrounding tissues, and the raised area in the normal saline group showed a tendency to expand obviously. The initial area was 188.56±9.47mm ² , and after 90 minutes, the area was 1.62 times of the initial area, 0.4%. The initial area of the sodium hyaluronate group was 161.96±8.34 mm ² , and the area was 1.46 times the initial area after 120 minutes. The area of the 2%-5% hydrogel group expanded relatively slowly. The initial areas were 135.71±11.04mm2, 137.59±3.92mm2, 116.22±7.52mm2, 95.68±2.33mm2. After 120 minutes, the areas expanded to the initial 1.33 , 1.34, 1.22, 1.19 times. Compared with the control group, the injections in the gel group maintained the raised morphology better in the submucosal membrane and diffused more slowly.

2.3 ESD stripping time of mucosal bulge in vitro

Take a fresh isolated pig stomach and fix it on a foam board, stick electrodes on it, and adjust the output power of the high-frequency electrosurgical knife to 80W; mark at least 8 points on a circle with a diameter of 3 cm in the simulated APC on the fundus mucosa of the pig stomach, and mark at least 8 points on the marked circle Inject 2%-5% La+GP hydrogel, 0.4% sodium hyaluronate and normal saline submucosally; inject different preparations in the marked circle until the mucosa is completely raised, and use high-frequency electric knife to simulate ESD operation, from the raised The peeling started on one side until the whole piece of mucosa was peeled off, and the complete peeling time of the mucosa was recorded, and each group was repeated 3 times.

Results: After the excised porcine gastric submucosal bulge was stripped by simulating ESD, the submucosal structure after stripping was shown in Figure 5. It can be seen that a small amount of injection is located in the submucosa, and the fibrous connective tissue of the submucosa connected with the mucosal layer can still be seen. In the 2%-5% gel group, after incision, it can be seen that the injection can still maintain a stable uplift shape and separate the mucosal layer from the submucosa layer.

The mucosa with the same diameter (3cm) was lifted by electrotome stripping, and the time required for complete stripping was recorded, as shown in Figure 6, compared with the normal saline group (7.78 ± 0.21min) and the sodium hyaluronate group (6.88 ± 0.12min). ), the average peeling time of the gel group was significantly reduced (P<0.05), and the peeling speed of the 4%La+GP (4.25±0.23min), 5%La+GP (3.94±0.05min) group was significantly lower than that of the 2 Compared with %La+GP (6.20±0.19min) and 3%La+GP (5.96±0.07min) groups, it was faster (P<0.05).

In the submucosal bulge experiment of isolated porcine stomach, compared with the normal saline group commonly used in clinical practice, the bulge heights of hydrogels of various concentrations were higher at each time point within 2 hours, and the area of submucosal bulge spread slowly. , indicating that the composite nanohydrogel of hectorite and sodium glycerophosphate can last longer in the submucosal tissue and form a more stable bulge than normal saline. This is further supported by the structure of the submucosa after stripping with the electric knife. It can be observed that the gel group can separate the mucosa from the submucosa better and maintain a complete gel form after complete stripping, while normal saline and 0.4% After sodium hyaluronate incision, it can be seen that the fibrous connective tissue is still connected to the mucosa, and only a small amount of liquid remains in the submucosa after peeling off. The time for the electric knife to completely strip the mucosa with a diameter of 3 cm was recorded. Compared with the normal saline group and the 0.4% sodium hyaluronate group, the stripping time of the ESD in the 2%-5% La+GP hydrogel group was also shorter.

