Method for marking collagen with quantum dots, collagen-quantum dot fluorescent probe and application
By preparing fluorescent probes using chemically modified collagen and CdSe/ZnS quantum dots, the problem of photobleaching with fluorescent dyes was solved, enabling dynamic, clear monitoring and long-term imaging of collagen in vivo.
Patent Information
- Application Number
- CN202511390549.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-26
- Publication Date
- 2025-11-21
AI Technical Summary
Existing fluorescent dyes are prone to photobleaching in vivo, resulting in insufficient imaging stability and making it impossible to achieve long-term visual monitoring of collagen in vivo.
A collagen-quantum dot fluorescent probe was prepared by chemically modifying CdSe/ZnS quantum dots with collagen and then using EDC and NHS to activate the carboxyl groups on the surface of the quantum dots to crosslink with the amino groups in the collagen.
It enables dynamic and clear monitoring of collagen in vivo, enhances the stability and selectivity of quantum dots, and supports long-term in vivo imaging.
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Figure CN120992574A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of nanobiomedicine, and more particularly relates to a method for labeling collagen with quantum dots, a collagen-quantum dot fluorescent probe and application. BACKGROUND
[0002] In clinical medicine, in vivo imaging, as an important branch of optical imaging, is often used for disease diagnosis, monitoring, drug delivery, etc. However, most fluorescent dyes are prone to photobleaching in vivo, and the imaging stability is insufficient, which is not conducive to long-term imaging. Studies have found that CdSe / ZnS quantum dots have unique optical properties and great potential in the imaging field, and have characteristics such as not prone to photobleaching and high fluorescence intensity, and are widely used in in vivo imaging.
[0003] Collagen is the most abundant structural protein in mammals, and is widely present in tissues and organs such as skin, bone and Achilles tendon. It is also one of the main components of processing waste in aquatic products, livestock and other agricultural and sideline industries. As a biological macromolecule, collagen has good biodegradability and biocompatibility, and has wide application prospects in the field of biomedicine. For example, it is used as a drug delivery system, a tissue scaffold material, etc., and is widely used in the medical field of disease diagnosis and treatment and tissue regeneration. However, collagen itself does not have fluorescence, and cannot realize the visualization of the diagnosis and treatment process, which brings certain challenges to the implementation of in situ dynamic visualization diagnosis and treatment.
[0004] Therefore, it is urgent to propose a method for labeling collagen with quantum dots and a collagen-quantum dot fluorescent probe and application. SUMMARY
[0005] The present application aims at the deficiencies of the prior art, and proposes a method for labeling collagen with quantum dots and a collagen-quantum dot fluorescent probe and application. The present application realizes the dynamic and clear monitoring of the in vivo distribution of collagen, and improves the in vivo visualization of collagen.
[0006] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a method for labeling collagen with quantum dots, which comprises the following steps:
[0007] S1: mixing and stirring collagen sponge with an organic solution to obtain a collagen dissolution solution, and dialyzing to obtain a collagen solution;
[0008] S2: mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and phosphate buffer to obtain an activation solvent; mixing the CdSe / ZnS quantum dot solution with the activation solvent to perform an activation reaction, thereby obtaining a carboxyl activation product; mixing the carboxyl activation product with the collagen solution to perform a cross-linking reaction, and washing to obtain the collagen-quantum dot fluorescent probe.
[0009] In the present application, the method of the present application activates the carboxyl groups on the surface of the CdSe / ZnS quantum dots by EDC and NHS, and then the activated carboxyl groups on the surface of the CdSe / ZnS quantum dots are cross-linked with the amino groups in the collagen, so as to chemically modify the collagen by using the CdSe / ZnS quantum dots, thereby obtaining the collagen-quantum dot fluorescent probe of the present application.
[0010] In the present application, the collagen sponge can be a collagen sponge prepared from collagen of various sources to adapt to different biomedical application scenarios. According to the present application, preferably, the collagen source of the collagen sponge is at least one of fish collagen, mammalian collagen and bullfrog collagen.
[0011] According to the present application, preferably, the collagen sponge is at least one of grass carp collagen sponge, bovine tendon collagen sponge and bullfrog collagen sponge.
[0012] According to the present application, preferably, the organic solution is an aqueous acetic acid solution with a concentration of 0.01-0.05 moL / L.
