X-ray excited nano-particles with organic photosensitizer wrapped by lipidosome as well as preparation method and application of nano-particles

Abstract

The invention provides X-ray excited nano-particles with lipidosome coated with an organic photosensitizer. The nano-particles comprise polymyxin B, a lipidosome carrier, an X-ray sensitizer and the organic photosensitizer. The invention also provides a preparation method and application of the nanoparticles. The nano photosensitizer provided by the invention realizes efficient resistance to drug-resistant bacteria under low-dose irradiation, and can be used for deep tissue drug-resistant bacteria infection treatment.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of nanomedicine and radiation biology, specifically to an X-ray excited liposome-encapsulated organic photosensitizer nanoparticle, its preparation method, and its application. Background Technology

[0002] Bacterial infections, especially those caused by drug-resistant bacteria or deep tissue biofilms, pose a significant challenge in clinical treatment. Traditional antibiotic therapies face problems such as increasing drug resistance, poor biofilm permeability, and systemic toxicity.

[0003] Photodynamic therapy (aPDT) is a promising strategy that uses photosensitizers to generate reactive oxygen species (ROS) under specific wavelengths of light to kill pathogens. However, traditional aPDT relies on ultraviolet-visible or near-infrared (NIR) light as the excitation source, which has limited tissue penetration depth (usually less than 1 cm), making it difficult to effectively treat deep tissue infections.

[0004] X-rays possess extremely strong tissue-penetrating capabilities, reaching deep into the human body. However, conventional organic photosensitizers are insensitive to X-rays and cannot directly utilize their energy. In recent years, X-ray activated photodynamic therapy (X-PDT) has become a research hotspot. Its core strategy is to use high atomic number (Z) nanomaterials as energy conversion media. These materials can effectively absorb X-ray photons and generate ultraviolet / visible light through scintillation or Cherenkov effects, thereby activating the photosensitizers bound to them. However, existing X-PDT nanosystems often suffer from problems such as complex preparation, poor biocompatibility, low photosensitizer loading, and insufficient ROS yield, resulting in high required X-ray doses and significant potential radiation risks.

[0005] Liposomes, as a classic nanocarrier, possess excellent biocompatibility, biodegradability, and ease of surface modification, making them an ideal platform for drug delivery. Therefore, developing a nanophotosensitizer system that is simple to prepare, biocompatible, highly X-ray responsive, and capable of achieving efficient bactericidal activity under low-dose X-ray irradiation has significant clinical implications and application value. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies and provide an X-ray activated liposomal photosensitizer, its preparation method, and its applications. This nanosystem integrates an X-ray energy conversion unit and a photosensitizer, enabling efficient utilization of penetrating X-rays to generate large amounts of reactive oxygen species (ROS), achieving low-dose, high-efficiency killing of deep-seated bacteria and biofilms.

[0007] The present invention first provides an X-ray excited liposome-encapsulated organic photosensitizer nanoparticle, which includes polymyxin B, a liposome carrier, an X-ray sensitizer and an organic photosensitizer; The polymyxin B is linked to the lipid carrier (DSPE-PEG) via an amide bond. 2000 -NHS) are covalently linked in a molar ratio of 1:1; The liposome carrier is composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), and DSPE-PEG. 2000 and DSPE-PEG 2000 -NHS is composed of a molar ratio of 25-55:12.5-30:2.5-8:1; preferably, the molar ratio is 50-55:25-30:5-8:1; The organic photosensitizer and X-ray sensitizer are encapsulated in the liposome carrier, and the molar ratio of X-ray sensitizer:organic photosensitizer:liposome carrier is 9-9.9:0.1-1:90.

[0008] In one embodiment of the invention, the X-ray sensitizer is 1,3,5-trifluoro-2,4,6-triiodobenzene (IFB).

[0009] In one embodiment of the present invention, the organic photosensitizer is tetraphenylporphyrin (TPP).

[0010] In one embodiment of the invention, the molar ratio of the X-ray sensitizer to the organic photosensitizer is selected from any one of 90:10, 95:5, 97:3, and 99:1.

