RECOMBINANT LIPOPROTEIN MODIFIED BY MONOSIALOTETRAHEXOSYL GANGLIOSIDE AND APPLICATION THEREOF

Abstract

The present invention discloses a reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside, an application in preparing a drug carrier thereof, and an application in preparing a drug for treating or preventing a disease associated with A deposition thereof. Specifically, the present invention discloses an application of an appropriate amount of monosialoteterahexosyl ganglioside in modifying the reconstituted lipoprotein to increase an affinity of the reconstituted lipoprotein with amyloid -protein (A), and facilitating clearance of A. Meanwhile, the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside is used as a multi-mode nano carrier for preventing and treating central nervous system diseases, and particularly Alzheimer's disease.

Claims

1. A reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside, comprising monosialoteterahexosyl ganglioside, lipid and an apolipoprotein, wherein the monosialoteterahexosyl ganglioside accounts for 1%-30% of a total lipid molar fraction.

2. The reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to claim 1, wherein the monosialoteterahexosyl ganglioside accounts for 1%-20% of the total lipid molar fraction.

3. The reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to claim 1, wherein the mass of the apolipoprotein accounts for 1-60% of prescription content.

4. The reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to claim 3, wherein the mass of the apolipoprotein accounts for 1-50% of the prescription content.

5. The reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to claim 1, wherein the lipid is one or more of egg lecithin, fabaceous lecithin, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, phosphatidyl glycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, lysophosphatide, sphingosine, ceramide, sphingomyelin, cerebroside, cholesterol, cholesteryl ester, glyceride and derivatives thereof.

6. The reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to claim 1, wherein the lipid does not include cholesterol or cholesteryl ester.

7. The reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to claim 1, wherein the apolipoprotein is one or more of ApoE and mimic peptides thereof, ApoA-I and mimic peptides thereof, ApoA-II and mimic peptides thereof and ApoC and mimic peptides thereof.

8. An application of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside of claim 1 in preparing a drug carrier.

9. The application of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside in preparing a drug carrier according to claim 8, wherein the drug comprises one or more of small-molecule chemicals, macromolecular polypeptides, proteins and gene drugs.

10. The application of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside in preparing a drug carrier according to claim 8, wherein the drug is a drug for treating or preventing a disease of the central nervous system.

11. An application of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside of claim 1 in preparing a drug for treating or preventing a disease associated with deposition of A.

12. The application of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside in preparing the drug for treating or preventing a disease associated with deposition of A according to claim 11, wherein the disease associated with the deposition of A is the Alzheimer's disease.

13. The application of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside in preparing the drug for treating or preventing a disease associated with deposition of A according to claim 11, wherein the drug is nasal administrated.

Description

DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 illustrates the transmission electron microscope image of (A) a reconstituted lipoprotein unmodified by the monosialoteterahexosyl ganglioside; (B) a reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside, scale bar: 20 nm;

[0038] FIG. 2 illustrates (A) the binding curves of reconstituted lipoprotein with A1-42 monomer; (B) the binding curves of a reconstituted lipoprotein with A1-42 oligomer; (C) the binding curves of reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside with A1-42 monomer; and (D) the binding curves of the reconstituted lipoprotein modified by monosialotetetrahexosyl ganglioside with A 1-42 oligomer;

[0039] FIG. 3 illustrates (A) A1-42 uptake, (B) A1-42 degradation in primary microglial cells in the presence of the reconstituted lipoprotein or the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside, and (C) co-location of the reconstituted lipoprotein or the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside with A1-42 in primary microglial cells; *p<0.05,**p<0.01,***p<0.0001 represent there are significant differences between the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside and the reconstituted lipoprotein;

[0040] FIG. 4 illustrates the cellular uptake of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside in a 16HBE cell line;

[0041] FIG. 5 shows effects of a reconstituted lipoprotein and a reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside on the brain clearance of As following nasal drug delivery in a A intra-cerebral injected mouse model: (A) percentage of the amount of free A1-42 in the mouse brain (that is, the amount of degraded A1-42) to the total injection amount; and (B) the amount of A1-42 in the mouse brain transported to periphery via blood brain barriers, represented as the brain excretion index; n=3-5, *p<0.05,**p<0.01, significantly different from that of the control group;

