ROS-responsive cationic liposome-loaded tryptanthrin as well as preparation method and application thereof

Abstract

The invention provides ROS (reactive oxygen species)-responsive cationic liposome-loaded tryptanthrin as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The ROS response cationic liposome loaded tryptanthrin disclosed by the invention comprises an ROS response cationic liposome and tryptanthrin which is entrapped in the ROS response cationic liposome; the ROS response cationic liposome is prepared from the following raw materials: egg yolk lecithin, cholesterol, DSPE-TK-PEG (distearoyl phosphatidylcholine) and DOTAP (dioctyl phthalate). In view of significant increase of intestinal local active oxygen under the ECLS condition, the ROS-responsive cationic liposome is adopted to wrap tryptanthrin, so that tryptanthrin is selectively released in an intestinal injury area, whole body exposure is reduced, and meanwhile, the protection effect on the intestinal tract is enhanced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a ROS-responsive cationic liposome ketone, its preparation method, and its application. Background Technology

[0002] Extracorporeal life support (ECLS) is an important treatment method widely used in the clinical treatment of critical illnesses such as cardiopulmonary failure. However, while maintaining circulation and oxygenation, ECLS is often accompanied by serious complications, among which intestinal injury is particularly prominent. Existing studies have shown that ECLS can lead to intestinal hemodynamic disturbances, barrier function disruption, and inflammatory response activation, ultimately resulting in damage to the intestinal mucosal structure and increased permeability. Previous studies have found that ADAMTS4 protein is significantly upregulated during ECLS, promoting VCAN degradation and leading to extracellular matrix destruction and inflammatory cell infiltration, which is an important molecular mechanism of intestinal injury. Further high-throughput virtual screening and experimental validation have determined that the natural product tryptophan can bind to and inhibit ADAMTS4, thereby alleviating intestinal injury.

[0003] However, free tryptophan has problems such as poor solubility, low bioavailability, and insufficient targeting. Although routine intravenous administration of tryptophan can reduce intestinal damage to some extent, it requires a large systemic dose and is prone to adverse reactions such as sedation and cardiovascular complications. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a ROS-responsive cationic liposome-loaded ketone, its preparation method, and its application. The ROS-responsive cationic liposome-loaded ketone provided by this invention can achieve targeted release of ketone in the ECLS-related high ROS inflammatory microenvironment, significantly enhancing therapeutic efficacy and reducing toxic side effects.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a ROS-responsive cationic liposome carrying tryptophan, comprising a ROS-responsive cationic liposome and tryptophan loaded in the ROS-responsive cationic liposome; The raw materials for preparing the ROS-responsive cationic liposomes include egg yolk lecithin, cholesterol, DSPE-TK-PEG, and DOTAP.

[0006] Preferably, the mass ratio of egg yolk lecithin, cholesterol, DSPE-TK-PEG and DOTAP is (8~12):(4~6):(2~4):(1.5~3).

[0007] Preferably, the ROS-responsive cationic liposomes also include a fluorescent dye.

[0008] Preferably, in the ROS-responsive cationic liposome loaded with tryptophan, the drug loading of tryptophan is 3-10%.

[0009] Preferably, the particle size of the ROS-responsive cationic liposome ketone is 100~150 nm.

[0010] This invention provides a method for preparing the above-mentioned ROS-responsive cationic liposome ketone, comprising the following steps: Egg yolk lecithin, cholesterol, DSPE-TK-PEG, DOTAP, tryptophan, and organic solvent are mixed and evaporated to form a film, thus obtaining the membrane material. The membrane material was mixed with water and subjected to ultrasound, liposome extrusion, dialysis, and freeze-drying in sequence to obtain ROS-responsive cationic liposomes with tryptophan.

[0011] Preferably, the evaporation film formation temperature is 40~50℃, the pressure is 150~300 mbar, and the time is 25~40 min; The power of the ultrasound is 80~120 W; the duration is 8~15 min.

[0012] Preferably, the liposome extruder is a polycarbonate membrane with a pore size of 100 nm.