2.4 Gastric submucosal injection experiment in rats

The rats were fasted for 24 hours, and the rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium 40 mg/kg. After skin preparation and disinfection, incisions were made layer by layer along the midline of the abdomen from under the xiphoid process to fully expose the abdominal cavity. Lift the gastric body out with forceps and cover it with sterile gauze soaked in saline. The stomach is cut open along the anterior wall of the stomach near the greater curvature, and the contents of the stomach are removed and rinsed with normal saline;

NS and 4% La+GP hydrogel were loaded into 30G insulin needles respectively, and 0.1ml of different injections were randomly injected submucosally on both sides of the gastric body. The stomach wall and abdominal wall were sutured after the operation; food and water were fasted for 1 day after the operation, and penicillin (40,000 U/kg) was injected intraperitoneally for 3 consecutive days. Three rats in each group were sacrificed on day 0 and day 7 after operation, and routine H&E pathological examination was performed.

result:

0.1 ml of normal saline and 4% La+GP hydrogel were randomly injected into the submucosa of the front and rear gastric walls of the rats. Compared with the saline group, the area of the bulge formed in the hydrogel group was smaller, and the height of the bulge was significantly higher.

H&E pathological sections also confirmed that compared with normal saline, submucosal injection of 4% La+GP hydrogel can form a thicker submucosal cushion, which can more fully separate the submucosa from the muscular layer (Figure 7).

The H&E pathological sections were made after the normal saline and 4% La+GP submucosal bulge were maintained in the stomach of rats for 7 days. The normal saline had been completely absorbed by the tissue after 7 days, and there was no bulge in the submucosa. In the 4%La+GP group, a small amount of hydrogel remained in the submucosa, and no inflammatory cell infiltration and cell necrosis were observed in the tissue at the injection site.

2.5 Exploration of Hectorite Composite Nano-hydrogels to Promote Blood Coagulation in Vitro

Blood was collected from the eyeballs of the mice and put into EP tubes filled with sodium citrate. Composite nano hydrogels with different concentrations of laponite (2%, 3%, 4%, 5%) and 0.3% sodium glycerophosphate were prepared. The 0.3% GP solution without La and the 2%-5% La+GP hydrogel were placed in a 96-well plate with a syringe and paved (n=3), 100 microliters per well. Add 150 μl coagulation-activated blood (50 μl 0.1M calcium chloride (CaCl 2 ) + 100 μl anticoagulant blood containing sodium citrate) to each well.

At selected time points, each well was washed with saline solution to stop clotting. Immediately aspirate the fluid and repeat the wash until the solution is clear, indicating that all soluble blood components have been removed, and the clotting time is recorded.

result:

The hemostatic ability of 2%-5% La+GP hydrogel was evaluated by measuring the clotting time of whole blood in contact with the surface of the hydrogel material in a 96-well plate to leave behind uncoagulated blood with saline at fixed time points. The condition of the blood clot below is used as the basis of the coagulation time (Figure 8). Under normal circumstances, human blood begins to coagulate within 5-6 minutes. In the control well without La+GP hydrogel material, it was observed that blood clots began to appear at 3 minutes, and the coagulation time of whole blood was 5.6±0.3 minute. In the 0.3% sodium glycerophosphate (GP) group without adding laponite (La), a small clot appeared at about 3 minutes, and a large area of clot appeared at the 5th minute. The time and change of the appearance of the clot All were similar to the control group, and the final coagulation time was 5.52±0.3 minutes, which was not significantly different from the control group (P>0.05).

In the 2% La+GP hydrogel group, blood clots began to form at 3 minutes, and a large area of blood clots gradually began to appear at 4 minutes, but the final clotting time (5.2±0.3min) was not statistically significant compared with the control group Significant difference (P>0.05). With the increase of hectorite concentration in the hydrogel, the coagulation time decreased significantly. In the first minute, a small area of blood clot appeared in the pores of the 3% La+GP hydrogel, and 4% La+GP In the hydrogel group, blood clots with a medium area appeared, and the 5% La+GP hydrogel produced a large area of blood clots.

Compared with the control group, the final coagulation time of whole blood was shortened by 27%, 35% and 54% with 3%, 4% and 5% concentration of composite nano hydrogel respectively, and the difference was statistically significant (P< 0.01), and compared with the 0.3% sodium glycerophosphate group, the coagulation time of each concentration hydrogel group was also significantly reduced, and the difference was statistically significant (P<0.01). Among the hydrogels of 2%-5%La+GP groups, there are also significant differences in coagulation time, and the coagulation speed is positively correlated with the concentration of hectorite (Figure 9).

