pH-responsive degradable amphiphilic dextran block polymers, cationic antimicrobial peptide nanomedicines and applications

Abstract

The application provides a pH-responsive disintegrating amphiphilic dextran block polymer, a cationic antibacterial peptide nanodrug and application, utilizes the pH-responsive disintegrating amphiphilic dextran block polymer and the nanometer AMPs to utilize the electrostatic force, the N-B coordination bond force and the guanyl-π electron force to self-assemble into the cationic antibacterial peptide nanodrug, realizes the effective capture of the nanometer AMPs, not only improves the drug loading of the amphiphilic dextran block polymer, but also can protect the free amino group in the nanometer AMPs residue, improves the stability of the nanometer AMPs to protease, reduces the toxicity to normal cells and reduces the risk of hemolysis; and the cationic antibacterial peptide nanodrug gradually hydrolyzes under the weak acid environment of the infection site, so that the drug is enriched at the infection site and the bioavailability is improved, and a new solution is provided for overcoming the increasingly serious drug-resistant bacterial infection problem in clinic.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, in particular to a pH-responsive decomposable amphiphilic dextran block polymer, a cationic antimicrobial peptide nanomedicine and application. BACKGROUND

[0002] Antimicrobial peptides (AMPs) are important immune molecules in eukaryotes to protect the host from invading pathogens. Unlike traditional antibiotics, which act by destroying cell membranes, interrupting DNA replication or protein synthesis mechanisms, AMPs can act on non-specific targets in multiple ways, making them less likely to develop drug resistance. These characteristics make AMPs a powerful weapon against most drug-resistant bacteria and are considered the most promising new generation of antimicrobial drugs in the post-antibiotic era. However, although more than 5000 AMPs have been isolated and identified, only a few have entered clinical applications. The large number of cationic amino acids (lysine, arginine) in AMPs are easily the target site of proteases, resulting in poor circulation stability of AMPs in animals and even the risk of hemolysis. Due to these limitations, most AMPs are limited to topical use, such as ear infections (Gramicidin S), eye infections, etc. Therefore, improving the circulation stability of AMPs in animals is a bottleneck problem for AMPs to fully enter clinical applications.

[0003] To this end, scientists have invented extremely inspiring strategies including chemical modification, nanoparticle drug delivery systems, and hydrogel embedding, which have successfully improved the protease stability of AMPs and reduced their cytotoxicity. However, chemical modification often involves changes to the chemical structure (primary and secondary) of AMPs, making it difficult to guarantee their original antimicrobial activity and mechanism of action. Some nanoparticles (metals, carbon nanotubes, etc.) are difficult to degrade after entering the animal body, and most hydrogels cannot be administered systemically by intravenous injection due to stability reasons, which greatly limits the clinical application of AMPs drug delivery systems. SUMMARY

[0004] To solve the problems in the prior art, the present application provides a pH-responsive decomposable amphiphilic dextran block polymer, a cationic antimicrobial peptide nanomedicine and application. The antimicrobial peptide nanomedicine prepared has pH hypersensitivity, solving the key problems of poor circulation stability and low bioavailability of AMPs biological active molecules.

[0005] In order to achieve the above object, the present application provides the following technical scheme: an amphiphilic dextran block polymer with pH-responsive decomposition, comprising a carrier chain main body: dextran; a drug-carrying functional group: phenylboronic acid group; and a pH-responsive element: ortho ester group, wherein the amphiphilic dextran block polymer with pH-responsive decomposition is obtained by co-dissolving dextran, aminophenylboronic acid and ortho ester amine in DMSO and performing condensation coupling reaction through dicarbonyl carbonate.

[0006] Further, the molar ratio of the use amount of dextran, aminophenylboronic acid and ortho ester amine is 1:1.3:0.1.

[0007] Further, the ortho ester amine includes methyl ortho ester amine, butyl ortho ester amine or octyl ortho ester amine, and the aminophenylboronic acid includes 3-aminophenylboronic acid or 2-aminophenylboronic acid, etc.

