A composition for tissue transparency and a method for preparing the same, a kit and a method for tissue transparency treatment

Abstract

The application discloses a kind of tissue transparentization compositions and preparation method thereof, wherein composition includes: transparentization medium, basic neutralizing agent, aqueous solvent, amphiphilic transition solvent and refractive index regulator, the transparentization medium is any one or several of surifenone, surifenone salt, p-xylylene dicyclohexanesulfonic acid and its salt or 2-phenyl benzimidazole-5-sulfonic acid and its salt, basic neutralizing agent is used to neutralize acidic group in transparentization medium to form clear homogeneous system;Refractive index regulator is used to adjust the refractive index of composition to the range matched with the target refractive index of the tissue to be treated.The composition of the application can realize the rapid transparentization of living tissue, and after flushing with PBS buffer, the transparentization effect can realize a certain degree of reversal, and the whole process does not cause tissue staining, which is beneficial to subsequent imaging analysis.

Description

Technical Field

[0001] This invention belongs to the field of tissue transparency technology, and particularly relates to a tissue transparency composition, its preparation method, kit, and tissue transparency treatment method. Background Technology

[0002] Although biological specimens are inherently three-dimensional, the obscuring effect of light scattering hinders high-resolution imaging of deep tissues. One approach to visualizing thick tissues is to sequentially slice them into thin sections and then reconstruct a three-dimensional image using computational methods. However, this method is not only laborious but also limited when the true three-dimensional properties of the tissue cannot be determined from the thin sections. Investigations of fine tissue structures, such as those in non-nervous, non-bone tissues or organs, are best performed within intact tissue.

[0003] To preserve the true three-dimensional structure of tissues, interest in developing tissue clearing agents and techniques has surged. Tissue clearing techniques directly make tissues transparent, allowing imaging at depth. By using microscopes capable of imaging selectively planes in depth (i.e., optical sectioning of tissues), three-dimensional (3D) images can be rapidly obtained without the cutting artifacts or sample damage associated with sequential sectioning methods. Effective optical clearing methods facilitate deep tissue bioimaging by reducing in-situ light scattering while preserving tissue integrity for accurate signal reconstruction.

[0004] Traditional tissue clearing methods are mostly used for ex vivo fixed tissues and often rely on dehydration, organic solvents, high refractive index immersion solutions, or long-term treatment. They are not suitable for rapid, reversible, and low-staining treatment of living tissue surfaces. Although recent absorption dye clearing can create transparent windows on living skin, many dyes have strong absorption in the visible light region, which can easily cause tissue staining. Furthermore, the stability of the formulation and the residual color after reversible rinsing still limit subsequent imaging and clinical translation. Summary of the Invention

[0005] In view of this, the present invention provides a tissue clearing composition and its preparation method, kit and tissue clearing treatment method, which can achieve rapid clearing of living tissues, and the clearing effect can be reversed to a certain extent after rinsing with PBS buffer, and the whole process does not cause tissue staining, which is beneficial to subsequent imaging analysis.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The first aspect of the present invention provides a tissue-transparent composition, comprising: a transparentizing agent, an alkaline neutralizing agent, an aqueous solvent, an amphiphilic transition solvent, and a refractive index modifier; The clearing medium is any one or more of benzophenone-4, benzophenone salts (such as benzophenone sodium-5), terephthalimide dicamphor sulfonic acid (TDSA) and its salts, or 2-phenylbenzimidazole-5-sulfonic acid (PBSA) and its salts. The alkaline neutralizing agent is used to neutralize the acidic groups in the clearing medium to form a clear and homogeneous system. The refractive index adjuster is used to adjust the refractive index of the composition to a range that matches the target refractive index of the tissue to be treated.

