A multi-site dendritic boric acid functionalized magnetic nanomaterial and its preparation method and application

Abstract

The present invention discloses a multi-site dendritic boronic acid functionalized magnetic nanomaterial and its preparation method and application, belonging to the field of biomaterial technology. The multi-site dendritic boronic acid functionalized magnetic nanomaterial is formed by polyethylenimine being coated on the surface of triferric oxide magnetic nanoparticles modified with sodium tripolyphosphate or sodium polyphosphate through electrostatic action to form polyethylenimine-modified triferric oxide magnetic nanoparticles, and then 4-carboxylphenylboronic acid is grafted onto the surface of the triferric oxide magnetic nanoparticles modified with polyethyleneimine through an amide reaction. Compared with the low binding ability of a single boronic acid site, the multi-site dendritic boronic acid functionalized magnetic nanomaterial in the present invention can more sensitively bind to sialic acid overexpressed by CTCs, thereby achieving a high capture rate of CTCs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a multi-site dendritic boric acid functionalized magnetic nanomaterial and a preparation method and application thereof. Background Art

[0002] Circulating tumor cells (CTCs) are cancer cells that have detached from the primary tumor or metastatic site and survive in the circulating blood. Detecting CTCs in blood samples has important clinical significance for early cancer diagnosis, cancer treatment evaluation, and monitoring cancer recurrence, and is considered an important tumor biomarker. However, CTCs are present in very low abundance in the blood, with only 1-100 CTCs per milliliter of blood, along with approximately several million white blood cells and several billion red blood cells. These large numbers of red blood cells and highly adherent white blood cells act as non-target cells, interfering with the sensitivity and purity of CTC isolation.

[0003] Currently, a variety of CTCs separation platforms have been established based on antigen-antibody binding or by utilizing the differences in size, deformability, density, and dielectric properties between CTCs and blood cells. One of the most common surface molecules is the epithelial cell adhesion molecule (EpCAM). However, epithelial-mesenchymal transition occurs during tumor metastasis, resulting in the loss of epithelial antigenicity of CTCs, which in turn significantly downregulates EpCAM expression. As a result, the capture efficiency targeting EpCAM is greatly reduced, and even false negative results may occur.

[0004] Phenylboronic acid is a Lewis acid that is often used as an effective sensor for carbohydrates, glycoproteins, and dopamine. Numerous studies have shown that boronic acid can covalently bind to the cis-diol molecules of sialic acid overexpressed on cancer cells to form five- or six-membered ring boronic acid esters. Based on the formation of boronic acid ester covalent bonds, boronic acid-modified materials are widely used to capture and separate biomolecules and cancer cells containing diols. In addition, compared with commonly used antibodies and aptamers, sialic acid and boronic acid have broad-spectrum recognition capabilities and are suitable for capturing CTCs in various cancers. However, the existing technology uses a single phenylboronic acid binding site to capture CTCs, resulting in a low capture rate of CTCs. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention proposes a multi-site dendritic boronic acid functionalized magnetic nanomaterial, its preparation method and application. Compared with the low binding ability of a single boronic acid site, dendritic boronic acid has more sialic acid binding sites, thereby significantly improving the capture efficiency of CTCs.

[0006] In a first aspect, the present invention provides a multi-site dendritic boronic acid functionalized magnetic nanomaterial, wherein the multi-site dendritic boronic acid functionalized magnetic nanomaterial is obtained by electrostatically coating polyethyleneimine on the surface of sodium tripolyphosphate or sodium polyphosphate modified ferroferric oxide magnetic nanoparticles to form polyethyleneimine modified ferroferric oxide magnetic nanoparticles, and then grafting 4-carboxyphenylboronic acid on the surface of the polyethyleneimine modified ferroferric oxide magnetic nanoparticles through an amide reaction.

[0007] Using the above scheme, the ferroferric oxide magnetic nanoparticles are pretreated with an inorganic polyanion, sodium tripolyphosphate or sodium polyphosphate. The sodium tripolyphosphate or sodium polyphosphate can form stable PO-Fe bonds on the ferroferric oxide magnetic nanoparticles, imparting a strong negative charge to the surface of the ferroferric oxide magnetic nanoparticles. The sodium tripolyphosphate or sodium polyphosphate-modified ferroferric oxide magnetic nanoparticles are more dispersed and stable, facilitating electrostatic interaction with polyethyleneimine to form magnetic nanoparticles.