[0008] The present application also provides application of the above-mentioned amphiphilic dextran block polymer as a cationic antibacterial peptide carrier.

[0009] The present application also provides application of the above-mentioned amphiphilic dextran block polymer in preparation of a cationic antibacterial peptide nanodrug.

[0010] The present application also provides a cationic antibacterial peptide nanodrug, which comprises a cationic antibacterial peptide and the amphiphilic dextran block polymer according to any one of claims 1-3, wherein the cationic antibacterial peptide nanodrug is obtained by self-assembly of the cationic antibacterial peptide and the amphiphilic dextran block polymer through N-B coordination bond force and piperidyl-π electron effect.

[0011] Further, the use amount ratio of the amphiphilic dextran block polymer and the cationic antibacterial peptide is 1:(0.1-0.4), the cationic antibacterial peptide and the functionalized dextran block polymer material are co-dissolved in DMSO, stirred, dialyzed and freeze-dried to obtain the cationic antibacterial peptide nanodrug.

[0012] Further, the cationic antibacterial peptide nanodrug disintegrates to release antibacterial peptide bioactive components under the condition of pH value 5.5-6.5.

[0013] The present application also provides application of the cationic antibacterial peptide nanodrug as a medicine for treating infectious diseases caused by gram-negative drug-resistant bacteria.

[0014] The present application also provides a medicine for treating infectious diseases caused by gram-negative drug-resistant bacteria, characterized in that the medicine comprises the above-mentioned cationic antibacterial peptide nanodrug.

[0015] Compared with the prior art, the present application has at least the following beneficial effects:

[0016] The application provides a cationic antibacterial peptide nanomedicine, which is prepared by modifying dextran with an ortho ester group and an aminophenylboronic acid through a CDI chemical coupling reaction to obtain a pH-responsive decomposition amphiphilic dextran block polymer, and the pH-responsive decomposition amphiphilic dextran block polymer and nano-AMPs are self-assembled into the cationic antibacterial peptide nanomedicine by using electrostatic force, N-B coordination force and aziridine group-π electron force, wherein the amphiphilic dextran block polymer takes a negative macromolecule Dextran as a basic skeleton, has the advantages of safety, non-toxicity and difficulty in being phagocytosed by a mononuclear phagocyte system (MPS), and can also load positive nano-AMPs through electrostatic force; the aminophenylboronic acid is used as a drug loading functional group, and the boron atom and the benzene ring in the aminophenylboronic acid group can produce nitrogen-boron coordination and aziridine group-π electron action with the amino group and the aziridine group in the antibacterial peptide residue, so that the nano-AMPs can be effectively grabbed, the drug loading capacity of the amphiphilic dextran block polymer is improved, the free amino group in the nano-AMPs residue is protected, the stability of the nano-AMPs to protease is improved, the toxicity to normal cells is reduced, and the risk of hemolysis is reduced; the ortho ester group is used as a hydrophobic source of a drug delivery system and a pH-responsive element, the chemical structure is stable under a neutral pH condition, and is gradually hydrolyzed under an acidic environment at an infection site, so that the drug can be enriched at the infection site and the bioavailability is improved.

[0017] Further, the application can also adjust the amphiphilic property of the functionalized amphiphilic dextran block polymer by adjusting the length of the ortho ester group fatty chain, so as to adjust the morphology of the amphiphilic dextran block polymer. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a preparation process of the cationic antibacterial peptide nanomedicine;

[0019] Figure 2 It is a preparation route diagram of Dex-PBA-OM;

[0020] Figure 3 It is a DLS analysis of THANPs with different Thanatin contents;

[0021] Figure 4 It is an analysis diagram of the encapsulation efficiency and drug loading capacity of THANPs

[0022] Figure 5 It is the particle size change of THANPs-3 under the conditions of pH 7.4, 6.5 and 5.5 (n=3);

[0023] Figure 6 It is the drug release curve of THANPs-3 under the conditions of pH 7.4, 6.5 and 5.5. DETAILED DESCRIPTION

[0024] The application will be further described in conjunction with the accompanying drawings and specific embodiments.

