Poly(n-butyl cyanoacrylate) nanoparticle with dual modifications, preparation method and use thereof

Abstract

The invention relates to a poly(n-butyl cyanoacrylate) nanoparticle with dual modifications, a drug delivery system comprising the nanoparticle, and a method for preparing the nanoparticle or the drug delivery system. The nanoparticle is modified with a first modifier and a second modifier on the surface, the first modifier is a hydrophilic polymer, and the second modifier is an amino acid and/or a lipid. The invention further relates to a use of the nanoparticle in promoting drug penetration across the blood brain barrier in a subject.

Claims

1. A nanoparticle, comprising poly(n-butyl cyanoacrylate), wherein the nanoparticle is modified with a first modifier and a second modifier on the surface, wherein the first modifier is polyethylene glycol, and the second modifier is lecithin or cholesterol; and wherein the polyethylene glycol has a number-average molecular weight of 2000-20000.

2. A method for preparing the nanoparticle according to claim 1, comprising the following steps: Step 1: n-butyl cyanoacrylate monomers are subjected to a polymerization reaction in an acidic medium comprising a first modifier; Step 2: a base is added to the reaction mixture of Step 1 until the reaction mixture is neutral, the reaction mixture is filtrated, and the filtrate is lyophilized; Step 3: the lyophilized product is dispersed in a buffer, a second modifier is added, and incubation is performed; and Step 4: nanoparticles are separated from the mixture of Step 3.

3. A drug delivery system comprising the nanoparticle according to claim 1, wherein the nanoparticle is loaded with a drug.

4. A method for preparing the drug delivery system according to claim 3, comprising the following steps: Step 1: n-butyl cyanoacrylate monomers are subjected to a polymerization reaction in an acidic medium comprising a first modifier; Step 2: a drug is added to the reaction mixture, and then the polymerization reaction is further performed; Step 3: a base is added to the reaction mixture until the reaction mixture is neutral, the reaction mixture is filtrated, and the filtrate is lyophilized; Step 4: the lyophilized product is dispersed in a buffer, a second modifier is added, and incubation is performed; and Step 5: nanoparticles are separated from the mixture of Step 4.

5. A pharmaceutical composition, comprising the nanoparticle according to claim 1, and a pharmaceutically acceptable carrier and/or excipient, wherein the nanoparticle is loaded with a drug.

6. A method for enhancing the ability of a drug to penetrate across the blood brain barrier in a subject, comprising loading the nanoparticle according to claim 1 with the drug.

7. A method for promoting drug penetration across the blood brain barrier in a subject, comprising loading the nanoparticle according to claim 1 with a drug, and administering the drug to the subject.

8. The nanoparticle according to claim 1, having one or more features selected from the following: (1) the nanoparticle has an average particle size of 50-800 nm; (2) the nanoparticle has a polydispersity index of 0.100-0.300; (3) the nanoparticle has a Zeta potential of −100-0 mV; (4) the first modifier and the polycyanoacrylate have a mass ratio of 0.5%-5%; (5) the second modifier and the polycyanoacrylate have a mass ratio of 0.05%-2%; (6) the concentration of the second modifier on the surface of the nanoparticle is 5×10.sup.−7 ng/nanoparticle-1×10.sup.−6 ng/nanoparticle; (7) the nanoparticle is prepared by a method comprising the following steps: Step 1: n-butyl cyanoacrylate monomers are subjected to a polymerization reaction in an acidic medium comprising a first modifier; Step 2: a base is added to the reaction mixture of Step 1 until the reaction mixture is neutral, the reaction mixture is filtrated, and the filtrate is lyophilized; Step 3: the lyophilized product is dispersed in a buffer, a second modifier is added, and incubation is performed; and Step 4: nanoparticles are separated from the mixture of Step 3.

9. The drug delivery system according to claim 3, having one or more features selected from the following: (1) the drug is encapsulated in the nanoparticle; (2) in the drug delivery system, the nanoparticle has a drug entrapment efficiency of 80%-100%; (3) the drug delivery system has a drug loading rate of 1%-10%; and (4) the drug delivery system is prepared by a method comprising the following steps: Step 1: n-butyl cyanoacrylate monomers are subjected to a polymerization reaction in an acidic medium comprising a first modifier; Step 2: a drug is added to the reaction mixture, and then the polymerization reaction is further performed; Step 3: a base is added to the reaction mixture until the reaction mixture is neutral, the reaction mixture is filtrated, and the filtrate is lyophilized; Step 4: the lyophilized product is dispersed in a buffer, a second modifier is added, and incubation is performed; and Step 5: nanoparticles are separated from the mixture of Step 4.

10. The drug delivery system according to claim 3, wherein the drug is a paclitaxel drug or a fluorescent substance.

11. A nanoparticle, comprising poly(n-butyl cyanoacrylate), wherein the nanoparticle is modified with a first modifier and a second modifier on the surface, wherein the first modifier is polyethylene glycol, and the second modifier is aspartic acid or leucine; wherein the polyethylene glycol has a number-average molecular weight of 2000-20000; and wherein the nanoparticle excludes polysorbate 80.

12. A drug delivery system comprising the nanoparticle according to claim 11, wherein the nanoparticle is loaded with a drug.

13. The drug delivery system according to claim 12, wherein the drug is a paclitaxel drug or a fluorescent substance.

14. A pharmaceutical composition, comprising the nanoparticle according to claim 11, and a pharmaceutically acceptable carrier and/or excipient, wherein the nanoparticle is loaded with a drug.

15. The nanoparticle according to claim 11, having one or more features selected from the following: (1) the nanoparticle has an average particle size of 50-800 nm; (2) the nanoparticle has a polydispersity index of 0.100-0.300; (3) the nanoparticle has a Zeta potential of −100-0 mV; (4) the first modifier and the polycyanoacrylate have a mass ratio of 0.5%-5%; (5) the second modifier and the polycyanoacrylate have a mass ratio of 0.05%-2%; (6) the concentration of the second modifier on the surface of the nanoparticle is 5×10.sup.−7 ng/nanoparticle-1×10.sup.−6 ng/nanoparticle; and (7) the nanoparticle is prepared by a method comprising the following steps: Step 1: n-butyl cyanoacrylate monomers are subjected to a polymerization reaction in an acidic medium comprising a first modifier; Step 2: a base is added to the reaction mixture of Step 1 until the reaction mixture is neutral, the reaction mixture is filtrated, and the filtrate is lyophilized; Step 3: the lyophilized product is dispersed in a buffer, a second modifier is added, and incubation is performed; and Step 4: nanoparticles are separated from the mixture of Step 3.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the result of the measurement for particle size (A) and Zeta potential (B) of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 1. As seen from the figure, the nanoparticle had a narrow particle size distribution, an average particle size of 185.4 nm, and a Zeta potential of −0.66 mV.

