RU2802348C1 - Method for purification of single-layer carbon nanotubes - Google Patents

Abstract

FIELD: nanotechnology.

SUBSTANCE: invention relates to a method for cleaning and "soft" oxidation of the surface of single-walled carbon nanotubes (SWCNTs). The invention can be used to clean the surface of nanotubes for their subsequent functionalization with various biologically active molecules for the purpose of biomedical applications for the therapy of a number of pathologies, as well as for the use of SWCNTs in the manufacture of accurate diagnostic instruments and probes. The method for cleaning single-walled carbon nanotubes (SWCNTs) consists in dispersing SWCNTs in water, then they are diluted in a buffer at neutral pH to a concentration of 100 mcg/ml, after which the treatment of SWCNTs with hypochlorite is started, adding it to a concentration of 50-250 mcM, at the same time, oxidation is monitored by instrumental measurement of optical absorption spectra not earlier than 7-8 hours after each addition of hypochlorite, which is carried out until the amplitude of the suspension spectrum in the near infrared region decreases by 12-20% relative to the control sample, to which an equivalent water volume is added instead of hypochlorite.

EFFECT: increased efficiency of purification of SWCNTs.

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Description

The field of technology to which the invention belongs

The invention relates to the field of nanotechnology, namely to a method for cleaning and "soft" oxidation of the surface of single-walled carbon nanotubes (SWCNTs). The invention can be used to clean the surface of nanotubes for their subsequent functionalization with various biologically active molecules for the purpose of biomedical applications for the therapy of a number of pathologies, as well as for the use of SWCNTs in the manufacture of accurate diagnostic instruments and probes.

State of the art

In the last decade, there has been significant progress in the use of nanomaterials for biomedicine, especially in the field of carbon nanotubes (CNTs). A carbon nanotube is a cylinder made of one layer of graphene (single-walled nanotubes, SWCNTs), or several coaxial cylinders (multilayer). Due to their structure and size, carbon nanotubes have unique physical and chemical properties. They are 20 times stronger than steel, chemically inert, electrical conductivity is 1000 times higher than that of copper, have special conductive properties, etc. This makes them promising for use in various fields of high technologies. In addition, a high surface area to length ratio, high thermal conductivity, and good ability to penetrate into cells make CNTs promising nanoparticles for medical applications for targeted drug delivery, tissue engineering, bioimaging, biosensors, and tumor therapy (Thakur A., et al. Carbon nanotubes: Types, synthesis, cytotoxicity and applications in biomedical Materials Today: Proceedings, 2022; 50(5): 2256-2268 doi: 10.1016/j.matpr.2021.10.002). In order to improve the biocompatibility of SWCNTs, they are functionalized, that is, the surface of nanotubes is chemically modified to improve their solubility, reduce toxicity, and impart special properties for use in targeted therapy. CNTs are oxidized with strong acids, modified with polyglycols, etc. Attaching target molecules and drugs to the surface of nanoparticles makes it possible to provide targeted therapy for various local pathologies. The functionalization of nanotubes can be covalent, then various molecules are chemically attached to them that improve their biocompatibility (PEG, PEI), as well as drugs or target molecules. In the case of non-covalent functionalization, biomolecules, surfactants, polymers, and other chemicals are sorbed on the CNT surface. Non-covalent bonding occurs through adsorption forces such as electrostatic force, hydrogen bonds, van der Waals forces, and π-stacking interactions (Murjani, BO, et al. Carbon nanotubes in biomedical applications: current status, promises, and challenges Carbon Lett 2022; 32:1207-1226 doi:10.1007/s42823-022-00364-4). On the other hand, the same forces ensure the sorption of various foreign substances on the surface of nanotubes, such as proteins, lipids, bacterial lipopolysaccharides, etc. For example, the binding of proteins to CNTs is very strong - the dissociation constants of protein complexes with nanotubes lie in the range of 1–0.1 μM. Since sterility is not observed in the production of nanotubes and in further laboratory work with them, various active substances can be adsorbed on the surface of nanoparticles, which change the properties of nanoparticles. The removal of organics, as well as impurity residues of amorphous carbon left after the catalytic processes of CNT synthesis, is possible using a number of physical methods, as well as using solvents such as alcohol and H ₂ O ₂ (Aleksashina EV et al. Acid activation of carbon nanotubes. Condensation Environment and interphase boundaries 2009; 11(2): 101-105; Zherlitsin AG et al. Obtaining carbon nanotubes from natural gas. Bulletin of Science of Siberia 2012; 3(4): 30-36). However, these substances are difficult to remove from nanotubes; they can remain in the inner cavity of nanoparticles and affect their properties.

Chemists also know the standard methodical approach for cleaning, modifying and shortening nanotubes - this is their oxidation as a result of sonication or boiling in a mixture of hydrogen peroxide and a strong acid (HNO ₃ , HCl) (Strong, KL, et al. Purification process for single-wall carbon nanotubes Carbon 2003 41(8): 1477-1488 DOI: 0.1016 / S0008-6223(03)00014-9). Oxidation of nanotubes makes it possible to get rid of metal impurities and amorphous carbon, and also leads to the appearance of charged carbonyl and hydroxyl groups on their surface, which improves the solubility and biocompatibility of nanoparticles. This purification procedure is carried out for most nanotubes in their commercial production.

