A myeloperoxidase-responsive fluorescent probe liposome and a preparation method and application thereof

Abstract

This invention discloses a myeloperoxidase-responsive fluorescent probe liposome, its preparation method, and its applications. The raw materials for this fluorescent probe liposome include lecithin, cholesterol, collagen, luminol, and Cy5. These raw materials are prepared using a thin-film dispersion method to obtain the fluorescent probe liposome. This invention combines luminol with Cy5, solving the problem of low sensitivity or even undetectability of luminol to deep inflammatory foci in body tissues. It enables imaging detection of deep inflammatory cells in the body, which has significant application value in evaluating inflammatory responses caused by tumors, epilepsy, etc., and is of great significance for guiding clinical treatment of tumors and epilepsy and assessing efficacy.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a myeloperoxidase-responsive fluorescent probe liposome, its preparation method, and its application. Background Technology

[0002] Liposomes are artificial microscopic vesicles with a lipid bilayer spherical structure and hydrophilic cavities. The hydrophilic heads of the bilayer form the inner and outer surfaces of the membrane, while the lipophilic tails are located in the middle of the membrane. This unique structure gives liposomes great potential for applications in medical imaging, but traditional liposomes often have drawbacks such as low stability and insufficient in vivo circulation time.

[0003] Myeloperoxidase (MPO) is a heme protein primarily found in neutrophils. MPO serves as a functional and activation marker for neutrophils, and its level and activity represent the function and state of neutrophils. Under normal circumstances, MPO-mediated hypochlorous acid participates in the body's immune response, playing a role in resisting pathogens, bacteria, and viruses. However, when the body's reactive oxygen species exceed a certain limit, the resulting oxidative stress damages biomolecules such as phospholipids, proteins, and DNA in normal cells, further activating cellular inflammatory pathways caused by tumors and epilepsy, triggering a vicious cycle in tumor and epilepsy development. Therefore, the activity of MPO in the body can represent the degree of active inflammatory response in the progression of tumors and epilepsy. By detecting MPO activity, the inflammatory process caused by tumors and epilepsy can be evaluated, guiding clinical treatment of tumors and epilepsy and assessing efficacy.

[0004] Current methods for detecting MPO typically use luminol as a chemiluminescent reagent. Luminol is a chemiluminescent reagent that can detect MPO activity at both the cellular and in vivo levels. It specifically interacts with hypochlorous acid produced by MPO catalysis to generate blue light, which can be detected using optical imaging techniques. However, luminol has a relatively short emission wavelength (λ). max At a wavelength of 425 nm, luminol has weak penetration into tissues, making its luminescent signal difficult to detect. When used in in vivo imaging, luminol is only sensitive to superficial inflammatory foci, and its sensitivity to inflammatory foci in deeper tissues is low or even undetectable.

[0005] Therefore, there is an urgent need to develop a fluorescent probe liposome that can detect inflammatory foci in deep tissues and has good stability. Summary of the Invention

[0006] To efficiently detect MPO in the body, thereby more effectively evaluating the inflammatory process caused by tumors and epilepsy, and guiding clinical treatment of tumors and epilepsy to assess efficacy, this invention provides the following technical solution.

[0007] In a first aspect, the present invention provides a fluorescent probe liposome, the raw materials of which include lecithin, cholesterol, collagen, luminol, and Cy5. The fluorescent probe liposome is prepared by means of a thin-film dispersion method.

[0008] Cy5 is a commonly used fluorescent dye with a maximum absorption wavelength of approximately 650 nm and a maximum emission wavelength of approximately 670 nm. Biological organisms absorb and scatter light in the near-infrared (NIR) region (especially 700–900 nm) more weakly, making NIR photons more likely to penetrate biological tissues and be detected. Therefore, the fluorescence properties in the near-infrared region make Cy5 particularly suitable for bioimaging.

