Chitosan oligosaccharide-based piceatannol nano delivery particle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and provides a piceatannol nano delivery particle based on chitosan oligosaccharide as well as a preparation method and application of the piceatannol nano delivery particle. The piceatannol nano delivery particle disclosed by the invention is a chitosan oligosaccharide nano drug-loaded particle modified by hyaluronic acid and D-tyrosine; the chitosan oligosaccharide nano drug-loaded particles comprise a chitosan oligosaccharide nano particle carrier and piceatannol wrapped in the chitosan oligosaccharide nano particle carrier. The hyaluronic acid and the D-tyrosine are adopted to carry out functional modification on the chitosan oligosaccharide nano drug-loaded particles, the hyaluronic acid realizes targeted delivery of MRSA infected parts, and the D-tyrosine has a biological membrane dispersing capacity, destroys a biological membrane structure, promotes a drug to deeply permeate into a biological membrane and improves an MRSA infection resisting effect; the antibacterial and anti-biofilm activity of piceatannol to MRSA (Methicillin Resistant Staphylococcus Aureus) is enhanced, and stronger antibacterial ability is shown; the lung bacterial load can be obviously reduced, the inflammatory lung injury can be relieved, and the treatment effect of resisting MRSA infection can be enhanced.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a chitosan oligosaccharide-based paclitaxel nanoparticle, its preparation method, and its application. Background Technology

[0002] Staphylococcus aureus is a common zoonotic opportunistic pathogen that causes a variety of diseases, including skin infections, abscesses, impetigo, necrotizing pneumonia, sepsis, catheter-related endocarditis, and osteomyelitis. Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen evolved from Staphylococcus aureus, known for its multidrug resistance, high infection rate, and high mortality rate, posing a significant challenge to clinical treatment. Furthermore, MRSA's ability to form biofilms protects the bacteria from host immune defenses and antibiotic treatment, significantly increasing the complexity of infection management. This leads to a vicious cycle of persistent infection and increased healthcare costs, making clinical treatment exceptionally difficult. There is an urgent need to develop simpler, more effective synthetic strategies and safer antimicrobial agents to combat MRSA.

[0003] Currently, antibiotics are the primary strategy for treating bacterial infections. However, long-term and irrational use of antibiotics has led to the emergence of bacterial resistance, with multidrug-resistant and pan-drug-resistant bacteria posing a serious threat to public health and safety. Against this backdrop, natural compounds have attracted widespread attention due to their diverse antibacterial mechanisms, lower potential for inducing resistance, and multi-target pharmacological properties. Piceatannol (PIC) has emerged as a promising candidate drug. PIC is a structural analog of resveratrol, commonly found in blueberries, grapes, and passion fruit seeds, and possesses various biological activities, including antibacterial, anti-inflammatory, and antioxidant effects. PIC's anti-inflammatory effect is primarily mediated by the inhibition of key pro-inflammatory factors such as TNF-α and IL-6, highlighting its therapeutic potential in various pathologies, including liver injury, cardiac injury, cancer, and skin diseases. Studies have shown that PIC can inhibit the growth of MRSA and biofilm formation. However, the clinical application of PIC is limited by its low solubility, poor bioavailability, and insufficient pathogen-targeting specificity. Therefore, improving the solubility, bioavailability, and clinical efficacy of PIC formulations remains an important focus of current research.

[0004] Nanoparticles, due to their small particle size, large specific surface area, unique physicochemical properties, and enhanced reactivity, show great promise for disease treatment. In recent years, nanocarriers have become one of the most promising drug delivery systems. Among numerous nanocarriers, chitosan oligosaccharide (COS), as a natural cationic polymer, is an ideal candidate material due to its ease of preparation into nanoparticles (COS-NPs) and its excellent biocompatibility, inherent antibacterial activity, and potential active targeting ability against bacteria. These properties enable COS-NPs to reduce the required drug dosage and improve overall antibacterial efficacy. However, the main limitation of COS-NPs lies in their strongly positively charged surface. While this is beneficial for their interaction with bacteria, it also promotes electrostatic adhesion to negatively charged biofilm matrices, leading to nanoparticle retention, hindering deep penetration, and thus limiting biofilm clearance.

[0005] Therefore, researching the use of chitosan oligosaccharide nanoparticles as a carrier for paclitaxel to establish a pathogen-responsive drug delivery mechanism acting on the site of MRSA infection is of great significance for anti-MRSA drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a chitosan oligosaccharide-based paclitaxel nanoparticle delivery system, its preparation method, and its application, addressing the shortcomings of existing technologies.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a chitosan oligosaccharide-based paclitaxel nanoparticle delivery system, wherein the paclitaxel nanoparticle delivery system is a chitosan oligosaccharide nanoparticle drug-carrying particle modified with hyaluronic acid and D-tyrosine. The chitosan oligosaccharide nanoparticles comprise a chitosan oligosaccharide nanoparticle carrier and tamarind encapsulated within the chitosan oligosaccharide nanoparticle carrier.

[0008] Preferably, the size of the paclitaxel nanoparticles is 230-270 nm, and the size of the chitosan oligosaccharide nanoparticles is 190-210 nm.

