Glycosylation modified polyethyleneimine and its use as an antituberculosis drug

Abstract

The application discloses a glycosylation modified polyethylene imine and application thereof as an anti-tuberculosis drug. The glycosylation modified polyethylene imine provided by the application is obtained by reacting polyethylene imine with gluconolactone, and one or more amino groups on the polyethylene imine are modified with a gluconolactone group L (HOCH2(CHOH)4CO-). Further, the anti-tuberculosis activity, cytotoxicity and stability of the glycosylation modified polyethylene imine are verified through a series of experiments, and the experimental results show that the toxicity of the gluconolactone modified polyethylene imine to normal cells is obviously reduced, the polyethylene imine has high inhibitory activity to tubercle bacillus, and the polyethylene imine has good stability, so the polyethylene imine has great clinical application potential as a new anti-tuberculosis drug.

Description

Technical Field

[0001] This invention relates to a glycosylated modified polyethyleneimine and its application as an anti-tuberculosis drug, belonging to the field of biomedical technology. Background Technology

[0002] Although the invention of antibiotics has somewhat reduced people's fear of tuberculosis, it remains a significant public health concern. For example, a large number of asymptomatic patients still exist in my country and globally, and drug resistance of Mycobacterium tuberculosis remains a prominent issue, making the development of novel anti-tuberculosis agents particularly urgent. Polycationic antibiotics have a different bactericidal mechanism than traditional antibiotics, thus avoiding drug resistance. However, due to their high charge, polycations are easily precipitated by proteins in blood and tissues, and this high charge also poses a significant toxicity to normal cells. Polyethyleneimine (PEI) is the gold standard for non-viral gene carriers and has recently been used in antibacterial materials, but its application has not been widespread due to its toxicity to normal tissues. PEI contains a large number of amino groups, which give it a crucial characteristic: a high positive charge. Polyethyleneimine (PEI) is a novel polycationic compound with water solubility. Based on its molecular structure, polyethyleneimine can be divided into two types: linear (L-PEI) and branched (B-PEI), as shown in the following molecular formula:

[0003]

[0004] Therefore, it is particularly important to find a way to effectively reduce cytotoxicity to normal tissues and retain antibacterial activity by modifying PEI in a convenient way, and it is of great significance to further apply it to Mycobacterium tuberculosis, which is a major hazard. Summary of the Invention

[0005] The purpose of this invention is to address the problems of drug resistance in existing antibiotic treatments for tuberculosis and the high toxicity of cationic antibacterial materials to normal tissues. This invention provides a glycosylated modified polyethyleneimine (named MCPI1) and its application as an anti-tuberculosis drug. MCPI1 is a PEI that is rapidly modified with gluconolactone using a quick and convenient modification method. Experimental results show that MCPI1 has significantly reduced toxicity to normal cells, but has high inhibitory activity against Mycobacterium tuberculosis and good stability. As a novel anti-tuberculosis drug, it has great potential for clinical application.

[0006] To address the aforementioned problems, the present invention provides a glycosylated polyethyleneimine. This glycosylated polyethyleneimine is a branched polyethyleneimine that, after reacting with gluconolactone, has one or more amino groups on its surface modified with gluconolactone groups L(HOCH2(CHOH)4CO-). The chemical structural formula of the gluconolactone groups L is shown below:

[0007]

[0008] Preferably, the glycosylated polyethyleneimine has the following chemical structural formula:

[0009]

[0010] Where n represents the degree of polymerization of polyethyleneimine.

[0011] Preferably, the method for preparing the glycosylated polyethyleneimine includes: dissolving branched polyethyleneimine and gluconolactone in an organic solvent, adding an organic base as an alkaline organic catalyst, and carrying out a stirring reaction under heating conditions.

[0012] Preferably, the organic solvent is an alcohol-based organic solvent, and the organic base is selected from at least one of triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), and diethylamine (DEA).

[0013] Preferably, the alcoholic organic solvent is selected from at least one of methanol, ethanol, and isopropanol. Methanol is the most preferred.

[0014] Preferably, the reaction further includes a purification step: first, soluble impurities and raw materials are precipitated in methanol to remove them, and then dialyzed in double-distilled water to remove small molecule impurities with a molecular weight ≤3KDa, thereby obtaining the purified product of gluconolactone-modified polyethyleneimine.

[0015] The present invention also provides the application of the above-mentioned glycosylated modified polyethyleneimine in the preparation of anti-tuberculosis drugs.

[0016] Preferably, the dosage form of the drug includes injections, tablets, powders, suspensions, capsules, pills, or syrups.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention provides a rapid and convenient modification method to quickly modify PEI with gluconolactone, resulting in a gluconolactone-modified PEI. This gluconolactone-modified PEI is characterized by high efficiency, safety, and stability, solving the technical problem of high toxicity to normal tissues in existing cationic antibacterial materials. The gluconolactone-modified PEI of this invention has significantly reduced toxicity to normal cells, but exhibits high inhibitory activity against Mycobacterium tuberculosis and good stability, showing great potential for clinical application as a novel anti-tuberculosis drug. Attached Figure Description

[0019] Figure 1 This is a route diagram for the preparation of PEI glycosylation modification;

[0020] Figure 2 The NMR spectrum of PEI glycosylation modification;

[0021] Figure 3 Results of anti-tuberculosis activity tests for glycosylated modified PEI: (A) lactosylation modification of PEI; (B) gluconolactone modification of PEI; (C) maltylation modification of PEI;

[0022] Figure 4 The results of cytotoxicity assays for PEI after glycosylation modification are as follows: (a) lactosylation modification of PEI; (b) gluconolactone modification of PEI; (c) maltylation modification of PEI.

