Abeta11 and T80 double-ligand modified core-shell nanoparticles, and preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an A beta 11 and T80 double-ligand modified core-shell nanoparticle as well as a preparation method and application of the A beta 11 and T80 double-ligand modified core-shell nanoparticle. The main technical scheme of the invention is as follows: a polymer nanoparticle core for entrapment of drugs and a lipid material wrapping the surface of the polymer nanoparticle core are provided, and A beta 11 and T80 are added into the lipid material for modification. The double-ligand-modified core-shell nanoparticles disclosed by the invention are excellent in the aspects of particle size, potential and encapsulation efficiency data, and are beneficial to delivery of nanoparticle drugs; the stability is better, and the transportation and storage of the entrapped medicine are more facilitated; the antibacterial agent can remarkably penetrate through the blood brain barrier and has a remarkable and better antibacterial effect; the compound has remarkable effects in pharmacokinetic power, pharmacodynamic power and safety, and has a very good application prospect in the aspect of intracranial anti-infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to a core-shell nanoparticle modified with Aβ11 and T80 double ligands, a preparation method thereof and uses thereof. Background Art

[0002] Intracranial infection is an acute inflammatory disease caused by pathogenic microorganisms invading the brain. Common infection sources include bacteria, fungi, viruses and parasites, and usually manifest as headache, fever, mental state changes and behavioral changes. Among them, bacteria are the most important pathogenic microorganisms causing intracranial infection clinically. Although certain progress has been made clinically at present, intracranial infection still has a high incidence rate, and its incidence is 0.34%-3.1%. Intracranial infection can be community-acquired or hospital-acquired, and patients with neurosurgery, severe nerve trauma or a history of indwelling cerebrospinal fluid drainage have a higher risk of nosocomial infection. At present, the existing technology for the drug treatment of intracranial infection usually adopts systemic antibacterial therapy, and among them, vancomycin, penicillin, rifampicin and the third-generation cephalosporins are common drugs for the treatment of intracranial infection.

[0003] The blood-brain barrier (BBB) is a barrier existing between the blood and the brain, formed by the perivascular endings of a layer of tightly closed endothelial cells, pericytes and astrocytes. The barrier function of the BBB strictly regulates the transport of various substances from the blood to the brain, and can prevent most drug molecules from entering to protect the brain. Since drugs cannot effectively penetrate the BBB to reach the therapeutic concentration in the brain parenchyma, the difficult treatment of brain diseases has always been a technical problem to be solved urgently.

[0004] In recent years, nanotechnology has been widely applied in the medical field, and nanocarriers have also been developed into drug delivery systems to achieve targeted, sustained and controlled release drug therapy. However, drug delivery using nanocarriers still fails to achieve good therapeutic effects in improving the penetration of drug components through the blood-brain barrier (BBB) for intracranial treatment. Currently, the nanotechnologies disclosed in the prior art include using brain-targeting polypeptide rabies virus glycoprotein (RVG) 29 and glucose transporter 1 (GLUT1) modified polymeric nanoparticles to treat intracranial infections caused by bacteria and fungi. There is also photothermal therapy based on gold nanoparticles for treating brain infections. However, these technologies disclosed in the prior art have the defects of low permeability through the BBB, and intravenous administration at conventional doses cannot achieve effective drug treatment concentrations in cerebrospinal fluid, resulting in poor therapeutic effects and high likelihood of drug resistance. The method of intraventricular injection used in the prior art to compensate for the above defects has technical defects such as high treatment costs in clinical practice, invasive operations being more likely to cause secondary infections, and repeated administration increasing the pain of patients. Therefore, its clinical application is greatly restricted. In addition, the prior art has also disclosed research by existing researchers on using Aβ11 as a brain-targeting peptide to modify liposomes to increase the concentration of doxorubicin in the brain for treating mouse glioblastoma. And there are also related studies on Tween80 (T80) as an ionic surfactant enhancing the accumulation of nanoparticles in the brain in BBB endothelial cells.

[0005] Although the prior art has disclosed some theoretical studies on the use of Aβ11 and T80 as described above to penetrate the blood-brain barrier for intracranial anti-infection, there are no reports on how to apply them in nanoparticle delivery, nor is it disclosed how to prepare nanoparticle delivery loads specifically in combination with antibacterial drugs. Therefore, the prior art for treating intracranial anti-infection by nanoparticle delivery through the blood-brain barrier is still in the theoretical research stage, and there is a need for application optimization. Summary of the Invention

[0006] Based on the above technical problems to be solved by the present invention in penetrating the blood-brain barrier and improving the antibacterial treatment of intracranial anti-infection drugs.

[0007] The first aspect of the present invention provides a core-shell nanoparticle modified with Aβ11 and T80 double ligands; the core-shell nanoparticle modified with double ligands includes a polymeric nanoparticle core encapsulating a drug and a lipid material coating the surface of the polymeric nanoparticle core; wherein, the lipid material is modified with Aβ11 and T80.

[0008] Further, for the core-shell nanoparticles modified with Aβ11 and T80, the polymer carrier used for the polymer nanoparticle core is one or more of mPEG-PLGA(75 / 25), PLGA(50 / 50), PLGA(75 / 25), PLGA-PEG-PLGA(50 / 50), and PLGA-PEG-PLGA(75 / 25);

[0009] Further, for the core-shell nanoparticles modified with Aβ11 and T80, the polymer carrier used is a mixed polymer carrier of mPEG-PLGA(75 / 25) and PLGA(75 / 25);

[0010] Further, for the core-shell nanoparticles modified with Aβ11 and T80, in the mixed polymer carrier, the mass ratio of mPEG-PLGA(75 / 25) to PLGA(75 / 25) is 1:0.1 to 1:2;

[0011] Further, for the core-shell nanoparticles modified with Aβ11 and T80, in the mixed polymer carrier, the mass ratio of mPEG-PLGA(75 / 25) to PLGA(75 / 25) is 1:1;

[0012] Further, for the core-shell nanoparticles modified with Aβ11 and T80, Aβ11 accounts for 1 to 20% of the total lipid molar amount in the lipid material;

[0013] Further, for the core-shell nanoparticles modified with Aβ11 and T80, the percentage content of T80 in the lipid material is 0.5% to 10%;

[0014] Further, for the core-shell nanoparticles modified with Aβ11 and T80, the drug is selected from one or more of proteins, polypeptides, small molecules, plasmids, siRNA, and micRNA;

[0015] Further, for the core-shell nanoparticles modified with Aβ11 and T80, the drug is a protein;

[0016] Further, for the core-shell nanoparticles modified with Aβ11 and T80, the drug is a polypeptide;

[0017] Further, for the core-shell nanoparticles modified with Aβ11 and T80, the drug is a small molecule;

[0018] Further, for the core-shell nanoparticles modified with Aβ11 and T80, the drug is a plasmid;

[0019] Furthermore, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, the drug is siRNA;

[0020] Furthermore, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, the drug is micRNA;

[0021] Furthermore, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, the drug is an antibacterial drug;

[0022] Furthermore, the antibacterial drug is one or more of methicillin, penicillin, vancomycin, and tigecycline;

[0023] Furthermore, the drug is tigecycline;

[0024] Furthermore, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, the polymer nanoparticle core is prepared by the nanoprecipitation method or the double emulsion method; preferably by the double emulsion method;

[0025] Furthermore, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, the lipid material is wrapped on the surface of the PLGA nanoparticle core by the thin film hydration method;

[0026] In a specific embodiment of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, one or more of polyvinyl alcohol, soybean lecithin, and cholesterol are further included in the polymer nanoparticle core and the lipid material;

[0027] In a specific implementation scheme of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, Aβ11 accounts for 1% of the total lipid molar amount in the lipid material;

[0028] In another specific implementation scheme of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, Aβ11 accounts for 5% of the total lipid molar amount in the lipid material;

[0029] In another specific implementation scheme of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, Aβ11 accounts for 10% of the total lipid molar amount in the lipid material;

[0030] In another specific implementation scheme of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, Aβ11 accounts for 15% of the total lipid molar amount in the lipid material;

[0031] In a specific implementation scheme of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80 ligands, the percentage content of T80 in the lipid material is 0.5%%;

[0032] In another specific embodiment of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80, the percentage content of T80 in the lipid material is 1%;

[0033] In another specific embodiment of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80, the percentage content of T80 in the lipid material is 1.5%;

[0034] In another specific embodiment of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80, the percentage content of T80 in the lipid material is 2%;

[0035] In another specific embodiment of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80, the percentage content of T80 in the lipid material is 5%;

[0036] In another specific embodiment of the present invention, for the core-shell nanoparticles modified with Aβ11 and T80, the percentage content of T80 in the lipid material is 8%;

[0037] On the other hand, the present invention also provides a method for preparing core-shell nanoparticles modified with Aβ11 and T80, including preparing a polymer nanoparticle core loaded with a drug, and then wrapping a lipid material containing Aβ11 and T80 on the surface of the polymer nanoparticle core to obtain core-shell nanoparticles modified with Aβ11 and T80;

[0038] Further, for the method for preparing core-shell nanoparticles modified with Aβ11 and T80, the polymer nanoparticle core is as defined above;

[0039] Further, for the method for preparing core-shell nanoparticles modified with Aβ11 and T80, the lipid material is as defined above;

[0040] Further, the specific steps of the method for preparing the double-ligand-modified nanoparticles include:

[0041] Step 1: Prepare a polymer nanoparticle core containing a drug: Dissolve the polymer material in an organic solvent of dichloromethane and acetone as the oil phase (O), and the aqueous solution of the loaded drug as the inner aqueous phase (W1). Slowly drip the aqueous phase into the oil phase and ultrasonically obtain the primary emulsion (W1 / O); Drip the primary emulsion (W1 / O) into the PVA outer aqueous phase (W2) and ultrasonically obtain the double emulsion (W1 / O / W2); Remove the organic solvents dichloromethane and acetone from the double emulsion under reduced pressure to obtain a polymer nanoparticle core loaded with a drug;

[0042] Step 2, lipid material encapsulation: PEG2000-Chol, Aβ11-PEG2000-Chol, PLS100, and Chol are dissolved in absolute ethanol. Tween80 is added, and after uniform mixing, the organic solvent is removed under reduced pressure to form a film. The polymer nanoparticle core solution loaded with the drug prepared in Step 1 is added to the film, and hydrated until the film completely detaches. The hydrated sample is sonicated to obtain the core-shell nanoparticles modified with Aβ11 and Tween80 (Aβ11 / T80@CSs-drug).

[0043] Furthermore, the drug is an antibacterial drug;

[0044] In a specific embodiment of the present invention, the antibacterial drug is TGC.