[0042] FIG. 6 illustrates degradation of A in brains following intravenous injection of the reconstituted lipoprotein or the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside in a A intra-cerebral injected mouse model, represented by percentage of the amount of free A1-42 in the mouse brain (that is, the amount of degraded A1-42) to the total injection amount;

[0043] FIG. 7 illustrates the neuroprotective effect the NAP-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside; primary neurons are co-incubated with A1-42 oligomers and the NAP fusion peptide solution, the NAP-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside, and the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside or the culture medium (the blank control) for 48 h; (A) the count of neuron cells, (B) mean neurite length and (C) mean branch-point count are recorded. *p<0.05,**p<0.01,***p<0001, ****p<0.0001 significantly different with that of the A1-42 only control;

[0044] FIG. 8 illustrates influences of two-week daily injection of the reconstituted lipoprotein, the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside and the NAP-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside on the escape latency of AD model mice investigated by the Morris water maze test; *p<0.05,***p<0.001 indicate that there is a significant difference from the blank control group; and #p<0.05 indicates that there is a significant difference from the reconstituted lipoprotein group modified by monosialoteterahexosyl ganglioside.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0045] The present invention is further described below in combination with specific embodiments. If not specially described, experimental methods used in embodiments below are all conventional methods. If not specially described, materials, reagents and the like used in embodiments below may be all commercially available. It should be understood that, these embodiments are used for describing the present invention only, rather than limiting a scope of the present invention.

Embodiment 1. Method for Increasing Monodispersity of a Reconstituted Lipoprotein Through the Modification with Monosialoteterahexosyl Ganglioside

[0046] (1) Preparation

[0047] steps: doping monosialoteterahexosyl ganglioside which accounts for 5%, 10% and 20% of a molar ratio of total lipids into dimyristoyl phosphatidylcholine which accounts for 95%, 90% and 80% of the molar ratio of the total lipids, dissolving with chloroform, performing decompression evaporation to remove the organic solvent, hydrating lipid membranes in phosphate buffer at a pH of 7.4, and ultrasonically homogenizing to obtain liposome (the total mass of the lipid of 4 mg) modified by the monosialoteteterahexosyl ganglioside; and adding 0.8 mg of ApoE, slightly mixing to be uniform, and incubating in a orbital shaker at 100 rpm at 37 C. for 36 h, thereby obtaining the reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside.

[0048] (2) Characterization

[0049] Particle sizes and zeta potentials are determined by dynamic laser scattering. Results show that, the particle size of liposome modified by the monosialoteterahexosyl ganglioside which is not incubated with the ApoE is 55.175.11 nm, the particle size of ApoE-incubated reconstituted lipoproteins modified by the monosialoteterahexosyl ganglioside (the monosialoteterahexosyl ganglioside accounting for 5%, 10% and 20% of the molar ratio of the total lipids) is decreased to be less than 25 nm, and the zeta potentials are 14.200.66 mV, 20.200.36 mV and 26.410.42 mV, respectively, while the particle size of the reconstituted lipoprotein unmodified by the monosialoteterahexosyl ganglioside is 24.643.59 nm, and the zeta potential is 8.060.78 mV.

[0050] Phosphotungstic acid negative staining is performed, and morphology is observed by a transmission electron microscope. Results show that (FIG. 1), the reconstituted lipoprotein unmodified by the monosialoteterahexosyl ganglioside has the particle size of about 20 nm and is partially stacked into a silkworm pupa shape, while the reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside also has the particle size of about 20 nm but has better dispersity, and stacked lipoproteins are not observed. The reasons may be that the zeta potential on the surface of the reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside is more negative, particles are difficulty aggregated due to electrostatic repulsion and the monodispersity is better.

