Chiral luminescent carbon nanoparticles and method for their preparation

Abstract

FIELD: nanotechnology.SUBSTANCE: chiral luminescent carbon nanoparticle for photoluminescent imaging of biological objects has been disclosed, which has optical transitions and emission in the red region of the spectrum, the surface of the nanoparticle is functionalized with R- or S-α-methylbenzyl isocyanate with the formation of a covalent bond between the chiral isocyanate and the surface of the nanoparticle. A method for its synthesis is also disclosed, which consists of post-synthetic functionalization of the surface of R- or S- carbon nanoparticles. α-methylbenzyl isocyanate followed by purification using dialysis bags.EFFECT: functionalization of the surface of carbon nanoparticles with chiral molecules and the emergence of circular dichroism signals in the spectral region from 300 nm while maintaining luminescent characteristics.2 cl, 1 dwg, 1 ex

Description

[0001] Field of technology to which the invention relates

[0002] The invention relates to the field of nanotechnology and nanomaterials, namely to the production of chiral luminescent carbon nanoparticles for targeted interaction with chiral biological objects and obtaining their images.

[0003] Technology Level

[0004] To visualize biological objects and processes, as well as to sensor biological molecules, which are in most cases chiral, it is necessary to create materials that meet the following criteria: possess optical transitions in the red region of the spectrum to avoid the registration of autoluminescence of biological objects in the blue-green region of the spectrum; have a chiral surface for selective interaction with biological objects; have circular dichroism signals in the region greater than 300 nm.

[0005] Carbon nanoparticles with red photoluminescence are known, synthesized according to the protocol described in the patent “Carbon nanoparticles and the method for their manufacture” (patent for invention No. 2841407 A61K 49 / 06, B82Y 40 / 00, C01B 32 / 15, C09K 11 / 77), according to which the precursors: formamide, citric acid and gadolinium salts (Gd(NO3)3), taken in a molar ratio of 19:1:0.01, are mixed for 5 minutes under the influence of ultrasound until completely dissolved, heated at 180 ° C for 8 hours, after which the mixture is cooled, dissolved in alcohol and centrifuged to isolate the precipitate for 15 minutes at 6000 rpm, after which the supernatant remove, and with the sediment, repeat the dissolution in alcohol and centrifugation, isolating the sediment each time and removing the newly formed supernatant until it acquires a yellow tint,After dialysis using dialysis bags with a molecular weight cutoff of 3500 Da against water, the carbon nanoparticles are dissolved in water and the precipitate is re-isolated under the same centrifugation parameters. The resulting precipitate is dried by lyophilization to obtain dark powders of carbon nanoparticles. The resulting synthesis yields carbon nanoparticles consisting of 59.9% C, 20.5% O, 19.1% N, and 0.5% Gd (atomic %), the long-wave photoluminescence band of which is located at 640 nm, the relaxivity r1 is equal to 20.99 l×mmol, -1 ×с -1 The disadvantages of the analogue include the lack of selectivity when interacting with biological objects due to the absence of a chiral / functionalized surface, which can lead to an increase in false signals in bioimaging and a decrease in sensitivity in biosensing.

