CN106398681B - Silica-based pH-sensitive fluorescent nano material, and preparation method and application thereof - Google Patents
Silica-based pH-sensitive fluorescent nano material, and preparation method and application thereof Download PDFInfo
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 86
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 43
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 36
- 239000002105 nanoparticle Substances 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 25
- 235000012239 silicon dioxide Nutrition 0.000 claims abstract description 19
- 239000000126 substance Substances 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000002245 particle Substances 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- 238000003756 stirring Methods 0.000 claims description 17
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- 238000000799 fluorescence microscopy Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 8
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 7
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- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 3
- 239000000298 carbocyanine Substances 0.000 claims description 3
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
- QWYZFXLSWMXLDM-UHFFFAOYSA-M pinacyanol iodide Chemical compound [I-].C1=CC2=CC=CC=C2N(CC)C1=CC=CC1=CC=C(C=CC=C2)C2=[N+]1CC QWYZFXLSWMXLDM-UHFFFAOYSA-M 0.000 claims description 3
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 3
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 claims description 2
- 210000001163 endosome Anatomy 0.000 claims description 2
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- 239000012265 solid product Substances 0.000 claims description 2
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- KWQQHTNSJIJFBO-UHFFFAOYSA-N 3-[methyl-bis(trimethylsilyloxy)silyl]propan-1-amine Chemical compound C[Si](C)(C)O[Si](C)(O[Si](C)(C)C)CCCN KWQQHTNSJIJFBO-UHFFFAOYSA-N 0.000 description 1
- PJURIXUDYDHOMA-UHFFFAOYSA-N 3-[tris[2-(2-methoxyethoxy)ethoxy]silyl]propan-1-amine Chemical compound COCCOCCO[Si](CCCN)(OCCOCCOC)OCCOCCOC PJURIXUDYDHOMA-UHFFFAOYSA-N 0.000 description 1
- SWDDLRSGGCWDPH-UHFFFAOYSA-N 4-triethoxysilylbutan-1-amine Chemical compound CCO[Si](OCC)(OCC)CCCCN SWDDLRSGGCWDPH-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
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- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- YVWPNDBYAAEZBF-UHFFFAOYSA-N trimethylsilylmethanamine Chemical compound C[Si](C)(C)CN YVWPNDBYAAEZBF-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
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Abstract
A pH-sensitive fluorescent nano material based on silicon dioxide, a preparation method and application thereof. The fluorescent dye is connected on the surface of the silica nanoparticle through a pH sensitive chemical bond through a chemical reaction and is applied to the field of biomedicine, the particle size of the fluorescent nanomaterial is within the range of 30-200nm, the inner core of the fluorescent nanomaterial is the silica nanoparticle, and the surface of the silica nanoparticle is connected with the fluorescent dye through a pH sensitive chemical bond. The fluorescent nano material has uniform size, controllable particle size and structure and good stability, does not generate fluorescence or only emits very weak fluorescence in the circulating process, and emits strong fluorescence in a specific pH environment, thereby having the effect of contrast enhancement. The method has the advantages of simple process, high generation efficiency, easy realization of large-scale production and good clinical application potential.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a fluorescent nano material based on silicon dioxide, a preparation method thereof and application thereof in living body fluorescence imaging.
Background
The death rate of malignant tumors is rising year by year, and is one of the major diseases seriously threatening the health of human beings. Investigation has shown that about 200 million new cases occur each year in China, and 130 million people die of malignant tumors, which is the second leading cause of death of Chinese people. There are many current cancer treatments, including surgical resection, chemotherapy, and radiation therapy. The surgical resection is mainly based on years of clinical practice experience of doctors, but the experience has great errors and limitations for judging the cancer boundary. Therefore, how to accurately locate the cancer boundary is the key to successful surgery, and the discovery of the micro-focus is also beneficial to effectively eliminate the cancer and reduce the possibility of relapse.