Embodiment 2. Method for Increasing Binding Affinity to A with a Reconstituted Lipoprotein Through the Modification with Monosialoteterahexosyl Ganglioside

[0051] Preparation

[0052] steps: doping monosialoteterahexosyl ganglioside which accounts for 5%, 10% and 20% of a molar ratio of total lipids into dipalmitoyl phosphatidyl choline which accounts for 95%, 90% and 80% of the molar ratio of the total lipids (the total mass of the lipid is 4 mg), adding 0.8 mg of ApoE, and preparing a reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside by using the same method in embodiment 1;

[0053] doping cardiolipin which accounts for 5% of the molar ratio of the total lipids into the dipalmitoyl phosphatidyl choline which accounts for 95% of the molar ratio of the total lipids (the total mass of the lipid is 4 mg), adding 0.8 mg of ApoE, and preparing a reconstituted lipoprotein modified by the cardiolipin according to the above method;

[0054] and doping sulfatide which accounts for 10% of the molar ratio of the total lipids into the dipalmitoyl phosphatidyl choline which accounts for 90% of the molar ratio of the total lipids (the total mass of the lipid is 4 mg), adding 0.8 mg of ApoE, and preparing a reconstituted lipoprotein modified by the sulfatide according to the above method.

[0055] (2) A surface plasmon resonance (SPR) experiment verifies A-binding affinity to the reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside.

[0056] An A monomer or oligomer is fixed on a CM5 chip via amine coupling: activating the surface of the chip by using 0.2 M EDC and 0.05 M NHS, diluting A monomer or oligomer in sodium acetate buffer solution at pH 4.0, enabling the concentration of A to be 23 M, injecting at a speed of 30 l/min, and blocking with ethanol amine at pH 8.5; and directly blocking with the ethanol amine after the reference channel is activated. The affinity test is detected in a dual-channel mode: diluting the reconstituted lipoprotein in 10 mM PBS at a pH of 7.4, and injecting into the reference channel and the A-fixed channel at a speed of 30 l/min, wherein contact time is 100 s or 300 s, and dissociation time is 400 s. Results are analyzed using the Biacore T200Evaluation Software program, and affinity value is calculated using 1:1 binding model. The results show that, the reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside accounting for 5%, 10% and 20% of the molar ratio of the total lipids is of high-binding affinity (FIG. 2) with A1-42 monomer or oligomer, and affinity constants KD of the reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside accounting for 5% of the molar ratio of the total lipids with A1-42 monomer or oligomer are calculated by thedynamic method, are (1.71.90)10-10 and (1.510.02)10-10 M, respectively, and are respectively increased by 58 times and 62 times compared with affinity between the unmodified reconstituted lipoprotein and the A1-42 monomer and oligomer (the affinity constants KD of (9.972.81)10-9 and (9.474.37)10-9, respectively), which indicates that the affinity characteristic between the reconstituted lipoprotein and A is increased by the modification of the monosialoteterahexosyl ganglioside.

[0057] Affinity of lipidosome modified by the monosialoteterahexosyl ganglioside which is not incubated with ApoE, and ApoE alone with the A1-42 monomer and oligomer is also detected. Results show that, the affinity constants of the liposome modified by the monosialoteterahexosyl ganglioside which is not incubated with the ApoE, with the A1-42 monomer and oligomer are respectively 1.5010-8 M and 4.0410-8 M, the affinity constant of the ApoE alone with the A1-42 monomer is 2.9510-8 M, and the affinity constant of the ApoE protein with the A1-42 oligomer is difficult to be calculated due to the weak binding signal. The affinity constants of the reconstituted lipoprotein modified by the 5% of cardiolipin prepared according to the same method with A1-42 monomer and oligomer are (14.962.51)10-9 and (6.064.66)10-9 M, respectively; and the affinity constants of the reconstituted lipoprotein modified by the 10% of sulfatide prepared according to the same method with A1-42 monomer and oligomer are 68.2610-9 and (18.157.34)10-9 M, respectively. The above results indicate that the affinity of the reconstituted lipoprotein to A is specifically increased by the modification of the monosialoteterahexosyl ganglioside.

