Double-ligand modified nano delivery system

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a double-ligand modified nano delivery system. The main technical scheme of the invention is as follows: polymer nanoparticles for entrapment of drugs and a lipid material wrapping the surfaces of the polymer nanoparticles are provided, and A beta 11 and T80 are added into the lipid material for modification. According to the double-ligand modified nano delivery system disclosed by the invention, the obtained core-shell nanoparticles (A beta11 / T80 atCSs-drug) are excellent in the aspects of particle size, potential and encapsulation efficiency data, and the delivery of the nanoparticle drug is facilitated; 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; and the method has a very good application prospect in the aspect of intracranial anti-infection.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a dual-ligand modified nano delivery system. Background Art

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

[0003] The blood-brain barrier (BBB) ​​is a barrier between the blood and the brain, formed by a layer of tightly closed perivascular endings of 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 therapeutic concentrations in the brain parenchyma, the difficulty in treating brain diseases has always been a technical problem that needs 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, the drug delivery system of 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 polymer nanodelivery systems modified with brain-targeting polypeptide rabies virus glycoprotein (RVG)29 and glucose transporter 1 (GLUT1) for treating 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 BBB permeability, inability to achieve effective drug treatment concentration in cerebrospinal fluid with conventional intravenous administration, poor therapeutic effects, and easy development of drug resistance. The intraventricular injection administration method adopted in the prior art to make up for the above defects has technical defects such as high treatment cost in clinical practice, invasive operation being more likely to cause secondary infection, and repeated administration increasing the pain of patients. Therefore, its clinical application is greatly limited. In addition, the prior art also discloses the research of existing researchers using Aβ11 as a brain-targeting peptide to modify liposomes to increase the concentration of doxorubicin in the brain for treating mouse glioblastoma. And the related research on Tween80 (T80) as an ionic surfactant enhancing the accumulation of nanoparticles in the brain in BBB endothelial cells.

[0005] Although some theoretical studies on the use of the above-mentioned Aβ11 and T80 to penetrate the blood-brain barrier for intracranial anti-infection have been disclosed in the prior art, there are no reports on how to apply them in a nanodelivery system, nor is it disclosed how to prepare a nanodelivery load specifically with antibacterial drugs. Therefore, the prior art for treating intracranial anti-infection through the blood-brain barrier using a nanodelivery system 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 nanodelivery system modified with dual ligands, and the dual-ligand modification is Aβ11 and T80 modification;

[0008] Furthermore, the nanodelivery system modified with dual ligands includes polymer nanoparticles encapsulating drugs and a lipid material coated on the surface of the polymer nanoparticles; wherein, the lipid material is modified with Aβ11 and T80.

[0009] Furthermore, for the dual-ligand modified nano-delivery system, the polymer carrier used for the polymer nanoparticles 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);

[0010] Furthermore, for the dual-ligand modified nano-delivery system, the polymer carrier used is a mixed polymer carrier of mPEG-PLGA(75 / 25) and PLGA(75 / 25);

[0011] Furthermore, for the dual-ligand modified nano-delivery system, in the mixed polymer carrier, the mass ratio of mPEG-PLGA(75 / 25) to PLGA(75 / 25) is 1:0.1 to 1:2;

[0012] Furthermore, for the dual-ligand modified nano-delivery system, in the mixed polymer carrier, the mass ratio of mPEG-PLGA(75 / 25) to PLGA(75 / 25) is 1:1;

[0013] Furthermore, for the dual-ligand modified nano-delivery system, Aβ11 accounts for 1 to 20% of the total lipid molar amount in the lipid material;

[0014] Furthermore, for the dual-ligand modified nano-delivery system, the percentage content of T80 in the lipid material is 0.5% to 10%;

[0015] Furthermore, the drug delivered by the dual-ligand modified nano-delivery system is one or more of proteins, polypeptides, small molecules, plasmids, siRNA, and micRNA; it is an antibacterial drug; the drug delivered by the dual-ligand modified nano-delivery system

[0016] Furthermore, the drug is an antibacterial drug;

[0017] Furthermore, the antibacterial drug is selected from one or more of methicillin, penicillin, vancomycin, and tigecycline;

[0018] Furthermore, the drug is tigecycline;

[0019] Furthermore, for the dual-ligand modified nano-delivery system, the PLGA nanoparticles are prepared by the nanoprecipitation method and the double emulsion method; preferably, the double emulsion method is used;

[0020] Furthermore, for the dual-ligand modified nano-delivery system, the lipid material is wrapped on the surface of the PLGA nanoparticles by the thin film hydration method;

[0021] In a specific embodiment of the present invention, the dual-ligand modified nano-delivery system further comprises one or more of polyvinyl alcohol, soybean lecithin, and cholesterol;