In addition to the advantages of submucosal bulge height and maintenance rate, La+GP hydrogel also showed accelerated hemostasis performance compared with clinically used normal saline. The whole blood coagulation experiment in vitro showed that, compared with normal saline and 0.3% sodium glycerophosphate, the hydrogel added with hectorite could obviously promote the generation of blood clot. It is expected to exert a better hemostatic effect than normal saline during endoscopic surgery.

3. Hydrogel safety and cytocompatibility

3.1 cck-8 method to detect the effect of La+GP hydrogel on the proliferation activity of GES-1

The extraction solution was prepared according to the cytotoxicity test standard for medical devices (GB/T 16886.5/ISO 10993-5:2009). Take 2g of 5% La+GP hydrogel to freeze-dry for 24 hours, sterilize by ultraviolet light for 24 hours, soak in 37°C 1640 complete medium and incubate in an incubator, centrifuge after 24 hours to get the supernatant, the preparation concentration is 0.2g/ ml of extract.

Add GES-1 cell suspension (cell density: 3000 cells/well) prepared with culture medium to a 96-well plate, 100 μl per well, culture in a _CO2 cell incubator at 37°C for 12 hours, discard the medium, and discard the culture medium for each well. Add 100 μL of extract, and add 1640 complete medium to the control group.

Discard the original culture medium at 0h, 12h, 24h, and 48h respectively, add 10 μl CCK8 and 100 μl 1640 complete medium to each well, and add 10 μl CCK8 and 100 μl medium to the blank wells for 3 wells each as a blank group.

After incubation for 2 hours, the absorbance was measured at 450 nm with a microplate reader.

Results: As shown in Figure 10, the results of the CCK-8 experiment showed that at 4 time points within 48 hours of adding the extract, the cell viability of the hydrogel extract group was 105%, 102%, and 102%, respectively, compared with the control group. 110%, 95%, the difference in the absorbance value of the two groups at each time point was not statistically significant ( FIG. 10 ), indicating that the La/GP hydrogel has good cytocompatibility.

3.2 Verification of long-term safety after subcutaneous injection of 5% La+GP hydrogel in mice

Five mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium 50mg/kg;

Determine the injection point on the back of the mouse, remove the surrounding hair, and then subcutaneously inject 5% La+GP hydrogel (3ml);

The mice were sacrificed 4 weeks after the operation, and the tissues of the injection site and major organs (heart, lung, liver, kidney, spleen) were collected for pathological examination.

result:

After the highest subcutaneous concentration on the back of the mouse, after 4 weeks, the subcutaneous local bulge formed by 5% La+GP hydrogel had no obvious absorption. All the mice survived well, without weight loss, listlessness, etc., and no obvious decrease in the volume of the dorsal bulge. The histological examination of major organs and skin after 4 weeks is shown in Figure 11. No local inflammatory infiltration, major organ infarction, air embolism, etc. were found.

In summary, the effect of the extract of the highest concentration of 5% La+GP hydrogel in the experimental group on the proliferation of GES-1 cells was detected by the CCK-8 method. Compared with the control group, the 5% La+GP hydrogel The extract had no effect on cell proliferation. Long-term observation of subcutaneous injection in mice also showed no effect on important organs.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (6)

1. A submucosal injection, characterized in that, comprises sodium glycerophosphate, hectorite powder and water. 2. The submucosal injection according to claim 1, wherein the submucosal injection contains 0.28-0.32W/V% sodium glycerophosphate and 2-5W/V% hectorite. 3. The submucosal injection according to claim 1, further comprising a dye. 4. The submucosal injection according to claim 3, characterized in that the content of the dye is 0.04-0.1W/V%. 5. The submucosal injection according to claim 4, wherein the dyeing agent is indigo carmine or methylene blue. 6. The preparation method of the submucosal injection according to any one of claims 1-5, characterized in that, the hectorite powder and sodium glycerophosphate powder are mixed evenly, and deionized water is added to fully dissolve, and the dyeing agent is dripped into , stirred, and the formed hydrogel is the submucosal injection.

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