[0009] Collagen is a structural protein with functional properties such as low viscosity, high biocompatibility, and low antigenicity. The inventors ingeniously utilized these properties to prepare liposomes with stable results and high drug loading capacity, overcoming the shortcomings of traditional liposomes.

[0010] Preferably, the mass ratio of the lecithin, cholesterol, collagen, luminol, and Cy5 is (1-30):(1-5):(30-50):(1-5):(1-5). More preferably, it is (1-25):(1-5):(35-45):(1-5):(1-5), for example: 1:1:35:5:1, 10:3:35:4:1, 15:4:40:3:1, 20:5:40:2:1, 25:5:45:1:2, 25:5:45:1:5.

[0011] Preferably, the mass ratio of luminol to Cy5 is (5-1):(1-5), for example: 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5.

[0012] Preferably, the mass ratio of lecithin to cholesterol is (1-6):1, for example: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1.

[0013] Secondly, the present invention provides a method for preparing fluorescent probe liposomes, comprising the following steps:

[0014] (1) Dissolve lecithin and cholesterol in an organic solvent and rotary evaporate to form a thin film;

[0015] (2) Add luminol, Cy5, collagen and pH buffer, and rotate to hydrate until the membrane detaches, forming a milky white liquid;

[0016] (3) The milky white liquid is sonicated and filtered to obtain the liposomes.

[0017] Preferably, in step (1), the mass ratio of lecithin to cholesterol is (1-6):1, for example: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1.

[0018] Preferably, in step (1), the organic solvent is an ether solvent, such as: diethyl ether, dipropyl ether, ethyl butyl ether, dibutyl ether, dipentyl ether, diisopentyl ether, dihexyl ether. More preferably, it is diethyl ether.

[0019] Preferably, in step (2), the amount of luminol added is 0.01 to 0.5 mg / mL g / mL buffer solution, for example: 0.01 mg / mL, 0.03 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL.

[0020] Preferably, in step (2), the amount of Cy5 added is 0.01-0.5 g / mL buffer solution, for example: 0.01 mg / mL, 0.03 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL.

[0021] Preferably, in step (2), the amount of collagen added is 2 to 5 mg / mL of buffer solution, for example: 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL.

[0022] Preferably, the pH of the pH buffer solution is 7.0 to 7.5, for example: 7.0, 7.2, 7.4, 7.5.

[0023] Preferably, the pH buffer solution is a phosphate buffer solution.

[0024] Preferably, in step (3), the ultrasound time is 20 to 40 minutes, for example: 20 minutes, 25 minutes, 30 minutes, 35 minutes, or 40 minutes.

[0025] Preferably, in step (3), the temperature of the ultrasound is 20 to 30°C, for example: 20°C, 23°C, 25°C, 27°C, 30°C.

[0026] Preferably, in step (3), the ultrasonic power is 50 to 200W, for example: 50W, 70W, 100W, 120W, 150W, 170W, 200W.

[0027] Thirdly, the present invention provides the use of the fluorescent probe liposomes described in the first aspect or the fluorescent probe liposomes prepared according to the preparation method described in the second aspect in the preparation of products for detecting myeloperoxidase.

[0028] The beneficial effects of this invention are:

[0029] I. This invention ingeniously combines luminol with Cy5 and employs the bioluminescent resonance energy transfer (BRET) method. It utilizes the luminol molecule's response to MPO and releases photons of a certain frequency. The wavelength of these photons is similar to the excitation wavelength of Cy5 fluorescent molecules, further exciting the Cy5 fluorescent molecules (i.e., energy resonance transfer occurs) to produce fluorescence. This solves the problem of luminol's low sensitivity or even undetectability to inflammatory foci in deep tissues.

[0030] Second, this invention enables imaging detection of deep inflammatory cells in the body, which has important application value in evaluating inflammatory responses caused by tumors, epilepsy, etc., and is of great significance for guiding clinical tumor and epilepsy-related treatments and evaluating efficacy.