[0022] In a specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, Aβ11 accounts for 1% of the total lipid molar amount in the lipid material;

[0023] In another specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, Aβ11 accounts for 5% of the total lipid molar amount in the lipid material;

[0024] In another specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, Aβ11 accounts for 10% of the total lipid molar amount in the lipid material;

[0025] In another specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, Aβ11 accounts for 15% of the total lipid molar amount in the lipid material;

[0026] In a specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, the percentage content of T80 in the lipid material is 0.5%;

[0027] In another specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, the percentage content of T80 in the lipid material is 1%;

[0028] In another specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, the percentage content of T80 in the lipid material is 1.5%;

[0029] In another specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, the percentage content of T80 in the lipid material is 2%;

[0030] In another specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, the percentage content of T80 in the lipid material is 5%;

[0031] In another specific embodiment of the present invention, for the dual-ligand modified nano-delivery system, the percentage content of T80 in the lipid material is 8%;

[0032] On the other hand, the present invention also provides a method for preparing a dual-ligand modified nanoparticle, which includes preparing a polymer nanoparticle loaded with a drug, and then wrapping a lipid material containing Aβ11 and T80 on the surface of the polymer nanoparticle to obtain a core-shell nanoparticle modified with Aβ11 and T80 dual ligands;

[0033] Furthermore, the method for preparing the dual-ligand modified nanoparticle specifically includes the following steps:

[0034] Step 1: Preparation of drug-containing polymer: Dissolve the polymer material in an organic solvent of dichloromethane and acetone as the oil phase (O), and the aqueous solution of the encapsulated drug as the inner aqueous phase (W1). Slowly drop the aqueous phase into the oil phase and obtain the primary emulsion (W1 / O) by ultrasonic treatment; Drop the primary emulsion (W1 / O) into the outer aqueous phase (W2) of PVA and obtain the multiple emulsion (W1 / O / W2) by ultrasonic treatment; Remove the organic solvents dichloromethane and acetone from the multiple emulsion under reduced pressure to obtain the polymer nanoparticles encapsulating the drug.

[0035] Step 2: Lipid material coating: Dissolve PEG2000-Chol, Aβ11-PEG2000-Chol, PLS100 and Chol in absolute ethanol, add Tween80, uniformly mix and then remove the organic solvent under reduced pressure to form a thin film; Add the solution of the polymer nanoparticles encapsulating the drug prepared in Step 1 to the thin film and hydrate until the thin film completely peels off. Ultrasonic treatment of the hydrated sample gives the core-shell nanoparticles (Aβ11 / T80@CSs-drug) modified with Aβ11 and Tween80 dual ligands.

[0036] Furthermore, the drug is an antibacterial drug;

[0037] In the specific embodiment of the present invention, the antibacterial drug is TGC.

[0038] The beneficial effects of the present invention are as follows:

[0039] The dual-ligand modified nano-delivery system disclosed by the present invention uses 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 dual ligands. The obtained core-shell nanoparticles (Aβ11 / T80@CSs-drug) are excellent in terms of particle size, zeta potential, and encapsulation efficiency data, which is beneficial to the delivery of nanoparticle drugs;

[0040] The dual-ligand modified nano-delivery system disclosed by the present invention has better stability for the obtained core-shell nanoparticles (Aβ11 / T80@CSs-drug), which is more beneficial to the transportation and storage of the encapsulated drug;

[0041] Through in vitro antibacterial experiments on mice and intrathecal injection experiments in vivo, the core-shell nanoparticles (Aβ11 / T80@CSs-drug) of the dual-ligand modified nano-delivery system of the present invention have significant penetration through the blood-brain barrier and significantly better antibacterial effects; it has very good application prospects in intracranial anti-infection. Description of the Drawings

[0042] Figure 1 Particle size diagram of the polymer nanoparticles encapsulating TGC of F1 to F7 in Example 1;

[0043] Figure 2Potential diagram of polymer nanoparticles encapsulating TGC for F1 - F7 in Example 1;

[0044] Figure 3 Encapsulation efficiency diagram of polymer nanoparticles encapsulating TGC prepared with different polymer carrier materials in Example 1;

[0045] Figure 4 Fluorescence intensity diagram of polymer nanoparticles encapsulating DiD fluorescence label in mouse brain prepared with different polymer carrier materials in Example 1;

[0046] Figure 5 Fluorescence distribution diagram of CSs modified with different Aβ11 molar ratios in mouse brain in Example 2;

[0047] Figure 6 Fluorescence quantification diagram of CSs modified with different Aβ11 molar ratios in mouse brain in Example 2;