TABLE-US-00001 TABLE 1 Comparison of affinity of different reconstituted lipoproteins to A A.sub.1-42 monomer A.sub.1-42 oligomer K.sub.ass K.sub.ass (M.sup.1s.sup.1) K.sub.diss (s.sup.1) K.sub.D(M) (M.sup.1s.sup.1) K.sub.diss (s.sup.1) K.sub.D(M) Reconstituted (11.38 3.27) (1.72 1.69) (0.17 0.190) (6.58 4.98) (9.93 7.30) (1.51 0.02) lipoprotein modified by 10.sup.5 10.sup.4 10.sup.9 10.sup.5 10.sup.5 10.sup.10 5% of monosialoteterahexosyl ganglioside Reconstituted (7.48 0.32) (11.15 1.40) (14.96 2.51) (19.57 14.7) (8.44 0.21) (6.06 4.66) lipoprotein modified by 10.sup.4 10.sup.4 10.sup.9 10.sup.4 10.sup.4 10.sup.9 5% of cardiolipin Reconstituted 4.19 10.sup.3 2.86 10.sup.3 68.26 (5.30 1.79) (8.89 2.72) (18.15 7.34) lipoprotein modified by 10.sup.9 10.sup.4 10.sup.4 10.sup.9 10% of sulfatide Unmodified (12.55 6.08) (11.66 2.53) (9.97 2.81) (9.30 3.66) (8.00 0.66) (9.47 4.37) reconstituted 10.sup.4 10.sup.4 10.sup.9 10.sup.4 10.sup.4 10.sup.9 lipoprotein

Embodiment 3. Method for Increasing Microglial Cells-Mediated Uptake and Degradation Following the Treatment with Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside

[0058] (1) Preparation

[0059] steps: adding monosialoteterahexosyl ganglioside which accounts for 5% of a molar ratio of total lipids into a mixture of phosphatidylcholine and phosphatidic acid accounting for 95% of the molar ratio of the total lipids (the total mass of the lipid is 4 mg), adding 0.4 mg of ApoE, and preparing a reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside by using the same method in embodiment 1.

[0060] (2) Facilitation of a Uptake in Primary Microglial Cells by the Reconstituted Lipoprotein Modified with Monosialoteterahexosyl Ganglioside

[0061] steps: adding FAM fluorescently-labeled A1-42 into 96-well plates cultured with microglial cells, diluting the reconstituted lipoprotein and the reconstituted lipoprotein modified with the monosialoteterahexosyl ganglioside with DMEM and adding into 96-well plates cultured with primary microglial cells until the final concentrations are 0, 0.01, 0.05, 0.5 and 2 g/mL, wherein the final concentration of the FAM fluorescently-labeled A1-42 is 2 g/mL; co-incubating in a 37 C. cell incubator for 4 h; fixing in 3.7% of formaldehyde at 37 C. for 10 min, performing Hoechst nucleus staining for 8 min, washing with PBS for 3 times, and performing shooting and quantitative analysis by virtue of a high-content TargetActivation program. Results are shown in FIG. 3A, and the uptake of the primary microglial cells to the FAM fluorescently-labeled A1-42 is obviously increased by the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside.

[0062] (3) Investigating the influence of reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside to A degradation in primary microglial cells via ELISA.

[0063] steps: diluting A1-42 to 4 g/ml with DMEM, adding into the 24-well plates cultured with microglial cells, adding the reconstituted lipoprotein or the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside diluted by the DMEM to the final concentrations 0, 1, 10, 100 g/mL, wherein the final concentration of tA1-42 is 2 g/mL; and incubating at 37 C. for 4 h, lysing and scraping cells, and performing subsequent detection or preserving at 80 C.;

[0064] taking a 1 mg/ml of human A1-42 standard stock solution stored at 80 C., diluting to 0, 6.25, 12.5, 25, 50, 100 and 200 pg/ml with dilution buffer to serve as a standard curve; diluting a sample by 80 times with the dilution buffer; and performing the experiment and result determination according to an ELISA product specification, and applying BCA analysis to determine the total protein amount in cell lysate. The result is represented as the ratio of the A1-42 concentration in the cell lysate to the total protein concentration. The result is shown in FIG. 3B, and indicates that, the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside can increase A1-42 degradation in the primary microglial cells in a concentration-dependent manner, and due to an addition of the GM1, the content of the ApoE may be obviously decreased.

