CN113730596A - Microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection and preparation method thereof - Google Patents

Microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection and preparation method thereof Download PDF

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CN113730596A
CN113730596A CN202111079949.1A CN202111079949A CN113730596A CN 113730596 A CN113730596 A CN 113730596A CN 202111079949 A CN202111079949 A CN 202111079949A CN 113730596 A CN113730596 A CN 113730596A
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respiratory tract
microenvironment
lower respiratory
delivery system
drug delivery
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CN113730596B (en
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徐福建
任慧敏
段顺
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Beijing University of Chemical Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Abstract

The invention discloses a preparation method of a microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection, which comprises the following preparation steps: preparing a hydroformylation natural polysaccharide; dissolving hydroformylation natural polysaccharide in alkaline buffer solution, adding into poor solvent, stirring to completely disperse and mix well, adding aminoglycoside antibiotics and hydrophilic PEG with amino, stirring at high speed, and reacting at 20-80 deg.C for 0.5-48 h; the ratio of aldehyde groups in the hydroformylation natural polysaccharide to amino groups in the aminoglycoside antibiotic is 3:1-2:1, and the ratio of amino groups in the aminoglycoside antibiotic to hydrophilic PEG with amino groups is 12:1-1: 12; and repeatedly centrifuging and ultrasonically dispersing the reaction product until the residual reactant is cleaned, and freeze-drying to obtain the self-adaptive nano-drug. Can realize the administration according to the requirement according to the infection condition of the lower respiratory tract, and at the serious infection part, the acid substances such as lactic acid and the like generated by bacteria stimulate Schiff base bond acid to respond and break to release antibiotics, and carry out rapid sterilization.

Description

Microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection and preparation method thereof
Technical Field
The invention belongs to the field of pharmaceutical preparations, relates to natural polysaccharide, aminoglycoside antibiotics, aerosol inhalation administration, a drug carrier, and a preparation method and application thereof, and particularly relates to a microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection and a preparation method thereof.
Background
Serious lower respiratory tract infection and drug resistance caused by bacteria are one of the problems of bacterial infection which are urgently to be solved. Antibiotic therapy remains the mainstream way of clinically treating lower respiratory tract infections, so that the establishment of reasonable treatment strategies and the realization of effective treatment of lower respiratory tract bacterial infections by reasonable use of antibiotics through proper administration modes are urgent and difficult challenges.
The issue of Proc. Quadrature Prof.A. 2020 in ACS Nano journal 14, Vol.5, pp.5686-5699, discloses a report on the electrostatic transformation of the acid response of nanoparticles to achieve charge reversal and size reduction and the intravenous treatment of pulmonary infections. However, the complex blood environment may cause adsorption and aggregation of nanoparticles within the blood vessels, and intravenous drugs are less available than inhalation administration. Therefore, the stability of the nanoparticles in blood transportation and the utilization efficiency of the drug in the lung are unknown.
Nowa company invented a TOBI Podhaler inhalation formulation, which physically wraps tobramycin in 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) to be inhaled in the form of dry powder for administration, and a patent publication No. CN112891327A discloses a liquid formulation for Reidesvir atomization inhalation, and a patent publication No. CN111840569A discloses a pH-responsive drug-loaded nanoparticle, and pH response is realized by electrostatic interaction of a quaternary ammonium salt modified pectin/polycaprolactone graft copolymer and a drug. However, due to the special physiological environment of the lung, the presence of a thick, viscous and dense biofilm in the trachea of a patient can block drug delivery through hydrophobic and electrostatic interactions, and these formulations may not achieve efficient delivery to the lung. On the other hand, these formulations generally carry the drug in an electrostatically-acting or physically-coated form, are unstable and do not allow on-demand administration.
Patent publication No. CN108478792A discloses a pH responsive macromolecule gold nano micelle capable of permeating mucus and a preparation method thereof, and patent publication No. CN109589418A also discloses a mesoporous silica drug-loaded nanoparticle coated by Schiff base copolymer with pH responsiveness and a preparation method thereof.