[0048] Figure 7 Fluorescence distribution diagram of CSs modified with different Tween80 volume ratios in mouse brain in Example 2;

[0049] Figure 8 Fluorescence quantification of CSs modified with different Tween80 volume ratios in mouse brain in Example 2;

[0050] Figure 9 Morphology diagram of Aβ11 / T80@CSs - TGC and CSs - TGC solutions under natural light conditions in Example 3;

[0051] Figure 10 Tyndall phenomenon diagram of Aβ11 / T80@CSs - TGC and CSs - TGC solutions in Example 3;

[0052] Figure 11 Particle size distribution diagram of Aβ11 / T80@CSs - TGC in Example 3;

[0053] Figure 12 Potential distribution diagram of Aβ11 / T80@CSs - TGC in Example 3;

[0054] Figure 13 Transmission electron microscopy diagram of Aβ11 / T80@CSs - TGC in Example 3;

[0055] Figure 14 Diagram of changes in encapsulation efficiency and particle size during the sample stability test in Example 3;

[0056] Figure 15 Diagram of the appearance of the solution during the sample stability test in Example 3;

[0057] Figure 16 Release curve of Aβ11 / T80@CSs-TGC in Example 3 in PBS;

[0058] Figure 17 Observation graph of the growth of bacteria in TGC and Aβ11 / T80@CSs-TGC in Test Example 5;

[0059] Figure 18 Histogram of OD600 values in Test Example 5;

[0060] Figure 19 Plate coating results of cerebrospinal fluid after intrathecal injection of different groups of preparations in Test Example 6;

[0061] Figure 20 Histogram of the number of bacteria in Test Example 6. Detailed implementation manners

[0062] Explanation of abbreviations: As shown in Table 1,

[0063]

[0064] The implementation of the technical solution of the present invention will be specifically described below through specific implementation manners.

[0065] Materials, instruments and animals used in specific examples:

[0066] (1) Materials

[0067] TGC (purity > 98%, Beijing Solarbio Science & Technology Co., Ltd.);

[0068] mPEG-PLGA (PLGA, Mw = 15kD; LA / GA = 75:25, PEG, Mw = 2000, Shandong Daigang Biotechnology Co., Ltd.);

[0069] PLGA (Mw = 15kD, LA / GA = 75:25, Shandong Daigang Biotechnology Co., Ltd.);

[0070] PLGA (Mw = 15kD, LA / GA = 50:50, Shandong Daigang Biotechnology Co., Ltd.);

[0071] PLGA-PEG-PLGA (PLGA, Mw = 7.5kD; LA / GA = 75:25, PEG, Mw = 2000, Shandong Daigang Biotechnology Co., Ltd.);

[0072] PLGA-PEG-PLGA (PLGA, Mw = 7.5kD; LA / GA = 50:50, PEG, Mw = 2000, Shandong Daigang Biotechnology Co., Ltd.);

[0073] Polyvinyl alcohol (PVA; MW = 30000 - 70000, purity > 80%, Sigma-Aldrich, USA);

[0074] Cell membrane far-infrared fluorescence probe DiD (purity > 98%, Dalian Meilun Biotechnology Co., Ltd.);

[0075] Aβ11 (purity > 98%, Qiangyao Biotechnology Co., Ltd.);

[0076] Tween80 (T80, Guangzhou Saiguo Biotechnology Co., Ltd.);

[0077] Aβ11-PEG2000-Chol (purity > 98%, State Key Laboratory of Biotherapy, Sichuan University);

[0078] Soybean lecithin (LIPOID S100, batch number: 790587 - 4 / 904, LIPOID, Germany);

[0079] Cholesterol (batch number: 2020010101, Chengdu Kelong Chemical Co., Ltd.);

[0080] Diammonium hydrogen phosphate (CAS: 7783 - 28 - 0, Chengdu Kelong Chemical Co., Ltd.);

[0081] Triethylamine (batch number: 20220412, Chengdu Jinshan Chemical Reagent Co., Ltd.);

[0082] Methanol (CAS: 67 - 52 - 1, Norsic Chengdu Kelong Chemical Co., Ltd.).