[0007] The transparentizing agent is used to regulate the optical properties of the dense collagen structure within the tissue and achieve uniformity of refractive index within the tissue; the alkaline neutralizing agent is used to neutralize the acidic groups in the transparentizing agent and improve the water solubility of the transparentizing agent and the stability of the composition system; the aqueous solvent is used to provide an environment for ion hydration and maintain the homogeneity of the composition system; the amphiphilic transition solvent is used to regulate the polarity of the system and achieve transition and compatibility between the aqueous phase and the organic phase; the refractive index regulator is used to adjust the overall refractive index of the composition system so that the refractive index of the composition reaches the preset target value.

[0008] Preferably, the mass fraction of the transparentizing medium is 0.5-15 w / v%. Transparentizing mediums within this range are within the safe range of use and can meet the requirements of tissue optical transparentization treatment. The mass fraction of the transparentizing medium is preferably 1-8 w / v; more preferably 1.5-5.5 w / v; and even more preferably 1.85-4.65 w / v.

[0009] Preferably, the alkaline neutralizing agent comprises an inorganic base and / or an organic base; The inorganic base is selected from any one or more of sodium hydroxide, potassium hydroxide, or sodium bicarbonate; the organic base is selected from any one or more of L-arginine, L-lysine, tromethamine, triethanolamine, and 2-amino-2-methyl-1-propanol.

[0010] Preferably, the molar ratio of the alkaline neutralizing agent to the acidic groups in the clearing medium is 0.8-2.0:1. Using a slightly excessive amount of alkaline neutralizing agent is beneficial for the full dissolution and dissociation of the clearing medium, while avoiding potential damage to living tissue caused by high pH values. The molar ratio of the alkaline neutralizing agent to the acidic groups in the clearing medium is preferably 0.95-1.5:1; more preferably 1.0-1.2:1; and even more preferably 1.05-1.07:1.

[0011] Preferably, the refractive index modifier is used to adjust the refractive index of the system so that the refractive index of the composition is adjusted to a range that matches the target refractive index of the tissue to be treated. The composition of the present invention can achieve 1.35-1.55, preferably 1.40-1.48; more preferably 1.42-1.45; and even more preferably 1.430-1.438.

[0012] Preferably, the refractive index modifier is selected from one or more of PEG-200, PEG-400, PEG-600, glycerol, sorbitol, xylitol, sucrose, and glucose.

[0013] Preferably, the aqueous solvent is selected from one or more of deionized water, purified water, PBS, Tris buffer, HEPES buffer, and physiological saline.

[0014] Preferably, the amphiphilic transition solvent is selected from one or more of propylene glycol, glycerol, 1,3-propanediol, butanediol, polyethylene glycol 200 / 300, and diethylene glycol monoethyl ether.

[0015] Preferably, the volume fraction of the aqueous solvent is 10%-60%, and the volume fraction of the amphiphilic transition solvent is 5%-40%. The aqueous solvent is used to provide the basic solvent environment, and the amphiphilic transition solvent is used to adjust the polarity of the system and promote the compatibility and transition between the aqueous phase and the organic phase.

[0016] Preferably, the volume fraction of the aqueous solvent is 20%-45%, more preferably 25%-35%, and even more preferably 29.6%-30.2%.

[0017] Preferably, the volume fraction of the amphiphilic transition solvent is 10%-30%; more preferably 12%-22%; and even more preferably 14.8%-18.6%.

[0018] A second aspect of the present invention also provides a method for preparing a tissue-clearing composition, characterized by comprising the following steps: To prepare an alkaline solution: Mix an aqueous solvent and an alkaline neutralizing agent to prepare an alkaline solution; Aqueous phase dissociation of active components: A transparentizing agent is added to the alkaline solution to dissociate the transparentizing agent and form a homogeneous clear solution; Then, an amphiphilic transition solvent is added to the clarified solution to obtain a mixed solution. A refractive index modifier is then added to the mixed solution to bring its refractive index to a predetermined target value. Using this order of addition yields a transparent and homogeneous solution.