(2) FIG. 2 shows the TEM photographs of coumarin-6-loaded PBCA nanoparticles with single modification by cholesterol (FIG. 2 (A)) and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol (FIG. 2 (B)) in Example 2. As seen from the figures, for both PBCA nanoparticles with single modification by cholesterol and PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, the nanoparticles were uniform in size, had a particle size of about 200 nm, and had good dispersion and no phenomenon of large-scale aggregation, and the two nanoparticles had little difference in morphology, indicating that dual modifications by PEG.sub.20000-cholesterol had little effect on the morphology of nanoparticles.

(3) FIG. 3 shows the result of the structural characterization of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 2, wherein, FIGS. 3(A)-3(C) show the DSC thermograms of mPEG.sub.20000, cholesterol and coumarin-6. As seen from the figure, mPEG.sub.20000, cholesterol and coumarin-6 had a melting peak at 68.21° C., 149.98° C. and 208.29° C., respectively. FIG. 3(D) shows the DSC thermogram of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol. In the figure, the melting peaks of mPEG.sub.20000 and cholesterol were still present, indicating that PEG.sub.20000 and cholesterol were not entrapped in the inner of the nanoparticles, and instead, were involved in the modification of nanoparticles on the surface; the melting peak of coumarin-6 disappeared, indicating that coumarin-6 was encapsulated into a nanoparticle.

(4) FIG. 4 (A) shows the comparison of the in vitro release curves of common nanoparticles loaded with coumarin-6, coumarin-6-loaded nanoparticles with single modification by cholesterol and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 4 (n=6, n is the number of times for parallel tests, and n below has the same meanings). As shown in the figure, as compared with the common nanoparticles and the nanoparticles with single modification by cholesterol, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a significant sustained-release property and an improved release stability.

(5) FIG. 4 (B) shows the comparison of the in vitro release curves of common nanoparticles loaded with coumarin-6, coumarin-6-loaded nanoparticles with single modification by PEG.sub.20000, and coumarin-6-loaded nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 4 (n=6). As shown in the figure, as compared with the unmodified nanoparticles and the nanoparticles with single modification by PEG.sub.20000, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a more significant sustained-release property.

(6) FIG. 5 shows the blood concentration—time curves of a coumarin-6 solution (a free drug), coumarin-6-loaded nanoparticles with single modification by cholesterol and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 5 (n=6). As shown in the figure, the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol could be released in vivo for a period of 12 h, and had a longer release time, and had a higher blood concentration at the same time, as compared with the free drug solution and the nanoparticles with single modification by cholesterol.

(7) FIG. 6 (A) shows the content of coumarin-6 in the brain tissue at different time points after the rats were intravenously injected with a coumarin-6 solution (a free drug), coumarin-6-loaded nanoparticles with single modification by cholesterol, and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 6, respectively (n=3). In the figure, for the nanoparticles with dual modifications by PEG.sub.20000-cholesterol, P was less than 0.01, as compared with the free drug solution and the nanoparticles with single modification by cholesterol. As shown in the figure, the coumarin-6-loaded nanoparticles with dual modifications by PEG.sub.20000-cholesterol could significantly enhance the content of coumarin-6 in the brain tissue of rats (which was significantly higher than that in the brain tissue of rats injected with a coumarin-6 solution (a free drug) and that in the brain tissue of rats injected with coumarin-6-loaded nanoparticles with single modification by cholesterol), and exhibited a sustained-release process.

(8) FIG. 6 (B) shows the content of coumarin-6 in the brain tissue of the rats injected with different samples in Example 6, wherein the samples from left to right were a free drug solution, common nanoparticles, PBCA nanoparticles with single modification by PEG.sub.20000, and PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, respectively. In the figure, **: p<0.05 VS a free drug solution group; ##: p<0.05 VS a common nanoparticle group; &&: p<0.05 VS a group of nanoparticles with single modification by PEG. As shown in the figure, the PBCA nanoparticles with single modification by PEG.sub.20000 had a significantly increased content of coumarin-6 in the brain tissue, as compared with the free drug solution and the common PBCA nanoparticles (P<0.01). The PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol according to the invention had a significantly increased content of coumarin-6 in the brain tissue, as compared with the PBCA nanoparticles with single modification by PEG.sub.20000 (P<0.01). The result showed that the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had good BBB permeability, and could significantly promote the passage of drug through blood-brain barrier.

(9) FIG. 7 shows the concentration of coumarin-6 at different time points in the tissues of rats from different groups in Example 7. As seen from the figure, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol were not significantly accumulated in various tissues and visceral organs as compared with the nanoparticles with single modification by cholesterol, and had the content of coumarin-6 in liver and spleen significantly lower than that of the nanoparticles with single modification. The result showed that the coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had better safety in vivo.

(10) FIG. 8 shows the cell viability of bEnd.3 cells after administration of (A) cholesterol, (B) PEG.sub.20000, (C) PBCA, (D) blank PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, and (E) coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 8, respectively. In the figure, the abscissa represents the administration concentration, and the ordinate represents the cell viability. As shown in the figure, cholesterol, PEG.sub.20000, PBCA, blank nanoparticles and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had no significant cytotoxicity for bEnd.3 cells with the experimental concentration range (cell viability >80%). The result showed that the blank carrier and the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had good safety in vitro.

(11) FIG. 9 shows the blood concentration—time curves after the rats were intravenously administered with a free drug solution of docetaxel and docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 9 (n=6), respectively. As shown in the figure, the docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a longer release time and a higher blood concentration in vivo.

(12) FIG. 10 shows the content of docetaxel in the brain tissue of rats after the rats were intravenously administered with a docetaxel solution (a free drug) and docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 10 (n=3), respectively. As shown in the figure, docetaxel (a free drug) could hardly penetrate across the blood-brain barrier, and docetaxel was not detected in the brain; however, the docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol could penetrate across the blood-brain barrier, and enabled docetaxel to be released slowly in the brain.

(13) FIG. 11 shows the cell viability of mouse breast cancer 4T1 cells after separate administration of docetaxel (a free drug) and docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in Example 11. In the figure, the abscissa represents the administration concentration (calculated as the concentration of docetaxel), and the ordinate represents the cell viability; n=5; for the nanoparticles with dual modifications by PEG.sub.20000-cholesterol, P was less than 0.01, as compared with the free drug solution. Fig. (A) and Fig. (B) show the results of incubation for 24 h and 48 h after administration, respectively. As shown in the figures, the docetaxel-loaded nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a stronger inhibitory effect on the proliferation of tumor cells as compared with free docetaxel at the same administration concentration for the same incubation time, and were dose-dependent and time-dependent to some extent.

