Patent Publication Number: US-2017368207-A1

Title: Self-assembled nanostructure and method for preparing the same

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the priority benefit of Taiwan application serial no. 105119828, filed on Jun. 24, 2016. The entirety of the above-mentioned patent application is hereby incorporated by reference herein and made a part of this specification. 
     BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The invention relates to a nanostructure and a method for preparing the same, particularly to a self-assembled nanostructure having contrasting ability and a method for preparing the same. 
     Description of Related Art 
     Magnetic resonance imaging (MRI), because of its non-invasiveness and non-radiation, is an important tool for diagnosing a disease and identifying the stage of development of the disease. In MRI, contrast agents are usually utilized for improving resolution of a tissue image. 
     In the prior art, a metal-containing compound, such as a superparamagnetic iron oxide or a derivative thereof, is often used as a contrast agent. However, such iron oxide is likely to accumulate in tissue of an organism due to its longer half-life, and the issue about the occurrence of chronic toxicity over time is raised. 
     SUMMARY OF THE INVENTION 
     In view of the above, the invention provides a self-assembled nanostructure and a method for preparing the same, and the prepared self-assembled nanostructure has excellent contrasting ability and has no biological toxicity. 
     A self-assembled nanostructure including an amphiphilic chitosan and a contrast agent compound is provided. The contrast agent compound is grafted to the amphiphilic chitosan. 
     In an embodiment of the invention, the amphiphilic chitosan includes carboxymethyl hexanoyl chitosan (CHC), deoxycholic acid modified carboxymethylated chitosan (DCMC), lauroyl sulfated chitosan (LSC) or methylpyrrolidone chitosan (MPC). 
     In an embodiment of the invention, the contrast agent compound includes a magnetic resonance imaging (MRI) contrast agent compound. 
     In an embodiment of the invention, the MRI contrast agent compound includes gadodiamide, gadopentetate dimeglumine, gadoterate meglumine or a combination thereof. 
     In an embodiment of the invention, the amphiphilic chitosan has a weight average molecular weight of about 1,000 to 60,000. 
     In an embodiment of the invention, the self-assembled nanostructure is entangled to form a spherical nanoparticle having a particle size of about 5 nm to 500 nm. 
     In an embodiment of the invention, the contrast agent compound is in an amount of about 1 part by weight to 30 parts by weight based on 100 parts by weight of the self-assembled nanostructure. 
     In an embodiment of the invention, the self-assembled nanostructure encapsulates a fluorescence contrast agent compound. 
     In an embodiment of the invention, the fluorescence contrast agent compound includes fluorescein isothiocyanate (FITC), C 83 H 95 N 13 O 23 S 2 Zn 2  or C 57 H 58 N 14 O 18 SZn 2 . 
     In an embodiment of the invention, the self-assembled nanostructure encapsulates a drug molecule. 
     In an embodiment of the invention, the drug molecule includes cis-diamminedichloridoplatinum(II) or a derivative thereof, gemcitabine or a derivative thereof; or demethoxycurcumin or a derivative thereof. 
     The invention provides a method for preparing a self-assembled nanostructure, including the following steps. An amphiphilic chitosan-containing solution is prepared. A contrast agent compound is added to the solution. A cross-linking agent is added to the solution. A cross-linking reaction is caused between the amphiphilic chitosan and the contrast agent compound. The solution is purified. The solution is dried. 
     In an embodiment of the invention, the cross-linking agent includes N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC•HCl), N-hydroxysuccinimide (NHS) or a combination thereof. 
     In an embodiment of the invention, a reaction temperature of the cross-linking reaction ranges from about 4° C. to 60° C. 
     In an embodiment of the invention, a reaction time of the cross-linking reaction ranges from about 4 hours to 24 hours. 
     In an embodiment of the invention, a surface of the self-assembled nanostructure is further modified with a protein molecule having specificity. 
     In an embodiment of the invention, the protein molecule includes an epidermal growth factor receptor (EGFR) or CD133 protein. 
     In an embodiment of the invention, in preparing the amphiphilic chitosan-containing solution, the content of the amphiphilic chitosan is about 0.05 part by weight to 1 part by weight based on 100 parts by weight of the solution. 
     In an embodiment of the invention, the contrast agent compound includes a magnetic resonance imaging (MRI) contrast agent compound. 
     In an embodiment of the invention, the MRI contrast agent compound includes gadodiamide, gadopentetate dimeglumine, gadoterate meglumine or a combination thereof. 
     Based on the above, in the self-assembled nanostructure of the invention, the contrast agent compound is grafted to the amphiphilic chitosan, and due to chemical bonding between these two, the contrast agent compound is prevented from being released into tissue of an organism to cause any harm to the organism. In addition, the bonding between the amphiphilic chitosan and the contrast agent compound has a synergistic effect, which further improves the contrasting ability of the contrast agent compound. In addition, the self-assembled nanostructure of the invention may be modified with a protein molecule having specificity, so that the self-assembled nanostructure of the invention exhibits recognition specificity, and efficiency in drug administration is improved. 
     To make the above features and advantages of the invention more comprehensible, embodiments accompanied with drawings are explained in detail as follows. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic diagram illustrating a self-assembled nanostructure according to an embodiment of the invention. 
         FIG. 2  is a schematic diagram illustrating encapsulating a drug molecule by a self-assembled nanostructure according to an embodiment of the invention. 
         FIG. 3  is a schematic structural diagram illustrating encapsulating a drug molecule and a fluorescence contrast agent compound by a self-assembled nanostructure according to another embodiment of the invention. 
         FIG. 4  is a flowchart illustrating a method for preparing a self-assembled nanostructure according to an embodiment of the invention. 
         FIG. 5  is an infrared spectrum of gadodiamide, carboxymethyl hexanoyl chitosan and carboxymethyl hexanoyl chitosan having gadodiamide grafted thereto according to an embodiment of the invention. 
         FIG. 6  illustrates a comparison of encapsulation rates of a self-assembled nanostructure for different drug molecules according to an embodiment of the invention. 
         FIG. 7  illustrates a comparison of cell endocytosis amounts between a self-assembled nanostructure modified with an epidermal growth factor receptor (EGFR) and a self-assembled nanostructure without being modified with an EGFR. 
     