[0019] Since sulphone, sulphone salt, TDSA, and TDSA salt have poor solubility in organic solvents, if the transparentizing agent, amphiphilic transition solvent, and refractive index regulator are mixed at the same time, or if the transparentizing agent is mixed with the refractive index regulator first and then the alkaline neutralizer is added, the system will become turbid, crystallize, separate into layers, or the transparentizing effect will be reduced.

[0020] A third aspect of the present invention provides a tissue clearing kit, comprising a first container and a second container, the first container containing a clearing medium, and the second container containing one or more of an alkaline neutralizer, an aqueous solvent, an amphiphilic transition solvent, and a refractive index modifier; the kit is used to prepare the tissue clearing composition before use.

[0021] The fourth aspect of the present invention provides a method for clearing the surface tissue of live animals for purposes other than disease diagnosis or treatment, wherein the tissue clearing composition is applied to the surface of an ex vivo tissue sample or infiltrated into the ex vivo tissue sample, thereby increasing the transparency of the ex vivo tissue sample within 5-30 min; optionally, the sample is rinsed with PBS, physiological saline or buffer solution to at least partially reverse the clearing effect.

[0022] Because the present invention adopts the above technical solution, it has the following advantages and positive effects compared with the prior art: (1) Achieving controllable adjustment and matching of the system's refractive index: The tissue-clearing composition provided by this invention contains a specific clearing medium, which is a water-soluble ultraviolet absorber with good biocompatibility. It has strong absorption characteristics in the ultraviolet region (280-360 nm) and contains acidic groups. Its absorption cutoff is located at the edge of the visible light region, and its coloring is weak in the visible light region. Therefore, the refractive index in the visible light region can be increased without causing significant coloring. The clearing medium is neutralized by an alkaline agent to form an aqueous ultraviolet-absorbing clearing medium. At the same time, it is compounded with an amphiphilic transition solvent and a refractive index regulator, so that the clearing medium containing strong polar groups can be stably dissolved and uniformly dispersed, thereby obtaining a clearing working solution with good uniformity and long-term stability.

[0023] (2) Possesses the potential for further optimization of biocompatibility and reversibility of processing: The tissue-transparent composition provided by this invention has low irritation to tissues and can effectively penetrate tissues to achieve the transparency effect. At the same time, the processing can be reversibly controlled through the PBS buffer system, which is beneficial to maintaining the integrity of tissue structure and basic physiological characteristics, thereby expanding its application scope in in vivo tissue imaging.

[0024] (3) Improved tissue optical transparency and enhanced imaging quality: The composition provided by this invention can effectively reduce light scattering caused by refractive index mismatch within tissues, improve the penetration depth and signal quality of optical imaging, and facilitate obtaining clearer information on deep tissue structures under imaging conditions such as confocal microscopy and two-photon microscopy. Furthermore, both sulpiride and TDSA can achieve a relatively significant transparency effect within approximately 10 minutes, making them suitable for rapid transparency processing. After rinsing with PBS buffer, the transparency effect can be reversed to a certain extent, and the entire process does not cause tissue staining, which is beneficial for subsequent imaging analysis.

[0025] (4) This invention achieves controllable adjustment and matching of the refractive index of the system by optimizing the order of addition of each component: First, an alkaline solution is prepared by mixing an aqueous solvent with an alkaline neutralizing agent. Then, a water-soluble transparentizing agent is mixed with the alkaline solution to dissociate the transparentizing agent and form a homogeneous clear solution. Next, an amphiphilic transition solvent is introduced into the clear solution to adjust the polarity of the system and achieve the transition from the aqueous phase system to the organic phase system. On this basis, a refractive index regulator is introduced step by step to control the refractive index of the system. The refractive index of the composition system can be adjusted to be close to the target tissue according to actual needs, which is beneficial to improving the propagation characteristics of light in the tissue and reducing interface reflection and scattering caused by refractive index mismatch. Attached Figure Description