To date, most nano-drugs have problems in treating severe respiratory infections, and it is difficult to achieve satisfactory therapeutic effects. The problems of biological safety, stability of nano particles, mucus and biological membrane permeability, controllable drug release and the like hinder the further development and application of nano carriers in the field of serious lower respiratory tract infection. Therefore, the inhalation administration treatment strategy which selects raw materials with excellent biocompatibility and biodegradability and couples the drugs to the carrier material by utilizing the stimulus-responsive covalent bond to realize the effect of penetrating mucus and biomembrane and has good microenvironment responsiveness of lower respiratory tract infection is a new breakthrough of nano drugs in the field of treatment of severe lower respiratory tract infection. The treatment strategy can realize high drug concentration in lung, reduce administration dosage, avoid first pass effect, greatly improve drug utilization rate, and reduce toxic and side effects of aminoglycoside antibiotics such as ototoxic nephrotoxicity.
Disclosure of Invention
In view of the above, the invention provides a preparation method of a microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection. The invention specifically provides the following technical scheme:
1. A preparation method of a microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection comprises the following preparation steps:
1) preparing a hydroformylation natural polysaccharide;
2) dissolving the hydroformylation natural polysaccharide obtained in the step 1) in an alkaline buffer solution, then adding a poor solvent, stirring until the mixture is completely dispersed and uniformly mixed, simultaneously adding aminoglycoside antibiotics and hydrophilic PEG with amino, stirring at a high speed, and reacting for 0.5-48h at the temperature of 20-80 ℃; the ratio of aldehyde group in the hydroformylation natural polysaccharide to amino group in the aminoglycoside antibiotic is 3:1-2:1, and the ratio of amino group in the aminoglycoside antibiotic to hydrophilic PEG with amino group is 12:1-1: 12.
3) Repeatedly centrifuging and ultrasonically dispersing the reaction product obtained in the step 2) until the residual reactant is cleaned and freeze-dried to obtain the self-adaptive nano-drug.
Further, the volume ratio of the alkaline buffer solution and the poor solvent in the step 2) is 2:1-1: 2.
Further, the natural polysaccharide in the step 1) is natural polysaccharide containing ortho-dihydroxy.
Further, methoxy polyethylene glycol-amino group in step 2).
Further, the natural polysaccharide is one or two of dextran, starch, hyaluronic acid, sodium alginate, glycyrrhiza polysaccharide, xanthan gum, cellulose, bletilla polysaccharide, chondroitin sulfate and heparin sodium.
Further, the hydroformylation degree of the hydroformylation natural polysaccharide in the step 1) is 10-100%.
Further, the pH value of the alkaline buffer solution in the step 2) is 7.4-11.
Further, the poor solvent in the step 2) is one or two of ethanol, methanol, acetone and diethyl ether.
Further, the aminoglycoside antibiotics in the step 2) are one or two of gentamicin, tobramycin, amikacin, streptomycin, kanamycin, etimicin, ribostamycin, netilmicin and micronomicin.