[0013] According to the present application, preferably, the stirring is performed on a low-temperature stirring table, the temperature of the stirring is-2℃ to 0℃, and the time of the stirring is 6-8h.
[0014] According to the present application, preferably, the dialysis uses a dialysate which is a phosphate buffer with a pH of 7.2-7.4.
[0015] According to the present application, preferably, the temperature of the dialysis is-2℃ to 0℃, and the time of the dialysis is 2-3d.
[0016] According to the present application, preferably, the pH of the collagen solution is 7.2-7.4.
[0017] According to the present application, preferably, the CdSe / ZnS quantum dot solution is prepared by mixing CdSe / ZnS quantum dots and a phosphate buffer, and the concentration of the CdSe / ZnS quantum dots in the CdSe / ZnS quantum dot solution is 0.02-0.04μmol / L, preferably 0.04μmol / L.
[0018] According to the present application, preferably, the temperature of the activation reaction and the cross-linking reaction is independently 20-30℃;
[0019] The time of the activation reaction is 25-35min;
[0020] The time of the cross-linking reaction is 3.5-4.5h.
[0021] The second aspect of the present application provides the collagen-quantum dot fluorescent probe prepared by the method of labeling collagen with quantum dots.
[0022] The third aspect of the present application provides the application of the collagen-quantum dot fluorescent probe in preparing in vivo imaging agents.
[0023] In the present application, the living body is an experimental mouse.
[0024] The beneficial effects of the technical solution of the present application are as follows:
[0025] 1. The present application uses quantum dots to modify collagen, which 'lights up' collagen with high quality without destroying the activity of collagen.
[0026] 2. Meanwhile, the present application can further enhance the stability (prolong the metabolic cycle) and selectivity of quantum dots, because quantum dots are zero-dimensional nanomaterials, which are easily metabolized by the reticuloendothelial system and the kidney in vivo. Collagen is an important component of the extracellular matrix, and has certain targeting properties, which makes the coupling of collagen and quantum dots form a new whole, which is conducive to improving its in vivo stability, and thus plays a long-term imaging role in vivo.
[0027] 3. The present application realizes the dynamic and clear monitoring of collagen distribution in vivo. The present application is expected to solve the inherent technical barriers such as the inability to visualize tracking in the diagnosis and treatment process of drug delivery and tissue regeneration.
[0028] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS
[0029] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:
[0030] Figure 1a The ultraviolet spectrum of the CdSe / ZnS quantum dot solution used in Example 1 of the present application is shown.
[0031] Figure 1bThe fluorescence spectrum of the CdSe / ZnS quantum dot solution used in Example 1 of the present application is shown.
[0032] Figure 2a The fluorescence spectrum of the collagen-quantum dot fluorescent probe obtained in Example 1 of the present application is shown (in the present application, "QDs" refers to CdSe / ZnS quantum dots).
[0033] Figure 2b The fluorescence spectrum of the collagen-quantum dot fluorescent probe obtained in Example 2 of the present application is shown.
[0034] Figure 2c The fluorescence spectrum of the collagen-quantum dot fluorescent probe obtained in Example 3 of the present application is shown.
[0035] Figure 3a The particle size test result of the CdSe / ZnS quantum dot solution used in Example 1 of the present application is shown.
[0036] Figure 3b The Zeta potential test result of the CdSe / ZnS quantum dot solution used in Example 1 of the present application is shown.
[0037] Figure 4 The hemolysis test result of the CdSe / ZnS quantum dot solution used in Example 1 of the present application is shown.
[0038] Figure 5 The dynamic distribution of the collagen-quantum dot fluorescent probe obtained in Example 1 of the present application in mice over time and the imaging of the collagen-quantum dot fluorescent probe in the heart, liver, spleen, lung and kidney of mice are shown.
[0039] Figure 6 The dynamic distribution of the collagen-quantum dot fluorescent probe obtained in Example 2 of the present application in mice over time and the imaging of the collagen-quantum dot fluorescent probe in the heart, liver, spleen, lung and kidney of mice are shown.
[0040] Figure 7 The dynamic distribution of the collagen-quantum dot fluorescent probe obtained in Example 3 of the present application in mice over time and the imaging of the collagen-quantum dot fluorescent probe in the heart, liver, spleen, lung and kidney of mice are shown.
