Dendritic macromolecule functionalized fluorescent carbon nanoparticles, preparation method and application thereof

Abstract

The invention discloses dendrimer functionalized fluorescent carbon nanoparticles, a preparation method and application thereof. First, citric acid and triaminobenzene are hydrothermally synthesized into fluorescent carbon nanoparticles FCNs, and then an EDC coupling method is used to form an amide bond between the carboxyl group of the fluorescent carbon nanoparticles and the amino group of PAMAM, and PAMAM is coupled to the surface of the fluorescent carbon nanoparticles through covalent functionalization to obtain dendrimer modified carbon nanoparticles PAMAM@FCNs. It has the two characteristics of excitation light independence and long wavelength emission, can effectively deliver siRNA across biological membranes into cells, and has potential in the research of new imaging tracing gene delivery nanocarriers. Compared with the prior art, the preparation method of the invention is simple, easy to operate, low in cost, and suitable for promotion.

Description

Technical Field

[0001] The invention belongs to the field of nano materials, and in particular relates to a method for preparing dendritic macromolecule functionalized fluorescent carbon nanoparticles. Background Art

[0002] As a new type of nanomaterial, fluorescent carbon nanoparticles (FCNs) have become a hot topic in current research and have been widely used in many fields such as photocatalysis, chemical analysis, biosensing, bioimaging and drug delivery (A. Simpson, RRPandey, et al. Carbon, 2018 (127): 122-130; Hu Shengliang et al. "Fluorescent Carbon Nanoparticles: New Progress and Technological Challenges" "Progress in Chemistry", 2010. Vol. 22. No 2 / 3).

[0003] There are a large number of oxygen- or nitrogen-containing groups on the surface of fluorescent carbon nanoparticles (FCNs), which can be combined with functional substances to form composite materials. While achieving surface functionalization, it can also improve surface defects and increase fluorescence quantum yield, which is beneficial to the application of FCNS (H. Ali, SKBhunia, et al. ACS Appl. Mater. Interfaces, 2016 (8): 9305-9313).

[0004] Polyamide amine dendrimer (PAMAM) has a unique structure and excellent performance, and is often used as a drug carrier material. PAMAM contains a large number of hydrophobic spaces inside, which can be used to embed drugs. At the same time, its ends contain a large number of amino and carboxyl groups with positive and negative charges, which can be connected to drugs through electrostatic interaction or covalent connection (Z. Zhang, YA Lin, et al. J. Neuroinflamm., 2020, 17(1): 319).

[0005] Therefore, the synthesis of PAMAM@FCNs (dendrimer functionalized carbon nanoparticles) by modifying polyamidoamine dendrimers onto the surface of fluorescent carbon nanoparticles has attracted the attention of more and more researchers. PAMAM@FCNs is expected to become a multifunctional drug delivery system that integrates imaging, targeting and therapy (I. Matai, A. Sachdev, et al. ACS Appl. Mater. Interfaces, 2015(7): 11423-11435; D. Li, Y. Fan, et al. J. Mater. Chem. B, 2019(7): 277-285; D. Li, LZ Lin, et al. Bioact. Mater. 2021(6): 729-739).

[0006] However, the PAMAM@FCNs reported so far all have short-wavelength (blue-green, 440-570nm) fluorescence emission, which is easily interfered by the biological autofluorescence background and causes a certain degree of damage to biological tissues. Long-wavelength (yellow-red, 570-760nm) fluorescence emission can not only reduce the interference of autofluorescence and damage to biological tissues, but also has stronger tissue penetration ability, which is conducive to the research of tracer imaging drug delivery systems (ZJ Zhu, YL Zhai, et al. Mater. Today, 2019 (30): 52-79; MR Zhang, RG Su, et al. Nano Res. 2019 (12): 815-821).

