CN107596368B

CN107596368B - Preparation of bacteria-targeted nano particles and application of bacteria-targeted nano particles in inhibition and killing of bacteria

Info

Publication number: CN107596368B
Application number: CN201710940630.0A
Authority: CN
Inventors: 胡祥龙; 邢达; 肖凤峰; 曹冰
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2017-10-11
Filing date: 2017-10-11
Publication date: 2020-02-14
Anticipated expiration: 2037-10-11
Also published as: CN107596368A

Abstract

The invention discloses preparation of a bacterium-targeted nano particle and application of the bacterium-targeted nano particle in inhibiting and killing bacteria. The bacteria targeted nano particle is prepared by self-assembling amphiphilic polymer with targeted fragments, the structure of which is shown in a formula (I). The invention uses photosensitizer and dimethylaminoethyl methacrylate to prepare poly (N, N-dimethylaminoethyl methacrylate) -copolymerization-photosensing agent, then uses the photosensitizer as macromolecular chain transfer agent and N-butyl methacrylate as hydrophobic units to carry out second block polymerization, activates carboxyl thereof and connects with bacteria targeting peptide, and forms nano particles soluble in water through self-assembly. The obtained nano particles can be selectively combined with bacteria through the bacteria targeting peptide, so that the safety of the material is improved; the antibacterial activity of the material is improved by combining the photodynamic action and the physical action in an antibacterial mode, and meanwhile, the drug resistance of bacteria is not easy to generate, so that the material can be widely applied to the bacteriostatic and bactericidal fields.

Description

Preparation of bacteria-targeted nano particles and application of bacteria-targeted nano particles in inhibition and killing of bacteria

Technical Field

The invention belongs to the technical field of nano material antibiosis, and particularly relates to preparation of a bacterium targeting nano particle and application of the bacterium targeting nano particle in bacterium inhibition and killing.

Background

Bacteria are ubiquitous and non-porous, wherein pathogenic microorganisms are more various and have rapid variation, and the spread of the pathogenic microorganisms seriously threaten the health of people. Because pathogenic microorganisms are subject to variation, antibiotic therapy using a single target poses a significant challenge. In the game of antibiotics and pathogenic microorganisms, antibiotics no longer have overwhelming advantages, and the pathogenic microorganisms sensitive to antibiotics have drug resistance phenomenon before, so that the infection caused by pathogens is more serious. Although researchers are always developing new antibiotics, the research and development speed is far from the mutation speed of pathogenic microorganisms; more serious is that the problem of drug resistance is not solved at all, but rather becomes more and more serious, and even "superbacteria" with multidrug resistance, which are resistant to a variety of new antibiotics, have emerged, causing panic worldwide. Therefore, it is urgent to develop new antibacterial agents against drug-resistant bacteria without causing the bacteria to develop drug resistance.

The current development of multidrug resistance by pathogenic microorganisms is through mutations that arise due to the accumulation of resistance genes in their plasmids. The resistance mechanism generally comprises: (1) producing a drug-modified enzyme to modify the drug; (2) producing a drug-degrading enzyme to degrade the drug; (3) the drug discharge pump removes the drug that has entered the interior of the bacteria. The traditional antibiotics have single action target and antibacterial mechanism and are easy to cause the drug resistance of bacteria, so that the drug resistance problem needs to be overcome by developing drugs with multiple targets and multiple antibacterial mechanisms.

The photodynamic antibacterial therapy is an antibacterial method which combines active oxygen generated by photosensitizer molecules and oxygen under the irradiation of light to destroy macromolecules such as pathogenic microorganism proteins, nucleic acid and the like and lead the death of pathogenic microorganisms. Generally, neutral or negatively charged photosensitizers have antibacterial effects against gram-positive bacteria but are weak against gram-negative bacteria, and the differences in antibacterial effects are mainly due to differences in the structure of their cell walls. There is therefore a need to combine physical antimicrobial methods to enhance photodynamic antimicrobial effects.

The physical antibacterial method is a mechanical antibacterial method that antibacterial peptide or polymer is combined with the membrane through electrostatic interaction and is subjected to co-assembly, so that the bacterial membrane is depolarized, the bacterial membrane is disturbed, and the endosolve flows out, and finally the bacteria die. Although the physical destruction of the antibacterial peptide does not cause the drug resistance of bacteria, the application of the antibacterial peptide in clinic is limited by the defects that the antibacterial peptide has high toxicity and is easy to degrade by protease and the like. It is necessary to develop an amphiphilic polymer with low toxicity and high stability to replace the antibacterial peptide. The physical destruction firstly destroys the bacterial cell membrane, and then combines the photodynamic to quickly inactivate macromolecules such as bacterial protein, lipid, nucleic acid and the like, so that the physical and photodynamic combined antibacterial mode can solve the problem of bacterial drug resistance.