[0045] On the other hand, the present invention provides the use of the core-shell nanoparticles modified with Aβ11 and T80 in the preparation of drugs for crossing the blood-brain barrier;

[0046] Furthermore, the drug is an antibacterial drug;

[0047] Furthermore, the antibacterial drug is selected from one or more of antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones, and glycylcyclines;

[0048] Furthermore, the antibacterial drug is selected from one or more of penicillin, potassium penicillin V, oxacillin, cloxacillin, ampicillin, benzathine penicillin, furbenicillin, amoxicillin, mezlocillin, nafcillin, azlocillin, piperacillin, meropenem, cefalexin, cefotiam, cefadroxil, cefoxitin, cefazolin, cefradine, cefaclor, cefuroxime, cefpiramide, cefathiamidine, cefprozil, ceftriaxone, cefixime, cefminox, ceftazidime, cefdinir, latamoxef, ceftezole, cefmetazole, cefotaxime, cefoperazone, cefamandole, cefodizime, cefpirome, cefepime, cefozopran, amoxicillin clavulanate potassium, amoxicillin sulbactam, mezlocillin sulbactam, cefoperazone sulbactam, piperacillin sulbactam, cefoperazone tazobactam, imipenem cilastatin, panipenem betamipron, amikacin, gentamicin, amikacin, streptomycin, netilmicin, tobramycin, etimicin, spectinomycin, isepamicin, kanamycin, paromomycin, chloramphenicol, thiamphenicol, vancomycin, norvancomycin, teicoplanin, erythromycin, erythromycin ethylsuccinate, kitasamycin, acetylkitasamycin, roxithromycin, clarithromycin, azithromycin, tetracycline, doxycycline, minocycline, tigecycline, sulfamethoxazole, trimethoprim, ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, fleroxacin, enoxacin, lomefloxacin, gatifloxacin, sparfloxacin, moxifloxacin, pazufloxacin, nitrofurantoin, furazolidone, nystatin, clotrimazole, bifonazole, terbinafine, ketoconazole, flucytosine, fluconazole, miconazole, itraconazole, amphotericin B, metronidazole, benzyimidazole metronidazole, tinidazole, ornidazole;

[0049] Furthermore, the antibacterial drug further includes one or more of the above antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones in combination with one or more other drugs;

[0050] Furthermore, the other drugs include, but are not limited to, one or more of anti-tumor drugs, anti-inflammatory drugs, and immune drugs.

[0051] The beneficial effects of the present invention are as follows:

[0052] The core-shell nanoparticles modified with Aβ11 and T80 ligands disclosed in the present invention use an inner core with an optimized polymer ratio to encapsulate the drug, and the outer layer includes a lipid material modified with Aβ11 and Tween80 ligands. The obtained core-shell nanoparticles (Aβ11 / T80@CSs-drug) are excellent in terms of particle size, zeta potential, and encapsulation efficiency, which is beneficial for the delivery of nanoparticle drugs;

[0053] The core-shell nanoparticles modified with Aβ11 and T80 dual ligands disclosed by the present invention, namely the obtained core-shell nanoparticles (Aβ11 / T80@CSs-drug), have better stability and are more conducive to the transportation and storage of the encapsulated drug;

[0054] Through in vitro antibacterial experiments on mice and intrathecal injection experiments in vivo, the core-shell nanoparticles (Aβ11 / T80@CSs-drug) of the Aβ11 and T80 dual ligand-modified core-shell nanoparticles of the present invention have remarkable ability to penetrate the blood-brain barrier and have significantly better antibacterial effects; they have significantly excellent effects in pharmacokinetic tests, pharmacodynamic tests and safety tests, and have very good application prospects in intracranial anti-infection. Description of the Drawings

[0055] Figure 1 Particle size diagram of the polymer nanoparticles encapsulating TGC of F1-F7 in Example 1;

[0056] Figure 2 Potential diagram of the polymer nanoparticles encapsulating TGC of F1-F7 in Example 1;

[0057] Figure 3 Encapsulation efficiency diagram of the polymer nanoparticles encapsulating TGC prepared from different polymer carrier materials in Example 1;

[0058] Figure 4 Fluorescence intensity diagram of the polymer nanoparticles encapsulating DiD fluorescence-labeled prepared from different polymer carrier materials in Example 1 in the brains of mice;

[0059] Figure 5 Fluorescence distribution diagram of CSs modified with different molar ratios of Aβ11 in the brains of mice in Example 2;

[0060] Figure 6 Fluorescence quantification diagram of CSs modified with different molar ratios of Aβ11 in the brains of mice in Example 2;

[0061] Figure 7 Fluorescence distribution diagram of CSs modified with different volume ratios of Tween80 in the brains of mice in Example 2;

[0062] Figure 8 Fluorescence quantification of CSs modified with different volume ratios of Tween80 in the brains of mice in Example 2;

[0063] Figure 9 Morphology diagram of Aβ11 / T80@CSs-TGC and CSs-TGC solutions under natural light conditions in Example 3;

[0064] Figure 10Tyndall phenomenon diagrams of Aβ11 / T80@CSs-TGC and CSs-TGC solutions for Example 3;

[0065] Figure 11 Particle size distribution diagram of Aβ11 / T80@CSs-TGC for Example 3;

[0066] Figure 12 Potential distribution diagram of Aβ11 / T80@CSs-TGC for Example 3;

[0067] Figure 13 Transmission electron microscope image of Aβ11 / T80@CSs-TGC for Example 3;

[0068] Figure 14 Graph of changes in encapsulation efficiency and particle size during the sample stability test for Example 3;

[0069] Figure 15 Appearance diagram of the solution during the sample stability test for Example 3;

[0070] Figure 16 Release curve of Aβ11 / T80@CSs-TGC in PBS for Example 3;

[0071] Figure 17 Observation diagram of the growth of bacteria in TGC and Aβ11 / T80@CSs-TGC for Test Example 5;

[0072] Figure 18 Histogram of OD600 values for Test Example 5;

[0073] Figure 19 Plate coating result diagram of cerebrospinal fluid after intrathecal injection of different groups of preparations for Test Example 6;

[0074] Figure 20 Histogram of bacterial numbers for Test Example 6;

[0075] Figure 21 Laser confocal microscope photograph for Test Example 7;

[0076] Figure 22 Fluorescence result diagram of the uptake of different preparations by bEnd.3 cells for Test Example 7;

[0077] Figure 23 In vitro cumulative transcellular release amount curve graph for Test Example 7;

[0078] Figure 24 Apparent permeability coefficient (Papp) diagram of in vitro trans-monolayer cell membrane for Test Example 7;

[0079] Figure 25 Fluorescence distribution diagram in rats for Test Example 8;

[0080] Figure 26 It is the fluorescence quantitative graph of the brain tissue distribution of the rats in Test Example 8;

[0081] Figure 27 It is the concentration graph of TGC in the cerebrospinal fluid of the rats in Test Example 8;

[0082] Figure 28 It is the blood drug concentration-time curve graph of TGC in Test Example 9;

[0083] Figure 29 It is the colony graph of MDR-AB at 60 mg / kg for 6 h in Test Example 10;

[0084] Figure 30 It is the colony graph of MDR-AB in the cerebrospinal fluid after multi-dose administration in Test Example 10;

[0085] Figure 31 It is the in vitro hemolysis phenomenon graph in Test Example 11;

[0086] Figure 32 It is the columnar graph of in vitro hemolysis quantitative calculation in Test Example 11;

[0087] Figure 33 It is the cytotoxicity detection graph of Free-TGC (A), CSs-TGC (B) and Aβ11 / T80@CSs-TGC (C) in Test Example 11;

[0088] Figure 34 It is the H&E staining graph of the heart, liver, spleen, lung and kidney of the rats in Test Example 11. Detailed implementation manners

[0089] Explanation of abbreviations: As shown in Table 1,

[0090] Table 1

[0091]

[0092] The implementation of the technical solution of the present invention will be specifically described below through specific implementation manners.

[0093] Materials, instruments and animals used in specific examples:

[0094] (1) Materials

[0095] TGC (purity > 98%, Beijing Solarbio Science & Technology Co., Ltd.);

[0096] mPEG-PLGA (PLGA, Mw = 15 kD; LA / GA = 75:25, PEG, Mw = 2000, Shandong Daigang Biotechnology Co., Ltd.);

[0097] PLGA (Mw = 15 kD, LA / GA = 75:25, Shandong Daigang Biotech Co., Ltd.);

[0098] PLGA (Mw = 15 kD, LA / GA = 50:50, Shandong Daigang Biotech Co., Ltd.);

[0099] PLGA-PEG-PLGA (PLGA, Mw = 7.5 kD; LA / GA = 75:25, PEG, Mw = 2000, Shandong Daigang Biotech Co., Ltd.);

[0100] PLGA-PEG-PLGA (PLGA, Mw = 7.5 kD; LA / GA = 50:50, PEG, Mw = 2000, Shandong Daigang Biotech Co., Ltd.);

[0101] PVA (MW = 30000 - 70000, purity > 80%, Sigma-Aldrich, USA);

[0102] Polyvinyl alcohol (PVA; MW = 30000 - 70000, purity > 80%, Sigma-Aldrich, USA);

[0103] Cell membrane far-infrared fluorescence probe DiD (purity > 98%, Dalian Meilun Biotechnology Co., Ltd.);

[0104] Aβ11 (purity > 98%, Qiangyao Biotech Co., Ltd.);

[0105] Tween80 (T80, Guangzhou Saiguo Biotech Co., Ltd.);

[0106] Aβ11-PEG2000-Chol (purity > 98%, State Key Laboratory of Biotherapy, Sichuan University);

[0107] Soybean lecithin (LIPOID S100, batch number: 790587 - 4 / 904, LIPOID, Germany);

[0108] Cholesterol (batch number: 2020010101, Chengdu Kelong Chemical Co., Ltd.);

[0109] Coumarin 6 (purity > 98%, Sigma-Aldrich, USA);

[0110] DMEM medium (Biosharp, Beijing Lanjieke Technology Co., Ltd.);

[0111] Fetal bovine serum (Biosharp, Beijing Lanjieke Technology Co., Ltd.);

[0112] Trypsin (State Key Laboratory of Biotherapy, Sichuan University);

[0113] MTT (Chengdu RuiAo Biotechnology Co., Ltd.);

[0114] HBSS buffer (Gibco, Life Technologies, USA);

[0115] Sterile PBS (Biosharp, Beijing LanJieKe Technology Co., Ltd.);

[0116] DMSO (Chengdu Jinshan Chemical Reagent Co., Ltd.);

[0117] Ammonium hydrogen phosphate (CAS: 7783 - 28 - 0, Chengdu Kelong Chemical Co., Ltd.);

[0118] Triethylamine (Batch No.: 20220412, Chengdu Jinshan Chemical Reagent Co., Ltd.);

[0119] Methanol (CAS: 67 - 52 - 1, NuoErShi Chengdu Kelong Chemical Co., Ltd.).