[0011] The present invention also provides a method for preparing the above-mentioned nanoparticles, comprising: 1) Add IFB, TPP, and DSPE-PEG 2000 DSPE-PEG 2000 NHS, DPPC, and cholesterol are added together to a container, followed by vector distillation of THF (tetrahydrofuran). The mixture is stirred until all components are completely dissolved, forming a homogeneous organic phase solution. The solution contains dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), and DSPE-PEG. 2000 and DSPE-PEG 2000 The molar ratio of -NHS is 25-55:12.5-30:2.5-8:1; preferably, the molar ratio is 50-55:25-30:5-8:1; 2) The homogeneous organic phase solution obtained in step 1) is slowly added dropwise to the Milli-Q ultrapure water in the ultrasonication process. The addition is continued until a clear and transparent mixed solution is formed, so that the organic phase is uniformly dispersed in the aqueous phase and particle agglomeration is avoided. Preferably, after the addition is completed, ultrasonic treatment is continued for 5 min to further refine the size of the nanoparticles and improve the dispersion stability, so as to obtain nanoparticles with a particle size of 90-120 nm. 3) Place the mixed solution obtained in 2) on a stirrer, stir continuously and introduce compressed air to accelerate the evaporation of THF solvent, and concentrate to obtain a crude dispersion containing NHS-IFBTPP NPs; 4) Use 0.22 µ The coarse dispersion was filtered using a mPTFE membrane syringe filter. The filtered NHS-IFBTPP NPs solution was then added to a Merck Millipore centrifuge filter unit with a molecular weight cutoff of 50 kDa and centrifuged at 6000 rpm for 10 min. The concentrated particles were then washed several times with Milli-Q ultrapure water and concentrated to a concentration of 0.625 mg / ml. -1 NHS-IFBTPP NPs solution; 5) Take an appropriate amount of polymyxin B aptamer precipitate, add HEPES buffer (pH=8.2), and gently shake to completely dissolve the precipitate to obtain a polymyxin B aptamer solution; 6) Add the purified NHS-IFBTPP NPs solution to the polymyxin B aptamer solution, adjust the total volume of the reaction system so that the molar ratio of aptamer to NHS-IFBTPP NPs is 1.2:1, mix the reaction system evenly, and incubate the mixed reaction system at 4°C for more than 1.5 h. 7) After the reaction was complete, the reaction solution was transferred to a Merck Millipore centrifuge filter unit with a molecular weight cutoff of 100 kDa, and washed several times with ultrapure water to obtain a concentration of 0.1 mg / ml. -1 The PMB-IFBTPP NPs solution is obtained.

[0012] The present invention further provides a pharmaceutical composition comprising the above-described nanoparticles and pharmaceutically acceptable excipients.

[0013] Another aspect of the present invention provides the use of the above-described nanoparticles or pharmaceutical compositions in the preparation of medicaments for treating drug-resistant bacterial infections.

[0014] In one embodiment of the invention, the drug-resistant bacterial infection is a deep tissue MRSA infection.

[0015] In one embodiment of the invention, the drug-resistant bacterial infection is MRSA lung infection.

[0016] In one embodiment of the invention, the application includes irradiating the infected tissue area with low-dose X-rays after applying the nanoparticles as described in any one of claims 1-4 or the pharmaceutical composition as described in claim 6; preferably, the low-dose X-ray irradiation is X-ray irradiation of 50 kV-25 mA.

[0017] The beneficial effects of the above-described technical solution of the present invention are as follows: The X-ray-excited liposome-encapsulated organic photosensitizer nanoparticles provided by this invention exhibit excellent low-dose bactericidal activity both in vitro and in vivo. A 99% in vitro bactericidal effect was achieved at an irradiation dose of 2.5 Gy. Therefore, PMB-IFBTPP NPs have the potential for further development into photosensitizers for treating deep-seated drug-resistant bacterial infections. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the composition of the nanoparticles provided by the present invention.

[0019] Figure 2 The singlet reactive oxygen species generation capacity of nanoparticles (PMB-IFBTPP NPs) with different core photosensitizer ratios is shown. Figure 3 Transmission electron microscopy (TEM) morphology characterization images of nanoparticles (PMB-IFBTPP NPs) with different core photosensitizer ratios; Figure 4 The image shows the in vitro bactericidal effect of nanoparticles (PMB-IFBTPP NPs) with different core photosensitizer ratios under different X-ray irradiation doses. Detailed Implementation

[0020] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0021] Unless otherwise specified, all reagents used in this embodiment are of analytical grade, and the progress of all chemical reactions is detected by thin-layer chromatography.