(14) FIG. 12 shows the in vitro release curves of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-leucine, and unmodified PBCA nanoparticles loaded with coumarin-6 in Example 13 (n=6). As shown in the figure, the nanoparticles with dual modifications by PEG.sub.20000-leucine had a more significant sustained-release property, as compared with the unmodified nanoparticle.

(15) FIG. 13 shows the in vitro release curves of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-aspartic acid, and unmodified PBCA nanoparticles loaded with coumarin-6 in Example 15 (n=6). As shown in the figure, the nanoparticles with dual modifications by PEG.sub.20000-aspartic acid had a more significant sustained-release property, as compared with the unmodified nanoparticles.

(16) FIG. 14 the in vitro release curves of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin, and unmodified PBCA nanoparticles loaded with coumarin-6 in Example 17 (n=6). As shown in the figure, the nanoparticles with dual modifications by PEG.sub.20000-lecithin had a more significant sustained-release property, as compared with the unmodified nanoparticles.

(17) FIG. 15 shows the content of coumarin-6 in the brain tissue of the rats injected with different samples in Example 18, wherein the samples from left to right were a free drug solution, common nanoparticles, PBCA nanoparticles with dual modifications by PEG.sub.20000-leucine, PBCA nanoparticles with dual modifications by PEG.sub.20000-aspartic acid, and PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin, respectively. For the three nanoparticles with dual modifications, P<0.01 as compared with the free drug solution and the common nanoparticles. As shown in the figure, as compared with the free drug solution and the common PBCA nanoparticles, the three nanoparticles with dual modifications according to the invention might have a significantly increased content of coumarin-6 in the brain tissue, and enhance the BBB permeability of drug.

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

(18) The embodiments of the invention are illustrated in detail by reference to the following examples. However, it is understood by those skilled in the art that the examples are used only for the purpose of illustrating the invention, rather than limiting the protection scope of the invention. In the case where the concrete conditions are not indicated in the examples, the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. The agents or instruments of which the manufacturer are not indicated are regular products that can be purchased on the market.

Example 1. Preparation and Evaluation of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(19) (1) Method for preparing coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol: the stabilizer Dex70 (1%, w/v) and methoxypolyethylene glycol having a number-average molecular weight of 20000 (mPEG.sub.20000) (1.5%, w/v) were dissolved in a HCl medium (pH 1.0), and BCA monomers (1%, v/v) were slowly added dropwise at room temperature under magnetic stirring. After stirring at 500 rpm for 4 h, coumarin-6 (1%, w/v) was added, and the stirring was performed at 750 rpm for 2.5 h. Then, the system was neutralized with NaOH to pH of 6-7, and was further stirred for 1 h so that BCA monomers were sufficiently polymerized, thereby obtaining PEG-PBCA nanoparticles.

(20) After filtration through a filtration membrane (0.45 μm), the PEG-PBCA nanoparticles obtained were lyophilized, and then were re-dissolved in PBS, and mixed homogeneously for 30 min. Cholesterol (1%, w/v) was added, and incubation was performed for 0.5 h. The resultant mixture was filtrated, and centrifuged at 20000 rpm for 30 min. The supernatant was removed, and the precipitate was re-suspended in a suitable amount of double distilled water, and then lyophilized, thereby obtaining coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, which were stored in a drier.

(21) Method for preparing coumarin-6-loaded PBCA nanoparticles with single modification by PEG.sub.20000: the stabilizer Dex70 (1%, w/v) and mPEG.sub.20000 (1.5%, w/v) were dissolved in a HCl medium (pH 1.0), and then BCA monomers (1%, v/v) were slowly added dropwise at room temperature under magnetic stirring. After stirring at 500 rpm for 4 h, coumarin-6 (1%, w/v) was added, and the stirring was performed at 750 rpm for 2.5 h. Then, the system was neutralized with NaOH to pH of 6-7, and was further stirred for 1 h so that BCA monomers were sufficiently polymerized, thereby obtaining coumarin-6-loaded PBCA nanoparticles with single modification by PEG.sub.200000. After filtration through a filtration membrane (0.45 μm), the nanoparticles obtained were lyophilized, which were stored in a drier, as an experimental control.

(22) By the method above, common unmodified PBCA nanoparticles comprising coumarin-6, and coumarin-6-loaded nanoparticles with single modification by cholesterol, were also prepared, as control.

(23) (2) Measurement and Result of particle size and potential: coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol were diluted with deionized water to a suitable concentration, and ultrasonically treated for 30 min, so as to mix the solution sufficiently. After filtration through a microfiltration membrane (0.45 μm), the filtrate was measured for particle size and potential at 25° C., and the result was shown in FIG. 1. FIG. 1 (A) showed the measurement result of the particle size of nanoparticles, and FIG. 1 (B) showed the measurement result of the Zeta potential of nanoparticles. As seen from the figure, the coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a narrow particle size distribution, an average particle size of 185.4 nm, a PDI of 0.133, and a Zeta potential of −0.66 mV.

(24) By the method above, coumarin-6-loaded PBCA nanoparticles with single modification by PEG.sub.20000 were measured for particle size and Zeta potential, which had an average particle size of 194.3 nm, a PDI of 0.159, and a Zeta potential of −8.04 mV.

(25) (3) Measurement and Result of entrapment efficiency and drug loading rate: a suspension (200 μl) of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol was added in a Vivaspin ultrafiltration centrifuge tube (MWCO: 2000, Sartorius company, Germany) and centrifuged at 4000 rpm for 20 min, and the free-form drug, which was not encapsulated into nanoparticles, was separated into the lower layer of the ultrafiltration centrifuge tube. The concentration of coumarin-6 in the lower layer was determined by Fluorescence/Chemiluminescence Analyzer, and the entrapment efficiency was calculated.

(26) To a suspension of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol (20 μl), acetonitrile (1980 μl) was added, and the nanoparticle structure was destructed by shaking for 30 s, so as to release the drug. The concentration of coumarin-6 was determined by Fluorescence/Chemiluminescence Analyzer, and the drug loading rate was calculated.

(27) Result: coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had an entrapment efficiency of 97.8%, and a drug loading rate of 2.07%.

(28) By the method above, the entrapment efficiency and the drug loading rate of coumarin-6-loaded PBCA nanoparticles with single modification by PEG.sub.20000 were determined, which were 98.6% and 2.03%, respectively.

Example 2. Morphological and Structural Characterization of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(29) (1) Morphological Characterization

(30) Method: Transmission Electron Microscope (TEM) was used for morphological characterization of coumarin-6-loaded PBCA nanoparticles with single modification by cholesterol and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol. A given amount of lyophilized nanoparticles was weighed, and diluted with pure water until the nanoparticles had a concentration of 0.5 mg/ml. The resultant solution was mixed homogenously by shaking for 100 times, 5 μl solution was pipetted and added dropwise to a Formvar-coated copper grid, and a drop of 1% (w/v) phosphotungstic acid aqueous solution was added for staining when the grid was almost dry. After staining for 2 min, most of the liquid was pipetted off carefully, and only a liquid film was left. After the liquid film was completely dried, it was observed by TEM.

