Airway basal cell-N-acetylcysteine conjugate and application thereof

Abstract

The invention discloses an airway basal cell-N-acetylcysteine conjugate, which is prepared by the following steps: adding DMPE-PEG-maleimide and N-acetylcysteine into a solution, incubating at room temperature, and centrifuging, so as to obtain a DMPE-PEG-maleimide-N-acetylcysteine compound; and adding the DMPE-PEG-maleimde-N-acetylcysteine compound into a cell suspension of human airway basal cells, and incubating at room temperature to obtain the human airway basal cell suspension. According to the invention, DMPE-PEG-maleimide is used as a non-invasive carrier, and airway basal cells and a drug-N-acetylcysteine are coupled; during treatment, inflammatory response can be controlled while lung injury is effectively repaired, cells can play a homing role, and the medicine is carried to reside at the injured part for long-acting treatment. The research of the invention is of great significance to the treatment of pneumonia.

Description

Technical Field

[0001] This invention relates to an airway basal cell-N-acetylcysteine ​​conjugate and its application in the preparation of a drug for treating pneumonia, belonging to the field of cell therapy and drug therapy technology. Background Technology

[0002] As is well known, the lung epithelium, exposed to the external environment, is crucial for coordinating the body's initial immune response to pathogens. In lung lesions, alveolar epithelial cells are often lost. This impairment of epithelial integrity prevents newly differentiated epithelial cells from effectively repairing damaged areas, thereby disrupting the maintenance of an effective physical barrier. The alveolar structure is then replaced by excessively proliferating fibroblasts and inflammatory cells, ultimately leading to serious consequences. Traditional drugs are ineffective in intervening in this process, necessitating the development of novel treatments.

[0003] The proximal airways in humans, including the trachea and main bronchi, are lined with columnar epithelium composed of basal cells and luminal cells. Basal cells function as stem cells within this epithelium, capable of self-renewal and differentiating into ciliated and rod-shaped cells. Irreversible damage to the bronchi and alveoli can lead to a variety of incurable lung diseases. Research has demonstrated that airway basal cells, located in the airway epithelium, can rebuild functional tissue, offering hope for reversing damage and curing such diseases. Researchers are increasingly interested in tissue regeneration as a novel treatment strategy for lung diseases, and adoptive transfer techniques, which transplant cultured cells from vitro into the host, have shown promising clinical results. However, these cell-based therapies face a common challenge: the inability to effectively modulate cell function to maximize therapeutic benefits, while traditional drug treatments often cannot directly target the affected area and maintain their effects for extended periods.

[0004] N-acetylcysteine ​​is a chemical substance that has been found to have multiple effects, including improving respiratory function, anti-oxidation, enhancing immune function, and reducing the number of airway bacteria. It protects lung structure through mechanisms such as stimulating the secretion of alveolar surfactant and inhibiting elastase from damaging alveolar elastin, making it a valuable adjuvant drug in the comprehensive treatment of pneumonia. Summary of the Invention

[0005] To address the aforementioned limitations of existing technologies, this invention provides an airway basal cell-N-acetylcysteine ​​conjugate and its application in the preparation of drugs for treating pneumonia. Addressing the issues of drugs failing to target the affected area, short retention time, and rapid metabolism, this invention modifies mesenchymal stem cells and uses chemical conjugation technology to directly anchor drug particles to the cell membrane, thereby achieving continuous drug stimulation at the transplantation site in vivo and enhancing targeted homing effects.

[0006] This invention is achieved through the following technical solution: A basal cell-N-acetylcysteine ​​conjugate for airways is prepared by the following method: DMPE-PEG-maleimide and N-acetylcysteine ​​are added to a solution, incubated at room temperature for 1.5–2.5 hours, and centrifuged to obtain a DMPE-PEG-maleimide-N-acetylcysteine ​​complex; the DMPE-PEG-maleimide-N-acetylcysteine ​​complex is added to a suspension of human basal cells for airways, and incubated at room temperature for 1–4 minutes to obtain the basal cell-N-acetylcysteine ​​conjugate for airways.

[0007] The DMPE-PEG-maleimide is a functionalized molecule composed of dimyristoyl phospholipid (DMPE), polyethylene glycol (PEG), and maleimide. It is widely used in drug delivery, biomarking, and nanomaterials and is readily available for purchase.

[0008] Further, the concentration of DMPE-PEG-maleimide is 0.1–1.0 mg / ml, preferably 0.5 mg / ml; the concentration of N-acetylcysteine ​​is 20–60 μM, preferably 40 μM.

[0009] Furthermore, in the cell suspension of the human airway basal cells, the density of the human airway basal cells is 0.5 × 10⁻⁶. 6 ~2×10 6 Cells / mL, preferably 1×10⁻⁶ 6 Cells / mL

[0010] The application of the airway basal cell-N-acetylcysteine ​​conjugate in the preparation of a drug for treating pneumonia.