2. The microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection is prepared according to the preparation method.
The hydroformylation natural polysaccharide obtained by the preparation method is used as a nano-drug carrier, and the aminoglycoside antibiotics pass through the matThe coupling of the fujine bond and the polysaccharide as the self-adaptive antibacterial component relies on mPEG-NH2A microenvironment adaptive nano-drug system for treating serious lower respiratory tract infection, which forms a hydrophilic layer on the surface of nano particles, is applied to the treatment of serious lower respiratory tract infection. The principle of constructing the microenvironment adaptive nano-drug system for severe lower respiratory tract infection is as follows: in a poor solvent, molecular chains of two water-soluble compounds are curled and mutually close to and intertwined, aldehyde groups on the hydroformylation polysaccharide obtained after hydroformylation of the natural polysaccharide and amino groups on aminoglycoside antibiotics can form acid-responsive Schiff base bonds in an alkaline buffer solution, nanoparticles are formed under the condition of magneton high-speed stirring, added hydrophilic PEG with amino groups can also be connected to the nanoparticles through Schiff base reaction, and a relatively hydrophilic surface is formed on the surfaces of the nanoparticles, so that the stability of the nanoparticles is improved, and the nanoparticles are prevented from agglomerating. The nano-drug with high drug loading capacity loaded by two modes of covalent bond and non-covalent bond is finally obtained by the method. The nano-drug can be applied according to the lower respiratory tract infection condition, acidic substances such as lactic acid and the like generated by bacteria stimulate Schiff base bond acid to respond to rupture and release antibiotics at the serious infection part, and on the other hand, the nano-drug is released due to the rupture of covalent bonds, so that the antibiotics wrapped by non-covalent bonds in the material are also released rapidly, and the material can rapidly release all the loaded antibiotics to perform rapid sterilization. When the bacterial infection is not serious, the material only releases the part loaded with the antibiotics through the non-covalent bond for sterilization, so that the ototoxic nephrotoxicity of the medicine to a human body can be relatively reduced, the utilization rate of the medicine is improved, and the generation of drug-resistant bacteria is reduced.
The invention has the beneficial effects that:
1. good biocompatibility
The nanometer medicine has natural polysaccharide as raw material, good biocompatibility, and can be digested and degraded by lysozyme or phagocyte, etc., and compared with inorganic compound, the nanometer medicine does not cause accumulation and residue of carrier material.
2. Good self-adaptation performance
The surface of the nano-drug is positively charged and can be attracted into the negatively charged mucus layer, and the hydrophilic layer on the surface can help the nano-particles to avoid the hydrophobic effect of the mucus layer and rapidly penetrate through the mucus layer. The nanoparticles carry the medicine by two loading modes of covalent bond and non-covalent bond, the obtained nanoparticles have high medicine loading capacity, and the Schiff base bonds in the nanoparticles can be broken in pH response, so that the medicine loss is basically avoided in the penetration process, and the medicine can be administered according to different infection degrees after reaching the infected part to carry out self-adaptive sterilization. The treatment strategy can effectively improve the utilization rate of the medicine, reduce the dosage of the medicine, reduce the ototoxicity and the nephrotoxicity of the aminoglycoside, and reduce the generation of drug-resistant bacteria.
3. Can realize the pulmonary drug delivery through the aerosol inhalation
The hydration layer formed by hydrophilic PEG with amino exists on the surface of the nano-drug, so that the nano-drug has good dispersibility, can effectively penetrate mucus, has good atomization performance, and can effectively deliver the nano-particles to the deep lung to kill and kill bacteria and biological membranes planted in the lower layer of the mucus. And the nanoparticle is formed by connecting covalent bond Schiff base bonds, has more stable structure compared with the nanoparticle formed by non-covalent action, can realize pulmonary administration by atomization inhalation, can form high drug concentration in the lung compared with oral administration or intravenous administration, avoids first pass effect, reduces drug dosage, reduces drug toxic and side effects, and is more suitable for treating serious lower respiratory tract infection.
Drawings
In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:
FIG. 1 is a flow chart of a process for synthesizing one of the nano-drugs.
FIG. 2 is a TEM photograph of the nano-drug.
FIG. 3 is a graph of the result of N1 s peak separation by X-ray photoelectron spectroscopy (XPS) of the nano-drug.
Fig. 4 is a schematic diagram of artificial simulation of lung mucus by nano-drug permeation.
FIG. 5 is a graph showing the antibacterial effect of the nano-drug.
FIG. 6 is a graph of H & E staining results of a nano-drug animal in vivo lung treatment experiment.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. FIG. 1 is a flow chart of a process for synthesizing one of the nano-drugs, wherein soluble starch is completely dissolved by hot water, and then is hydroformylated by sodium periodate to obtain the hydroformylation soluble starch. Dissolving the hydroformylation starch in sodium carbonate buffer solution, adding the solution into ethanol after completely dissolving, stirring until the solution is uniformly dispersed, and simultaneously adding tobramycin and methoxy polyethylene glycol-amino (mPEG-NH) into the mixed solution2) And forming the nano particles through Schiff base reaction.
