Rhamnolipid-modified inflammatory macrophage-targeting nanocarrier and its application after loading magnoflorine

Abstract

The application is a rhamnolipid modified inhalation type nanocarrier loaded with magnoflorine, which is selected from one of liposomes, solid lipid nanoparticles, polymer micelles, nanostructured lipid carriers or albumin nanoparticles; the nanocarrier is wrapped with magnoflorine inside; the application first discloses and utilizes the high tropism of rhamnolipid (RLS) to inflammatory (M1 type) macrophages. Experiments prove that the RLS modified nanocarrier (RLS-NPs) shows specific uptake to M1 type macrophages in vivo and in vitro, and effectively avoids the accumulation of drugs in non-target cells. The loaded magnoflorine (MAG) can effectively down-regulate M1 type macrophage markers (such as CD86) and up-regulate M2 type macrophage markers (such as CD206) after being targetedly taken up by inflammatory macrophages, promote the polarization of macrophages from pro-inflammatory M1 type to anti-inflammatory / repair M2 type, and thus block the liver inflammation storm from the source.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical manufacturing, and more particularly to a rhamnolipid-modified inflammatory macrophage-targeting nanocarrier and its application after loading magnoflorine. Background Technology

[0002] Drug-induced liver injury (DILI) is a common and critical condition in clinical practice. It is liver damage caused by drugs and their metabolites, a common serious adverse drug reaction, and a significant cause of acute liver failure. Acetaminophen (APAP) is a common antipyretic and analgesic; overdose can lead to severe DILI, with mechanisms involving metabolic activation to produce hepatotoxic substances, glutathione depletion, oxidative stress, and mitochondrial dysfunction. In the process of APAP-induced liver injury, the activation of Kupffer cells in the liver and the large-scale recruitment and polarization of peripheral monocytes / macrophages into the liver as pro-inflammatory M1 macrophages are key pathological steps leading to persistent hepatic inflammatory storms and hepatocellular necrosis. Currently, the main effective drug for treating APAP-induced liver injury is N-acetylcysteine ​​(NAC), but it has drawbacks such as a short therapeutic window, limited efficacy, and the potential to cause allergic reactions.

[0003] Magnolia alkaloid is a natural isoquinoline alkaloid with anti-inflammatory, antioxidant and immunomodulatory effects, but its low bioavailability and insufficient in vivo targeting limit its clinical application.

[0004] Rhamnolipids are a class of natural glycolipid biosurfactants with good biocompatibility and membrane affinity. The inventors discovered that rhamnolipid-modified nanocarriers exhibit a significant attraction to inflammatory macrophages, enhancing their accumulation at inflammatory sites. Therefore, using rhamnolipids to construct a targeted nanodelivery system for inflammatory macrophages, and loading it with magnoflorine, holds promise for achieving precise treatment of diabetic inflammatory lymphadenopathy (DILI).

[0005] Therefore, developing an inhaled, inflammatory macrophage-targeting nanoformulation is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide an inhaled nanocarrier modified with rhamnolipid and loaded with magnoflorine. This carrier can actively attract inflammatory macrophages, improving the accumulation efficiency of magnoflorine in inflamed liver tissue, thereby achieving highly effective treatment for drug-induced liver injury. To achieve the above objective, this invention adopts the following technical solution: An inhaled nanocarrier modified with rhamnolipid and loaded with magnoflorine, selected from liposomes, solid lipid nanoparticles, polymer micelles, nanostructured lipid carriers or albumin nanoparticles; The nanocarrier contains magnolia alkaloids. The surface of the nanocarrier is covalently coupled or physically adsorbed with a rhamnolipin-modified layer. This invention provides the application of rhamnolipid-modified nanocarriers loaded with magnoflorine in the preparation of treatments and / or preventive measures against drug-induced liver injury.

[0007] The drug is administered via a non-oral route, more preferably via pulmonary delivery, and even more preferably via tracheal infusion.

[0008] The drug-induced liver injury was induced by hepatotoxic drugs such as acetaminophen.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to reveal and utilize the high tropism of rhamnolipids (RLS) for inflammatory (M1 type) macrophages. Experiments have shown that RLS-modified nanocarriers (RLS-NPs) exhibit specific uptake of M1 type macrophages both in vivo and in vitro, effectively avoiding drug accumulation in non-target cells.

[0010] After being targeted and taken up by inflammatory macrophages, the loaded magnoflorine (MAG) can effectively downregulate M1 macrophage markers (such as CD86) and upregulate M2 macrophage markers (such as CD206), promoting macrophage polarization from the pro-inflammatory M1 type to the anti-inflammatory / repairing M2 type, thereby blocking the liver inflammatory storm at its source.