The optical imaging diagnosis has the advantages of no damage to human body, no wound, no ionizing radiation, small volume, convenient carrying, rapid imaging and high sensitivity. To improve the resolution and contrast of fluorescence imaging, a diagnostic aid, a fluorescent dye, is introduced. Traditional fluorescent dyes are mainly small molecules which are non-specifically distributed in vivo, and only can image blood vessels or certain specific organs, so that the application of the fluorescent dyes in disease diagnosis is limited. With the development of nanotechnology and fluorescence imaging equipment, in recent years, the fluorescence excitation imaging in optical imaging is more and more widely applied to the fields of tumor boundaries and intraoperative navigation. However, the current applications of fluorescence excitation in intraoperative navigation are mostly limited to superficial tumors such as breast cancer, and the applications of the fluorescence excitation in some internal tumors are rarely reported. This is mainly due to the background noise of fluorescence excitation imaging is large, imaging sensitivity is low, and the like, so that the diagnosis and treatment of cancer are not accurate enough. In order to improve this situation. Researchers modify fluorescent dyes, mainly adopt the technology of physical embedding and chemical bonding to nanoparticles, although the enrichment of the fluorescent dyes in tumor sites is increased to a certain extent, the fluorescence of most fluorescent dyes in an aggregation state is quenched to a certain extent, so that the method cannot effectively improve the fluorescence intensity of the tumor sites. Therefore, how to increase the fluorescence intensity of the tumor site and reduce the background noise is an urgent problem to be solved in the current fluorescence imaging technology.
Disclosure of Invention
In view of the above, the main objective of the present invention is to provide a preparation method and application of a silica-based pH-sensitive fluorescent nanomaterial to overcome the disadvantages of poor stability and in vivo nonspecific distribution of the existing fluorescent dyes.
In order to accomplish the above objects, as one aspect of the present invention, there is provided a silica-based pH-sensitive fluorescent nanomaterial having a particle size ranging from 30 to 200 nm; and
the inner core of the fluorescent nano material is a silica nanoparticle, and the surface of the silica nanoparticle is connected with the fluorescent dye through a chemical bond sensitive to pH.
As another aspect of the present invention, the present invention also provides a method for preparing a silica-based pH-sensitive fluorescent nanomaterial, comprising the steps of:
step 1, mixing ethanol, ammonia water and deionized water, and stirring for 0.5-1 h at the temperature of 30 ℃;
and 3, dispersing the fluorescent dye in dimethyl sulfoxide, dropwise adding the fluorescent dye into the silicon dioxide nano particles prepared in the step 2, adding different catalysts according to needs to react for 0-24h, and separating and purifying to obtain a final product.
Preferably, the preparation method further comprises:
and 4, dispersing the final product of the step 3 into deionized water or phosphate buffer solution with the pH value of 7.4, and connecting the target molecule to the surface of the final product through EDC/NHS reaction.
As a further aspect of the invention, the invention also provides the application of the pH-sensitive fluorescent nano-material based on silicon dioxide in vivo fluorescence imaging.
Based on the technical scheme, the method and the fluorescent nano material prepared by the method have the following beneficial effects: the raw materials involved in the invention are cheap, the reaction conditions are mild, and the operability and controllability are strong; the prepared fluorescent nano material has uniform and controllable particle size and good stability, does not generate fluorescence or only emits very weak fluorescence in the circulation process, and emits strong fluorescence in a weak acid environment, so that the contrast enhancement effect is achieved, mainly because the fluorescence is quenched when dye molecules are connected to the surfaces of the nano particles in an aggregation state under the neutral pH condition, chemical bonds between the dye and the nano particles are broken in the weak acid environment, and the dye is dissociated out, so that the strong fluorescence is displayed, and the fluorescent nano material has a good application prospect in the fields of pathological tissue fluorescence imaging and combination with other imaging modes or treatment modes; the method has the advantages of simple process, high generation efficiency, easy realization of large-scale production and good clinical application potential.
Drawings
FIG. 1 is a schematic view of a production process of the present invention;
FIG. 2 is a scanning electron microscope image of nanoparticles of the present invention exemplified by Cy7 fluorescent dye with amino group;
FIG. 3 is a fluorescence emission spectrum of the nanoparticle of the present invention, which is exemplified by Cy7 fluorescent dye with amino group;
FIG. 4 is a graph showing the distribution of the particle size of the nanoparticles of the present invention, which is exemplified by the Cy7 fluorescent dye having an amino group.
