CN115337410A

CN115337410A - Tissue staining agent for endoscopic screening of canceration and preparation method thereof

Info

Publication number: CN115337410A
Application number: CN202211057928.4A
Authority: CN
Inventors: 胡兵; 高远; 叶连松; 徐小鹏; 彭强; 李佳昌; 向世帅
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-08-30
Filing date: 2022-08-30
Publication date: 2022-11-15

Abstract

The invention relates to the technical field of disease screening, in particular to a tissue staining agent for endoscopic screening of canceration and a preparation method thereof. The tissue stain includes: 1 to 4 parts of polyvinylpyrrolidone iodine and 0.5 to 1 part of iodized salt. The invention solves the problem of strong irritation of the tissue staining agent in the prior art. The tissue staining agent has the advantages of wide applicable patient population, high effectiveness, simplicity in operation, high safety and small stimulation.

Description

Tissue staining agent for endoscopic screening of canceration and preparation method thereof

Technical Field

The invention relates to the technical field of disease screening, in particular to a tissue staining agent for endoscopic screening of canceration and a preparation method thereof.

Background

Esophageal cancer is one of the most common malignant tumor diseases in China and is also a high-incidence malignant tumor in the world. Early esophageal cancer has no obvious clinical manifestation, most of the esophageal cancer is in the middle and late stages when symptoms appear, patients in the early stage and without clinical manifestation can be found through routine gastroscopy, and the incidence rate and the fatality rate of esophageal cancer can be reduced through timely intervention or treatment. The diagnosis of early esophageal cancer is therefore particularly important. More than 90% of esophageal cancer in China is squamous cell carcinoma, and early Esophageal Squamous Cell Carcinoma (ESCC) refers to squamous cell carcinoma limited to mucosal layer.

At present, common endoscope inspection techniques for early cancer include common white light endoscopes, pigment endoscopes, electronic pigment endoscopes, magnifying endoscopes, confocal laser microscopy and the like. The pigment endoscope is characterized in that various dyes are scattered or sprayed on the surface of esophageal mucosa, so that the color of a focus is clearly contrasted with that of a normal mucosa, the focus range can be more clearly displayed, and indicative biopsy is convenient to carry out.

The iodine stain (Lugol's liquid) which is clinically used most frequently at present has the advantages of fast color development and clear boundary, and the basic principle is that iodine simple substance reacts with glycogen particles in esophageal squamous epithelium to form brown tea color, while cancer epithelium and intraepithelial neoplasia epithelium contain little glycogen to form light stain or no stain, thereby forming contrast (the specific effect is referred to as figure 1 below). However, iodine staining agents have many adverse reactions including hypersensitivity, gum swelling, sore throat burning, stomach discomfort or vomiting and diarrhea. Therefore, the research and development of the stain which is suitable for wide patient population, high in effectiveness, simple to operate, high in safety and small in stimulation is of great clinical significance for early esophageal cancer screening.

Disclosure of Invention

The invention relates to a tissue stain for endoscopic screening in canceration and a preparation method thereof, and solves the problem of poor safety of the tissue stain in the prior art. The tissue staining agent has the advantages of wide applicable patient population, high effectiveness, simplicity in operation, high safety and small stimulation.

The invention provides a tissue stain for endoscopic screening of canceration, which comprises the following components in parts by weight: 1 to 4 parts of polyvinylpyrrolidone iodine and 0.5 to 1 part of iodized salt.

Polyvinylpyrrolidone (PVP) is a non-ionic polymeric compound that has been widely used due to its excellent unique properties. PVP is a synthetic water-soluble polymer compound, and has the general properties of a water-soluble polymer compound, such as colloid protection, film-forming property, cohesiveness, hygroscopicity, solubilization or coacervation. PVP has excellent physiological inertia, does not participate in human metabolism, has excellent biocompatibility and does not cause any stimulation to skin, mucous membrane, eyes and the like. The pharmaceutical grade PVP is one of three new medicinal auxiliary materials advocated internationally, and can be used as a binder for tablets and granules, a cosolvent for injection and a glidant for capsules; antidotes for eye drugs, retarding agents, lubricants, film-forming coating agents, dispersants for liquid preparations, stabilizers for enzymes and heat-sensitive drugs, and cryopreservative agents. PVP is widely used as an adjuvant for pharmaceutical preparations. In addition, PVP has good food safety, can form a complex with a specific polyphenol compound (such as tannin), and is mainly used as a food clarifying agent and a stabilizing agent for beer, fruit juice, wine and the like in the aspect of food processing. In synthetic polymers, like PVP, PVP is soluble in water and most organic solvents, and has low toxicity and good physiological compatibility.