[0009] The present invention also provides a method for preparing the aforementioned chitosan-based paclitaxel nanoparticles, comprising the following steps: 1) Mix paclitaxel solution, chitosan oligosaccharide solution and sodium tripolyphosphate solution, and crosslink them to obtain chitosan oligosaccharide nanoparticles; 2) The activated hyaluronic acid solution was added to the chitosan oligosaccharide nanoparticles for coupling reaction, and then D-tyrosine solution was added for reaction to obtain chitosan oligosaccharide-based paclitaxel nanoparticles.

[0010] Preferably, the concentration of the chitosan oligosaccharide solution in step 1) is 1~3 mg / mL, the concentration of the sodium tripolyphosphate solution is 0.5~1.5 mg / mL, and the concentration of the paclitaxel solution is 0.5~1.5 mg / mL; The volume ratio of the chitosan oligosaccharide solution to the sodium tripolyphosphate solution is 5~8:1; The volume ratio of the paclitaxel solution to the chitosan oligosaccharide solution is 1:4~10.

[0011] Preferably, the crosslinking temperature in step 1) is 20~30℃ and the crosslinking time is 5~15min.

[0012] Preferably, the concentration of the hyaluronic acid solution in step 2) is 0.5~1.5 mg / mL, and the concentration of the D-tyrosine solution is 1~3 mg / mL.

[0013] Preferably, the volume ratio of the hyaluronic acid solution in step 2) to the chitosan oligosaccharide solution in step 1) is 1:38~43, and the volume ratio of the D-tyrosine solution in step 2) to the chitosan oligosaccharide solution in step 1) is 1:1~3.

[0014] Preferably, the reagents used for activation in step 2) include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The activation temperature is 35~40℃, and the activation time is 2~4h.

[0015] Preferably, the temperature of the coupling reaction in step 2) is 35~40℃, and the time of the coupling reaction is 20~26h; The reaction temperature in step 2) is 35~40℃, and the reaction time is 2~4h.

[0016] The present invention also provides the application of the aforementioned chitosan-based oligosaccharide-based paclitaxel nanoparticles in the preparation of anti-MRSA drugs.

[0017] The beneficial effects of this invention are: This invention utilizes hyaluronic acid and D-tyrosine to functionalize chitosan oligosaccharide nanoparticles. Hyaluronic acid enables targeted delivery to the MRSA infection site, while D-tyrosine possesses biomembrane dispersibility, disrupting biomembrane structures, promoting deeper drug penetration into the biomembrane, and enhancing the anti-MRSA infection effect. This strengthens the antibacterial and anti-biomembrane activity of paclitaxel against MRSA, exhibiting stronger antibacterial capabilities. In a mouse model of MRSA pneumonia, it significantly reduces lung bacterial load and alleviates inflammatory lung injury. Combining strong antibacterial activity, improved biomembrane permeability, and bacterial targeting ability, these three characteristics work synergistically to enhance the therapeutic effect against MRSA infection. Attached Figure Description

[0018] Figure 1 The characterization results of D-Tyr / HA / PIC-COS-NPs are shown below. A is the particle size distribution of D-Tyr / HA / PIC-COS-NPs, B is the Zeta potential distribution of D-Tyr / HA / PIC-COS-NPs, C is the scanning electron microscope image of D-Tyr / HA / PIC-COS-NPs, D is the infrared spectrum of PIC-COS-NPs, E is the particle size stability curve of D-Tyr / HA / PIC-COS-NPs, F is the Zeta potential stability curve of D-Tyr / HA / PIC-COS-NPs, and G is the PIC release curve of D-Tyr / HA / PIC-COS-NPs. Figure 2 The in vitro antibacterial performance curves of paclitaxel nanoparticles are shown, where A is the growth curve of D-Tyr / HA / PIC-COS-NPs against MRSA, B is the growth curve of PIC against MRSA, C is the bactericidal curve of D-Tyr / HA / PIC-COS-NPs against MRSA, and D is the bactericidal curve of PIC against MRSA. Figure 3 The figures show the in vitro anti-biofilm activity curves, where A is the inhibition curve against MRSA biofilm and B is the scavenging effect curve against MRSA biofilm. Figure 4 The results represent biocompatibility assessments, where A is the cell viability curve, B is the organ index curve, C is the blood index, and D is a representative H&E staining image of the organ. In the figure, NPs represent D-Tyr / HA / PIC-COS-NPs. Figure 5 This figure shows the biofilm clearance effect in vivo. A represents the colony count results, B is a scanning electron microscope image of the MRSA biofilm, and C is a representative H&E staining image. NPs in the figure represent D-Tyr / HA / PIC-COS-NPs. Figure 6 This study analyzes the inflammation and damage caused by an in vivo anti-MRSA-induced pneumonia model. In the figure, A represents the wet-to-dry weight ratio of lung tissue, B represents the average colony count of lung tissue, C represents the expression level of IL-6 mRNA, D represents the expression level of IL-1β mRNA, E represents the expression level of TNF-α mRNA, and F represents a representative H&E staining image. Figure 7 The particle size, polydispersity index (PDI), and zeta potential of the chitosan oligosaccharide nanoparticles prepared in Examples 1-3 are shown. Detailed Implementation

[0019] This invention provides a chitosan oligosaccharide-based paclitaxel nanoparticle delivery system, wherein the paclitaxel nanoparticle delivery system is a chitosan oligosaccharide nanoparticle drug-carrying particle modified with hyaluronic acid and D-tyrosine. The chitosan oligosaccharide nanoparticles comprise a chitosan oligosaccharide nanoparticle carrier and tamarind encapsulated within the chitosan oligosaccharide nanoparticle carrier.