[0041] Figure 8 The dynamic distribution of the fluorescent probe of Comparative Example 1 of the present application in mice over time and the imaging of the collagen-quantum dot fluorescent probe in the heart, liver, spleen, lung and kidney of mice are shown. DETAILED DESCRIPTION
[0042] Preferred embodiments of the present application will be described in more detail below. Although the following describes preferred embodiments of the present application, it is to be understood that the present application can be carried out in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and fully convey the scope of the present application to those skilled in the art.
[0043] In the following various examples and comparative examples:
[0044] CdSe / ZnS quantum dots were purchased from Wuhan Jia Yuan Quantum Dot Technology Development Co., Ltd., Q2605;
[0045] Grass carp collagen sponge, bovine tendon collagen sponge and bullfrog collagen sponge are commercially available;
[0046] The CAS number of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) is 25952-53-8;
[0047] The CAS number of N-hydroxysuccinimide (NHS) is 6066-82-6;
[0048] The PBS solution is the same PBS solution as in step (1).
[0049] Example 1
[0050] The present embodiment provides a method for labeling collagen with quantum dots, comprising the following steps:
[0051] (1) 2.5 g of sodium dihydrogen phosphate (NaH2PO4·H2O), 29.5 g of disodium hydrogen phosphate (Na2HPO4·H2O) and 43.875 g of sodium chloride (NaCl) were weighed with an analytical balance to prepare 5 L of PBS solution (phosphate buffer solution). The above prepared PBS solution was used to dilute 8 μmol / L CdSe / ZnS quantum dots to obtain 4 mL of CdSe / ZnS quantum dot solution with a concentration of 0.04 μmol / L.
[0052] (2) A dialysis bag with a molecular weight cut-off of 1400 and a length of 10-20 cm was cut and placed in a beaker. 10 g of NaHCO3 was weighed and dissolved in 500 mL of ultrapure water to obtain a NaHCO3 solution; another 0.1862 g of EDTA (ethylene diamine tetraacetic acid) was also dissolved in 500 mL of ultrapure water to obtain an EDTA solution. The above dialysis bag-containing beaker was added with the above NaHCO3 solution to submerge the dialysis bag, and an appropriate amount of EDTA solution was added, then boiled for 10 min, washed with ultrapure water, and then placed in the remaining EDTA solution and boiled for another 10 min. After cooling, the treated dialysis bag was placed in acetic acid for standby;
[0053] Reconfigure PBS solution 5L, put in the refrigerator for cold storage. Take 10 mg of grass carp collagen sponge in a 100 mL beaker, add 100 mL of 0.01 M aqueous acetic acid to the beaker containing the grass carp collagen sponge, and place it on a low-temperature stirring table for thorough stirring (the stirring temperature is -2°C to 0°C). The uniformly stirred collagen solution is placed in the above-processed dialysis bag, checked for leaks, and then placed in the above-mentioned PBS solution for cold storage. Then place it in the refrigerator for dialysis for three days (the dialysis temperature is -2°C to 0°C), and change the dialysate every day. When the collagen solution is neutral, as indicated by the pH test paper, the dialysis is complete. The collagen solution is transferred to a test tube and stored in the refrigerator.
[0054] (3) Take 1 mL of the CdSe / ZnS quantum dot solution with a concentration of 0.04 μmol / L obtained in step (1), centrifuge (1200 r / min, 6 min) with PBS solution for 1 time, then ultrasonically disperse the washed quantum dot solution into 600 μL of PBS solution to obtain an ultrasonic dispersion liquid; take 10 mg of EDC and 5 mg of NHS, respectively dissolve them with 200 μL of PBS solution, then quickly add them to the above ultrasonic dispersion liquid, place it on a shaker for 200 rpm for 30 min to obtain a carboxyl activation product. Centrifuge the carboxyl activation product with PBS solution (1200 r / min, 6 min) for 1 time, then ultrasonically disperse it in 1 mL of PBS solution, then add 50 μL of 50 μg / mL of the collagen solution of step (2), place it on a shaker for 150 rpm for 4 h to obtain a crosslinked product. Centrifuge the crosslinked product with PBS solution for 1 time to remove the unreacted collagen protein, and obtain a collagen-quantum dot fluorescent probe.