[0011] By using inhalation, the drug can be rapidly delivered into the systemic circulation through the alveolar microvascular system. This not only avoids the gastrointestinal degradation and first-pass effect of oral administration, but also has the advantages of being non-invasive and having good patient compliance compared to intravenous injection, providing a new approach for outpatient emergency care and routine treatment of acute liver injury. Attached Figure Description

[0012] Figure 1 Graph showing the effect of magnoflorine (MAG) on macrophage polarization and in vivo intervention in APAP-induced liver injury.

[0013] The chart on the left shows that under LPS stimulation, different concentrations of MAG intervention resulted in downregulation of CD86+ expression and upregulation of CD206+ expression in macrophages, exhibiting a concentration-dependent M1 to M2 phenotype conversion. The flow cytometry and statistical plots on the right side show that in APAP model mice, the APAP+MAG group significantly reduced the number of recruited macrophages in the liver and effectively regulated the proportion of Kupffer cells, with better results than the clinical control drug NAC.

[0014] Figure 2 The in vivo fluorescence distribution map and cell uptake statistics of the nanocarrier of the present invention in an APAP-induced mouse drug-induced liver injury model.

[0015] The figure above shows extremely high fluorescence accumulation signals in the liver of APAP-injured mice at 1h, 3h, 6h, and 24h after drug administration, while the distribution in the healthy control group (Control) is less. The figure also shows the results of in vitro cellular uptake experiments of rhamnolipid-modified nanocarriers (RLS-NP).

[0016] Figure A shows the effect of LPS stimulation on the proportion of receptors on the surface of macrophages. Figure B shows the uptake of fluorescently labeled RLS-NP(Cy5) in RAW264.7 macrophages. After 24 hours of culture, the uptake of the nanocarrier by the LPS-induced inflammatory macrophage group (M1 type) was significantly higher than that by the unstimulated control group (Control) and the IL-4-induced group (M2 type) (**** p<0.0001), which fully validates the specific tactic uptake effect of rhamnolipid modification on inflammatory macrophages. Detailed Implementation

[0017] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this invention are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or end that includes a series of steps or modules is not limited to the listed steps or modules, but may optionally include steps or modules not listed, or may optionally include other steps or modules inherent to these processes, methods, products, or ends.

[0019] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments and test examples of this invention are commercially available; and all methods used in the embodiments and test examples of this invention are conventional methods unless otherwise specified. Example 1

[0021] Intratracheal infusion of magnoflorine improves drug-induced liver injury by regulating macrophage polarization and liver inflammatory immune recruitment. 1. Experimental Methods like Figure 1 As shown in A, in this embodiment, c57 mice were randomly divided into four groups: control group, acetaminophen group, acetaminophen + tracheal infusion of magnolia alkaloid group, and acetaminophen + acetylcysteine ​​group, with three mice in each group.

[0022] After acclimatization, mice in the rhamnolipid-modified magnoflorine-loaded treatment group were administered the drug via intratracheal infusion at a dose of 5 mg / kg, given once every 24 hours for a total of three administrations. One hour after the last administration, 300 mg / kg of acetaminophen was injected intraperitoneally into the mice. Twelve hours after modeling, the mice were anesthetized and their livers were perfused; the livers were then harvested for flow cytometry analysis.

[0023] like Figure 1 As shown in C, this embodiment uses macrophages, which are divided into five groups: control group, LPS group, LPS+0.1 μm MAG group, LPS+1 μm MAG group and LPS+0.10 μm MAG group. After 24 hours of LPS modeling, different concentrations of drugs are administered. After 24 hours of drug administration, cells are collected for flow cytometry to detect the expression of M1 and M2 types.

[0024] 2. Experimental Results like Figure 1 As shown in Figure A, the distribution of liver macrophage subsets in the control group mice was normal. Flow cytometry scatter plots showed that Kupffer cells (liver-resident macrophages, F4 / 80^high CD11b^low) and recruited macrophages (F4 / 80^low CD11b^high) were at a low basal level.