[0008] Multi-site dendritic boronic acid-functionalized magnetic nanomaterials are based on dendritic polyethyleneimine-functionalized magnetic nanoparticles, surface-modified with boronic acid ligands. The boronic acid used for modification is a compound containing carboxyl and boronic acid groups, preferably carboxylphenylboronic acid. In its molecular structure, the carboxyl group condenses with the amino group to form an amide bond, allowing the boronic acid group to bind to the sugar group in sialic acid. The multi-site dendritic boronic acid groups enable more sensitive capture of CTCs.

[0009] In a second aspect, the present invention provides a method for preparing a multi-site dendritic boronic acid functionalized magnetic nanomaterial, comprising the following steps:

[0010] S1, dispersing ferroferric oxide magnetic nanoparticles into a sodium tripolyphosphate or sodium polyphosphate solution to obtain sodium tripolyphosphate or sodium polyphosphate modified ferroferric oxide magnetic nanoparticles;

[0011] S2, dispersing polyethyleneimine in acidic phosphate buffer to obtain a polyethyleneimine solution;

[0012] S3, adding the sodium tripolyphosphate or sodium polyphosphate modified ferroferric oxide magnetic nanoparticles to the polyethyleneimine solution to obtain polyethyleneimine modified ferroferric oxide magnetic nanoparticles after reaction;

[0013] S4. The polyethyleneimine-modified ferroferric oxide magnetic nanoparticles are reacted with 4-carboxyphenylboronic acid through an amide reaction to obtain a multi-site dendritic boronic acid functionalized magnetic nanomaterial.

[0014] Furthermore, in step S1, the mass ratio of the ferroferric oxide magnetic nanoparticles to the solute in the sodium tripolyphosphate or sodium polyphosphate solution is (10.8-11.3): (0.9-1.1); the concentration of the sodium tripolyphosphate or sodium polyphosphate solution is 1.4-1.6 mg / mL.

[0015] Furthermore, in step S2, the amount of polyethyleneimine and the acidic phosphate buffer added is 1 g: 10-14 mL, and the pH of the acidic phosphate buffer is 6.4-6.6.

[0016] Furthermore, in step S3, the mass ratio of the polyethyleneimine in the polyethyleneimine solution to the sodium tripolyphosphate or sodium polyphosphate modified ferrosoferric oxide magnetic nanoparticles is (4-6):1.

[0017] Furthermore, step S3 is:

[0018] S31, adding the sodium tripolyphosphate or sodium polyphosphate modified ferroferric oxide magnetic nanoparticles to the polyethyleneimine solution, then adding acidic phosphate buffer, and sonicating for 25 to 35 minutes to obtain a mixed solution;

[0019] S32, reacting the mixed solution at 55-65° C. for 22-26 hours to obtain polyethyleneimine-modified ferrosoferric oxide magnetic nanoparticles.

[0020] Furthermore, step S4 is:

[0021] S41, sonicating the polyethyleneimine-modified ferroferric oxide magnetic nanoparticles in dimethyl sulfoxide to obtain a dispersion;

[0022] S42, dissolving 4-carboxyphenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide, and stirring at room temperature in the dark to obtain an activated mixed solution;

[0023] S43, adding the activated mixed solution to the dispersion solution, stirring at room temperature in the dark to obtain a multi-site dendritic boronic acid functionalized magnetic nanomaterial.

[0024] Furthermore, the mass ratio of the polyethyleneimine-modified ferrosoferric oxide magnetic nanoparticles, the 4-carboxyphenylboronic acid, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and the N-hydroxysuccinimide is (7-9):(8-10):(11-13):(5-7).

[0025] Using this approach, the surface of ferroferric oxide magnetic nanoparticles was anchored by forming a stable PO-Fe chemical bond. Pretreatment with sodium tripolyphosphate or sodium polyphosphate resulted in more dispersed and surface-active ferroferric oxide-modified nanoparticles. Polyethyleneimine was then used as a scaffold to increase the number of boronic acid groups, thereby providing more CTC binding sites. This resulted in a multi-site dendritic boronic acid-functionalized magnetic nanomaterial with rapid magnetic response. Compared to the low binding capacity of a single boronic acid site, the dendritic boronic acid possesses more sialic acid binding sites, significantly improving the capture efficiency of CTCs.