[0013] This invention provides the application of the above-mentioned ROS-responsive cationic liposome ketone in the preparation of drugs for treating intestinal damage.

[0014] Preferably, the intestinal injury is in vitro life support-related intestinal injury.

[0015] This invention provides a ROS-responsive cationic liposome carrying tryptophan, comprising a ROS-responsive cationic liposome and tryptophan encapsulated within the ROS-responsive cationic liposome; the raw materials for preparing the ROS-responsive cationic liposome include egg yolk lecithin, cholesterol, DSPE-TK-PEG, and DOTAP. Given the significant increase in local reactive oxygen species (ROS) in the intestine under ECLS conditions, this invention uses ROS-responsive cationic liposomes to encapsulate tryptophan, enabling its selective release in the intestinal injury area, thereby enhancing the protective effect on the intestine while reducing systemic exposure. Specifically, this invention uses DSPE-TK-PEG as the liposome component, which possesses ROS-responsive properties, and DOTAP, which has cationic properties, to provide the liposome with a persistent and stable positive charge. This invention uses cationic liposomes as a drug carrier, which can promote cellular uptake, increase the total amount of drug at the target site, and simultaneously regulate the release site, promoting endosome escape. Furthermore, this invention uses egg yolk lecithin, cholesterol, DSPE-TK-PEG, and DOTAP as liposome raw materials, which can improve the loading stability of the drug.

[0016] The results of the examples show that, under ROS-free conditions, the release rate of tryptophan from the cationic liposomes of the present invention was less than 20% after 24 h, while under the condition of adding H2O2 (100 μM), the release rate reached 82% after 48 h, demonstrating good ROS-triggered release characteristics. In the human small intestinal organoid OGD / R model, the cationic liposome delivery group of the present invention significantly reduced ADAMTS4 expression and VCAN degradation more than free tryptophan, improved barrier integrity by about 35%, and increased the reduction of inflammatory factors by 15-20%. In the ECLS rat model, the intestinal tissue damage score of the cationic liposome delivery group of the present invention was reduced by 60% compared with the model group, and plasma permeability decreased by 55%, while no significant abnormalities were observed in liver and kidney function indicators, indicating that the system has good efficacy and safety in vivo.

[0017] This invention provides a method for preparing ROS-responsive cationic liposomes loaded with tryptophan. This method is simple to operate, the encapsulation rate of tryptophan is higher than 70%, and it is easy to achieve industrial-scale mass production. Attached Figure Description

[0018] Figure 1 This is a transmission electron microscope image of the ROS-responsive cationic liposome ketone obtained in Example 1; Figure 2 The HPLC standard chromatogram of tryptophan is shown below. Figure 3 The HPLC spectrum of ROS-responsive cationic liposome ketone; Figure 4 The UV absorption spectrum of the ROS-responsive cationic liposome ketone obtained in Example 1; Figure 5 The fluorescence spectrum of the ROS-responsive cationic liposome ketone obtained in Example 1; Figure 6 The physical stability of the ROS-responsive cationic liposome ketone obtained in Example 1 under different environments; Figure 7 The ROS-responsive cationic liposome ketone obtained in Example 1 is shown to be released under ROS conditions. Figure 8 The levels of I-FABP and DAO in the serum of each group; Figure 9 The levels of typical inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 in the serum of each group were measured. Figure 10 HE staining results for each group; Figure 11 HE staining scores were assigned to each group. Detailed Implementation

[0019] The present invention provides a ROS-responsive cationic liposome carrying tryptophan, comprising a ROS-responsive cationic liposome and tryptophan loaded in the ROS-responsive cationic liposome; The raw materials for preparing the ROS-responsive cationic liposomes include egg yolk lecithin, cholesterol, DSPE-TK-PEG, and DOTAP. In this invention, TK represents ketithiolide, PEG is polyethylene glycol, and the molecular weight of PEG is preferably 2000. In this invention, the Chinese name for DOTAP is (2,3-dioleoyl-propyl)-trimethylamine sulfate.