[0083] (2) Instruments

[0084] Electronic analytical balance (BSM - 120.4, Shanghai Zhuojing Electronic Technology Co., Ltd.);

[0085] Circulating vacuum pump (SHZ - D(Ⅲ), Zhengzhou Zhichuang Machinery Co., Ltd.);

[0086] Ultrasonic cell crusher (SCIENTZ - Ⅲ, Ningbo Xinzhi Biotechnology Co., Ltd.);

[0087] Malvern particle size analyzer (Nano ZS ZEN 36000, Malvern Instruments, UK);

[0088] Rotary evaporator (RE - 201D, Zhengzhou Shengsheng Instrument Co., Ltd.);

[0089] Centrifuge (Sorvall ST16, Thermo Scientific, USA);

[0090] High performance liquid chromatograph (SIL-20AC, Shimadzu, Japan);

[0091] Small animal in vivo imager (IVIS Lumina III, PerkinElmer, USA);

[0092] Chromatographic column (InertSustain C18, Shimadzu, Japan);

[0093] Dialysis bag (Mw = 3500, Shanghai Yuanye Bio-Technology Co., Ltd.);

[0094] Transmission electron microscope (HF-3300, Hitachi);

[0095] Milli-Q Integral ultrapure water machine (Millipore, USA);

[0096] Carbon dioxide incubator (LRH-70F, Shanghai Yiheng Instrument Co., Ltd.);

[0097] Microplate reader (BioTek, USA);

[0098] Bacterial culture dish (BS-90-D90mm, Beijing Lanjieke Technology Co., Ltd.);

[0099] 4 °C centrifuge (Sorvall Legend Micro 17, Thermo, USA).

[0100] (3) Animals

[0101] SPF-grade male Balb / c mice, 6 - 8 weeks old, 22 - 25 g.

[0102] (4) Strains

[0103] Multidrug-resistant Acinetobacter baumannii strain.

[0104] Example 1. Preparation of polymer nanoparticle formulation

[0105] (1) Preparation of polymer nanoparticles loaded with TGC

[0106] 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).

[0107] The double emulsion was placed in a 37 °C water bath and the organic solvents dichloromethane and acetone were removed by rotary evaporation under reduced pressure to obtain polymer nanoparticles loaded with TGC.

[0108] (2) Determination of particle size and potential

[0109] According to the above method, 7 kinds of TGC-loaded nanoparticles encapsulated with different polymer materials were prepared, and the prescription compositions of each group of preparations are shown in Table 2;

[0110] Table 2

[0111]

[0112] Take 100 μL of the polymer nanoparticles loaded with TGC of F1 - F7 prepared above into a 1.5 mL EP tube, and add 900 μL of water from a Milli-Q Integral water purifier and mix evenly. The particle size and potential of each group of preparations were measured with a Malvern particle size analyzer, and each sample was measured in parallel 3 times and the average value was taken.

[0113] 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 potential diagram of the polymer nanoparticles loaded with TGC of F1 - F7;

[0114] From Figure 1 it can be seen that the particle sizes of F1 - F7 nanoparticles prepared with different polymer materials are all less than 200 nm,

[0115] From Figure 2 it can be seen that the Zeta potential of F1 - F7 nanoparticles prepared with different polymer materials shows negative charges.

[0116] There are certain differences in the particle sizes and potentials of 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.

[0117] It shows that the polymer nanoparticles loaded with TGC of F1 - F7 prepared in this example are beneficial to cross the blood - brain barrier in terms of particle size and potential data.

[0118] (3) Determination of encapsulation efficiency

[0119] Take 100 μL of the polymer nanoparticles encapsulating TGC prepared above for F1 - F7 and add them to 10 times the volume of methanol. Sonicate to break the emulsion 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 the 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 measurement.

[0120] Centrifuge all 1.5 - mL EP tubes containing the solution to be measured 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 chromatograph (HPLC). Repeat the operation 3 times for each preparation to obtain the total amount of drug in the supernatant. 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.

[0121]

[0122] The results are as Figure 3 shown, Figure 3 which is the encapsulation efficiency diagram of the polymer nanoparticles encapsulating TGC prepared with different polymer carrier materials;

[0123] As can be seen from Figure 3 it, for the polymer nanoparticles encapsulating TGC prepared above for 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%;

[0124] This 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 their proportion - mixed polymers have high encapsulation efficiency and excellent preparation quality.

[0125] (4) Mouse distribution experiment

[0126] Using the same operation as in the preparation of the polymer nanoparticles encapsulating TGC in (1) above, replace TGC with DiD fluorescent dye to prepare polymer nanoparticles encapsulating DiD - fluorescent - labeled with different polymer materials;

[0127] The experimental group randomly divided Balb / c mice into 9 groups. Using the tail vein injection method, the above-mentioned polymer nanoparticles loaded with DiD fluorescent labels were administered to the mice. An enzyme-linked immunosorbent assay (ELISA) reader was used to measure the DiD content loaded in the delivery system, and the concentration of DiD in each group was adjusted to 50 μg / mL with PBS; the administration volume for each mouse was 200 μL; the blank group (Control) was injected with the same volume of normal saline, and the control group was administered free DiD fluorescent dye (Free-DiD) that was not encapsulated. After 4 hours of administration, the mice were sacrificed, and the brain tissues were isolated. A small animal in vivo imager was used to detect the fluorescence intensity of the brain tissues, and the distribution of the polymer nanoparticles loaded with DiD fluorescent labels of different polymer materials in the mouse brain was analyzed.