[0020] In this invention, the design principle of using hyaluronic acid for functional modification is as follows: Staphylococcus aureus can secrete hyaluronidase, which can degrade hyaluronic acid, promote the local release of drugs at the site of infection, and help to establish a pathogen-responsive drug delivery mechanism that precisely targets the site of MRSA infection.

[0021] In this invention, the particle size of the paclitaxel nanoparticles is preferably 230-270 nm, more preferably 240-260 nm, and even more preferably 250 nm; the particle size of the chitosan oligosaccharide nanoparticles is preferably 190-210 nm, more preferably 195-205 nm, and even more preferably 200 nm.

[0022] The present invention also provides a method for preparing the aforementioned chitosan-based paclitaxel nanoparticles, comprising the following steps: 1) Mix paclitaxel solution, chitosan oligosaccharide solution and sodium tripolyphosphate solution, and crosslink them to obtain chitosan oligosaccharide nanoparticles; 2) The activated hyaluronic acid solution was added to the chitosan oligosaccharide nanoparticles for coupling reaction, and then D-tyrosine solution was added for reaction to obtain chitosan oligosaccharide-based paclitaxel nanoparticles.

[0023] In this invention, the concentration of the chitosan oligosaccharide solution in step 1) is preferably 1-3 mg / mL, more preferably 1.5-2.5 mg / mL, and even more preferably 2 mg / mL; the concentration of the sodium tripolyphosphate solution is preferably 0.5-1.5 mg / mL, more preferably 0.8-1.2 mg / mL, and even more preferably 1 mg / mL; the concentration of the leucopicrin solution is preferably 0.5-1.5 mg / mL, more preferably 0.8-1.2 mg / mL, and even more preferably 1 mg / mL. The volume ratio of the chitosan oligosaccharide solution to the sodium tripolyphosphate solution is preferably 5-8:1, more preferably 6-7:1, and even more preferably 6.5:1; The volume ratio of the leucopicrin solution to the chitosan oligosaccharide solution is preferably 1:4 to 10, more preferably 1:5 to 9, and even more preferably 1:6 to 8.

[0024] In this invention, the crosslinking temperature in step 1) is preferably 20~30℃, more preferably 22~28℃, and even more preferably 25℃; the crosslinking time is preferably 5~15min, more preferably 8~12min, and even more preferably 10min.

[0025] In this invention, the concentration of the hyaluronic acid solution in step 2) is preferably 0.5~1.5 mg / mL, more preferably 0.8~1.2 mg / mL, and even more preferably 1 mg / mL; the concentration of the D-tyrosine solution is preferably 1~3 mg / mL, more preferably 1.5~2.5 mg / mL, and even more preferably 2 mg / mL.

[0026] In this invention, the volume ratio of the hyaluronic acid solution in step 2) to the chitosan oligosaccharide solution in step 1) is preferably 1:38~43, more preferably 1:39~42, and even more preferably 1:40; the volume ratio of the D-tyrosine solution in step 2) to the chitosan oligosaccharide solution in step 1) is preferably 1:1~3, more preferably 1:1.5~2.5, and even more preferably 1:2.

[0027] In this invention, the reagent used for activation in step 2) preferably includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The activation temperature is preferably 35~40℃, more preferably 36~39℃, and even more preferably 37~38℃; the activation time is preferably 2~4h, more preferably 2.5~3.5h, and even more preferably 3h.

[0028] In this invention, the temperature of the coupling reaction in step 2) is preferably 35~40℃, more preferably 36~39℃, and even more preferably 37~38℃; the time of the coupling reaction is preferably 20~26h, more preferably 22~24h, and even more preferably 23h. The reaction temperature in step 2) is preferably 35~40℃, more preferably 36~39℃, and even more preferably 37~38℃; the reaction time is preferably 2~4h, more preferably 2.5~3.5h, and even more preferably 3h.

[0029] The present invention also provides the application of the aforementioned chitosan-based oligosaccharide-based paclitaxel nanoparticles in the preparation of anti-MRSA drugs.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] The materials used in the embodiments of this invention are as follows: chitosan oligosaccharide (COS) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (item number C302935), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (item number E106172), N-hydroxysuccinimide (NHS) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (item number H109330), picratecan (PIC) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (item number S31383), and sodium tripolyphosphate (TPP) was used. The following substances were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (item number S30235), D-tyrosine (D-Tyr) (item number S20088), hyaluronic acid (HA) (item number H874944), TSB medium (item number 024048), TSA medium (item number 028074), and 4% paraformaldehyde fixative (item number G1101) from Wuhan Saiweier Biotechnology Co., Ltd.