[0020] In this invention, the structural formula of the tryptophan ketone is shown in Formula I: Formula I.

[0021] In this invention, the preferred mass ratio of egg yolk lecithin, cholesterol, DSPE-TK-PEG, and DOTAP is (8~12):(4~6):(2~4):(1.5~3), more preferably 10:5:3:2. By controlling the mass ratio of egg yolk lecithin, cholesterol, DSPE-TK-PEG, and DOTAP, this invention can ensure the particle size and uniformity of ROS-responsive cationic liposomes with tryptophan, thereby ensuring membrane stability and therapeutic efficacy.

[0022] In this invention, the ROS-responsive cationic liposomes preferably also include a fluorescent dye, and the fluorescent dye preferably includes Cy5.5.

[0023] In this invention, the drug loading of tryptophan in the ROS-responsive cationic liposome is preferably 3-10%, more preferably 5.2%, and the drug loading of the fluorescent dye is preferably 1-4%, more preferably 2.2%.

[0024] In this invention, the particle size of the ROS-responsive cationic liposome ketone is preferably 100-150 nm, more preferably 120-140 nm.

[0025] This invention provides a method for preparing the above-mentioned ROS-responsive cationic liposome ketone, comprising the following steps: Egg yolk lecithin, cholesterol, DSPE-TK-PEG, DOTAP, tryptophan, and organic solvent are mixed and evaporated to form a film, thus obtaining the membrane material. The membrane material was mixed with water and subjected to ultrasound, liposome extrusion, dialysis, and freeze-drying in sequence to obtain ROS-responsive cationic liposomes with tryptophan.

[0026] This invention involves mixing egg yolk lecithin, cholesterol, DSPE-TK-PEG, DOTAP, tryptophan, and an organic solvent, followed by evaporation to form a membrane material. In this invention, the organic solvent preferably includes chloroform and / or methanol. This invention does not have specific requirements regarding the amount of the organic solvent, as long as it is sufficient to dissolve the aforementioned components. When the ROS-responsive cationic liposome carrying tryptophan contains a fluorescent dye, this invention preferably mixes the fluorescent dye along with the liposome.

[0027] In this invention, the evaporation film formation temperature is preferably 40~50℃, more preferably 45℃, the pressure is preferably 150~300 mbar, more preferably 200 mbar, and the time is preferably 25~40 min, more preferably 30 min.

[0028] This invention involves mixing the membrane material with water, followed by sequential ultrasonication, extrusion using a liposome extruder, dialysis, and lyophilization to obtain ROS-responsive cationic liposomes with tryptophan. In this invention, the ultrasonic power is preferably 80-120 W, more preferably 100 W; the time is preferably 8-15 min, more preferably 10 min. In this invention, the liposome extruder is preferably a polycarbonate membrane with a pore size of 100 nm. This invention preferably uses a nanodialysis device for dialysis, and the dialysis membrane of the nanodialysis device is preferably a polycarbonate membrane with a pore size of 10 nm. This invention does not have special requirements for the lyophilization method; any lyophilization method well known to those skilled in the art can be used.

[0029] This invention provides the application of the aforementioned ROS-responsive cationic liposome-based ketone in the preparation of drugs for treating intestinal injury. In this invention, the intestinal injury is preferably in vitro life support-related intestinal injury.

[0030] The following detailed description, in conjunction with embodiments, illustrates the ROS-responsive cationic liposome ketone, its preparation method, and its applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] In the following examples, egg yolk lecithin, cholesterol, DSPE-TK-PEG2K, DOTAP, and cy5.5 were purchased from Xi'an Ruixi Biotechnology Co., Ltd., and tryptophan was purchased from Beijing Innocare Technology Co., Ltd.