[0026] Figure 1 These are comparative images of the appearance of the composition of Example 1 of the present invention after being stored at room temperature for 7 days, wherein (a) is a photograph of the composition just prepared and (b) is a photograph of the composition after being stored for 7 days; Figure 2 These are comparative images of the appearance of the composition of Example 2 of the present invention after being stored at room temperature for 7 days. Figure (a) is a photograph of the composition just prepared, and (b) is a photograph of the composition after being stored for 7 days. Figure 3 The images shown are before and after the composition of Example 1 of the present invention was applied to the abdomen of a mouse, and after the optical transparency was reversed. In Figure (a), the original image of the mouse abdomen is shown; (b), the image after the composition was applied to the abdomen of the mouse is shown; and (c), the image after the optical transparency of the mouse abdomen was reversed. Figure 4 The images shown are before and after the composition of Example 2 of the present invention was applied to the abdomen of a mouse, and after the optical transparency was reversed. In Figure (a), the original image of the mouse abdomen is shown; (b), the image after the composition was applied to the abdomen of the mouse is shown; and (c), the image after the optical transparency of the mouse abdomen was reversed. Detailed Implementation

[0027] The following detailed description, in conjunction with the accompanying drawings and specific embodiments, provides a tissue-clearing composition, its preparation method, kit, and tissue-clearing treatment method according to the present invention. The advantages and features of the present invention will become clearer from the following description.

[0028] All chemical reagents used in the following examples and application cases were commercially available, and the mouse experiments all met animal testing standards.

[0029] Example 1: Tissue-clearing composition with a refractive index of 1.432 Formula ratio: Sulphone powder: 0.25g; 1M sodium hydroxide aqueous solution: 0.85ml; deionized water: 3.15ml; pure propylene glycol: 2.0ml; pure PEG400: 7.5ml (for final volume adjustment and refractive index adjustment).

[0030] Specific preparation process steps: Step 1: In a clean solution preparation container, add 3.15 ml of deionized water and 0.85 ml of 1 M sodium hydroxide solution in sequence, shake thoroughly to mix, and obtain an alkaline solution with a total volume of about 4.0 ml; Step 2: Weigh 0.25g of sulphone powder and add it directly to the above alkaline aqueous system. Mix under stirring or vortex conditions and heat in a water bath at about 55°C until the solid is completely dissolved to form a clear solution. Then cool to room temperature for later use. Step 3: Add 2.0 ml of propylene glycol to the above clear solution and mix thoroughly to form a homogeneous system, thereby achieving the transition adjustment of the system's polarity; Step 4: First, add about 7.0 ml of PEG400 to the system and mix thoroughly. Then, adjust the system by adding the remaining PEG400 in portions or dropwise, mixing thoroughly after each addition, and measuring the refractive index of the system using an Abbe refractometer until it stabilizes at 1.432.

[0031] Example 2: Tissue-clearing composition with a refractive index of 1.436 Formula ratio: TDSA powder: 0.2g; 1M sodium hydroxide aqueous solution: 0.4ml; 1M arginine aqueous solution: 0.36ml; deionized water: 0.54ml; pure propylene glycol: 0.8ml; pure PEG400: 2.2ml (for final volume adjustment and refractive index adjustment).

[0032] Specific preparation process steps: Step 1: In a clean solution preparation container, add 0.54 ml of deionized water, 0.4 ml of 1M sodium hydroxide solution and 0.36 ml of 1M arginine aqueous solution in sequence, shake thoroughly to mix, and obtain a composite alkaline aqueous solution with a total volume of about 1.3 ml.

[0033] Step 2: Add 0.2g of TDSA powder to the above alkaline aqueous system, mix under stirring or vortex conditions, and heat in a water bath at about 55°C until a homogeneous and clear solution is formed.

[0034] Step 3: Add 0.8 ml of propylene glycol to the obtained clear solution and mix thoroughly to construct a transition solvent system; Step 4: Add about 2.0 ml of PEG400 to the system and mix well. Then, finely adjust the system by adding the remaining PEG400 dropwise and repeatedly measuring the refractive index until the refractive index of the system stabilizes at 1.436.

