Berberine derivative photosensitive nanoparticle, and preparation method and application thereof

Abstract

The present application relates to a kind of berberine derivative photosensitive nanoparticles and its preparation method and application, belong to the field of biological medicine.The technical problem that bacteria is resistant to photodynamic therapy to cationic berberine derivative, the present application provides a kind of photosensitive nanoparticles formed by the co-assembly of berberine derivative and rhamnolipid, and the particle size is 80 nm to 320 nm.The nanoparticle overcomes the PDT resistance of methicillin-resistant staphylococcus aureus to berberine derivative B-12;Significantly improve PDT antibacterial activity, under the illumination of 3.125 μg / mL (B-12 component) can make MRSA reduce more than 3 log CFU, photosensitizer demand reduces 96.875%;And no antibacterial activity in dark conditions, with light control safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a photosensitive nanoparticle of berberine derivative, its preparation method and application. Background Technology

[0002] With the increasing threat of antibiotic resistance globally, the need for alternative therapies is becoming more urgent. Against this backdrop, photodynamic therapy (PDT) has emerged as one of the most promising options. Unlike traditional antibiotics, PDT uses light-activated photosensitizers (PS) to generate reactive oxygen species (ROS). These ROS rapidly damage multiple cellular structures simultaneously, thereby killing bacteria. However, recent studies have shown that bacteria can also develop resistance to PDT. For example, a clinical study found significant differences in the sensitivity of different clinical isolates of Staphylococcus aureus to PDT. This difference within the same bacterial species indicates that PDT resistance does indeed exist under specific conditions. Other studies have shown that some photosensitizers can be expelled from cells by bacterial efflux pumps, leading to resistance. Therefore, we should pay attention to bacterial PDT resistance and develop alternative solutions.

[0003] Several mechanisms have been proposed to explain how bacteria acquire photodynamic tolerance, primarily including the following three: First, the upregulation of innate antioxidant defense systems such as catalase and superoxide dismutase may neutralize the generated ROS; second, physical barriers such as biofilms can prevent photosensitizers from effectively binding to or entering cells; and third, multidrug efflux pumps can actively expel photosensitizers from cells. While these factors contribute to bacterial survival, they must be considered in conjunction with the physical limitations of the photodynamic process itself. Because ROS have an extremely short lifespan and a diffusion distance of less than 2 μm, oxidative damage occurs only near the photosensitizer. Therefore, the proximity of the photosensitizer to bacterial cells is crucial to the therapeutic effect. For example, before entering cells, rose red (RB) binds to specific phospholipid regions in the membranes of Gram-negative and Gram-positive bacteria. Once inside the cell, Gram-positive bacteria (such as Staphylococcus aureus) take up RB at higher levels, leading to bacterial inactivation. Similarly, toluidine blue (TBO) can effectively cross the cell membrane and enter the cell to exert its effects, participating in ROS generation or cell membrane disruption. These findings suggest that ensuring the effective binding and entry of photosensitizers into bacteria is key to overcoming photodynamic tolerance.

[0004] Cationic photosensitizers are generally considered to have high antibacterial activity because their positive charge can bind to the negatively charged bacterial surface through electrostatic attraction. However, bacteria can also develop resistance to cationic drugs by altering their membrane charge, and there are currently no strategies to target bacteria resistant to cationic photosensitizers. Summary of the Invention

[0005] In view of this, berberine derivative B-12 is a cationic photosensitizer with significant photosensitive photosensitizer (PDT) activity; however, its PDT effect on some Staphylococcus aureus strains, such as methicillin-resistant Staphylococcus aureus (MRSA), is extremely weak. The purpose of this invention is to provide photosensitizing nanoparticles of berberine derivative B-12 that enhance PDT activity and successfully overcome the cationic photosensitizer resistance of MRSA.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides berberine derivative photosensitive nanoparticles: the berberine derivative photosensitive nanoparticles are formed by co-assembling berberine derivatives and rhamnolipids, wherein the structural formula of the berberine derivative is shown below: ; Preferably, the particle size of the photosensitive nanoparticles is between 80 nm and 320 nm; Preferably, the zeta potential of the photosensitive nanoparticles is between -20 mV and -40 mV; Preferably, the weight ratio of berberine derivative to rhamnolipin is 2:1 to 1:5; Preferably, the weight ratio of the berberine derivative to rhamnolipin is 1:4; Furthermore, the application of the berberine derivative photosensitive nanoparticles in the preparation of antibacterial agents; Preferably, the bacteria are PDT-resistant bacteria; The present invention also provides a photosensitive antibacterial preparation comprising the aforementioned berberine derivative photosensitive nanoparticles.

