Hybrid polyhedral oligomeric silsesquioxane as well as preparation method and application thereof

Abstract

The invention discloses hybrid polyhedral oligomeric silsesquioxane and a preparation method thereof, belongs to the technical field of biomedicine, and solves the problems that in perfusion type blood vessel marking in the prior art, the uniformity of exogenous fluorescent substances is not ideal, and the compatibility of the exogenous fluorescent substances and a tissue transparency method is not ideal. The method comprises the following steps: dissolving a fluorescent dye and a corresponding silane coupling agent in methanol, and stirring at room temperature for 1-4 hours to generate a coupling reaction; adding KH-550 into the reaction system, dropwise adding concentrated hydrochloric acid, and continuously stirring at 25 DEG C for 7 days; and filtering, collecting filter residues, adding tetrahydrofuran into the filtrate, collecting separated solids, combining the filter residues and the separated solids, and drying to obtain the hybrid polyhedral oligomeric silsesquioxane. The hybridized polyhedral oligomeric silsesquioxane provided by the invention has the advantages of low cost, simple operation flow, low toxicity, certain design freedom, replaceable luminous core and tolerance to common tissue transparency flow.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more particularly to a hybrid cage-like polyhedral oligomeric silsesquioxane and its preparation method and applications. Background Art

[0002] As a class of efficient and intuitive visualization research means, fluorescence imaging technology has been widely applied in the biomedical field in recent years and shows broad prospects in aspects such as real-time monitoring of physiological processes such as gene expression, drug delivery tracing, and metabolism. Compared with traditional imaging technologies, fluorescence imaging technology has significant advantages such as high sensitivity, low biological damage, high theoretical imaging resolution, and real-time monitoring. However, the actual resolution of fluorescence microscopy imaging is related to the stability of the optical path between the excitation light source-fluorescent label-receiver during the imaging process and the intensity of the effective signal transmitted, and is thus restricted by the limited penetration depth of the excitation and emission light of the label in optically inhomogeneous large biological tissues. Especially when the excitation light is visible light, even though visible light band fluorescent probes with good stability and high quantum yield under physiological conditions are already very mature in industrial production and commercial sales, their applications in biological fluorescence microscopy imaging still have many inconveniences. In cases where fine physiological structures need to be observed, tissue section imaging and then stitching and modeling are often required, but this process of acquiring and processing imaging data is very cumbersome and there is a possibility of structural errors caused by mechanical damage during sectioning.

[0003] The emergence and popularization of tissue clearing technology have revolutionary significance for fluorescence microscopy imaging of large tissues. It is an auxiliary imaging technology that washes away some lipids and proteins in tissues by means of soaking in different solutions and then fills them with hydrogels or liquid organic reagents with refractive indices close to the average value of the tissues, so that the tissues reach a state where the refractive indices of each part are relatively uniform. Thus, this technology enables visible light to penetrate centimeter-scale large tissues, with very low cost, simple process, and good repeatability. Through tissue clearing technology, researchers can capture more information about macroscopic fine structures through intuitive and high-resolution fluorescence microscopy imaging. Currently, the combined strategy of tissue clearing technology and fluorescence microscopy imaging has been widely applied to the research and application of physiological and pathological models.

[0004] Blood vessels, as important physiological structures and objects of observation and research, have a unique cavity structure that has given rise to two fluorescence labeling ideas: filling-type labeling and blood vessel wall labeling. According to the specific labeling substances used, they can be further divided into three categories, namely: enabling mouse blood vessel wall cells to carry fluorescent proteins based on gene editing technology, antibody staining of blood vessels using tomato lectin, etc., and pouring blood vessels by injecting a hydrogel precursor solution containing exogenous fluorescent substances through cardiac perfusion. For existing clearing methods, the high-level structure of fluorescent proteins is easily damaged in the soaking of clearing reagents, resulting in fluorescence quenching. Most commercially available exogenous fluorescent substances (such as organic small molecule dyes and quantum dots) have good solubility in organic solvents commonly used as clearing reagents and are easily washed away during tissue clearing, causing significant losses of the fluorescence signals provided by these labeling substances during common tissue clearing processes. Therefore, specific research involving the combination of tissue clearing technology and fluorescence microscopy imaging often aims to achieve an ideal balance between the degree of clearing and the retention of fluorescence signals, and compromise to obtain the required information; correspondingly, how to balance clear and stable fluorescence labeling and deep tissue clearing has also become a hot topic in the field of imaging methodology.

