Bionic responsive multifunctional nano-agent as well as preparation method and application thereof

Abstract

The invention relates to a bionic responsive multifunctional nano agent as well as a preparation method and application thereof, and belongs to the field of biological medicines. According to the nano agent, an anti-inflammatory drug, a contrast agent metal ion and dopamine are self-assembled to form a particle core, and an outer layer wraps a cell membrane to serve as a shell layer. The membrane has excellent biocompatibility, can realize long-term circulation in vivo, avoids immune clearance, and selectively targets an inflammatory site. The nano-agent creatively integrates the characteristics of cell membrane targeting, ROS responsiveness, multifunctional treatment, imaging monitoring and the like, not only provides a multi-target collaborative treatment and precise diagnosis strategy for AP, but also opens up a new thought for development of nano-drugs for other inflammation-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to biomimetic responsive multifunctional nanoparticles, their preparation methods, and applications. Background Technology

[0002] Acute pancreatitis (AP) is a life-threatening inflammatory disease that progresses rapidly, often developing into systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Ulinastatin (U) is a clinically used broad-spectrum anti-inflammatory drug that exhibits significant anti-inflammatory and antioxidant effects in the treatment of AP by inhibiting various hydrolases, reducing the release of inflammatory mediators, and mitigating damage caused by oxidative stress. However, its limited efficacy is restricted by its single function, rapid clearance rate, and insufficient targeting.

[0003] Therapeutic strategies play a synergistic role in promoting early disease diagnosis, monitoring the distribution of targeted drugs, and assessing treatment response. Among numerous imaging techniques, magnetic resonance imaging (MRI) has become an important tool for medical diagnosis due to its high spatial resolution, excellent soft tissue contrast, and lack of ionizing radiation. Therefore, MRI-guided treatment of acute pancreatitis can provide real-time feedback on treatment effectiveness, thereby optimizing treatment plans and improving treatment outcomes. Summary of the Invention

[0004] In view of this, the present invention aims to provide a biomimetic responsive multifunctional nanoparticle, a second objective is to provide a biomimetic responsive multifunctional nanoparticle and a preparation method thereof, and a third objective is to provide an application of the biomimetic responsive multifunctional nanoparticle.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a biomimetic responsive multifunctional nanoparticle, which consists of a core nanoparticle encapsulated by a cell membrane shell; the cell membrane shell is selected from neutrophil membrane, macrophage membrane, mesenchymal stem cell membrane, erythrocyte membrane, platelet membrane, or a mixture thereof; the core nanoparticle is composed of contrast agent metal ions, dopamine, and a drug.

[0007] Preferably, the contrast agent metal ion is any one of gadolinium, manganese, and iron;

[0008] Preferably, the drug is the anti-inflammatory drug ulinastatin;

[0009] Preferably, the mass ratio of the cell membrane shell to the core nanoparticles is 1:1;

[0010] Preferably, the molar ratio of anti-inflammatory drug, dopamine and contrast agent metal ions in the core nanoparticles is 150-180:280-320:0.9-1.1, mg:mg:mM;

[0011] Furthermore, the preparation method of the aforementioned biomimetic responsive multifunctional nanoparticles comprises the following steps:

[0012] Preparation of core nanoparticles: Anti-inflammatory drugs, dopamine and contrast agent metal ions were dissolved in deionized water at a mass molar ratio of 150-180:280-320:0.9-1.1 (mg:mg:mM) and stirred evenly. Then ammonia water was slowly added and stirred for 24 hours. The core nanoparticles were collected, centrifuged and washed to obtain the core nanoparticles.

[0013] Cell membrane preparation: Culture and collect any one or more of neutrophils, macrophages, mesenchymal stem cells, erythrocytes, and platelets, add PBS containing protease inhibitors and phosphatase inhibitors, repeatedly freeze and thaw in liquid nitrogen, centrifuge at 700g for 15 minutes at 4°C to remove organelles, take the supernatant and continue to centrifuge at 14000g for 30 minutes, remove the supernatant, and the remaining precipitate is the cell membrane;

[0014] Assembly of multifunctional nanoparticles: Core nanoparticles and cell membranes were mixed at a mass ratio of 1:1 and sonicated at 42 kHz and 100 W for 5 minutes. The solution was then transferred to a liposome extruder and repeatedly extruded through polycarbonate porous membranes of 800 nm, 400 nm, and 200 nm, respectively, to obtain the multifunctional nanoparticles.