Embodiment 4. The Uptake of Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside by an In Vitro Nasal Mucosa Cell Model

[0065] A 16HBE cell line serves as the in vitro nasal mucosa cell model, and cellular uptake of the reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside are investigated.

[0066] (1) Preparation

[0067] The reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside is prepared according to the same method in embodiment 1.

[0068] (2) Cellular Uptake of the Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside in the 16HBE Cell Line.

[0069] Steps: inoculating 16HBE into 96-well plates, culturing for 24 h, and conducting the experiment; diluting the formulation to 50, 100, 250, 500 and 800 g/m with DMEM, adding the material into the above 96-well plates, and incubating at 37 C. for 4 h; fixing the cells in 3.7% of formaldehyde, performing Hoechst nucleus staining after PBS washing, and performing quantitative analysis using a high-content analysis. The result indicates that, the cellular uptake amount of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside accounting for 5% of the molar ratio of the total lipids is equivalent to that of an unmodified reconstituted lipoprotein (FIG. 4).

Embodiment 5. Intra-Cerebral Delivery Efficiency of a Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside Via Nasal Delivery

[0070] Reconstituted lipoproteins modified at a monosialoteterahexosyl ganglioside modification degree increased from 5% of the molar ratio of the total lipids to 10%, 20%, 30% and 40% are prepared according to the same method in embodiment 1.

[0071] 125I is labeled onto the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside via Bolton-Hunter method. Results show that, following nasal administration in ICR mice, the maximal concentration (Cmax) of the 125I-labeled reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside accounting for 5% of the molar ratio of the total lipids in the cortex and hippocampus is 0.0434% ID/g, and is 75% higher than that of the 125I-labeled unmodified reconstituted lipoprotein; and the AUC all in the cortex and hippocampus is 0.2956% ID/g.Math.h, which is 85% higher than that of the unmodified reconstituted lipoprotein; and the AUC all in blood is 9.8650% ID/g.Math.h, which is 80% higher than that of the unmodified reconstituted lipoprotein. However, the AUCCortex+Hippocampus/AUCblood of the two preparations is close to each other, which indicates that the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside has higher nasal mucosa absorption efficiency than the unmodified reconstituted lipoprotein. But along with an increase of the monosialoteterahexosyl ganglioside modification degree from 5% to 10%, 20%, 30% and 40%, the intra-cerebral delivery characteristic of the formulations is not obviously increased, but shows a certain decline trend. Intra-cerebral distribution of a high-density 125I-labeled reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside accounting for 20% of the molar ratio of the total lipids is slightly higher than that of an unmodified high-density reconstituted lipoprotein (increased by 25.9%), the intra-cerebral distribution of a 125I-labeled high-density reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside accounting for 30% of the molar ratio of the total lipids is equivalent to that of the unmodified high-density reconstituted lipoprotein (102.3%), and the intra-cerebral distribution of a high-density 125I-labeled reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside accounting for 40% of the molar ratio of the total lipids is lower than that of the unmodified high-density reconstituted lipoprotein (87.2%).

TABLE-US-00002 TABLE 2 Pharmacokinetic parameters of the unmodified reconstituted lipoprotein and the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside in mouse following nasal delivery Reconstituted lipoprotein Unmodified modified by reconstituted monosialoteterahexosyl lipoprotein ganglioside Cortex + hippocampus Cortex + hippocampus Blood Blood T.sub.max (h) 4 4 2 4 C.sub.max (% ID/g) 0.0248 0.8074 0.0434 1.333 AUC.sub.all (% ID/g) 0.1596 5.4945 0.2956 9.8650 AUC.sub.Cortex+Hippocampus/ 0.0291 0.0300 AUC.sub.blood

Embodiment 6. Method for Facilitating Clearance of A in Mouse Brains by a Reconstituted Lipoprotein Modified with Monosialoteterahexosyl Ganglioside

[0072] (1) Preparation

[0073] steps: adding monosialoteterahexosyl ganglioside which accounts for 5% of a molar ratio of total lipids into a mixture of phosphatidylcholine and phosphatidylcholine which accounts for 95% of the molar ratio of the total lipids (a total mass of the lipid is 4 mg), adding 0.8 mg of ApoE and preparing a reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside by using the same method in embodiment 1.