[0031] Third, the fluorescent probe liposomes prepared by this invention have a high absolute value of Zeta potential (-30.41mV), a large repulsive force between liposome particles, and strong stability, which to a certain extent overcomes the defects of traditional liposomes such as low stability and insufficient in vivo circulation time. Attached Figure Description

[0032] Figure 1 The diagram shown is a structural diagram of the luminol-Cy5 fluorescent probe liposome of the present invention.

[0033] Figure 2 The figure shows the effect of the luminol:Cy5 ratio on the encapsulation efficiency of the luminol-Cy5 fluorescent probe liposomes;

[0034] Figure 3 The results shown are the response surface methodology results of the interaction of various factors on the encapsulation efficiency of luminol in Example 3. AB is the response surface plot of the interaction between the luminol:Cy5 ratio and the membrane material ratio; CD is the response surface plot of the interaction between the luminol:Cy5 ratio and the hydration temperature; and EF is the response surface plot of the interaction between the membrane material ratio and the hydration temperature.

[0035] Figure 4The results shown are the response surface methodology results of the interaction of various factors on the encapsulation efficiency of Cy5 in Example 3. AB is the response surface plot of the interaction between the luminol:Cy5 ratio and the membrane material ratio; CD is the response surface plot of the interaction between the luminol:Cy5 ratio and the hydration temperature; and EF is the response surface plot of the interaction between the membrane material ratio and the hydration temperature.

[0036] Figure 5 The image shows the quality evaluation results of luminol-Cy5 fluorescent probe liposomes, where AB represents the morphology of the fluorescent probe liposomes; C represents the particle size distribution of the fluorescent probe liposomes; and D represents the zeta potential of the fluorescent probe liposomes.

[0037] Figure 6 The images show the excitation and emission spectra of luminol and Cy5 molecules, where A is the excitation spectrum of luminol; B is the emission spectrum of luminol; C is the excitation spectrum of Cy5; and D is the emission spectrum of Cy5.

[0038] Figure 7 The emission fluorescence spectra of luminol-Cy5 fluorescent probe solution and luminol-Cy5 fluorescent probe liposomes at 459 nm are shown.

[0039] Figure 8 The images show the cell imaging results (×200) of luminol-Cy5 fluorescent probe liposomes. AB represents the cell imaging results of RAW264.7 cells co-cultured with luminol-Cy5 fluorescent probe liposomes without LPS stimulation; CD represents the cell imaging results of RAW264.7 cells co-cultured with luminol after LPS stimulation; EF represents the cell imaging results of RAW264.7 cells co-cultured with Cy5 after LPS stimulation; and GH represents the cell imaging results of RAW264.7 cells co-cultured with luminol-Cy5 fluorescent probe liposomes after LPS stimulation. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0041] It should be noted that, unless otherwise specified, the reagents and experimental methods used in the embodiments of the present invention are all conventional reagents and experimental methods in the art.

[0042] The main materials and instruments used in the embodiments of this invention are as follows:

[0043] Sulfonated Cy5 NHS active ester (Cy5) (analytical grade, Wuhan Duoying Biotechnology Co., Ltd.); Luminol (analytical grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); Lecithin and cholesterol (analytical grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); Collagen (biotechnology grade, Shanghai Maclean Biochemical Technology Co., Ltd.); Dextran gel G-25 (biotechnology grade, Shanghai Maclean Biochemical Technology Co., Ltd.); UV-Vis spectrophotometer (UV2700, Shimadzu Corporation, Japan); Malvern Panalytical particle size analyzer (Zetasizer Pro, Malvern Panalytical, UK); Ultrasonic cleaner (KQ5200DV, Kunshan Ultrasonic Instrument Co., Ltd.); Rotary evaporator (RE-2000A, Shanghai Yarong Biochemical Instrument Factory); Electronic analytical balance (BSA224S, Shanghai Ohaus Instrument Co., Ltd.); Shimadzu fluorescence spectrometer (RF6000, Shimadzu Corporation, Japan).