[0026] In a third aspect, the present invention provides a use of the multi-site dendritic boronic acid functionalized magnetic nanomaterial or the multi-site dendritic boronic acid functionalized magnetic nanomaterial prepared by the preparation method in capturing circulating tumor cells.

[0027] By adopting the above scheme, the beneficial effects of the present invention are:

[0028] The present invention pre-treats synthesized ferroferric oxide magnetic nanoparticles using sodium tripolyphosphate or sodium polyphosphate. The sodium tripolyphosphate or sodium polyphosphate can form a stable PO-Fe bond with ferroferric oxide and anchor it on the surface of the ferroferric oxide magnetic nanoparticles, thereby improving the dispersibility and surface activity of the ferroferric oxide magnetic nanoparticles, which is beneficial for subsequent polyethyleneimine coating.

[0029] When the pH value of the polyethyleneimine solution is 6.5, the electrostatic interaction between polyethyleneimine and sodium tripolyphosphate or sodium polyphosphate modified ferroferric oxide magnetic nanoparticles is the strongest, the coating effect is the best, and the dispersibility is better.

[0030] Polyethyleneimine, with its dendritic network structure and abundant amino groups, can be used to encapsulate sodium tripolyphosphate- or sodium polyphosphate-modified ferroferric oxide magnetic nanoparticles and then coupled with 4-carboxyphenylboronic acid to form a multi-site dendritic boronic acid-functionalized magnetic nanomaterial. This significantly increases the number of sialic acid binding sites, thereby greatly improving the capture rate. Polyethyleneimine carries a large number of amino groups and can serve as a scaffold to expand the number of boronic acid groups, thereby providing more CTC binding sites. Compared to the low binding capacity of a single boronic acid site, dendritic boronic acid can more sensitively bind to the sialic acid overexpressed by CTCs, thereby achieving a high capture rate of CTCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the preparation process of the multi-site dendritic boric acid functionalized magnetic nanomaterial of the present invention.

[0032] Figure 2The figure is a transmission electron microscope image of magnetic nanomaterials; among them, A is naked ferroferric oxide magnetic nanoparticles, B is polyethyleneimine-modified magnetic nanoparticles, and C is 4-carboxyphenylboronic acid-modified magnetic nanoparticles.

[0033] Figure 3 The following are Fourier transform infrared spectra of the naked ferroferric oxide magnetic nanoparticles, the polyethyleneimine modified nanoparticles and the 4-carboxyphenylboronic acid modified nanoparticles of the present invention.

[0034] Figure 4 This is the X-ray energy spectrum of the naked ferroferric oxide magnetic nanoparticles of the present invention, the polyethyleneimine modified nanoparticles and the 4-carboxyphenylboronic acid modified nanoparticles.

[0035] Figure 5 This is the hysteresis loop of the naked ferroferric oxide magnetic nanoparticles, the polyethyleneimine modified nanoparticles and the 4-carboxyphenylboronic acid modified nanoparticles.

[0036] Figure 6 The capture rates of 4T1 cells by multi-site dendritic boronic acid-modified nanomaterials and single-site boronic acid-modified nanomaterials.

[0037] Figure 7 The capture efficiency of boric acid-functionalized magnetic nanomaterials on different numbers of 4T1 cells in PBS buffer and simulated blood samples. DETAILED DESCRIPTION

[0038] The nanomagnetic affinity material of the present invention and its preparation and application methods are introduced below in conjunction with specific implementation cases, but it is not intended to be a specific limitation of the method of the present invention.