[0032] Example 1 The preparation of ROS-responsive cationic liposomes with ketamine follows these steps: 15.0 mg egg yolk lecithin, 7.5 mg cholesterol, 4.5 mg DSPE-TK-PEG2K, 3.0 mg DOAPT, 0.68 mg cy5.5, and 1.78 mg tryptophan were dissolved together in 5 mL of chloroform. After mixing, the mixture was evaporated under reduced pressure at 45 °C and 200 mbar in a sample vial for 30 min. Deionized water was added for hydration. The mixture was then sonicated at 100 W for 10 min and extruded using a liposome extruder (polycarbonate membrane, 100 nm pore size). The membrane was dialyzed using a nanodialysis device (polycarbonate membrane, 10 nm pore size). Deionized water was added to bring the volume to 5 mL. After lyophilization, ROS-responsive cationic liposomes carrying tryptophan were obtained and designated as the TRYP@ROS-lipo group.

[0033] Comparative Example 1 The difference from Example 1 is that tryptophan was not added, resulting in blank ROS-responsive cationic liposomes.

[0034] Structural characterization (1) The morphology of the liposomes was observed by transmission electron microscopy (TEM). The TEM image of the ROS-responsive cationic liposomes ketone obtained in Example 1 is shown below. Figure 1 As shown in the figure. The results show that the prepared liposomes have a uniform spherical structure, concentrated particle size distribution, smooth surface, and intact structure, with an average diameter of about 100 nm, which is consistent with the morphological characteristics of nanoscale drug delivery systems, indicating that the prepared liposomes have stable structure and good dispersibility.

[0035] (2) The particle size and potential test results of the ROS-responsive cationic liposomes ketone obtained in Example 1 are shown in Table 1.

[0036] Table 1. Particle size and potential of ROS-responsive cationic liposomes containing ketamine

[0037] (3) The HPLC standard chromatogram of tryptophan is shown below. Figure 2 As shown, the HPLC spectrum of ROS-responsive cationic liposome ketone is as follows. Figure 3 As shown. HPLC conditions were: C18 column ( The column was used for HPLC analysis of the sample using a mobile phase of solvent A (methanol) and solvent B (acetonitrile). Gradient elution was performed as follows: 7 min, 20% solvent A and 80% solvent B. The flow rate was 1 mL / min, the column temperature was 25 °C, and the measurement wavelength was 252 nm.

[0038] Depend on Figure 2 and Figure 3 It can be seen that tryptophanone was successfully loaded onto the lipid carrier.

[0039] (4) The optical properties of liposomes and drug encapsulation were verified by detecting ultraviolet absorption spectroscopy and fluorescence spectroscopy. Ultraviolet absorption spectroscopy is shown below. Figure 4 As shown, the fluorescence spectrum is as follows Figure 5 As shown.

[0040] UV absorption spectroscopy showed that the ROS-responsive liposomes encapsulating tryptophan and Cy5.5 in Example 1 exhibited significant absorption peaks in the 200–800 nm range. Compared to the liposomes encapsulating only Cy5.5 in Comparative Example 1, the overall absorption intensity was enhanced, indicating that tryptophan was successfully encapsulated into the liposome and interacted with the lipid membrane structure. Fluorescence spectroscopy results showed that both types of liposomes exhibited emission peaks around 700 nm. The fluorescence intensity of the liposomes encapsulating tryptophan was slightly lower, indicating that the drug had a certain quenching effect on the probe signal after entering the liposome, further confirming that the drug was successfully loaded.

[0041] The drug loading was calculated according to formula (1), and the loading rate was calculated according to formula (2), wherein the drug mass was calculated based on the ultraviolet absorbance or fluorescence intensity. Testing showed that in the ROS-responsive cationic liposome loaded with tryptophan obtained in Example 1, the drug loading of tryptophan was 5.2%, and the drug loading of Cy5.5 was 2.0%; the encapsulation efficiency of tryptophan was 94.5%, and the encapsulation efficiency of Cy5.5 was 95.2%.

[0042] Drug loading (%) = drug mass / carrier mass × 100% Equation (1); Loading rate (%) = drug quality / total drug quality put in × 100% Equation (2).

