Preparation method of photoacoustic nanoprobe for targeted recognition of M1 type macrophages
By preparing the photoacoustic nanoprobe Lipo@NM-Glu for targeting and recognizing M1 macrophages, the problems of insufficient imaging sensitivity and subtype selectivity in the existing technology were solved, and precise targeting and high-sensitivity imaging of M1 macrophages were achieved.
Patent Information
- Application Number
- CN202510931564.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-10-31
AI Technical Summary
Existing photoacoustic probes are rapidly metabolized in vivo and are difficult to accumulate on M1 macrophages for extended periods, resulting in insufficient imaging sensitivity and an inability to distinguish between M1 and M2 macrophages, thus lacking subtype selectivity in the inflammatory microenvironment.
Lipo@NM-Glu, a photoacoustic nanoprobe designed to target and recognize M1 macrophages, was prepared by synthesizing a small molecule compound c that responds to nitric oxide and combining it with soybean lecithin and DSPE-PEG2000-Glucose. This process enhanced the specificity and sensitivity of the nanoprobe to M1 macrophages.
It achieves precise targeted identification of M1 macrophages, improves the sensitivity and specificity of photoacoustic imaging, and enables dynamic monitoring of inflammatory responses in vivo, meeting the needs of dynamic monitoring of NO levels in vivo.
Smart Images
Figure CN120860265A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine, specifically to a method for preparing a photoacoustic nanoprobe that targets and identifies M1 macrophages. Background Technology
[0002] M1 macrophages are specific biomarkers reflecting the level of inflammation in the body. By detecting their infiltration in tissues in an in situ and non-invasive manner, the strength of the inflammatory response in vivo can be directly assessed. Photoacoustic (PA) imaging technology combines the high sensitivity of optical imaging with the deep penetration advantage of ultrasound imaging. It can generate light-excited ultrasound signals in vivo, thereby achieving high-contrast imaging of deep tissues. At the same time, when combined with photoacoustic probes with molecular recognition and response functions, PA imaging can provide dynamic and precise visualization and monitoring of specific physiological or pathological molecular events.
[0003] Currently available photoacoustic probes for imaging M1 macrophages in vivo are mostly small-molecule probes. Due to their rapid metabolism in vivo, they are difficult to accumulate on target cells for extended periods, thus limiting their ability to target and recognize M1 macrophages and resulting in insufficient imaging sensitivity. While a few reported photoacoustic nanoprobes utilize the high phagocytic capacity of macrophages for nanoparticle imaging, these methods only reflect the total distribution of macrophages and cannot distinguish between different subtypes such as M1 and M2, thus lacking subtype selectivity in the inflammatory microenvironment.
[0004] Therefore, we propose a method for preparing photoacoustic nanoprobes that target and recognize M1 macrophages, which can accurately target and respond to M1 macrophages in vivo, thereby improving the sensitivity and specificity of PA imaging in inflammation monitoring. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and meet practical needs by providing a method for preparing photoacoustic nanoprobes for targeted recognition of M1 macrophages. This addresses the problem that most currently available photoacoustic probes for imaging M1 macrophages in vivo are small-molecule probes, which are rapidly metabolized in vivo and difficult to accumulate in target cells for extended periods, thus limiting their targeted recognition ability for M1 macrophages and resulting in insufficient imaging sensitivity. While a few reported photoacoustic nanoprobes utilize the high phagocytic capacity of macrophages for nanoparticle imaging, these methods only reflect the total distribution of macrophages and cannot distinguish between different subtypes such as M1 and M2, thus lacking the technical problem of subtype selectivity in the inflammatory microenvironment.
[0006] To achieve the objectives of this invention, the technical solution adopted is as follows: a method for preparing a photoacoustic nanoprobe that targets and recognizes M1 macrophages, comprising the following steps: S1. Synthesize small molecule compound c that responds to nitric oxide.
[0007] S2. Preparation of photoacoustic nanoprobes Lipo@NM-Glu that target and recognize M1 macrophages.