In summary, it would be of great interest to develop a combination of physical and photodynamic antibacterial means, and drugs that can target bacteria and reduce material toxicity, to overcome the problem of bacterial resistance.

Disclosure of Invention

Aiming at the problems, the inventor of the invention finds that a photosensitizer modified by small molecules is efficiently polymerized with N, N-dimethylaminoethyl methacrylate (DMAEMA) to form a hydrophilic segment, the formed hydrophilic segment is polymerized with a hydrophobic monomer N-Butyl Methacrylate (BMA) to form an amphiphilic block polymer, and finally, a bacterial targeting peptide is connected, and finally, after the amphiphilic polymer with the targeting peptide is self-assembled in an aqueous solution, the combined antibacterial nano-particle which can target bacteria and can be jointly antibacterial through photodynamic and physical damage can be formed, and the combined antibacterial nano-particle can overcome the problem of bacterial drug resistance.

The primary object of the present invention is to provide a nanoparticle that can target bacteria through photodynamic and physical actions without causing the bacteria to develop drug resistance, i.e., the bacteria-targeted nanoparticle.

The invention also aims to provide a preparation method of the bacteria-targeted nano-particle.

It is a further object of the present invention to provide the use of the above-described bacteria-targeting nanoparticles.

The purpose of the invention is realized by the following technical scheme:

a bacterial targeted nanoparticle is prepared by self-assembling an amphiphilic polymer with a targeted segment, which has a structure shown in a formula (I):

in the formula (I), each R₁Independently H, CH₃Or CH₂CH₃(ii) a Each R is₂Independently H, CH₃Or CH₂CH₃；R₃A bacterial targeting peptide, preferably Ubiqicidin (UBI), HLFpeptides, hNP-1 or Depsipeptide; PS is a photosensitizer, preferably phenothiazine photosensitizers (phenothiazinium photosensizers), organic dye photosensitizers, porphyrin photosensitizers (porlyrin), phthalocyanine photosensitizers (phthalocyanines), or the like; each P' is independently

R₄Independently is-C₆H₅、-CH₂C₆H₅、-CH₂CH₂COOH、-CH₂CH₂OH or-CH₂OH; q is independently-CHNCH₃CH₂CH₂COOH, or-CH₂CH₂COOH；m＝2-200；n＝2-200；g＝2-200；h＝1-10；i＝1-10。

The phenothiazine photosensitizer comprises methylene blue (methylene blue)

Rose color (rosebengal)

Or toluidine blue (tolulidine blue O)

Etc.;

the organic dye photosensitizer comprises eosin (eosin)

Or rhodamine B (rodamine B)Etc.;

the structural formula of the porphyrin photosensitizer is shown in the specification

The structural formula of the phthalocyanine photosensitizerIs composed of

The amino acid sequence of the bacterial targeting peptide UBI is TGRAKRRMQYNRR (specifically Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg); HLFpeptides having an amino acid sequence as disclosed in publication CN 101395180A; the amino acid sequence of hNP-1 is ACYCRIPACIAGERRYGTCIYQGRLWAFCC [ Ala-Cys-Tyr-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Tyr-Gly-Thr-Cys-Ile-Tyr-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys (Disulfides Cys2-Cys30, Cys4-Cys19, and Cys 9-29) ]; hNP-1 and Depsipeptide (the name in the literature: N- (11, 23-diisobutyl-14-isopropyl-7-methyl-5, 9,12,15,21, 24-hexaoxadocosane-7H, 17H-dipyridazino [6,1-c:6,1-i ] [1,4,7,10,13,16] oxapentaazanonacydo-8-yl) -2- (carboxamido) -3-methylpentaneamide) are available directly from the market.

Preferably, h is 2, i is 2, and P' is C₆H₅CSS-, at the moment, the bacteria targeted nano particle is prepared by self-assembling amphiphilic polymer with targeted fragments, the structure of which is shown in a formula (II):

r in the formula (II)₁、R₂、R₃PS, S, m, n and g are as defined for formula (I), i.e.: each R is₁Independently H, CH₃Or CH₂CH₃(ii) a Each R is₂Independently H, CH₃Or CH₂CH₃；R₃A bacterial targeting peptide, preferably UBI, HLFpeptides, hNP-1 or Depsipeptide; PS is a photosensitizer, preferably phenothiazine photosensitizer, organic dye photosensitizer, porphyrin photosensitizer or phthalocyanine photosensitizer and the like; q is independently-CHNCH₃CH₂CH₂COOH, or-CH₂CH₂COOH；m＝2-200；n＝2-200；g＝2-200。

Preferably, P' in formula (I) is C₆H₅CSS-, Q is-CHCNCH₃CH₂CH₂COOH, and PS is eosin; at the moment, the bacteria targeted nano particle is prepared by self-assembling amphiphilic polymer with targeted fragments, the structure of which is shown as a formula (III):

in the formula (III), R₁、R₂、R₃H, i, m, n and g are as defined for formula (I), i.e.: each R is₁Independently H, CH₃Or CH₂CH₃(ii) a Each R is₂Independently H, CH₃Or CH₂CH₃；R₃A bacterial targeting peptide, preferably UBI, HLFpeptides, hNP-1 or Depsipeptide; g is 2-200; m is 2-200; n is 2-200; 1-10; h is 1-10.