[0011] The airway basal cell-N-acetylcysteine ​​conjugate of this invention uses DMPE-PEG-maleimide as a non-invasive carrier to conjugate airway basal cells and the drug N-acetylcysteine. During treatment, the N-acetylcysteine ​​conjugate in airway basal cells can effectively repair lung damage while further controlling the inflammatory response. Furthermore, the cells can exhibit homing activity, carrying the drug to the damaged site for long-term therapeutic effect, thus providing drug therapy simultaneously with cell therapy. This invention has significant implications for the treatment of pneumonia. Attached Figure Description

[0012] Figure 1 Microscopic photograph of isolated and cultured airway basal cells.

[0013] Figure 2 Photograph of fluorescently labeled airway basal cells.

[0014] Figure 3 Flow cytometry results. Detailed Implementation

[0015] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0016] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0017] Example 1: Acquisition and identification of basal cells of human airway Obtaining human airway basal cells: Human airway epithelial tissue was obtained using a bronchial brush. The bronchial brush was rinsed with sterile physiological saline, and cells were collected. The collected cells were centrifuged at 400×g for 5 minutes and then resuspended in complete bronchial epithelial cell growth medium (BEGM, purchased from Lonza Group Ltd.) to a final volume of 5×10⁻⁶. 5 cells / mL, followed by 1×10 5 Cells were seeded at a density of 100 cells / mL in collagen-coated tissue culture plates and cultured. After two passages, the cells were digested and collected for subsequent experiments.

[0018] Identification of human airway basal cells: After passage culture of the above-mentioned human airway basal cells, P3 generation cells were taken, washed three times with PBS, fixed with 4% paraformaldehyde solution at room temperature for 10 minutes, and permeabilized in 0.1% Triton X-100 at room temperature for 10 minutes. Cells were washed three times with PBS and blocked with 2% BSA at room temperature for 60 minutes. The blocked cells were incubated with human Krt5 monoclonal antibody and human p63 monoclonal antibody at 37°C for 2 hours. The cells were washed three times with PBS and incubated with fluorescent Alexa Fluor488-labeled secondary antibody at room temperature in the dark for 1 hour. The cells were washed with PBS, incubated with DAPI for 10 minutes, and images of the fluorescently labeled cells were captured using a fluorescence microscope.

[0019] Results: After 7 days of culture in collagen-coated tissue culture plates, cuboidal cell clusters with round nuclei located in the center and sparse cytoplasm could be observed under a microscope; these were identified as airway basal cells. Figure 1 As shown. After incubating cells with human Krt5 monoclonal antibody and human p63 monoclonal antibody, and then binding with fluorescent secondary antibody, positive results were observed under a fluorescence microscope, as shown. Figure 2As shown, the cultured cells are confirmed to be basal epithelial cells with stem cell function.

[0020] Example 2: Coupling of human airway basal cells with N-acetylcysteine Dissolve 1 mg of DMPE-PEG-maleimide in 1 ml of PBS, then add 10 mg / ml of FITC (a green fluorescein derivative, effective against pH and Cu). 2+ (Sensitive) solution. Incubate gently with shaking at room temperature in the dark for 1 hour. Remove excess FITC using a dye removal column. Add N-acetylcysteine ​​to the solution to a concentration of 40 μM, incubate at room temperature for 2 hours, and centrifuge at 400 g for 10 minutes to remove impurities, yielding the DMPE-PEG-maleimide-FITC-N-acetylcysteine ​​complex for later use.

[0021] Human airway basal cells cultured in Example 1 were taken and suspended in phenol red-free culture medium (BEGM medium), and the cell density was adjusted to 1×10⁻⁶. 6 Cells were cultured at a density of 100 cells / mL to obtain a cell suspension. The prepared DMPE-PEG-maleimide-FITC-N-acetylcysteine ​​complex was added to 1 mL of the cell suspension and incubated at room temperature for 2 minutes. After incubation, cells were collected, washed, and resuspended in HBSS buffer (Hank's balanced salt solution, a cell culture buffer), adjusting the cell density to 1 × 10⁻⁶ cells / mL. 6 Cells / mL

[0022] Identification of cell-drug conjugates: The percentage of FITC-positive cells was determined using flow cytometry. Results are as follows: Figure 3 As shown, 84.21% of cells were successfully conjugated with the carrier drug, indicating a high success rate.