[0128] The results are as Figure 4 shown, Figure 4 which are the fluorescence intensity diagrams of the polymer nanoparticles loaded with DiD fluorescent labels prepared from different polymer carrier materials in the mouse brain;

[0129] It can be seen from Figure 4 that the fluorescence intensity of the nanoparticles in all experimental groups in the mouse brain was 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 was significantly better than that of the other groups in the experimental group.

[0130] In summary, based on the experimental results of the particle size, zeta potential, encapsulation efficiency, and the fluorescence intensity distribution in the mouse brain, the polymer nanoparticles loaded with TGC prepared from the mixed material of mPEG-PLGA(75 / 25) and PLGA(75 / 25) with a mass ratio of 1:1 as the polymer carrier material in Example 1 of the present invention have a significant effect of penetrating the blood-brain barrier.

[0131] Example 2: Preparation of a dual-ligand modified nanoparticle formulation encapsulating DiD

[0132] (1) Preparation of core-shell nanoparticles (CSs) with different molar ratios of Aβ11

[0133] Using the same operation as in (1) the preparation of the polymer nanoparticles loaded with TGC in Example 1 above, replace TGC with DiD fluorescent dye, and the polymer carrier material used is a mixed material of mPEG-PLGA(75 / 25) and PLGA(75 / 25) with a mass ratio of 1:1. Polymer nanoparticles loaded with DiD fluorescent labels were prepared; reserved;

[0134] Then, the thin film hydration method was used to coat the surface of the above polymer nanoparticles loaded with DiD fluorescently labeled with a lipid material (including Aβ11) 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 in 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 above-prepared polymer nanoparticle solution loaded with DiD fluorescent label, and hydrate it in a water bath at 40 °C until the thin film completely peels off. The hydrated sample was sonicated for 6 min under ice bath conditions to obtain Aβ11-modified DiD core-shell nanoparticles (Aβ11@CSs-DiD).

[0135] At the same time, using the above operation method, an equimolar amount of PEG2000-Chol was used to replace Aβ11-PEG2000-Chol to prepare core-shell nanoparticles (CSs-DiD) not modified with Aβ11 as a control (as shown in the CSs column in Table 3).

[0136] Table 3

[0137]

[0138] The fluorescence intensity of the brain distribution of the above-obtained DiD core-shell nanoparticles (Aβ11@CSs-DiD) modified with different molar ratios of Aβ11 was detected according to the same operation as 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 and the core-shell nanoparticles (CSs-DiD) not modified with Aβ11;

[0139] The results are as Figure 5 and Figure 6 shown, Figure 5 which are the fluorescence distribution maps of CSs modified with different molar ratios of Aβ11 in the mouse brain, Figure 6 and the 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;

[0140] From Figure 5 and Figure 6It can be seen that the fluorescence intensity of the nanoparticles modified by 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 the drug to enter the brain.

[0141] 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.

[0142] (2) Preparation of dual-ligand modified core-shell nanoparticles (CSs) modified with different volume ratios of Tween80

[0143] Using the same operation as in the preparation of the polymer nanoparticles encapsulating TGC in Example 1(1), 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 labeled with DiD fluorescence are prepared and reserved;

[0144] 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 of 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 labeled with DiD fluorescence prepared above, and hydrate at 40 °C until the film completely peels off. The hydrated sample is ultrasonically treated 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.

[0145] The dual-ligand modified DiD core-shell nanoparticles (Aβ11 / T80@CSs-DiD) modified with different volume ratios of Tw5een80 obtained above are detected for the brain distribution of fluorescence intensity according to the same operation 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) without encapsulation and the core-shell nanoparticles (CSs-DiD) not modified by Aβ11 and T80, and the core-shell nanoparticles (5% Aβ11) modified with a 5% molar ratio of Aβ11 and not modified by T80 in the above (1);

[0146] The results are as Figure 7 and Figure 8 shown, Figure 7Fluorescence distribution maps of CSs modified with different volume ratios of Tween80 in the mouse brain Figure 8 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

[0147] From Figure 7 and Figure 8 it can be seen that the fluorescence intensities of the core-shell nanoparticles modified with both Aβ11 and Tween80 in the brain are significantly higher than those of the nanoparticles modified with Aβ11 alone (5% Aβ11), and are significantly higher than those of the unmodified nanoparticles (CSs-DiD) and the free Free-DiD not encapsulated