[0055] Example 2
[0056] This example provides a method for labeling collagen with quantum dots. The difference between this example and Example 1 is that this example uses bovine tendon collagen sponge to obtain a collagen-quantum dot fluorescent probe.
[0057] Example 3
[0058] This example provides a method for labeling collagen with quantum dots. The difference between this example and Example 1 is that this example uses bullfrog collagen sponge to obtain a collagen-quantum dot fluorescent probe.
[0059] Examples 4-7
[0060] Examples 4-7 provide a method for labeling collagen with quantum dots. The difference between Examples 4-7 and Example 1 is that:
[0061] Example 4, step (1) obtained a CdSe / ZnS quantum dot solution with a concentration of 0.02 μmol / L, step (3) obtained a collagen-quantum dot fluorescent probe using the same method as in Example 1 and the CdSe / ZnS quantum dot solution with a concentration of 0.02 μmol / L;
[0062] Example 5, step (1) obtained a CdSe / ZnS quantum dot solution with a concentration of 0.016 μmol / L, step (3) obtained a collagen-quantum dot fluorescent probe using the same method as in Example 1 and the CdSe / ZnS quantum dot solution with a concentration of 0.016 μmol / L;
[0063] Example 6, step (1) obtained a CdSe / ZnS quantum dot solution with a concentration of 0.01 μmol / L, step (3) obtained a collagen-quantum dot fluorescent probe using the same method as in Example 1 and the CdSe / ZnS quantum dot solution with a concentration of 0.01 μmol / L;
[0064] Example 7, step (1) obtained a CdSe / ZnS quantum dot solution with a concentration of 0.008 μmol / L, step (3) obtained a collagen-quantum dot fluorescent probe using the same method as in Example 1 and the CdSe / ZnS quantum dot solution with a concentration of 0.008 μmol / L.
[0065] Comparative Example 1
[0066] This comparative example used a CdSe / ZnS quantum dot solution with a concentration of 0.04 μmol / L as a fluorescent probe.
[0067] Test Example 1, measurement of the particle size and Zeta potential of CdSe / ZnS quantum dots
[0068] This test example took an appropriate amount of the CdSe / ZnS quantum dot solution with a concentration of 0.04 μmol / L obtained in Example 1 into a sample cell, covered the sample cell with a cover, and placed the sample cell into a nanoparticle size and Zeta potential analyzer to measure the particle size and Zeta potential of the CdSe / ZnS quantum dots.
[0069] By Figure 3a It can be seen that the results of three particle size measurements were 54.98 nm, 61.11 nm and 64.06 nm, and the average value was 60.05 ± 4.55 nm. The particle size distribution range was relatively narrow, indicating that the CdSe / ZnS quantum dots had good particle size uniformity, were suitable for mouse tail vein injection, and were expected to have a good imaging effect in a living body.
[0070] By Figure 3bIt can be seen that the results obtained by the three potential tests are -25.64 mV, -25.81 mV and -25.34 mV, and the average value is -25.60 ± 0.24 mV. All the test values are stable at about -25 mV, the negative charge intensity is high, which indicates that the CdSe / ZnS quantum dot solution of the application has good stability, and the negative charge is derived from the carboxyl groups on the surface of the CdSe / ZnS quantum dots, which provides sufficient active sites for the subsequent cross-linking reaction based on collagen.
[0071] Test Example 2, Measurement of Hemolysis Rate of CdSe / ZnS Quantum Dots
[0072] The hemolysis experiment was performed on the CdSe / ZnS quantum dot solution with a concentration of 0.04 μmol / L obtained in Example 1, as follows.
[0073] A 10 mL centrifuge tube was taken, and anticoagulated blood and PBS solution were added in a volume ratio of 4:5, and then gently shaken to prepare diluted blood.
[0074] An appropriate amount of CdSe / ZnS quantum dot solution with a concentration of 0.04 μmol / L was taken and added to 5 mL of PBS solution as an experimental group, and three groups of samples were prepared in parallel.
[0075] 5 mL of PBS solution was used as a negative control, and three groups of samples were prepared in parallel.
[0076] 5 mL of ultrapure water was used as a positive control, and three groups of samples were prepared in parallel.
[0077] The nine groups of samples were placed in a constant temperature water bath at 37℃ and incubated for 30 min.