[0023] Preparation of the airway basal cell-N-acetylcysteine ​​conjugate: 1 mg of DMPE-PEG-maleimide was dissolved in 1 ml of PBS, and N-acetylcysteine ​​was added to a concentration of 40 μM. The mixture was incubated at room temperature for 2 hours and centrifuged at 400 g for 10 minutes to obtain the DMPE-PEG-maleimide-N-acetylcysteine ​​complex. Human airway basal cells cultured in Example 1 were taken and suspended in BEGM complete bronchial epithelial cell growth medium, and the cell density was adjusted to 1 × 10⁶ cells / mL. 6 Cells were collected at a density of 100 cells / mL to obtain a cell suspension. The prepared DMPE-PEG-maleimide-N-acetylcysteine ​​complex was added to 1 mL of the cell suspension and incubated at room temperature for 2 minutes. After incubation, cells were collected, washed, and resuspended in HBSS buffer to adjust the cell density to 1 × 10⁻⁶ cells / mL. 6One cell / mL yields the airway basal cell-N-acetylcysteine ​​conjugate.

[0024] Experiment 1: Modeling, treatment, and efficacy detection of pneumonia in rats Thirty SD rats (28 days old, weighing 220±10 g, half male and half female) were used and instilled with Klebsiella pneumoniae bacterial solution (1.2×10⁻⁶ g) via tracheal instillation. 14 A pneumonia model was established by administering 0.3 mL of cfu / L (0.3 mL / rat) to each rat. The model was considered successful if the rats exhibited sluggishness or rapid breathing.

[0025] Rats that successfully developed the model were randomly divided into a coupled therapy group, a cell therapy group, and a control group (n=10 per group). Rats in the coupled therapy group were administered 1×10⁻⁶ cells / mL. 6 1 cell / kg body weight of airway basal cell-N-acetylcysteine ​​conjugate (prepared in Example 2) was infused into the lung lobe via bronchoscopy. Rats in the cell therapy group received 1×10⁻⁶ cells / kg body weight. 6 Human airway basal cell suspension (prepared in Example 2) was infused into the lung lobes via bronchoscopy at a rate of 1 cell / kg body weight. The control group received the same volume of physiological saline in the same manner.

[0026] Before treatment and 14 days after treatment, blood was collected from the tail vein of rats, and serum was collected by centrifugation and then tested by serum ELISA.

[0027] Results: The results of serum inflammatory marker detection in rats before and after treatment are shown in Table 1. ELISA results of serum inflammatory markers showed that serum CRP (C-reactive protein) decreased by an average of 5.3 in the conjugate treatment group, decreased by 4.9 in the cell therapy group, and increased by 2.4 in the control group; ESR (electrodesorption / reduction ratio) decreased by 8.1 in the conjugate treatment group, decreased by 6.8 in the cell therapy group, and decreased by 2.6 in the control group; LDH (lactate dehydrogenase) decreased by 23 in the conjugate treatment group, decreased by 9 in the cell therapy group, and increased by 1 in the control group.

[0028] Table 1. Results of serum inflammatory markers in rats before and after treatment. Group detection indicators Before treatment After treatment p-value Coupled therapy group CRP 7.6 2.3 0.003 ESR 14.9 6.8 <0.001 LDH 362 339 0.004 Cell therapy group CRP 8.0 3.1 <0.001 ESR 16.2 9.4 <0.001 LDH 359 350 <0.001 control group CRP 6.0 8.4 0.039 ESR 15.3 12.7 0.085 LDH 355 356 0.489 Based on the data in Table 1, it can be inferred that both the conjugate therapy group and the cell therapy group, compared with the control group, can produce beneficial effects in reducing inflammatory markers in rats. The conjugate therapy group has a better therapeutic effect than the cell therapy group and can better control inflammation.

[0029] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An airway basal cell-N-acetylcysteine conjugate, characterized in that, The DMPE-PEG-maleimide-N-acetylcysteine conjugate is prepared by the following method: adding DMPE-PEG-maleimide and N-acetylcysteine into the solution, incubating at room temperature for 1.5-2.5 hours, centrifuging to obtain DMPE-PEG-maleimide-N-acetylcysteine complex; adding the DMPE-PEG-maleimide-N-acetylcysteine complex into the cell suspension of human airway basal cells, incubating at room temperature for 1-4 minutes to obtain the airway basal cell-N-acetylcysteine conjugate.

2. The airway basal cell-N-acetylcysteine conjugate of claim 1, wherein: The concentration of the DMPE-PEG-maleimide is 0.1-1.0 mg / ml; the concentration of the N-acetylcysteine is 20-60 μ.

3. The airway basal cell-N-acetylcysteine conjugate of claim 1, wherein: The density of the human airway basal cells in the cell suspension of the human airway basal cells is 0.5 x 10 6 ~ 2 x 10 6 cells per milliliter.

4. Use of the airway basal cell-N-acetylcysteine conjugate of any one of claims 1-3 in the preparation of a medicament for treating pneumonia.

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