(31) Result: the TEM photographs of the two samples were shown in FIG. 2. FIGS. 2(A) and (B) showed the TEM photographs of coumarin-6-loaded PBCA nanoparticles with single modification by cholesterol and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol. As seen from the figure, for both PBCA nanoparticles with single modification by cholesterol and PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, the nanoparticles were uniform in size, had a particle size of about 200 nm, had good dispersion and no phenomenon of large-scale aggregation, and the two nanoparticles had little difference in morphology, indicating dual modifications by PEG.sub.20000-cholesterol had little effect on the morphology of nanoparticles.

(32) (2) Structural Characterization

(33) Method: Differential Scanning Calorimetry (DSC) was used for structural characterization of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol. Coumarin-6 (about 5 mg), mPEG.sub.20000 (about 5 mg), cholesterol (about 5 mg) and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol (about 5 mg) were separately weighed, and placed in T zero aluminum trays (Waters company, USA), each of which was covered with a lid to seal. The reference tray was a blank T zero aluminum tray. The temperature was decreased to 0° C., kept at 0° C. for 5 min, and then increased at a rate of 10° C./min.

(34) Result: the DSC thermograms of the above four samples were shown in FIG. 3.

(35) FIGS. 3(A)-(C) showed the DSC thermograms of mPEG.sub.20000, cholesterol and coumarin-6. As seen from the figure, mPEG.sub.20000, cholesterol and coumarin-6 had a melting peak at 68.21° C., 149.98° C. and 208.29° C., respectively.

(36) FIG. 3(D) showed the DSC thermogram of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol. In the figure, the melting peaks of mPEG.sub.20000 and cholesterol were still present, indicating that PEG.sub.20000 and cholesterol were not entrapped into nanoparticles, and instead were involved in the modification of nanoparticles on the surface; the melting peak of coumarin-6 disappeared, indicating that coumarin-6 was encapsulated into a nanoparticle. In addition, it could be seen from the figure that poly(n-butyl cyanoacrylate) was crosslinked from about 300° C., and had a significant endothermic peak at 316.45° C.

Example 3 Determination of the Cholesterol Concentration on the Surface of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(37) (1) Method: Cholesterol Quantification Kit (Sigma-Aldrich, St. Louis, Mo., USA)) was used to determine the mass of cholesterol on the nanoparticle surface. The average number (N) of nanoparticles was calculated by the following formula (Olivier et al., 2002):

(38) $N=\frac{6\times W\times {10}^{-3}}{\pi \times {\left(D\times {10}^{-7}\right)}^3\times \rho }$

(39) wherein, W represents the mass of nanoparticles, D represents the number of nanoparticles calculated by the average particle size of nanoparticles, and p represents density, i.e. the mass of nanoparticles in a unit volume, which is 1.1 g/cm.sup.3.

(40) The cholesterol concentration on the nanoparticle surface was calculated by the following formula:

(41) the cholesterol concentration on the nanoparticle surface=the mass of cholesterol on the nanoparticle surface/the average number of nanoparticles.

(42) (2) Result: as calculated, the cholesterol concentration was (8.5±0.4)×10.sup.7 ng/nanoparticle on the surface of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol.

Example 4. Study on the In Vitro Release of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(43) (1) Method: a suspension (1 ml) of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol was placed in a dialysis bag (MWCO: 20000, USA), and the dialysis bag was placed in 200 ml pH7.4 phosphate-buffered saline (PBS), under stirring at 100 rpm in dark in a 37° C. thermostatic water bath. The release medium (200 μl) was taken at different time points, and meanwhile PBS was supplemented. The concentration of coumarin-6 in the release medium was determined by Fluorescence/Chemiluminescence Analyzer, with blank PBS used as control, and for each time point, 6 samples were determined in parallel. By the same method, the in vitro release curves were determined for common nanoparticles loaded with coumarin-6, nanoparticles with single modification by cholesterol and PBCA nanoparticles with single modification by PEG.sub.20000, as control. (2) Result: the release curves for the samples were shown in FIGS. 4 (A) and 4 (B).

(44) FIG. 4 (A) showed the comparison of in vitro release behavior of common nanoparticles loaded with coumarin-6, nanoparticles with single modification by cholesterol and nanoparticles with dual modifications by PEG.sub.20000-cholesterol. As seen from the figure, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol were significantly different from the common nanoparticles in terms of in vitro release behavior (f.sub.2=25.89, <50)

(45) (f.sub.2 equation is a common criterion for evaluating the release behavior of two formulations, and its formula is as follows:

(46) $f_2=50\log \left\{{\left[1+\frac{1}{n}\underset{t=1}{\overset{n}{.Math.}}{\left(R_t-T_t\right)}^2\right]}^{-0.5}\times 100\right\}$

(47) wherein, R.sub.t represents the release degree of a reference sample at t time; T.sub.t represents the release degree of a test sample at t time; and n is the number of sampling points.

(48) As prescribed by FDA, when f.sub.2 is between 50 and 100, it is believed that the two formulations are not different in terms of drug release behavior under the same drug release conditions, and when f.sub.2 is less than 50, it is believed that the two are significantly different.)

(49) The common nanoparticles had the release finished within 24 h, and exhibited a high release degree in a short time. The nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a release time of above 48 h, and no burst release occurred in a short time. On one hand, it indicated that coumarin-6 was substantively encapsulated into nanoparticles with dual modifications, and on the other hand, it indicated that nanoparticles with dual modifications had a significant sustained-release property. As compared with the nanoparticles with single modification by cholesterol, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a lower release degree within the same period, indicating a more stable release behavior in vitro.

(50) The result showed that as compared with the unmodified common nanoparticles or the nanoparticles with single modification by cholesterol, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a significant sustained-release property and a better release stability.

(51) FIG. 4 (B) showed the comparison of the in vitro release behavior of common nanoparticles, nanoparticles with single modification by PEG.sub.20000 and nanoparticles with dual modifications by PEG.sub.20000-cholesterol, all of which were loaded with coumarin-6. As seen from the figure, in terms of in vitro release behavior, the nanoparticles with single modification by PEG.sub.20000 were significantly different from the common nanoparticles (f.sub.2=29.69, <50), and were not significantly different from the nanoparticles with dual modifications by PEG.sub.20000-cholesterol (f.sub.2=73.93, >50); however, 12 h later, the nanoparticles with dual modification had a better sustained-release property in vitro. The result showed that as compared with the unmodified nanoparticles and the nanoparticles with single modification by PEG.sub.20000, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a more significant sustained-release property.