[0022] Reagents: 1,3,5-Trifluoro-2,4,6-triiodobenzene (IFB) (CAS:84322-56-5), tetraphenylporphyrin (TPP) (CAS:917-23-7), dipalmitoylphosphatidylcholine (DPPC) (CAS:63-89-8), cholesterol (Chol) (CAS:57-88-5), and polymyxin B (PMB) (CAS:1404-26-8) were purchased from Beijing Innocare Biotechnology Co., Ltd.

[0023] DSPE-PEG 2000(CAS:147867-65-0), DSPE-PEG 2000 -NHS (CAS: 1445723-73-8) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0024] The water mentioned in this invention is distilled water, and the organic solvents are all commercially available analytical grade polar or non-polar solvents, such as tetrahydrofuran. Example 1

[0025] Take a 20 ml round-bottom flask and add IFB (0.92 mg, 1.80 mg). µ mol), TPP (0.12 mg, 0.19 mg) µ mol), DSPE-PEG-2000 (6.8 mg, 2.42 µ mol), DSPE-PEG-2000-NHS (1 mg, 0.35 µ mol), DPPC (13.8 mg, 18.7 µ mol) and cholesterol (3.62 mg, 9.35 mg) µ Add 10 ml of distilled THF (tetrahydrofuran) to the mol of THF and stir until all components are completely dissolved to obtain a homogeneous organic phase solution.

[0026] Prepare 90 ml of ultrapure water in a 250 ml round-bottom flask and place it in an ultrasonic reaction vessel. Use 200... µ Using a pipette, the homogeneous organic phase solution obtained above was slowly added dropwise to the ultrapure water in the ultrasonic chamber. The addition continued until a clear and transparent mixed solution was formed, ensuring that the organic phase was uniformly dispersed in the aqueous phase and preventing particle aggregation. After the addition was complete, ultrasonic treatment was continued for 5 minutes to further refine the nanoparticle size and improve dispersion stability. The resulting mixed solution was placed on a stirrer, continuously stirred, and compressed air was introduced to evaporate the THF solvent, yielding 80 ml of a coarse dispersion containing NHS-IFBTPP NPs.

[0027] Use 0.22 µ The 80 ml coarse dispersion was filtered through a PTFE membrane syringe filter. 5 ml of the filtered NHS-IFBTPP NPs solution was then added to a Merck Millipore centrifuge filter unit with a 50 kDa molecular weight cutoff. The solution was centrifuged using a Hermle Z 306 centrifuge at 4... o C was centrifuged at 6000 rpm for 10 min, and the concentrated particles were washed three times with ultrapure water, finally concentrated to 100. µL yielded a high-purity NHS-IFBTPP NPs solution with a concentration of 0.625 mg / ml. -1 .

[0028] Take 17.1 nmol of polymyxin B sulfate aptamer precipitate and add 171 µ Add L HEPES buffer (pH=8.2) and gently shake to completely dissolve the precipitate, obtaining a polymyxin B aptamer solution. Add 78.7 g of [unspecified substance] to the above polymyxin B aptamer solution. µ L purified NHS-IFBTPP NPs solution, followed by 242 mg / L supplementation. µ L HEPES buffer (pH=8.2), with a molar ratio of aptamer to NHS-IFBTPP NPs of 1.2:1. The mixed reaction system was incubated at 4°C for at least 1.5 h to ensure that the NHS active ester groups on the surface of NHS-IFBTPP NPs fully undergo amidation with the amino groups at the ends of the polymyxin B aptamer, forming stable amide bonds and achieving covalent linkage between the aptamer and the nanocarrier. After the reaction, the reaction solution was transferred to a Merck Millipore centrifuge filter unit with a molecular weight cutoff of 100 kDa (the molecular weight cutoff is greater than that of NHS-IFBTPP NPs to ensure that the nanoparticles are retained and the free aptamer is removed). The solution was washed three times with ultrapure water to thoroughly remove unreacted free polymyxin B aptamers and other impurities. The final solution volume was adjusted to 492 mL. µ L, to obtain a PMB-IFBTPP NPs solution with a concentration of 0.1 mg / mL. -1 The structure of the nanoparticles provided by this invention is as follows: Figure 1 As shown: Figure 2 Transmission electron microscopy (TEM) morphology characterization of nanophotosensitive agents (PMB-IFBTPP NPs) with different core photosensitizer ratios is shown. The figures reveal nanoparticles with nanosphere structures, exhibiting good uniformity with particle sizes ranging from 90 to 120 nm.