[0006] Chiral carbon nanoparticles with red photoluminescence are known, synthesized according to the postsynthetic processing protocol described in the publication of Vedernikov AA et al. Green and red emissive N, O-doped chiral carbon dots functionalized with L-cysteine ​​ / / The Journal of Physical Chemistry Letters. - 2023. - Vol. 15. - No. 1. - Pp. 113-120, according to which, in the first stage, carbon nanoparticles are synthesized from a mixture of 2 g of citric acid and 1 g of urea in 10 ml of dimethylformamide by autoclaving at 180 ° C for 8 hours, the resulting product is cooled and purified using dialysis bags with a molecular weight cutoff of 3500 Da against water for 72 hours. In the second step, the surface of carbon nanoparticles is functionalized with chiral L- / D-cysteine ​​molecules by forming an amide bond, for which 0.2 mmol EDC is first added to 5 ml of a carbon nanoparticle solution with a concentration of 2.8 mg / ml and left with constant stirring for 1 hour.Next, 0.5 mmol of NHS is added to the mixture and the mixture is stirred for 20 min. Then, 1.3 mmol of cysteine ​​molecules is added and the mixture is stirred for 12 hours. Finally, the mixture is dialyzed against water for 5 hours using dialysis bags with a molecular weight cutoff of 500 Da. The resulting carbon nanoparticles exhibit green and red photoluminescence, with the long-wavelength photoluminescence band maximum at 630 nm upon excitation at 550 nm. The circular dichroism signal is observed in the 200–300 nm range with a maximum dissymmetry factor of 6.15 × 10. -5 (at 220 nm) and 2.3 × 10 -4(at 260 nm). A disadvantage of the analog is that the presented material exhibits circular dichroism signals in the blue region of the spectrum (up to 300 nm), which can overlap with signals from biological objects (chiral molecules contained in the analyzed samples), which in turn complicates the sensing of chiral biological objects and the processes in which they are involved.

[0007] The prototype of the claimed invention is carbon nanoparticles functionalized with fluorescein, synthesized according to the protocol described in the publication of Sun S. et al. Toward high-efficient red emissive carbon dots: facile preparation, unique properties, and applications as multifunctional theranostic agents / / Chemistry of Materials. - 2016. - Vol. 28. - No. 23. - Pp. 8659-8668, according to which, in the first stage, carbon nanoparticles are obtained by autoclaving 2.8 g of citric acid in 50 ml of formamide in a microwave reactor at 160 ° C (400 W) for 1 hour and at 120 ° C (400 W) for 1 hour.The resulting product is then cooled to room temperature, the carbon nanoparticles are precipitated from the solution by adding 250 ml of acetone and cooling in a freezer at -20°C overnight, the precipitate is then washed with 50 ml of acetone and a methanol / acetone mixture in a ratio of 10 / 90, the precipitate is then dispersed in methanol and filtered through a membrane with a pore size of 0.22 μm. The functionalization of carbon nanoparticles occurs as follows: 2 mg of fluorescein isothiocyanate and 10 mg of carbon nanoparticles are added to 20 mL of anhydrous methanol in a flask, then 20 μL of triethylamine are added, the resulting mixture is stirred for 24 hours at room temperature, after which the mixture is diluted 5 times with 0.1 M NaHCO3 and purified using dialysis bags with a molecular weight cutoff of 1000 Da against 0.1 M NaHCO3 for 2 days, then against deionized water for 2 days.The synthesis results in carbon nanoparticles with a maximum emission intensity at 640 nm with excitation at 540 nm, a photoluminescence quantum yield of 22.9%, and a surface functionalized with fluorescein isothiocyanate, which is a common fluorescent dye for labeling biomolecules.

[0008] The disadvantages of the prototype include the following properties of carbon nanoparticles: (1) low reactivity of isothiocyanates compared to isocyanates, which reduces the efficiency of their surface functionalization; (2) the lack of chirality of isothiocyanates, therefore, the carbon nanoparticles from the prototype also do not have surface chirality, which leads to low selectivity when interacting with biological objects, limiting their use for luminescence imaging.

[0009] The problem to be solved by the proposed invention is the creation of carbon nanoparticles with the ability to selectively bind to a biological object of interest for effective luminescent imaging or sensing.

[0010] The stated problem is solved by achieving a technical result consisting in the functionalization of the surface of carbon nanoparticles with chiral molecules and the emergence of circular dichroism signals in the spectral region from 300 nm while maintaining their luminescent characteristics.

[0011] This technical result is achieved by the fact that carbon nanoparticles with the property of photoluminescent visualization of biological objects in the red region of the spectrum, with sizes of 1 - 15 nm, consisting of carbon, oxygen and nitrogen, are distinguished by the fact that the carbon nanoparticles have a chiral surface, selective interaction with biological objects, signals in the circular dichroism spectra in the region of more than 300 nm and responses in magnetic resonance imaging.