The tissue coloring agent comprises polyvinylpyrrolidone iodine and iodine salt compound. PVP energy and I ₂ Form complex PVP-I, the product is stable, non-irritant, completely water soluble and free I ₂ And (4) effectively preserving. Free iodine makes the esophagus epithelium contain glycogen to be brown, and the tumor and the xenobiotic parts show slight or no staining due to low glycogen content. Based on the principle, the purpose of staining is achieved, the range of lesions is displayed, and clinical marking of the lesions is guided. And free I ₂ After the consumption, iodine in the PVP-I complex is supplemented in time, and the content of effective iodine simple substances is ensured.

The purpose of adding a proper amount of iodine salt into the tissue staining agent is mainly to ensure that PVP and I are mixed ₂ Better binding and more stable product.

In one or more embodiments of the present invention, the tissue stain comprises the following components in parts by weight: 1 to 3 parts of polyvinylpyrrolidone iodine and 0.5 to 0.8 part of iodized salt.

In one or more embodiments of the present invention, the tissue stain comprises the following components in parts by weight: 1.5 to 2.5 parts of polyvinylpyrrolidone iodine and 0.5 to 0.7 part of iodized salt.

In one or more embodiments of the present invention, the iodine salt is potassium iodide, sodium iodide.

In one or more embodiments of the present invention, the tissue stain comprises the following components in parts by weight: 1.8 to 2.2 parts of polyvinylpyrrolidone iodine and 0.6 to 0.7 part of potassium iodide.

In one or more embodiments of the present invention, the tissue stain comprises the following components in parts by weight: 2 parts of polyvinylpyrrolidone iodine and 0.65 part of potassium iodide.

In one or more embodiments of the present invention, the tissue stain comprises the following components in parts by weight: 1.8 to 2.2 parts of polyvinylpyrrolidone iodine and 0.55 to 0.65 part of sodium iodide.

In one or more embodiments of the present invention, the tissue stain comprises the following components in parts by weight: 2 parts of polyvinylpyrrolidone iodine and 0.6 part of sodium iodide.

In a second aspect of the present invention, there is provided a method for preparing the tissue stain for endoscopic screening of cancer, including: uniformly mixing polyvinylpyrrolidone, iodine simple substance, iodized salt and solvent according to the weight part ratio to obtain the tissue coloring agent.

In one or more embodiments of the invention, the solvent is water.

Advantageous effects

The staining agent simultaneously satisfies the effect of prompting early esophageal cancer by utilizing the classical principle of reaction of iodine solution and glycogen for color development, and the PVP (polyvinyl pyrrolidone) high polymer material is innovatively added, so that the staining agent has obvious effect and mild action, and mucous membrane stimulation symptoms caused by a strong oxidant iodine simple substance are avoided.

Drawings

Fig. 1 is a diagram showing a biological sample 1 stained with the tissue stain of example 1.

FIG. 2 is a real diagram of a biological sample 1 dyed with Lugol's solution at a content of 1%.

Fig. 3 is a diagram showing a biological sample 2 after being stained with the tissue stain of example 2.

FIG. 4 is a real object diagram of the biological sample 2 dyed with 1% Lugol's solution.

Fig. 5 is a diagram showing a biological sample 3 after being stained with the tissue stain of example 3.

FIG. 6 is a real object diagram of the biological sample 3 dyed with 1% Lugol's solution.

Fig. 7 is a diagram showing a biological sample 4 stained with the tissue stain of example 4.

FIG. 8 is a real image of a biological sample dyed in Lugol's solution at a content of 1% in 4%.

Fig. 9 is a diagram showing a biological sample 5 stained with the tissue stain of example 5.

FIG. 10 is a real image of biological sample 5 dyed in Lugol's solution (1%).

Fig. 11 is a diagram showing a biological sample 6 after being stained with the tissue stain of example 6.

FIG. 12 is a drawing showing a biological sample 6 stained with Lugol's solution (content: 1%).

Fig. 13 is a diagram showing a biological sample 7 stained with the tissue stain of example 7.

