Preparation process of targeting nanoparticles for early hepatic fibrosis diagnosis

Abstract

The invention discloses a preparation process of targeting nanoparticles for early hepatic fibrosis diagnosis. The preparation process comprises the following steps: S01, dissolving dipalmitoyl phosphatidylcholine, phospholipid polyethylene glycol amino, 1, 2-distearoyl-sn-glycerol-3-phosphoryl glycerol and cholesterol in trichloromethane according to a certain mass ratio to form a mixed solution A; s02, the mixed solution A is placed in a rotary evaporator and evaporated for 30-45 min at the temperature of 35-50 DEG C, the organic solvent in the mixed solution is removed, and a mixture film is formed; s03, after the mixture is subjected to thin film elution treatment, a perfluoropentane solution is added for ultrasonic treatment and centrifugal treatment, and amino nanoparticles are obtained; and S04, mixing the amino nanoparticles, the specific binding protein of the IV-type collagen and a buffer solution, oscillating, and centrifugally washing to obtain the targeted nanoparticles. The targeted nanoparticles prepared by the process can effectively realize detection and diagnosis of hepatic fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a preparation process of targeted nanoparticles for the diagnosis of early liver fibrosis. Background Art

[0002] Liver fibrosis is a chronic inflammatory, autoimmune, and biliary disease caused by hepatitis B or C virus infection, and is also a key factor in the progressive development of liver diseases such as alcoholic, non-alcoholic, and fatty hepatitis. In addition, advanced liver fibrosis is generally considered irreversible, and the only curative treatment is transplantation, which highlights the necessity of early detection and intervention of liver fibrosis.

[0003] Currently, the clinical diagnosis of liver fibrosis mainly relies on invasive liver biopsy, which has problems such as high operation risk, large sampling error, and difficulty in dynamically monitoring the progression of fibrosis. Existing non-invasive imaging methods (such as ultrasound, MRI, etc.) have insufficient sensitivity and specificity for early liver fibrosis (F1-F3 stages), and lack technical solutions for accurately quantifying the degree of fibrosis. Therefore, there is an urgent need to develop a new type of nanomaterial that can target and identify specific markers of liver fibrosis and enhance image contrast to improve the accuracy and reliability of non-invasive liver fibrosis diagnosis. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a preparation process of targeted nanoparticles for the diagnosis of early liver fibrosis, aiming to solve the above technical problems.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A near-infrared second-region fluorescent molecule, whose structural formula is:

[0006] S01: Dissolve dipalmitoyl phosphatidylcholine, phospholipid polyethylene glycol amine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol in chloroform according to a certain mass ratio to form a mixed solution A;

[0007] S02: Place the mixed solution A in a rotary evaporator and evaporate for 30-45 min at a temperature of 35°C - 50°C to remove the organic solvent in the mixed solution and form a mixture film;

[0008] S03: After eluting and treating the mixture film, add a perfluoropentane solution for ultrasonic and centrifugal treatment to obtain amino nanoparticles;

[0009] S04: Mix, oscillate, and centrifuge and wash the amino nanoparticles, the specific binding protein of type IV collagen, and a buffer solution to obtain targeted nanoparticles.

[0010] Further, in the step S01: the mass ratio of dipalmitoyl phosphatidylcholine, phospholipid polyethylene glycol amine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol is (15-20):(6-8):(2-4):(3-5).

[0011] Further, the step S03 includes:

[0012] S031: Suspending the mixture film on phosphate buffered saline for elution treatment, and then adding perfluoropentane solution for ultrasonic treatment at 3-6 °C for 2-6 min;

[0013] S032: Placing the mixed solution in step S031 in a centrifuge and centrifuging at 3-6 °C for 4-8 min to obtain amino nanoparticles.

[0014] Further, the volume ratio of the phosphate buffered saline to the perfluoropentane solution is 30:1.

[0015] Further, the buffer solution is 2-(N-morpholino)ethanesulfonic acid.

[0016] Further, the step S04 includes:

[0017] S041: Suspending the amino nanoparticles in 2-(N-morpholino)ethanesulfonic acid for standby;

[0018] S042: Weighing a certain amount of 2-(N-morpholino)ethanesulfonic acid and adjusting the pH value of 2-(N-morpholino)ethanesulfonic acid to weakly acidic; then dissolving water-soluble EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) in this 2-(N-morpholino)ethanesulfonic acid according to a certain mass ratio, adding a specific binding protein solution of type IV collagen and incubating with shaking, and adjusting the pH of the solution to 7-9;

[0019] S043: Adding the solution in step S041 to step S042, shaking and centrifuging and washing several times at 3-6 °C to obtain targeted nanoparticles.