[0025] This reflects the homeostatic balance of the liver's immune microenvironment under normal physiological conditions, where resident macrophages dominate and peripherally derived inflammatory cells infiltrate very little. APAP (acetaminophen) treatment significantly disrupted macrophage subsets in mouse livers. Compared to the control group, the proportion of Kupffer cells decreased to 10.8%, suggesting a reduction in the number or phenotypic changes of resident macrophages under drug toxicity; simultaneously, the proportion of recruited macrophages increased dramatically (approximately 3.7 times that of the control group), indicating a large influx of peripheral inflammatory monocytes / macrophages into the liver. This pattern of "reduced resident macrophages and increased recruitment" is a typical immunopathological feature of APAP-induced acute liver injury. After MAG intervention, the disordered macrophage subsets in mouse livers were significantly improved. Specifically, the proportion of Kupffer cells significantly recovered to 24.6% (not only returning to normal levels but even higher than the control group), suggesting that MAG may promote the survival, proliferation, or phenotypic stability of resident macrophages; meanwhile, the proportion of recruited macrophages decreased to 11.4%. This bidirectional regulatory effect indicates that MAG can effectively restore the homeostasis of hepatic macrophage subsets, inhibit the pathological infiltration of peripheral inflammatory cells, and protect and restore the integrity of hepatic innate immune cells. Treatment with the positive control drug NAC (N-acetylcysteine, a classic glutathione prodrug antioxidant and clinical antidote) did not show significant improvement in hepatic macrophage subsets. The proportion of Kupffer cells was similar to that in the APAP model group, with no significant recovery; more notably, the proportion of recruited macrophages further increased. This result suggests that under these experimental conditions, the protective mechanism of NAC may not primarily be achieved through regulating macrophage subset balance, or its antioxidant effect may not effectively block the inflammatory signaling pathways driving the recruitment of peripheral macrophages to the liver. Furthermore, the sustained increase in macrophage recruitment after NAC treatment may also reflect a response in the later repair phase of injury, or may be related to the timing and dosage of administration.

[0026] like Figure 1 As shown in Figure B, flow cytometry analysis of liver macrophages confirmed that, for Kupffer cells (left figure), the number was normal in the control group; the number in the APAP model group was significantly lower than that in the control group (approximately 1 / 3); and the number rebounded significantly after MAG treatment. For recruited macrophages (right figure), the number was significantly higher in the APAP model group than in the control group; and the number decreased significantly after MAG treatment, approaching normal levels.

[0027] like Figure 1 As shown in Figure C, compared with the control group, the proportion of pro-inflammatory M1 macrophages in the LPS model group was significantly increased, but decreased significantly after MAG treatment. Conversely, the proportion of M2 macrophages showed the opposite pattern, significantly increasing after MAG treatment.

[0028] This in vitro result is highly consistent with the in vivo effects of "MAG reducing macrophage infiltration and improving liver inflammation" observed in the previous APAP liver injury model, providing direct cellular-level evidence for the molecular mechanism by which MAG exerts its organ-protective effect by regulating macrophage polarization. Example 2

[0029] In vivo targeted distribution of rhamnolipid-modified nanocarriers in a drug-induced liver injury model after inhalation 1. Experimental Methods like Figure 2 As shown in Figure A, in this embodiment, C57 mice were divided into four groups: a high / low dose control group and a high / low dose APAP model group, with three mice in each group. After one week of acclimatization, both groups were given rhamnolipid-modified Cy5.5 dye (100 μg / mouse and 200 μg / mouse) via intratracheal infusion. APAP modeling was then initiated in the APAP group at 1-hour intervals. In vivo imaging was performed at 1, 3, 6, and 24 hours after drug administration.

[0030] like Figure 2 As shown in Figure B, RAW264.7 cells were evenly seeded into 6 cm culture dishes at an appropriate density and divided into two groups: an untreated control group (no-LPS 24h) and an LPS-stimulated group (LPS 24h). After cell adhesion, RLS was administered at concentrations of 1 μM, 10 μM, and 50 μM, respectively. Cells were collected after 24 hours. The ratio of CD86⁺ to CD206⁺ was analyzed by flow cytometry.

[0031] like Figure 2 As shown in Figure C, RAW264.7 cells were evenly seeded into 6 cm culture dishes at an appropriate density and divided into control, LPS, and IL-4 groups. After cell adhesion, each group of cells was co-incubated with Cy5-labeled rhamnolipid nanoparticles [RLS-NP(Cy5)]. The number of Cy5-positive cells was detected by flow cytometry at 6 and 24 hours.

[0032] 2. Experimental Results like Figure 2 As shown in A, a significant fluorescent signal could be detected in the abdomen after 1 hour. The APAP group was mainly confined to the liver area, and no obvious nonspecific diffusion throughout the body was observed.

[0033] During the 3-6 hour period, the liver fluorescence signal remained stable, suggesting that the liver was the primary target organ for the marker in damaged livers.

[0034] By 24 hours, the differentiation in clearance kinetics between the two dose groups became more pronounced: the 200 μg high-dose group maintained a strong signal in the liver, indicating a longer retention half-life; while the 100 μg low-dose group showed significantly attenuated signal or even only a weak signal. The fluorescent signal remaining in the liver at 24 hours suggests that the marker may have been taken up and internalized by local liver cells (especially macrophages), thereby prolonging its retention time at the site of injury.