    
    
     DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS 
       FIG. 1  is a schematic diagram illustrating a self-assembled nanostructure according to an embodiment of the invention. 
     Referring to  FIG. 1 , a self-assembled nanostructure  100  of the invention includes an amphiphilic chitosan  102  and a contrast agent compound  104 , wherein the contrast agent compound  104  is grafted to the amphiphilic chitosan  102 . In other words, there is chemical bonding instead of physical adsorption between the amphiphilic chitosan  102  and the contrast agent compound  104 . As shown in  FIG. 1 , in an embodiment, in an appropriate environment (e.g., an aqueous solution), the amphiphilic chitosan  102  having the contrast agent compound  104  grafted thereto is entangled and self-assembles into a spherical nanoparticle. More specifically, the amphiphilic chitosan  102  of the invention has both hydrophilic and hydrophobic functional groups, and has a self-assembly property in aqueous solution, and is therefore capable of forming the self-assembled nanostructure  100  of the invention by having the contrast agent compound  104  grafted thereto. 
     In an embodiment, the amphiphilic chitosan  102  of the invention is, for example, carboxymethyl hexanoyl chitosan (CHC), deoxycholic acid modified carboxymethylated chitosan (DCMC), lauroyl sulfated chitosan (LSC) or methylpyrrolidone chitosan (MPC). The amphiphilic chitosan  102  of the invention, by having the contrast agent compound  104  grafted thereto, has a critical micelle concentration of between about 0.010 mg/ml and 0.030 mg/ml, such as about 0.026 mg/ml to 0.028 mg/ml. That is, the amphiphilic chitosan  102  of the invention, by having the contrast agent compound  104  grafted thereto, is capable of self-assembly at extremely low concentration to form the self-assembled nanostructure  100  of the invention. 
     The contrast agent compound  104  includes a magnetic resonance imaging (MRI) contrast agent compound. In an embodiment, the MRI contrast agent compound is, for example, a gadolinium complex, a manganese complex, a chromium complex or an iron complex. The gadolinium complex is, for example, gadodiamide, gadopentetate dimeglumine, gadoterate meglumine or a combination thereof. In an embodiment, as shown in  FIG. 1 , the contrast agent compound  104  of the invention is located inside the self-assembled nanostructure  100 . Accordingly, the probability of the contrast agent compound  104  contacting an organism is reduced. Therefore, the self-assembled nanostructure  100  of the invention has extremely low toxicity. In  FIG. 1 , all the contrast agent compounds  104  are located inside the self-assembled nanostructure  100 . However, the invention is not limited thereto. In other embodiments (not illustrated), some of the contrast agent compounds  104  may be located outside the self-assembled nanostructure  100 . 
     In an embodiment, the amphiphilic chitosan  102  has a weight average molecular weight of about 1,000 to 60,000, such as about 5,000 to 60,000, about 10,000 to 50,000 or about 20,000 to 40,000. The weight average molecular weight of the amphiphilic chitosan  102  affects the particle size of the self-assembled nanostructure  100 . In an embodiment, the self-assembled nanostructure  100  of the invention has a particle size of about 5 nm to 500 nm, such as about 10 nm to 500 nm, about 50 nm to 400 nm or about 100 nm to 300 nm. If the particle size of the self-assembled nanostructure  100  is smaller than about 5 nm, the self-assembled nanostructure  100  is easily carried away by the blood. If the particle size of the self-assembled nanostructure  100  is larger than about 500 nm, the self-assembled nanostructure  100  has difficulty entering tumor cells in the organism. 