Detailed Description
In order to more clearly illustrate the present invention, the present invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
The invention provides a fluorescent nano material based on silicon dioxide, a preparation method and application thereof, aiming at solving the defects of poor stability and in-vivo nonspecific distribution of the existing fluorescent dye. The invention relates to a silica-based pH-sensitive fluorescent nano material prepared by connecting a fluorescent dye to the surface of a silica nanoparticle through a pH-sensitive chemical bond through a chemical reaction, and the silica-based pH-sensitive fluorescent nano material is applied to the field of biomedicine. The silica nano-particle of the invention can be prepared by changing the raw material components to make the surface thereof carry some active groups which are easy to modify, such as amino, hydroxyl, carboxyl and the like; the prepared silicon dioxide nano particles have the advantages of good stability, uniform and controllable particle size, low toxicity, high contrast of in vivo fluorescence imaging and the like, and have good clinical application value in the field of pathological tissue fluorescence imaging.
More specifically, the invention discloses a preparation method of a fluorescent nano material based on silicon dioxide, which comprises the following steps:
step 1: mixing ethanol, ammonia water and deionized water in a certain proportion in a container, such as a flask, and stirring for 0.5-1 h at 30 ℃;
step 2: adding a certain volume of silane reagent quickly, continuing to stir vigorously for 15 min-24 h, centrifugally washing after the reaction is finished, for example, centrifuging at 10000rpm, washing with ethanol for three times, and finally ultrasonically dispersing in ethanol;
and step 3: dispersing fluorescent dye in dimethyl sulfoxide (DMSO), dropwise adding into the prepared silica nanoparticles, adding different catalysts according to the needs to react for 0-24h, finally removing free dye by centrifugation or dialysis or ultrafiltration, and dispersing the particles into deionized water or phosphate buffer salt solution with pH of 7.4;
and 4, step 4: and connecting the target molecule to the surface of the nano particle through EDC/NHS reaction to obtain the required fluorescent nano material based on the silicon dioxide.
Wherein, the target molecule comprises small molecules and antibody target molecules.
In the reaction, the particle size of the nano material can be adjusted by changing the proportion of ethanol, ammonia water, deionized water and silane reagent and the reaction time.
In the above reaction, the fluorescent dye is selected from one or more of fluorescein, rhodamine, cyanine and carbocyanine fluorescent dyes, for example, Cy7 fluorescent dye with amino group.
In the above reaction, the chemical bond sensitive to pH may be selected from one or more of an imine bond, a maleic acid dimethyl ester bond, a hydrazone bond, an oxime bond, an acylhydrazone bond, an acetal/ketal bond, and an orthoester bond. The above chemical bond can be formed between the silica nanoparticle and the fluorescent dye by a fine organic synthesis route design method, and the above chemical bond can be obtained by reacting the amino group-containing Cy7 fluorescent dye with an acid or a base, for example.
In the reaction, the reaction condition is mild, the targeting property of the nano particles can be increased after the target molecules are connected, the enrichment of the nano particles in target tissues is increased, and the contrast between the target tissues and normal tissues is increased.
In the reaction, the density of fluorescent molecules on the surface of the silicon dioxide is changed, so that the dye is subjected to fluorescence quenching, and strong fluorescence is emitted if and only if the pH sensitive chemical bond is broken after the nano material reaches a target group and enters a cell lysosome or an endosome.
The invention also discloses the fluorescent nano material based on silicon dioxide prepared by the preparation method.
The invention also discloses application of the fluorescent nano material based on silicon dioxide in preparation of a living body fluorescence imaging developer, so that focus tissues are diagnosed.
The technical solution and effects of the present invention will be further described with reference to specific preferred embodiments. The following embodiments are merely examples, and the technical solution of the present invention is not limited to the following embodiments, and may be any combination of various embodiments.