[0023] Figure 5 The results of stability tests on PEI glycosylation modified products are as follows: (a) lactylation modification of PEI; (b) gluconolactone modification of PEI; (c) maltylation modification of PEI. Detailed Implementation

[0024] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The polyethyleneimine used in the examples is branched polyethyleneimine, purchased from Shanghai Mairui Chemical Technology Co., Ltd., with a molecular weight of 10000 Da.

[0026] Example

[0027] This embodiment provides a glycosylated modified polyethyleneimine and its application as an anti-tuberculosis agent:

[0028] 1. Preparation and characterization of PEI modified with gluconolactone

[0029] Preparation route as follows Figure 1As shown, polyethyleneimine (3.3 g) and gluconolactone (3.3 g) were dissolved in methanol (60 mL), and DIPEA (3.23 mL) was added. The mixture was stirred at 30 °C for 72 hours. PEI was conveniently modified by gluconolactone in methanol. The crude product precipitated in methanol, and after dialyzing in double-distilled water (molecular weight cutoff MWCO = 3 kDa), it was precipitated again to obtain the final product. NMR characterization confirmed successful modification (e.g., Figure 2 The gluconolactone-modified PEI was named MCPI1, also simply referred to as glycosylated PEI. For comparison, lactosylated and maltosylated PEIs were prepared using the same method with lactose and maltose, respectively.

[0030] 2. Anti-tuberculosis activity test of gluconolactone-modified PEI (glycosylated PEI, MCPI1)

[0031] The anti-tuberculosis activity of the successfully prepared gluconolactone-modified PEI was evaluated. *Mycobacterium bovis Bacille Calmette-Guérin* (BCG) or *Mycobacterium tuberculosis* (Mtb) were cultured in Middlebrook 7H9 liquid medium (hereinafter referred to as 7H9 medium) containing 10% OADC at 37°C using a shaker. OD600 was observed and measured daily. When the OD600 value was approximately 1 (in the logarithmic growth phase), bacterial suspensions were collected for antibacterial experiments. Nanomaterials were serially diluted using 7H9 medium and added to 96-well U-bottom plates. Simultaneously, BCG / Mtb bacterial suspensions were diluted using 7H9 medium to adjust the initial OD to 0.001 and added to 96-well plates containing different concentrations of nanomaterials, with a total volume of 200 μl / well. The concentration gradients of nanomaterials were 1 mg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.25 μg / ml, 15.625 μg / ml, and 7.8125 μg / ml. Wells without nanomaterials served as negative controls. The culture plates were incubated at 37°C for one week. During this period, the bacterial concentration in each well was observed every other day, and 100 μl of each plate was spread onto Middlebrook 7H11 agar plates containing 10% OADC (10-fold dilution, 3-4 concentration gradients). After incubation at 37°C for three weeks, colony counting was performed, and the bacterial load was calculated based on the colony count and dilution concentration. Figure 3 As shown, gluconolactone-modified PEI exhibits significant antibacterial activity, and lactosylated, gluconolactone-modified, and maltose-modified PEIs also show similar antibacterial activities.

[0032] 3. Safety testing of PEI modified with gluconolactone

[0033] Safety is crucial for antimicrobial materials. The cytotoxicity of three glycosylated PEIs was determined using the MTT assay, with conventional somatic 293T cells used as the evaluation model. Figure 4 As shown, the cytotoxicity of PEI was significantly reduced after three glycosylation modifications, with gluconolactone-modified PEI exhibiting the lowest cytotoxicity.

[0034] 4. Stability test of PEI modified with gluconolactone

[0035] The stability of antibacterial agents is also very important. The stability of PEI modified by lactosylation, gluconic acid lactone modification, and maltylation was determined. PEI modified by all three methods showed good solubility in aqueous solution. After being placed at room temperature for 3 months, gluconic acid lactone modified PEI was still soluble in water, while PEI modified by the other two methods was no longer soluble in water, which should be due to cross-linking.

[0036] Based on a comprehensive evaluation of its anti-tuberculosis activity, cytotoxicity, and stability, gluconolactone-modified PEI is a promising anti-tuberculosis drug.

[0037] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of a glycosylated polyethyleneimine in the preparation of anti-tuberculosis drugs, characterized in that, The glycosylated polyethyleneimine is a branched polyethyleneimine that, after reacting with gluconolactone, has one or more amino groups L modified onto it. The chemical structural formula of the gluconolactone group L is shown below: 。 2. The application as described in claim 1, characterized in that, The glycosylated polyethyleneimine has the following chemical structural formula: ； Where n represents the degree of polymerization of polyethyleneimine.

3. The application as described in claim 2, characterized in that, The method for preparing the glycosylated modified polyethyleneimine includes: dissolving branched polyethyleneimine and gluconolactone in an organic solvent, adding an organic base as an alkaline organic catalyst, and carrying out a stirring reaction under heating conditions.

4. The application as described in claim 3, characterized in that, The organic solvent is an alcohol-based organic solvent, and the organic base is selected from at least one of triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), and diethylamine (DEA).

5. The application as described in claim 4, characterized in that, The alcoholic organic solvent is selected from at least one of methanol, ethanol and isopropanol.

6. The application as described in claim 3, characterized in that, After the reaction is completed, a purification step is also included: first, soluble impurities and raw materials are removed by precipitation in methanol, and then dialyzed in double-distilled water to remove small molecule impurities with a molecular weight ≤3KDa, thereby obtaining the purified product of gluconolactone-modified polyethyleneimine.

7. The application as described in claim 1, characterized in that, The dosage forms of the drug include injections, tablets, powders, suspensions, capsules, pills, or syrups.

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