[0025] As shown in Figure 1 The present application provides a kind of pH response decomposition amphiphilic dextran block polymer, including chain carrier main body: dextran (Dextran), drug carrying functional group: amino phenyl boric acid (PBA), for providing reliable binding site for AMP, and hydrophobic acid response chemical group;Oxime ester amine (OM), for being used as hydrophobic source and pH response element, can promote the formation of nano drug-loaded micelles, dextran is functionally modified by amino phenyl boric acid and oxime ester amine using dicarbonyl carbonate CDI chemical coupling reaction, and amphiphilic dextran block polymer is obtained.

[0026] The present application also provides a kind of cationic antimicrobial peptide nano drug, nano AMPs are co-dissolved in DMSO with the above-mentioned amphiphilic dextran block polymer, nano drug solution is prepared by dialysis method, then it is freeze-dried, and cationic antimicrobial peptide nano drug is obtained, the above-mentioned amphiphilic dextran block polymer is loaded with nano AMPs by N-B coordination bond force and guanyl-π electron effect, and oxime ester structure is used as acid pH response functional group to promote the drug release of nano micelles in acidic microenvironment response.

[0027] Further, the particle size of the prepared cationic antimicrobial peptide nano drug is about 200nm, PDI is less than 0.2, the encapsulation efficiency is not less than 65%, the drug loading capacity is not less than 15%, the active ingredient of AMPs is released in response to the acid environment with pH value of 5.5-6.5, the drug sustained release time is 1 hour when pH is 5.5, the drug release time is 4 hours when pH is 6.5, the cumulative drug release rate is higher than 90%, and no drug is released under neutral conditions.

[0028] Cationic amino acids (lysine, arginine) are commonly present in AMPs (such as Thanatin, Spinigerin, Protegrin-1, Termicin, Tachyplesin I, etc.), which are important constituent units for AMPs to exhibit cation-related properties (antibacterial, antitumor, antiviral).

[0029] As shown in Figure 2 The present application provides a kind of pH response decomposition amphiphilic dextran block polymer, including chain carrier main body: dextran (Dextran), drug carrying functional group: amino phenyl boric acid (PBA), for providing reliable binding site for AMP, and hydrophobic acid response chemical group;Oxime ester amine (OM), for being used as hydrophobic source and pH response element, can promote the formation of nano drug-loaded micelles, dextran is functionally modified by amino phenyl boric acid and oxime ester amine using dicarbonyl carbonate CDI chemical coupling reaction, and amphiphilic dextran block polymer is obtained.

[0030] Under argon protection, dextran with molecular weight of 20KDa is dissolved in anhydrous DMSO dimethyl sulfoxide (concentration of 20-30mg / mL), then dicarbonyl imidazole (CDI) is added, and stirring is carried out at room temperature for 2h to fully activate the hydroxyl groups in dextran.

[0031] Then, the orthoester amine is added, and after 2 hours of reaction, the amino phenyl boric acid (PBA) is added, and the reaction is continued for 24 hours.

[0032] After the reaction is completed, the mixed solution is dialyzed in a dialysis bag with a molecular weight cut-off of 3500 Da for 48 hours, and after freeze-drying, a series of amputated Dex-PBA-OM containing different orthoester amines are obtained, that is, cationic antimicrobial peptide nano-drug-loaded micelles are obtained.

[0033] Preferably, the orthoester amine includes methyl orthoate amine, butyl orthoate amine or octyl orthoate amine.

[0034] Preferably, the amino phenyl boric acid includes 3-amino phenyl boric acid, 2-amino phenyl boric acid and other amino-substituted phenyl boric acid compounds.