Example 1
1. Synthesis of hydroformylation soluble starch
5g of soluble starch was dissolved in 50mL of deionized water, 3.3g of sodium periodate was added, and 1M of H was used 2SO4Adjusting the pH to be about 3, reacting for 4h at 40 ℃ in a dark place, transferring the reaction product into a 3500D dialysis bag after the reaction is finished, dialyzing for 3 days, and freeze-drying to obtain the hydroformylation degree of the obtained hydroformylation starch: 50 percent.
2. Synthesis of nanoparticles
Dissolving 25mg of hydroformylation starch in 5.6mL of sodium carbonate buffer solution with pH of 8, adding into 11mL of anhydrous ethanol after completely dissolving, stirring for 30min, and adding 6.15mg of tobramycin and 36.9mg of mPEG-NH simultaneously2(Mw＝5000g mol-1) And reacting at room temperature for 2 h. After the reaction is finished, supernatant is removed by centrifugation at 12000rpm, then the mixture is added with ionized water, washed by ultrasonic waves and centrifuged, repeated for three times and freeze-dried, and the obtained material is named as STP 21.
3. Pulmonary mucus penetration behavior of nanoparticles
Firstly, preparing artificial mucus (CF-AM), wherein the formula is as follows: 500mg DNA, 250mg mucin, 0.295mg DPTA, 1mL RPMI 1640 amino acid solution, 250. mu.L egg yolk emulsion, 250mg NaCl, 110mg KCl were added to 50mL nuclease-free water and mixed, and the pH was adjusted to neutral with 1M NaOH. 400 mul of agarose gel is placed at the lower layer of a 2mL sample bottle, 600 mul of CF-AM is placed at the upper layer, 50 mul of nanoparticle dispersion liquid dyed by Coomassie brilliant blue is placed at the upper layer, and the time of the blue nanoparticle layer penetrating through the artificial mucus layer is recorded.
4. Antibacterial property
Samples were first serially diluted in 96-well plates to 2-2048. mu.g/mL, 70. mu.L per well in triplicate. Add 70. mu.L of 2X 10 to each well5The suspension of Staphylococcus aureus in CFU/mL was incubated for 12h in a shaker. OD was read with a microplate reader.
5. Experiment on treatment of pulmonary infection in animal body
Experiments were performed in four groups using BALB/c female mice, about 20g at 8 weeks of age. The following four groups were inoculated by nasal drip. Healthy control group: inoculated with 20 μ L of sterile PBS, untreated group: inoculation with 20. mu.L of 1X 1010Staphylococcus aureus in CFU/mL, ensuring the inoculation concentration of 2X 10 in mice8CFU/mouse, after inoculation, the mice are placed vertically for 5min to ensure that bacteria can fully enter the lungs through the trachea, and the subsequent group is not treated. STP21 group: inoculation 2X 108CFU/mouse Staphylococcus aureus, nebulized STP 21. The dosage of the medicine is 30 mg/kg. The aerosol administration is carried out by a household atomizer, the aerosol administration time is controlled by a timer according to the body weight of a mouse, and the aerosol treatment is carried out once a day and twice in total. The lungs of the mice were removed and the right lung was fixed with a tissue fixative and then subjected to H&E staining characterization, observation of inflammatory conditions in tissues.
Example 2
1. Synthesis of hydroformylation soluble starch
5g of soluble starch was dissolved in 50mL of deionized water, 3.3g of sodium periodate was added, and 1M of H was used2SO4Adjusting pH to about 3, reacting at 40 deg.C for 4h, transferring into 3500D dialysis bag, dialyzing for 3 days, and lyophilizing. The degree of hydroformylation of the resulting hydroformylation starch was: 50 percent.