     In an embodiment, based on 100 parts by weight of the self-assembled nanostructure  100 , the contrast agent compound  104  is in an amount of about 1 part by weight to 30 parts by weight, such as about 1 part by weight to 20 parts by weight, about 5 parts by weight to 20 parts by weight or about 10 parts by weight to 15 parts by weight. If the contrast agent compound  104  is in an amount of less than about 1 part by weight, the self-assembled nanostructure  100  has insufficient contrasting ability. If the contrast agent compound  104  is in an amount of more than about 30 parts by weight, the self-assembled nanostructure  100  has difficulty self-assembling into a spherical shape in aqueous solution. 
     The self-assembled nanostructure  100  of the invention can be used to encapsulate a drug molecule, so that the drug molecule is protected by the self-assembled nanostructure  100  during the transportation inside the organism. In addition, by means of the contrasting ability of the self-assembled nanostructure  100  of the invention, the position of the drug molecule encapsulated by the self-assembled nanostructure  100  in the organism is easily observed. 
       FIG. 2  is a schematic diagram illustrating encapsulating a drug molecule by a self-assembled nanostructure according to an embodiment of the invention.  FIG. 3  is a schematic structural diagram illustrating encapsulating a drug molecule and a fluorescence contrast agent compound by a self-assembled nanostructure according to another embodiment of the invention. 
     Referring to  FIG. 2  and  FIG. 3 , the self-assembled nanostructure  100  encapsulates a drug molecule  106 . More specifically, the entangled network structure of the self-assembled nanostructure  100  traps the drug molecule  106  therein. In an embodiment, the drug molecule is, for example, cis-diamminedichloridoplatinum(II) or a derivative thereof, gemcitabine or a derivative thereof, or demethoxycurcumin or a derivative thereof. It is noted that the drug molecule  106  is physically encapsulated in the self-assembled nanostructure  100 . That is, there is no chemical bonding between the drug molecule  106  and the self-assembled nanostructure  100 . Therefore, the drug molecule  106  is more easily released from the self-assembled nanostructure  100  so as to be absorbed by the organism. 
     In an embodiment, the self-assembled nanostructure  100  further encapsulates a fluorescence contrast agent compound  108  so as to further improve the contrasting ability, as shown in  FIG. 3 . More specifically, the entangled network structure of the self-assembled nanostructure  100  traps the fluorescence contrast agent compound  108  therein. In an embodiment, the fluorescence contrast agent compound  108  is, for example, fluorescein isothiocyanate (FITC), C 83 H 95 N 13 O 23 S 2 Zn 2  (e.g., PSVue™794) or C 57 H 58 N 14 O 18 SZn 2  (e.g., PSVue™480). 
     It is noted that the fluorescence contrast agent compound  108  is also physically encapsulated in the self-assembled nanostructure  100 . That is, there is no chemical bonding between the fluorescence contrast agent compound  108  and the self-assembled nanostructure  100 . 
     In an embodiment, the contrast agent compound of the invention is, for example, an FDA certified metal complex. Therefore, there is an extremely low probability of the self-assembled nanostructure of the invention causing serious allergic reaction in the human body. 
       FIG. 4  is a flowchart illustrating a method for preparing a self-assembled nanostructure according to an embodiment of the invention. 
     Referring to  FIG. 4 , in step S 100 , an amphiphilic chitosan-containing solution is first prepared. In an embodiment, the amphiphilic chitosan is, for example, carboxymethyl hexanoyl chitosan (CHC), deoxycholic acid modified carboxymethylated chitosan (DCMC), lauroyl sulfated chitosan (LSC) or methylpyrrolidone chitosan (MPC). In an embodiment, in preparing the amphiphilic chitosan-containing solution, based on 100 parts by weight of the solution, the content of the amphiphilic chitosan is about 0.05 part by weight to 1 part by weight, such as about 0.1 part by weight to 1 part by weight, about 0.15 part by weight to 0.85 part by weight or about 0.2 part by weight to 0.7 part by weight. 