[0032] Example 1

[0033] A chitosan oligosaccharide solution with a concentration of 2 mg / mL, a sodium tripolyphosphate solution with a concentration of 1 mg / mL, a paclitaxel solution with a concentration of 1 mg / mL, and a D-tyrosine solution with a concentration of 2 mg / mL were prepared. A hyaluronic acid solution with a concentration of 1 mg / mL was activated at 37°C for 3 h using 20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 30 mg of N-hydroxysuccinimide.

[0034] A paclitaxel solution and a chitosan oligosaccharide solution were mixed at a volume ratio of 1:6, and then sodium tripolyphosphate solution was added. Crosslinking was carried out at 25°C for 10 hours to form chitosan oligosaccharide nanoparticles, labeled PIC-COS-NPs. The volume ratio of chitosan oligosaccharide solution to sodium tripolyphosphate solution was 8:1. Activated hyaluronic acid solution was added to the chitosan oligosaccharide nanoparticles, and a coupling reaction was carried out at 37°C for 24 hours. Then, D-tyrosine solution was added, and the reaction was carried out at 37°C for 3 hours to obtain paclitaxel nanoparticles based on chitosan oligosaccharide, labeled D-Tyr / HA / PIC-COS-NPs. The volume ratio of chitosan oligosaccharide solution to hyaluronic acid solution was 40:1, and the volume ratio of chitosan oligosaccharide solution to D-tyrosine solution was 2:1.

[0035] Experiment 1 Characterization of Paclitaxel Nanoparticles

[0036] The particle size, polydispersity index (PDI), and zeta potential of the leucocele nanoparticles were characterized using a nanoparticle size potentiometer (Zetasizer Advance Lab, Malvern Instruments Ltd.). The leucocele content was determined by high-performance liquid chromatography (HPLC). The HPLC detection conditions were as follows: 2998 PDA detector, Sample Manager FTN-R injector, Quaternary Solvent Manager-R quaternary solvent manager, Supersil ODS2-C18 column (5 μm, 250 mm × 4.6 mm), mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution, flow rate was 1 mL / min, injection volume was 10 µL, detection wavelength was 324 nm, column temperature was 30 °C, and the gradient elution program is shown in Table 1.

[0037] Table 1 Gradient elution program for high performance liquid chromatography

[0038] Drug encapsulation efficiency (EE) and drug loading (LC) are calculated using the following formulas: . In this embodiment, the encapsulation efficiency (EE) of D-Tyr / HA / PIC-COS-NPs is 87.25±0.04%, and the drug loading (LC) is 7.77±0.48%.

[0039] The in vitro PIC release behavior of paclitaxel nanoparticles was evaluated using a dialysis method. Specifically, paclitaxel nanoparticles containing 20 mg of PIC were placed in a dialysis bag (molecular weight cutoff of 1000 Da). The dialysis bag was then immersed in 30 mL of PBS buffer at pH 7.4 and pH 5.5, respectively. Release experiments were conducted at 37°C and 100 rpm. pH 7.4 simulated the physiological environment, and pH 5.5 simulated the acidic microenvironment of sites of inflammation or bacterial infection. 2 mL of release medium was collected at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, and 48 h at the start of the release experiment, and an equal volume of PBS buffer was added. The release medium was quantified using high-performance liquid chromatography (HPLC), and the cumulative PIC release percentage was calculated.

[0040] The paclitaxel nanoparticles were stored at 4°C, and the particle size and zeta potential were measured on days 1, 4, 7, 14, and 21.

[0041] Figure 1To characterize the results, A is the particle size distribution of D-Tyr / HA / PIC-COS-NPs, B is the zeta potential distribution of D-Tyr / HA / PIC-COS-NPs, C is the scanning electron microscope image of D-Tyr / HA / PIC-COS-NPs, D is the infrared spectrum of PIC-COS-NPs, E is the particle size stability curve of D-Tyr / HA / PIC-COS-NPs, F is the zeta potential stability curve of D-Tyr / HA / PIC-COS-NPs, and G is the PIC release curve of D-Tyr / HA / PIC-COS-NPs. Figure 1 It can be seen that the particle size of D-Tyr / HA / PIC-COS-NPs is 239.7 ± 2.33 nm, and the PDI is 0.149 ± 0.02, indicating uniform particle size distribution. The Zeta potential decreased to 24.58 ± 0.50 mV (the Zeta potential of PIC-COS-NPs is 31.71 ± 0.35 mV), indicating that HA and D-Tyr have been successfully adsorbed onto the surface of nanoparticles. D-Tyr / HA / PIC-COS-NPs are spherical, exhibiting good dispersibility and uniformity. The infrared spectrum shows that the characteristic absorption peak of amino groups in chitosan oligosaccharide (COS) increases from 1526 cm⁻¹. -1 Moved to 1534cm -1 This indicates that the amino group has undergone protonation (-NH3). + ), and undergoes electrostatic interaction with the phosphate groups of sodium tripolyphosphate (TPP); simultaneously, in the range of 1213~1012 cm⁻¹ -1 Characteristic absorption peaks corresponding to P=O and POC bonds in TPP were observed within the range; these results confirm the incorporation of TPP and the formation of ionic cross-linked structures, with PIC encapsulated within COS-TPP. Particle size stability and Zeta potential stability curves indicate that D-Tyr / HA / PIC-COS-NPs maintain excellent stability over 21 days; the PIC release curve shows that the cumulative release rate of PIC is relatively low at pH 7.4 (30.7%), while it has a higher release rate at pH 5.5 (78.4%), indicating stability under physiological conditions and pH-responsive release characteristics in slightly acidic environments such as inflammation.