[0078] According to the proportion of 0.2 mL of diluted blood / 10 mL of PBS solution, the diluted blood was added to the nine groups of samples after incubation for 30 min, and then gently mixed, and then incubated at 37℃ in a constant temperature water bath for 60 min. After incubation, the test tube was gently mixed, and then centrifuged at 800 times gravity acceleration for 5 min, and the supernatant was aspirated, and the absorbance OD at 545 nm was measured. The hemolysis rate calculation formula is as follows:
[0079] Hemolysis rate = (OD (实验组) - OD (阴性对照) ) / (OD (阳性对照) - OD (阴性对照) )*100%;
[0080] According to Figure 4 It can be seen that the hemolysis rate of the CdSe / ZnS quantum dot solution under the concentration of Example 1 is 3.67 ± 0.14%, which is lower than the safety threshold of 5%, and has good biocompatibility.
[0081] Test Example 3: Detection of Fluorescent Probe Imaging Effect
[0082] In this test case, the fluorescent probes of Examples 1-3 and Comparative Example 1 were injected into mice via the tail vein. Using a small animal imaging system, photographs were taken of the mice at time points of 0h, 3h, 6h, 9h, 20h, 24h, 30h, 44h, 48h, and 56h (to capture images of the dynamic distribution process of the fluorescent probes). Subsequently, the mice were dissected, and their hearts, livers, spleens, lungs, and kidneys were removed and photographed using the imaging system.
[0083] Depend on Figures 5-8 A clear distribution of fluorescence signals was observed within the mice, with strong fluorescence signals observed in the liver, spleen, and kidneys, among which the liver showed the highest fluorescence intensity. Furthermore, fluorescence signals remained in the mice after 52 hours of physiological metabolism, indicating that the collagen-quantum dot fluorescent probe prepared in this invention is not easily metabolized by mice, providing a solid theoretical basis for subsequent long-term dynamic visualization in vivo imaging.
[0084] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.
Claims
1. A method for quantum dot labeling collagen, characterized in that, The method includes the following steps: S1: Mix and stir the collagen sponge with an organic solution to obtain a collagen solution, and then dialyze it to obtain a collagen solution; S2: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and phosphate buffer are mixed to obtain an activation solvent; CdSe / ZnS quantum dot solution is mixed with the activation solvent to carry out an activation reaction to obtain a carboxyl-activated product; the carboxyl-activated product is mixed with the collagen solution to carry out a crosslinking reaction, and after washing, a collagen-quantum dot fluorescent probe is obtained.
2. The method for quantum dot labeling collagen according to claim 1, wherein, The collagen source of the collagen sponge is at least one of fish collagen, mammalian collagen, and bullfrog collagen.
3. The method for quantum dot labeling collagen according to claim 2, wherein, The collagen sponge is at least one of grass carp collagen sponge, bovine Achilles tendon collagen sponge, and bullfrog collagen sponge.
4. The method for quantum dot labeling collagen according to claim 1, wherein, The organic solution is an aqueous solution of acetic acid with a concentration of 0.01-0.05 mol / L.
5. The method for quantum dot labeling collagen according to claim 1, wherein, The stirring is carried out on a low-temperature stirring table, the stirring temperature is -2℃ to 0℃, and the stirring time is 6-8 hours.
6. The method for quantum dot labeling collagen according to claim 1, wherein, The dialysate used in the dialysis was a phosphate buffer solution with a pH of 7.2-7.4; The dialysis temperature is from -2°C to 0°C, and the dialysis time is 2-3 days; The pH of the collagen solution is 7.2-7.
4.
7. The method for quantum dot labeling collagen according to claim 1, wherein, The CdSe / ZnS quantum dot solution was prepared by mixing CdSe / ZnS quantum dots and phosphate buffer, and the concentration of CdSe / ZnS quantum dots in the CdSe / ZnS quantum dot solution was 0.02-0.04 μmol / L.
8. The method for quantum dot labeling collagen according to claim 1, wherein, The activation reaction and cross-linking reaction are each carried out at temperatures of 20-30°C independently. The activation reaction takes 25-35 minutes. The cross-linking reaction takes 3.5-4.5 hours.
9. A collagen-quantum dot fluorescent probe prepared by the method for quantum dot labeling collagen according to any one of claims 1-8.
10. The application of the collagen-quantum dot fluorescent probe of claim 9 in the preparation of in vivo imaging agents.