[0043] (5) The physical stability of the ROS-responsive cationic liposome ketone obtained in Example 1 under different environments was investigated by acid-base stability test. The results are as follows: Figure 6 As shown in the figure. The results showed that the absorbance of the liposomes remained relatively stable over time under pH=2 and pH=8 conditions, with no significant changes, indicating that the liposomes have good structural stability in both acidic and alkaline environments and are not prone to aggregation or rupture.

[0044] (6) The ROS-responsive cationic liposomes carrying tryptophan under ROS conditions were evaluated by in vitro release experiments. Specifically, TRYP@ROS-lipo was placed in two release media (PBS containing 1 mM H2O2 to simulate a high ROS environment, and ordinary PBS as a control) and incubated with shaking at 37°C. Samples were taken at preset time points, and the concentration of tryptophan in the media was determined by high-performance liquid chromatography (HPLC), and the cumulative release rate was calculated. The responsive drug release characteristics of the liposomes under ROS conditions were verified by comparing the two release curves. The results are as follows: Figure 7 As shown.

[0045] The results showed that under conditions containing oxidants, the cumulative release of liposomes gradually increased over time, reaching a release rate of over 82% at approximately 48 hours, exhibiting significant time dependence and ROS response characteristics. In contrast, under normal conditions, drug release was slow, with a 24-hour tryptophan release rate of less than 20%. These results indicate that the liposomes can be triggered to release drugs in environments with elevated oxidative stress levels, demonstrating good ROS responsiveness and controlled-release characteristics.

[0046] In summary, the ROS-responsive cationic liposomes prepared in this invention have uniform particle size, good dispersibility, and stability under different pH conditions. They can achieve specific drug release in ROS environments and have good physicochemical properties and biocompatibility, providing a structural and functional basis for their targeted release of tryptophan and reduction of intestinal damage during in vitro life support.

[0047] Performance testing (1) In the extracorporeal life support (ECLS)-related intestinal injury experiment, healthy adult SD rats were randomly divided into three groups: sham operation group (Sham group), ECLS control group (Vehicle group), and ECLS+ROS-responsive cationic liposome azotocin treatment group (TRYP@ROS-lipo group). The Sham group was only anesthetized and vascularized, without being connected to extracorporeal circulation; the ECLS control group was under general anesthesia with arterial and venous catheters, connected to a miniature ECLS device, and maintained extracorporeal life support for a preset time; the treatment group was based on the same modeling as the ECLS control group, and was given ROS-responsive cationic liposome azotocin by intravenous injection. The administration time was before or immediately after ECLS was started, and the dosage was calculated based on body weight, with a dosage of 19.2 mg / kg. The animals were euthanized immediately after ECLS was completed by exsanguination, and serum and small intestinal tissue were collected for subsequent testing.

[0048] Serum I-FABP and DAO were used as representative markers of intestinal mucosal injury and intestinal barrier disruption. Serum I-FABP and DAO levels in each group were measured using enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 8 As shown in the figure. The results showed that the I-FABP and DAO levels in the ECLS control group were significantly higher than those in the Sham group, suggesting that ECLS can significantly damage the small intestinal mucosa and disrupt the intestinal barrier; while the I-FABP and DAO levels in the ECLS+ROS-responsive cationic liposome azotocin group were significantly lower than those in the ECLS control group, and were partially close to or approached the levels in the Sham group, indicating that the present invention can effectively alleviate ECLS-induced small intestinal damage and reduce the increase in intestinal damage markers.

[0049] (2) To evaluate its anti-inflammatory effect, the serum levels of typical inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 were measured. The results are as follows: Figure 9As shown in the results, the levels of the aforementioned inflammatory factors in the ECLS control group were significantly higher than those in the Sham group, indicating a significant systemic inflammatory response during ECLS. In contrast, in the ECLS+ROS-responsive liposome ketone group, the concentrations of inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-8 were significantly lower than those in the ECLS control group, suggesting that this preparation can inhibit the excessive release of inflammatory factors during ECLS, thereby reducing inflammation-mediated secondary intestinal damage.