[0012] Disclosure of the essence of the invention

[0013] A method for postsynthetic functionalization of the surface of carbon nanoparticles, which consists in forming carbon nanoparticles having optical transitions in the red region of the spectrum, with amines or hydroxyls on the surface, in which a mixture of carbon nanoparticles and isothiocyanate is added to an anhydrous solvent with the addition of triethylamine, the mixture is left to react, and then purified using dialysis bags, characterized in that chiral isocyanates, such as R- or S-α-methylbenzyl, are used instead of isothiocyanate, anhydrous dimethylformamide is used as an anhydrous solvent, the resulting mixture is left for 2 hours to undergo a reaction of covalent binding of the isocyanate to the surface of the nanoparticles at room temperature, then purified using dialysis bags with a molecular weight cutoff of 3500 Da against water for 48 hours.

[0014] These chiral carbon nanoparticles retain the optical and magnetic properties (absorption and intense long-wavelength photoluminescence, as well as magnetic resonance imaging responses) of the original achiral nanoparticles: the excitation wavelength is 530–650 nm, and the photoluminescence signal is detected in the range of 580–700 nm. Such spectral responses improve the signal-to-noise ratio during bioimaging. This effect is due to a reduced probability of autofluorescence in biological tissues, as well as an increased penetration depth due to the use of a source for excitation and detection of nanoparticle photoluminescence in the long-wavelength range.

[0015] The presence of chiral isocyanates covalently bound to the surface of carbon nanoparticles enables selective interaction with chiral biological objects, such as chiral molecules (RNA, glutathione, etc.) and cell membranes, during the visualization of biological processes. Moreover, the responses in the circular dichroism spectra of the resulting chiral carbon nanoparticles are in the region greater than 300 nm, increasing the sensitivity of the measurements, as signals from biological objects, particularly chiral molecules, are present in the region below 300 nm. In addition to the unique optical properties of chiral nanoparticles, the proposed method offers the following advantages: synthesis at room temperature, high reactivity of isocyanates, and simple purification of the product by dialysis.

[0016] To use the developed chiral carbon nanoparticles as tissue imaging labels, an aqueous solution of the nanoparticles is prepared at a concentration of no more than 100 mg / ml with the addition of 5% (v / v) dimethyl sulfoxide. The solution is then sonicated to improve dispersibility of the nanoparticles in water. This solution is added to the cell culture medium or injected into the tissue.

[0017] The following example reveals the essence of the invention. Carbon nanoparticles were synthesized from 11.7 mg of Gd(NO3)3⋅6H2O and 0.5 g of citric acid in 2 ml of formamide.

[0018] Step 1. A mixture of gadolinium salt and citric acid in formamide is heated in an autoclave at 180°C for 6 hours. After the reaction, the autoclave is naturally cooled to room temperature. The reaction mixture is precipitated by adding 200 ml of ethanol and centrifuging for 15 minutes at 6000 rpm. The precipitate is then dispersed in water and purified using dialysis bags with a molecular weight cutoff of 3500 Da for 5 days. The resulting product is lyophilized to a violet-red powder.

[0019] Step 2. Nanoparticle surface functionalization. 4 mg of red carbon nanoparticles are dispersed in 1 mL of anhydrous dimethylformamide, 10 μL of chiral R- or S-α-methylbenzyl isocyanate and 5 μL of triethylamine are added. The mixture is left to react for 2 hours at room temperature, then purified using dialysis bags with a molecular weight cutoff of 3500 Da for 48 hours.

[0020] Chiral carbon nanoparticles exhibit long-wavelength photoluminescence with a band maximum at 640 nm and a quantum yield of 21 ± 2%, circular dichroism signals are in the region of 300 - 650 nm with a maximum dissymmetry factor of 1.0 × 10 -4 at 350 nm.