[0008] Preferably, step S1 includes the following sub-steps: S1.1 A mixture of 5-bromothiophene-2-boronic acid, 4-(diphenylamino)phenylboronic acid, Pd(PPh3)4 and K2CO3 was dissolved in H2O / 1,4-dioxane 1:1 in 20.0 mL and reacted at 80 °C for 12 h under N2 atmosphere. The product a was purified by silica gel column chromatography. S1.2. A mixture of product a, 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole, Pd(PPh3)4 and K2CO3 was dissolved in 5.0 mL of H2O / 1,4-dioxane at a ratio of 1:1. The mixture was stirred overnight at 80 °C under a N2 atmosphere and purified by silica gel column chromatography to obtain product b. S1.3 Dissolve the mixture of product b, B2(OH)4 and 4,4′-bipyridine in DMF, stir at room temperature for 10 min, and purify by gradient silica column chromatography to obtain small molecule compound c.
[0009] Preferably, in S1.1, the amount of 5-bromothiophene-2-boronic acid is 2.5 mmol, the amount of 4-(diphenylamino)phenylboronic acid is 5.0 mmol, the amount of Pd(PPh3)4 is 0.25 mmol, and the amount of K2CO3 is 5.0 mmol.
[0010] Preferably, in S1.2, the amount of product a is 1.25 mmol, the amount of 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole is 0.5 mmol, the amount of Pd(PPh3)4 is 0.1 mmol, and the amount of K2CO3 is 2.0 mmol.
[0011] Preferably, in S1.3, the amount of product b is 0.3 mmol, the amount of B2(OH)4 is 3.0 mmol, the amount of 4,4′-bipyridine is 0.15 mmol, and the amount of DMF is 5.0 mL.
[0012] Preferably, step S2 includes the following sub-steps: S2.1 Dissolve small molecule compound c, soybean lecithin and DSPE-PEG2000-Glucose in chloroform at a mass ratio of 2:25:5, remove chloroform by rotary evaporation, and vacuum dry for 4 hours.
[0013] S2.2 The film is hydrated with ultrapure water and ultrasonically treated until a transparent colloidal solution is formed.
[0014] S2.3. The colloidal solution is centrifuged and filtered, washed three times with ultrapure water, and resuspended in an aqueous medium to obtain Lipo@NM-Glu nanoprobes.
[0015] Preferably, in step S2.1, the rotary evaporation temperature is 40°C and the rotation speed is 120 rpm.
[0016] Preferably, in step S2.2, the ultrasonic power is 150W and the ultrasonic time is 5min.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. By employing a novel synthesis method, this invention not only reduces the number of synthesis steps for small molecule compound c and improves synthesis efficiency, but also effectively reduces the preparation cost of the probe.
[0018] 2. This invention endows the probe with the ability to target M1 macrophages in vivo by overexpressing glucose transporter receptors and selectively responding to the biomarker of M1 macrophages, such as nitric oxide, thereby improving the probe's ability to target and recognize M1 macrophages, and thus improving the sensitivity and specificity of in vivo photoacoustic imaging of M1 macrophages. Attached Figure Description
[0019] Figure 1 This is a schematic diagram illustrating the probe morphology, hydration dynamics diameter, and zeta potential characterization of the present invention.
[0020] Figure 2 This is a schematic diagram illustrating the response performance, selectivity, and stability of the probe of the present invention.
[0021] Figure 3 This is a schematic diagram illustrating the in vitro targeting and recognition of M1 macrophages by the probe of the present invention.
[0022] Figure 4 This is a schematic diagram illustrating the in vivo targeting and recognition of M1 macrophages by the probe of the present invention.
[0023] Figure 5 This is a schematic diagram of Embodiment 1 of the present invention.
[0024] Figure 6 This is a schematic diagram of the synthetic route for compound c of the present invention.
[0025] Figure 7 This is a schematic diagram of the probe Lipo@NM-Glu preparation method of the present invention.
[0026] Figure 8 This is a schematic diagram of the preparation method of the present invention.
[0027] Figure 9 This is a schematic diagram of the synthesis process of compound c of the present invention.
[0028] Figure 10 This is a schematic diagram of the preparation process of the probe Lipo@NM-Glu of the present invention. Detailed Implementation
[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments: A method for preparing a photoacoustic nanoprobe that targets and recognizes M1 macrophages, see [link to documentation]. Figures 1 to 10 This includes the following steps: S1. Synthesize small molecule compound c that responds to nitric oxide.
[0030] S2. Preparation of photoacoustic nanoprobes Lipo@NM-Glu that target and recognize M1 macrophages.