FIG. 14 is a real object diagram of the biological sample 7 dyed with 1% Lugol's solution.

Fig. 15 is a diagram showing a biological sample 8 stained with the tissue stain of example 8.

FIG. 16 is a real object diagram of the biological sample 8 dyed with 1% Lugol's solution.

Fig. 17 is a diagram showing a biological sample 9 stained with the tissue stain of example 9.

FIG. 18 is a real image of a biological sample 9 dyed with Lugol's solution at a content of 1%.

Fig. 19 is a schematic diagram of dissecting esophagus and gastric mucosa after mouse a01 is subjected to intragastric lavage for 2 hours by using physiological saline.

FIG. 20 is a schematic diagram of dissecting esophagus and gastric mucosa after mouse A02 is subjected to intragastric lavage with normal saline for 2 h.

Fig. 21 is a schematic diagram of dissecting the esophagus and gastric mucosa after mouse a03 is subjected to intragastric administration with physiological saline for 1 d.

Fig. 22 is a schematic diagram of dissecting the esophagus and gastric mucosa after mouse a04 is subjected to gastric lavage treatment with physiological saline for 1 d.

FIG. 23 is a schematic diagram of dissecting esophagus and gastric mucosa after mouse A05 is treated for 3 days by lavage with physiological saline.

FIG. 24 is a schematic diagram of dissecting the esophagus and gastric mucosa of a mouse A06 after gastric lavage with physiological saline for 3 days.

Fig. 25 is a schematic diagram of dissected esophageal and gastric mucosa of mouse B01 after 2h of intragastric gavage treatment with the tissue stain of example 1.

Fig. 26 is a schematic diagram of dissecting esophagus and gastric mucosa of mouse B02 after 2h of intragastric administration with the tissue stain of example 1.

Fig. 27 is a schematic diagram of dissected esophageal and gastric mucosa of mouse B03 after intragastric administration for 1d with the tissue stain of example 1.

Fig. 28 is a schematic diagram of dissected esophageal and gastric mucosa of mouse B04 after intragastric treatment for 1d with the tissue stain of example 1.

Fig. 29 is a schematic diagram of dissecting the esophagus and gastric mucosa 3d after mouse B05 was gavaged with the tissue stain of example 1.

Fig. 30 is a schematic diagram of dissected esophagus and gastric mucosa of mouse B06 after 3d of gavage treatment with the tissue stain of example 1.

FIG. 31 is a schematic diagram showing the dissected esophagus and gastric mucosa after the gavage treatment of mouse A01 using a 1% Lugol's solution for 2 h.

FIG. 32 is a schematic diagram showing dissected esophageal and gastric mucosa after lavage of mouse A02 with 1% Lugol's solution for 2 h.

FIG. 33 is a schematic view of dissected esophagus and gastric mucosa of mouse A03 after intragastric treatment with 1% Lugol's solution.

FIG. 34 is a schematic view of dissected esophageal and gastric mucosa after gastric lavage of mouse A04 with 1% Lugol's solution for 1 d.

FIG. 35 is a schematic view of the dissected esophagus and gastric mucosa after lavage of mouse A05 with 1% Lugol's solution for 3 d.

FIG. 36 is a schematic view of the dissected esophagus and gastric mucosa after lavage of mouse A06 with a solution of 1% Lugol's for 3 d.

Fig. 37 shows normal esophageal mucosa under endoscope.

FIG. 38 is a schematic diagram showing endoscopic esophageal precancer coloration after Lugol's solution spraying (lightly stained part is a diseased tissue).

FIG. 39 is a sample of esophageal precancer in vitro without stain sprayed.

FIG. 40 is a schematic view of the sample of FIG. 39 sprayed with the tissue stain of the present invention, showing the sample of early esophageal cancer in vitro (the middle lightly stained lesion tissue).

FIG. 41 is a schematic diagram of the specimen of FIG. 39 sprayed with a conventional iodine staining reagent to obtain an in vitro esophageal precancer specimen (the middle lightly stained lesion tissue).

FIG. 42 is an unstained esophageal lesion specimen.

FIG. 43 is a specimen of esophageal lesions stained with the tissue stain of example 10.

FIG. 44 is the tissue stain stained esophageal lesion specimen of example 11.

FIG. 45 is the tissue stain stained esophageal lesion specimen of example 12.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present invention.