[0074] (2) Evaluation of the Influence of the Reconstituted Lipoprotein Modified by the Monosialoteterahexosyl Ganglioside on A Clearance in Brain Via a Mouse Intra-Cerebral Injection Model

[0075] steps: randomly dividing ICR mice into 3 groups, administrating the mice with reconstituted lipoproteins or reconstituted lipoproteins modified by monosialoteterahexosyl ganglioside through nasal delivery or intravenous injection, and giving the control animals with PBS; performing intracerebral A injection at 30 min after drug delivery, i.e., mixing 125I-labeled A1-42 and 14C-containing inulin ([14C]inulin), diluting with artificial cerebrospinal fluid, and accurately injecting the material into the mouse hippocampal area; sacrificing the mice at 10 min and 30 min after the intracerebral injection, and collecting the mice brains; weighing half of the brain tissues, detecting the radiation quantity of the 125I-labeled A1-42 on a -ray counter, adding 10% of trichloroacetic acid (TCA) at the volume of 3 times of the weight of brain tissues to precipitate the tissues, centrifuging to remove the supernatant, and detecting the radiation intensity of the un-degraded 125I-labeled A1-42. FIG. 5A shows that the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside obviously facilitates degradation of A1-42 in the brain at a short time after nasal delivery; and FIG. 5B shows that the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside obviously facilitates the brain-to-peripheral clearance of A1-42 following nasal delivery. By contrast, the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside does not show an obvious facilitation effect on the degradation of A1-42 in the brain immediately (5 min) after intravenous injection, while the unmodified high-density reconstituted lipoprotein slightly facilitates the degradation of A1-42 in the brain at this time point (FIG. 6). It is supposed that, shortly following intravenous injection, the intra-cerebral distribution of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside could be less than that of the unmodified high-density reconstituted lipoprotein, and the A degradation facilitation effect of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside needs be achieved at the longer time points (e.g., 30 min). In contrast, the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside increase the degradation of A1-42 and facilitate brain-to-periphery transport of A1-42 shortly after nasal administration.

Embodiment 7. Neuroprotective Effect of a Drug-Loaded Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside

[0076] (1) Preparation

[0077] steps: adding monosialoteterahexosyl ganglioside which accounts for 5%, 10% and 20% of a molar ratio of total lipids into dimyristoyl phosphatidylcholine which accounts for 95%, 90% and 80% of the molar ratio of the total lipids, dissolving with chloroform, performing decompression evaporation to remove the organic solvent, hydrating lipid membranes in phosphate buffer at pH 7.4, and ultrasonically homogenizing to obtain liposome (a total mass of the lipid of 4 mg) modified by the monosialoteterahexosyl ganglioside; adding 0.05-1 mg of NAP fusion peptide, incubating overnight at 4 C., adding 0.5-5 mg of ApoE3, and continuously performing ultrasonic treatment for 50 min; and cooling to a room temperature, and incubating overnight, thereby obtaining the NAP-loaded reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside.

[0078] (2) Neuroprotective Effect of the NAP-Loaded Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside

[0079] steps: diluting A1-42 oligomer to 20 M with neuron culture medium, adding the oligomer into 96-well plates cultured with primary neurons, adding the NAP fusion peptide solution, the NAP-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside or a reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside, enabling the final concentration of the A1-42 oligomer to be 10 M, and only adding the neuron culture medium into the blank control group; co-incubating at 37 C. for 48 h, fixing the cells with PBS containing 37% of formaldehyde, adding a TritonX-100 permeable membrane for 15 min, blocking at 37 C. by using PBS with 1% of BSA for 30 min, adding the MAP2 antibody to incubate overnight at 4 C., adding an Alexa Fluor 488 fluorescent-conjugated secondary antibody, incubating at 37 C. for 1 h, and performing Hoechst nucleus staining; and detecting the primary neuron cells by using a high-content analysis, and analyzing the data via the NeuroProfiling program. Results are shown in FIGS. 7A-7C, and the NAP-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside obviously reverses A1-42 oligomer-induced neurotoxicity, thereby increasing the quantity of neurons, the mean neurite length and the mean branch-point counts.