[0035] As shown in Figure 2 The application also provides a preparation method of the cationic antimicrobial peptide nano-drug, and the specific steps are as follows:

[0036] The nano AMPs of the application select Thanatin (denoted as THA), which is an AMPs derived from hemipteran insects, has high inhibitory activity on bacteria including various gram-negative drug-resistant bacteria, and contains four primary amine groups and three pyrrolyl groups in the amino acid residues.

[0037] Dex-PBA-OM and THA are co-dissolved in DMSO at a mass ratio of 1:(0.1-0.4), and stirred at 25℃ for 1 hour (8000 rmp / min). After dialysis in deionized water (containing 0.1% NEt3) for 48 hours, Thanatin nano-drug (THANPs) is obtained after freeze-drying.

[0038] The THANPs prepared by the application are administered by intravenous injection, the EPR effect of the nanoparticles can make them be directionally enriched in the infected site, and the acidic microenvironment of the infected site promotes the disintegration of the nanoparticles to release the active ingredients of AMPs, which will help to improve the circulation stability and bioavailability of AMPs, and can be used for the treatment of systemic infection diseases caused by gram-negative drug-resistant bacteria.

[0039] Specifically, the gram-negative drug-resistant bacteria are carbapenem-resistant Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and the like.

[0040] Example 1

[0041] A preparation method of a pH-responsive disintegrating amphiphilic dextran block polymer, and the specific steps are as follows:

[0042] Under argon protection, dextran with molecular weight of 20 KDa was dissolved in anhydrous DMSO (concentration of 20 mg / mL), then carbonyldiimidazole (CDI) was added, and stirred at room temperature for 2 h to fully activate the hydroxyl groups in dextran;

[0043] Then, methyl laurate amine was added, and after 2 h of reaction, 3-aminobenzoic acid was added, and the reaction was continued for 24 h.

[0044] After the reaction was completed, the mixture was dialyzed in a dialysis bag with a molecular weight cut-off of 3500 Da for 48 h, and then freeze-dried to obtain the functionalized dextran block polymer Dex-PBA-OM-Methyl.

[0045] Example 2

[0046] A preparation method of a pH-responsive decomposable amphiphilic dextran block polymer, the specific steps are as follows:

[0047] Under argon protection, dextran with molecular weight of 20 KDa was dissolved in anhydrous DMSO (concentration of 20 mg / mL), then carbonyldiimidazole (CDI) was added, and stirred at room temperature for 2 h to fully activate the hydroxyl groups in dextran;

[0048] Then, butyl laurate amine was added, and after 2 h of reaction, 2-aminobenzoic acid was added, and the reaction was continued for 24 h.

[0049] After the reaction was completed, the mixture was dialyzed in a dialysis bag with a molecular weight cut-off of 3500 Da for 48 h, and then freeze-dried to obtain the functionalized dextran block polymer Dex-PBA-OM-Butyl.

[0050] Example 3

[0051] A preparation method of a pH-responsive decomposable amphiphilic dextran block polymer, the specific steps are as follows:

[0052] Under argon protection, dextran with molecular weight of 20 KDa was dissolved in anhydrous DMSO (concentration of 20 mg / mL), then carbonyldiimidazole (CDI) was added, and stirred at room temperature for 2 h to fully activate the hydroxyl groups in dextran;

[0053] Then, octyl laurate amine was added, and after 2 h of reaction, 3-aminobenzoic acid was added, and the reaction was continued for 24 h.

[0054] After the reaction was completed, the mixture was dialyzed in a dialysis bag with a molecular weight cut-off of 3500 Da for 48 h, and then freeze-dried to obtain the functionalized dextran block polymer Dex-PBA-OM-Octyl.

[0055] Example 4

[0056] A preparation method of a cationic antibacterial peptide nanomedicine, the specific steps are as follows:

[0057] The dextran block polymer Dex-PBA-OM-Butyl prepared in Example 2 is used;

[0058] 20mg Dex-PBA-OM-Butyl is dissolved in 5mL DMSO, mixed with 1mL DMSO containing 2mg THA respectively, and stirred vigorously for 1.0h. Dialysis is performed with deionized water (containing 0.1% NEt3) for 48h, and thanatin nanomedicine THANPs-1 with an encapsulation efficiency of 93% and a drug loading of 8% is obtained after freeze-drying.