Example 5. Study on the Pharmacokinetics of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(52) (1) Experimental purpose: to study the sustained-release property of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in animal.

(53) (2) Method: 6- to 8-week old male Wistar rats were randomly divided into 3 groups, with 6 rats for each group. The rats were anesthetized by intraperitoneal injection of a pentobarbital solution (40 mg/kg), and jugular vein catheterization was performed. The rats were recovered for at least 12 h after the operation, and were subjected to tail vein injection with an equimolar amount of a coumarin-6 solution (a free drug), coumarin-6-loaded nanoparticles with single modification by cholesterol and PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol as prepared in Example 1, respectively. At different time points (5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h) after administration, blood (200 μl) was collected at the site where jugular vein catheterization was performed. After treatment, the content of coumarin-6 in the blood was determined.

(54) (3) Result: FIG. 5 showed the blood concentration—time curves of various samples. In the figure, the ordinate represents the concentration of coumarin-6 in blood, and the abscissa represents the time. As seen from the figure, different samples had significantly different blood concentration—time curves. After the administration of the free drug solution, the drug could be detected in blood only within 4 h, the release time was short, and there was no sustained-release effect; after administration of the nanoparticles with single modification by cholesterol, the release time was 8 h, longer than that of the free drug solution; the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol (CLS-PEG NPs) could sustain the release for 12 h in vivo, and had a longer release time and a higher blood concentration as compared with the free drug solution and the nanoparticles with single modification by cholesterol (CLS NPs). The pharmacokinetic parameters were shown in Table 1.

(55) TABLE-US-00001 TABLE 1 Coumarin-6 Parameter Free drug CLS NPs CLS-PEG NPs C.sub.0 (ng/ml) 828.4 ± 139.0 853.5 ± 143.8 906.6 ± 175.5 t.sub.1/2, λ.sub.z (min) 60.1 ± 24.0 106.8 ± 21.7** 275.6 ± 117.4** AUC.sub.0-t 15.8 ± 2.4   22.9 ± 3.0**  35.2 ± 4.4** (μg .Math. min/ml) AUC.sub.0-∞ 16.0 ± 2.3   23.3 ± 3.2**  39.9 ± 5.3** (μg .Math. min/ml) V.sub.d, λ.sub.z (l) 0.6 ± 0.3  0.6 ± 0.1  1.0 ± 0.4* CL (ml/min) 6.4 ± 0.9  4.4 ± 0.6**  2.5 ± 0.4** F (%) — 144.9 212.5 *p < 0.05, **p < 0.01 vs. free-form drug group

(56) The result showed that as compared with the free drug solution and the nanoparticles with single modification by cholesterol, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a significantly longer release time, a higher blood concentration, a better release behavior and a higher bioavailability in animal.

Example 6. Study on the Brain Tissue Distribution of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(57) Research objective: to study the brain-targeting property of PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in animal.

(58) Experiment 1

(59) (1) Method: 6- to 8-week old male Wistar rats were randomly divided into 3 groups, with 21 rats for each group. The rats were subjected to tail vein injection with an equimolar amount of a coumarin-6 solution (a free drug), coumarin-6-loaded nanoparticles with single modification by cholesterol and PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol as prepared in Example 1, respectively. The rats were killed at different time points (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h) after administration, brain tissues were taken, and the content of coumarin-6 in brain was determined.

(60) (2) Result: FIG. 6 (A) showed the concentration of coumarin-6 at different time points in the brain tissues of rats from different groups. As seen from the figure, the rats injected with the free drug solution had a low content of coumarin-6 in the brain tissue, which did not exceed 50 ng/g brain tissue, and no coumarin-6 was detected in the brain tissue 8 h after administration, indicating that free coumarin-6 had a low BBB permeability, and did not have a sustained-release property. The rats injected with coumarin-6-loaded nanoparticles with single modification by cholesterol had a much higher content of coumarin-6 in the brain tissue than that of the rats injected with free coumarin-6, however, no coumarin-6 was detected in the brain 8 h after administration. The rats injected with coumarin-6-loaded nanoparticles with dual modifications by PEG.sub.20000-cholesterol had the content of coumarin-6 in the brain tissue further enhanced, as compared with the rats injected with nanoparticles with single modification by cholesterol, and coumarin-6 could be still detected in the brain 12 h after administration.

(61) The result showed that as compared with free coumarin-6 and the coumarin-6-loaded nanoparticles with single modification by cholesterol, the coumarin-6-loaded nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a good BBB permeability, could significantly promote the passage of drug through blood-brain barrier, and had a sustained-release process.

(62) Experiment 2

(63) (1) Method: 6- to 8-week old male Wistar rats were randomly divided into 4 groups, with 3 rats for each group. The rats were subjected to tail vein injection with an equimolar amount of a coumarin-6 solution (a free drug), common nanoparticles loaded with coumarin-6, coumarin-6-loaded PBCA nanoparticles with single modification by PEG.sub.20000 and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, respectively. The rats were killed 30 min after administration, the brain tissues were taken, and the content of coumarin-6 in brain was determined.

(64) (2) Result: FIG. 6 (B) showed the content of coumarin-6 in the brain tissue of rats, which from left to right was the content of coumarin-6 in the brain tissue of rats injected with a free drug solution, common nanoparticles, PBCA nanoparticles with single modification by PEG.sub.20000 and PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, respectively.

(65) As shown in the figure, as compared with the free drug solution and the common PBCA nanoparticles, the PBCA nanoparticles with single modification by PEG.sub.20000 had the content of coumarin-6 in the brain tissue significantly increased (P<0.01). However, the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol according to the invention could have the content of coumarin-6 in the brain tissue significantly increased, as compared with the PBCA nanoparticles with single modification by PEG.sub.20000 (P<0.01). The result showed that as compared with free coumarin-6 and the coumarin-6-loaded nanoparticles with single modification by PEG.sub.20000, the coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a good BBB permeability, and could promote the passage of drug through blood-brain barrier.

Example 7. Study on the Tissue Distribution of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(66) (1) Experimental purpose: to study the tissue distribution of PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol in animal, and to study the toxicity in vivo.

(67) (2) Method: 6- to 8-week old male Wistar rats were randomly divided into 3 groups, with 21 rats for each group. The rats were subjected to tail vein injection with an equimolar amount of a coumarin-6 solution (a free drug), coumarin-6-loaded nanoparticles with single modification by cholesterol and PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol as prepared in Example 1, respectively. The rats were killed at different time points (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h) after administration, hearts, livers, spleens, lungs, and kidney tissues were taken, and the content of coumarin-6 therein was determined, respectively.