[0039] Example 1: Preparation of multi-site dendritic boronic acid functionalized magnetic nanomaterials

[0040] like Figure 1 As shown, the preparation method of the multi-site dendritic boric acid functionalized magnetic nanomaterial of the present invention comprises the following steps:

[0041] (1) Pretreatment of ferroferric oxide nanoparticles: 0.5 g of ferroferric oxide magnetic nanoparticles were sonicated in 30 mL of pure water containing 45 mg of sodium tripolyphosphate for 40 minutes to obtain sodium tripolyphosphate or sodium polyphosphate-modified ferroferric oxide magnetic nanoparticles;

[0042] This step uses ultrasound to improve dispersibility on the one hand, and on the other hand, sodium tripolyphosphate can form a stable PO-Fe chemical anchor on the surface of the ferroferric oxide magnetic nanoparticles by reacting with the ferroferric oxide magnetic nanoparticles, so that the surface of the ferroferric oxide magnetic nanoparticles has a strong negative charge.

[0043] (2) Modification of polyethyleneimine: 2.5 g of polyethyleneimine was ultrasonicated in 30 mL of pH 6.5 phosphate buffer for 60 minutes to obtain a polyethyleneimine solution. Then, sodium tripolyphosphate or sodium polyphosphate modified ferroferric oxide magnetic nanoparticles were added to the polyethyleneimine solution and supplemented with 30 mL of pH 6.5 phosphate buffer, and the mixed solution was ultrasonicated for 30 minutes. Finally, the ultrasonicated solution was maintained at 60°C for 24 hours under vigorous stirring, and the polyethyleneimine-coated precipitate was magnetically collected, washed with pure water, and then dried in an oven at 40°C for 12 hours to obtain polyethyleneimine-modified ferroferric oxide magnetic nanoparticles;

[0044] This step involves ultrasonically dispersing polyethyleneimine into an acidic phosphate buffer solution. This not only improves dispersibility, but also, under acidic conditions, the amino groups on the polyethyleneimine molecular chains become protonated, resulting in a more positive surface charge. This allows the sodium tripolyphosphate- or sodium polyphosphate-modified ferroferric oxide magnetic nanoparticles to have a negative surface charge, resulting in the strongest electrostatic interaction and the best coating effect.

[0045] (3) Grafting 4-carboxyphenylboronic acid: 80 mg of polyethyleneimine-modified ferroferric oxide magnetic nanoparticles were dissolved in 4 mL of dimethyl sulfoxide and ultrasonically dispersed for 20 minutes to obtain a dispersion of polyethyleneimine-modified ferroferric oxide magnetic nanoparticles. Then, 90 mg of 4-carboxyphenylboronic acid, 120 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 60 mg of N-hydroxysuccinimide were dissolved in 3 mL of dimethyl sulfoxide and stirred at room temperature in the dark for 30 minutes to obtain an activated mixed solution. Finally, the activated mixed solution was slowly added to the dispersion of polyethyleneimine-modified ferroferric oxide magnetic nanoparticles and stirred at room temperature in the dark for another 24 hours. After magnetic separation, a multi-site dendritic boronic acid functionalized magnetic nanomaterial was obtained and placed in a drying oven at 40°C for 1 hour.

[0046] Example 2: Characterization of magnetic materials

[0047] (1) Morphological characterization

[0048] The morphology of the multi-site dendritic boric acid functionalized magnetic nanomaterial prepared in Example 1 was observed using a transmission electron microscope. Figure 2 As can be seen, the ferroferric oxide magnetic nanoparticles are spherical in shape, with a particle size of 11.30 ± 4.38 nm. Polyethyleneimine modification did not significantly change the size, remaining at 12.61 ± 4.01 nm. This may be due to the amorphous nature of polyethyleneimine modification, making it difficult to observe using a transmission electron microscope. Modification with 4-carboxyphenylboronic acid increased the particle size to 15.65 ± 4.59 nm, with no significant change in size, which may be related to the smaller size of the nanoparticles themselves.

[0049] (2) Characterization of chemical composition and elements

[0050] Fourier transform infrared spectrum Figure 3 As shown in the figure, the absorption band of the spectrum after polyethyleneimine modification is at 3430 cm -1 The enhanced -1 A new absorption peak appeared, and after further modification with 4-carboxyphenylboronic acid, it peaked at 1120 cm -1 The absorption peak of BO bond appears at Figure 4 As shown, the four peaks appearing in the spectrum have binding energies of 191.6, 284.8, 400.1 and 532.4 eV, corresponding to B1s, C1s, N1s and O1s, respectively. These peaks also indicate that 4-carboxyphenylboronic acid is successfully connected.