[0005] Currently, the vast majority of research results in this field focus on the development of clearing methods that can provide a relatively mild environment for fluorescently labeled antibodies while maintaining a high clearing power. Although filling-type labeling greatly reduces costs and shortens the labeling cycle compared to antibody labeling, the solubility problems, loss of fluorescence signals, aggregation, or diffusion outside blood vessels of the commercially available fluorescent probes used in it are relatively serious during tissue clearing. If a general medium that can uniformly and stably retain exogenous fluorescent substances in the aqueous solution used for perfusion and the formed hydrogel can be introduced, it will greatly expand the application scope of perfusion-based blood vessel labeling and even become a new low-cost general solution for blood vessel labeling based on tissue clearing technology. Summary of the Invention

[0006] The present invention provides a hybrid cage-like polyhedral oligomeric silsesquioxane, its preparation method and application, to solve the problems of insufficient uniformity of exogenous fluorescent substances and their compatibility with tissue clearing methods in perfusion-based blood vessel labeling in the prior art.

[0007] In the first aspect, the present invention provides a preparation method of a hybrid cage-like polyhedral oligomeric silsesquioxane, comprising the following steps: dissolving a fluorescent dye and a corresponding silane coupling agent in methanol, and stirring at room temperature for 1 - 4 h to carry out a coupling reaction; adding KH-550 to the reaction system ， dropwise adding concentrated hydrochloric acid, and continuing to stir at 25 °C for 7 days; filtering, collecting the filter residue, adding tetrahydrofuran to the filtrate and collecting the precipitated solid, combining the filter residue and the precipitated solid, and drying to obtain the hybrid cage-like polyhedral oligomeric silsesquioxane.

[0008] As a possible implementation, the fluorescent dye is a small organic fluorescent molecule, which has any one or several of amino group, mercapto group, acryloyl group, isocyanato group, isothiocyanato group, succinimide, maleimide, carboxyl group; the silane coupling agent is one or several of KH-550 and 3-isocyanatopropyltriethoxysilane.

[0009] As a possible implementation, by volume, the addition amount of KH-550 is 7 to 50 times that of the silane coupling agent; and / or, by volume, the ratio of the addition amounts of methanol, the sum of KH-550 and the silane coupling agent, and concentrated hydrochloric acid is 24:3:4.

[0010] As a possible implementation, when the dye is a thiol, the corresponding silane coupling agent has a methacryloxy group, and the conditions for the coupling reaction are light, room temperature, and stirring for 1 h; when the dye is an isothiocyanate or succinimide, the corresponding silane coupling agent has an amino group, and the conditions for the coupling reaction are room temperature and stirring for 4 h; when the dye is an isothiocyanate or maleimide, the corresponding silane coupling agent has a mercapto group, and the conditions for the coupling reaction are room temperature and stirring for 4 h; when the dye is an amine or a thiol, the corresponding silane coupling agent has an isocyanato group, and the conditions for the coupling reaction are room temperature and stirring for 4 h.

[0011] In a second aspect, the present invention provides a hybrid cage-like polyhedral oligomeric silsesquioxane prepared by the preparation method according to any one of the possible implementations of the first aspect.

[0012] In a third aspect, the present invention provides an application of the hybrid cage-like polyhedral oligomeric silsesquioxane according to any one of the possible implementations of the second aspect in vascular imaging technology.

[0013] As a possible implementation, the application method includes: mixing gelatin, buffer solution, and deionized water and stirring until the gelatin is dissolved, adding the hybrid cage-like polyhedral oligomeric silsesquioxane, and adding an aqueous solution of paraformaldehyde for the first time under stirring conditions after fully dissolving to obtain working solution A; using the gelatin solution as working solution B; sequentially perfusing PBS buffer solution, an aqueous solution of paraformaldehyde for the second time, and a mixture of working solution A and working solution B; obtaining the target tissue, fixing it for 24 h, and performing vascular imaging.

[0014] As a possible implementation, the buffer solution is 10×PBS buffer solution; and / or, the mass fraction of the aqueous solution of paraformaldehyde for the first time is 16%; and / or, the mass fraction of the aqueous solution of paraformaldehyde for the second time is 4% and the pH is 7.4.

[0015] As a possible implementation, the method for preparing the first paraformaldehyde aqueous solution includes the steps of: mixing formaldehyde and deionized water at a mass ratio of 16:90 and heating to 60 °C; dropping in sodium hydroxide with a concentration of 5 mol / L to adjust the pH of the reaction system to 9; supplementing deionized water until the total addition ratio of formaldehyde and deionized water is 16:100.