2. Synthesis of nanoparticles
Dissolving 25mg of hydroformylation starch in 5.6mL of sodium carbonate buffer solution with pH of 8, adding into 11mL of anhydrous ethanol after completely dissolving, stirring for 30min, and adding 3.075mg of tobramycin and 36.9mg of mPEG-NH simultaneously2(Mw＝2000g mol-1) And reacting at room temperature for 2 h. Centrifuging at 12000rpm after the reaction is finished to remove supernatant, adding ionized water, ultrasonically cleaning, centrifuging,this was repeated three times and lyophilized and the resulting material was designated STP 41.
3. Antibacterial property
Samples were first serially diluted in 96-well plates to 2-2048. mu.g/mL, 70. mu.L per well in triplicate. Add 70. mu.L of 2X 10 to each well5The suspension of Staphylococcus aureus in CFU/mL was incubated for 12h in a shaker. OD was read with a microplate reader.
Example 3
1. Synthesis of hydroformylation sodium alginate
Dissolving 2g of sodium alginate in 100mL of deionized water, adding 1.1g of sodium periodate, reacting for 4h at 40 ℃ in a dark place, stopping the reaction by using excessive ethylene glycol, transferring the mixture into a 3500D dialysis bag after the reaction is finished, dialyzing for 3 days, and freeze-drying. The degree of hydroformylation of the resulting hydroformylation starch was: 60 percent.
2. Synthesis of nanoparticles
Dissolving 25.5mg of sodium alginate hydroformylation in 5.6mL of sodium carbonate buffer solution with pH of 8, adding into 11mL of absolute ethanol after completely dissolving, stirring for 30min, and simultaneously adding 6.15mg of tobramycin and 36.9mg of mPEG-NH2(Mw＝3000g mol-1) And reacting at room temperature for 2 h. After the reaction is finished, supernatant is removed by centrifugation at 12000rpm, then the mixture is added with ionized water, washed by ultrasonic waves and centrifuged, repeated for three times and freeze-dried, and the obtained material is named as ATP 21.
3. Pulmonary mucus penetration behavior of nanoparticles
Firstly, preparing artificial mucus (CF-AM), wherein the formula is as follows: 500mg DNA, 250mg mucin, 0.295mg DPTA, 1mL RPMI 1640 amino acid solution, 250. mu.L egg yolk emulsion, 250mg NaCl, 110mg KCl were added to 50mL nuclease-free water and mixed, and the pH was adjusted to neutral with 1M NaOH. 400 mul of agarose gel is placed at the lower layer of a 2mL sample bottle, 600 mul of CF-AM is placed at the upper layer, 50 mul of nanoparticle dispersion liquid dyed by Coomassie brilliant blue is placed at the upper layer, and the time of the blue nanoparticle layer penetrating through the artificial mucus layer is recorded.
4. Antibacterial property
Samples were first serially diluted in 96-well plates to 2-2048. mu.g/mL, 70. mu.L per well in triplicate. Add 70. mu.L of 2X 10 to each well5The suspension of Staphylococcus aureus in CFU/mL was incubated for 12h in a shaker. OD was read with a microplate reader.
5. Experiment on treatment of pulmonary infection in animal body
Experiments were performed in four groups using BALB/c female mice, about 20g at 8 weeks of age. The following four groups were inoculated by nasal drip. Healthy control group: inoculated with 20 μ L of sterile PBS, untreated group: inoculation with 20. mu.L of 1X 1010Staphylococcus aureus in CFU/mL, ensuring the inoculation concentration of 2X 10 in mice8CFU/mouse, after inoculation, the mice are placed vertically for 5min to ensure that bacteria can fully enter the lungs through the trachea, and the subsequent group is not treated. ATP21 group: inoculation 2X 108CFU/mouse Staphylococcus aureus, nebulized ATP21 treatment. The dosage of the medicine is 30 mg/kg. The aerosol administration is carried out by a household atomizer, the aerosol administration time is controlled by a timer according to the body weight of a mouse, and the aerosol treatment is carried out once a day and twice in total. The lungs of the mice were removed and the right lung was fixed with a tissue fixative and then subjected to H&E staining characterization, observation of inflammatory conditions in tissues.