[0042] Experiment 2: In vitro antibacterial activity

[0043] The minimum inhibitory concentrations (MICs) of D-Tyr / HA / PIC-COS-NPs and PIC against MRSA were determined using the microbroth dilution method, and the minimum bactericidal concentrations (MBCs) of D-Tyr / HA / PIC-COS-NPs and PIC against MRSA were determined using the plate colony counting method. The results showed that the MIC of D-Tyr / HA / PIC-COS-NPs against MRSA was 64 μg / mL, and the MBC was 256 μg / mL; the MIC of PIC against MRSA was 128 μg / mL, and the MBC was 256 μg / mL. The MICs of D-Tyr / HA / PIC-COS-NPs against MRSA were significantly lower than those of PIC, indicating that D-Tyr / HA / PIC-COS-NPs could enhance the inhibitory effect on airborne MRSA and improve the antibacterial activity of PIC.

[0044] Figure 2 The images show the in vitro antibacterial performance curves of paclitaxel nanoparticles. In these curves, A represents the growth curve of D-Tyr / HA / PIC-COS-NPs against MRSA, B represents the growth curve of PIC against MRSA, C represents the bactericidal curve of D-Tyr / HA / PIC-COS-NPs against MRSA, and D represents the bactericidal curve of PIC against MRSA. Figure 2 It can be seen that the inhibitory effect of D-Tyr / HA / PIC-COS-NPs on MRSA growth becomes more significant with increasing concentration. Compared with PIC, D-Tyr / HA / PIC-COS-NPs at a concentration of 64 μg / mL showed significant antibacterial activity, effectively inhibiting the growth and proliferation of MRSA throughout the culture period. In contrast, the control group at a concentration of 0 entered the logarithmic growth phase within 4–12 hours of culture, resulting in rapid bacterial proliferation, followed by the stationary phase. D-Tyr / HA / PIC-COS-NPs at a concentration of 128 μg / mL showed significant bactericidal activity with prolonged exposure time, while PIC only showed some bactericidal effect at 256 μg / mL and did not show any bactericidal activity at a concentration of 128 μg / mL. The results indicate that D-Tyr / HA / PIC-COS-NPs significantly enhanced the antibacterial activity of PIC.

[0045] Experiment 3: In vitro anti-biofilm activity

[0046] Chitosan oligosaccharide solution and sodium tripolyphosphate solution were mixed at a volume ratio of 8:1 and crosslinked at 25°C for 10 h to obtain chitosan oligosaccharide nanoparticle carriers. Activated hyaluronic acid solution was added to the chitosan oligosaccharide nanoparticle carriers, and a coupling reaction was carried out at 37°C for 24 h. Then, D-tyrosine solution was added, and the reaction was carried out at 37°C for 3 h to obtain chitosan oligosaccharide nanoparticle delivery particles, labeled D-Tyr / HA / COS-NPs, as the blank carrier group. The volume ratio of chitosan oligosaccharide solution to hyaluronic acid solution was 1:40, and the volume ratio of chitosan oligosaccharide solution to D-tyrosine solution was 2:1.

[0047] The anti-MSRA biofilm activity and scavenging effect on MRSA biofilms of D-Tyr / HA / PIC-COS-NPs, D-Tyr / HA / COS-NPs and PIC were evaluated using crystal violet staining.

[0048] Figure 3 These are in vitro anti-biofilm activity curves, where A represents the inhibition curve against MRSA biofilm, and B represents the scavenging effect curve against MRSA biofilm. Figure 3 It can be seen that within the concentration range of 32~128 μg / mL, the biofilm inhibition rate of D-Tyr / HA / PIC-COS-NPs was significantly higher than that of other groups (p<0.05), indicating that D-Tyr / HA / PIC-COS-NPs has a significant inhibitory effect on the formation of MRSA biofilm. At a concentration of 32 μg / mL, D-Tyr / HA / PIC-COS-NPs showed significant MRSA biofilm clearance ability, with a clearance rate of 67.38%, which was better than D-Tyr / HA / COS-NPs (61.49%) and PIC (55.59%). When the concentration reached 128 μg / mL, D-Tyr / HA / PIC-COS-NPs achieved almost complete biofilm clearance, with a clearance rate of 87.97%. At any concentration, D-Tyr / HA / PIC-COS-NPs always showed stronger anti-biofilm activity than D-Tyr / HA / COS-NPs and PIC. This result indicates that D-Tyr / HA / PIC-COS-NPs themselves possess inherent anti-biofilm properties.