[0021] Implementation of the invention

[0022] X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy of the carbon nanoparticle samples described in the example showed the presence of oxygen- and nitrogen-doped carbon matrix, and the formation of N'-C(=O)-N'' and N'-C(=O)-O bonds with chiral isocyanates was confirmed. Circular dichroism spectroscopy revealed signals in the wavelength range greater than 300 nm. Absorption and photoluminescence spectroscopy revealed optical transitions in the wavelength range greater than 550 nm. The circular dichroism and absorption spectra for the chiral carbon nanoparticles from the example are shown in the figure.

[0024] The selective interaction of chiral carbon nanoparticles from Example 1 with chiral molecules is demonstrated using tryptophan enantiomers as an example. Circular dichroism spectroscopy revealed that the enantiomers of chiral carbon nanoparticles interact with L- / D-tryptophan differently: the circular dichroism spectrum of chiral carbon nanoparticles with the S-enantiomer of isocyanate on the surface during interaction with D-tryptophan showed a narrowing of the main band while maintaining the peak at 345 nm, while with L-tryptophan, the intensity of the peak at 345 nm decreased and a new band appeared at 425 nm.

[0025] The resulting chiral carbon nanoparticles almost completely retain the spectral characteristics of the original nanoparticles: they have long-wavelength emission with a band in the range of 600-660 nm when excited by radiation with a wavelength of 550 - 600 nm. The photoluminescence quantum yield, measured with excitation at a wavelength of 550 nm, for carbon nanoparticles is 21%. Chiral carbon nanoparticles have chiral properties, which is manifested in the form of selective interaction with chiral molecules and the presence of signals in the circular dichroism spectra in the wavelength range of more than 300 nm with a dissymmetry factor of over 1.0 × 10 -4 .

[0026] Thus, the proposed carbon nanoparticles have enantioselective interaction with chiral biological objects, as well as efficient photoluminescence in the long-wavelength region of the emission spectrum, which is important for biovisualization of biological processes.

[0027] Figure. Spectral characteristics of chiral carbon nanoparticles from the example: 1 - circular dichroism spectrum of the S-enantiomer of chiral carbon nanoparticles, 2 - circular dichroism spectrum of the R-enantiomer of chiral carbon nanoparticles, 3 - absorption spectrum of the S-enantiomer of chiral carbon nanoparticles, 4 - absorption spectrum of the R-enantiomer of chiral carbon nanoparticles.

Claims

1. A chiral luminescent carbon nanoparticle for photoluminescent visualization of biological objects, consisting of carbon, oxygen and nitrogen, which exhibits optical transitions and emission in the red region of the spectrum, characterized in that the surface of the nanoparticle is functionalized, wherein the substance for functionalizing the surface of the carbon nanoparticle is a chiral isocyanate R- or S-α-methylbenzyl isocyanate with the formation of a covalent bond between the chiral isocyanate and the surface of the nanoparticle.

2. A method for synthesizing chiral luminescent carbon nanoparticles, which consists of postsynthetic functionalization of the surface of carbon nanoparticles with amines and hydroxyls on the surface obtained on the basis of citric acid and formamide, characterized in that a chiral isocyanate R- or S-α-methylbenzyl isocyanate is taken as a substance for functionalizing the surface of carbon nanoparticles, anhydrous dimethylformamide is used as a solvent, and consists of dispersing 4 mg of red carbon nanoparticles in 1 ml of anhydrous dimethylformamide, adding 10 μl R- or S-α-methylbenzyl isocyanate and 5 μl triethylamine, carrying out the reaction at room temperature for 2 hours, cleaning using dialysis bags with a molecular weight cutoff of 3500 Da against water for 48 hours, wherein the resulting nanoparticles have optical transitions and radiation in the red region of the spectrum, consist of carbon, oxygen and nitrogen,have a covalent bond between R- or S-α-methylbenzyl isocyanate and the surface of the nanoparticles.

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