[0059] Example 5

[0060] A preparation method of a cationic antibacterial peptide nanomedicine, the specific steps are as follows:

[0061] The dextran block polymer Dex-PBA-OM-Butyl prepared in Example 2 is used;

[0062] 20mg Dex-PBA-OM-Butyl is dissolved in 5mL DMSO, mixed with 1mL DMSO containing 4mg THA respectively, and stirred vigorously for 1.0h. Dialysis is performed with deionized water (containing 0.1% NEt3) for 48h, and thanatin nanomedicine THANPs-2 with an encapsulation efficiency of 87% and a drug loading of 14% is obtained after freeze-drying.

[0063] Example 6

[0064] A preparation method of a cationic antibacterial peptide nanomedicine, the specific steps are as follows:

[0065] The dextran block polymer Dex-PBA-OM-Butyl prepared in Example 2 is used;

[0066] 20mg Dex-PBA-OM-Butyl is dissolved in 5mL DMSO, mixed with 1mL DMSO containing 6mg THA respectively, and stirred vigorously for 1.0h. Dialysis is performed with deionized water (containing 0.1% NEt3) for 48h, and thanatin nanomedicine THANPs-3 with an encapsulation efficiency of 73% and a drug loading of 16.8% is obtained after freeze-drying.

[0067] Example 7

[0068] A preparation method of a cationic antibacterial peptide nanomedicine, the specific steps are as follows:

[0069] The dextran block polymer Dex-PBA-OM-Butyl prepared in Example 2 is used;

[0070] THANPs-4: 20 mg Dex-PBA-OM-Butyl was dissolved in 5 mL DMSO, mixed with 1 mL DMSO containing 8 mg THA, and stirred vigorously for 1.0 h. Dialysis was performed in deionized water (containing 0.1% NEt3) for 48 h, and the freeze-dried product had an encapsulation efficiency of 60% and a drug loading of 17%.

[0071] Example 8

[0072] A method for preparing a cationic antimicrobial peptide nanomedicine, the specific steps being as follows:

[0073] The dextran block polymer Dex-PBA-OM-Butyl prepared in Example 2 was used;

[0074] THANPs-5: 20 mg Dex-PBA-OM-Butyl was dissolved in 5 mL DMSO, mixed with 1 mL DMSO containing 10 mg THA, and stirred vigorously for 1.0 h. Dialysis was performed in deionized water (containing 0.1% NEt3) for 48 h, and the freeze-dried product had an encapsulation efficiency of 52% and a drug loading of 15%.

[0075] The antimicrobial agent THANPs prepared in the above examples was tested for properties, as follows:

[0076] (1) Dynamic light scattering experiment (DLS) was performed on the nanomedicine.

[0077] Instrument: Malvern particle size analyzer, test conditions 25°C.

[0078] Results analysis: As shown in Figure 3 , with the increase of THA content, the particle size of THANPs first decreased and then increased. When the mass ratio of Thanatin to Dex-PBA-OM-Butyl was 3:10, the particle size of the obtained nanomedicine was 185.1 nm, PDI: 0.185, and Zeta potential -30.2 mV. When the mass ratio of THA to drug-loaded material was 4:10, the particle size of the obtained nanomedicine was 253 nm, PDI: 0.2, and Zeta potential -27.2 mV.

[0079] (2) High performance liquid chromatography (HPLC) standard curve method was used to determine the drug loading and encapsulation efficiency.

[0080] Sample preparation: 1 mg of each THANP with different drug loadings was dissolved in 1 mL of an aqueous solution containing 0.1% TFA (trifluoroacetic acid). After shaking for 2 hours, the solution was filtered through a 0.4 μm filter. The resulting solution was then subjected to HPLC analysis. HPLC conditions: C18 column, 25℃; detection wavelength 214 nm; gradient elution: 0.01 min 5% acetonitrile / 95% water (0.1% TFA), 25 min 50% acetonitrile / 50% water (0.1% TFA); retention time t = 13.98 min. The concentration of thanatin in each sample was calculated by referring to the thanatin concentration-absorption peak standard curve.