[0049] Experiment 4 Biocompatibility

[0050] In vitro biocompatibility was assessed using the MLE-12 cell line as a model, and cytotoxicity was detected using the CCK-8 assay.

[0051] In vivo biocompatibility: Thirty 6-week-old SPF-grade male BALB / c mice were randomly divided into three groups of 10 each: a blank control group, a D-Tyr / HA / PIC-COS-NPs group, and a PIC group. Mice in the blank control group were injected intraperitoneally with saline, while mice in the D-Tyr / HA / PIC-COS-NPs and PIC groups were injected intraperitoneally with the corresponding drugs once daily for 7 days at a dose of 40 mg / kg (based on PIC). On day 8, all mice were euthanized, and blood was collected for complete blood count (CBC). Hearts, lungs, kidneys, livers, and spleens were collected for Hematologic & Escherichia coli (H&E) staining (n=10, scale bar=50 μm).

[0052] Figure 4 The results represent biocompatibility assessments, where A is the cell viability curve, B is the organ index curve, C is the blood index, and D is a representative H&E staining image of the organ. NPs in the figure represent D-Tyr / HA / PIC-COS-NPs. Figure 4 As can be seen from the cell viability curve, the cell viability increased from 2 μg / mL to 128 μg / mL. The concentration of D-Tyr / HA / PIC-COS-NPs and PIC did not significantly decrease the cell viability, but it remained above 80%, indicating sufficient safety. Organ index curves showed no significant differences in organ indices among the three groups (p>0.05), indicating that neither D-Tyr / HA / PIC-COS-NPs nor PIC caused significant changes in organ indices. Blood parameters showed that the levels of white blood cells, red blood cells, and hemoglobin were comparable among the three groups, with no statistically significant differences (p>0.05). H&E staining images showed no significant pathological changes in the heart, liver, lungs, kidneys, and spleen in both the D-Tyr / HA / PIC-COS-NPs and PIC groups: no signs of myocardial fibrosis were observed in the heart tissue; hepatocytes were arranged in an orderly manner without necrosis or degeneration; the lung tissue structure remained intact with clear outlines; the kidney tissue cell structure was normal, without swelling or necrosis; the white and red pulp of the spleen were clearly demarcated, with abundant lymphocytes and no abnormal morphological findings. The above results indicate that D-Tyr / HA / PIC-COS-NPs and PIC did not cause acute toxicity to the hematopoietic system and major organs of mice, and have good biosafety.

[0053] Experiment 5: In vivo MRSA biofilm clearance effect

[0054] A 10mm diameter sterile silica wafer was placed in a 24-well plate and mixed with an MRSA bacterial suspension (concentration 1×10⁻⁶). 6CFU / mL, TSB medium) was incubated at 37℃ for 24 h to form a biofilm. Forty 8-week-old SPF-grade male SD rats were used, divided into groups of 10. The rats were anesthetized and the hair in the interscapular region was removed. A subcutaneous air sac was created by subcutaneously injecting 10 mL of sterile air. A 1 cm longitudinal incision was made, and a biofilm-containing silicon wafer was implanted into one group of rats. A sterile silicon wafer was implanted into one group of rats, and the incision was sutured with sterile sutures. Rats with implanted sterile silicon wafers were injected intraperitoneally with saline as a sham control group; rats with implanted biofilm silicon wafers were injected intraperitoneally with saline as a model control group; the remaining two groups of rats were injected intraperitoneally with D-Tyr / HA / PIC-COS-NPs and PIC, respectively, once daily for 3 days, at a dose of 20 mg / kg (based on PIC). On day 4, rats were anesthetized with isoflurane and the silicon wafers were removed. The clearance of the biofilm was observed and evaluated by scanning electron microscopy. The removed silicon wafers were sonicated for 20 minutes to dissociate the biofilm, and then quantitative analysis was performed using the plate count method. Tissue around the implant was collected for H&E staining and immunofluorescence analysis.

[0055] Figure 5 This figure shows the biofilm clearance effect in vivo. A represents colony count results, B is a scanning electron microscope image of the MRSA biofilm, and C is a representative H&E staining image. NPs in the figure represent D-Tyr / HA / PIC-COS-NPs. Figure 5As can be seen from the colony count results, there was a statistically significant difference (p<0.01) between the colony counts of D-Tyr / HA / PIC-COS-NPs and the model control group, indicating that administration of D-Tyr / HA / PIC-COS-NPs demonstrated enhanced antibacterial activity, reduced inflammatory response, and decreased tissue damage. Scanning electron microscopy revealed that the model control group exhibited a dense and structurally intact MRSA biofilm with high bacterial density, tight adhesion, and well-preserved morphology. The PIC group showed a reduced bacterial load, but the three-dimensional structure of the biofilm remained largely intact, and bacterial adhesion was still detectable. In contrast, the biofilm in the D-Tyr / HA / PIC-COS-NPs group was extensively disrupted, with the membrane structure almost completely removed, and both bacterial adhesion and number significantly reduced. These results are consistent with the in vitro anti-biofilm activity verification results, confirming that D-Tyr / HA / PIC-COS-NPs has a strong disruptive and scavenging effect on MRSA biofilms. H&E staining images showed that tissue sections in the model control group exhibited structural abnormalities, including sparse skin structure with edema, extensive collagen fiber loss, severe connective tissue hyperplasia (indicated by black arrows), extensive lymphocyte / granulocyte infiltration (indicated by green arrows), and multifocal hemorrhage (indicated by red arrows). The PIC group showed severe connective tissue hyperplasia, mild lymphocyte / granulocyte infiltration, and multifocal hemorrhage. In contrast, the D-Tyr / HA / PIC-COS-NPs group showed a significant antibacterial effect, with essentially normal skin tissue structure and only a very small amount of inflammatory cell infiltration. These results indicate that D-Tyr / HA / PIC-COS-NPs significantly alleviated MRSA infection by reducing inflammatory cell infiltration and tissue edema.