(68) (3) Result: FIG. 7 showed the concentration of coumarin-6 at different time points in the tissues of rats from different groups. As seen from the figure, the nanoparticles with dual modifications by PEG.sub.20000-cholesterol were not significantly accumulated in various tissues and organs as compared with the nanoparticles with single modification by cholesterol, and had a significantly lower content in the liver and spleen as compared with the nanoparticles with single modification. The result showed that the coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had better safety in vivo.

Example 8. Study on the In Vitro Safety of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(69) (1) Experimental purpose: to study the cytotoxicity of PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol

(70) (2) Experimental method: MTT method was used to determine the cell viability.

(71) The particular steps were as followed: bEnd.3 cells (mouse brain microvascular endothelial cells) in the logarithmic growth phase were digested and counted, and then seeded to a 96-well plate at a density of 5×10.sup.4/well. After incubation in a 5% CO.sub.2, 37° C. incubator for 24 h, the old medium was pipetted off, and media (200 μL) containing cholesterol, PEG.sub.20000 and PBCA at a series of concentrations (5, 10, 20, 50, 100, 200, 500 and 1000 μg/ml), and media (200 μL) containing blank PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol at a series of concentrations (1, 2, 5, 10, 20, 50, 100 and 200 μg/ml) were added, respectively. After further incubation for 24 h and 48 h, respectively, the old medium was pipetted off, and a medium containing 0.5 mg/ml MTT was added to each well. After further incubation for 4 h, the medium was pipetted off, and washing with PBS was performed twice. DMSO (200 μl) was added to dissolve the formazan crystal, and shaking was performed in dark for 10 min; and then the OD value of each well at 490 nm was determined by ELISA instrument. The OD value of the cells incubated without the addition of drug was used as control (100%), and the cell viability for each drug groups were calculated, so as to study the proliferation state of cells, wherein 5 parallel wells were set for each group.

(72) (3) Experimental result: FIG. 8 showed the cell viability of bEnd.3 cells after administration of (A) cholesterol, (B) PEG.sub.20000, (C) PBCA, (D) blank PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, and (E) coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, respectively. In the figure, the abscissa represents the administration concentration, and the ordinate represents the cell viability.

(73) As shown in the figure, cholesterol, PEG.sub.20000, PBCA, blank nanoparticles and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had no significant cytotoxicity for bEnd.3 cells within the experimental concentration range (cell viability >80%). The result showed that the blank nanoparticles and the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had good safety in vitro.

Example 9. Study on the Pharmacokinetics of Docetaxel-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(74) (1) Experimental purpose: docetaxel, a medicine for treating breast cancer and non-small cell lung cancer, was generally administered via intravenous drop infusion in clinic, and could not penetrate across blood-brain barrier easily.

(75) The purpose of this experiment was to load the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol according to the invention with a drug that could not penetrate across blood-brain barrier easily, so as to observe the sustained-release of the drug after intravenous injection, wherein docetaxel was used as an example.

(76) (2) Method: by the method as described in Example 1, docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol were prepared and characterized. The docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a narrow particle size distribution, an average particle size of 200.7 nm, a PDI of 0.122, and a Zeta potential of −2.11 mV; and the entrapment efficiency was 98.8%, and the drug loading rate was 2.13%.

(77) 6- to 8-week old male Wistar rats were subjected to jugular vein catheterization. The rats were recovered for at least 12 h after the operation, and then was randomly divided into 2 groups, with 6 rats for each group. The rats were administered with a docetaxel solution (a free drug) and docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, respectively. Blood (200 μl) was collected at the site where jugular vein catheterization was performed, at different time points after administration. After treatment, the content of docetaxel in the blood was determined by HPLC.

(78) (3) Result: FIG. 9 showed the blood concentration—time curves of a free drug solution of docetaxel and docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol after intravenous administration in rats. As seen from the figure, they were significantly different. The docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol could achieve the sustained-release in vivo for 8 h, whereas the free drug solution could not be detected in the blood 2 h after administration the free drug. Moreover, the docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a higher blood concentration in vivo.

Example 10. Study on the Brain Tissue Distribution of Docetaxel-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol

(79) (1) Experimental purpose: the purpose of this experiment was to load the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol according to the invention with a drug that could not penetrate BBB easily, so as to observe the entry of the drug into the brain tissue via penetration across BBB after intravenous injection, wherein docetaxel was used as an example. If the concentration of docetaxel was increased in the brain tissue, it indicated that the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol according to the invention had brain-targeting property, and could deliver a drug that could not easily penetrate across BBB to the brain tissue.

(80) (2) Method: 6- to 8-week old male Wistar rats were subjected to jugular vein catheterization. The rats were recovered for at least 12 h after the operation, and then was randomly divided into 2 groups, with 6 rats for each group. The rats were subjected to tail vein injection with a docetaxel solution (a free drug), and docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, respectively. The rats were killed at different time points after administration, respectively, and the brain tissues were taken, washed with physiological saline and weighed. After treatment, the content of docetaxel in the brain tissue was determined by HPLC.

(81) (3) Result: FIG. 10 showed the content of docetaxel in the brain tissue of rats at different time points after intravenous administration. As shown in the figure, after intravenous administration, docetaxel (a free drug) could hardly penetrate across blood-brain barrier, and could not be detected in the brain; however, the docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol could penetrate across blood-brain barrier, so as to significantly increase the content of docetaxel in the brain tissue, and the drug could be stilled detected in the brain tissue 8 h after administration. The result showed that the PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol according to the invention could promote the penetration of docetaxel across blood-brain barrier, and could achieve the sustained-release of drug in the brain.

Example 11. The Inhibitory Effect of Docetaxel-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Cholesterol on Proliferation of Tumor Cells In Vitro

(82) (1) Experimental purpose: the purpose of this experimental is to observe the pharmacodynamical effect of a drug loaded to PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol by using docetaxel as an example.

(83) (2) Method: MTT method was used to determine the proliferation state of tumor cells. The particular steps were as followed: 4T1 cells (mouse breast cancer cells) in the logarithmic growth phase were digested and counted, and then seeded to a 96-well plate at a density of 5×10.sup.4/well. After incubation in a 5% CO.sub.2, 37° C. incubator for 24 h, the old medium was pipetted off, and media (200 μL) containing docetaxel (a free drug) and docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol (the administration concentration was calculated based on the amount of the loaded docetaxel) at a series of concentrations (0.0001, 0.001, 0.01, 1, 10 and 100 nmol/ml) were added, respectively. After further incubation for 24 h and 48 h, respectively, the old medium was pipetted off, and a medium containing 0.5 mg/ml MTT was added to each well. After further incubation for 4 h, the medium was pipetted off, and washing with PBS was performed twice. DMSO (200 μl) was added to dissolve the formazan crystal, and shaking was performed in dark for 10 min; and then the OD value of each well at 490 nm was determined by ELISA instrument. The OD value of the cells incubated without the addition of drug was used as control (100%), and the cell viability for each drug groups were calculated, so as to study the proliferation state of tumor cells, wherein 5 parallel wells were set for each group.