[0031] Specifically, step S1 includes the following sub-steps: S1.1 A mixture of 5-bromothiophene-2-boronic acid, 4-(diphenylamino)phenylboronic acid, Pd(PPh3)4 and K2CO3 was dissolved in H2O / 1,4-dioxane 1:1 in 20.0 mL. The mixture was reacted at 80 °C for 12 h under N2 atmosphere and purified by silica gel column chromatography to obtain product a.
[0032] S1.2. A mixture of product a, 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole, Pd(PPh3)4 and K2CO3 was dissolved in 5.0 mL of H2O / 1,4-dioxane at a ratio of 1:1. The mixture was stirred overnight at 80 °C under a N2 atmosphere and purified by silica gel column chromatography to obtain product b.
[0033] S1.3 Dissolve the mixture of product b, B2(OH)4 and 4,4′-bipyridine in DMF, stir at room temperature for 10 min, and purify by gradient silica column chromatography to obtain small molecule compound c.
[0034] Furthermore, in S1.1, the amount of 5-bromothiophene-2-boronic acid is 2.5 mmol, the amount of 4-(diphenylamino)phenylboronic acid is 5.0 mmol, the amount of Pd(PPh3)4 is 0.25 mmol, and the amount of K2CO3 is 5.0 mmol.
[0035] It is worth noting that in S1.2, the amount of product a was 1.25 mmol, the amount of 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole was 0.5 mmol, the amount of Pd(PPh3)4 was 0.1 mmol, and the amount of K2CO3 was 2.0 mmol.
[0036] It is worth noting that in S1.3, the amount of product b was 0.3 mmol, the amount of B2(OH)4 was 3.0 mmol, the amount of 4,4′-bipyridine was 0.15 mmol, and the amount of DMF was 5.0 mL.
[0037] It is worth noting that step S2 includes the following sub-steps: S2.1 Dissolve small molecule compound c, soybean lecithin and DSPE-PEG2000-Glucose in chloroform at a mass ratio of 2:25:5, remove chloroform by rotary evaporation, and vacuum dry for 4 hours.
[0038] S2.2. Hydrate the membrane with ultrapure water and sonicate it until a transparent colloidal solution is formed.
[0039] S2.3. The colloidal solution was centrifuged and filtered, washed three times with ultrapure water, and resuspended in an aqueous medium to obtain the Lipo@NM-Glu nanoprobe.
[0040] It is worth noting that in step S2.1, the rotary evaporation temperature is 40℃ and the rotation speed is 120rpm.
[0041] It is worth emphasizing that the ultrasonic power in step S2.2 is 150W and the ultrasonic time is 5min.
[0042] Example 1 In vivo photoacoustic imaging and validation of Lipo@NM-Glu probe in diabetic mouse models at different disease stages.
[0043] like Figure 5 As shown: Baseline photoacoustic imaging acquisition.
[0044] Before injecting the probe into the tail vein, basic photoacoustic imaging was performed on diabetic mice at different disease stages to obtain the initial photoacoustic signal distribution in the liver of each group of model mice.
[0045] Probe drug delivery and imaging.
[0046] The probe was slowly injected into the model mouse through the tail vein. Six hours after injection, photoacoustic imaging was performed again to collect photoacoustic signals from the liver of the model mouse and to analyze the changes in signals before and after injection.
[0047] Signal amplification analysis.
[0048] Based on the increase in photoacoustic signal in the liver region of model mice in different disease course groups, the infiltration level of M1 macrophages in each group was assessed, and the relationship between signal intensity and disease progression was analyzed to verify the sensitivity of the probe to M1 macrophage infiltration.
[0049] Histological and biochemical marker detection.
[0050] After imaging, liver tissue was taken and analyzed using the iNOS kit, and H&E staining and CD86 immunofluorescence staining were performed respectively.
[0051] Comparison of imaging and histological results.
[0052] By comparing histological (H&E) and immunofluorescence (CD86) results with in vivo photoacoustic imaging signals, the function of probe targeting and recognizing M1 macrophages and its application value in evaluating the progression of steatohepatitis were clarified.
[0053] Among them, such as Figure 1 As shown, the obtained Lipo@NM-Glu probe has a hydration kinetic diameter of 225±46nm and a potential of approximately –18mV.
[0054] like Figure 2 As shown, the probe is extremely sensitive to nitric oxide (NO), with a detection limit of 11 nmol / L. It also exhibits excellent photoacoustic signal stability and selective response in different media, meeting the needs of dynamic monitoring of NO levels in vivo and accurately reflecting the inflammatory microenvironment.