[0148] Among them, the fluorescence intensity of the brain distribution of the core-shell nanoparticles modified with both 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

[0149] It shows that the effect of the core-shell nanoparticles modified with double ligands 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 Aβ11 single ligand (5% Aβ11)

[0150] Example 3. Preparation of a double-ligand modified nanoparticle preparation encapsulating TGC

[0151] Take 10 mg of mPEG-PLGA (75 / 25) and 10 mg of PLGA (75 / 25) and dissolve them in 1 mL of an organic solvent mixture of dichloromethane and 200 μL of acetone as the oil phase (O). The aqueous TGC solution is used as the inner aqueous phase (W1) and is slowly added dropwise to the oil phase. Under ice bath conditions, a 100 W probe sonicator is used to obtain the primary emulsion (W1 / O). The primary emulsion is slowly added dropwise to 4 mL of the outer aqueous phase of 1% PVA (W2), and sonication is performed again under ice bath conditions to obtain the multiple emulsion (W1 / O / W2). The multiple emulsion is transferred to a 250 mL eggplant-shaped flask, and under the conditions of a 37 °C water bath, the organic solvent is removed by rotary evaporation under reduced pressure to obtain TGC-loaded PLGA 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 Tween 80, 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 it at 40 °C in a water bath until the film completely detaches. The hydrated sample is sonicated at 80 W for 6 min under ice bath conditions, with 3 s of sonication followed by 3 s of pause, to obtain the Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC).

[0152] Place the prepared solution of Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC) in a vial. Using Mili-Q water as the Control and the unmodified Aβ11 / T80 TGC core-shell nanoparticles (CSs-TGC) as the control, directly observe the morphology of each solution with the naked eye under natural light conditions, and observe the Tyndall phenomenon of the solution by irradiating it with a laser pointer in the dark;

[0153] The results are as Figure 9 and Figure 10 shown. Figure 9 Figure Figure 10 is the morphology diagram of the solution under natural light conditions, and

[0154] Figure Figure 9 is the morphology diagram of the solution under laser conditions in the dark; Figure 10 As can be seen from

[0155] (1) Determination of particle size and potential

[0156] Take 100 μL of the Aβ11 and T80 dual-ligand modified TGC core-shell nanoparticles (Aβ11 / T80@CSs-TGC) prepared above 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.

[0157] The results are as Figure 11 and Figure 12 shown, Figure 11 Figure is the particle size distribution diagram of Aβ11 / T80@CSs-TGC; Figure 12 Figure is the zeta potential distribution diagram of Aβ11 / T80@CSs-TGC;

[0158] From Figure 11 and Figure 12 it can be seen that the average particle size of the Aβ11 / T80@CSs-TGC prepared in this example is 158.0 ± 3.1 nm, and the zeta potential is -11.7 ± 0.6 mV;

[0159] It shows that the particle size distribution of the Aβ11 / T80@CSs-TGC prepared in this example is uniform, showing a negatively charged unimodal distribution, meeting the requirements of the particle size and zeta potential for the nanodelivery system.

[0160] (2) Determination of encapsulation efficiency

[0161] 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 for measuring the total amount of drug in the preparation. Place the above solution in an ultrafiltration tube, centrifuge at 3500 rpm for 15 min to separate the polymer nanoparticles and free drug, and 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.

[0162] 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 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 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 Equation 1 and calculate the drug loading according to the following formula 2:

[0163]

[0164] 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.

[0165] (3) Transmission electron microscopy (TEM)

[0166] 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 tweezers 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.

[0167] The results were as Figure 13 shown, Figure 13 which was the transmission electron micrograph of Aβ11 / T80@CSs-TGC. It could 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 uniformly distributed, had a good appearance, and showed a spherical 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 with the cell membrane, enhancing cell uptake.

[0168] (4) Stability detection

[0169] 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 respectively detected 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.

[0170] 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 [graph], 1 is Free-TGC, and 2 is Aβ11 / T80@CSs-TGC;

[0171] From Figure 14 it can be seen that for the Aβ11 / T80@CSs-TGC solution stored at 4°C, from the start 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 the 7th day of storage, the particle size increased significantly and the encapsulation efficiency decreased significantly.

[0172] 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.

[0173] 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.

[0174] (5) In vitro release detection

[0175] Using a similar operation method as above, unmodified core-shell nanoparticles (CSs-TGC) without Aβ11 and T80 were prepared as a control, and 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:

[0176] Dialysis bags (Mw = 3.5KDa) were cut into small sections about 10 cm long, boiled in an induction cooker until the dialysis bags were completely softened, and after cooling, they were repeatedly rinsed with Mili-Q water. 1 mL of Aβ11 / T80@CSs-TGC, CSs-TGC, and Free-TGC were respectively taken into the dialysis bags, the dialysis bags were tied tightly with a thin 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

[0177] 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:

[0178]

[0179] Release%=Qn / W×100

[0180] 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.