[0035] like Figure 1-2 As shown, the compositions of Examples 1 and 2, after preparation, presented optically homogeneous and clear solutions. After being stored at room temperature in a dark and sealed environment for 7 days, no significant changes were observed in their appearance; the system remained clear, and no crystallization, liquid phase separation, or turbidity was observed. Furthermore, periodic monitoring showed no significant change in the refractive index of the solution during storage. These results indicate that under the stated formulation conditions, the composition possesses good physical and optical stability, meeting the requirements for laboratory use and storage within a certain period.

[0036] Application Example: Skin Transparency Treatment of Live Mice Step 1: Preparation of animal subjects BALB / c mice (approximately 2–3 weeks old, weighing 9–11 g) were selected as experimental subjects; these conditions facilitated better observation of skin transparency. Differences in subcutaneous fat content and skin diffusion characteristics among individuals may affect the transparency effect.

[0037] After anesthetizing the animal by inhalation or intraperitoneal injection, the target abdominal area is treated for hair removal. First, apply Veet hair removal cream to the target area, and after approximately 5 minutes, wipe away the hair and cream with an alcohol swab. Veet hair removal cream can be applied repeatedly (1–2 minutes) as needed to remove any remaining hair.

[0038] During the treatment, ensure the hair removal cream remains on the skin for no more than 10 minutes to avoid skin irritation or burns. After treatment, ensure the skin is clean and dry. The skin should be pink after hair removal.

[0039] Step Two: Transparency Processing Method The transparent compositions prepared in Examples 1 and 2 were used for topical treatment of mouse skin. The solution was applied to the hairless skin using a cotton swab, and gentle massage was performed to promote uniform distribution and penetration of the solution on the skin surface.

[0040] After approximately 5 minutes of continuous treatment, the transparency of the treated area increased. Further extending the treatment time (to approximately 10 minutes) yielded a significant transparency effect.

[0041] like Figure 3 As shown in (b) of 4 and (b) of 4, the clearing compositions prepared in Examples 1 and 2 exhibited significant clearing effects when applied to the abdomen of mice, without any change in the surface volume of the mouse abdomen. After applying PBS solution to the area coated with the clearing composition for 5 minutes, the clearing effect was partially reversed, and no staining occurred on the tissue. This indicates that the clearing compositions do not cause tissue staining. Figure 3 (c) and Figure 4 (c) shows that the skin is intact and there are no adverse effects such as skin irritation, which is beneficial for subsequent imaging analysis.

[0042] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, if these changes fall within the scope of the claims of the present invention and their equivalents, they shall still fall within the protection scope of the present invention.

Claims

1. A composition for tissue transparency, characterized in that, include: Transparenting agents, alkaline neutralizers, aqueous solvents, amphiphilic transition solvents, and refractive index modifiers; The clearing medium is any one or more of sulphone, sulphone salt, terephthalimide dicamphor sulfonic acid and its salt, or 2-phenylbenzimidazole-5-sulfonic acid and its salt; the alkaline neutralizing agent is used to neutralize the acidic groups in the clearing medium to form a clear and homogeneous system; the refractive index adjuster is used to adjust the refractive index of the composition to a range that matches the target refractive index of the tissue to be treated.

2. The tissue-clearing composition according to claim 1, characterized in that, The transparentizing agent has a mass-volume fraction of 0.5-15 w / v.

3. The tissue-clearing composition according to claim 2, characterized in that, The transparentizing agent has a mass-volume fraction of 1-8 w / v.

4. The tissue-clearing composition according to claim 3, characterized in that, The transparentizing agent has a mass-volume fraction of 1.5-5.5 w / v.

5. The tissue-clearing composition according to claim 4, characterized in that, The transparentizing agent has a mass-volume fraction of 1.85-4.65 w / v.