[0035] like Figure 2 As shown in Figure B, in the untreated control group, the CD86⁺ / CD206⁺ ratio was approximately 12.5. In the RLS-treated group, the ratios at concentrations of 1 μM, 10 μM, and 50 μM were approximately 16.5, 8.5, and 20.0, respectively. Although the ratios fluctuated slightly compared to the control group, statistical analysis showed no significant differences between the groups or compared to the control group (P>0.05). In the LPS-stimulated group, LPS treatment increased the CD86⁺ / CD206⁺ ratio to approximately 15.5, indicating a significant pro-inflammatory effect of macrophages.

[0036] When 1 μM RLS was applied, the ratio changed only slightly to about 16.5, a fold change of 0.93 compared to the LPS-only group.

[0037] After administration of 10 μM RLS, the ratio decreased significantly to approximately 6.0, which is equivalent to a reduction of 2.61 times (P<0.05).

[0038] After administration of 50 μM RLS, the ratio decreased to approximately 7.8, a reduction of 1.97-fold (P<0.05). These results indicate that both 10 μM and 50 μM RLS can significantly reverse the LPS-induced increase in the CD86⁺ / CD206⁺ ratio, with the effect being more pronounced at 10 μM.

[0039] like Figure 2 As shown in Figure C, at 6 hours, the Cy5⁺ cell count was approximately 1.2 × 10⁸ cells in the control group, approximately 5.5 × 10⁷ cells in the LPS group, and approximately 7.5 × 10⁷ cells in the IL-4 group. All groups showed low uptake of RLS-NP (Cy5) and no significant inflammatory bias.

[0040] At 24 hours, the number of Cy5⁺ cells in the control group was approximately 2.5 × 10⁸ cells, while the number in the LPS group was significantly increased to 1.9 × 10⁹ cells, and the number in the IL-4 group was approximately 6.0 × 10⁸ cells. The uptake rate of RLS-NP (Cy5) by cells was significantly increased.

[0041] like Figure 2As shown in D, acetaminophen can induce liver swelling and a noticeable granular texture on the liver surface. These liver changes were significantly improved after treatment with magnolol endotracheal infusion.

[0042] Under basal conditions, RLS does not alter the polarization state of macrophages; however, under LPS-induced inflammatory conditions, RLS exhibits concentration-dependent immunomodulatory effects. Specifically, 10 μM and 50 μM RLS significantly reduced the CD86⁺ / CD206⁺ ratio, suggesting that RLS can modulate macrophages from a pro-inflammatory phenotype to an anti-inflammatory one. In other words, RLS preferentially regulates macrophage polarization in an inflammatory environment. The uptake of RLS nanoparticles by macrophages showed time-dependent and inflammatory state-dependent effects.

[0043] In the early 6 hours, uptake was low and not biased towards inflammatory cells; however, after 24 hours, LPS-induced inflammatory macrophages showed significantly enhanced uptake of RLS-NP (Cy5), which was 7–8 times that of the control group and 3 times that of the IL-4-treated group.

[0044] This indicates that the inflammatory state can delay but significantly promote the uptake of RLS nanoparticles by macrophages, suggesting that RLS nanoparticles may have the potential for targeted delivery to inflammatory macrophages.

[0045] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. The various components mentioned in this invention are common technologies in the existing field. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. An inhalation nanocarrier modified with rhamnolipid and loaded with magnoflorine, characterized in that, It is selected from one of liposomes, solid lipid nanoparticles, polymer micelles, nanostructured lipid carriers, or albumin nanoparticles.

2. The inhalation nanocarrier modified with rhamnolipid and loaded with magnoflorine according to claim 1, characterized in that, The nanocarrier contains magnolia alkaloids. The surface of the nanocarrier is covalently coupled or physically adsorbed with a rhamnolipin-modified layer.

3. An inhaled nanocarrier modified with rhamnolipid and loaded with magnoflorine according to claim 1, characterized in that... The application of magnoflorine loaded on rhamnolipid-modified nanocarriers in the preparation of drugs for the treatment and / or prevention of drug-induced liver injury.

4. An inhalation nanocarrier modified with rhamnolipid and loaded with magnoflorine according to claim 3, characterized in that, The drug is administered via non-oral route.

5. An inhalation nanocarrier modified with rhamnolipid and loaded with magnoflorine according to claim 4, characterized in that, The drug is administered via pulmonary delivery.

6. An inhalation nanocarrier modified with rhamnolipid and loaded with magnoflorine according to claim 5, characterized in that, The lung delivery method is tracheal infusion.

7. An inhalation nanocarrier modified with rhamnolipid and loaded with magnoflorine according to any one of claims 1-6, characterized in that, The drug-induced liver injury was induced by hepatotoxic drugs such as acetaminophen.

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