     Next, in step S 102 , a contrast agent compound is added to the solution. In an embodiment, the contrast agent compound includes a magnetic resonance imaging (MRI) contrast agent compound. The MRI contrast agent compound is, for example, a gadolinium complex, a manganese complex, a chromium complex or an iron complex. The gadolinium complex is, for example, gadodiamide, gadopentetate dimeglumine or gadoterate meglumine. 
     Then, in step S 104 , a cross-linking agent is added to the solution. In an embodiment, the cross-linking agent is, for example, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide or a combination thereof. 
     Then, in step S 106 , a cross-linking reaction is caused between the amphiphilic chitosan and the contrast agent compound. In an embodiment, the reaction temperature of the cross-linking reaction is, for example, about 4° C. to 60° C., such as about 10° C. to 60° C., about 20° C. to 60° C. or about 40° C. to 60° C. The reaction time of the cross-linking reaction is, for example, about 4 hours to 24 hours, such as about 8 hours to 24 hours, about 12 hours to 24 hours or about 18 hours to 24 hours. In the invention, by controlling parameters such as the amount of the cross-linking agent, the reaction temperature and the reaction time of the cross-linking reaction, self-assembled nanostructures having different contents of contrast agents are prepared. 
     Subsequently, in step S 108 , the solution is purified. In an embodiment, the purification method is, for example, purification by dialysis. The solution used for dialysis is, for example, water or ethanol. 
     Next, in step S 110 , the solution is dried to obtain yellow powder. In an embodiment, the drying method is, for example, drying by baking or freeze-drying. Then, the yellow powder is dissolved in an aqueous solution to obtain the self-assembled nanostructure  100  of the invention. 
     In an embodiment, the surface of the self-assembled nanostructure  100  is further modified with a protein molecule having specificity. The protein molecule is, for example, an epidermal growth factor receptor (EGFR) or CD133 protein. After the surface of the self-assembled nanostructure  100  of the invention is modified with the protein molecule having specificity, the self-assembled nanostructure  100  exhibits recognition specificity for different targets. For example, the protein molecule having specificity is an antibody and is used to modify the surface of the self-assembled nanostructure  100  of the invention. In this case, the self-assembled nanostructure  100  of the invention has specificity for a corresponding antigen. 
     The self-assembled nanostructure of the invention is prepared by a simple process, which reduces the difficulty of mass production. Most of the solvents used in the production process of the self-assembled nanostructure of the invention are solvents having lower toxicity, such as water, methanol or isopropyl alcohol, etc., which increases safety of the self-assembled nanostructure. 
     EMBODIMENTS 
     The self-assembled nanostructure of the invention is prepared, for example, in the following manner. 0.5 g of carboxymethyl hexanoyl chitosan, 0.2 g of gadodiamide and 0.65 g of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride were prepared and dissolved in 100 ml of phosphate buffer. The phosphate buffer had a pH of 7.4. After the resultant was stirred at a low temperature (4° C.) for 30 minutes, 0.39 g of N-hydroxysuccinimide was added and the resultant was stirred at a low temperature (4° C.) for 24 hours. After the reaction was completed, the solution was subjected to dialysis with pure water for 48 hours and then with ethanol for 24 hours. Next, the solution was dried by baking and then crushed by stirring, so as to obtain yellow powder. The yellow powder was dissolved in an aqueous solution to obtain the self-assembled nanostructure of the invention. 
     In an embodiment, as shown in Table 1 below, by adjusting the reaction time of the cross-linking reaction, self-assembled nanostructures having different contents of contrast agents were obtained. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 Content of 
                 Content of 
               