[0007] The beneficial effects of this invention are as follows: 1. Berberine derivative photosensitive nanoparticles overcome bacterial tolerance to PDT of cationic berberine derivatives. The berberine derivative photosensitive nanoparticles (BR NPs) prepared by this invention can effectively kill methicillin-resistant Staphylococcus aureus (MRSA) that is resistant to photodynamic therapy to free berberine derivative B-12, successfully overcoming the problem of cationic photosensitizer resistance in MRSA.

[0008] 2. Significantly enhances photodynamic antibacterial activity At the same concentration, the photodynamic antibacterial activity mediated by BR NPs was significantly better than that of free B-12 and the control photosensitizer Ce6. It reduced colony forming units (CFU) by more than 3 log against Staphylococcus aureus and MRSA, reaching the sensitivity standard.

[0009] 3. Reduce the effective dosage of photosensitizer For MRSA, the effective concentration of B-12 in berberine derivative photosensitive nanoparticles is only 3.125 μg / mL, while free B-12 requires more than 100 μg / mL to produce a weak bactericidal effect. Berberine derivative photosensitive nanoparticles reduce the required amount of photosensitizer B-12 by 96.875%.

[0010] 4. Light-controlled sterilization properties Berberine derivative photosensitive nanoparticles (BR NPs) show no significant antibacterial activity in the dark, but exhibit highly efficient bactericidal activity in a concentration-dependent manner under light, demonstrating photocontrolled safety.

[0011] 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

[0012] 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: Figure 1 Characterization images of berberine derivative photosensitive nanoparticles: a, electron microscopy image of nanoparticles; b, DLS size of nanoparticles; c, Zeta potential; d, UV-Vis absorption spectrum; e, fluorescence spectrum; f, infrared spectrum; g, assembly mechanism.

[0013] Figure 2 Figure 1 shows the B-12-mediated PDT tolerance experiment of MRSA: a, concentration-dependent effect of B-12 on the bactericidal activity of S. aureus PDT; b, concentration-dependent effect of B-12 on the bactericidal activity of MRSA PDT.

[0014] Figure 3 PDT activity diagrams of berberine derivative nanoparticles: comparison of PDT activity of ab, BR NP and reference photosensitizer Ce6 in S. aureus and MRSA; concentration-dependent antibacterial activity of cf, BR NP under dark and light conditions. Detailed Implementation

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Example 1: Preparation of berberine derivative B-12 photosensitive nanoparticles First, 5 mg of berberine derivative (B-12) was dissolved in 1 mL of DMSO, and then mixed with 20 mg of rhamnolipid (RHL) to form a 1:4 mass ratio solution. Next, 1 mL of the mixed DMSO solution was slowly added dropwise to 10 mL of ultrapure water, and the reaction was carried out with continuous stirring at 800 r / min and room temperature for 30 min. The final solution was incubated at 4 °C for 24 h, and then centrifuged at 8000 r / min for 20 min. The precipitate was berberine derivative photosensitive nanoparticles (BR NPs), which were spherical with a particle size of approximately 170 nm. Figure 1 a, 1b), Zeta potential approximately -28.9 mV ( Figure 1 c).