Preferably, R₁、R₂Is H, H-2, i-2, P' is C₆H₅CSS-, Q is-CHCNCH₃CH₂CH₂COOH, PS are eosin, and the bacterial targeting peptide R₃When the target gene is UBI, the bacteria-targeted nano particle is prepared by self-assembling an amphiphilic polymer with a targeted segment, the structure of which is shown as a formula (IV):

in the formula (IV), TGRAKRRMQYNRR is an amino acid sequence of a bacterial targeting peptide UBI, and m is 2-200; n is 2-200; g is 2-200.

The self-assembly method of the bacteria-targeted nano particle comprises the following steps: dissolving the amphiphilic polymer with the targeting fragment shown in the formula (I) in an organic solvent, quickly adding the amphiphilic polymer into stirred deionized water, and removing the organic solvent by dialysis or ultrafiltration to obtain the target-specific amphiphilic polymer; the organic solvent is preferably one or more of dimethyl sulfoxide, dimethylformamide and tetrahydrofuran.

Preferably, the amphiphilic polymer with the targeting segment is prepared by the following steps: dissolving the amphiphilic block polymer in an organic solvent, azeotropically removing water with toluene for 0.5-2h, and reacting with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), N-hydroxysuccinimide (NHS) in a molar ratio of 1: 2: 2, reacting for 6-24h in vacuum, adding the bacterial targeting peptide, reacting for 6-48h, and finally precipitating by anhydrous ether; the organic solvent is preferably one or more of 1, 4-dioxane, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran and toluene; the bacterial targeting peptide is preferably UBI, HLFpeptides, hNP-1 or Depsipeptide, most preferably UBI.

More preferably, the amphiphilic block polymer is prepared by the following steps: reacting a butyl methacrylate monomer with a macromolecular chain transfer agent in an organic solvent at the temperature of 50-120 ℃ for 1-100h in the presence of an initiator, and then precipitating by using petroleum ether to prepare the amphiphilic block polymer; the initiator is preferably at least one of Azobisisobutyronitrile (AIBN), Azobisisoheptonitrile (ABVN), dimethyl Azobisisobutyrate (AIBME), most preferably AIBN; the organic solvent is preferably one or more of 1, 4-dioxane, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran and toluene; wherein the structure of the butyl methacrylate monomer is shown as a formula (1), and the structure of the macromolecular chain transfer agent is shown as a formula (2):

in formula (2): each R is₂Independently H, CH₃Or CH₂CH₃(ii) a Each P is independently

R₄Independently is-C₆H₅、-CH₂C₆H₅、-CH₂CH₂COOH、-CH₂CH₂OH or-CH₂OH; q is independently-CHNCH₃CH₂CH₂COOH, or-CH₂CH₂COOH；n＝2-200；g＝2-200。

More preferably, the macromolecular chain transfer agent is prepared by the following steps: reacting a micromolecular chain transfer agent, N-dimethylaminoethyl methacrylate and a micromolecular modified photosensitizer in an organic solvent at the temperature of 50-120 ℃ for 1-100h in the presence of an initiator, and then precipitating by using petroleum ether to obtain the modified photosensitizer; the initiator is preferably at least one of Azobisisobutyronitrile (AIBN), Azobisisoheptonitrile (ABVN), dimethyl Azobisisobutyrate (AIBME), most preferably AIBN; the organic solvent is preferably one or more of 1, 4-dioxane, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran and toluene; the structure of the micromolecule chain transfer agent is shown as a formula (3), the structure of the N, N-dimethylaminoethyl methacrylate is shown as a formula (4), and the photosensitizer is a phenothiazine photosensitizer, an organic dye photosensitizer, a porphyrin photosensitizer or a phthalocyanine photosensitizer;

in the preparation process of the macromolecular chain transfer agent, if the photosensitizer does not have a functional group capable of performing polymerization reaction (such as a double bond, a triple bond, a hydroxyl group, a carboxyl group and the like), the photosensitizer needs to be modified by using a small molecular compound (for example, eosin needs to be modified by using p-chloromethyl styrene in advance, and the like), and then the modified photosensitizer is polymerized with N, N-dimethylaminoethyl methacrylate; if the photosensitizer itself is provided with a functional group capable of undergoing polymerization, it does not need to be modified in advance. Methods of modifying photosensitizers are generally available using techniques conventional in the art.