[0044] Example 1: Preparation and Encapsulation Efficiency Measurement of Luminol-Cy5 Fluorescent Probe Liposome Samples

[0045] 1.1 Weigh 20 mg of lecithin and 5 mg of cholesterol, dissolve them in 20 mL of ether, and remove the ether by rotary evaporation under reduced pressure to form a uniform film. Add 10 mL of collagen solution containing 2 mg of luminol, 1 mg of Cy5 molecules, and 4 mg / mL, and 20 mL of pH 7.2 phosphate buffer for rotary hydration until the film completely detaches and forms a milky white liquid. Sonicate for 30 min (room temperature 25℃, 100 W), and filter three times through a 0.45 μm filter to obtain the fluorescent probe liposome sample (structure shown in...). Figure 1 ), stored at 4°C.

[0046] 1.2 The fluorescent probe liposomes prepared in 1.1 were loaded into a dextran gel glass chromatography column and eluted with phosphate buffer (pH 7.2). 2 mL of eluent was collected per tube, for a total of 40 tubes. The absorbance was measured using a spectrophotometer, and the concentrations of luminol and Cy5 were calculated. The encapsulation efficiency of luminol and Cy5 was then calculated using the following formula:

[0047] Encapsulation rate = Encapsulated drug content ÷ (Encapsulated drug content + Free drug content) × 100%.

[0048] Example 2: Effect of the mass ratio of luminol to Cy5 on the encapsulation efficiency of luminol-Cy5 fluorescent liposomes

[0049] Following the preparation and measurement methods described in Example 1, and using the encapsulation efficiency of fluorescent liposomes as the evaluation index, a single-factor study was conducted on the luminol:Cy5 mass ratio during liposome preparation. The masses of luminol and Cy5 were set to 3 mg and 1 mg, 2 mg and 1 mg, 1 mg and 1 mg, 1 mg and 2 mg, and 1 mg and 3 mg, respectively. With other conditions remaining constant, the encapsulation efficiencies of luminol and Cy5 were measured. The results are shown below. Figure 2 .

[0050] Depend on Figure 2 It can be seen that as the mass ratio of luminol to Cy5 increases, the encapsulation efficiency of luminol and Cy5 first increases and then decreases. The encapsulation efficiency is the highest when the mass ratio of luminol to Cy5 is 2:1, with an encapsulation efficiency of 65.6% for luminol and 70.2% for Cy5.

[0051] Example 3: Response surface methodology for the encapsulation efficiency of luminol-Cy5 fluorescent probe liposomes

[0052] Based on the experimental results of Example 2, the Box-Behnken experimental design principle in Design Expert software was applied. With encapsulation rate as the response value, a three-factor, three-level response surface methodology was designed and carried out, including luminol:Cy5 mass ratio, membrane material ratio (lecithin:cholesterol mass ratio), and ultrasonic time. The experimental design is shown in Tables 1 and 2.

[0053] Table 1 Response surface methodology for liposome encapsulation efficiency of luminol-Cy5 fluorescent probe.

[0054] Table 1 Response surface experiment on encapsulation efficiency ofLuminol-Cy5 fluorescent probe liposomes

[0055]

[0056] Table 2 Response surface methodology for luminol-Cy5 fluorescent probe liposome encapsulation efficiency.

[0057] Table 2 Experimental Design ofLuminol-Cy5 Fluorescent Probe LiposomeEncapsulation Rate Response Surface Methodology

[0058]

[0059]

[0060] The experimental results are shown in Figure 3 and Figure 4 .

[0061] Depend on Figure 3 and Figure 4 It can be seen that the best encapsulation efficiency was achieved when the luminol:Cy5 mass ratio was 1.99:1, the membrane material ratio was 4.10:1, and the hydration temperature was 39.88℃, with a luminol encapsulation efficiency of 58.07% and a Cy5 encapsulation efficiency of 61.68%. Based on the response surface methodology results, a luminol:Cy5 mass ratio of 2:1, a membrane material ratio of 4:1, and a hydration temperature of 40℃ were selected to prepare luminol-Cy5 fluorescent probe liposomes. The encapsulation efficiency of the luminol-Cy5 fluorescent probe was verified, and the results showed that the luminol encapsulation efficiency was 57.26% and the Cy5 encapsulation efficiency was 60.22%, which is consistent with the response surface methodology results of the luminol-Cy5 fluorescent probe liposome encapsulation efficiency.