[0120] (2) Instruments

[0121] Electronic analytical balance (BSM - 120.4, Shanghai Zhuojing Electronic Technology Co., Ltd.);

[0122] Circulating vacuum pump (SHZ - D(Ⅲ), Zhengzhou Zhichuang Machinery Co., Ltd.);

[0123] Ultrasonic cell disruptor (SCIENTZ - Ⅲ, Ningbo Xinzhi Biotechnology Co., Ltd.);

[0124] Laminar flow hood (Sujing Antai AIRTECH);

[0125] Cell incubator (Thermo Scientific, USA);

[0126] Cell culture dish (LABSELECT, Beijing LanJieKe Technology Co., Ltd.);

[0127] Cell culture plate (LABSELECT, Beijing LanJieKe Technology Co., Ltd.);

[0128] Flow cytometer (2060R, NovoCyte, ACEA Biosciences, USA);

[0129] Malvern particle size analyzer (Nano ZS ZEN 36000, Malvern Instruments, UK);

[0130] Rotary evaporator (RE - 201D, Zhengzhou Shengsheng Instruments Co., Ltd.);

[0131] Centrifuge (Sorvall ST16, Thermo Scientific, USA);

[0132] High-performance liquid chromatograph (SIL-20AC, Shimadzu, Japan);

[0133] Small animal in vivo imager (IVIS Lumina III, PerkinElmer, USA);

[0134] Chromatographic column (InertSustain C18, Shimadzu, Japan);

[0135] Dialysis bag (Mw = 3500, Shanghai Yuanye Bio-Technology Co., Ltd.);

[0136] Transmission electron microscope (HF-3300, Hitachi);

[0137] Milli-Q Integral ultrapure water machine (Millipore, USA);

[0138] Carbon dioxide incubator (LRH-70F, Shanghai Yiheng Instrument Co., Ltd.);

[0139] Microplate reader (BioTek, USA);

[0140] Laser confocal microscope (LSM880, Zeiss, Germany);

[0141] Transwell cell culture plate (24-well, LABSELECT, Beijing LanJieKe Technology Co., Ltd.);

[0142] Confocal dish (LABSELECT, Beijing LanJieKe Technology Co., Ltd.);

[0143] Bacterial culture dish (BS-90-D90mm, Beijing LanJieKe Technology Co., Ltd.);

[0144] 4°C centrifuge (Sorvall Legend Micro 17, Thermo, USA);

[0145] Pathological section scanner (Pannoramic MIDI);

[0146] 96-well plate (LABSELECT, Beijing LanJieKe Technology Co., Ltd.).

[0147] (3) Animals

[0148] SPF-grade male Balb / c mice, 6 - 8 weeks old, 22 - 25 g;

[0149] SPF-grade Sprague-Dawley female rats, 6 - 8 weeks old, weighing 200 - 250 g.

[0150] (4) Strains

[0151] Multidrug-resistant Acinetobacter baumannii strains.

[0152] (5) Cells

[0153] Mouse brain endothelial cells bEnd.3.

[0154] Example 1. Preparation of polymer nanoparticle formulations

[0155] (1) Preparation of polymer nanoparticles loaded with TGC

[0156] Take 20 mg of polymer material and dissolve it in an organic solvent containing 1 mL of dichloromethane and 200 μL of acetone as the oil phase (O), and 0.5 mg / mL of TGC aqueous solution as the inner aqueous phase (W1). Slowly add the aqueous phase dropwise to the oil phase and ultrasonicate for 3 min under ice bath conditions to obtain the primary emulsion (W1 / O); slowly add the primary emulsion dropwise to 4 mL of 1% PVA outer aqueous phase (W2), and ultrasonicate for 3 min under ice bath to obtain the double emulsion (W1 / O / W2).

[0157] Vacuum rotary evaporate the organic solvents dichloromethane and acetone from the double emulsion at 37 °C in a water bath to obtain polymer nanoparticles loaded with TGC;

[0158] (2) Determination of particle size and zeta potential

[0159] According to the above method, prepare TGC nanoparticles loaded with 7 different polymer materials. The prescription compositions of each group of preparations are shown in Table 2;

[0160] Table 2

[0161]

[0162] Take 100 μL of the polymer nanoparticles loaded with TGC of F1 - F7 prepared above into a 1.5 mL EP tube, add 900 μL of water from a Milli-Q Integral water purifier and mix evenly. Use a Malvern particle size analyzer to measure the particle size and zeta potential of each group of preparations, and measure each sample in parallel 3 times and take the average value.

[0163] The results are as Figure 1 and Figure 2 shown, Figure 1 is the particle size diagram of the polymer nanoparticles loaded with TGC of F1 - F7, Figure 2 is the zeta potential diagram of the polymer nanoparticles loaded with TGC of F1 - F7;

[0164] FromFigure 1 It can be seen from [reference] that the particle sizes of the nanoparticles F1 to F7 prepared using different polymer materials are all less than 200 nm.

[0165] From Figure 2 it can be seen that the Zeta potential of the nanoparticles F1 to F7 prepared using different polymer materials shows a negative charge.

[0166] There are certain differences in the particle sizes and potentials of the nanoparticles of different materials. Among them, F3 has the largest particle size, approaching 200 nm, and the particle sizes of F4 and F5 are 97.1 ± 5.9 nm and 110.9 ± 12.0 nm respectively, while the Zeta potential is close to 0 mV.

[0167] It shows that the polymer nanoparticles F1 to F7 loaded with TGC prepared in this example are beneficial for crossing the blood-brain barrier in terms of particle size and potential data.

[0168] (3) Determination of encapsulation efficiency

[0169] Take 100 μL of the polymer nanoparticles F1 to F7 loaded with TGC prepared above and add 10 times the amount of methanol. Sonicate to demulsify for measuring the total amount of drug in the preparation. Place the above solution in an ultrafiltration tube and centrifuge at 3500 rpm for 15 min to separate the polymer nanoparticles and free drug. The free TGC is at the bottom of the ultrafiltration tube. Take 100 μL of the lower-layer free drug and dilute it 10 times with methanol for testing.

[0170] Centrifuge all 1.5 mL EP tubes containing the solution to be tested at 12000 rpm for 3 min. Take 200 μL of the supernatant and place it in a dry injection vial for detecting the content of TGC using a high performance liquid chromatography (HPLC). Repeat the operation 3 times for each preparation to obtain the total amount of drug in the lower clear liquid. The specific chromatographic conditions are as follows: mobile phase: diammonium hydrogen phosphate - triethylamine - methanol (50:1:49, adjusted to pH = 6.30 with phosphoric acid); flow rate: 1 mL / min; column temperature: 30 °C; detection wavelength: 246 nm; injection volume: 50 μL. Calculate the encapsulation efficiency according to the following formula 1.

[0171]

[0172] The results are as Figure 3 shown, Figure 3 which is the encapsulation efficiency diagram of the polymer nanoparticles loaded with TGC prepared using different polymer carrier materials;

[0173] From Figure 3As can be seen, for the polymer nanoparticles encapsulating TGC prepared above, namely F1 - F7, the encapsulation efficiencies of F1 - F3 and F5 - F7 are all above 50%; among them, the encapsulation efficiencies of the two groups of nanoparticles, F2 and F7, are higher than 80%.

[0174] It shows that the polymer nanoparticles encapsulating TGC prepared with mPEG - PLGA (LA / GA = 75:25), PLGA (LA / GA = 75:25), PLGA (LA / GA = 50:50) and the polymers obtained by mixing them in proportion have high encapsulation efficiency and excellent quality of the preparation.

[0175] (4) Mouse distribution experiment

[0176] Using the same operation as in the preparation of the above - mentioned (1) polymer nanoparticles encapsulating TGC, replace TGC with DiD fluorescent dye to prepare polymer nanoparticles encapsulating DiD fluorescent - labeled with different polymer materials.

[0177] The experimental group randomly divided Balb / c mice into 9 groups. By the method of tail - vein injection, administer the above - mentioned polymer nanoparticles encapsulating DiD fluorescent - labeled to the mice. Use an enzyme - linked immunosorbent assay (ELISA) reader to measure the content of DiD encapsulated in the delivered nanoparticles, and adjust the concentration of DiD in each group to 50 μg / mL with PBS; the administration volume for each mouse is 200 μL; the blank group (Control) injects the same volume of normal saline, and the control group administers free DiD fluorescent dye (Free - DiD) that is not encapsulated. After 4 hours of administration, sacrifice the mice, isolate the brain tissue, and use a small - animal in - vivo imager to detect the fluorescence intensity of the brain tissue and analyze the distribution of the polymer nanoparticles encapsulating DiD fluorescent - labeled with different polymer materials in the mouse brain.

[0178] The results are as Figure 4 shown, Figure 4 which is the fluorescence intensity map of the polymer nanoparticles encapsulating DiD fluorescent - labeled prepared with different polymer carrier materials in the mouse brain.

[0179] As can be seen from Figure 4 it, the fluorescence intensity of the nanoparticles in the mouse brain in all experimental groups is significantly higher than that in the Free - DiD group and the Control group; among them, the fluorescence intensity of the nanoparticles in the F7 and F1 groups in the brain is significantly better than that of the other groups in the experimental group.

[0180] In summary, considering the experimental results of particle size, zeta potential, encapsulation efficiency and the distribution of fluorescence intensity in the mouse brain, the polymer nanoparticles encapsulating TGC prepared in Example 1 of the present invention using the mixed material of mPEG - PLGA (75 / 25) and PLGA (75 / 25) with a mass ratio of 1:1 as the polymer carrier material have a significant effect of penetrating the blood - brain barrier.