[0007] There are two types of long-wavelength fluorescent carbon nanoparticles (FCNs): excitation light dependent and excitation light independent. The emission wavelength of excitation light-dependent fluorescent carbon nanoparticles changes with the change of the excitation wavelength, and when the excitation wavelength increases, its emission wavelength red-shifts. Therefore, in order to obtain long-wavelength emission, the excitation wavelength is usually increased, but this will cause a sharp drop in fluorescence intensity, which is not conducive to biological imaging (HJZhao, JKDuan, et al.Chem.Mater.2018(30):3438-3453). On the contrary, for excitation light-independent FCNs, when the excitation wavelength changes, its fluorescence emission wavelength remains unchanged, so it can be excited at its optimal excitation wavelength to obtain the strongest fluorescence emission, with high sensitivity and convenient for biological imaging (XWHua, YWBao, et al.ACS Appl.Mater.Interfaces,2019(11):32647-32658).

[0008] At present, the research on PAMAM@FCNs at home and abroad is still in its infancy, and there is no report on the synthesis of PAMAM@FCNs that have both the characteristics of excitation light independence and long-wavelength emission. Summary of the invention

[0009] The purpose of the present invention is to provide dendrimer functionalized fluorescent carbon nanoparticles, a preparation method and applications thereof in view of the above-mentioned problems. The dendrimer functionalized fluorescent carbon nanoparticles (PAMAM@FCNs) are obtained by modifying the surface of fluorescent carbon nanoparticles (FCNs) with polyamide-amine dendrimers (PAMAM), and have two characteristics: excitation light independence and long-wavelength emission. Because PAMAM itself does not emit fluorescence, the synthesized fluorescent carbon nanoparticles (FCNs) are required to be not only excitation light independent and emit long-wavelength fluorescence, but also contain groups that can be coupled with PAMAM. The preparation method is simple, easy, low-cost, suitable for promotion, batch and green production. The synthesized PAMAM@FCNs can be used as a new type of imaging tracer gene delivery nanocarrier.

[0010] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0011] Dendrimer-functionalized fluorescent carbon nanoparticles are prepared by modifying the surface of fluorescent carbon nanoparticles (FCNs) with polyamidoamine dendrimers (PAMAM), which have the characteristics of excitation light independence and long-wavelength emission.

[0012] The maximum emission wavelength of the PAMAM@FCNs is 585 nm.

[0013] The preparation method of dendrimer-functionalized fluorescent carbon nanoparticles comprises the following steps:

[0014] (1) citric acid and triaminobenzene are ultrasonically dissolved and then transferred to a high-pressure reactor to synthesize fluorescent carbon nanoparticles (FCNs) by a hydrothermal method, and the obtained fluorescent carbon nanoparticle stock solution is dialyzed, concentrated, precipitated, washed, and dried to obtain fluorescent carbon nanoparticle solids;

[0015] In step (1), the concentration of triaminobenzene is 0.015-0.15 mol / L, and the concentration ratio of citric acid to triaminobenzene is 1:1-1:80.

[0016] (2) Take an appropriate amount of fluorescent carbon nanoparticles, dissolve them in ultrapure water, add NHS (N-hydroxysuccinimide), EDC (1-(3-dimethylamino)-3-ethylcarbodiimide hydrochloride), and PAMAM (polyamide-amine dendritic macromolecules) in sequence, adjust the pH value, and use the EDC coupling method to couple PAMAM to the surface of the fluorescent carbon nanoparticles to obtain dendritic macromolecule-modified carbon nanoparticles PAMAM@FCN. The obtained PAMAM@FCNs stock solution is dialyzed, concentrated, and freeze-dried to obtain PAMAM@FCNs solid.

[0017] In step (2), NHS is added to control the pH in the range of 4.0-7.0 to activate the carboxyl groups on the surface of the fluorescent carbon nanoparticles; EDC is added to adjust the pH in the alkaline range of 7.4-9.0 to carry out coupling between the fluorescent carbon nanoparticles and PAMAM.

[0018] The concentration of fluorescent carbon nanoparticles is 0.2-2.0 mg / mL; the generation of PAMAM is 0.0-5.0; and the concentration of PAMAM is 0.15-1.50 mmol / L.