[0016] As a possible implementation, on a g:mL:mL basis, the addition ratio of the gelatin, the buffer solution, and the deionized water is 0.2:1:3.84; and / or, the mass ratio of the hybrid cage-like polyhedral oligomeric silsesquioxane and the gelatin is 1:2.

[0017] As a possible implementation, the preparation process of the working solution A is carried out under the temperature condition of 37 °C; the fixation includes: soaking the target tissue in a 4% paraformaldehyde aqueous solution and fixing it at 4 °C for 24 h.

[0018] The fluorescence signal donor provided by the present invention can be uniformly and stably dispersed in the perfusion aqueous solution, with strong and stable fluorescence signals and adjustable wavelengths. The fluorescence signals can be uniformly anchored in the perfusion hydrogel through formaldehyde, and the provided pre-crosslinking step can effectively prevent the leakage of fluorescence signals outside the blood vessels during perfusion; the tissue samples obtained after blood vessel casting by the method provided by the present invention can withstand including uDISCO (ultimate 3D imaging of solvent-cleared organs), FDISCO (3D imaging of solvent-cleared organs with superior fluorescence preserving capability), PEGASUS (polyethylene glycol (PEG)-associated solvent system), CUBIC (clear unobstructed brain imaging cocktails and computational analysis), MACS (m-xylylenediamine (MXDA)-based Aqueous Clearing System). After being washed by the tissue clearing methods based on organic / water-soluble cleaning agents, the fluorescence signals of the labeled blood vessels are still well retained, with low labeling cost and low toxicity, and have potential practical value in the fields of in vivo imaging, disease diagnosis, and pathological model research. Brief Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 It is an imaging diagram of the fluorescence signals of blood vessels labeled with conventional blood vessel labeling materials before and after different clearing methods. Among them, the conventional blood vessel labeling materials are Gel-Dex-FITC, Dil, LEL-D649, and CD31-A647 respectively. Before represents before clearing, and After represents after clearing; Figure 1Cited from the literature [Jingtan Zhu, Yating Deng, Tingting Yu, Xiaomei Liu, Dongyu Li, Dan Zhu. Optimal combinations of fluorescent vessel labeling and tissue clearing methods for three-dimensional visualization of vasculature. Neurophotonics. Vol. 9, Issue 4, 045008 (November 2022). Address: https: / / doi.org / 10.1117 / 1.NPh.9.4.045008].

[0021] Figure 2 This is a hybrid cage-like polyhedral oligomeric silsesquioxane prepared from different dyes as raw materials in the embodiments of the present invention, and is applied to the vascular imaging diagram of mouse tissue sections. Among them, Brain represents the brain, Liver represents the liver, and Kidney represents the kidney; PyMal represents N-(1-pyrene) maleimide; MC-NHSE represents 7-methoxycoumarin-3-carboxylic acid succinimidyl ester, FITC represents fluorescein isothiocyanate, and RbITC represents rhodamine isothiocyanate.

[0022] Figure 3 This is a transmission electron micrograph of hybrid cage-like polyhedral oligomeric silsesquioxane Ⅰ provided in the embodiments of the present invention.

[0023] Figure 4 This is the cerebral vascular imaging diagram of a mouse using hybrid cage-like polyhedral oligomeric silsesquioxane Ⅰ as a raw material in the embodiments of the present invention, and the mouse tissue sections are transparently imaged by uDISCO, FDISCO, PEGASUS, CUBIC, and MACS methods. Among them, Brain represents the brain, Liver represents the liver, and Kidney represents the kidney.

[0024] Figure 5 This is the vascular imaging diagram of tissue sections of the brain, kidney, and liver of a mouse when hybrid cage-like polyhedral oligomeric silsesquioxane Ⅰ is used as a raw material in the embodiments of the present invention. Among them, A, B, and C represent the brain, liver, and kidney respectively.

[0025] Figure 6 This is the three-dimensional vascular imaging diagram of the mouse brain when hybrid cage-like polyhedral oligomeric silsesquioxane Ⅱ is used as a raw material in the embodiments of the present invention. Detailed implementation manners

[0026] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0027] To solve the problems in the prior art that the homogeneity of exogenous fluorescent substances and their compatibility with tissue clearing methods in perfusion vascular labeling are not ideal enough, this embodiment provides a hybrid cage-like polyhedral oligomeric silsesquioxane and its preparation method and application.