[0181] Example 2: Preparation of a dual-ligand modified nanoparticle formulation encapsulating DiD

[0182] (1) Preparation of core-shell nanoparticles (CSs) with different molar ratios of Aβ11

[0183] Using the same procedure as in the preparation of the polymer nanoparticles encapsulating TGC in Example 1(1), replace TGC with the DiD fluorescent dye. The polymer carrier material used is a mixed material with a mass ratio of mPEG-PLGA(75 / 25) to PLGA(75 / 25) of 1:1. The polymer nanoparticles encapsulating the DiD fluorescent label are prepared and set aside;

[0184] Then, the lipid material (including Aβ11) is encapsulated on the surface of the above-mentioned polymer nanoparticles encapsulating the DiD fluorescent label by the thin-film hydration method to obtain core-shell nanoparticles modified with different molar ratios of Aβ11. The specific operation is as follows: Weigh Aβ11-PEG2000-Chol, PEG2000-Chol, soybean lecithin (PLS100), and cholesterol (Cholesterol, Chol) according to the molar ratio formula in Table 3 into an eggplant-shaped flask, add 4 mL of absolute ethanol to dissolve it, and after uniform mixing, obtain an ethanol solution with a total material concentration of 20 mg / mL. Remove the organic solvent by rotary evaporation under reduced pressure at 37 °C to form a thin film; add 4 mL of the solution of the polymer nanoparticles encapsulating the DiD fluorescent label prepared above, and hydrate at 40 °C in a water bath until the thin film completely peels off. The hydrated sample is sonicated for 6 min under ice bath conditions to obtain Aβ11-modified DiD core-shell nanoparticles (Aβ11@CSs-DiD).

[0185] At the same time, core-shell nanoparticles (CSs-DiD) not modified with Aβ11, which are prepared by replacing Aβ11 with an equal amount of PEG2000-Chol, are used as a control.

[0186] Table 3

[0187]

[0188] The fluorescence intensity of the brain distribution of the above-mentioned DiD core-shell nanoparticles (Aβ11@CSs-DiD) modified with different molar ratios of Aβ11 is detected according to the same procedure as in the mouse distribution experiment in Example 1(4); the blank group (Control) is injected with the same volume of normal saline, and the control groups are the free DiD fluorescent dye (Free-DiD) not encapsulated and the core-shell nanoparticles (CSs-DiD) not modified with Aβ11;

[0189] The results are as Figure 5 and Figure 6 shown, Figure 5Fluorescence distribution maps of CSs modified with different molar ratios of Aβ11 in the mouse brain. Figure 6 Fluorescence quantification maps of CSs modified with different molar ratios of Aβ11 in the mouse brain; in the figure, **p < 0.01, ***p < 0.001.

[0190] From Figure 5 and Figure 6 it can be seen that the fluorescence intensity of the nanoparticles modified with Aβ11 in the brain is significantly higher than that of the unmodified nanoparticles (CSs-DiD) and the free Free-DiD group without encapsulation, showing a significant difference; indicating that Aβ11 can target the blood-brain barrier and assist drugs in entering the brain.

[0191] Among them, especially when the molar ratio of Aβ11 is 5%, the fluorescence intensity is the strongest, and the effect of crossing the blood-brain barrier and reaching the mouse brain is the best.

[0192] (2) Preparation of dual-ligand modified core-shell nanoparticles (CSs) modified with different volume ratios of Tween80

[0193] Using the same operation as in the preparation of the polymer nanoparticles encapsulating TGC in Example 1(1) above, replace TGC with DiD fluorescent dye, and the polymer carrier material used is a mixed material with a mass ratio of mPEG-PLGA(75 / 25) and PLGA(75 / 25) of 1:1. The polymer nanoparticles encapsulating DiD fluorescent label are prepared; for later use.

[0194] Weigh 6.5 mg of PEG2000-Chol, 7.5 mg of Aβ11-PEG2000-Chol, 20 mg of PLS100 and 5 mg of Chol into a 250 mL eggplant-shaped flask, add 4 mL of absolute ethanol to dissolve the materials, and then add 0.5%, 1%, 1.5% and 2% (v / v) of Tween80 in the total volume respectively. After uniform mixing, remove the organic solvent by rotary evaporation under reduced pressure at 37 °C to form a film; add 4 mL of the polymer nanoparticle solution encapsulating DiD fluorescent label prepared above, and hydrate at 40 °C until the film completely peels off. The hydrated sample is sonicated for 6 min under ice bath conditions to obtain dual-ligand modified DiD core-shell nanoparticles (Aβ11 / T80@CSs-DiD) modified with different volume ratios of Tween80.

[0195] The differently volume ratio of Tween80-modified dual-ligand modified DiD core-shell nanoparticles (Aβ11 / T80@CSs-DiD) obtained above were detected for the brain distribution of fluorescence intensity according to the same operation as in the (4) mouse distribution experiment in Example 1; the blank group (Control) was injected with the same volume of normal saline, and the control groups were the free DiD fluorescent dye (Free-DiD) not encapsulated by the drug and the core-shell nanoparticles (CSs-DiD) not modified by Aβ11 and T80, and the core-shell nanoparticles (5% Aβ11) modified with a molar ratio of 5% of Aβ11 in the above (1) and not modified by T80;

[0196] The results are as Figure 7 and Figure 8 shown, Figure 7 are the fluorescence distribution maps of CSs modified with different volume ratios of Tween80 in the mouse brain, Figure 8 are the fluorescence quantification of CSs modified with different volume ratios of Tween80 in the mouse brain; in the figure, *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0197] From Figure 7 and Figure 8 it can be seen that the fluorescence intensities of the core-shell nanoparticles modified with the dual ligands of Aβ11 and Tween80 in the brain are significantly higher than those of the group modified with the single ligand of Aβ11 (5% Aβ11), and are significantly higher than those of the unmodified nanoparticles (CSs-DiD) and the free Free-DiD not encapsulated;

[0198] Among them, the fluorescence intensity of the brain distribution of the core-shell nanoparticles modified with the dual ligands of 5% Aβ11 + 0.5% T80 is the best, which is 3 times that of the unmodified group (CSs-DiD) and 1.3 times that of the 5% Aβ11 group.

[0199] It shows that the effect of the dual-ligand modified core-shell nanoparticles prepared in Example 2 of the present invention in penetrating the blood-brain barrier is significantly higher than that of the unmodified nanoparticles (CSs-DiD) and the core-shell nanoparticles modified with the single ligand of Aβ11 (5% Aβ11).

[0200] Example 3, Preparation of Dual-Ligand Modified Nanoparticle Preparation Encapsulating TGC

[0201] 10 mg of mPEG-PLGA(75 / 25) and 10 mg of PLGA(75 / 25) were dissolved in 1 mL of an organic solvent mixture of dichloromethane and 200 μL of acetone to form the oil phase (O). An aqueous TGC solution was used as the internal aqueous phase (W1) and was slowly added dropwise to the oil phase. Under ice bath conditions, 100 W probe sonication was performed to obtain the primary emulsion (W1 / O). The primary emulsion was slowly added dropwise to 4 mL of an external aqueous phase of 1% PVA (W2), and sonication was performed again under ice bath conditions to obtain the multiple emulsion (W1 / O / W2). The multiple emulsion was transferred to a 250 mL eggplant-shaped flask, and under the conditions of a 37 °C water bath, the organic solvents were removed by rotary evaporation under reduced pressure to obtain TGC nanoparticles encapsulated with PLGA. 7.5 mg of Aβ11-PEG2000-Chol, 6.5 mg of PEG2000-Chol, 20 mg of PLS100, and 5 mg of Chol were weighed into a 250 mL eggplant-shaped flask. 4 mL of absolute ethanol was added to dissolve the materials, and then 20 μL of Tween80 was added. After uniform mixing, the organic solvents were removed by rotary evaporation under reduced pressure at 37 °C to form a film. 4 mL of the prepared PLGA nanoparticle solution was added, and hydration was carried out at 40 °C in a water bath until the film completely peeled off. The hydrated sample was sonicated at 80 W for 6 min under ice bath conditions, with 3 s of sonication followed by 3 s of pause, to obtain Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC).

[0202] The solution of Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC) prepared was placed in a vial. Using Mili-Q water as the Control and unmodified Aβ11 / T80 TGC core-shell nanoparticles (CSs-TGC) as the control, the morphology of each solution was directly observed with the naked eye under natural light conditions, and the Tyndall phenomenon of the solution was observed by irradiating with a laser pen in the dark;

[0203] The results are as Figure 9 and Figure 10 shown. Figure 9 Figure [X] is the morphology diagram of the solution under natural light illumination conditions, Figure 10 and Figure [X] is the morphology diagram of the solution under laser conditions in the dark;

[0204] As can be seen from Figure 9 , under natural light illumination conditions, the Aβ11 / T80@CSs-TGC and CSs-TGC solutions are clear and transparent, accompanied by a faint blue opalescence; as can be seen from Figure 10 , after irradiating with a laser pen in the dark, except for the blank group, both Aβ11 / T80@CSs-TGC and CSs-TGC showed an obvious Tyndall phenomenon, indicating that the Aβ11 / T80@CSs-TGC nanoformulation is a homogeneous colloidal solution.

[0205] (1) Determination of particle size and zeta potential

[0206] Take 100 μL of the Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC) prepared in this example into a 1.5 mL EP tube, and add 900 μL of water from a Milli-Q Integral water purifier and mix evenly. Measure the particle size and its zeta potential with a Malvern particle size analyzer, and measure each sample in parallel 3 times.

[0207] The results are as Figure 11 and Figure 12 shown, Figure 11 is the particle size distribution diagram of Aβ11 / T80@CSs-TGC; Figure 12 is the zeta potential distribution diagram of Aβ11 / T80@CSs-TGC;

[0208] From Figure 11 and Figure 12 it can be seen that the average particle size of Aβ11 / T80@CSs-TGC prepared in this example is 158.0 ± 3.1 nm, and the zeta potential is -11.7 ± 0.6 mV;

[0209] It shows that the Aβ11 / T80@CSs-TGC prepared in this example has a uniform particle size distribution, showing a negatively charged unimodal distribution, meeting the requirements of nanoparticle delivery for particle size and zeta potential.

[0210] (2) Determination of encapsulation efficiency

[0211] Take 100 μL of the Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC) prepared above and add 10 times the amount of methanol, and ultrasonically demulsify it for measuring the total amount of drugs in the preparation. Put the above solution in an ultrafiltration tube, centrifuge at 3500 rpm for 15 min to separate the polymer nanoparticles and free drugs. The free TGC is at the bottom of the ultrafiltration tube. Take 100 μL of the lower layer of free drug and dilute it 10 times with methanol for testing.

[0212] Centrifuge all 1.5 mL EP tubes containing the solution to be tested at 12000 rpm for 3 min. Take 200 μL of the supernatant and put it into a dry injection vial, and detect the content of TGC with a high performance liquid chromatography (HPLC). Repeat the operation 3 times for each preparation to obtain the total amount of drugs in the lower clear liquid. The specific chromatographic conditions are as follows: mobile phase: diammonium hydrogen phosphate-triethylamine-methanol (50:1:49, adjusted to pH = 6.30 with phosphoric acid); flow rate: 1 mL / min; column temperature: 30 °C; detection wavelength: 246 nm; injection volume: 50 μL. Calculate the encapsulation efficiency according to Equation 1 and calculate the drug loading according to the following formula 2:

[0213]

[0214] The results were as follows: the encapsulation efficiency of the Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles Aβ11 / T80@CSs prepared in this example was 84.2±1.3%, and the drug loading was 8.13±0.25%; the high encapsulation efficiency and high drug loading were beneficial for subsequent applications.