[0020] Further, the mass ratio of the water-soluble EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) in the step S042 is 8:3.

[0021] Further, the incubation time with shaking in the step S042 is 1.5-3 h.

[0022] Further, after the step S04, it also includes: modifying the nanoparticles with DiI; modifying the specific binding protein of type IV collagen with FITC fluorescent probe.

[0023] The beneficial effects of the present invention are as follows: Compared with the prior art: (1) The targeted nanoparticles prepared in the present invention use type IV collagen-specific binding protein as a molecular probe, making the nanoparticles have targeting properties, recognizing type IV collagen in liver fibrosis, and enabling the detection and diagnosis of liver fibrosis; (2) The prepared targeted nanoparticles have good biocompatibility and excellent safety; (3) Through the targeted nanoparticles, ultrasound imaging can be used to achieve the targeted imaging effect of liver fibrosis, achieving the effect of high-efficiency recognition and detection.

[0024] Other advantages, objectives, and features of the present invention will be described in the subsequent specification, and to some extent, will be obvious to those skilled in the art, or those skilled in the art can obtain teachings from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0026] Figure 1 It is an electron micrograph of the targeted nanoparticles observed by low-voltage transmission electron microscopy (LV-TEM);

[0027] Figure 2 It is the potential map and particle size map of the targeted nanoparticles;

[0028] Figure 3 It is the stability analysis diagram of the targeted nanoparticles;

[0029] Figure 4 It is the ultrasonic image, H&E staining, masson staining, PSR staining, fluorescence microscopy images of Dil and FITC-labeled targeted nanoparticles of the rat liver after intervention with different concentrations of CCL4 after injection of the targeted nanoparticles;

[0030] Figure 5 It is a schematic diagram of the correlation between the contrast intensity value of ultrasound (CEUS) and the collagen content of liver tissues at different stages of liver fibrosis;

[0031] Figure 6 It is the preparation flow chart of the preparation process of a targeted nanoparticle for the diagnosis of early liver fibrosis of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] Please refer to Figure 1 as shown, the present application includes a preparation process of a targeted nanoparticle for the diagnosis of early liver fibrosis, including the following steps:

[0033] S01: Dissolve dipalmitoyl phosphatidylcholine, phospholipid-polyethylene glycol amine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol in chloroform according to a certain mass ratio to form a mixed solution A;

[0034] S02: Place the mixed solution A in a rotary evaporator and evaporate it at a temperature of 35°C - 50°C for 30 - 45 minutes to remove the organic solvent in the mixed solution and form a mixture film;

[0035] S03: After eluting and treating the mixture film, add a perfluoropentane solution for ultrasonic treatment and centrifugation to obtain amino nanoparticles;

[0036] S04: Mix, oscillate, and centrifuge and wash the amino nanoparticles, the specific binding protein of type IV collagen, and a buffer solution to obtain targeted nanoparticles.

[0037] In this application, by mixing and proportioning dipalmitoyl phosphatidylcholine, phospholipid-polyethylene glycol amine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol; by selecting phospholipid compounds, it is beneficial to form nanoparticles with better biocompatibility and good safety; after eluting and treating the mixture film, adding it to a perfluoropentane solution for ultrasonic treatment, so that the phase transition temperature of perfluoropentane itself is closer to the physiological conditions of the human body, avoiding or reducing the phase transition of liquid fluorocarbon nanoparticles at non-target sites; by mixing amino nanoparticles, the specific binding protein of type IV collagen, and a buffer solution, the specific binding protein of type IV collagen can be connected to the surface of the amino nanoparticles, so that the specific binding protein of type IV collagen can recognize type IV collagen in liver fibrosis, realizing the detection and diagnosis of liver fibrosis. In addition, the targeted nanoparticles can also be used for ultrasonic imaging to achieve the targeted imaging effect of liver fibrosis and achieve the effect of efficient recognition and detection.

[0038] Preferably, in step S01: the mass ratio of the dipalmitoyl phosphatidylcholine, phospholipid-polyethylene glycol amine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol is (15 - 20):(6 - 8):(2 - 4):(3 - 5). Specifically, the mass ratio of the dipalmitoyl phosphatidylcholine, phospholipid-polyethylene glycol amine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol can be 15:6:2:3, 17:7:3:3, 18:7:2:3, 20:7:3:4, 20:7:4:5, etc.