[0019] In the synthesis of FCNs, the present invention adopts a hydrothermal method with simple equipment and mild conditions; in terms of raw materials, CA (citric acid) and TAB (triaminobenzene) are selected. Citric acid is rich in carboxyl groups, which can provide coupling groups for the subsequent modification of fluorescent carbon nanoparticles. Triaminobenzene is rich in amino groups, and N-doped fluorescent carbon nanoparticles (FCNs) can be synthesized. N acts as an n-type impurity that provides excess electrons, which changes the Fermi level and optical properties of the fluorescent carbon nanoparticles. At present, there is no report on the combination of citric acid and triaminobenzene as precursors to synthesize fluorescent carbon nanoparticles.

[0020] Before surface modification of PAMAM, the present invention performs post-treatment on the fluorescent carbon nanoparticle (FCNs) stock solution. First, a dialysis-precipitation method is used to separate and obtain a precipitate; then, the precipitate is washed; and finally, vacuum drying is performed. Compared with the traditional dialysis method, the dialysis-precipitation method shortens the dialysis time from more than 48 hours to 3 hours, reduces losses, and improves the yield. Secondly, surface modification using purified fluorescent carbon nanoparticles can eliminate the side reactions between PAMAM and the remaining raw materials and improve the reaction efficiency.

[0021] When modifying PAMAM on the surface, the present invention adopts the EDC (1-(3-dimethylamino)-3-ethylcarbodiimide hydrochloride) coupling method to obtain covalently functionalized PAMAM@FCNs by forming an amide bond between the carboxyl group of the fluorescent carbon nanoparticles and the amino group of PAMAM. First, NHS (N-hydroxysuccinimide) is added to control the pH in the acidic range of 4.0-7.0 to activate the carboxyl group on the surface of the fluorescent carbon nanoparticles; then, EDC is added to adjust the pH in the alkaline range of 7.4-9.0 for coupling.

[0022] Dendrimer macromolecule-functionalized fluorescent carbon nanoparticles are used as imaging tracer gene delivery nanocarriers. PAMAM@FCNs can combine with siRNA to form a stable complex, effectively delivering siRNA across the biomembrane into the cell to exert a gene silencing effect.

[0023] Beneficial Effects

[0024] (1) The present invention adopts the EDC coupling method to synthesize PAMAM@FCNs, which has simple equipment, mild conditions, green environmental protection, low cost and high yield.

[0025] (2) The synthesized PAMAM@FCNs have excellent optical properties (independent of excitation light, emission of long-wavelength yellow fluorescence, and high quantum yield), high water solubility, good biocompatibility, low cytotoxicity, and strong cellular uptake ability, and can effectively deliver siRNA into cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 UV-visible absorption spectra and fluorescence emission spectra of FCNs prepared in Example 16 and PAMAM@FCNs prepared in Example 34.

[0027] FCNs have two absorption peaks at 257nm and 501nm. The absorption peak at 257nm is caused by the π-π* transition of the conjugated double bond C=C; the absorption peak at 501nm is caused by the n-π* transition of C=N, CNC, C=O and CO. Figure 1 A).

[0028] After PAMAM@FCNs were modified on the surface of FCNs, the absorption band at 257 nm of FCNs remained basically unchanged, while the absorption band at 501 nm moved significantly to the short-wave direction, and the absorption wavelength decreased from 501 to 443 nm, blue-shifting by 58 nm ( Figure 1 A).

[0029] The fluorescence emission spectra show that the maximum emission wavelengths of FCNs and PAMAM@FCNs are 623nm and 585nm, respectively, both in the long wavelength range (570-760nm). Compared with FCNs, the emission wavelength of PAMAM@FCNs is significantly reduced, blue-shifted by 38nm, which may be caused by surface defects. The proportion of oxygen functional groups in PAMAM@FCNs is reduced, resulting in a larger HOMO-LUMO band gap ( Figure 1 B).

[0030] Figure 2 The fluorescence properties of the FCNs prepared in Example 16 and the PAMAM@FCNs prepared in Example 34 under light excitation of different wavelengths.

[0031] Depend on Figure 2 As can be seen, the emission wavelengths of FCNs and PAMAM@FCNs remain unchanged as the excitation wavelength changes, indicating that both are independent of excitation light and have excellent luminescence properties, which can be applied to biological imaging research. However, as the excitation wavelength changes, the fluorescence intensity of FCNs and PAMAM@FCNs changes significantly ( Figure 2 B).