[0051] (3) Characterization of magnetic properties

[0052] Hysteresis loop such as Figure 5 As shown in the figure, the coercive force and residual magnetization of the three are all zero, and the magnetic nanoparticles modified with 4-carboxyphenylboronic acid are superparamagnetic. The saturation magnetization value of the unmodified magnetic nanoparticles is 48.05emu g -1 After modification with non-magnetic polyethyleneimine and 4-carboxyphenylboronic acid, the magnetization intensity values ​​decreased to 46.52emu g -1 and 43.98emu g -1 . It shows that the modified material has good magnetic response performance and can achieve rapid separation under an external magnetic field.

[0053] Example 3: Capture efficiency of 4T1 cells by multi-site dendritic boronic acid-modified magnetic nanomaterials and single-site boronic acid-modified nanomaterials

[0054] 75 μl (2 mg mL -1 ) The multi-site dendritic boronic acid functionalized magnetic nanomaterial and the single-site boronic acid modified nanomaterial prepared in Example 1 were added with 925 μl of 1.0×10 5 The cells were then incubated in a PBS solution containing 4T1 cells for 20 minutes. Finally, the supernatant was magnetically separated and the unbound cells in the supernatant were counted under a microscope. The capture efficiency was defined as: (total cell number - number of uncaptured cells in the supernatant) / total cell number.

[0055] Depend on Figure 5 It can be seen that the capture efficiency of 4T1 cells by single-site boronic acid-modified nanomaterials can only reach 62.9%, while the capture efficiency of multi-site dendritic boronic acid-functionalized magnetic nanomaterials can reach 93.1%.

[0056] Example 4: Capture efficiency of multi-site dendritic boronic acid functionalized magnetic nanomaterials for different numbers of 4T1 cells in PBS buffer and simulated blood samples.

[0057] 75 μl (2 mg mL -1 To prepare simulated clinical samples, the multi-site dendritic boronic acid-functionalized magnetic nanomaterials were added to 925 μl of PBS containing 20, 50, 100, 500, or 1000 4T1 cells pre-stained with a red fluorescent dye and erythrocyte-depleted mouse blood. After incubation with the cells for 20 minutes, the supernatant was magnetically separated, and unbound cells were counted under a fluorescence microscope. Capture efficiency was defined as (total cell number - number of uncaptured cells in the supernatant) / total cell number.

[0058] Depend on Figure 6 The capture rate in PBS solution was approximately 83%, and in artificial blood samples, approximately 80%. Therefore, the prepared multi-site dendritic boronic acid-functionalized magnetic nanomaterial has high sensitivity. This demonstrates that it can effectively capture low-concentration CTCs even in complex physiological environments and has great application potential in the isolation of CTCs in clinical blood samples.

[0059] In summary, the present invention provides a method for preparing a multi-site dendritic boronic acid-functionalized magnetic nanomaterial for highly efficient capture of circulating tumor cells. First, after pretreatment with sodium tripolyphosphate, a more stable and dispersible magnetic nanoparticle is formed. Next, a polyethyleneimine-modified material is obtained through electrostatic interaction. Finally, 4-carboxyphenylboronic acid is grafted onto the material through an amide reaction to yield a multi-site dendritic boronic acid-functionalized magnetic nanomaterial. This modified material is specific for sialic acid-overexpressing 4T1 cells, achieving a capture rate of up to 93.1%, and even reaching an 80% capture rate in simulated clinical samples.

[0060] It should be noted that the attached Figure 1 In the present invention, PEI is polyethyleneimine, 4-CPBA is 4-carboxyphenylboronic acid, MNP is ferroferric oxide magnetic nanoparticles modified with sodium tripolyphosphate, MNP@PEI is ferroferric oxide magnetic nanoparticles modified with polyethyleneimine, and MNP@PEI@4-CPBA is ferroferric oxide magnetic nanoparticles modified with 4-carboxyphenylboronic acid (i.e., the multi-site dendritic boronic acid functionalized magnetic nanomaterial described in the present invention).