[0039] Further, step S03 includes:

[0040] S031: Suspend the mixture film on the phosphate buffered saline solution for elution treatment, and then add the perfluoropentane solution and sonicate it at 3 - 6 °C for 2 - 6 min;

[0041] S032: Place the mixed solution in step S031 in a centrifuge and centrifuge it at 3 - 6 °C for 4 - 8 min to obtain amino nanoparticles.

[0042] In this application, the sonication temperature in step S031 can be 3 °C, 4 °C, 5 °C, 6 °C, etc.; the sonication time can be 2 min, 3 min, 4 min, 5 min, 6 min, etc.

[0043] In this application, the centrifugation temperature in step S032 can be 3 °C, 4 °C, 5 °C, 6 °C, etc., and the centrifugation time can be 4 min, 5 min, 6 min, 7 min, 8 min, etc. Of course, the above sonication time and centrifugation time can be adjusted accordingly according to the actual situation, and are not uniquely limited here.

[0044] Preferably, the volume ratio of the phosphate buffered saline to the perfluoropentane solution is 30:1. For example, when the volume of perfluoropentane is 100 μL, 200 μL, 300 μL, etc., correspondingly, the phosphate buffered saline is 3 mL, 6 mL, 9 mL, etc.

[0045] Preferably, the buffer solution is 2 - morpholinoethanesulfonic acid. This buffer liquid has good chemical stability and biocompatibility and will not be toxic to biological samples.

[0046] Furthermore, step S04 includes:

[0047] S041: Suspend the amino nanoparticles in 2 - morpholinoethanesulfonic acid for standby;

[0048] S042: Weigh a certain amount of 2 - morpholinoethanesulfonic acid and adjust the pH value of 2 - morpholinoethanesulfonic acid to weakly acidic; immediately dissolve water - soluble EDC (1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride), NHS (N - hydroxysuccinimide) in this 2 - morpholinoethanesulfonic acid according to a certain mass ratio, add the specific binding protein solution of type Ⅳ collagen and incubate it with shaking, and adjust the pH of the solution to 7 - 9;

[0049] S043: Add the solution in step S041 to step S042, shake and centrifuge and wash it several times at 3 - 6 °C to obtain targeted nanoparticles.

[0050] In this application, in step S041, ensure that the amino nanoparticles are evenly suspended in 2 - morpholinoethanesulfonic acid for effective reactions in subsequent steps. The concentration of the suspension can be adjusted according to specific needs to achieve the best preparation effect.

[0051] In step S042, 2-morpholinoethanesulfonic acid is accurately weighed and its pH value is adjusted to the weakly acidic range. Subsequently, water-soluble EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are dissolved in the adjusted 2-morpholinoethanesulfonic acid according to a predetermined mass ratio. These compounds, as crosslinking agents, can promote the coupling reaction between the specific binding protein of type IV collagen and the amino nanoparticles. After adding the specific binding protein solution, oscillation incubation is carried out to ensure that the protein reacts fully with the crosslinking agent. Finally, the pH value of the solution is adjusted to 7-9 to create a suitable environment for the subsequent coupling reaction.

[0052] In step S043, the amino nanoparticle suspension obtained in step S041 is added to the reaction solution obtained in step S042. By oscillating at 3-6 °C, the binding of the targeting molecule to the nanoparticle is promoted; by oscillating at this temperature, the volatilization of perfluoropentane is avoided. Subsequently, centrifugal washing is carried out several times to remove unbound molecules and impurities, and finally pure targeting nanoparticles are obtained. These targeting nanoparticles have the ability to specifically recognize type IV collagen and can be used for the diagnosis of early liver fibrosis.

[0053] Furthermore, the mass ratio of the water-soluble EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) in step S042 is 8:3. By setting this ratio, the aim is to optimize the crosslinking effect and ensure the formation of a stable and efficient coupling between the specific binding protein and the amino nanoparticles. If the mass ratio is too high, it may lead to over-crosslinking, affecting the stability and biocompatibility of the nanoparticles; if the mass ratio is too low, it may not be sufficient to promote a full coupling reaction and reduce the targeting efficiency.