6. The tissue-clearing composition according to claim 1, characterized in that, The alkaline neutralizing agent includes inorganic bases and / or organic bases; The inorganic base is selected from any one or more of sodium hydroxide, potassium hydroxide, or sodium bicarbonate; the organic base is selected from any one or more of L-arginine, L-lysine, tromethamine, triethanolamine, and 2-amino-2-methyl-1-propanol.

7. The tissue-clearing composition according to claim 1, characterized in that, The molar ratio of the alkaline neutralizing agent to the acidic groups in the transparentizing agent is 0.8-2:

1.

8. The tissue-clearing composition according to claim 4, characterized in that, The molar ratio of the alkaline neutralizing agent to the acidic groups in the transparentizing agent is 0.95-1.5:

1.

9. The tissue-clearing composition according to claim 4, characterized in that, The molar ratio of the alkaline neutralizing agent to the acidic groups in the transparentizing agent is 1.0-1.2:

1.

10. The tissue-clearing composition according to claim 4, characterized in that, The molar ratio of the alkaline neutralizing agent to the acidic groups in the transparentizing agent is 1.05-1.07:

1.

11. The tissue-clearing composition according to claim 1, characterized in that, The refractive index modifier is selected from one or more of PEG-200, PEG-400, PEG-600, glycerol, sorbitol, xylitol, sucrose, and glucose.

12. The tissue-clearing composition according to claim 1, characterized in that, The aqueous solvent is selected from one or more of deionized water, purified water, PBS, Tris buffer, HEPES buffer, and physiological saline. The amphiphilic transition solvent is selected from one or more of propylene glycol, glycerol, 1,3-propanediol, butanediol, polyethylene glycol 200 / 300, and diethylene glycol monoethyl ether.

13. The tissue-transparent composition according to claim 1, characterized in that, The volume fraction of the aqueous solvent is 10%-60%, and the volume fraction of the amphiphilic transition solvent is 5%-40%.

14. The tissue-clearing composition according to claim 13, characterized in that, The volume fraction of the aqueous solvent is 20%-45%, and the volume fraction of the amphiphilic transition solvent is 10%-30%.

15. The tissue-clearing composition according to claim 14, characterized in that, The volume fraction of the aqueous solvent is 25%-35%, and the volume fraction of the amphiphilic transition solvent is 12%-22%.

16. The tissue-clearing composition according to claim 15, characterized in that, The volume fraction of the aqueous solvent is 29.6%-30.2%, and the volume fraction of the amphiphilic transition solvent is 14.8%-18.6%.

17. The tissue-clearing composition according to any one of claims 1-8, characterized in that, The refractive index of the composition is 1.35-1.

55.

18. A method for preparing a tissue-clearing composition as described in any one of claims 1-17, characterized in that, Includes the following steps: To prepare an alkaline solution: Mix an aqueous solvent and an alkaline neutralizing agent to prepare an alkaline solution; Aqueous phase dissociation of active components: A transparentizing agent is added to the alkaline solution to dissociate the transparentizing agent and form a homogeneous clear solution; Then, an amphiphilic transition solvent is added to the clarified solution to obtain a mixed solution. A refractive index regulator is then added to the mixed solution to make the refractive index of the mixed solution reach the preset target value.

19. A tissue clearing kit, characterized in that, The kit includes a first container and a second container, the first container containing a clearing medium, and the second container containing one or more of an alkaline neutralizer, an aqueous solvent, an amphiphilic transition solvent, and a refractive index modifier; the kit is used to prepare the tissue clearing composition according to any one of claims 1-17 before use.

20. A method for transparentizing the surface tissue of a live animal not for disease diagnosis or treatment purposes, characterized in that, The composition of any one of claims 1-17 is applied to the surface of or immerses the isolated tissue sample, thereby increasing the transparency of the isolated tissue sample within 5-30 minutes.

21. The method for transparentizing the surface tissue of a live animal for purposes other than disease diagnosis or treatment, as described in claim 20, is characterized in that... Rinse with PBS, saline, or buffer to at least partially reverse the clearing effect.

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