More preferably, the macromolecular chain transfer agent represented by formula (2), i.e., poly (N, N-dimethylaminoethyl methacrylate) -co-photosensitizer, has a molecular weight of 420-40300, i.e., N-2-200, g-2-200.

In one preferred embodiment, the amphiphilic block polymer has a structural formula as shown in formula (V):

wherein m is 2-200, n is 2-200, and g is 2-200.

The bacteria-targeted nano particles provided by the invention are applied to the field of bacteriostasis or sterilization.

The invention uses a reversible addition fragmentation chain transfer polymerization (RAFT) method to copolymerize a photosensitizer modified by small molecules and N, N dimethyl ethyl methacrylate (DMAEMA) to obtain a hydrophilic polymer (P (DMAEMA-co-PS)) containing the photosensitizer; and polymerizing a hydrophobic compound n-Butyl Methacrylate (BMA) and the synthesized hydrophilic polymer by using a reversible addition fragmentation chain transfer polymerization (RAFT) method again to obtain an amphiphilic block polymer (P (DMAEMA-co-PS) -b-PBMA) containing a photosensitizer, and finally modifying the amphiphilic block polymer by using a bacterial targeting peptide. Finally, the amphiphilic polymer with the targeting segment obtained after the modification of the bacterial targeting peptide is assembled into the nano particle by a self-assembly method, and the nano particle can target and kill bacteria through photodynamic and physical destruction without causing the drug resistance of the bacteria.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the photosensitizer is polymerized with the hydrophobic and hydrophilic compound in a covalent connection mode and self-assembled into the nano particles, so that the photosensitizer is stable, can be gathered, generates more ROS and kills bacteria efficiently.

(2) The nano particles are combined with two antibacterial modes with different action mechanisms of photodynamic and physics for the first time, so that the drug-resistant bacteria can be killed efficiently, the drug resistance of the bacteria can not be generated, and the problem of the drug resistance of the bacteria can be solved.

(3) The bacterial targeting peptide is used as a targeting segment, so that the nano particles can be selectively targeted to the surface of bacteria, specifically kill the bacteria without generating toxicity on normal cells, and the safety of the material can be improved.

Drawings

Fig. 1 is a nuclear magnetic resonance hydrogen spectrum of the macromolecular chain transfer agent prepared in example 1, namely poly (N, N-dimethylaminoethyl methacrylate) -co-eosin P (DMAEMA-co-EOS) in deuterated DMSO (N ═ 14).

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of amphiphilic block polymer P (DMAEMA-co-EOS) -b-PBMA of example 2 in deuterated DMSO.

Figure 3 is the results of dynamic light scattering and TEM characterization based on eosin photodynamic and physically targeted bactericidal-inhibiting nanoparticles of example 4.

FIG. 4 shows the eosin-based photodynamic and physically targeted bacteriostatic nanoparticles of example 4 (i.e., UBI-P (DMAEMA)₂₀-co-EOS_0.8)-b-PBMA₁₆Self-assembled nanoparticles) at different concentrations, and their effect on the activity of staphylococcus aureus, MRSA, escherichia coli, pseudomonas aeruginosa and their zone of inhibition at 10 μ M for staphylococcus aureus and pseudomonas aeruginosa.

FIG. 5 is the confocal results of example 4 based on the selective inhibition and killing of bacteria by eosin photodynamic and physical targeting antibacterial nanoparticles, but without affecting normal cells.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

The invention relates to an amphiphilic block polymer which takes Polybutylmethacrylate (PBMA) as a hydrophobic chain and takes a macromolecular chain copolymerized by N, N-dimethylaminoethyl methacrylate and a photosensitizer as a hydrophilic chain, and is then connected with a bacterial targeting peptide. Such as UBI-P (DMA-co-EOS) -b-PBMA and nanoparticles obtained by self-assembly thereof. The method comprises the steps of carrying out reversible addition fragmentation chain transfer polymerization (RAFT) on a photosensitizer and N, N-dimethylaminoethyl methacrylate (DMAEMA) to obtain a hydrophilic polymer, carrying out polymerization on the hydrophilic polymer and Butyl Methacrylate (BMA) to obtain an amphiphilic block polymer P (DMAEMA-co-PS) -b-PBMA, connecting bacteria targeting peptide through activated carboxyl and dehydration condensation to form an amphiphilic polymer with a targeting segment, and carrying out self-assembly on the finally formed amphiphilic polymer with the targeting segment to form nanoparticles which target and kill bacteria through photodynamic and physical effects and do not enable the bacteria to generate drug resistance. The nano particles can efficiently and targetedly resist bacteria, solve the problems of dispersion of photosensitizer and unconcentration of generated ROS, and simultaneously overcome the problems of toxicity and drug resistance of antibacterial materials; has high-efficiency broad-spectrum antibacterial activity, has the capability of inhibiting and killing drug-resistant bacteria, and is difficult for bacteria to generate drug resistance after being used. Belongs to the field of medicinal chemistry and biomedical polymer.