[0215] (3) Transmission electron microscopy (TEM)

[0216] The preparation of the above-mentioned Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC) was characterized by transmission electron microscopy. A copper mesh with a carbon support film on the surface was picked up with forceps and placed with its front side facing up. Aβ11 / T80@CSs-TGC was diluted with deionized water to 0.1 mg / mL, and 5 μL of the solution was pipetted onto the copper mesh. After standing for 10 min, the remaining liquid on the copper mesh was blotted with a filter paper with a frayed edge; after standing for 5 min, 5 μL of 2% phosphotungstic acid staining solution (pH = 7.0) was pipetted onto the copper mesh. After standing for 8 min, the remaining liquid on the copper mesh was blotted with a filter paper with a frayed edge, and after drying at room temperature, it was placed in a transmission electron microscope for observation and photography.

[0217] The results were as Figure 13 shown, Figure 13 as the transmission electron micrograph of Aβ11 / T80@CSs-TGC. It can be seen from the figure that the Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles Aβ11 / T80@CSs-TGC obtained in this example were evenly distributed, had a good appearance, and were spherical in structure; the preparation had a core-shell structure, with PLGA nanoparticles as the core and a ligand-modified lipid membrane as the shell to wrap the PLGA nanoparticles. This structure not only successfully modified the ligands on the nanoparticles but also combined the dual advantages of polymer materials and lipid membranes. The polymer carrier material provided a high surface area ratio and mechanical stability, increasing the encapsulation efficiency of the drug, and the biocompatibility of the lipid shell enhanced the affinity of the nanoparticles for cell membranes and enhanced cell uptake.

[0218] (4) Stability detection

[0219] The above-mentioned Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC) were stored in the dark at 4 °C in a refrigerator. Samples were taken on the 0th, 1st, 3rd, 5th, and 7th days, and the particle size and encapsulation efficiency of the preparation were detected separately according to the methods of the above-mentioned (1) determination of particle size and potential and (2) determination of encapsulation efficiency; the appearance of the TGC core-shell nanoparticle solution was not observed.

[0220] The results were as Figure 14 and 15 shown,Figure 14 It is a graph showing the changes in encapsulation efficiency and particle size, Figure 15 and it is a graph of the appearance of the solution; among them, Figure 15 in [the graph], 1 is Free-TGC, and 2 is Aβ11 / T80@CSs-TGC;

[0221] From Figure 14 it can be seen that for the Aβ11 / T80@CSs-TGC solution stored at 4 °C from the beginning to the 3rd day, there were no significant changes in the particle size and encapsulation efficiency data; after 3 days, the particle size began to increase and the encapsulation efficiency began to decrease; after 7 days of storage, the particle size increased significantly and the encapsulation efficiency decreased significantly.

[0222] From Figure 15 it can be seen that at the same TGC concentration, for the free Free-TGC solution not encapsulated, the color of the solution changed on the 1st day and the drug was oxidized and decomposed; while Aβ11 / T80@CSs-TGC still showed a light yellow color on the 5th day.

[0223] It shows that preparing TGC into nanoparticles can not only enhance the ability to cross the blood-brain barrier, but also increase its stability and facilitate storage.

[0224] (5) In vitro release detection

[0225] The core-shell nanoparticles (CSs-TGC) without Aβ11 and T80 modification were prepared by a method similar to that of this example as a control, and the free Free-TGC not encapsulated in nanoparticles was used as a control. Together with the TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC) modified with Aβ11 and T80 ligands prepared in this example, an in vitro release experiment was carried out; PBS with the same osmotic pressure as blood was selected as the release medium, and the specific operation was as follows:

[0226] The dialysis bag (Mw = 3.5KDa) was cut into small sections about 10 cm long, boiled in an induction cooker until the dialysis bag was completely softened, and after the dialysis bag cooled, it was repeatedly rinsed with Mili-Q water. 1 mL of Aβ11 / T80@CSs-TGC, CSs-TGC, and Free-TGC were respectively taken into the dialysis bag, the dialysis bag was tied tightly with a string and placed in a BD tube containing 30 mL of PBS, and then placed in a constant temperature shaker at 37 °C and 100 rpm. 1 mL of the sample was taken from the release medium at time points of 0, 1, 2, 3, 4, 6, 8, and 12 hours, and the volume was replenished with 1 mL of fresh PBS. Each group was operated in parallel for 3 replicates. The samples were centrifuged at 12,000 rpm for 3 min, and 200 μL of the supernatant was taken into the injection vial; the control group was the free TGC (Free-TGC) not encapsulated in the drug and the core-shell nanoparticles (CSs-TGC) not modified with Aβ11 and T80.

[0227] Operate according to the method for measuring the encapsulation efficiency described above (2) to detect the concentration of the sampled TGC. Calculate the cumulative release amount according to the following formula (1):

[0228]

[0229] Release%=Qn / W×100 (1)

[0231] In the formula, Qn is the cumulative release amount at each time point, Release% is the cumulative release percentage at each time point, n is the nth time point, i is the (n - 1)th time point, Cn is the actually measured drug concentration at the nth sampling time point, Ci is the actually measured drug concentration at the ith sampling time point, W is the total amount of the drug, V0 is the total volume of the dissolution medium, and Vi is the sampling volume.

[0232] The results are as Figure 16 shown. Within the first 1 h, approximately 50% of the TGC was released from the nanoparticles for Free-TGC, CSs-TGC, and Aβ11 / T80@CSs-TGC, and the release continued with the extension of time; the cumulative drug release amounts of CSs-TGC and Aβ11 / T80@CSs-TGC within 12 h were 85 ± 2.3% and 80.5 ± 4.8% respectively, and the drug releases of the two groups were similar; the free drug had no material coating. When it was just put into the release medium, due to the large difference in the TGC concentration on both sides of the dialysis bag, TGC quickly penetrated from the dialysis bag into PBS and was almost completely released within 3 h;

[0233] It indicates that the modification of the ligand has no effect on the release of TGC from the nanoparticles.

[0234] Example 4 Preparation of a dual-ligand modified nanoparticle formulation encapsulating siRNA

[0235] Dissolve 10 mg of mPEG-PLGA(75 / 25) and 10 mg of PLGA-PEG-PLGA(75 / 25) in 1 mL of an organic solvent mixture of dichloromethane and 200 μL of acetone to form the oil phase (O). The aqueous solution of TP53-siRNA as the inner aqueous phase (W1) is slowly added dropwise to the oil phase, and 5 primary emulsions (W1 / O) are obtained by ultrasonic treatment with a 100 W probe under ice bath conditions. The primary emulsion is slowly added dropwise to 4 mL of the outer aqueous phase of 1% PVA (W2), and a double emulsion (W1 / O / W2) is obtained by ultrasonic treatment again under ice bath conditions. The double emulsion is transferred to a 250 mL eggplant-shaped flask, and the organic solvent is removed by rotary evaporation under reduced pressure in a 37 °C water bath to obtain PLGA-encapsulated siRNA nanoparticles. Weigh 7.5 mg of Aβ11-PEG2000-Chol, 6.5 mg of PEG2000-Chol, 20 mg of PLS100, and 5 mg of Chol into a 250 mL eggplant-shaped flask, add 4 mL of absolute ethanol to dissolve the materials, then add 20 μL of Tween80, and after uniform mixing, the organic solvent is removed by rotary evaporation under reduced pressure at 37 °C to form a film. Add 4 mL of the prepared PLGA nanoparticle solution, and hydrate at 40 °C in a water bath until the film completely detaches. The hydrated sample is ultrasonically treated at 80 W for 6 min under ice bath conditions, with 3 s of ultrasound and 3 s of pause, to obtain Aβ11 and T80 dual-ligand modified siRNA core-shell nanoparticles (Aβ11 / T80@CSs-siRNA).

[0236] (1) Determination of particle size and zeta potential

[0237] Take 100 μL of the Aβ11 and T80 dual-ligand modified TP53-siRNA core-shell nanoparticles (Aβ11 / T80@CSs-siRNA) prepared above into a 1.5 mL EP tube, and add 900 μL of water from a Milli-Q Integral water purifier for uniform mixing. The particle size and its zeta potential are measured using a Malvern particle size analyzer. Each sample is measured in parallel 3 times. The results show that the average particle size is 143.4 ± 1.2 nm and the zeta potential is -9.4 ± 1.8 mV. It is shown that the Aβ11 / T80@CSs-siRNA prepared in this example has a uniform particle size distribution, showing a negatively charged unimodal distribution, meeting the requirements of the particle size and zeta potential for the nano-delivery system.

[0238] (2) Determination of encapsulation efficiency

[0239] Add 100 μL of the Aβ11 and T80 dual-ligand modified siRNA core-shell nanoparticles (Aβ11 / T80@CSs-siRNA) prepared above to 10 times the amount of methanol, and sonicate to break the emulsion for measuring the total amount of the drug in the preparation. Place the above solution in an ultrafiltration tube and centrifuge at 3500 rpm for 15 min to separate the polymer nanoparticles and the free drug. The free siRNA is at the bottom of the ultrafiltration tube. Take 100 μL of the lower layer of the free drug and dilute it 10 times with methanol for testing.

[0240] Centrifuge all 1.5 mL EP tubes containing the solution to be tested at 12000 rpm for 3 min. Take 200 μL of the supernatant and place it in a dry injection vial for detecting the content of siRNA by a high performance liquid chromatography (HPLC). Repeat the operation 3 times for each preparation to obtain the total amount of the drug in the lower clear liquid. The specific chromatographic conditions are as follows: mobile phase: A: 0.1 mol / L TEAA in water, mobile phase B: 0.1 mol / L TEAA / acetonitrile (75 v:25 v), column temperature: 50 °C, flow rate: 0.4 mL / min; detection wavelength: 260 nm; injection volume: 20 μL. Calculate the encapsulation efficiency according to Equation 1 and calculate the drug loading according to the following formula 2:

[0241]

[0242] The results are as follows: The encapsulation efficiency of the Aβ11 and T80 dual-ligand modified siRNA core-shell nanoparticles Aβ11 / T80@CSs prepared in this example is 79.51 ± 0.87%, and the drug loading is 9.88 ± 0.51%; the encapsulation efficiency is high and the drug loading is high, which is beneficial for subsequent applications.

[0243] Test Example 5: In vitro antibacterial activity test

[0244] (1) Preparation of MHB medium

[0245] Weigh 12 g of MHB medium powder, dissolve it in 500 mL of distilled water by heating in a water bath, wrap it well with kraft paper and seal it, and then autoclave at 121 °C for 40 min.