Embodiment 8. Method for Decreasing Expression of -Secretase in SH-SY5Y Cells by BACE-siRNA-Loaded Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside

[0080] steps: preparing a reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to the same method in embodiment 1, enabling BACE-siRNA to link with cholesterol, and inserting into a lipid membrane; culturing the SH-SY5Y cells to reach confluence of 80%, and transfecting the cells with 1 M siRNA; and detecting the expression of -secretase in the cells by Western blot at 48 h after transfection, and statistically quantifying the gray value of the band by virtue of Image J software. Results indicate that the BACE-siRNA-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside decreases the expression of the f-secretase in the SH-SY5Y cells by 40%.

Embodiment 9. Method for Alleviating Inflammations of Microglial Cells Caused by a Cu2+-A Complex by Curcumin-Loaded Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside

[0081] steps: dissolving curcumin and lipids in a chloroform-methanol mixed solvent according to a certain ratio, and preparing a reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to the same method in embodiment 1; incubating Cu2+-A at 37 C. at a molar ratio of 1:1 for 24 h to prepare a complex; adding primary microglial cells into 5 M of the Cu2+-A complex to incubate for 24 h, increasing content of TNF- by 100%, and adding the Cu2+-A complex and the curcumin-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside to simultaneously incubate the primary microglial cells, wherein the content of the TNF- is equivalent to that in an untreated group. It is indicated that, the curcumin-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside effectively alleviates the inflammations of the microglial cells caused by the Cu2+-A complex.

Embodiment 10. Method for Synergistically Improving Spatial Learning and Memory Ability of AD Model Animals by the Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside and the Loaded Drug

[0082] (1) Preparation

[0083] An NAP-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside is prepared according to the same method in embodiment 7.

[0084] (2) Synergistic Improvement of the Spatial Learning and Memory Ability of the AD Model Animals by the Reconstituted Lipoprotein Modified by Monosialoteterahexosyl Ganglioside and the Loaded Drug

[0085] Improvement effects of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside and the NAP-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside on the space learning-memory capabilities of the AD model animals are investigated by the Morris water maze test. The AD model mice are randomly divided into groups; each group includes 7-8 mice; and the mice are subjected to nasal delivery according to the following manners: the blank control group and the AD model group: giving PBS solution, 10 l/mouse per day; the AD model group: giving PBS solution, 10 l/mouse per day; the drug solution group: giving an NAP fusion peptide solution, 24 g/kg per day; the reconstituted lipoprotein group: giving there constituted lipoprotein solution, 5 mg/kg per day (lipid concentration); the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside group: giving the lipoprotein modified by monosialoteterahexosyl ganglioside solution, 5 mg/kg per day (lipid concentration); and the drug-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside group: giving the solution of the NAP-loaded reconstituted lipoprotein modified by monosialoeterahexosyl ganglioside, 5 mg/kg per day (lipid concentration). The mice are subjected to continuous nasal delivery per day for 14 days.

[0086] The Morris water maze has a pool diameter of 120 cm, a height of 50 cm, a water depth of 25 cm and a water temperature of 221 C., Four start points are equally divided along a circumference of the pool; a circular pool is equally divided into four quadrant areas I, II, III and IV by virtue of connecting lines of the four start points, and a 9 cm black platform is arranged in a center of the quadrant area I. The platform is about 1 cm lower than a water surface. A bottom of the pool, the platform and four walls are painted black by food dyes, so that the platform is invisible. Swimming trajectories of the mice are monitored and recorded by the Morris water maze video analysis system 2.0. A hidden platform test is used for training and measuring the spatial learning abilities of the mice, lasting for 5 days. The platform is fixed in the center of the quadrant area I; the mice are put into water towards pool walls from the start points in the quadrant areas I, II, III and IV according to a random principle; and routes of the mice from a moment of entering the water and searching to a moment of finding and climbing up the black platform, the needed time (escape latency), swimming speeds and the like are monitored and recorded by a computer. If a mouse does not find the platform within 60s, the mouse should be led to the platform and stopped for 30s, and the escape latency is recorded as 60s. Each mouse is trained by 4 times per day, and a training interval is 30s each time.