[0032] The optimal excitation wavelengths of FCNs and PAMAM@FCNs are 420 nm and 440 nm, respectively.

[0033] Figure 3 It is the Fourier infrared spectra of FCNs prepared in Example 16 and PAMAM@FCNs prepared in Example 34.

[0034] Since both FCNs and PAMAM@FCNs contain -NH2, the asymmetric stretching vibration of NH (ν NH as ) and symmetric stretching vibration (ν NH s ) absorption peak. However, the peak position has changed, and the ν NH as and ν NH s Located at 3313 and 3192 cm respectively -1 , while the ν of PAMAM@FCNs NH as and ν NH s Located at 3357 and 3300 cm -1, moving toward the high frequency direction. This is because the -NH2 in FCNs comes from TAB, which undergoes a p-π conjugation effect with the benzene ring, and the absorption frequency decreases, while the -NH2 in PAMAM@FCNs comes from PAMAM, which is an aliphatic primary amine and has a higher absorption frequency.

[0035] Although both FCNs and PAMAM@FCNs contain -CH2, the CH stretching vibration peak (ν CH ), which does not appear in the infrared spectrum of FCNs, but only appears in the infrared spectrum of PAMAM@FCNs. This is because CA contains -CH2 in the synthetic raw materials of FCNs, but its dosage is extremely low (n CA :n TAB 1:60), the infrared analysis sensitivity is not high, and no detection was made; however, the content of -CH2 in PAMAM is high, and after it is modified on the surface of FCNs, the -CH2 -1 Appearance CH .

[0036] In the infrared spectrum of FCNs, 1625cm -1 and 1516cm -1 Skeletal stretching vibration of benzene ring, 1310 cm -1 It is the CN stretching vibration of aromatic amine, 1198cm -1 It is the out-of-plane bending vibration of the CH of the aromatic ring. However, these four absorption peaks disappeared in the infrared absorption graph of PAMAM@FCNs. Instead, three characteristic peaks of secondary amide appeared: C=O stretching vibration (1645cm -1 ), NH in-plane bending vibration (1557cm -1 ) and CN stretching vibration (1252cm -1 ), this is because PAMAM contains a large number of secondary amide groups.

[0037] The above results confirmed that PAMAM was modified on the surface of FCNs and PAMAM@FCNs were successfully synthesized.

[0038] Figure 4 are transmission electron micrographs of FCNs prepared in Example 21 and PAMAM@FCNs prepared in Example 21. FCNs are severely aggregated ( Figure 4 A), while PAMAM@FCNs have good dispersion. PAMAM@FCNs are spherical with a particle size of about 80nm ( Figure 4 B).

[0039] Figure 5The figure is a schematic diagram of the effect of PAMAM@FCNs prepared in Example 34 on the migration of siRNA. Free siRNA is negatively charged and will move from the negative electrode to the positive electrode under the action of the electric field, generating an obvious siRNA migration band in the gel.

[0040] When the weight ratio of PAMAM@FCNs to siRNA was 0.625:1 or 1.25:1, a bright band was observed at the same position as that of free siRNA, indicating that PAMAM@FCNs had little effect on the migration of siRNA.

[0041] When the mass ratio of PAMAM@FCNs to siRNA reached 2.5:1, bright siRNA bands were observed in the sample wells, and there were siRNA bands migrating downward, indicating that PAMAM@FCNs did not completely block siRNA.

[0042] When the mass ratio of PAMAM@FCNs to siRNA increased to above 3.75:1, there were bright bands in the sample wells and no downward migrating bands, indicating that siRNA was completely blocked by PAMAM@FCNs.

[0043] These results suggest that an appropriate amount of PAMAM@FCNs can bind to siRNA to form a stable complex and thus has the potential to serve as a carrier of siRNA.

[0044] Figure 6 It is a schematic diagram of the cytotoxicity of PAMAM@FCNs prepared in Example 34. After incubation of PC12 cells with PAMAM@FCNs in the concentration range of 0μg / ml-100μg / ml for 48h, the cell viability was above 85%, indicating that PAMAM@FCNs had no cytotoxicity within the experimental concentration range.