[0054] Furthermore, the oscillation incubation time in step S042 is 1.5-3 h. The purpose of the oscillation incubation in this application is to promote the full reaction between the water-soluble EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), and the specific binding protein. By controlling within the range of 1.5-3 h, it can ensure that the reaction reaches the optimal state, neither resulting in incomplete reaction due to too short a time nor causing unnecessary side reactions due to too long a time. The setting of this time range is based on the optimization results of experimental data, aiming to improve the preparation efficiency and quality of the targeting nanoparticles. Specifically, the oscillation incubation time can be 1.5 h, 2 h, 2.5 h, 3 h, etc., and the specific incubation time can be determined according to the actual situation.

[0055] Furthermore, after the step S04, the following steps are also included: modifying the nanoparticles with DiI; modifying the specific binding protein of type IV collagen with FITC fluorescent probe. By modifying with DiI and FITC fluorescent probes, the targeted nanoparticles carry DiI and FITC fusion fluorescence signals, enabling the specific binding protein of type IV collagen to perform targeted fluorescence labeling on the liver fibrosis site. This kind of fluorescence labeling not only improves the visualization degree of the nanoparticles in the organism, but also helps with subsequent observation and analysis. In specific operations, by adjusting the dosage of the DiI fluorescent probe, the intensity and duration of the fluorescence labeling can be further controlled to meet different experimental requirements. The addition of this step provides a more convenient and intuitive means for the application of targeted nanoparticles in the diagnosis of liver fibrosis.

[0056] Example:

[0057] Dissolve dipalmitoyl phosphatidylcholine, phospholipid-polyethylene glycol amine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol in 15 mL of chloroform according to a mass ratio of 20:7:3:4; then, use a rotary evaporator (RE-52A, Yarong, Shanghai, China) to evaporate for 30 minutes at 45 °C; suspend the mixture film in 6 mL of PBS (Phosphate Buffered Saline), add 200 μL of PFP (Perfluoropentane), and sonicate for 3 minutes at 4 °C (condition: 5-second on / off pulse, 45% intensity). Then, centrifuge at 4 °C (8000 rpm, 5 minutes) using a refrigerated centrifuge (Eppendorf, Hamburg, Germany) to obtain amino nanoparticles; suspend the obtained amino nanoparticles above in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.1 M, pH = 8.0); subsequently, weigh EDC and NHS according to a mass ratio of 8:3, dissolve them in MES (0.1 M, pH = 5.2) buffer, immediately add 600 microliters of the specific binding protein solution of type IV collagen, oscillate and incubate at room temperature for 2 h to activate the carboxyl groups on the specific binding protein of type IV collagen, then adjust its pH to 8.0, mix it with the amino nanoparticle solution, oscillate and incubate at 3 - 6 °C, and after centrifuging and washing 3 times, obtain targeted nanoparticles.

[0058] Figure 1 The figure shows the electron micrograph of the targeted nanoparticles observed by low-voltage transmission electron microscopy (LV-TEM). It can be seen that the prepared targeted nanoparticles are spherical-like with a smooth surface. Figure 2It is the particle size diagram of the targeted nanoparticles. Measured by a Malvern laser particle size and zeta potential analyzer, the average particle size of the targeted nanoparticles is (307.90 ± 4.17) nm, and the zeta potential is (-20.21 ± 1.75) mV. In this way, by forming a similar spherical shape and corresponding particle size, it can enter the liver well.

[0059] Figure 3 It is the stability analysis diagram of the targeted nanoparticles. As can be seen from Figure 3 it, when stored at 4 °C, the particle size of the nanoparticles does not change significantly within two weeks, showing good stability.

[0060] In order to further illustrate the detection effect of the targeted nanoparticles, the present application specifically prepares the following four groups of experiments, namely, including a control group, and three comparison experiments with 30% CCL4, 40% CCL4, and 50% CCL4. As Figure 4 shown, through the comparative analysis of the four groups of experiments, it can be seen that the percentage of collagen area in the liver fibrosis area increases with the increase of the CCL4 concentration. Among them, the percentage of collagen area in the liver fibrosis area shown in the control group is 1.92% ± 0.59%; the percentage of collagen area in the liver fibrosis area shown in 30% CCL4 is 15.72% ± 1.53%; the percentage of collagen area in the liver fibrosis area shown in 40% CCL4 is 23.56% ± 2.25%; the percentage of collagen area in the liver fibrosis area shown in 50% CCL4 is 33.14% ± 1.79%. This result clearly reveals the positive correlation between the CCL4 concentration and the degree of liver fibrosis.