In one embodiment, an eosin photosensitizer is used, the eosin is first modified with p-chloromethylstyrene, then, the poly-P-chloromethylstyrene modified eosin amphiphilic polymer (P (DMAEMA-co-EOS)) is polymerized together with N, N-dimethylaminoethyl methacrylate monomer to obtain a P-chloromethylstyrene modified eosin amphiphilic polymer (P (DMAEMA-co-EOS)), the macromolecular chain transfer agent (P (DMAEMA-co-EOS)) and Butyl Methacrylate (BMA) are used for preparing an amphiphilic block polymer (P (DMAEMA-co-EOS) -b-PBMA) which is subjected to combined physical and photodynamic antibacterial action, then UBI is added to prepare an amphiphilic polymer (UBI-P (DMAEMA-co-EOS) -b-PBMA) with a targeting segment, and finally UBI-P (DMAEMA-co-EOS) -b-PBMA is subjected to self-assembly to obtain the bacterial targeting nanoparticles. In the case of an Eosin (EOS) amphiphilic polymer modified with p-chloromethylstyrene, the polymerization degree g of an EOS monomer and the polymerization degrees N and m of poly (N, N-dimethylaminoethyl methacrylate) and poly (butyl methacrylate) are determined in accordance with the polymerization degree and conversion rate set experimentally, and are usually in the range of g ═ 2 to 200, m ═ 2 to 200, and N ═ 2 to 200.

In the above embodiment, poly (N, N-dimethylaminoethyl methacrylate) -co-poly eosin (P (DMAEMA-co-EOS)) is used as the macromolecular chain transfer agent, which is obtained by co-polymerizing P-chlorostyrene modified Eosin (EOS) with N, N-dimethylaminoethyl methacrylate monomer using RAFT polymerization method; the preparation method of the macromolecular chain transfer agent P (DMAEMA-co-EOS) is RAFT polymerization reaction shown as a reaction formula (a):

the specific steps of the formula (a) are as follows: using a small molecular Chain Transfer Agent (CTA) and N, N-dimethylaminoethyl methacrylate, dimethyl sulfoxide as an organic solvent, controlling the temperature to be 50-120 ℃ in the presence of an initiator Azobisisobutyronitrile (AIBN), reacting for 1-100h, and then precipitating by petroleum ether to obtain a final product.

In the above embodiment, the preparation method of amphiphilic block polymer, i.e., P (DMAEMA-co-EOS) -b-PBMA, through physical and photodynamic combined antibacterial is as shown in the reaction formula (b) RAFT polymerization:

the specific steps of the formula (b) are as follows: using macromolecular chain transfer agent (P (DMAEMA-co-EOS)) and Butyl Methacrylate (BMA), dimethyl sulfoxide and 1, 4-dioxane as organic solvents, controlling the temperature at 50-120 ℃ in the presence of initiator Azobisisobutyronitrile (AIBN), reacting for 1-100h, and then precipitating by petroleum ether to obtain the final product.

In the above embodiment, the amphiphilic polymer with a targeting segment, i.e., UBI-poly (N, N-dimethylaminoethylmethacrylate) -co-poly (eosin) -block-polybutylmethacrylate (UBI-P (DMAEMA-co-EOS) -b-PBMA) is prepared as shown in equation (c):

the specific steps of the formula (c) are as follows: firstly, an amphiphilic block polymer (P (DMAEMA-co-EOS) -b-PBMA)) which is subjected to combined physical and photodynamic antibacterial action is dissolved in DMSO, toluene is subjected to azeotropic dehydration for 0.5-2h, carboxyl is activated for 6-24h by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl) and N-hydroxysuccinimide (NHS), and then the carboxyl is subjected to dehydration condensation reaction with bacterial targeting peptide UBI (amino acid sequence TGRAKRRMQYNRR) and is subjected to reaction for 6-48h at normal temperature, and then petroleum ether is used for settling to obtain a final product.

In the above specific embodiment, the bacteria-targeted nanoparticles are prepared by a "bottom-up" self-assembly method: UBI-P (DMAEMA-co-EOS) -b-PBMA was dissolved in an organic solvent, deionized water was added thereto, stirred for a while, and the organic solvent was removed by dialysis or ultrafiltration.