[0045] Figure 7 This is a schematic diagram of the HDAC2 protein expression level of PC12 cells 6 hours after transfection of the nanocomplex formed by PAMAM@FCNs prepared in Example 34 and histone deacetylase 2 (HDAC2)-specific siRNA (siHDAC2) into the cells.

[0046] Compared with the untransfected blank control group, naked siHDAC2 transfection of PC12 cells had almost no effect on their HDAC2 expression. After transfection with PAMAM@FCNs / siHDAC2 composite nanoparticles formed by 50μg / ml or 100μg / ml PAMAM@FCNs and siHDAC2, the expression of HDAC2 in PC12 cells was significantly reduced, especially the nanocomplex formed by PAMAM@FCNs and siHDAC2 at a concentration of 100μg / ml. The experimental results show that PAMAM@FCNs can effectively deliver siRNA across the biomembrane into cells within a certain concentration range and exert a gene silencing effect. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments:

[0048] The abbreviated Chinese notes involved in the technical solution of the present invention are as follows:

[0049] FCNs (fluorescent carbon nanoparticles);

[0050] CA (citric acid, MW196.081, 95%, Shanghai MacLean Biochemical Co., Ltd.);

[0051] TAB (triaminobenzene, MW192.13, 99.5%, Sinopharm Chemical Reagent Co., Ltd.);

[0052] PAMAM (Polyamide amine dendrimer, polyamide-amine dendrimer, 0.0-5.0 generations, concentration 0.15-1.50 mmol / L, pH range 4.0-9.0, Shanghai Hansi Chemical Co., Ltd.);

[0053] PAMAM@CNPs (dendrimer-functionalized carbon nanoparticles);

[0054] NHS (N-hydroxysuccinimide, MW 115.09, 98%, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0055] EDC (1-(3-dimethylamino)-3-ethylcarbodiimide hydrochloride, MW 191.70, 98%, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0056] Ethyl acetate (MW88.11, 99.5%, Sinopharm Chemical Reagent Co., Ltd.);

[0057] All reagents used above were of analytical grade. Ultrapure water used in the laboratory was homemade.

[0058] Example

[0059] The preparation method of dendrimer-functionalized fluorescent carbon nanoparticles comprises the following steps:

[0060] (1) Weigh TAB (triaminobenzene) in a centrifuge tube, add appropriate amount of CA (citric acid) solution and ultrapure water, control the total volume to 7 mL, vortex for 30 seconds, and ultrasonicate to fully dissolve the raw materials. Transfer the solution to a 30 ml high pressure reactor, react at 110-150 ° C in a drying oven for 2-6 hours to obtain dark brown fluorescent carbon nanoparticles (FCNs), take out, and cool to room temperature.

[0061] The above FCNs stock solution was placed in a dialysis bag of MD 500 specification and dialyzed with ultrapure water for 3 hours. After being concentrated to a quarter of the total volume by a rotary evaporator at 60°C, it was precipitated with ethyl acetate. The obtained precipitate was centrifuged at 10,000 rpt for 10 minutes, the supernatant was discarded, and the precipitate was washed with ethyl acetate 3 times. Finally, the precipitate was vacuum dried to obtain a brown FCNs solid.

[0062] The concentration of TAB in the reaction system is 0.015-0.15 mol / L.

[0063] The concentration ratio of CA:TAB is 1:1 to 1:80;

[0064] (2) Take an appropriate amount of FCNs, dissolve it in ultrapure water, add NHS solution, and adjust the pH. Add EDC solution, place it in a shaker, and activate it at 37°C for 0.5 to 3 hours. Adjust the pH, add PAMAM, place it in a shaker, and react at 37°C for 3 to 10 hours. Put the PAMAM@FCNs stock solution into a dialysis bag of MD 500 specifications, dialyze it with ultrapure water for 1 hour, concentrate it to one-fourth of the total volume of the solution on a rotary evaporator at 60°C, freeze-dry it, and weigh it.