(84) (3) Result: FIG. 11 showed the cell viability of mouse breast cancer 4T1 cells after administration of docetaxel (a free drug) and docetaxel-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-cholesterol, respectively. In the figure, the abscissa represents the administration concentration (calculated as the concentration of docetaxel), and the ordinate represents the cell viability. Fig. (A) and Fig. (B) showed the results of incubation for 24 h and 48 h after administration, respectively.

(85) As shown in the figure, after administration of docetaxel-loaded nanoparticles with dual modifications by PEG.sub.20000-cholesterol at a concentration of 0.0001 nmol/ml for 24 h, the cell viability of the tumor cells could be decreased to about 90%, whereas after the administration of docetaxel (a free drug) for 24 h, the tumor cells had no significant change in the cell viability. The docetaxel-loaded nanoparticles with dual modifications by PEG.sub.20000-cholesterol had a stronger inhibitory effect on the proliferation of the tumor cells than free docetaxel at the administration concentration for the same incubation time, and were dose-dependent and time-dependent to some extent. A higher administration concentration and a longer the incubation time indicated a stronger inhibitory effect on the proliferation of the tumor cells.

(86) The result showed that as compared with free docetaxel, the docetaxel-loaded nanoparticles with dual modifications by PEG.sub.20000-cholesterol could inhibit the proliferation of the tumor cells better. As compared with the free drug solution, the drug, which was loaded to the PBCA nanoparticles with dual modification according to the invention had a better therapeutic effect, instead of a reduced efficacy.

Example 12. Preparation and Evaluation of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Leucine

(87) (1) Preparation method: the stabilizer Dex70 (1%, w/v) and mPEG.sub.20000 (1.5%, w/v) were dissolved in a HCl medium (pH 1.0), and then BCA monomers (1%, v/v) were slowly added dropwise at room temperature under magnetic stirring. After stirring at 500 rpm for 4 h, coumarin-6 (1%, w/v) was added, and the stirring was performed at 750 rpm for 2.5 h. Then, the system was neutralized with NaOH to pH of 6-7, and was further stirred for 1 h so that BCA monomers were polymerized sufficiently, thereby obtaining PEG-PBCA nanoparticles. After filtration through a filtration membrane (0.45 μm), the nanoparticles obtained were lyophilized, and then re-dissolved in PBS, and mixed homogeneously for 30 min. Leucine (1%, w/v) was added, and incubation was performed for 0.5 h. After filtration and lyophilization, coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-leucine were obtained and stored in a drier.

(88) By the method above, common unmodified PBCA nanoparticles loaded with coumarin-6 were also prepared, as control.

(89) (2) Measurement and Result of particle size and potential: by the method as described in Example 1, the coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-leucine were measured for particle size and Zeta potential. The nanoparticles had an average particle size of 167.3 nm, a PDI of 0.138, and a Zeta potential of −9.63 mV.

(90) (3) Measurement and Result of entrapment efficiency and drug loading rate: as measured by the method as described in Example 1, the entrapment efficiency and drug loading rate of coumarin-6 were 98.1% and 2.04%, respectively.

Example 13. Study on the In Vitro Release of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Leucine

(91) (1) Method: PBCA nanoparticles with dual modifications by PEG.sub.20000-leucine (1 ml) were placed in a dialysis bag (MWCO: 20000, USA), and the dialysis bag was placed in 200 ml pH7.4 phosphate-buffered saline (PBS), under stirring at 100 rpm in dark in a 37° C. thermostatic water bath. The release medium (200 μl) was taken at different time points within 24 h, and meanwhile PBS was supplemented. The concentration of coumarin-6 in the release medium was determined with blank PBS used as control. For each time point, 6 samples were determined in parallel. By the same method, unmodified PBCA nanoparticles loaded with coumarin-6 were determined.

(92) (2) Result: FIG. 12 showed the in vitro release curve of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-leucine, and the in vitro release curve of unmodified PBCA nanoparticles loaded with coumarin-6. As seen from the figure, the nanoparticles with dual modifications by PEG.sub.20000-leucine were significantly different from the common nanoparticles in terms of in vitro release behavior (f2=32.88, <50). As compared with the unmodified nanoparticles, the nanoparticles with dual modifications by PEG.sub.20000-leucine had a more significant sustained-release property, indicating a better stability, which was favorable for a better BBB permeability in vivo.

Example 14. Preparation and Evaluation of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Aspartic Acid

(93) (1) Preparation method: the stabilizer Dex70 (1%, w/v) and mPEG.sub.20000 (1.5%, w/v) were dissolved in a HCl medium (pH1.0), and then BCA monomers (1%, v/v) were slowly added dropwise at room temperature under magnetic stirring. After stirring at 500 rpm for 4 h, coumarin-6 (1%, w/v) was added, and the stirring was performed at 750 rpm for 2.5 h. Then, the system was neutralized with NaOH to pH of 6-7, and was further stirred for 1 h so that BCA monomers were sufficiently polymerized, thereby obtaining PEG-PBCA nanoparticle. After filtration through a filtration membrane (0.45 μm), the nanoparticles obtained were lyophilized, and then re-dissolved in PBS, and mixed homogeneously for 30 min. L-aspartic acid (1%, w/v) was added, and incubation was performed for 0.5 h. After filtration and lyophilization, PBCA nanoparticles with dual modifications by PEG.sub.20000-aspartic acid were obtained and stored in a drier.

(94) By the method above, common unmodified PBCA nanoparticles loaded with coumarin-6 were also prepared, as control.

(95) (2) Measurement and Result of particle size and potential: by the method as described in Example 1, coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-aspartic acid were measured for particle size and Zeta potential. The nanoparticles had an average particle size of 146.2 nm, a PDI of 0.119, and a Zeta potential of −6.78 mV.

(96) (3) Measurement and Result of entrapment efficiency and drug loading rate: as measured by the method as described in Example 1, the entrapment efficiency and drug loading rate of coumarin-6 were 96.9% and 1.98%, respectively.

Example 15. Study on the In Vitro Release of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Aspartic Acid

(97) (1) Method: coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-aspartic acid (1 ml) were placed in a dialysis bag (MWCO: 20000, USA), and the dialysis bag was placed in 200 ml pH7.4 phosphate-buffered saline (PBS), under stirring at 100 rpm in dark in a 37° C. thermostatic water bath. The release medium (200 μl) was taken at different time points within 24 h, and meanwhile PBS was supplemented. The concentration of coumarin-6 in the release medium was determined with blank PBS used as control. By the same method, the unmodified PBCA nanoparticles loaded with coumarin-6 were determined.