               
                   
                   
                 Gadodiamide 
                 Gadolinium 
               
               
                   
                   
                 in Self- 
                 in Self- 
               
               
                   
                   
                 assembled 
                 assembled 
               
               
                   
                   
                 Nano- 
                 Nano- 
               
               
                 Reaction 
                   
                 structure 
                 structure 
               
               
                 Time 
                 Gadodiamide:EDC•HCl:NHS 
                 (wt %) 
                 (wt %) 
               
               
                   
               
             
            
               
                  4 hours 
                 1:2:2 
                 15.86 wt % 
                 4.77 wt % 
               
               
                  8 hours 
                 1:2:2 
                 12.39 wt % 
                 3.62 wt % 
               
               
                 12 hours 
                 1:2:2 
                 13.36 wt % 
                 3.94 wt % 
               
               
                 18 hours 
                 1:2:2 
                  9.92 wt % 
                 2.84 wt % 
               
               
                 24 hours 
                 1:2:2 
                 17.10 wt % 
                 5.20 wt % 
               
               
                   
               
            
           
         
       
     
     In an embodiment, as shown in Table 2 below, by adjusting the reaction temperature of the cross-linking reaction, self-assembled nanostructures having different contents of contrast agents were obtained. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 Content of 
                 Content of 
               
               
                   
                   
                 Gadodiamide 
                 Gadolinium 
               
               
                   
                   
                 in Self-assembled 
                 in Self-assembled 
               
               
                 Reaction 
                 Reaction 
                 Nanostructure 
                 Nanostructure 
               
               
                 Time 
                 Temperature 
                 (wt %) 
                 (wt %) 
               
               
                   
               
             
            
               
                 4 hours 
                  4° C. 
                 15.98 wt % 
                 4.81 wt % 
               
               
                 4 hours 
                 Room temperature 
                 15.86 wt % 
                 4.77 wt % 
               
               
                 4 hours 
                 40° C. 
                 13.90 wt % 
                 4.12 wt % 
               
               
                 4 hours 
                 50° C. 
                 11.57 wt % 
                 3.36 wt % 
               