PVP: brand name: qlodine Prills,2821418916, K16-K18, average molecular weight 8000.

I ₂ : brand name: GENERAL-REAGENT, molecular weight 253.81, 99.8% or more, CAS 7553-56-2.

KI: brand name: GENERAL-REAGENT, AR ≥ 99%, MW =166, CAS 7681-11-0.

NaI: brand name: innochem, A24635-500G, AR,99%.

Example 1

A preparation method of a tissue stain for endoscopic screening of canceration specifically comprises the following steps:

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 3g of NaI, placing in a brown glass bottle;

step two, weighing 500ml of deionized water by using a measuring cup, shaking and uniformly mixing until the solute is completely dissolved, and storing the solution in a shade place to obtain the water-soluble film.

Example 2

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 2.5g of NaI, placing in a brown glass bottle;

and step two, weighing 500ml of deionized water by using a measuring cup, shaking and uniformly mixing until the solute is completely dissolved, and storing the solution in a shade place to obtain the water-soluble film.

Example 3

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 2g of NaI, placing in a brown glass bottle;

Example 4

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 3.5g of NaI, placing in a brown glass bottle;

Example 5

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 4g of NaI, placing in a brown glass bottle;

Example 6

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 3.3g of KI, and placing the KI in a brown glass bottle;

Example 7

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 3g of KI, placing the KI in a brown glass bottle;

Example 8

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 2.5g of KI, placing the KI in a brown glass bottle;

Example 9

step one, weighing 5g PVP and 5g I by an electronic scale ₂ And 4g of KI, placing the KI in a brown glass bottle;

Tissue stain concentration test:

example 10

PVP and I ₂ And KI is added into the deionized water according to the weight ratio in the embodiment 6 to prepare a tissue coloring agent, wherein the PVP-I weight concentration in the tissue coloring agent is 2 percent, and the mass concentration of KI is0.65％。

Example 11

The tissue stain is prepared according to the preparation method, wherein the PVP-I weight concentration is 4% and the KI mass concentration is 0.65%.

Example 12

The tissue stain is prepared according to the preparation method, wherein the PVP-I weight concentration is 6%, and the KI mass concentration is 0.65%.

The tissue stain of examples 10-12 was stained with the same esophageal lesion specimen, fig. 42 is an unstained esophageal lesion specimen, fig. 43 is an esophageal lesion specimen stained with the tissue stain of example 10, fig. 44 is an esophageal lesion specimen stained with the tissue stain of example 11, and fig. 45 is an esophageal lesion specimen stained with the tissue stain of example 12. The results show that: the PVP-I weight concentration of 2% can obviously outline the lesion boundary, the PVP-I weight concentration of 4% and the dyeing effect of 6% concentration have no obvious difference relative to 2%.

Dyeing effect verification experiment

For the dyeing effect of verifying novel dyeing agent, this application has designed clinical trial, through dyeing human esophagus ESD postoperative sample, compares the dyeing effect of novel dyeing agent and traditional Lugol's liquid.

The verification method comprises the following steps: and spraying the tissue staining agent on a human esophagus ESD postoperative sample, taking a picture by fixing a focal length by using a fixed camera after staining, and analyzing by using Image analysis software Image J after taking the picture. And then eluting by using a vitamin C solution, spraying the traditional Lugol's solution on the human esophagus ESD postoperative specimen after the elution is finished, and analyzing by using Image analysis software Image J after photographing. The order of use of Lugol's solution and the tissue stain of the present application can be interchanged.

The Image analysis software Image J is used for outlining the abnormal dyeing area, and the size difference of the lesion is large, so that the abnormal dyeing area is difficult to directly compare with the abnormal dyeing area, so that the difference ratio = (novel dyeing agent-iodine dyeing agent)/iodine dyeing agent is used for carrying out statistical analysis, and the dyeing effect of the tissue dyeing agent is compared with that of the traditional Lugol's solution. The tissue stain of the present application makes normal tissue appear dark brown, and diseased regions are lightly stained/not stained. Lugol's solution makes normal tissue appear dark brown to brown, and the diseased part is lightly stained/not stained.