[0061] In addition, we performed contrast imaging on the liver tissues of the four groups of experimental mice using the targeted nanoparticles. As Figure 5 shown, Figure 5 It is the schematic diagram of the correlation between the contrast-enhanced ultrasound (CEUS) intensity value and the collagen content in the liver tissue at different stages of liver fibrosis. This diagram shows that there is a strong positive correlation between the contrast-enhanced ultrasound (CEUS) intensity value and the collagen content in the liver tissue at the S1 stage (r = 0.847, P < 0.05), and the correlation between the collagen and the contrast-enhanced ultrasound (CEUS) echo intensity value at the S2 stage is (r = 0.773, P < 0.01). The correlation between the collagen and the contrast-enhanced ultrasound (CEUS) intensity value at the S3 stage is (r = 0.690, P < 0.05). This result indicates that the targeted nanoparticles can specifically bind to the fibrotic area and show stronger fluorescence signals in tissues with higher fibrosis degrees. This property makes the targeted nanoparticles not only applicable to the early diagnosis of liver fibrosis but also a potential tool for evaluating the fibrosis process and treatment effect.

[0062] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A preparation process of targeted nanoparticles for early diagnosis of liver fibrosis, characterized in that: The steps include: S01: dissolving dipalmitoylphosphatidylcholine, phospholipid polyethylene glycol amino, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol in chloroform according to a certain mass ratio to form a mixed solution A; S02: placing the mixed solution A in a rotary evaporator and evaporating at a temperature of 35° C.-50° C. for 30-45 min to remove the organic solvent in the mixed solution and form a mixture film; S03: After eluting the mixture film, adding a perfluoropentane solution to perform ultrasonic and centrifugal treatment to obtain amino nanoparticles; S04: The amino nanoparticles, the specific binding protein of type IV collagen, and the buffer solution are mixed, shaken, and centrifuged to obtain targeted nanoparticles.

2. The preparation process of targeted nanoparticles for early diagnosis of liver fibrosis according to claim 1, characterized in that: In step S01, the mass ratio of dipalmitoylphosphatidylcholine, phospholipid polyethylene glycol amino, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, and cholesterol is (15-20): (6-8): (2-4): (3-5).

3. The preparation process of targeted nanoparticles for early diagnosis of liver fibrosis according to claim 2, characterized in that: The step S03 comprises: S031: suspending the mixture film in a phosphate buffered saline solution for elution, and then adding a perfluoropentane solution for ultrasonic treatment at 3-6°C for 2-6 minutes; S032: Place the mixed solution in step S031 in a centrifuge and centrifuge at 3-6° C. for 4-8 minutes to obtain amino nanoparticles.

4. The preparation process of targeted nanoparticles for early diagnosis of liver fibrosis according to claim 3, characterized in that: The volume ratio of the phosphate buffer solution to the perfluoropentane solution is 30:

1.

5. The preparation process of targeted nanoparticles for early diagnosis of liver fibrosis according to claim 1, characterized in that: The buffer is 2-morpholineethanesulfonic acid.

6. The preparation process of targeted nanoparticles for early diagnosis of liver fibrosis according to claim 5, characterized in that: The step S04 comprises: S041: suspending the amino nanoparticles in 2-morpholineethanesulfonic acid for later use; S042: Weigh a certain amount of 2-morpholineethanesulfonic acid, adjust the pH value of the 2-morpholineethanesulfonic acid to weak acidity; then dissolve water-soluble EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) in the 2-morpholineethanesulfonic acid according to a certain mass ratio, add a solution of type IV collagen-specific binding protein, shake and incubate, and adjust the pH value of the solution to 7-9; S043: Add the solution of step S041 to step S042, shake and centrifuge and wash several times at 3-6°C to obtain targeted nanoparticles.

7. The preparation process of targeted nanoparticles for early diagnosis of liver fibrosis according to claim 6, characterized in that: The mass ratio of water-soluble EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to NHS (N-hydroxysuccinimide) in step S042 is 8:

3.

8. The preparation process of targeted nanoparticles for early diagnosis of liver fibrosis according to claim 6, characterized in that: The shaking incubation time in step S042 is 1.5-3 hours.

9. The preparation process of targeted nanoparticles for early diagnosis of liver fibrosis according to claim 1, characterized in that: The step S04 further includes: modifying the nanoparticles with Dil; and modifying the specific binding protein of type IV collagen with FITC fluorescent probe.

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