[0019] Example 2: MRSA tolerance to PDT mediated by berberine derivative photosensitizer (B-12) Staphylococcus aureus ATCC 25923 ( S. aureus ATCC 43300 (MRSA) was cultured in LB broth and LB agar. Pre-cultured bacterial cells were transferred to 20 mL of LB broth in a 150 mL flask and then incubated at 37°C and 180 rpm until the exponential phase. Approximately 10 8 CFU / mL bacteria were dispersed in 200 μL of PBS, and B-12 at a concentration of 50 μg / mL was added. After incubation for 30 min, the bacteria were irradiated with a custom-made LED lamp (405 nm, 1 mW / cm²) for 30 min. Bacterial viability was quantified using the plate count method. The dilution factor was 10. 1 , 10 2 , 10 3 , 10 4 , 10 5 Dilute the bacteria. Then, drop 3 μL of the bacterial solution onto an LB agar plate and incubate at 37°C for 18 h. CFU counts were logarithmized, and a decrease in log10 value >3 was considered sensitive. Figure 2 As shown in a and 2b, S. aureus It is sensitive to B-12-mediated photodynamic antibacterial activity alone, with a log10 value decreasing by >3 at a concentration of 25 μg / mL. In contrast, MRSA is resistant to B-12-mediated photodynamic antibacterial activity alone, with a log10 value decreasing by <3 at an increased concentration of 100 μg / mL, indicating an approximately 4-fold increase in resistantness.

[0020] Example 3: Photosensitive berberine derivative nanoparticles kill PDT-resistant bacteria MRSA Staphylococcus aureus ATCC 25923 ( S. aureus ATCC 43300 (MRSA) was cultured in LB broth and LB agar. Pre-cultured bacterial cells were transferred to 20 mL of LB broth in a 150 mL flask and then incubated at 37°C and 180 rpm until the exponential phase. Approximately 10 8 CFU / mL bacteria were dispersed in 200 μL PBS, and 50 μg / mL (B-12 component concentration) of nanoparticles were added. After incubation for 30 min, the bacteria were irradiated with a custom-designed LED lamp (405 nm, 1 mW / cm²) for 30 min. Bacterial viability was quantified using plate counting. The bacterial viability was determined using a dilution factor of 10. 1 , 10 2 , 10 3 , 10 4 , 10 5 Dilute the bacteria. Then, drop 3 μL of the bacterial solution onto an LB agar plate and incubate at 37°C for 18 h. CFU counts are logarithmized, and a decrease in log10 value >3 is considered sensitive. Figure 3As shown in a and 3b, S. aureus Both MRSA and BR NPs are sensitive to the photodynamic antibacterial activity mediated by BR NPs, with a CFU reduction of >3 log10, and their bactericidal effect is significantly better than that of B-12 and Ce6 at the same concentration.

[0021] Example 4: Concentration-dependent bactericidal effect of photosensitive berberine derivative nanoparticles The concentration dependence of BR NP under darkness and light conditions was tested. Figure 3 As shown in cf, BR NPs did not significantly reduce the CFU of Staphylococcus aureus in the dark, but at concentrations above 1.56 μg / mL and under light, they resulted in a reduction of >5 log CFU. In MRSA, BR NPs showed no antibacterial activity in the dark, but at 3.125 μg / mL and under light, they resulted in a reduction of >3 log CFU, with bactericidal activity increasing in a concentration-dependent manner. For MRSA, the effective concentration of B-12 in the nanoparticles was only 3.125 μg / mL, only 3.125% of that required when B-12 was used alone, reducing the required amount of photosensitizer B-12 by 96.875%.

[0022] 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. The application of berberine derivative photosensitive nanoparticles in the preparation of anti-PDT resistant bacterial agents, characterized in that: The berberine derivative photosensitive nanoparticles are formed by co-assembling berberine derivatives and rhamnolipids, wherein the structural formula of the berberine derivative is shown below: The resistant bacteria are methicillin-resistant Staphylococcus aureus.

2. The application according to claim 1, characterized in that: The particle size of the photosensitive nanoparticles is 80 nm to 320 nm.

3. The application according to claim 1, characterized in that: The zeta potential of the photosensitive nanoparticles is between -20 mV and -40 mV.

4. The application according to claim 1, characterized in that: The weight ratio of berberine derivatives to rhamnolipin is 2:1 to 1:

5.

5. The application according to claim 4, characterized in that: The weight ratio of the berberine derivative to rhamnolipin is 1:

4.

6. A photosensitive antibacterial agent, characterized in that: The photosensitive antibacterial preparation comprises berberine derivative photosensitive nanoparticles formed by co-assembling berberine derivatives and rhamnolipids in any one of the applications described in claims 1-5.

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