Example 4
1. Synthesis of hydroformylation soluble starch
5g of soluble starch was dissolved in 50mL of deionized water, 3.3g of sodium periodate was added, and 1M of H was used2SO4Adjusting pH to about 3, reacting at 40 deg.C for 4h, transferring into 3500D dialysis bag, dialyzing for 3 days, and lyophilizing. The degree of hydroformylation of the resulting hydroformylation starch was: 50 percent.
2. Synthesis of nanoparticles
Dissolving 25mg of hydroformylation starch in 5.6mL of sodium carbonate buffer solution with pH of 8, adding into 11mL of anhydrous ethanol after completely dissolving, stirring for 30min, and adding 12.3mg of tobramycin and 36.9mg of mPEG-NH simultaneously2(Mw＝5000g mol-1) And reacting at room temperature for 2 h. After the reaction is finished, supernatant is removed by centrifugation at 12000rpm, then the mixture is added with ionized water, washed by ultrasonic waves and centrifuged, repeated for three times and freeze-dried, and the obtained material is named as STP 12.
Example 5
1. Synthesis of hydroformylation soluble starch
5g solubleStarch was dissolved in 50mL of deionized water, 3.3g of sodium periodate was added, and 1M H was used2SO4Adjusting pH to about 3, reacting at 40 deg.C for 4h, transferring into 3500D dialysis bag, dialyzing for 3 days, and lyophilizing. The degree of hydroformylation of the resulting hydroformylation starch was: 30 percent.
2. Synthesis of nanoparticles
41.7mg of the hydroformylation starch was dissolved in 5.6mL of sodium carbonate buffer solution having pH 8, and after completion of the dissolution, the solution was added to 11mL of anhydrous ethanol, and after stirring for 30min, 6.15mg of tobramycin was added thereto, and the reaction was carried out at room temperature for 2 hours. After the reaction, the supernatant was removed by centrifugation at 12000rpm, washed with additional ionized water by ultrasound, centrifuged, repeated three times, and lyophilized to obtain ST 21.
3. Pulmonary mucus penetration behavior of nanoparticles
Firstly, preparing artificial mucus (CF-AM), wherein the formula is as follows: 500mg DNA, 250mg mucin, 0.295mg DPTA, 1mL RPMI 1640 amino acid solution, 250. mu.L egg yolk emulsion, 250mg NaCl, 110mg KCl were added to 50mL nuclease-free water and mixed, and the pH was adjusted to neutral with 1M NaOH. 400 mul of agarose gel is placed at the lower layer of a 2mL sample bottle, 600 mul of CF-AM is placed at the upper layer, 50 mul of nanoparticle dispersion liquid dyed by Coomassie brilliant blue is placed at the upper layer, and the time of the blue nanoparticle layer penetrating through the artificial mucus layer is recorded.
FIG. 2 is a schematic diagram showing the surface morphological characteristics of the nano-drugs, which shows that the nanoparticles of examples 1, 2 and 3 have good dispersibility, the nanoparticles of examples 4 and 5 have poor dispersibility, and the amino group and hydrophilic mPEG-NH with amino group in the aminoglycoside antibiotic of example 42Is 1:2 (not in the range of 3:1-2:1 as claimed in the invention), indicating that the amino group of the aminoglycoside antibiotic is present in the amino group of the hydrophilic mPEG-NH carrying an amino group2The molar ratio of (A) to (B) can influence the dispersibility of the nanoparticles, and the excessive amount of amino groups on the antibiotic can occupy mPEG-NH2Site of the upper amino group, resulting in mPEG-NH on reaction2Is reduced, which results in the failure to form a hydrophilic layer on the surface of the nanoparticles, example 5 no addition of mPEG-NH2And a hydrophilic layer can not be formed, so that the nanoparticles are crosslinked with each other and aggregated,agglomeration results in poor nanoparticle dispersibility, inability to effectively penetrate mucus, poor atomization, inability to effectively deliver nanoparticles to the deep lung, and inability to kill bacteria and biofilms colonized on the lower layers of mucus.