[0056] Experiment 6: In vivo anti-MRSA induced pneumonia model

[0057] Fifty 6-week-old SPF-grade male BALB / c mice were randomly divided into five groups of 10 mice each. One group served as a blank control group, while the remaining four groups were inoculated intratracheally with MRSA (10 μL, concentration 1×10⁻⁶). 9 Pneumonia was induced in mice using CFU / mL solutions. A blank control group was injected with saline. One group of mice induced with pneumonia received saline as the model group. The remaining three groups were injected with PIC solution, D-Tyr / HA / PIC-COS-NPs solution, and vancomycin (VAN) solution, respectively, as the three treatment groups. These treatments were administered once daily for 3 days. The dosage of PIC was 20 mg / kg, and the dosage of VAN was 5 mg / kg. Mice were sacrificed on day 4, and lung tissue was collected for analysis.

[0058] Using β-actin as an internal reference gene, the relative expression levels of key inflammatory factors were analyzed using the 2-ΔΔCt method.

[0059] Figure 6 This study analyzes inflammation and damage induced by an in vivo anti-MRSA-induced pneumonia model. In the in vivo model, A represents the wet-to-dry weight ratio of lung tissue, B represents the average bacterial count of lung tissue, C represents the expression level of IL-6 mRNA, D represents the expression level of IL-1β mRNA, E represents the expression level of TNF-α mRNA, and F represents a representative H&E staining image. Figure 6 It can be seen that, compared with the blank control group, the wet-to-dry weight ratio (W / D) of lung tissue in the model group was significantly increased (p<0.0001), indicating severe pulmonary edema. After treatment with PIC, D-Tyr / HA / PIC-COS-NPs, and vancomycin, respectively, pulmonary edema was significantly relieved in all three treatment groups. The mean bacterial count showed that, compared with the model group, the bacterial load in the lungs of the PIC, D-Tyr / HA / PIC-COS-NPs, and vancomycin treatment groups was significantly reduced (p<0.01). The D-Tyr / HA / PIC-COS-NPs treatment group showed a significantly stronger inhibitory effect on MRSA than the PIC treatment group. The expression levels of inflammatory factors showed that, compared with the blank control group, the levels of inflammatory factors IL-6, IL-1β, and TNF-α in the lung tissue of the model group were significantly increased, while the levels in all three treatment groups were significantly decreased compared with the model group (p<0.0001). The D-Tyr / HA / PIC-COS-NPs treatment group showed the highest level of these factors. The treatment group showed lower levels of inflammatory factors than PIC, indicating that D-Tyr / HA / PIC-COS-NPs have a stronger anti-inflammatory effect against MRSA-induced inflammation. These results suggest that D-Tyr / HA / PIC-COS-NPs can alleviate Staphylococcus aureus-induced lung tissue damage through their antibacterial and anti-inflammatory properties. H&E staining images showed that the lungs of the model group exhibited significant inflammatory lesions, specifically inflammatory cell infiltration and congestion, with necrotic cell debris observed around the alveoli and bronchioles, accompanied by occasional focal perivascular and peribronchiolar lymphocytic infiltration, perivascular edema, disordered connective tissue arrangement, widened interstitial spaces, and scattered punctate lymphocytic infiltration (indicated by black arrows), and visible erythrocyte proliferation (indicated by green arrows). In contrast, both the D-Tyr / HA / PIC-COS-NPs and vancomycin treatment groups showed significant improvement in pathological tissue damage and pulmonary inflammatory infiltration.

[0060] Note: In the accompanying drawings of this invention: This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0061] Example 2

[0062] The volume ratios of chitosan oligosaccharide solution and paclitaxel solution in Example 1 were modified to 2:1, 4:1, 8:1, and 10:1, respectively, while other aspects remained the same as in Example 1.

[0063] Example 3

[0064] The volume ratios of chitosan oligosaccharide solution and D-tyrosine solution in Example 1 were modified to 1:2, 1:1, and 3:1, respectively, while other aspects remained the same as in Example 1.