[0050] (3) To visually evaluate the histological changes in the intestines, HE staining was performed on the ileum or jejunum tissues of each group. The HE staining results are as follows: Figure 10 As shown, the HE staining score is as follows: Figure 11 As shown in the figure, the Sham group exhibited regular intestinal villi morphology, neat arrangement, and moderate height, with intact mucosal epithelium and no obvious edema or inflammatory cell infiltration in the lamina propria and submucosa. In the ECLS control group, significant edema, shortening, and even partial breakage of the intestinal villi were observed, along with epithelial cell shedding, necrotic exfoliated material on the luminal surface, extensive inflammatory cell infiltration in the lamina propria and submucosa, and significant interstitial edema, resulting in the highest pathological damage score. In contrast, the ECLS+ROS-responsive liposome-based azotamine group showed significantly improved intestinal villi structure, with better villi height and integrity than the ECLS control group. Epithelial shedding and edema were reduced, inflammatory cell infiltration in the lamina propria was significantly decreased, and the pathological score was significantly lower than the ECLS control group, more closely resembling that of the Sham group.

[0051] In summary, in the ECLS animal model, ROS-responsive cationic liposome ketone significantly reduced serum levels of intestinal injury markers such as I-FABP and DAO, downregulated the expression of multiple inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-8, and alleviated histological damage such as villous edema, epithelial shedding, and inflammatory cell infiltration in the small intestine under HE pathology. This indicates that the preparation has a clear protective effect against ECLS-related intestinal injury and can be used as a targeted and controlled-release drug delivery system for intestinal protection during ECLS.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A ROS-responsive cationic liposome-based ketone, characterized in that, Including ROS-responsive cationic liposomes, and tryptophan encapsulated in the ROS-responsive cationic liposomes; The raw materials for preparing the ROS-responsive cationic liposomes include egg yolk lecithin, cholesterol, DSPE-TK-PEG, and DOTAP.

2. The ROS-responsive cationic liposome ketone according to claim 1, characterized in that, The mass ratio of egg yolk lecithin, cholesterol, DSPE-TK-PEG and DOTAP is (8~12):(4~6):(2~4):(1.5~3).

3. The ROS-responsive cationic liposome ketone according to claim 1 or 2, characterized in that, The ROS-responsive cationic liposomes also include fluorescent dyes.

4. The ROS-responsive cationic liposome ketone according to claim 1 or 2, characterized in that, In the ROS-responsive cationic liposome loaded with tryptophan, the drug loading of tryptophan is 3-10%.

5. The ROS-responsive cationic liposome ketone according to claim 1 or 2, characterized in that, The ROS-responsive cationic liposomes containing ketamine have a particle size of 100-150 nm.

6. The method for preparing ROS-responsive cationic liposome ketone according to any one of claims 1 to 5, characterized in that, Includes the following steps: Egg yolk lecithin, cholesterol, DSPE-TK-PEG, DOTAP, tryptophan, and organic solvent are mixed and evaporated to form a film, thus obtaining the membrane material. The membrane material was mixed with water and subjected to ultrasound, liposome extrusion, dialysis, and freeze-drying in sequence to obtain ROS-responsive cationic liposomes with tryptophan.

7. The preparation method according to claim 6, characterized in that, The evaporation film formation temperature is 40~50℃, the pressure is 150~300 mbar, and the time is 25~40 min; The power of the ultrasound is 80~120 W; the duration is 8~15 min.

8. The preparation method according to claim 6, characterized in that, The liposome extruder is a polycarbonate membrane with a pore size of 100 nm.

9. The use of the ROS-responsive cationic liposome ketone according to any one of claims 1 to 5 or the ROS-responsive cationic liposome ketone according to any one of claims 6 to 8 prepared by the preparation method of any one of claims 1 to 5 in the preparation of drugs for treating intestinal injury.

10. The application according to claim 9, characterized in that, The intestinal injury mentioned refers to intestinal injury related to in vitro life support.

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