FIG. 3 is a graph showing the result of N1 s peak separation by X-ray photoelectron spectroscopy (XPS) of the nano-drug, from which it can be seen that the Schiff base bond peak positions of examples 1, 2, 3, 4 and 5 all appear around 399.8eV, this indicates that antibiotic loading was successful through Schiff base bonding, the peak areas were the largest and the amount of antibiotic loaded for examples 1 and 3, the peak area was relatively smaller for example 2, the amount of antibiotic loading was less, this is due to the fact that the molar amount of antibiotic added in example 2 is small, the molar ratio of aldehyde groups in the hydroformylated natural polysaccharide to amino groups in the aminoglycoside antibiotic in example 2 is 4:1 (outside the range of 3:1-2:1 claimed in the present invention), the amount of antibiotic on the reaction is greatly reduced, indicating that the molar ratio of aldehyde groups in the hydroformylation natural polysaccharide to amino groups in the aminoglycoside antibiotic affects the antibiotic loading.
FIG. 4 is a schematic diagram of artificial simulation of lung mucus by nano-drug permeation, and it can be seen that examples 1 and 3 have good mucus permeation performance, while example 5 has relatively poor mucus permeation performance due to the mPEG-NH on the surface of each of examples 1 and 32The hydrophilic layer formed is beneficial to the smooth penetration of the mucus layer by the nano particles, while the antibiotic is added in example 5 without adding mPEG-NH2Therefore, the surface does not have a hydrophilic layer, so that the effect of the nano particles on penetrating mucus is relatively poor, the delivery efficiency of the nano particles to infected parts of the lung is low, and the killing effect on bacteria and biological membranes planted in the lower layer of the mucus is poor.
Fig. 5 is a graph showing the antibacterial results of the nano-drugs, and it can be seen that examples 1 and 3 have better antibacterial activity against staphylococcus aureus than example 2, because examples 1 and 3 have a greater proportion of added antibiotics and a greater drug loading.
Fig. 6 is a graph of H & E staining results of a lung treatment experiment in a nano-drug animal, and it can be seen from the graph that compared with the untreated group, the alveolar structures of the two groups of examples 1 and 3 are relatively complete, the inflammation condition is greatly reduced, and the lung condition is close to that of the healthy control group, and the treatment effect is good, because the nanoparticles of examples 1 and 3 have good dispersibility and mucus permeability, the nanoparticles can smoothly penetrate the mucus to reach the infected part for sterilization after atomization, and the good treatment effect on lower respiratory tract infection is generated.
In conclusion, the two groups of nanoparticles in the examples 1 and 3 have the best comprehensive performance, and both have the advantages of good dispersibility, higher drug loading capacity, good mucus permeability, excellent antibacterial performance and good treatment effect on pulmonary infection in animals. Example 2 had a good dispersion effect, but the low drug loading level had a poor antibacterial effect, and examples 4 and 5 had poor dispersion and poor permeation. Therefore, the examples 2, 4 and 5 can not be used as microenvironment adaptive nano-drugs for severe lower respiratory tract infection.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (10)

1. A preparation method of a microenvironment adaptive nano-drug delivery system for severe lower respiratory tract infection is characterized by comprising the following preparation steps:
1) preparing a hydroformylation natural polysaccharide;
2) dissolving the hydroformylation natural polysaccharide obtained in the step 1) in an alkaline buffer solution, then adding a poor solvent, stirring until the mixture is completely dispersed and uniformly mixed, simultaneously adding aminoglycoside antibiotics and hydrophilic PEG with amino, stirring at a high speed, and reacting for 0.5-48h at the temperature of 20-80 ℃; the ratio of aldehyde groups in the hydroformylation natural polysaccharide to amino groups in the aminoglycoside antibiotic is 3:1-2:1, and the ratio of amino groups in the aminoglycoside antibiotic to hydrophilic PEG with amino groups is 12:1-1: 12;
3) Repeatedly centrifuging and ultrasonically dispersing the reaction product obtained in the step 2) until the residual reactant is cleaned and freeze-dried to obtain the self-adaptive nano-drug.