[0062] Example 4: Quality Evaluation of Luminol-Cy5 Fluorescent Probe Liposomes

[0063] The luminol-Cy5 fluorescent probe liposome sample prepared in Example 3 was examined under a microscope to observe its microstructure and morphology. The results are shown in the figure. Figure 5 The particle size distribution and zeta potential of liposomes were determined using a Malvern particle size analyzer. The results are shown in [Figure number missing]. Figure 6 .

[0064] Depend on Figure 5 As can be seen from A and B, the luminol-Cy5 fluorescent probe liposomes appear as dispersed, single spherical particles.

[0065] Depend on Figure 5 As can be seen from C, the average particle size of the luminol-Cy5 fluorescent probe liposomes is 205.01 nm, and the particle size distribution is uniform.

[0066] Depend on Figure 5 As can be seen from D, the Zeta potential of the luminol-Cy5 fluorescent probe liposome is -30.41mV, which is relatively large in absolute value, indicating a large repulsive force between liposome particles, thus meeting the potential requirements for the stability of the fluorescent probe liposome.

[0067] Example 5: Fluorescence spectroscopy analysis of luminol-Cy5 fluorescent probe liposomes

[0068] Take 2 mL of the luminol-Cy5 fluorescent probe liposome sample prepared in Example 3, place it in an RF-6000 fluorescence spectrometer, and measure its fluorescence spectrum using 459 nm as the excitation wavelength. The results are shown in [Figure Number]. Figure 6 and Figure 7 .

[0069] Depend on Figure 6It can be seen that the maximum excitation wavelength of luminol is 318 nm, and the maximum emission wavelength is 459 nm. The maximum excitation wavelength of Cy5 is 591 nm, and the maximum emission wavelength is 673 nm. Figure 6 Therefore, 459 nm was selected as the excitation wavelength for luminol-Cy5 fluorescent probe liposomes for cell imaging detection.

[0070] Depend on Figure 7 It can be seen that, under the condition of 459 nm as the excitation wavelength, the fluorescence intensity of the luminol-Cy5 molecule mixture solution is 20439 at 511 nm and 9797 at 669 nm; the fluorescence intensity of the luminol-Cy5 fluorescent probe liposome is 35385 at 535 nm and 12700 at 677 nm. The emission wavelength of the luminol-Cy5 fluorescent probe liposome exhibits a redshift. Therefore, 677 nm was selected as the emission wavelength of the luminol-Cy5 fluorescent probe liposome for cell imaging detection.

[0071] Example 6: Cell imaging detection of luminol-Cy5 fluorescent probe liposomes

[0072] RAW264.7 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and placed in a 37°C, 5% CO2 incubator. RAW264.7 cells in the logarithmic growth phase were collected and diluted to 1×10⁻⁶ cells. 5 Cells were cultured at a density of 2 mL / well in 6-well plates. Cells were incubated for 24 hours until adherence. The original culture medium was discarded. 100 μL of LPS solution (final concentration 5 μg / mL) was added to the experimental groups, and phosphate buffer (pH 7.2) was added to the control group. Cells were incubated at 37°C with 5% CO2 for 24 hours. Then, 500 μL of liposomes containing 0.2 mg / mL luminol, 0.1 mg / mL Lcy5, and luminol-Cy5 fluorescent probes were added to the experimental groups, and 500 μL of luminol-Cy5 fluorescent probe liposomes was added to the control group. Cells were incubated at 37°C for another 12 hours. Cells were then fixed onto slides and their imaging was performed using a laser confocal microscope. Results are shown in the figure below. Figure 8 .