               
                 4 hours 
                 60° C. 
                  9.03 wt % 
                 2.57 wt % 
               
               
                   
               
            
           
         
       
     
     In an embodiment, as shown in Table 3 below, by adjusting the proportion of the cross-linking agent, self-assembled nanostructures having different contents of contrast agents were obtained. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                 Content of 
                 Content of 
               
               
                   
                   
                 Gadodiamide 
                 Gadolinium 
               
               
                   
                   
                 in Self- 
                 in Self- 
               
               
                   
                   
                 assembled 
                 assembled 
               
               
                   
                   
                 Nano- 
                 Nano- 
               
               
                 Reaction 
                   
                 structure 
                 structure 
               
               
                 Time 
                 Gadodiamide:EDC•HCl:NHS 
                 (wt %) 
                 (wt %) 
               
               
                   
               
             
            
               
                 4 hours 
                 1:2:5 
                 14.57 wt % 
                 4.34 wt % 
               
               
                 4 hours 
                 1:2:1 
                 10.54 wt % 
                 3.04 wt % 
               
               
                 4 hours 
                 1:5:2 
                 15.94 wt % 
                 4.80 wt % 
               
               
                 4 hours 
                 1:4:4 
                 12.57 wt % 
                 3.68 wt % 
               
               
                   
               
            
           
         
       
     
       FIG. 5  is an infrared spectrum of gadodiamide, carboxymethyl hexanoyl chitosan and carboxymethyl hexanoyl chitosan having gadodiamide grafted thereto according to an embodiment of the invention. 
     Referring to  FIG. 5 , the C═O signal of carboxymethyl hexanoyl chitosan appears at 1668.93 cm −1 , and the C═O signal of carboxymethyl hexanoyl chitosan having gadodiamide grafted thereto appears at 1641.23 cm −1 . More specifically, the C═O signal is shifted after the gadodiamide is grafted to the carboxymethyl hexanoyl chitosan. The carboxymethyl hexanoyl chitosan with gadodiamide grafted thereto has a tertiary amine, while the carboxymethyl hexanoyl chitosan without gadodiamide grafted thereto only has a secondary amine. The electron-donating behavior of the alkyl group on the tertiary amine reduces the bond energy of C═O. Hence the C═O signal of the carboxymethyl hexanoyl chitosan with gadodiamide grafted thereto has a smaller wavenumber. It is thus proved that in the self-assembled nanostructure of the invention, there is chemical bonding between carboxymethyl hexanoyl chitosan and gadodiamide. 
     Relaxivities of a self-assembled nanostructure of the invention, gadodiamide, and carboxymethyl hexanoyl chitosan that physically encapsulates gadodiamide are as shown in Table 4. Hereinafter, the symbol “CHC+Gd” represents that there is only physical mixing between gadodiamide and carboxymethyl hexanoyl chitosan, while the symbol “CHC−Gd” represents that chemical bonding exists between gadodiamide and carboxymethyl hexanoyl chitosan (i.e., the self-assembled nanostructure of the invention). 
     
       
         
           
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Relaxivity (mM −1 s −1 ) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 CHC − Gd (Self-assembled 
                 4.2729 
               
               
                   
                 Nanostructure of 
               
               
                   
                 the Invention) 
               
               
                   
                 CHC + Gd 
                 1.2277 
               
               
                   
                 Gadodiamide 
                 1.8158 
               
               
                   
                   
               
            
           
         
       