In addition, for clarity of description, the difference comparison of examples 1 to 36 is shown in Table 1, and examples 37 to 108 are different only in step (5) as described later, but not shown in Table 1 for economy.
Example 1
(1) Weighing 330mL of ethanol, 8mL of ammonia water and 110mL of deionized water, mixing in a round-bottom flask, and stirring for 0.5h at the temperature of 30 ℃;
(2) weighing 20mL of a mixture of tetraethoxysilane and aminopropyltriethoxysilane (the volume ratio of the tetraethoxysilane to the aminopropyltriethoxysilane is 9: 1-0: 10), quickly adding the mixture into the mixed solution obtained in the step (1), continuously and strongly mechanically stirring for 15min, centrifuging (10000 rpm, 10min) after the reaction is finished, and washing for 2-3 times by using ethanol and deionized water respectively;
(3) dispersing the obtained silicon dioxide nano particles with amino on the surface in a phosphate buffer solution with the pH value of 8.0, dispersing a fluorescent dye containing amino in a DMSO solution, mixing the solutions according to the amino molar ratio of 10: 1-2: 1, and magnetically stirring for 0.5h at room temperature in a dark place;
(4) dripping 50 mu L of 0.25% glutaraldehyde solution into the mixed solution in the step (3), and continuously stirring for reaction for 12 hours;
(5) finally, the reaction solution was centrifuged at 10000rpm for 10min and washed three times with deionized water.
Example 2:
(1) weighing 330mL of ethanol, 8mL of ammonia water and 110mL of deionized water, mixing in a round-bottom flask, and stirring for 0.5h at the temperature of 30 ℃;
(2) weighing 20mL of a mixture of tetraethoxysilane and aminopropyltriethoxysilane (the volume ratio of the tetraethoxysilane to the aminopropyltriethoxysilane is 9: 1-0: 10), quickly adding the mixture into the mixed solution obtained in the step (1), continuously and strongly mechanically stirring for 15min, centrifuging (10000 rpm, 10min) after the reaction is finished, and washing for 2-3 times by using ethanol and deionized water respectively;
(3) dispersing the obtained silicon dioxide nano particles with amino on the surface in a phosphate buffer solution with the pH value of 8.0, dispersing a fluorescent dye containing aldehyde groups in a DMSO solution, mixing the solutions according to the molar ratio of the amino to the aldehyde groups of 10: 1-2: 1, and magnetically stirring at room temperature in a dark place for reaction for 24 hours;
(4) finally, the reaction solution was centrifuged at 10000rpm for 10min and washed three times with deionized water.
Example 3:
(1) weighing 330mL of ethanol, 8mL of ammonia water and 110mL of deionized water, mixing in a round-bottom flask, and stirring for 0.5h at the temperature of 30 ℃;
(2) weighing 20mL of a mixture of tetraethoxysilane and aminopropyltriethoxysilane (the volume ratio of the tetraethoxysilane to the aminopropyltriethoxysilane is 9: 1-0: 10), quickly adding the mixture into the mixed solution obtained in the step (1), continuously and strongly mechanically stirring for 15min, centrifuging (10000 rpm, 10min) after the reaction is finished, and washing for 2-3 times by using ethanol and deionized water respectively;
(3) dispersing the obtained silicon dioxide nano particles with amino on the surface in a phosphate buffer solution with the pH value of 8.0, dispersing a fluorescent dye containing carbonyl in a DMSO solution, mixing the solutions according to the molar ratio of the amino being 10: 1-2: 1, and magnetically stirring the mixed solution at room temperature for 0.5 h;
(4) adding DCC with 4 times of carbonyl equivalent into the mixed solution, and continuously stirring and reacting for 24 hours in a dark place;
(5) finally, the reaction solution was centrifuged at 10000rpm for 10min and washed three times with deionized water.
Examples 4 to 6:
the same as in embodiments 1 to 3, except that the tetraethoxysilane added in step (2) was partially replaced with a polyethylene glycol-modified silane reagent, the results were similar.