[0246] (2) Preparation of MHB agar medium

[0247] Weigh 24 g of MHB medium powder and 12 g of agar respectively, add and dissolve them in 1000 mL of distilled water in a water bath, wrap it well with kraft paper and seal it, and sterilize at 121 °C for 40 min. After sterilization, pour it into a bacterial culture dish and let it cool and solidify.

[0248] (3) Activation, culture, and counting of MDR-AB

[0249] Pick a monoclonal colony of MDR-AB from the agar slant medium with a disposable sterile inoculation loop, inoculate it into 20 mL of MHB liquid medium, and culture the bacteria for 20 h at 37 °C and 230 rpm in a constant-temperature bacterial shaker to activate the bacteria. Then, aspirate 200 μL of the bacterial suspension and inoculate it into 20 mL of fresh medium, and culture it in a constant-temperature shaker for 3 h. Measure the OD600 value of the bacterial suspension with an enzyme-linked immunosorbent assay (ELISA) reader, and dilute the bacterial suspension with sterile medium to an OD value of 0.6 for standby.

[0250] (4) MIC determination

[0251] The MIC values of TGC and Aβ11 / T80@CSs-TGC against MDR-AB were determined by the microdilution method.

[0252] The specific operation is as follows. Inoculate 100 μL of the suspension containing 10 5 CFU / mL bacteria into a sterile 96-well plate, and then add 100 μL of Free-TGC and Aβ11 / T80@CSs-TGC nano-solutions diluted with sterile medium at different concentrations to each well. The concentrations are 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, respectively. After culturing in a constant-temperature incubator at 37 °C for 24 h, visually observe the growth of bacteria in the wells, and measure the OD600 value with an ELISA reader. The Control group is without free TGC and nanoparticles encapsulating TGC;

[0253] The results are as Figure 17 and Figure 18 shown. Figure 17 is the observation diagram of the growth of bacteria in TGC and Aβ11 / T80@CSs-TGC, Figure 18 is the OD600 value;

[0254] From Figure 17 and Figure 18 it can be seen that when the concentration of TGC is 2 μg / mL, the culture in the incubator becomes clear and transparent, and the detected OD600 value is lower than 0.5, which is significantly lower than the OD600 value of the Control group, indicating that the minimum inhibitory concentrations of free TGC and Aβ11 / T80@CSs-TGC against MDR-AB are both 2 μg / mL, showing similar antibacterial activities.

[0255] It shows that compared with free Free-TGC, the TGC nanoparticles Aβ11 / T80@CSs-TGC modified with Aβ11 and T80 dual ligands prepared in the present invention have the same good antibacterial activity and do not affect the original antibacterial effect of the drug.

[0256] Test Example 6. Intrathecal injection activity experiment

[0257] (1) Establishment of intracranial infection model caused by MDR-AB

[0258] Cultivate bacteria according to the activation, culture and counting methods of MDR-AB in Example 5. Aspirate 1.5 mL of bacterial suspension, centrifuge at 4 °C and 3500 rpm for 5 min, discard the upper layer of old culture medium, add 1.5 mL of sterile normal saline to wash the bacteria twice, and dilute the bacterial solution with sterile normal saline to an OD600 value of 0.6 and a bacterial quantity of 108 CFU / mL for standby.

[0259] After each SD rat is anesthetized with isoflurane, inject 20 μL of MDR-AB bacterial solution into the intracisternal space through the occipital cistern with a microsyringe, and the rat intracranial infection model is obtained.

[0260] (2) Experimental grouping and drug administration

[0261] Randomly divide the rats with intracranial infection obtained in (1) above into 4 groups, namely Control group, Free-TGC group, CSs-TGC group and Aβ11 / T80@CSs-TGC group. Inject 20 μL of different preparations through the intrathecal route, with the concentration of TGC equivalent to 0.4 mg / mL. The Control group is injected with 20 μL of sterile normal saline.

[0262] (3) Changes in the number of bacteria in cerebrospinal fluid

[0263] After 24 h of drug administration, anesthetize the rats, and aspirate 20 μL of cerebrospinal fluid from the occipital cistern with a microsyringe into a sterile 0.5 mL EP tube. Take a certain amount of cerebrospinal fluid stock solution, dilute it 10 times with sterile normal saline, then aspirate 10 μL of the diluted solution with a pipette and spread it on a solid medium plate, and culture it in a 37 °C constant temperature incubator for 20 h and record the number of bacterial colonies.

[0264] The results are as Figure 19 and Figure 20 shown. Figure 19 It is the plate spreading result diagram of cerebrospinal fluid after intrathecal injection of preparations in different groups. Figure 20 It is the bar chart of the number of bacteria.

[0265] From Figure 19 and Figure 20 it can be seen that on the solid medium, a large number of bacteria can be observed growing in the Control group, while the number of bacteria in the free TGC (Free-TGC) and the nanoparticle groups loaded with drugs (CSs-TGC and Aβ11 / T80@CSs-TGC) is very small, and only a few scattered monoclonal colonies exist; for the statistical analysis of the number of bacteria, the number of bacteria in the Control group is 100 times that of the Free-TGC group, CSs-TGC group and Aβ11 / T80@CSs-TGC group.

[0266] It was shown that after intrathecal injection, TGC and its nanoparticle formulations could significantly inhibit the growth of bacteria in vivo, exerting an antibacterial effect, which proved that the selected TGC had good antibacterial activity both in vitro and in vivo.

[0267] As can be seen from the above, for the Aβ11 / T80@CSs-TGC, the core-shell nanoparticles modified with Aβ11 and T80 of the present invention, the particle size and zeta potential meet the delivery requirements, and it has good stability; the appearance of the solution is clear and transparent, accompanied by a faint blue opalescence, with obvious colloidal properties. The transmission electron microscope imaging results show that the nanoparticles are uniform spherical structures with good dispersion; they can penetrate the blood-brain barrier and reach the brain, which is beneficial to improving the antibacterial activity of the encapsulated drug in the brain. It is beneficial for the therapeutic application of intracranial anti-infection.

[0268] Experimental Example 7, bEnd.3 cell experiment

[0269] (1) Culture and passage of bEnd.3 cells

[0270] Take out bEnd.3 cells from the liquid nitrogen tank, place them in a 37°C water bath and heat until the cryopreservation solution is completely dissolved. Centrifuge at 800 rpm for 3 min, discard the upper cryopreservation solution, add 1 mL of cell culture medium, pipette to mix evenly, and then inoculate into a cell culture dish. Culture in a 37°C, 5% CO2 constant temperature incubator. When the density of bEnd.3 cells grows to about 90%, passage the cells. Use a pipette to aspirate the old culture medium in the cell culture dish, add 2 mL of sterile PBS to wash the cells; then add 2 mL of trypsin solution to fully digest the cells. After a few minutes, gently shake the culture dish and the cells will start to detach. Then add 4 mL of fresh cell culture medium containing 10% serum to stop digestion, and gently pipette the cells to make them completely detach to form a cell suspension. Transfer the cell suspension to a 15 mL BD tube, centrifuge at 800 rpm for 3 min, discard the supernatant, add 1 mL of fresh culture medium into the BD tube, resuspend the cells, and then take a certain proportion of the cell suspension and inoculate it into a new culture dish, and put it back into the incubator to continue culturing. (2) Preparation of coumarin 6-labeled nanoparticles and bEnd.3 cell uptake experiment

[0271] According to the operation of Example 3, replace the encapsulated antibacterial drug TGC with coumarin 6 (Cou6) to prepare Aβ11 / T80-modified Cou6 core-shell nanoparticles (Aβ11 / T80@CSs-Cou6);

[0272] Use unmodified Aβ11 / T80 Cou6 core-shell nanoparticles (CSs-Cou6) and unmodified T80 Cou6 core-shell nanoparticles (Aβ11@CSs-Cou6) as controls;

[0273] Take the bEnd.3 cell suspension cultured in (1) above and inoculate the cells in a confocal dish (size: 15 mm) at 1×104 cells / dish, and culture in an incubator for 24 hours until the cells are completely attached to the wall. Aspirate the old culture medium in the confocal dish, add 1 mL of sterile PBS to wash the cells; then add 1 mL of fresh culture medium (containing 10% FBS) to dilute CSs-Cou6, Aβ11@CSs-Cou6 and Aβ11 / T80@CSs-Cou6 to 1.6 μg / mL, respectively, and perform three replicates in each group. After the small dish was placed in the incubator for 4 hours, the culture medium in the confocal culture dish was discarded, the cells were washed with PBS and 500 μL of fixative was added. After fixation for 20 minutes, the cells were washed with PBS, and then 1×Intracellular Staining Permeabilization Wash Buffer containing 1% DAPI was added for staining for 10 minutes; the cells were washed with PBS, 1 mL of PBS buffer was added, the confocal dish was observed and photographed under a laser confocal microscope, and the fluorescence was quantified using ImageJ software.

[0274] The results are as follows Figure 21 The confocal image results show that from the green fluorescence display pictures of bEnd.3 cells, compared with the green fluorescence displayed by bEnd.3 cells in the CSs-Cou6 and Aβ11@CSs-Cou6 groups, the green fluorescence intensity displayed by bEnd.3 cells in the Aβ11 / T80@CSs-Cou6 group of the present invention's Aβ11 and T80 dual ligand modified core-shell nanoparticles encapsulating coumarin 6 is the strongest, and the green fluorescence is concentrated in the cytoplasm and partially attached to the cell membrane.

[0275] ImageJ software was used to quantify the fluorescence intensity of bEnd.3 cells. Figure 22 (**p<0.01, ***p<0.001) As shown in the data, the fluorescence intensity of bEnd.3 cells in the Aβ11 / T80@CSs-Cou6 group of core-shell nanoparticles modified with Aβ11 and T80 dual ligands and encapsulating coumarin 6 of the present invention is 3.9 times that of the CSs-Cou6 group and 1.65 times that of the Aβ11@CSs-Cou6 group, indicating that after the nanoparticles are modified with Aβ11 and Tween80, bEnd.3 can enhance the uptake of the nanoparticles, which is beneficial to enhance the penetration of the nanoparticles on the BBB.

[0276] (3) In vitro Transwell permeation experiment study

[0277] In vitro blood-brain barrier model construction:

[0278] Culture bEnd.3 cells according to the method (1) of Example 7 to a certain density. Take 1×105 cells per well and inoculate them on the polycarbonate membrane of a 24-well Transwell plate. Then add 1.5 mL of fresh medium to the outer chamber. Place the Transwell plate in an incubator at 37 °C and 5% CO2 and culture the cells for 20 days. When the transcellular resistance value measured by a cell resistance meter is about 200 Ω·cm2 and no longer increases, it indicates that the cells have differentiated into a monolayer with tight junctions, that is, the in vitro blood-brain barrier (BBB) model is successfully constructed and can be used for in vitro Transwell permeability experiments.