     
     Referring to Table 4, it is proved that the relaxivity of the self-assembled nanostructure of the invention is higher than the relaxivities of the single gadodiamide and CHC+Gd. As a molecule grafted to a paramagnetic metal of a contrast agent is larger, a rotation time between the paramagnetic metal and a solvent molecule becomes longer, so that the relaxivity of the contrast agent is enhanced. The higher the relaxivity of the contrast agent, the better the contrast effect, i.e., the more excellent the contrasting ability. In the self-assembled nanostructure of the invention, gadolinium (paramagnetic metal) has an amphiphilic chitosan (polymer) grafted thereto, and thus has a higher relaxivity than the relaxivities of the single gadodiamide and CHC+Gd. Therefore, the self-assembled nanostructure of the invention exhibits a more excellent contrast effect than that of the single gadodiamide or CHC+Gd. In other words, the contrast agent compound of the invention, after being grafted to the amphiphilic chitosan, produces a synergistic effect on the contrasting ability of the contrast agent compound. Moreover, this synergistic effect only occurs when chemical bonding is present between gadodiamide and carboxymethyl hexanoyl chitosan (i.e., the self-assembled nanostructure of the invention), and does not occur when there is only physical mixing between these two. 
       FIG. 6  illustrates a comparison of encapsulation rates of a self-assembled nanostructure for different drug molecules according to an embodiment of the invention. 
     The self-assembled nanostructure of the invention, after encapsulating a drug molecule in an aqueous solution for 12 hours, exhibits an excellent encapsulation rate for the drug molecule. As shown in  FIG. 6 , the self-assembled nanostructure of the invention, after encapsulating a drug molecule such as demethoxycurcumin, cis-diamminedichloridoplatinum (II) or gemcitabine, etc. in an aqueous solution for 12 hours, exhibits an excellent encapsulation rate for the aforementioned drug molecules. Therefore, the self-assembled nanostructure of the invention is valuable in application in the biomedical field. 
       FIG. 7  illustrates a comparison of cell endocytosis amounts between a self-assembled nanostructure modified with an epidermal growth factor receptor (EGFR) and a self-assembled nanostructure without being modified with an EGFR. 
     In an embodiment of the invention, the self-assembled nanostructure modified with an EGFR and the self-assembled nanostructure without being modified with an EGFR each encapsulate a fluorescence contrast agent compound (e.g., PSVue™794 or PSVue™480), and are then respectively placed in lung cancer cell lines (A549) for cell culture for 4 hours. In the invention, a self-assembled nanostructure encapsulating a fluorescence contrast agent compound is obtained, for example, in the following manner. Carboxymethyl hexanoyl chitosan having gadodiamide grafted thereto was dissolved in water. The concentration of the carboxymethyl hexanoyl chitosan having gadodiamide grafted thereto was 0.05 wt %. 500 g/ml of a fluorescence contrast agent compound (e.g., PSVue™794 or PSVue™480) was added to the solution and stirred for 12 hours. The solution was then subjected to dialysis with pure water to obtain the self-assembled nanostructure encapsulating the fluorescence contrast agent compound. 
     By comparing relative fluorescence intensities of the lung cancer cell lines (A549), cell endocytosis amounts of the lung cancer cell lines (A549) for the self-assembled nanostructure modified with an EGFR and the self-assembled nanostructure without being modified with an EGFR are obtained. As shown in  FIG. 7 , the lung cancer cell line (A549) has an excellent cell endocytosis amount for the self-assembled nanostructure modified with an EGFR. Therefore, the self-assembled nanostructure of the invention is valuable in application in the biomedical field. 
     In summary, in the self-assembled nanostructure of the invention, the contrast agent compound is grafted to the amphiphilic chitosan, and due to chemical bonding between these two, the contrast agent compound is prevented from being released into tissue of an organism to cause any harm to the organism. In addition, the bonding between the amphiphilic chitosan and the contrast agent compound has a synergistic effect, which further improves the contrasting ability of the contrast agent compound. In addition, the self-assembled nanostructure of the invention may be modified with a protein molecule having specificity, so that the self-assembled nanostructure of the invention exhibits recognition specificity, and the efficiency in drug administration is improved. 
     Although the invention has been disclosed with reference to the above embodiments, it will be apparent to persons of ordinary skill in the art that modifications to the described embodiments may be made without departing from the spirit of the invention. Accordingly, the scope of the invention will be defined by the attached claims and not by the above detailed descriptions.