2. The method for preparing the microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection according to claim 1, wherein the volume ratio of the alkaline buffer solution and the poor solvent in the step 2) is 2:1-1: 2.
3. The method for preparing the microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection according to claim 1, wherein the natural polysaccharide in the step 1) is a natural polysaccharide containing ortho-dihydroxy.
4. The method for preparing the microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection according to claim 1, wherein the methoxypolyethylene glycol-amino in the step 2).
5. The method for preparing the microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection according to claim 1, wherein the natural polysaccharide is one or two of dextran, starch, hyaluronic acid, sodium alginate, glycyrrhiza polysaccharide, xanthan gum, cellulose, bletilla polysaccharide, chondroitin sulfate and heparin sodium.
6. The method for preparing the microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection according to claim 1, wherein the hydroformylation degree of the hydroformylation natural polysaccharide in the step 1) is 10 to 100 percent.
7. The method for preparing the microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection according to claim 1, wherein the pH of the alkaline buffer in the step 2) is 7.4 to 11.
8. The method for preparing the microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection according to claim 1, wherein the poor solvent in the step 2) is one or two of ethanol, methanol, acetone and diethyl ether.
9. The method for preparing the microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection according to claim 1, wherein the aminoglycoside antibiotic of step 2) is one or two of gentamicin, tobramycin, amikacin, streptomycin, kanamycin, etimicin, ribostamycin, netilmicin, and micronomicin.
10. A microenvironment-adaptive nano-drug delivery system for severe lower respiratory tract infection prepared by the preparation method according to any one of claims 1 to 9.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014161085A1 (en) * 2013-04-02 2014-10-09 University Of Manitoba Schiff-based aldehydic hyaluronic acid-chitosan hydrogel compositions and uses thereof
CN110772664A (en) * 2019-11-25 2020-02-11 北京化工大学 Preparation method of surface intelligent coating of orthopedic temporary implant based on natural polysaccharide and product thereof
WO2020206348A1 (en) * 2019-04-04 2020-10-08 The Texas A&M University System Delivery devices for localized delivery of antimicrobial, anti-inflammatory, and antioxidant agents
CN112336685A (en) * 2020-10-29 2021-02-09 西南交通大学 Antibacterial micro-emulsion gel and preparation method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014161085A1 (en) * 2013-04-02 2014-10-09 University Of Manitoba Schiff-based aldehydic hyaluronic acid-chitosan hydrogel compositions and uses thereof
WO2020206348A1 (en) * 2019-04-04 2020-10-08 The Texas A&M University System Delivery devices for localized delivery of antimicrobial, anti-inflammatory, and antioxidant agents
CN110772664A (en) * 2019-11-25 2020-02-11 北京化工大学 Preparation method of surface intelligent coating of orthopedic temporary implant based on natural polysaccharide and product thereof
CN112336685A (en) * 2020-10-29 2021-02-09 西南交通大学 Antibacterial micro-emulsion gel and preparation method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JINGJING HU ET.AL.: "A smart aminoglycoside hydrogel with tunable gel degradation, on-demand drug release, and high antibacterial activity", 《JOURNAL OF CONTROLLED RELEASE》, vol. 247, pages 147 *
JUNG SOO SUK ET.AL.: "The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles", 《BIOMATERIALS》, vol. 30, no. 13, pages 2594 *
吴修利 等: "溶剂交换法制备淀粉纳米颗粒及表征", 《食品研究与开发》, vol. 38, no. 24, pages 7 *

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