[0065] The concentration of FCNs in the reaction system is 0.2-2.0 mg / mL, the generation of PAMAM is 0.0-5.0, the concentration of PAMAM is 0.15-1.50 mmol / L, and the pH range is 4.0-9.0.

[0066] The following Examples 1-36 investigate the synthesis conditions of FCNS and PAMAM@FCNs:

[0067] The synthesis of FCNS was investigated in terms of raw material ratio (Examples 1-6), raw material dosage (Examples 7-10), reaction temperature (Examples 11-14) and reaction time (Examples 15-18).

[0068] The synthesis of PAMAM@FCNs investigated the concentration of FCNs (Examples 19-22), the type of PAMAM (Examples 23-26), the amount of PAMAM (Examples 27-31) and the reaction time (Examples 32-36). The specific implementation scheme and experimental results are shown in Table 1.

[0069] Table 1

[0070]

[0071]

[0072]

[0073] From Examples 1-18, it can be seen that the optimal conditions for synthesizing FCNs are: nCA:nTAB=1:60, TAB 0.5mol, reaction temperature 130°C, reaction time 3h. Under these conditions, the fluorescence emission wavelength of the synthesized FCNs is 623nm, and the quantum yield is 9.4%. The results show that long-wavelength emission FCNs were successfully synthesized, providing a raw material with excellent performance for the next step of surface modification of FCNs with PAMAM.

[0074] Examples 19-36 show that as the synthesis conditions change, the fluorescence emission wavelength of PAMAM@FCNs remains almost unchanged, basically stable at around 585nm; while the fluorescence quantum yield of PAMAM@FCNs changes greatly. Under the same conditions, the smaller the PAMAM, the higher the fluorescence quantum yield of the modified PAMAM@FCNs. However, the smaller the PAMAM, the less drug loading it has.

[0075] Taking all factors into consideration, the optimal synthesis conditions of PAMAM@FCNs were: FCNs 0.95 mg / ml, concentration 0.045 μmol / ml, PAMAM 3.0 generation, and reaction time 7 h.

[0076] The above specific implementation manner does not limit the technical solution of the present invention in any form. Any technical solution obtained by equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A method for preparing dendrimer-functionalized fluorescent carbon nanoparticles, characterized in that: The dendrimer-functionalized fluorescent carbon nanoparticles are obtained by modifying the surface of fluorescent carbon nanoparticles with polyamidoamine dendrimers to obtain PAMAM@FCNs, which have both the characteristics of excitation light independence and long-wavelength emission; the maximum emission wavelength of the PAMAM@FCNs is 585nm; Here are the steps: (1) citric acid and triaminobenzene are ultrasonically dissolved and then transferred to a high-pressure reactor to synthesize fluorescent carbon nanoparticles by a hydrothermal method. The obtained fluorescent carbon nanoparticle stock solution is dialyzed, concentrated, precipitated, washed, and dried to obtain fluorescent carbon nanoparticle solids; The concentration of triaminobenzene is 0.015~0.15 mol / L, and the concentration ratio of citric acid to triaminobenzene is 1:1~1:80; (2) Take an appropriate amount of fluorescent carbon nanoparticles and dissolve them in ultrapure water at a concentration of 0.2~2.0 mg / mL. Add NHS to control the pH in the range of 4.0-7.0 to activate the carboxyl groups on the surface of the fluorescent carbon nanoparticles. Add EDC to adjust the pH in the alkaline range of 7.4-9.0 to couple the fluorescent carbon nanoparticles with PAMAM. Add PAMAM at 0.0-5.0 generations and a concentration of 0.15~1.50mmol / L. Use the EDC coupling method and react at 37°C for 3~10 h to form an amide bond between the carboxyl groups of the fluorescent carbon nanoparticles and the amino groups of PAMAM. Through covalent functionalization, PAMAM is coupled to the surface of the fluorescent carbon nanoparticles to obtain dendrimer-modified carbon nanoparticles PAMAM@FCNs. The obtained PAMAM@FCNs stock solution is dialyzed, concentrated, and freeze-dried to obtain PAMAM@FCNs solid.

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