[0279] Permeability experiment operation:

[0280] Use a pipette to aspirate and discard the old medium in each well, and wash the cells twice with HBSS buffer. Add 1.5 mL of HBSS buffer to the outside of the small chamber and 500 μL of HBSS buffer to the inside of the small chamber. Place the Transwell plate in a cell incubator and incubate for 30 min to equilibrate the cells. Aspirate and discard the buffer in each well. Add 500 μL of Free-TGC, CSs-TGC, Aβ11@CSs-TGC, and Aβ11 / T80@CSs-TGC diluted to 50 μg / mL with HBSS buffer (containing 10% FBS) prepared in Example 3 to the inside of the small chamber. Add 1.5 mL of blank HBSS buffer to the outside of the small chamber as the receiving solution. Culture the Transwell cell plate in a cell incubator at 37 °C. Take 200 μL of samples from the outside of the small chamber at 1 h, 2 h, 3 h, and 4 h, and then add an equal volume of blank HBSS to make up the volume. Detect the content of TGC in the samples collected at different time points by HPLC according to the method under "2.1.3" in Chapter 1. Calculate the cumulative permeation amount (Mn) and apparent permeability coefficient (Papp) of TGC on the outside of the small chamber according to the following formula (2).

[0281]

[0282]

[0283] In the formula,

[0284] Mn is the total cumulative permeation amount of TGC at the nth time point,

[0285] Cn is the measured concentration of TGC at the nth time point,

[0286] V is the total volume of the solution outside the small chamber,

[0287] Ci is the measured concentration of TGC at the ith time point,

[0288] Vi is the volume of the sampled solution taken at the ith time point;

[0289] dQ / dt is the cumulative release amount of the preparation transferred from the upper layer to the lower layer of the Transwell plate,

[0290] C0 is the initial concentration of the preparation in the upper layer,

[0291] A is the membrane area (cm2).

[0292] The results are as Figure 23 shown. From the in vitro cumulative release amount curve of the preparation across cells, and Figure 23 the histogram of the apparent permeability coefficient (Papp) of the in vitro across a single-layer cell membrane ( Figure 24 ( ** p < 0.01, *** p < 0.001)); it can be seen that the amount of Free-TGC passing through a single layer of bEnd.3 cells is the least, and the ability to cross the blood-brain barrier is the weakest. The amount of CSs-TGC passing through the BBB model within 4 h is less, and the permeation rate increases slowly in the last 3 h. The permeation ability of the single-ligand modified Aβ11@CSs-TGC and the double-ligand modified Aβ11 / T80@CSs-TGC of the present invention is significantly higher than that of the other Free-TGC group and CSs-TGC group; and the permeation effect of the double-ligand modified Aβ11 / T80@CSs-TGC of the present invention is the most significant, significantly higher than that of the single-ligand modified Aβ11@CSs-TGC.

[0293] It shows that the double-ligand modified Aβ11 / T80@CSs-TGC of the present invention has a better ability to penetrate the blood-brain barrier.

[0294] Test Example 8. Animal intracranial anti-infection experiment

[0295] (1) Detection of the in vivo distribution of Aβ11 / T80@CSs in animal models of intracranial infection

[0296] Rats with intracranial infection models were established according to the method in the intrathecal injection activity experiment of Test Example 6;

[0297] According to the preparation method of Example 3, the encapsulated TGC was replaced with a DiD fluorescent marker; unmodified CSs-DiD and the double-ligand modified Aβ11 / T80@CSs-DiD of the present invention were obtained, and immediately after modeling, they were injected via the tail vein, and the amount of DiD for each rat was 20 μg / rat (using free Free-DiD as the positive control); 4 h after administration, the rats were anesthetized with isoflurane and photographed in a small animal in vivo imager to detect the fluorescence intensity, and the in vivo distribution in animal models of intracranial infection was obtained;

[0298] The results are as Figure 25 shown, Figure 25It is the fluorescence distribution map in rats. The fluorescence intensity of Aβ11 / T80@CSs-DiD in the brain is significantly stronger than that in the Free-DiD and CSs-DiD groups;

[0299] Figure 26 ( ** p < 0.01) It is the fluorescence quantification of the distribution in rat brain tissue. The fluorescence intensity of Aβ11 / T80@CSs-DiD is 19 times that of Free-DiD and 8.6 times that of the CSs-DiD group, showing significant differences.

[0300] It shows that in the model rats infected with MDR-AB, Aβ11 / T80@CSs can penetrate the blood-brain barrier, improve the accumulation of drugs in the brain, and the well brain targeting property of the core-shell nanoparticles modified by Aβ11 and Tween80 can significantly improve the performance of penetrating the blood-brain barrier.

[0301] (2) Determination of the concentration of Aβ11 / T80@CSs in cerebrospinal fluid

[0302] According to the preparation method of Example 3, unmodified CSs-TGC and the double-ligand modified Aβ11 / T80@CSs-TGC of the present invention were prepared, and the free drug TGC was used as the positive control; an intracranial infection model was established according to the method of Test Example 6. The intracranially infected rats were randomly divided into 4 groups, namely the Control group, Free-TGC group, CSs-TGC group, and Aβ11 / T80@CSs-TGC group. They were administered by tail vein injection, and the drug was injected immediately after modeling, with a dose of 60 mg / kg. At 6 h after administration, 20 μL of cerebrospinal fluid was aspirated from the cisterna magna into a sterile 0.5 mL EP tube.

[0303] Precisely aspirate 5 μL of the rat cerebrospinal fluid sample, add 15 μL of acetonitrile to dissolve it, vortex and mix for 3 min, centrifuge at 4 °C and 12,000 rpm for 10 min, and take the upper layer solution. The content of TGC was detected by HPLC; (The detection conditions are as follows: mobile phase: diammonium hydrogen phosphate - triethylamine - methanol (50:1:49, adjusted to pH = 6.30 with phosphoric acid); flow rate: 1 mL / min; column temperature: 30 °C; detection wavelength: 246 nm; injection volume: 10 μL.)

[0304] The results are as Figure 27 ( * p < 0.5, ** p < 0.01) As shown, in the cerebrospinal fluid, the TGC concentration in the Aβ11 / T80@CSs-TGC group is the highest, which is 3.7 ± 0.4 μg / mL, about 2 times that of the free Free-TGC and CSs-TGC groups.

[0305] It is shown that the lipid nanoparticles modified with dual ligands of the present invention can target the brain, penetrate the blood-brain barrier and increase the concentration of drugs entering the cerebrospinal fluid.

[0306] In summary, bEnd.3 cells have the best uptake effect on the Aβ11 / T80@CSs-TGC modified with dual ligands of the present invention. In cells, the Aβ11 / T80@CSs-TGC modified with dual ligands of the present invention has the largest apparent permeability coefficient, the strongest permeability ability, and the strongest ability to penetrate through the BBB.

[0307] Experimental Example 9: Pharmacokinetic Experiment

[0308] (1) Prepare the preparations according to the method of Example 3 to obtain CSs-TGC and Aβ11 / T80@CSs-TGC nano-formulations.

[0309] Preparation of free drug: Dilute and dissolve a certain amount of TGC powder with normal saline to obtain a Free-TGC sample solution, and store it in a refrigerator at 4°C for later use.

[0310] (2) Randomly divide SD rats into 4 groups: blank group, Free-TGC group, CSs-TGC group and Aβ11 / T80@CSs-TGC group. Adopt the single-dose tail vein injection method for drug administration, and the administration dose is 12.5 mg / kg. The blank group is given the same volume of normal saline. At 15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 8 h after administration, take blood from the orbital cavity and place it in an EP tube treated with sodium heparin. Centrifuge the collected blood samples at 3500 rpm and 4°C for 10 min, take the upper serum and place it in a 1.5 mL EP tube, and store it at -20°C.

[0311] (3) Precisely pipette 50 μL of rat plasma sample, add 150 μL of methanol to dissolve it, vortex and mix well for 3 min, then centrifuge at 4°C and 13000 rpm for 10 min to take the supernatant. Determine the content of TGC according to the conditions in (3) for the determination of encapsulation efficiency. Draw a blood drug concentration-time curve with the blood sampling time as the abscissa and the plasma drug concentration as the ordinate.

[0312] The results are as Figure 28 shown. After intravenous administration, the blood drug concentration of Aβ11 / T80@CSs-TGC is the highest, significantly higher than that of Free-TGC, indicating that after preparing TGC into nanoparticles, the circulation time of free drugs can be improved and the bioavailability can be increased.

[0313] (4) Analyze the blood drug concentration of TGC using DAS2 software and calculate the pharmacokinetic parameters of each group after intravenous injection using a non-compartmental model. The main parameters include: peak drug concentration (Cmax), time to reach peak drug concentration (Tmax), area under the blood drug concentration-time curve within 8 hours of TGC (AUC0-8h), mean residence time (MRT), elimination half-life (T1 / 2), and clearance rate (CL).

[0314] The results are shown in Table 4 as follows:

[0315] Table 4

[0316]

[0317] n = 3. Compared with the Free-TGC group, * p < 0.5, ** p < 0.01, *** p < 0.001)

[0318] From the data in Table 4, it can be seen that after intravenous injection of Aβ11 / T80@CSs-TGC into rats, the Cmax and AUC increased significantly. The maximum blood drug concentration (Cmax) of Aβ11 / T80@CSs-TGC was 10.72 ± 2.26 μg / mL, which was

[0319] 2.9 times and 1.3 times that of the Free-TGC and CSs-TGC groups respectively. The area under the curve

[0320] (AUC0-8h) of Aβ11 / T80@CSs-TGC was 21.62 ± 1.79 μg / mL*h, which was 2.5 times and 1.3 times that of the Free-TGC and CSs-TGC groups respectively.

[0321] In addition, both the Cmax and AUC0-8h of CSs-TGC were about 2 times that of Free-TGC.

[0322] On the other hand, the clearance rate of TGC also decreased significantly. Compared with Free-TGC, the CL of CSs-TGC decreased by 2 times, and the CL of Aβ11 / T80@CSs-TGC decreased by 2.76 times;

[0323] Pharmacokinetic experiments showed that preparing TGC into nanoparticles and encapsulating them in the dual-ligand carrier material of the present invention can increase the blood drug concentration, slow down the clearance of TGC in the body, extend the action time of the drug in the blood, improve the stability of the drug, increase the bioavailability, allow more nanoparticles to have the opportunity to distribute to the brain tissue, have the function of targeted drug delivery, and exert antibacterial effects.