(98) (2) Result: FIG. 13 showed the in vitro release curve of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-aspartic acid, and the in vitro release curve of unmodified PBCA nanoparticles loaded with coumarin-6. As seen from the figure, the nanoparticles with dual modifications by PEG.sub.20000-aspartic acid were significantly different from the common nanoparticles (f2=34.32, <50). As compared with the unmodified nanoparticles, nanoparticles with dual modifications by PEG.sub.20000-aspartic acid had a more significant sustained-release property, indicating a better stability, which was favorable for a better BBB permeability in vivo.

Example 16. Preparation and Evaluation of Coumarin-6-Loaded PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Lecithin

(99) (1) Preparation method: the stabilizer Dex70 (1%, w/v) and mPEG.sub.20000 (1.5%, w/v) were dissolved in a HCl medium (pH 1.0), and then BCA monomers (1%, v/v) were slowly added dropwise at room temperature under magnetic stirring. After stirring at 500 rpm for 4 h, coumarin-6 (1%, w/v) was added, and the stirring was performed at 750 rpm for 2.5 h. Then, the system was neutralized with NaOH to pH of 6-7, and was further stirred for 1 h so that BCA monomers were sufficiently polymerized, thereby obtaining PEG-PBCA nanoparticle. After filtration through a filtration membrane (0.45 μm), the nanoparticles obtained were lyophilized, and then re-dissolved in PBS and mixed homogeneously for 30 min. Lecithin (1%, w/v) was added, and incubation was performed for 0.5 h. After filtration and lyophilization, coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin were obtained and stored in a drier.

(100) By the method above, common unmodified PBCA nanoparticles loaded with coumarin-6 were prepared, as control.

(101) (2) Measurement and Result of particle size and potential: by the method as described in Example 1, coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin were measured for particle size and Zeta potential. The nanoparticles had an average particle size of 344.2 nm, a PDI of 0.252, and a Zeta potential of −37.3 mV.

(102) (3) Measurement and Result of entrapment efficiency and drug loading rate: as measured by the method described in Example 1, the entrapment efficiency and drug loading rate of coumarin-6 were 97.4% and 1.95%, respectively.

Example 17. Study on In Vitro Release of PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Lecithin

(103) (1) Method: coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin (1 ml) were placed in a dialysis bag (MWCO: 20000, USA), and the dialysis bag was placed in 200 ml pH7.4 phosphate-buffered saline (PBS), under stirring at 100 rpm in dark in a 37° C. thermostatic water bath. The release medium (200 μl) was taken at different time points within 24 h, and meanwhile PBS was supplemented. B The concentration of coumarin-6 in the release medium was determined with blank PBS used as control. At each time point, 6 samples were determined in parallel.

(104) (2) Result: FIG. 14 showed the in vitro release curve of coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin, and the in vitro release curve of unmodified PBCA nanoparticles loaded with coumarin-6. As shown in the figure, the nanoparticles with dual modifications by PEG.sub.20000-lecithin were significantly different from the common nanoparticles in terms of in vitro release behavior (f2=30.98, <50). As compared with the unmodified nanoparticles, the nanoparticles with dual modifications by PEG.sub.20000-lecithin had a more significant sustained-release property, indicating a better stability, which was favorable for a better BBB permeability in vivo.

Example 18. Study on the Brain Tissue Distribution of PBCA Nanoparticles with Dual Modifications by Different Modifiers

(105) (1) Experimental purpose: to study the brain-targeting property of PBCA nanoparticles with dual modifications by different modifiers in animal.

(106) (2) Method: 6- to 8-week-old male Wistar rats were randomly divided into 5 groups, with 3 rats for each group. The rats were subjected to tail vein injection with an equimolar amount of a coumarin-6 solution (a free drug), common nanoparticles loaded with coumarin-6, coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-leucine, coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-aspartic acid and coumarin-6-loaded PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin, respectively. The rats were killed 30 min after administration, the brain tissues was taken, and the content of coumarin-6 in brain was determined.

(107) (3) Result: FIG. 15 showed the content of coumarin-6 in the brain tissue of rats, which from left to right was the content of coumarin-6 in the brain tissue of the rats injected with a free drug solution, common nanoparticles, PBCA nanoparticles with dual modifications by PEG.sub.20000-leucine, PBCA nanoparticles with dual modifications by PEG.sub.20000-aspartic acid and PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin, respectively.

(108) As shown in the figure, as compared with the free drug solution and the common PBCA nanoparticles, the three nanoparticles with dual modification according to the invention might have a significantly increased content of coumarin-6 in the brain tissue (P<0.01). The result showed that all of the three PBCA nanoparticles with dual modification had a good BBB permeability, and could significantly promote the passage of drug through blood-brain barrier

Example 19. Preparation of PBCA Nanoparticles with Dual Modifications by PEG.SUB.20000.-Lecithin

(109) The stabilizer Dex70 (1%, w/v) and mPEG.sub.20000 (1.5%, w/v) were dissolved in a HCl medium (pH 1.0), and then BCA monomers (1%, v/v) were slowly added dropwise at room temperature under magnetic stirring. After stirring at 500 rpm for 4 h, the system was neutralized with NaOH to pH of 6-7, and was further stirred for 1 h so that BCA monomers were sufficiently polymerized, thereby obtaining PEG-PBCA nanoparticles. After filtration through a filtration membrane (0.45 μm), the nanoparticles obtained were lyophilized, and then were re-dissolved in PBS, and mixed homogeneously for 30 min. Lecithin (1%, w/v) was added, and incubation was performed for 0.5 h. After filtration and lyophilization, PBCA nanoparticles with dual modifications by PEG.sub.20000-lecithin were obtained and stored in a drier.

(110) As seen from the experimental results in the Examples above, the PBCA nanoparticles with dual modification according to the invention had good BBB permeability. By loading the PBCA nanoparticles with dual modification according to the invention with a drug that could not penetrate across the BBB easily, it could significantly increase the content of the drug in the brain tissue of animal, and bring about a better sustained-release behavior, which was favorable for enhancing safety and efficacy of drugs. A drug delivery system comprising the drug-loaded nanoparticle could be used in manufacture of a brain-targeting formulation, and had a good application value in the diagnosis, prevention and/or treatment of a central nervous system disease (including but not limited to brain tumor).

(111) Although the embodiments of the invention have been described in detail, a person skilled in the art would understand that according to all the disclosed teachings, details can be amended and modified, and these alterations all fall into the protection scope of the invention. The scope of the invention is defined by the attached claims and any equivalent thereof.