Lesion area displayed by tissue stain and Lugol's liquid on esophagus staining

TABLE 1

The results of the study in Table 1 show that the tissue stain of the present invention has similar definition of the stained boundary of the esophagus to the conventional Lugol's fluid, as can be seen from a comparison of FIGS. 1-18. The stained area was measured by Imaje J software and tested using SPSS software using differential test method, showing P =0.053 (> 0.05), indicating that there was no difference between the two reagents in the "lesion area" outlined. The tissue staining agent has a staining effect no less than that of the conventional Lugol's solution, and is helpful for an endoscopist to identify early esophageal tumors.

Biosafety test

In order to verify the safety and the irritation of the novel staining agent, the unit designs an animal experiment.

The test protocol was as follows: 18 KM mice were randomly divided into 3 groups of 6 mice each, and esophageal perfusion was performed with physiological saline, 1% tissue stain (tissue stain prepared in example 1), and l% Lugol's solution, respectively. After successful gavage, 2 mice were sacrificed by cervical dislocation each time according to the time (2 hours, 1 day, 3 days) and the mice esophagus and stomach were obtained. Cutting off esophagus and stomach, turning the mucosa outwards, fixing and spreading with pin, observing and shooting, measuring the damage length of esophagus and stomach surface with ruler, and calculating the damage index (UI) of gastric mucosa. The gastric mucosal lesion index of each group of mice was calculated according to the Guth method (i.e., the sum of the gastric mucosal lesion scores of each mouse in each group was added). The punctate bleeding is 1 minute, the linear bleeding length is less than 1mm and is 2 minutes,

the content of the waste water is 3 minutes,

4 minutes and 5 minutes when the diameter is larger than 4 mm; a value of 2[ 2 ], [1 ] to 3 ] having a width of 2mm]。

Tissue stain and Lugol's solution of example 1 dissected gastric mucosal injury index (UI value) after gavage of mice

TABLE 2

The research result shows that the 3 groups of mice have no obvious abnormality of activity state, food intake and spirit after gastric lavage and have no abnormal death within 3 days. The degree of redness and swelling of the gastric mucosa of the mice reached the highest in the acute inflammatory phase (1 day), the average value of UI in the 1% tissue staining agent lavage group was lower than that in the Lugol's solution lavage group, and the mice gradually relieved at 3 days (the research results are shown in the following Table 2). The above results demonstrate the safety of the novel stain and the irritation of the oesophageal-gastric mucosa is less than that of Lugol's solution.

In addition, in terms of formula, the components contained in the tissue stain are stable and completely water-soluble, the irritation is extremely small, the safety is higher theoretically, and the mucosa irritation symptom caused by a strong oxidant iodine simple substance is avoided.

In conclusion, the tissue stain disclosed by the invention has the advantages of wide applicable patient population, high effectiveness, simplicity in operation, high safety and small stimulation. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. The tissue stain for endoscopic screening of canceration is characterized by comprising the following components in parts by weight: 1 to 4 parts of polyvinylpyrrolidone iodine and 0.5 to 1 part of iodized salt.

2. The tissue stain of claim 1, comprising the following components in parts by weight: 1 to 3 parts of polyvinylpyrrolidone iodine and 0.5 to 0.8 part of iodized salt.

3. The tissue stain of claim 1, comprising the following components in parts by weight: 1.5 to 2.5 parts of polyvinylpyrrolidone iodine and 0.5 to 0.7 part of iodized salt.

4. The tissue coloring agent according to any one of claims 1 to 3, wherein the iodine salt is potassium iodide or sodium iodide.

5. The tissue stain of claim 4, comprising the following components in parts by weight: 1.8 to 2.2 parts of polyvinylpyrrolidone iodine and 0.6 to 0.7 part of potassium iodide.

6. The tissue stain of claim 5, comprising the following components in parts by weight: 2 parts of polyvinylpyrrolidone iodine and 0.65 part of potassium iodide.

7. The tissue stain of claim 4, comprising the following components in parts by weight: 1.8 to 2.2 parts of polyvinylpyrrolidone iodine and 0.55 to 0.65 part of sodium iodide.

8. The tissue stain of claim 7, comprising the following components in parts by weight: 2 parts of polyvinylpyrrolidone iodine and 0.6 part of sodium iodide.

9. The method for preparing a tissue stain for endoscopic screening for cancer according to any one of claims 1 to 8, comprising: uniformly mixing polyvinylpyrrolidone, iodine simple substance, iodized salt and solvent to obtain the tissue coloring agent.

10. The method of claim 9, wherein the solvent is water.