The raw materials used in the following examples are illustrated below:

eosin (Eosin-y), p-chloromethylstyrene was purchased from Sigma-Aldrich and used without further purification. N, N-Dimethylaminoethyl methacrylate (DMAEMA) was purchased from Sigma-Aldrich and used with basic Al prior to use₂O₃Treating and purifying by a column; methylphenylene acid ester (BMA) was purchased from Sigma-Aldrich and purified by distillation under reduced pressure before use. Azobisisobutyronitrile (AIBN) was purchased from Acros and purified by recrystallization from 95% ethanol. The triethylamine was dried over 5A molecular sieves. The dichloromethane was dried over calcium hydride and then purified by distillation. Tetrahydrofuran was dried with sodium thread under reflux and distilled for use. Other reagents such as petroleum ether, anhydrous ether, ethyl acetate and the like are analytically pure, are purchased from chemical reagents of national drug group, Inc., and are directly used without further purification. The ultrapure water used in the experiment was prepared by a Milli-QSP smart ultrapure water system and had a resistivity of 18.4M Ω. cm. The small molecule chain transfer agent of formula (3) was prepared by the reference method (Lei Tao, Jingquan Liu, B.H.Tan, andMethohmas P.Davis macromolecules200942(14), 4960-. The para-chlorostyrene modified Eosin monomer used in the experiment was prepared from Eosin-y and para-chlorostyrene by reference to the literature (Liu, G.; Hu, J.; Zhang, G.; Liu, S., Radi Engineering plastics of Eosin-Conjugated reactive Polymeric nanoparticles Intracellular molecular pH gradients. bioconjugate chemistry 2015,26(7), 1328-38).

The invention will be further illustrated by the following examples, examples 1-7 employing, R₁And R₂＝-CH₃，R₃＝-CH₂CH₃And i is 2 and h is 2. Its purpose is merely to better understand the invention and not to limit its scope.

Example 1: preparation of P (DMAEMA-co-EOS) macromolecular chain transfer agent

Referring to the above reaction scheme (a), small molecule chain transfer agents CTA (13.9mg, 0.05mmol) of formula (3), N-dimethylaminoethylmethacrylate (DMAEMA, 353.7mg, 2.25mmol), p-chloromethylstyrene modified eosin (EOS, 191mg, 0.25mmol) and azobisisobutyronitrile AIBN (1.6)4mg, 0.01mmol) was dissolved in 1.5mL of 1, 4-dioxane in an ampoule. Placing the ampoule bottle in liquid nitrogen for freezing, then pumping air by using an oil pump, then closing the ampoule bottle, recovering to room temperature to melt a reaction mixture, then freezing and pumping air again, repeating the freezing and thawing cycle for three times, then sealing under vacuum, stirring at 70 ℃ for 20h, then stopping a polymerization reaction by using liquid nitrogen, opening the reaction bottle, precipitating the reaction mixture in petroleum ether, centrifuging, dissolving in dichloromethane, precipitating with a large amount of petroleum ether, repeating for three times, and finally drying the final product in a vacuum drying oven at room temperature overnight to obtain a red viscous solid (366mg, yield 67.2%). FIG. 1 shows the NMR spectrum of P (DMAEMA-co-EOS) prepared in this example, and the integrated area of the proton at the position above 7.0ppm on the benzene ring in the chain transfer agent and the integrated area of the proton at the position 2.3ppm on the methyl group in N, N-dimethylaminoethyl methacrylate are calculated by comparison, so that the polymerization degree of DMAEMA is 20, the polymerization degree of EOS is 0.8, and the molecular formula of the DMAEMA is P (DMAEMA-co-EOS)₂₀-co-EOS_0.8) The structural formula is shown as a formula (d).

Example 2: p (DMAEMA)₂₀-co-EOS_0.8)-b-PBMA₁₆Preparation of amphiphilic Block polymers

With reference to the above reaction formula (b), the macromolecular chain transfer agent P (DMAEMA) prepared in example 1₂₀-co-EOS_0.8) (302mg, 0.075mmol), butyl methacrylate (BMA, 213mg, 1.5mmol) and azobisisobutyronitrile AIBN (2.46mg, 0.015mmol) were dissolved in 1.1mL of 1, 4-dioxane and 0.9mL of DMSO in an ampoule. Freezing ampoule in liquid nitrogen, pumping air with oil pump, sealing ampoule, recovering to room temperature to melt reaction mixture, freezing, pumping air again, repeating the freezing and thawing cycle for three times, sealing under vacuum, stirring at 70 deg.C for 19 hr, stopping polymerization with liquid nitrogen, opening reaction flask, precipitating the reaction mixture in petroleum ether, centrifuging, dissolving in DMF, precipitating with petroleum ether, repeating for three times, and vacuum drying in vacuum drying oven to obtain final productDrying overnight at medium room temperature gave a red viscous solid (251mg, 48.7% yield). FIG. 2 shows the NMR spectrum of P (DMAEMA-co-EOS) -b-PBMA prepared in this example, where the integrated area of protons in the benzene ring in the chain transfer agent above 7.0ppm was calculated by comparison with the integrated area of protons in the methyl group in N, N-dimethylaminoethyl methacrylate at 2.3ppm, and a standard curve of eosin was used to obtain a BMA with a degree of polymerization of 16, which is expressed as P (DMAEMA)₂₀-co-EOS_0.8)-b-PBMA₁₆The structural formula is shown as a formula (e).