[0055] like Figure 3 As shown, in in vitro cell experiments, the Lipo@NM-Glu probe can be lit by M1 macrophages, demonstrating good targeting and recognition capabilities for the M1 subtype. It can be used for precise in vitro cell imaging and functional verification.
[0056] like Figure 4 As shown, the Lipo@NM-Glu probe can target and recognize M1 macrophages infiltrating acute hepatitis lesions in vivo.
[0057] In addition, all components designed in this invention are general standard parts or components known to those skilled in the art. Their structures and principles can be learned by those skilled in the art through technical manuals or conventional experimental methods. They can be fully implemented by those skilled in the art, so there is no need to elaborate. The content protected by this invention does not involve improvements to the internal structure and methods.
Claims
1. A method for preparing a photoacoustic nanoprobe that targets and recognizes M1 macrophages, characterized in that, Includes the following steps: S1. Synthesize small molecule compound c that responds to nitric oxide; S2. Preparation of photoacoustic nanoprobes Lipo@NM-Glu that target and recognize M1 macrophages.
2. The method for preparing the photoacoustic nanoprobe targeting and recognizing M1 macrophages as described in claim 1, characterized in that, Step S1 includes the following sub-steps: S1.1 A mixture of 5-bromothiophene-2-boronic acid, 4-(diphenylamino)phenylboronic acid, Pd(PPh3)4 and K2CO3 was dissolved in H2O / 1,4-dioxane 1:1 in 20.0 mL and reacted at 80 °C for 12 h under N2 atmosphere. The product a was purified by silica gel column chromatography. S1.
2. A mixture of product a, 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole, Pd(PPh3)4 and K2CO3 was dissolved in 5.0 mL of H2O / 1,4-dioxane at a ratio of 1:
1. The mixture was stirred overnight at 80 °C under a N2 atmosphere and purified by silica gel column chromatography to obtain product b. S1.3 Dissolve the mixture of product b, B2(OH)4 and 4,4′-bipyridine in DMF, stir at room temperature for 10 min, and purify by gradient silica column chromatography to obtain small molecule compound c.
3. The method for preparing the photoacoustic nanoprobe for targeting and recognizing M1 macrophages as described in claim 2, characterized in that, In S1.1, the amount of 5-bromothiophene-2-boronic acid is 2.5 mmol, the amount of 4-(diphenylamino)phenylboronic acid is 5.0 mmol, the amount of Pd(PPh3)4 is 0.25 mmol, and the amount of K2CO3 is 5.0 mmol.
4. The method for preparing the photoacoustic nanoprobe for targeting M1 macrophages as described in claim 2, characterized in that, In S1.2, the amount of product a is 1.25 mmol, the amount of 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole is 0.5 mmol, the amount of Pd(PPh3)4 is 0.1 mmol, and the amount of K2CO3 is 2.0 mmol.
5. The method for preparing the photoacoustic nanoprobe for targeting and recognizing M1 macrophages as described in claim 2, characterized in that, In S1.3, the amount of product b is 0.3 mmol, the amount of B2(OH)4 is 3.0 mmol, the amount of 4,4′-bipyridine is 0.15 mmol, and the amount of DMF is 5.0 mL.
6. The method for preparing the photoacoustic nanoprobe targeting and recognizing M1 macrophages as described in claim 1, characterized in that, Step S2 includes the following sub-steps: S2.1, Small molecule compound c, soybean lecithin and DSPE-PEG 2000 -Glucose was dissolved in chloroform at a mass ratio of 2:25:5, the chloroform was removed by rotary evaporation, and the mixture was dried under vacuum for 4 hours. S2.
2. The film is hydrated with ultrapure water and ultrasonically treated until a transparent colloidal solution is formed; S2.
3. The colloidal solution is centrifuged and filtered, washed three times with ultrapure water, and resuspended in an aqueous medium to obtain Lipo@NM-Glu nanoprobes.
7. The method for preparing the photoacoustic nanoprobe for targeting and recognizing M1 macrophages as described in claim 6, characterized in that, In step S2.1, the rotary evaporation temperature is 40℃ and the rotation speed is 120rpm.
8. The method for preparing the photoacoustic nanoprobe for targeting and recognizing M1 macrophages as described in claim 6, characterized in that, In step S2.2, the ultrasonic power is 150W and the ultrasonic time is 5min.