Examples 7 to 36:
the method is the same as the method of examples 1-6, except that aminopropyltriethoxysilane is replaced by one or more of aminopropyltrimethoxysilane, aminomethyltrimethylsilane, 3-aminopropylbis (trimethylsiloxy) methylsilane, 4-aminobutyltriethoxysilane, and 3-aminopropyltris (methoxyethoxyethoxy) silane in step (2), and the results are similar.
Examples 37 to 108:
the only difference from examples 1 to 36 is that, after step (5), the reaction proceeds according to the amino group: NHS activated antibody (examples 37-72) or small molecule target molecule (examples 73-108) is added into the activated target molecule at a molar ratio of 1: 1-100: 1 to obtain the targeted silica-based pH sensitive fluorescent nanoparticle.
TABLE 1
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A pH sensitive fluorescent nano material based on silicon dioxide is characterized in that: the particle size of the fluorescent nano material is in the range of 30-200 nm; and
the inner core of the fluorescent nano material is a silica nanoparticle, and the surface of the silica nanoparticle is connected with a fluorescent dye through a chemical bond sensitive to pH; the chemical bond sensitive to pH is selected from one or more of imine bond, maleic acid dimethyl ester bond, hydrazone bond, oxime bond, acylhydrazone bond, acetal/ketal bond and ortho ester bond; the fluorescent dye is selected from one or more of fluorescein, rhodamine, anthocyanin and carbocyanine fluorescent dyes; the fluorescent nanomaterial is capable of dissociating the fluorescent dye after the pH sensitive chemical bond is broken, thereby exhibiting strong fluorescence.
2. The fluorescent nanomaterial of claim 1, wherein the silica nanoparticles are prepared from one or more of ethyl orthosilicate, aminopropyltriethoxysilane, aminopropyltrimethoxysilane, and polyethylene glycol-linked silane.
3. The fluorescent nanomaterial of claim 1, wherein the surface of the silica nanoparticle is further bonded with target molecules comprising small molecules and antibody-like target molecules through EDC/NHS reaction.
4. The fluorescent nanomaterial of claim 3, wherein the chemical bond sensitive to pH is cleaved to emit intense fluorescence if and only after the fluorescent nanomaterial reaches the target group and enters the lysosome or endosome of the cell.
5. A preparation method of a pH sensitive fluorescent nano material based on silicon dioxide is characterized by comprising the following steps:
step 1, mixing ethanol, ammonia water and deionized water, and stirring for 0.5-1 h at the temperature of 30 ℃;
step 2, rapidly adding a silane reagent, continuously and violently mechanically stirring for 15 min-24 h, centrifugally washing after the reaction is finished, and dispersing a solid product in ethanol to obtain silicon dioxide nano particles;
step 3, dispersing fluorescent dye in dimethyl sulfoxide, dropwise adding the fluorescent dye into the silicon dioxide nano particles prepared in the step 2, adding different catalysts according to requirements to react for 0-24h, and separating and purifying to obtain a final product; the inner core of the final product is a silica nanoparticle, the surface of the silica nanoparticle is connected with a fluorescent dye through a chemical bond sensitive to pH, and the chemical bond sensitive to pH is selected from one or more of an imine bond, a maleic acid dimethyl ester bond, a hydrazone bond, an oxime bond, an acylhydrazone bond, an acetal/ketal bond and an orthoester bond; the fluorescent dye is selected from one or more of fluorescein, rhodamine, anthocyanin and carbocyanine fluorescent dyes; the fluorescent nanomaterial is capable of dissociating the fluorescent dye after the pH sensitive chemical bond is broken, thereby exhibiting strong fluorescence.
6. The method of claim 5, further comprising:
and 4, dispersing the final product of the step 3 into deionized water or phosphate buffer solution with the pH value of 7.4, and connecting the target molecule to the surface of the final product through EDC/NHS reaction.
7. The method of claim 6, wherein the target molecule comprises a small molecule and an antibody-based target molecule.
8. Use of the silica-based pH-sensitive fluorescent nanomaterial according to any one of claims 1 to 4 in the preparation of a developer for in vivo fluorescence imaging.
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