Example 3: (UBI-P (DMAEMA)₂₀-co-EOS_0.8)-b-PBMA₁₆) Preparation of

With reference to the above reaction formula (c), amphiphilic block polymer P (DMAEMA) prepared in example 2₂₀-co-EOS_0.8)-b-PBMA₁₆(31.5mg, 0.005mmol) was put into a 5mL ampoule and dissolved in 1mL of DMF, 3mL of toluene was added, after azeotropic removal of water from toluene for 0.5h, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl, 1.9mg, 0.01mmol) and N-hydroxysuccinimide (1.2mg, 0.01mmol) were added and the mixture was stirred hermetically at room temperature for 12h, i.e., amphiphilic block polymer P (DMAEMA)₂₀-co-EOS_0.8)-b-PBMA₁₆Activating terminal carboxyl, adding bacterial targeting peptide (UBI, 6.76mg, 0.004mmol), sealing and stirring at normal temperature for reaction for 24h, precipitating the reacted mixture in ether, centrifuging, dissolving in DMF, precipitating with a large amount of petroleum ether, repeating for three times, and drying the final product in a vacuum drying oven overnight at room temperature to obtain red viscous solid with a structural formula shown in formula (f). The sequences of the bacterial targeting peptides UBI added in this example were: TGRAKRRMQYNRR are provided.

Example 4 construction of Eosine-based photodynamic and physically targeted bacteriostatic and bactericidal nanoparticles

1mg of UBI-P (DMAEMA) prepared in example 3₂₀-co-EOS_0.8)-b-PBMA₁₆Fully dissolving in 0.5mL of DMF, quickly adding into 4mL of quickly stirred water phase, continuously stirring for 1.5h, and removing the organic solvent through a dialysis membrane with the molecular weight of 3.5 KDa; changing water every 2h, dialyzing for 6h, and obtaining the bacterial targeting nanoparticles (namely the eosin photodynamic-based and physical targeting bacteria inhibiting and killing nanoparticles) through self-assembly

The size of the bacteria-targeted nanoparticles formed by self-assembly in the aqueous phase was characterized using a particle sizer, UBI-P (DMAEMA)₂₀-co-EOS_0.8)-b-PBMA₁₆The average diameter of the self-assembled bacteria-targeted nanoparticles is about 28.2nm, and the PDI is 0.39. The results were further validated using TEM to characterize the bacteria-targeting nanoparticles as shown in figure 3. And the results of transmission electron microscopy TEM are slightly less than the results of dynamic light scattering. The reason is that when the particle size instrument is used for representing the size of the bacteria targeted nano particles in a water phase, a hydration layer is formed outside the micelle, so that the particle size is slightly larger than the TEM result.

Example 5 evaluation of antibacterial Properties of bacterium-targeting nanoparticles

The antibacterial experiments selected Staphylococcus aureus (ATCC 6538) and methicillin-resistant Staphylococcus MRSA (ATCC43300) as representative of gram-positive bacteria, and Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC27853) as representative of gram-negative bacteria. In the case of Staphylococcus aureus, a single colony of Staphylococcus aureus was first inoculated into MH broth and shaken overnight at 37 ℃. The broth was diluted to OD600 of 0.1 using MH broth, and then re-inoculated into MH broth for culture until a medium log phase was reached. Finally, the bacterial liquid is diluted to the bacterial liquid concentration of about 5 multiplied by 10 corresponding to the OD600 being 0.001⁵CFU mL^-1And adding the adjusted 100 mu L of bacterial liquid into 100 mu L of various bacteria-targeted nano particle solutions which are diluted by 2 times in advance by using sterilized deionized water in a 96-well plate. As a control group, bacteria were incubated with Broth medium. Measuring with enzyme-linked immunosorbent assayThe OD600 value of each well was defined as a value of 0 h. Shaking at 37 deg.C for 12h, and determining the OD600 value of each well again. The effect on bacterial activity at different concentrations of drug was calculated.