[0029] Example 2: Reactive oxygen species generation capacity test of PMB-IFBTPP NPs: 1. ABDA absorption attenuation method for detection 1 O2 Sample preparation: Dilute 20 mM ABDA stock solution to 20 mM. µ M, and 1 µ M PMB-IFBTPP NPs series samples were incubated in a DMSO / water (v:v=1:100) mixture.

[0030] Light irradiation: X-ray irradiation of 50 kV-25 mA, with the spectrum recorded once for every 0.5 Gy irradiation dose.

[0031] Absorption detection: A UV-Vis spectrophotometer was used to scan the absorption spectrum from 330 to 450 nm, and the decrease in absorbance at 378 nm was monitored to indirectly characterize the absorbance. 1 O2 generation rate (ABDA can be 1 O2 oxidation decomposition).

[0032] Figure 1 The singlet reactive oxygen species (ROS) generation capacity of nanophotosensors (PMB-IFBTPP NPs) with different core photosensitizer ratios is shown. As can be seen from the figure, the absorbance of ADBA decreases more rapidly with increasing doping ratio of the organic photosensitizer TPP, resulting in a higher generation rate. 1 An increase in O2 indicates that... 1 The O2 generation rate is positively correlated with the TPP doping concentration. 1 The O2 generation rate increases with increasing TPP doping concentration.

[0033] Figure 2 Transmission electron microscopy (TEM) morphology characterization of nanophotosensitive agents (PMB-IFBTPP NPs) with different core photosensitizer ratios is shown. The figures reveal nanoparticles with nanosphere structures, exhibiting good uniformity with particle sizes ranging from 90 to 120 nm.

[0034] Example 3: Bioactivity test of PMB-IFBTPP NPs: 1. Plate colony assay to evaluate the bactericidal ability of PMB-IFBTPP NPs First, prepare MRSA bacterial culture in the logarithmic growth phase (collected from the Intensive Care Unit of Southwest Hospital) (diluted to 1×10⁻⁶). 4 CFU•mL -1 ), LB solid culture plates and different concentrations (0 / 2 / 5 / 10), µ The PMB-IFBTPP NPs photosensitizer solution (M) was prepared, followed by the addition of 100 µL of bacterial culture and the corresponding concentration of photosensitizer solution (PBS was added to bring the volume to 500 mL) to a sterile EP tube. µ L), 37 o Incubate at C for 15 min to allow the photosensitizer to bind to the bacteria. Then, place the "light-illuminated group" samples under X-ray irradiation for different times (the light-protected group was protected from light throughout the process). Afterward, take 100 µL of the mixture and spread it on an LB agar plate at 37°C. oIncubate at a constant temperature of C for 12-16 hours. Finally, observe and count the number of colonies on each plate and calculate the sterilization rate. The entire process must be carried out under aseptic conditions in a clean bench, while ensuring that the photosensitizer is protected from light and that the experimental conditions are uniform.

[0035] Figure 3 The in vitro bactericidal effects of nano-photosensitizers (PMB-IFBTPP NPs) with different core photosensitizer ratios under different X-ray irradiation doses are shown. As can be seen from the figures, under 2.0 Gy X-ray irradiation, complete bacterial killing was achieved when the organic photosensitizer IFB:TPP ratio was 95:5 and 90:10. These results indicate that this nano-photosensitizer can achieve good antibacterial effects under X-ray irradiation of 2.0–2.5 Gy.

[0036] in conclusion: This invention relates to X-ray excited liposome-encapsulated organic photosensitizer nanoparticles, their preparation method, and their application in achieving highly efficient bactericidal activity under low-dose irradiation. Simplified formulations, represented by PMB-IFBTPP NPs, exhibit excellent low-dose bactericidal activity both in vitro and in vivo. A 99% in vitro bactericidal effect was achieved at an irradiation dose of 2.5 Gy. Therefore, PMB-IFBTPP NPs have the potential for further development into photosensitizers for treating deep-seated drug-resistant bacterial infections.