[0028] In the embodiments of the present invention, a hybrid cage-like polyhedral oligomeric silsesquioxane is prepared from a silane coupling agent and a fluorescent dye. In the preparation method claimed by the present invention, the type, dosage of the silane coupling agent and the reaction conditions of the coupling reaction are determined according to different riveting groups, as shown in Table 1 specifically:

[0029] Table 1 Corresponding relationship between raw materials and reaction conditions in the preparation method

[0030]

[0031]

[0032] The embodiments of the present invention provide imaging diagrams of the vascular fluorescence signals of conventional vascular labeling materials before and after different clearing methods as shown in Figure 1 ; It can be seen from Figure 1 that different degrees of fluorescence signal loss occurred in the conventional materials after being treated by several methods.

[0033] The embodiments of the present invention also provide hybrid cage-like polyhedral oligomeric silsesquioxanes prepared from different dyes and are used for vascular imaging of mouse brain tissue sections, and the results are as shown in Figure 2 .

[0034] Next, the technical solutions of the present invention will be further elaborated in conjunction with specific embodiments.

[0035] Example 1

[0036] This embodiment provides an experiment for preparing a hybrid cage-like polyhedral oligomeric silsesquioxane.

[0037] 624 mg of fluorescein isothiocyanate, 3 mL of KH-550, and 24 mL of methanol were added to a 50 mL flask, mixed, and continuously stirred at 60 °C for 12 h. 4 mL of concentrated hydrochloric acid was added to the reaction system and stirred at 25 °C for 7 days. The precipitated solid A was collected by filtration. 24 mL of tetrahydrofuran was added to the filtrate, centrifuged, and the precipitated solid B was collected. The obtained solid A and solid B were combined, dried under vacuum at 60 °C, and then ground into powder to obtain hybrid cage-like polyhedral oligomeric silsesquioxane I, which was sealed and reserved for use. The transmission electron micrograph of hybrid cage-like polyhedral oligomeric silsesquioxane I is as shown in Figure 3 shown.

[0038] 370 mg of coumarin 120, 400 μL of ureidopropyltriethoxysilane, and 24 mL of methanol were added to a 50 mL flask and continuously stirred at 60 °C for 12 h. 2.6 mL of KH-550 and 4 mL of concentrated hydrochloric acid were added to the reaction system and stirred at 25 °C for 7 days. The precipitated solid C was collected by filtration. 24 mL of tetrahydrofuran was added to the obtained filtrate, centrifuged, and the precipitated solid D was collected. The obtained solid C and solid D were combined, dried under vacuum at 60 °C, and then ground into powder to obtain hybrid cage-like polyhedral oligomeric silsesquioxane II, which was sealed and reserved for use.

[0039] Example 2

[0040] This example provides an application experiment of hybrid cage-like polyhedral oligomeric silsesquioxane.

[0041] 0.2 g of gelatin, 1 mL of 10×PBS buffer solution, and 3.84 mL of deionized water were taken and stirred at 37 °C until the gelatin was dissolved; 0.1 g of the hybrid cage-like polyhedral oligomeric silsesquioxane to be tested was added to the obtained solution. After being fully dissolved, 0.16 mL of 16% aqueous paraformaldehyde solution was added dropwise under stirring conditions for pre-crosslinking, and the original temperature was maintained and stirred for 1 h to obtain working solution A; 0.6 g of gelatin and 1.16 mL of deionized water were taken and stirred at 37 °C until the gelatin was dissolved to obtain working solution B.

[0042] 5 mL of PBS buffer solution and 5 mL of 4% aqueous paraformaldehyde solution were successively injected into the blood vessels of mice in the form of cardiac perfusion; working solution A and working solution B were mixed and degassed by ultrasound for 1 min to obtain a mixed working solution. The mixed working solution was injected into the blood vessels of mice, and the internal organs of the mice were harvested and immersed in 4% aqueous paraformaldehyde solution and fixed at 4 °C for 24 h. Three-dimensional vascular imaging was performed.

[0043] When using the hybrid cage-like polyhedral oligomeric silsesquioxane I prepared in Example 1 as the hybrid cage-like polyhedral oligomeric silsesquioxane to be tested, cerebral, renal, and hepatic blood vessel imaging maps of mice were obtained by using methods such as uDISCO, FDISCO, PEGASUS, CUBIC, and MACS for clearing, and the results are as shown in Figure 4The results shown are obtained from Figure 4 It can be seen that the tissue samples obtained after vascular perfusion with hybrid cage-like polyhedral oligomeric silsesquioxane I can withstand cleaning by tissue clearing methods based on organic / water-soluble cleaning agents, including uDISCO, FDISCO, PEGASUS, CUBIC, and MACS, and still retain the fluorescent signals marking blood vessels well after cleaning.