[0073] Depend on Figure 8 As shown in A and B, when using 459nm as the excitation wavelength and 677nm as the emission wavelength for cell imaging detection, RAW264.7 cells without LPS stimulation and cells co-cultured with luminol-Cy5 fluorescent probe liposomes did not exhibit any fluorescence.

[0074] Depend on Figure 8As shown in C and D, RAW264.7 cells stimulated with LPS exhibited enhanced myeloperoxidase activity. When co-cultured with luminol, luminol responded to myeloperoxidase activity by emitting blue fluorescence, but Cy5 molecules did not exhibit red fluorescence.

[0075] Depend on Figure 8 As can be seen from E and F, when RAW264.7 cells were co-cultured with Cy5 after LPS stimulation, although the activity of myeloperoxidase in the cells was enhanced, the Cy5 molecule did not show red fluorescence due to the lack of myeloperoxidase response to luminol.

[0076] Depend on Figure 8 As can be seen from G and H, when RAW264.7 cells were co-cultured with luminol-Cy5 fluorescent probe liposomes after LPS stimulation, the activity of myeloperoxidase was enhanced. Luminol was able to respond to myeloperoxidase activity by emitting blue fluorescence. At the same time, the blue fluorescence could excite Cy5 in the fluorescent probe liposomes to emit red fluorescence at 677nm, thus realizing the imaging detection of deep inflammatory cells.

[0077] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A fluorescent probe liposome, characterized in that: The fluorescent probe liposomes are made from lecithin, cholesterol, collagen, luminol, and sulfonated Cy5 NHS active ester. The mass ratio of the lecithin, cholesterol, collagen, luminol and sulfonated Cy5 NHS active ester is (1~30):(1~5):(30~50):(1~5):(1~5); The method for preparing the fluorescent probe liposomes includes the following steps: (1) The lecithin and cholesterol are dissolved in an organic solvent and then rotary evaporated to form a thin film; (2) Add the luminol, the sulfonated Cy5 NHS active ester, the collagen and pH buffer to the film, and rotate to hydrate until the film detaches to form a mixture; (3) The mixture is sonicated and filtered to obtain the fluorescent probe liposome.

2. The fluorescent probe liposome according to claim 1, characterized in that: The mass ratio of lecithin to cholesterol is (1~6):

1.

3. A method for preparing fluorescent probe liposomes, characterized in that: The preparation method includes the following steps: (1) Dissolve lecithin and cholesterol in an organic solvent and rotary evaporate to form a thin film; (2) Add luminol, sulfonated Cy5 NHS active ester, collagen and pH buffer to the film, and rotate to hydrate until the film detaches to form a mixture; (3) The mixture is sonicated and filtered to obtain the fluorescent probe liposomes; The mass ratio of the lecithin, cholesterol, collagen, luminol and sulfonated Cy5 NHS active ester is (1~30):(1~5):(30~50):(1~5):(1~5).

4. The preparation method according to claim 3, characterized in that: In step (1), the mass ratio of lecithin to cholesterol is (1~6):

1.

5. The preparation method according to claim 3, characterized in that: In step (2), the mass-to-volume ratio of luminol to the pH buffer is 0.01~0.5 mg / mL; and / or The mass-to-volume ratio of the sulfonated Cy5 NHS active ester to the pH buffer is 0.01~0.5 mg / mL; and / or The mass-to-volume ratio of collagen to the pH buffer is 2-5 mg / mL.

6. The preparation method according to claim 3, characterized in that: In step (2), the pH of the pH buffer solution is 7.0~7.

5.

7. The preparation method according to claim 3, characterized in that: In step (3), the ultrasound time is 20~40 min, the ultrasound temperature is 20~30℃, and the ultrasound power is 50~200 W.

8. The use of the fluorescent probe liposomes according to any one of claims 1 to 2 or the fluorescent probe liposomes prepared according to any one of claims 3 to 7 in the preparation of products for detecting myeloperoxidase.

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