[0037] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles 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 nanoparticle containing an organic photosensitizer encapsulated in an X-ray excited liposome, comprising polymyxin B, a liposome carrier, an X-ray sensitizer, and an organic photosensitizer; The polymyxin B is covalently linked to the lipid carrier via an amide bond at a molar ratio of 1:

1. The liposome carrier is composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), and DSPE-PEG. 2000 and DSPE-PEG 2000 -NHS is composed of a molar ratio of 25-55:12.5-30:2.5-8:1; preferably, the molar ratio is 50-55:25-30:5-8:1; The organic photosensitizer and X-ray sensitizer are encapsulated in the liposome carrier, and the molar ratio of X-ray sensitizer:organic photosensitizer:liposome carrier is 9-9.9:0.1-1:

90.

2. The nanoparticles as described in claim 1, wherein, The X-ray sensitizer is 1,3,5-trifluoro-2,4,6-triiodobenzene (IFB).

3. The nanoparticles as described in claim 1 or 2, wherein, The organic photosensitizer is tetraphenylporphyrin (TPP).

4. The nanoparticles according to any one of claims 1-3, wherein, The molar ratio of the X-ray sensitizer to the organic photosensitizer is selected from any one of 90:10, 95:5, 97:3, and 99:

1.

5. The method for preparing nanoparticles according to any one of claims 1-4, comprising: 1) Add IFB, TPP, and DSPE-PEG 2000 DSPE-PEG 2000 NHS, DPPC, and cholesterol are added together to a container, followed by vector distillation of THF (tetrahydrofuran). The mixture is stirred until all components are completely dissolved, forming a homogeneous organic phase solution. The solution contains dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), and DSPE-PEG. 2000 and DSPE-PEG 2000 The molar ratio of -NHS is 25-55:12.5-30:2.5-8:1; preferably, the molar ratio is 50-55:25-30:5-8:1; 2) Slowly add the homogeneous organic phase solution obtained in step 1) to the Milli-Q ultrapure water in the ultrasonication process, and continue adding until a clear and transparent mixed solution is formed, so that the organic phase is uniformly dispersed in the aqueous phase and particle agglomeration is avoided; preferably, after the addition is completed, continue ultrasonic treatment for 5 min to further refine the size of nanoparticles and improve dispersion stability, and obtain nanoparticles with a particle size of 90-120 nm. 3) Place the mixed solution obtained in 2) on a stirrer, stir continuously and introduce compressed air to accelerate the evaporation of THF solvent, and concentrate to obtain a crude dispersion containing NHS-IFBTPP NPs; 4) Use 0.22 µ The coarse dispersion was filtered using a mPTFE membrane syringe filter. The filtered NHS-IFBTPP NPs solution was then added to a Merck Millipore centrifuge filter unit with a molecular weight cutoff of 50 kDa and centrifuged at 6000 rpm for 10 min. The concentrated particles were then washed several times with Milli-Q ultrapure water and concentrated to a concentration of 0.625 mg / ml. -1 NHS-IFBTPP NPs solution; 5) Take an appropriate amount of polymyxin B aptamer precipitate, add HEPES buffer (pH=8.2), and gently shake to completely dissolve the precipitate to obtain a polymyxin B aptamer solution; 6) Add the purified NHS-IFBTPP NPs solution to the polymyxin B aptamer solution, adjust the total volume of the reaction system so that the molar ratio of aptamer to NHS-IFBTPP NPs is 1.2:1, mix the reaction system evenly, and incubate the mixed reaction system at 4°C for more than 1.5 h. 7) After the reaction was complete, the reaction solution was transferred to a Merck Millipore centrifuge filter unit with a molecular weight cutoff of 100 kDa, and washed several times with ultrapure water to obtain a concentration of 0.1 mg / ml. -1 The PMB-IFBTPP NPs solution is obtained.

6. A pharmaceutical composition comprising nanoparticles as described in any one of claims 1-4, and pharmaceutically acceptable excipients.

7. The use of the nanoparticles according to any one of claims 1-4 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating drug-resistant bacterial infections.

8. The application according to claim 7, wherein, The drug-resistant bacterial infection is a deep tissue MRSA infection.

9. The application according to claim 7 or 8, wherein, The drug-resistant bacterial infection was MRSA lung infection.

10. The application according to any one of claims 7-9, comprising irradiating the infected tissue area with low-dose X-rays after applying the nanoparticles according to any one of claims 1-4 or the pharmaceutical composition according to claim 6; preferably, the low-dose X-ray irradiation is X-ray irradiation of 50 kV-25 mA.

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