[0181] 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 was not coated with materials. When it was just put into the release medium, due to the large difference in 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;

[0182] It indicates that the modification of the ligand has no effect on the release of TGC from the nanoparticles.

[0183] Example 4 Preparation of a Dual - Ligand - Modified Nanoparticle Preparation Encapsulating siRNA

[0184] 10 mg of mPEG-PLGA (75 / 25) and 10 mg of PLGA-PEG-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). The aqueous solution of TP53-siRNA as the inner aqueous phase (W1) was slowly added dropwise to the oil phase, and 5 primary emulsions (W1 / O) were obtained by sonication with a 100 W probe under ice bath conditions. The primary emulsion was slowly added dropwise to 4 mL of 1% PVA (W2) outer aqueous phase, and double emulsions (W1 / O / W2) were obtained by sonication again under ice bath conditions. The double emulsion was transferred to a 250 mL eggplant-shaped flask, and the organic solvent was removed by rotary evaporation under reduced pressure in a 37 °C water bath to obtain siRNA nanoparticles encapsulated by 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 Tween 80 was added. After uniform mixing, the organic solvent was 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 the film was hydrated until it completely peeled off under 40 °C water bath conditions. 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 siRNA core-shell nanoparticles (Aβ11 / T80@CSs-siRNA).

[0185] (1) Determination of particle size and zeta potential

[0186] 100 μL of the above-prepared Aβ11 and T80 dual-ligand modified TP53-siRNA core-shell nanoparticles (Aβ11 / T80@CSs-siRNA) were taken into a 1.5 mL EP tube, and 900 μL of water from a Milli-Q Integral water purifier was added and mixed evenly. The particle size and its zeta potential were measured using a Malvern particle size analyzer. Each sample was measured in parallel 3 times. The results showed that the average particle size was 143.4 ± 1.2 nm and the zeta potential was -9.4 ± 1.8 mV. It was shown that the Aβ11 / T80@CSs-siRNA prepared in this example had a uniform particle size distribution, a negatively charged unimodal distribution, and met the requirements of the particle size and zeta potential for the nanodelivery system.

[0187] (2) Determination of encapsulation efficiency

[0188] Take 100 μL of the Aβ11 and T80 dual-ligand modified siRNA core-shell nanoparticles (Aβ11 / T80@CSs-siRNA) prepared above and add it to 10 times the amount of methanol. 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.

[0189] 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 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:

[0190]

[0191] 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 high encapsulation efficiency and high drug loading are beneficial for subsequent applications.

[0192] Test Example 5: In vitro antibacterial activity test

[0193] (1) Preparation of MHB medium

[0194] 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.

[0195] (2) Preparation of MHB agar medium

[0196] 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.

[0197] (3) Activation, culture, and counting of MDR-AB

[0198] 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 at 37 °C and 230 rpm in a constant-temperature bacterial shaker for 20 h 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.

[0199] (4) MIC determination

[0200] The MIC values of TGC and Aβ11 / T80@CSs-TGC against MDR-AB were determined by the microdilution method.

[0201] The specific operation is as follows. 100 μL of the suspension containing 10 5 CFU / mL bacteria was inoculated into a sterile 96-well plate, and then 100 μL of Free-TGC and the Aβ11 / T80@CSs-TGC nano-solution prepared in Example 3 diluted with sterile medium at different concentrations were added to each well. The concentrations were 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, respectively. After culturing in a 37 °C constant-temperature incubator for 24 h, the growth of bacteria in the wells was visually observed, and the OD600 value was measured with an ELISA reader. The Control group was without free TGC and nanoparticles encapsulating TGC;

[0202] The results are as Figure 17 and Figure 18 shown. Figure 17 Figure for observing the growth of bacteria in TGC and Aβ11 / T80@CSs-TGC, Figure 18 is the OD600 value;

[0203] From Figure 17 and Figure 18 it can be seen that when the concentration of TGC is 2 μg / mL, the observation 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.

[0204] 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.

[0205] Test Example 6, Intrathecal injection activity experiment

[0206] (1) Establishment of intracranial infection model caused by MDR-AB

[0207] Cultivate bacteria according to the activation, culture, and counting methods of MDR-AB in Example 5. Aspirate 1.5 mL of the bacterial suspension, centrifuge at 4°C and 3500 rpm for 5 min, discard the upper layer of the 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.

[0208] After each SD rat is anesthetized with isoflurane, inject 20 μL of the MDR-AB bacterial solution into the cisterna magna intrathecally with a micro syringe, and then the rat intracranial infection model is obtained.