[0015] Furthermore, the application of the aforementioned biomimetic responsive multifunctional nanoparticles in the preparation of drugs for acute pancreatitis;

[0016] Furthermore, the application of the aforementioned biomimetic responsive multifunctional nanoparticles in the preparation of developer.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention presents a biomimetic therapeutic nanoplatform (MDU@Mn) sensitive to the inflammatory microenvironment, designed for targeted therapy and imaging-based monitoring of acute pancreatitis (AP). This nanoparticle platform consists of a core (DU@Mn) formed by the self-assembly of uranium, manganese chloride, and dopamine, encapsulated by a macrophage membrane as a shell. This membrane exhibits excellent biocompatibility, allowing for long-term circulation in vivo, evading immune clearance, and selectively targeting inflammatory sites. Upon entering acinar cells, DU@Mn releases Mn under low pH and high ROS conditions. 2+ And U. In addition, Mn2 +By enhancing MRI signals to enable real-time monitoring of the treatment process, U works synergistically to alleviate the inflammatory response and promote tissue repair through multiple mechanisms, including inhibiting pancreatic enzyme activity, anti-inflammatory and antioxidant effects, and improving microcirculation. This platform innovatively integrates macrophage membrane targeting, ROS responsiveness, multifunctional therapy, and imaging monitoring, providing not only a multi-target synergistic therapy and precise diagnostic strategy for acute pancreatitis (AP), but also opening up new avenues for the development of nanomedicines for other inflammation-related diseases.

[0019] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0021] Figure 1 This is a schematic diagram of the MDU@Mn preparation process;

[0022] Figure 2 For the preparation and physicochemical characterization of MDU@Mn: (A) Transmission electron microscopy (TEM) image of DU@Mn, (B) Transmission electron microscopy (TEM) image of MDU@Mn, (C) Elemental mapping of DU@Mn, (D) Hydrodynamic diameters of DU@Mn and MDU@Mn (n=3), (E) Zeta potentials of MM, DU@Mn and MDU@Mn (n=3), (F) UV-Vis spectra of MM, DU@Mn and MDU@Mn, (G) SDS-PAGE analysis of protein bands of DU@Mn, MDU@Mn and MM, (H) Characteristic protein bands of MDU@Mn, MM and macrophages separated by Western blotting, (I) Fourier transform infrared spectra of PDA, U, DU@Mn and MnCl2;

[0023] Figure 3 Characterization of nanoparticles: (A) UV-Vis spectra of different concentrations of U, (B) Absorption spectra of U in supernatant and stock solution to measure U loading, (C) Drug release curves of MDU@Mn in phosphate-buffered saline (PBS) (pH 7.4, pH 5.4, pH 7.4 + 0.1 mM H2O2 and pH 5.4 + 0.1 mM H2O2; n = 3), (D) Particle size variation of MDU@Mn over seven days, n = 3. Data are expressed as standard deviation ± mean.

[0024] Figure 4 The relaxation rate of MDU@Mn is defined as follows: (A) the relaxation rate of MDU@Mn at 0.5T, 1.5T, and 3.0T; (B) the change in the relaxation rate of MDU@Mn under different treatments; and (C) the relaxation rate of Mn under different treatments. 2+ Release curves, (D) T1-weighted MRI images and pseudo-color maps of MDU@Mn under different treatment conditions;

[0025] Figure 5 Representative T1-weighted MRI images of AP group and normal rats at different time points after tail vein injection of DU@Mn or MDU@Mn. Data are expressed as standard deviation ± mean (n=3).

[0026] Figure 6 Evaluation of the therapeutic effect of MDU@Mn in AP rats: (A) AP rat model and treatment flowchart, (B) Ratio of pancreas to body weight in rats (n=3), (C) Survival rate of rats in different treatment groups (n=10), (DE) Lipase and amylase levels (n=3), (F) Representative images of pancreas in rats in different treatment groups, (G) Representative images of H&E staining of pancreatic samples, scale bar = 25 μm, (HJ) Pancreatic inflammatory infiltration, necrosis and edema assessed by double-blind method (n=3); *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0027] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0028] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0029] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0030] Example 1: Preparation of MDU@Mn nanoparticles

[0031] DU@Mn synthesis method:

[0032] Dissolve 16 mg U, 30 mg dopamine, and 0.1 mM manganese chloride in 16 mL of deionized water and stir for 30 minutes until fully dissolved. Then slowly add ammonia water and stir for 24 hours, during which the solution gradually changes from white to black. Collect the product in a centrifuge tube and centrifuge at 10,000 rpm for 15 minutes. Wash the product twice with deionized water and then disperse it in 2 mL of deionized water to obtain DU@Mn. Finally, freeze-dry using a vacuum dryer.