[0065] Experiment 7: Comparison of chitosan oligosaccharide concentration and volume

[0066] Chitosan oligosaccharide solution and sodium tripolyphosphate solution were mixed and cross-linked at 25°C for 10 h to form chitosan oligosaccharide nanoparticle carriers, labeled COS-NPs. The volume ratios of the chitosan oligosaccharide solution and sodium tripolyphosphate solution were 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1, respectively. The concentrations of the chitosan oligosaccharide solution were 1 mg / mL, 2 mg / mL, and 3 mg / mL, respectively, and the concentration of the sodium tripolyphosphate solution was 1 mg / mL.

[0067] Figure 7 The particle size, polydispersity index (PDI), and zeta potential of the chitosan oligosaccharide nanoparticle carrier are shown. Figure 7 As can be seen, in Example 1, when the concentration of chitosan oligosaccharide is 2 mg / mL and the volume ratio of chitosan oligosaccharide solution to sodium tripolyphosphate solution is 8:1, the chitosan oligosaccharide nanoparticle carrier with the best properties can be obtained, with a particle size of 184.03±5.96 nm, a polydispersity index (PDI) of 0.15±0.01, and a zeta potential of 29.92±0.58 mV.

[0068] The particle size, PDI, Zeta potential, and encapsulation efficiency (EE) of the chitosan oligosaccharide nanoparticles prepared in Examples 1 and 2 are shown in Table 2.

[0069] Table 2 Characterization of chitosan oligosaccharide nanoparticles from Examples 1 and 2

[0070] As can be seen from Table 2, when the volume ratio of chitosan oligosaccharide solution to leucine solution is 6:1 in Example 1, the optimal encapsulation efficiency of 92.13±2.27 can be obtained, while also exhibiting good particle size, PDI and Zeta potential.

[0071] The particle size, PDI, Zeta potential and encapsulation efficiency EE of the paclitaxel nanoparticles prepared in Examples 1 and 3 are shown in Table 3.

[0072] Table 3 Characterization of the paclitaxel nanoparticles in Examples 1 and 3

[0073] As can be seen from Table 3, when the volume ratio of chitosan oligosaccharide solution to D-tyrosine solution in Example 1 is 2:1, the biofilm removal efficiency can be improved while maintaining the good properties of nanoparticles.

[0074] 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 chitosan-based oligosaccharide-based paclitaxel nanoparticle delivery system, characterized in that, The paclitaxel nanoparticles are chitosan oligosaccharide nanoparticles modified with hyaluronic acid and D-tyrosine. The chitosan oligosaccharide nanoparticles comprise a chitosan oligosaccharide nanoparticle carrier and tamarind encapsulated within the chitosan oligosaccharide nanoparticle carrier.

2. The white paclitaxel nanoparticles based on chitosan oligosaccharide according to claim 1, characterized in that, The paclitaxel nanoparticles have a particle size of 230-270 nm, and the chitosan oligosaccharide nanoparticles have a particle size of 190-210 nm.

3. The method for preparing paclitaxel nanoparticles based on chitosan oligosaccharide according to claim 1 or 2, characterized in that, It includes the following steps: 1) Mix paclitaxel solution, chitosan oligosaccharide solution and sodium tripolyphosphate solution, and crosslink them to obtain chitosan oligosaccharide nanoparticles; 2) The activated hyaluronic acid solution was added to the chitosan oligosaccharide nanoparticles for coupling reaction, and then D-tyrosine solution was added for reaction to obtain chitosan oligosaccharide-based paclitaxel nanoparticles.

4. The preparation method according to claim 3, characterized in that, Step 1) The concentration of the chitosan oligosaccharide solution is 1~3 mg / mL, the concentration of the sodium tripolyphosphate solution is 0.5~1.5 mg / mL, and the concentration of the paclitaxel solution is 0.5~1.5 mg / mL; The volume ratio of the chitosan oligosaccharide solution to the sodium tripolyphosphate solution is 5~8:1; The volume ratio of the paclitaxel solution to the chitosan oligosaccharide solution is 1:4~10.

5. The preparation method according to claim 3 or 4, characterized in that, Step 1) The crosslinking temperature is 20~30℃ and the crosslinking time is 5~15min.

6. The preparation method according to claim 5, characterized in that, Step 2) The concentration of the hyaluronic acid solution is 0.5~1.5 mg / mL, and the concentration of the D-tyrosine solution is 1~3 mg / mL.

7. The preparation method according to claim 6, characterized in that, The volume ratio of the hyaluronic acid solution in step 2) to the chitosan oligosaccharide solution in step 1) is 1:38~43, and the volume ratio of the D-tyrosine solution in step 2) to the chitosan oligosaccharide solution in step 1) is 1:1~3.

8. The preparation method according to claim 6 or 7, characterized in that, Step 2) The activation reagents used include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The activation temperature is 35~40℃, and the activation time is 2~4h.

9. The preparation method according to claim 8, characterized in that, Step 2) The coupling reaction temperature is 35~40℃, and the coupling reaction time is 20~26h; Step 2) The reaction temperature is 35~40℃ and the reaction time is 2~4h.

10. The use of the chitosan-based oligosaccharide-based paclitaxel nanoparticles according to claim 1 or 2 in the preparation of anti-MRSA drugs.

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