[0087] The results are shown in FIG. 8. In a training process of 5 days, the escape latencys of the drug solution group and the reconstituted lipoprotein are not obviously decreased. The escape latency of the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside in the training on the 5th day is obviously lower than that of the AD model group, which indicates that the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside has a certain improvement effect on the learning-memory abilities of the AD model mice. After 14-day nasal delivery of the NAP-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside, the escape latency of the AD model mice in a spatial probe test have a decline trend on the 3rd day. In the training on the 5th day, compared with the AD model group, the escape latency is obviously shortened (33.92.1 s), and compared with a non-drug-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside, the escape latency is shortened by 12%. The results indicate that, the drug-loaded reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside could further enhance a disease modifying effect on the AD by virtue of the multi-mode effect of the reconstituted lipoprotein modified by monosialoeterahexosyl ganglioside and the loaded neuroprotective peptide.

Embodiment 11. Method for Maintaining High A Binding Affinity of Reconstituted Lipoproteins Via the Modification with Monosialoteterahexosyl Ganglioside Even at Decreased Amount of Apolipoproteins

[0088] steps adding monosialoteterahexosyl ganglioside which accounts for 10% of a molar ratio of total lipids into dimyristoyl phosphatidylcholine (4 mg) which accounts for 90% of the molar ratio of the total lipids, adding ApoE3 accounting for 2.5%, 5%, 10% or 20% of lipid mass (0.1, 0.2, 0.4 and 0.8 mg), and preparing the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside. A surface plasmon resonance (SPR) experiment is performed to detect the A-binding affinity of the reconstituted lipoprotein modified by the monosialoteterahexosyl ganglioside. The A binding affinity constants of the reconstituted lipoproteins modified by the monosialoteterahexosyl ganglioside with the ApoE3 content accounting for 2.5%, 5%, 10% and 20% of the lipid mass to A are 0.1910-9M, 0.6410-9M, 0.8010-9M and 0.5210-9M, respectively. The evidences indicate that, when monosialoteterahexosyl ganglioside is added, even if the mass ratio of apolipoprotein is greatly decreased, the binding affinity of the reconstituted lipoproteins to A may be still maintained or enhanced.

Embodiment 12. Method for Increasing Brain Delivery of A Polypeptide Vaccine by the Reconstituted Lipoprotein Mediated by Modification of Monosialoteterahexosyl Ganglioside

[0089] steps: preparing the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside according to the above method, adding an 125I-labeled A polypeptide vaccine and co-incubating for 5 min; performing intravenous injection of the mixture, collecting the brain tissues at 1 h after injection, and detecting the radioactive intensity of the 125I-labeled A polypeptide vaccine in the brain by a -ray counter. Results show that, compared with single injection of A polypeptide vaccine, the co-incubation with reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside increases the intra-cerebral amount of A polypeptide vaccine by 76.47%, while the co-incubation with unmodified reconstituted lipoprotein only increases the intra-cerebral amount of A polypeptide vaccine by 11.44%. The result indicates that, by virtue of simple co-incubation, the reconstituted lipoprotein modified by monosialoteterahexosyl ganglioside can effectively increase the intra-cerebral delivery of a polypeptide drug with an intra-cerebral transport characteristic, and such effect could be more striking for the brain delivery of those drugs that are difficult to enter the brain.

[0090] The above only describes preferred embodiments of the present invention. It should be noted that, those ordinary skilled in the art may make several improvements and modifications on premise of not deviating from a principle of the present invention. These improvements and modifications should also be regarded as a protection scope of the present invention.