[0081] Results Analysis: The test results are as follows: Figure 4 As shown, with the increase of THA dosage, the encapsulation efficiency decreased from 93% to 52%, while the drug loading showed a trend of first increasing and then decreasing. The drug loading was 16.8% when the ratio of THA to drug loading material was 3:10, and 17.1% when it was 4:10.

[0082] (3) pH response behavior of THANPs-3

[0083] The method for studying the pH response and drug release behavior of THANPs-3 is as follows: THANPs were dissolved in 1×PBS buffer at pH values ​​of 7.4, 6.5, and 5.5 at a concentration of 1.0 mg / mL. -1 Then, every 10 minutes, 100 μL of the nano-solution was added to 2 mL of distilled water, and the particle size change was detected using a dynamic light scattering instrument. At room temperature, 400 μL of the nano-solution was ultrafiltered and subjected to HPLC analysis at 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, and 10 hours to analyze the release characteristics of Thatin.

[0084] Results analysis: such as Figure 5 As shown, the particle size of THANPs-3 is approximately 190 nm, and remains almost unchanged at pH 7.4. However, the particle size increases significantly at pH 6.5, and the increase is even more pronounced at the lower pH 5.5, ultimately reaching over 1400 nm within 60 minutes. The drug release curves under different pH conditions are shown below. Figure 6 As shown, no drug release was detected in THANPs-3 at pH 7.4, indicating that the material has good stability under neutral conditions and will not cause drug leakage. However, the release is significant under acidic conditions. Specifically, the drug release was basically completed within 4 hours at pH 6.5 and within 1 hour at pH 5.5, with a cumulative release rate of 92%.

[0085] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application. Any simple modification, change and equivalent variation of the above embodiments according to the technical essence of the present application are still within the protection scope of the technical scheme of the present application.

Claims

1. A cationic antimicrobial peptide nanomedicine, characterized in that, The invention includes cationic antimicrobial peptides and amphiphilic dextran block polymers, wherein the amphiphilic dextran block polymers self-assemble with the cationic antimicrobial peptides through NB coordination bond forces and guanidinium-π electronic effects to obtain cationic antimicrobial peptide nanomedicines. The amphiphilic dextran block polymer includes a carrier chain main body: dextran; a drug-loaded functional group: 2-aminophenylboronic acid; and a pH-responsive element: butyl succinate. The pH-responsive dextran block polymer is obtained by co-dissolving dextran, 2-aminophenylboronic acid, and butyl succinate in DMSO and then performing a condensation coupling reaction with dicarbonyl imidazole. The molar ratio of dextran, 2-aminophenylboronic acid, and butyl succinate is 1:1.3:0.

1. The ratio of the amphiphilic dextran block polymer to the cationic antimicrobial peptide is 1:0.

3. The cationic antimicrobial peptide and the amphiphilic dextran block polymer are co-dissolved in DMSO, stirred, dialyzed, and freeze-dried to obtain the cationic antimicrobial peptide nanomedicine. The cationic antimicrobial peptide is Thanatin.

2. The cationic antimicrobial peptide nanomedicine according to claim 1, characterized in that, The cationic antimicrobial peptide nanomedicine disintegrates and releases its antimicrobial peptide bioactive components under pH conditions of 5.5-6.

5.

3. The use of any one of the cationic antimicrobial peptide nanomedicines according to claims 1 to 2 in the preparation of a medicine for treating infectious diseases caused by Gram-negative drug-resistant bacteria.

4. A medicine for treating infectious diseases caused by Gram-negative drug-resistant bacteria, characterized in that, A cationic antimicrobial peptide nanomedicine comprising any one of claims 1 to 2.

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