[0033] MDU@Mn synthesis method:

[0034] RAW264.7 cells were cultured, centrifuged, and collected in EP tubes. PBS containing protease inhibitors and phosphatase inhibitors was added, and the cells were repeatedly freeze-thawed in liquid nitrogen until 70-80% cell disruption was achieved. The cells were centrifuged at 700g for 15 minutes at 4°C to remove organelles. The supernatant was then centrifuged again at 14000g for 30 minutes, and the supernatant was removed. The remaining precipitate was the macrophage membrane (MM). The prepared MM was mixed with DU@Mn at a 1:1 mass ratio and sonicated at 42kHz and 100W for 5 minutes. The solution was then transferred to a liposome extractor and passed through polycarbonate porous membranes of 800nm, 400nm, and 200nm, respectively, and repeatedly squeezed 11 times to obtain cell membrane-encapsulated nanoparticles (MDU@Mn), which were stored at 4°C.

[0035] Example 2: Performance testing of MDU@Mn nanoparticles

[0036] Drug loading rate and encapsulation efficiency are important factors for drug efficacy. The drug encapsulation efficiency (DEE) and drug loading (DL) of MDU@Mn, determined by UV-Vis spectroscopy, were 66.7% and 11.8%, respectively. Figure 3(A, B). Furthermore, the release mechanism of MDU@Mn was investigated by simulating healthy and pathological conditions. Under healthy physiological conditions, MDU@Mn releases U at a relatively slow rate. Further decreasing the pH and / or adding H2O2 accelerated its release rate, which gradually plateaued after 24 hours. The cumulative release rates of MDU@Mn reached 50.7%, 62.9%, and 78.9%, respectively. Figure 3 C). To assess the stability of the nanoformulation, we analyzed its particle size in 1.0×PBS and 0.5×FBS. The results showed that the particle size of MDU@Mn did not change significantly over 7 days. Figure 3 D). The above results indicate that MDU@Mn has a high drug loading rate and encapsulation rate, and can rapidly release drugs at the site of inflammation.

[0037] Relaxation rate is crucial for imaging, and its main influencing factors include temperature, the physicochemical properties of the contrast agent, its concentration, and the magnetic field strength. The relaxation rates of MDU@Mn at magnetic field strengths of 0.5T, 1.5T, and 3.0T are 27.662, 12.008, and 4.761, respectively. Figure 4 A), indicating its excellent imaging capabilities. We simulated the pH and reactive oxygen species (ROS) concentrations in the inflammatory microenvironment and examined the relaxation rate of MDU@Mn in different environments. Figure 4 B). With decreasing pH and the addition of H2O2, the relaxation rate of MDU@Mn gradually increased, eventually reaching approximately 10.806. We speculate that this is due to the release of Mn from MDU@Mn in the inflammatory pathological microenvironment (low pH and high ROS). 2+ This improves its relaxation rate. Subsequently, we analyzed Mn under the same conditions. 2+ Release behavior ( Figure 4 C). Under physiological conditions, Mn 2+ The release rate was 10.25% after 24 hours. When the pH decreased to 5.4, the release rate increased to 31.86%. The addition of H2O2 accelerated the release of Mn. 2+ The release rate of Mn increased to 25.57% and 61.98% at pH 7.4 and 5.4, respectively. 2+ The release rate trend was consistent with the relaxation rate change of MDU@Mn. Furthermore, with increasing H2O2 concentration, the signal intensity of MDU@Mn in T1-weighted MRI imaging increased, especially at pH 5.4, which may be attributed to its redox-sensitive properties. Figure 4 D).

[0038] Example 3 Imaging analysis of MDU@Mn nanoparticles

[0039] Due to the excellent relaxation rate of MDU@Mn, we established AP rat models and sham-operated rat models, and evaluated their contrast agent properties via MRI by tail vein injection of MDU@Mn and DU@Mn. Figure 5 As shown, pancreatic tissue was more clearly distinguishable after injection, indicating that MDU@Mn and DU@Mn are suitable as MRI contrast agents. Two hours after MDU@Mn injection, the T1-weighted signal of pancreatic tissue in AP rats was the strongest (approximately 145), higher than that in the sham-operated group (approximately 91). The signal gradually decreased over time, returning to normal after 24 hours. Similarly, two hours after DU@Mn injection, the pancreatic signal in the AP group rats (approximately 106) was higher than that in the sham-operated group rats (approximately 71). It is speculated that these signal differences are mainly influenced by inflammatory chemotaxis, immune escape, and the inflammatory microenvironment.