[0209] (2) Experimental grouping and drug administration

[0210] Randomly divide the rats with intracranial infection obtained in (1) above into 4 groups, namely the Control group, Free-TGC group, CSs-TGC group, and Aβ11 / T80@CSs-TGC group. Inject 20 μL of different preparations intrathecally, with the concentration of TGC equivalent to 0.4 mg / mL. The Control group is injected with 20 μL of sterile normal saline.

[0211] (3) Changes in the number of bacteria in cerebrospinal fluid

[0212] After 24 h of drug administration, anesthetize the rats, and aspirate 20 μL of cerebrospinal fluid from the cisterna magna with a micro syringe into a sterile 0.5 mL EP tube. Take a certain amount of the original cerebrospinal fluid, 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. Culture it in a 37°C constant temperature incubator for 20 h and record the number of bacterial colonies.

[0213] 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.

[0214] 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, with only a few scattered monoclonal colonies existing; for the statistical analysis of the number of bacteria, the bacterial quantity in the Control group is 100 times that of the Free-TGC group, CSs-TGC group, and Aβ11 / T80@CSs-TGC group.

[0215] It shows that after intrathecal injection, TGC and its nanoparticle preparation can significantly inhibit the growth of bacteria in vivo, achieving an antibacterial effect, proving that the selected TGC has good antibacterial activity both in vitro and in vivo.

[0216] In summary, for the Aβ11 / T80@CSs-TGC of the dual-ligand modified nano-delivery system of the present invention, the particle size and 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 and is conducive to the treatment application of intracranial anti-infection.

Claims

1. A dual-ligand modified nano-delivery system, characterized in that: The double ligand modification is Aβ11 and T80 modification.

2. The nano-delivery system according to claim 1, characterized in that: It includes polymer nanoparticles loaded with drugs and lipid materials wrapped on the surface of the polymer nanoparticles; wherein, the lipid materials are modified with Aβ11 and T80.

3. The nano-delivery system according to claim 2, characterized in that: The polymer carriers used for the polymer nanoparticles are 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).

4. The nano-delivery system according to claim 3, characterized in that: For the double ligand modified nano-delivery system, the polymer carrier used is a mixed polymer carrier of mPEG-PLGA(75 / 25) and PLGA(75 / 25).

5. The nano-delivery system according to claim 4, characterized in that: For the double ligand modified nano-delivery system, for the mixed polymer carrier, the mass ratio of mPEG-PLGA(75 / 25) to PLGA(75 / 25) is 1:0.1 to 1:

2.

6. The nano-delivery system according to claim 2, characterized in that: The Aβ11 accounts for 1 to 20% of the total lipid molar amount in the lipid material.

7. The nano-delivery system according to claim 2, characterized in that: The percentage content of T80 in the lipid material is 0.5% to 10%.

8. The nano-delivery system according to claim 1, characterized in that: The drugs delivered by the double ligand modified nano-delivery system are one or more of proteins, polypeptides, small molecules, plasmids, siRNA, and micRNA.

9. The nano-delivery system according to claim 8, characterized in that: The drug is an antibacterial drug, and the antibacterial drug is selected from one or more of methicillin, penicillin, vancomycin, and tigecycline.

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

11. The preparation method according to claim 10, characterized in that: The specific steps include: Step 1, prepare a polymer 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 drop the aqueous phase into the oil phase and obtain a primary emulsion (W1 / O) by ultrasonic treatment; drop the primary emulsion (W1 / O) into the PVA outer aqueous phase (W2) and obtain a double emulsion (W1 / O / W2) by ultrasonic treatment; remove the organic solvents dichloromethane and acetone from the double emulsion under reduced pressure to obtain polymer nanoparticles loaded with drugs. Step 2, lipid material wrapping: Dissolve PEG2000-Chol, Aβ11-PEG2000-Chol, PLS100, and Chol in absolute ethanol, add Tween80, and after uniform mixing, remove the organic solvent under reduced pressure to form a film; add the polymer nanoparticle solution loaded with drugs prepared in Step 1 to the film and hydrate until the film completely peels off; ultrasonically treat the hydrated sample to obtain Aβ11 and Tween80 double ligand modified core-shell nanoparticles (Aβ11 / T80@CSs-drug).

12. An Aβ11 and Tween80 dual-ligand modified core-shell nanoparticle (Aβ11 / T80@CSs-drug) prepared by the preparation method of claim 11; characterized in that: The Aβ11 and Tween80 double ligand modified core-shell nanoparticles (Aβ11 / T80@CSs-drug) are used for delivering drugs to penetrate the blood-brain barrier.

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