[0044] When using the hybrid cage-like polyhedral oligomeric silsesquioxane I prepared in Example 1 as the hybrid cage-like polyhedral oligomeric silsesquioxane to be tested, tissue section vascular imaging was performed on the brains, kidneys, and livers of mice, respectively, and the results shown as A, B, and C in Figure 5 were obtained.

[0045] When using the hybrid cage-like polyhedral oligomeric silsesquioxane II prepared in Example 1 as the hybrid cage-like polyhedral oligomeric silsesquioxane to be tested, the mouse brain was taken out, the standard operation procedure of the tissue clearing method uDISCO was performed, and three-dimensional vascular imaging was carried out, and the results shown in Figure 6 were obtained. Comparing the Figure 1 , Figure 2 , Figure 4 , Figure 5 and Figure 6 provided by the present invention, it can be seen that the materials used in the present invention have excellent resistance to clearing cleaning.

[0046] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concepts. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0047] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A preparation method of a hybrid cage-like polyhedral oligomeric silsesquioxane, characterized in that, It includes the following steps: Dissolve the fluorescent dye and the corresponding silane coupling agent in methanol, and stir at room temperature for 1 - 4 h to carry out the coupling reaction; Add KH-550 to the reaction system, dropwise add concentrated hydrochloric acid, and continue to stir at 25 °C for 7 days; Filter, collect the filter residue, add tetrahydrofuran to the filtrate and collect the precipitated solid, combine the filter residue and the precipitated solid, and dry to obtain the hybrid cage-like polyhedral oligomeric silsesquioxane.

2. The preparation method according to claim 1, wherein, The fluorescent dye is a fluorescent organic small molecule, which has any one or several of amino group, mercapto group, acryloyl group, isocyanato group, isothiocyanato group, succinimide, maleimide, carboxyl group; The silane coupling agent is one or several of KH-550, 3-isocyanatopropyltriethoxysilane.

3. The preparation method according to claim 1, characterized in that, By volume ratio, the addition amount of KH-550 is 7 - 50 times that of the silane coupling agent; And / or, by volume ratio, the ratio of the sum of KH-550 and the silane coupling agent, methanol, and the addition amount of concentrated hydrochloric acid is 1:6 - 24:1 - 2.

4. The preparation method according to claim 1, characterized in that, When the dye is a thiol, the corresponding silane coupling agent has a methacryloyloxy group, and the conditions for the coupling reaction are light irradiation, room temperature, and stirring for 1 h; When the dye is an isothiocyanate or succinimide, the corresponding silane coupling agent has an amino group, and the conditions for the coupling reaction are room temperature and stirring for 4 h; When the dye is an isothiocyanate or maleimide, the corresponding silane coupling agent has a mercapto group, and the conditions for the coupling reaction are room temperature and stirring for 4 h; When the dye is an amine or a thiol, the corresponding silane coupling agent has an isocyanato group, and the conditions for the coupling reaction are room temperature and stirring for 4 h.

5. The hybrid cage-like polyhedral oligomeric silsesquioxane prepared by the preparation method according to any one of claims 1 - 4.

6. The application of the hybrid cage-like polyhedral oligomeric silsesquioxane according to claim 5 in vascular imaging technology.

7. The application according to claim 6, characterized in that The application method includes: Mix gelatin, buffer solution, and deionized water and stir until the gelatin is dissolved, add the hybrid cage-like polyhedral oligomeric silsesquioxane, and after fully dissolving, add the first paraformaldehyde aqueous solution under stirring conditions to obtain working solution A; Use the gelatin solution as working solution B; Perfuse PBS buffer solution, the second paraformaldehyde aqueous solution, and the mixed solution of working solution A and working solution B in sequence; Obtain the target tissue, fix it for 24 h, and perform vascular imaging.

8. The application according to claim 7, wherein The buffer solution is 10×PBS buffer solution; And / or, the mass fraction of the first paraformaldehyde aqueous solution is 16%; And / or, the mass fraction of the second paraformaldehyde aqueous solution is 4%.

9. The application according to claim 7, wherein By g:mL:mL, the addition ratio of gelatin, buffer solution, and deionized water is 0.2:1:3.84; And / or, the mass ratio of the hybrid cage-like polyhedral oligomeric silsesquioxane and gelatin is 1:

2.

10. The application according to claim 7, characterized in that The preparation process of working solution A is carried out at a temperature of 37 °C; The fixation includes: soaking the target tissue in a 4% paraformaldehyde aqueous solution and fixing it at 4 °C for 24 h.

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