[0040] Example 4: Application of MDU@Mn nanoparticles

[0041] We further investigated the therapeutic effects of MDU@Mn. First, we successfully established an experimental acute pancreatitis (AP) model in rats by retrograde injection of 3.5% sodium taurocholate into the pancreatic ducts. Subsequently, 20 mg / kg of MDU@Mn or DU@Mn were administered at 2 hours or 24 hours post-modeling. Figure 6 A). U, DU@Mn, and MDU@Mn not only significantly reduced the degree of pancreatic edema ( Figure 6 B) It also effectively reduced the mortality rate of mice with acute pancreatitis, with the survival rate of the MDU@Mn group reaching 90%. Figure 6 C). Amylase and lipase are important biochemical indicators for diagnosing AP23 and 24. Stimulation with sodium taurocholate triggers an inflammatory response, activating many proenzymes and leading to a significant increase in amylase and lipase levels. After treatment with MDU@Mn, amylase and lipase levels decreased significantly, with no significant difference compared to the control group (p>0.05). Figure 6 (D, E) Due to the pancreas's "autodigestion," many enzymes are consumed, and tissue damage leads to a decrease in amylase levels; therefore, amylase and lipase cannot objectively assess the severity of AP25. We collected pancreatic tissue from sacrificed rats for observation ( Figure 6 F). The pancreatic tissue in the model group showed severe edema, hemorrhage, and necrosis, which were alleviated to varying degrees after treatment. To objectively evaluate the therapeutic effect on the pancreas, we used histopathological analysis to examine the inflammation, necrosis, and edema of the pancreas. Samples from the AP group showed significant widening of the interstitial space (edema), infiltration of lymphocytes and neutrophils, and necrosis. After treatment with U and DU@Mn, the widening of the interstitial space and the infiltration of inflammatory cells were reduced. In the MDU@Mn group, the pancreatic histopathology tended to be normalized ( Figure 6G). Subsequent quantitative analysis of pancreatic tissue using the Schmidt scoring system revealed that, compared to DU@Mn and U, MDU@Mn exhibited superior therapeutic efficacy, with lower scores for granular necrosis, inflammatory cell infiltration, and interstitial edema. Figure 6 HJ).

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A biomimetic responsive multifunctional nanoparticle, characterized in that: The multifunctional nanoparticles consist of a core nanoparticle encapsulated by a cell membrane shell; the cell membrane shell is selected from neutrophil membranes, macrophage membranes, mesenchymal stem cell membranes, erythrocyte membranes, platelet membranes, or a mixture thereof; the core nanoparticles consist of contrast agent metal ions, dopamine, and drugs.

2. The biomimetic responsive multifunctional nanoparticle according to claim 1, characterized in that: The contrast agent metal ion is any one of gadolinium, manganese and iron, and the drug is the anti-inflammatory drug ulinastatin.

3. The biomimetic responsive multifunctional nanoparticle according to claim 2, characterized in that: The mass ratio of the cell membrane shell to the core nanoparticles is 1:

1.

4. The biomimetic responsive multifunctional nanoparticle according to claim 3, characterized in that: The core nanoparticles contain an anti-inflammatory drug, dopamine, and contrast agent metal ions in a molar ratio of 150-180:280-320:0.9-1.1 (mg:mg:mM).

5. The method for preparing the biomimetic responsive multifunctional nanoparticles according to any one of claims 1-4, characterized in that, The preparation steps are as follows: Preparation of core nanoparticles: Anti-inflammatory drugs, dopamine and contrast agent metal ions were dissolved in deionized water at a mass molar ratio of 150-180:280-320:0.9-1.1 (mg:mg:mM) and stirred evenly. Then ammonia water was slowly added and stirred for 24 hours. The core nanoparticles were collected, centrifuged and washed to obtain the core nanoparticles. Cell membrane preparation: Culture and collect any one or more of neutrophils, macrophages, mesenchymal stem cells, erythrocytes, and platelets, add PBS containing protease inhibitors and phosphatase inhibitors, repeatedly freeze and thaw in liquid nitrogen, centrifuge at 700g for 15 minutes at 4°C to remove organelles, take the supernatant and continue to centrifuge at 14000g for 30 minutes, remove the supernatant, and the remaining precipitate is the cell membrane; Assembly of multifunctional nanoparticles: Core nanoparticles and cell membranes were mixed at a mass ratio of 1:1 and sonicated at 42 kHz and 100 W for 5 minutes. The solution was then transferred to a liposome extruder and repeatedly extruded through 800 nm, 400 nm, and 200 nm polycarbonate porous membranes 11 times to obtain the multifunctional nanoparticles.

6. The application of the biomimetic responsive multifunctional nanoparticles according to any one of claims 1-4 in the preparation of drugs for acute pancreatitis.

7. The application of the biomimetic responsive multifunctional nanoparticles according to any one of claims 1-4 in the preparation of developer.

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