[0061] It should be noted that the attached Figure 6 In the present invention, APTES is a single-site boronic acid-modified nanomaterial obtained by using (3-aminopropyltriethoxysilane) as a scaffold, and PEI is a multi-site dendritic boronic acid-functionalized magnetic nanomaterial obtained by using polyethyleneimine as a scaffold.

[0062] It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical concept of the present application, and these all fall within the scope of protection of the present application.

Claims

1. A multi-site dendritic boric acid functionalized magnetic nanomaterial, characterized in that: The multi-site dendritic boric acid functionalized magnetic nanomaterial is obtained by coating polyethyleneimine on the surface of sodium tripolyphosphate-modified ferroferric oxide magnetic nanoparticles through electrostatic interaction to form polyethyleneimine-modified ferroferric oxide magnetic nanoparticles, and then grafting 4-carboxyphenylboronic acid on the surface of the polyethyleneimine-modified ferroferric oxide magnetic nanoparticles through an amide reaction; The method for preparing the multi-site dendritic boric acid functionalized magnetic nanomaterial comprises the following steps: S1, dispersing ferroferric oxide magnetic nanoparticles into sodium tripolyphosphate to obtain sodium tripolyphosphate-modified ferroferric oxide magnetic nanoparticles; S2, dispersing polyethyleneimine in acidic phosphate buffer to obtain a polyethyleneimine solution; S3, adding the sodium tripolyphosphate-modified ferroferric oxide magnetic nanoparticles to the polyethyleneimine solution to obtain polyethyleneimine-modified ferroferric oxide magnetic nanoparticles after reaction; S4. The polyethyleneimine-modified ferroferric oxide magnetic nanoparticles are reacted with 4-carboxyphenylboronic acid through an amide reaction to obtain a multi-site dendritic boronic acid functionalized magnetic nanomaterial.

2. The multi-site dendritic boronic acid functionalized magnetic nanomaterial according to claim 1, characterized in that: In step S1, the mass ratio of the ferroferric oxide magnetic nanoparticles to the solute in the sodium tripolyphosphate solution is (10.8-11.3): (0.9-1.1); the concentration of the sodium tripolyphosphate solution is 1.4-1.6 mg / mL.

3. The multi-site dendritic boronic acid functionalized magnetic nanomaterial according to claim 1, characterized in that: In step S2, the amount of the polyethyleneimine and the acidic phosphate buffer added is 1 g: 10-14 mL, and the pH of the acidic phosphate buffer is 6.4-6.

6.

4. The multi-site dendritic boronic acid functionalized magnetic nanomaterial according to claim 1, characterized in that: In step S3, the mass ratio of the polyethyleneimine in the polyethyleneimine solution to the sodium tripolyphosphate-modified ferrosoferric oxide magnetic nanoparticles is (4-6):

1.

5. The multi-site dendritic boronic acid functionalized magnetic nanomaterial according to claim 1, characterized in that: Step S3 is: S31, adding the sodium tripolyphosphate-modified ferroferric oxide magnetic nanoparticles to the polyethyleneimine solution, then adding acidic phosphate buffer, and sonicating for 25 to 35 minutes to obtain a mixed solution; S32, reacting the mixed solution at 55-65° C. for 22-26 hours to obtain polyethyleneimine-modified ferrosoferric oxide magnetic nanoparticles.

6. The multi-site dendritic boronic acid functionalized magnetic nanomaterial according to claim 1, characterized in that: Step S4 is: S41, sonicating the polyethyleneimine-modified ferroferric oxide magnetic nanoparticles in dimethyl sulfoxide to obtain a dispersion; S42, dissolving 4-carboxyphenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide, and stirring at room temperature in the dark to obtain an activated mixed solution; S43, adding the activated mixed solution to the dispersion solution, stirring at room temperature in the dark to obtain a multi-site dendritic boronic acid functionalized magnetic nanomaterial.

7. The multi-site dendritic boronic acid functionalized magnetic nanomaterial according to claim 6, characterized in that: The mass ratio of the polyethyleneimine-modified ferrosoferric oxide magnetic nanoparticles, the 4-carboxyphenylboronic acid, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and the N-hydroxysuccinimide is (7-9): (8-10): (11-13): (5-7).

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