Bacterial Activity [% DrugOD600_12h-DrugOD600_0h]/[BrothOD600_12h-BrothOD600_0h]×100％

Wherein drug OD600_0hAnd drug 600_12hRepresent the OD600 values at 0h and 12h for the bacteria-targeted nanoparticle treatment groups, respectively. Brothod600_0hAnd BrothoD600_12hRepresents the OD600 values of the medium blank at 0h and 12h, respectively.

UBI-P (DMAEMA) as shown in FIG. 4₂₀-co-EOS_0.8)-b-PBMA₁₆The bacteria targeted nano particles have strong antibacterial activity on gram-positive bacteria (staphylococcus aureus, methicillin-resistant staphylococcus aureus MRSA), escherichia coli and pseudomonas aeruginosa. UBI-P (DMAEMA)₂₀-co-EOS_0.8)-b-PBMA₁₆The minimum inhibitory concentration MIC of the self-assembled bacterial targeting nanoparticles to the self-assembled bacterial targeting nanoparticles is shown in table 1.

TABLE 1UBI-P (DMAEMA)₂₀-co-EOS_0.8)-b-PBMA₁₆Minimum inhibitory concentration of self-assembled bacteria-targeted nano particles

Example 6 confocal experiments to verify that bacterial targeting nanoparticles selectively target bacteria without damaging normal cells

Firstly, pseudomonas aeruginosa is put into a shaking table, activated and cultured for 12h at 37 ℃, then centrifuged for 5min at 3000r/min, washed twice by PBS, and the bacteria are diluted to 1 multiplied by 10 by PBS⁸CFU, 500. mu.L of bacterial suspension containing 1X 10⁵Adding 500 μ L DMEM medium containing 10% serum into a confocal dish containing bacteria, culturing in a refrigerator at 4 deg.C for 10min, centrifuging at 3000r/min for 3min to remove excessive drug, diluting bacteria to original concentration, digesting cells in the dish, mixing bacteria and cells, and adding 10 μ L bacteria and cell mixtureThe glass slide is covered with a cover glass, and the glass slide is immediately placed under confocal observation, wherein the excitation wavelength is 488 nm.

As shown in fig. 5, the bacterial surface has red fluorescence and the cell surface has no fluorescence, indicating that the drug can be selectively bound to the bacterial surface and the drug has the function of targeting pathogens.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The bacteria-targeted nanoparticle is characterized in that the bacteria-targeted nanoparticle is prepared by self-assembling an amphiphilic polymer with a targeted segment, the structure of which is shown in a formula (IV):

in the formula (IV), TGRAKRRMQYNRR is an amino acid sequence of a bacterial targeting peptide UBI; m is 2-200; n is 2-200; g is 2-200.

2. A method of self-assembly of bacteria-targeting nanoparticles comprising the steps of: the bacteria-targeted nano-particle is prepared by dissolving the amphiphilic polymer with the targeting segment of claim 1 in an organic solvent, adding the solution into stirred deionized water, and removing the organic solvent by dialysis or ultrafiltration.

3. The self-assembly method of bacteria-targeted nanoparticles according to claim 2, wherein the amphiphilic polymer with targeting segment is prepared by the following steps: dissolving the amphiphilic block polymer in an organic solvent, azeotropically removing water with toluene for 0.5-2h, and then reacting with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide in a molar ratio of 1: 2: 2, reacting for 6-24h in vacuum, adding the bacterial targeting peptide, reacting for 6-48h, and finally precipitating by anhydrous ether; the bacterial targeting peptide is UBI;

the structural formula of the amphiphilic block polymer is shown as the formula (V):

wherein m is 2-200, n is 2-200, and g is 2-200.

4. The self-assembly method of bacteria-targeted nanoparticles according to claim 3, wherein the amphiphilic block polymer is prepared by the following steps: reacting a butyl methacrylate monomer with a macromolecular chain transfer agent in an organic solvent at the temperature of 50-120 ℃ for 1-100h in the presence of an initiator, and then precipitating by using petroleum ether to prepare the amphiphilic block polymer; wherein the structure of the butyl methacrylate monomer is shown as a formula (1), and the structure of the macromolecular chain transfer agent is shown as a formula (2):

in formula (2): n is 2-200; g is 2-200.

5. The self-assembly method of bacteria-targeted nanoparticles according to claim 4, wherein the macromolecular chain transfer agent is prepared by the following steps: reacting a small-molecular chain transfer agent, N-dimethylaminoethyl methacrylate and p-chloromethyl styrene modified eosin in an organic solvent at the temperature of 50-120 ℃ for 1-100h in the presence of an initiator, and then precipitating by using petroleum ether; wherein the structure of the micromolecular chain transfer agent is shown as a formula (3), and the structure of the N, N-dimethylaminoethyl methacrylate is shown as a formula (4);

6. use of the bacteria-targeting nanoparticle of claim 1 for the preparation of a bacteriostatic or bacteriocidal medicament.