[0324] It has unique advantages in improving drug metabolism and enhancing bioavailability, can change the circulation time and distribution of drugs in the body, and improve the bioavailability of drugs.

[0325] Test Example 10, Pharmacodynamic Test

[0326] (1) Prepare the preparations according to the method in (1) of Test Example 9 to obtain Free-TGC samples, CSs-TGC, and Aβ11 / T80@CSs-TGC nanoparticles.

[0327] (2) Establish an intracranial infection model according to the method in Test Example 6. Randomly divide the intracranial infected rats into 4 groups, namely Control group, Free-TGC group, CSs-TGC group, and Aβ11 / T80@CSs-TGC group. Administer the drugs via the tail vein injection. Immediately inject the drugs after modeling, and the single-dose administration dose is 60 mg / kg. At 6 h after administration, aspirate 20 μL of cerebrospinal fluid from the cisterna magna into a sterile 0.5 mL EP tube. Take 10 μL of the cerebrospinal fluid stock solution, dilute it 10 times with sterile physiological saline, then take 10 μL of the diluted solution and evenly coat it on the solid medium with a sterile spreader, and culture it in a 37 °C constant temperature incubator for 20 h to observe the colony growth of each group.

[0328] The results are as Figure 29 shown. The number of bacteria in the cerebrospinal fluid of the Aβ11 / T80@CSs-TGC group is significantly less than that in the free group and the group without ligand modification, showing a significant effect of inhibiting bacterial growth.

[0329] (3) Use the same method as in (2) above, replacing single-dose administration with multi-dose administration;

[0330] Administer the drugs via the tail vein injection, and administer the drugs once at 0 h, 6 h, and 18 h after modeling, with each administration dose being 30 mg / kg. At 24 h after the last administration, aspirate 20 μL of cerebrospinal fluid from the cisterna magna into a sterile 0.5 mL EP tube. Take 5 μL of the cerebrospinal fluid stock solution, dilute it 10 times with sterile physiological saline, take 10 μL of the diluted solution and plate it on the solid medium, and culture it in a 37 °C constant temperature incubator for 20 h to observe the colony growth of each group.

[0331] The results are as Figure 30 shown. After intravenous injection of Aβ11 / T80@CSs-TGC, the growth of MDR-AB in the cerebrospinal fluid is inhibited by TGC, and the number of colonies is the least, significantly lower than that of the other groups. This indicates that the nanoparticles modified with dual ligands of the present invention can improve the distribution properties of TGC, help the drug penetrate the blood-brain barrier, and exert an antibacterial effect.

[0332] Test Example 11, Safety Test

[0333] (1) In vitro hemolysis experiment

[0334] Take a healthy female SD rat, and collect about 5 mL of whole blood by orbital blood collection. Add the blood to normal saline, stir clockwise with a glass rod and then centrifuge to remove the red supernatant. Wash the lower-layer red blood cells repeatedly with normal saline until the supernatant is clear and colorless, and red blood cells can be obtained. Dilute the red blood cells with normal saline to a 2% (v / v) red blood cell suspension for later use.

[0335] A total of 5 groups were set up in the experiment, namely the positive control group (PC), the negative control group (NC), Free-TGC, CSs-TGC, and Aβ11 / T80@CSs-TGC. After adding different solutions and 2% red blood cell suspension to 5 flow tubes respectively, place them in a constant temperature water bath at 37 °C and incubate for 3 h. Centrifuge the flow tubes at 2000 rpm and 4 °C for 5 min, take 200 μL of the supernatant solution of each sample, measure its absorbance at 545 nm with an enzyme-labeling instrument, and calculate the hemolysis rate of each group using formula (3).

[0336]

[0337] The results are as Figure 31 shown. After adding pure water (PC group), the red blood cells ruptured, hemolysis occurred, the solution turned red, and there were red blood cell precipitates at the bottom of the test tubes in the negative control group (NC) and the drug groups, and the supernatant was clear and transparent without hemolysis.

[0338] Figure 32 It is a column chart for quantitative calculation of hemolysis. It can be seen from Figure 32 this that the hemolysis rates of Free-TGC, CSs-TGC, and Aβ11 / T80@CSs-TGC are all lower than 5%. The above results indicate that the dual-ligand modified nanoparticles of the present invention do not cause hemolysis and are suitable for intravenous injection.

[0339] (2) bEnd.3 cell cytotoxicity test

[0340] The MTT experiment was used to investigate the toxicity of Aβ11 / T80@CSs-TGC to bEnd.3 cells. Collect bEnd.3 cells in the logarithmic growth phase, evenly spread them in a 96-well plate at a cell density of 1×104 cells / well, and culture them in an incubator at 37 °C and 5% CO2 for 24 h until the cells are completely adherent. Add 100 μL of drugs with different concentrations to each well, and repeat the operation 6 times for each group. Add the same volume of cell culture medium to the blank group. After continuing to incubate in the incubator for 24 h, add 20 μL of MTT solution (5 mg / mL) to each well above, and terminate the culture after incubating for 3 h. Remove the supernatant, add 150 mL of DMSO to each well and incubate for 15 min, then measure the absorbance of each well at 570 nm with an enzyme-labeling instrument, and calculate the cell survival rate according to the following formula (4).

[0341]

[0342] The results are as Figure 33 shown. The survival rate of bEnd.3 cells at different TGC concentrations is above 80%, indicating that the dual-ligand modified nanoformulation Aβ11 / T80@CSs-TGC of the present invention has no significant increase in cytotoxicity compared with the free drug Free-TGC and the unmodified CSs-TGC, and has good safety.

[0343] (3) H&E staining test

[0344] SPF-grade Sprague-Dawley female rats were randomly divided into 4 groups: Control group, Free-TGC, CSs-TGC and Aβ11 / T80@CSs-TGC groups, with 3 rats in each group. The administration was performed by single tail vein injection at a dose of 30 mg / kg, and the Control group was injected with an equal volume of normal saline. The rats were sacrificed on the second day after administration, and the heart, liver, spleen, lung and kidney tissues were taken. Then, the tissues were embedded, sectioned and stained with H&E. The obtained H&E-stained tissue sections were scanned and photographed with a pathological section scanner.

[0345] The results are as Figure 34 shown. H&E staining was performed on the heart, liver, spleen, lung and kidney collected in the Free-TGC, CSs-TGC and Aβ11 / T80@CSs-TGC groups, and no obvious pathological changes were observed in each group; indicating that Aβ11 / T80@CSs-TGC has good biosafety in vivo.

[0346] In summary, compared with Free-TGC and CSs-TGC, the dual-ligand modified Aβ11 / T80@CSs-TGC of the present invention can increase the blood drug concentration, prolong the blood circulation time of the drug, reduce the clearance rate, and significantly improve the bioavailability of TGC. Multiple-dose intravenous injection of Aβ11 / T80@CSs-TGC can reduce the bacterial count and treat intracranial infections caused by MDR-AB. The in vitro hemolysis and in vivo cell experiments show that the dual-ligand modified core-shell nanoparticles of the present invention have less cytotoxicity in vivo and in vitro and have good biosafety.

Claims

1. A core-shell nanoparticle modified with Aβ11 and T80 dual ligands, characterized in that: The double-ligand modified core-shell nanoparticles include a polymer nanoparticle core loaded with a drug and a lipid material coated on the surface of the polymer nanoparticle core; wherein, the lipid material is modified with Aβ11 and T80.

2. The core-shell nanoparticle according to claim 1, characterized in that: The polymer carrier used for the polymer nanoparticle core is one or more of mPEG-PLGA(75 / 25), PLGA(50 / 50), PLGA(75 / 25), PLGA-PEG-PLGA(50 / 50), PLGA-PEG-PLGA(75 / 25).

3. The core-shell nanoparticle according to claim 2, characterized in that: The polymer carrier used for the polymer nanoparticle core is a mixed polymer carrier of mPEG-PLGA(75 / 25) and PLGA(75 / 25).

4. The core-shell nanoparticle according to claim 1, characterized in that: The Aβ11 accounts for 1-20% of the total lipid molar amount in the lipid material; the percentage content of T80 in the lipid material is 0.5%-10%.

5. The core-shell nanoparticle modified with dual ligands according to claim 1, characterized in that: The drug is selected from one or more of proteins, polypeptides, small molecules, plasmids, siRNA, micRNA.

6. The core-shell nanoparticle according to claim 1, characterized in that: The drug is an antibacterial drug.

7. A preparation method of a core-shell nanoparticle modified with Aβ11 and T80 dual ligands, characterized in that: It includes preparing a polymer nanoparticle core loaded with a drug, and then coating the lipid material containing Aβ11 and T80 on the surface of the polymer nanoparticle core to obtain the double-ligand modified core-shell nanoparticles of Aβ11 and T80.

8. The preparation method according to claim 7, characterized in that: The specific steps include: Step 1, preparing a polymer containing a drug: dissolving the polymer material in an organic solvent of dichloromethane and acetone as the oil phase (O), the aqueous solution of the loaded drug as the inner aqueous phase (W1), slowly dropping the aqueous phase into the oil phase, and obtaining a primary emulsion (W1 / O) by ultrasonic treatment; dropping the primary emulsion (W1 / O) into the PVA outer aqueous phase (W2), and obtaining a double emulsion (W1 / O / W2) by ultrasonic treatment; removing the organic solvents dichloromethane and acetone from the double emulsion under reduced pressure to obtain a polymer nanoparticle core loaded with a drug. Step 2, lipid material coating: dissolving PEG2000-Chol, Aβ11-PEG2000-Chol, PLS100 and Chol in absolute ethanol, adding Tween80, uniformly mixing and then removing the organic solvent under reduced pressure to form a film; adding the polymer nanoparticle core solution prepared in Step 1 to the film, hydrating until the film completely peels off, and ultrasonically treating the hydrated sample to obtain the double-ligand modified core-shell nanoparticles of Aβ11 and Tween80 (Aβ11 / T80@CSs-drug).

9. The use of the core-shell nanoparticle according to any one of claims 1 to 6 and the core-shell nanoparticle modified with Aβ11 and T80 dual ligands prepared by the preparation method according to any one of claims 7 to 8 in the preparation of a drug for breaking through the blood-brain barrier, characterized in that: The double-ligand modified core-shell nanoparticles of Aβ11 and Tween80 (Aβ11 / T80@CSs-drug) are used for delivering drugs to penetrate the blood-brain barrier.

10. The use according to claim 9, characterized in that: The antibacterial drug is selected from one or more of antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones, glycylcyclines; or one or more of antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolone drugs are used in combination with one or more other drugs.

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