The inventors also conducted studies in which they studied the oxidation of the CNT surface with reactive oxygen species and hopohalic acids. It has been shown that the most effective oxidant capable of oxidizing and degrading (destroying) nanotubes is hypochlorous acid (HOCl), the main product of neutrophil myeloperoxidase. To prove the possibility of CNT biodegradation in vivo in the focus of inflammation, we carried out long-term treatment of CNTs, both single-layer (5-7 days) and multilayer (10-15 days), with high concentrations of NaOCl hypochlorite (1-10 mM) - the sodium salt of HOCl, which at neutral pH, it exists in solution in equilibrium with HOCl (Vlasova I.I. et al. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes. Toxicol Appl Pharmacol. 2012; 264(1):131 Masyutin AG, Bagrov D.V., Vlasova 1.1., et al., Wall Thickness of Industrial Multi-Walled Carbon Nanotubes Is Not a Crucial Factor for Their Degradation by Sodium Hypochlorite. 2018;8(9):715 doi: 10.3390/nano8090715).

In the works of other authors, hypochlorite was also used for the oxidation and degradation of nanotubes. Treatment of native (as produced) CNTs with hypochlorite to improve their solubility was previously proposed (application US 2009/0048386 Al, published Feb. 19, 2009. METHOD FOR TREATMENT OF CARBON NANOTUBES). The authors used extremely high concentrations of hypochlorite (0.1–10% or up to 1.2 M), and, consequently, alkaline solution pH and short incubation times (from minutes to 24 hours). At such high concentrations of NaOCl, the modification of CNTs occurs uncontrollably. The authors of the application measured the formation of charged groups on the surface of CNTs, however, this parameter alone is not a characteristic of the modification of nanotubes - as a result of oxidation, defects (holes) can form on their surface, and in the places of these defects, CNTs become brittle and lose their unique properties.

In the application US 2010/0190239 A1, publ. On July 29, 2010, the treatment of nanotubes with peroxidases - horseradish peroxidase or myeloperoxidase - in the presence of hydrogen peroxide was proposed (Star A., Kagan V.E., Allen B.L. DEGRADATION OF NANOMATERIALS, 2010, Pub. No.: US 2010/0190239 A1). Oxidants produced by enzymes react with nanotubes (Authors publication: Kagan VE, Konduru N.V., Feng W., Allen B.L., Conroy J., Volkov Y., Vlasova I.I. et al. Carbon nanotubes degraded by neutrophil myeloperoxidase induce less pulmonary inflammation. Nat Nanotechnol. 2010;5(5):354-359 doi: 10.1038/nnano.2010.44), modify their surface, which facilitates the degradation of CNTs in the body, and can also be used to remove CNTs accumulated in the environment at the place of their production. However, the added peroxidases themselves are biologically active materials and can remain on the NT surface and react with other molecules.

Other physicochemical approaches have also been used to purify and modify the properties of CNTs. The combined use of microwave radiation and Cl ₂ gas makes it possible to effectively remove impurities from transition metal catalysts used in the synthesis of CNTs (Gomez V, et al. Enhanced purification of carbon nanotubes by microwave and chlorine cleaning procedures. RSC advances. 2016; 6(14): 11895-11902. doi:10.1039/C5RA24854J). Patent RU 2296046 CI proposes heating nanotubes to a temperature of 1500-1600°C in zinc sulfide vapor to increase the sorption capacity of nanoparticles.

Hypochlorous acid is known to react with amorphous carbon. Activated carbon is used for water dechlorination (С*+HOCl=С*O+Cl-+Н+) (https://aquaboss.ru/poleznye-stati/udalenie-hlora-i-chloramina-na-aktivirovannom-ugle.html ), so it can be expected that amorphous carbon adsorbed on carbon nanotubes will be oxidized in the presence of hypochlorite.

It should be noted that all known methods of processing nanotubes do not allow solving the problem of increasing their biocompatibility, since they do not take into account the need for surface cleaning and there is no constant control over the process of oxidative modification of NTs, which is important for maintaining a balance between the properties of NTs and their solubility/biocompatibility. . Unpurified organic molecules sorbed on the surface of CNTs can have an adverse effect on cells and tissues, and especially on cells of the immune system.

Disclosure of the essence of the invention

The technical result of the invention is to increase the efficiency of purification of SWCNTs. As a result of the implementation of the method, nanotubes are obtained that do not contain organic impurities on the surface and accompanying amorphous carbon, with continuous monitoring of the process of oxidative modification of SWCNTs.

The technical result is achieved due to the fact that SWCNTs are dispersed in water, then they are diluted in a buffer at neutral pH to a concentration of 100 μg/ml, after which the SWCNTs are treated with hypochlorite, adding it to a concentration of 50-250 μM, while monitoring oxidation by instrumental measurement of optical absorption spectra not earlier than 7-8 hours after each addition of hypochlorite, which is carried out until the amplitude of the suspension spectrum in the near infrared region decreases by 12-20% relative to the control sample, to which an equivalent volume of water is added instead of hypochlorite.

In this case, for each type of nanotubes, it is necessary to select its own treatment time (the amount of additions of NaOCl to SWCNTs), so that the decrease in the amplitude of the absorption spectra of the SWCNT suspension is 12–20%.

The proposed method for cleaning the SWCNT surface is based on a difference of several orders of magnitude in the reaction rate of HOCl with amorphous carbon or organic substances and with strong SWCNTs. The rate constant of the reaction of NaOCl with most chemical compounds, including proteins and lipids, is ~ 1-10 ⁶ M ^-1 s ^-1 ) (Pattison DI, et al. Reactions of myeloperoxidase-derived oxidants with biological substrates: gaining chemical insight into human inflammatory diseases, Curr Med Chem., 2006; 13(27):3271-3290. doi: 10.2174/092986706778773095), which will make it possible to clean the CNT surface from possible impurities. Previously, in the work of the authors of the invention, it was shown that the rate of the reaction of the oxidizer with SWNTs is very slow, for example, for carboxylated SWNTs (c-SWNTs) it is (450 ± 50) nmol HOCl per hour per 1 mg of c-SWNTs (Vlasova I.I. Myeloperoxidase-induced degradation of single-walled carbon nanotubes is determined by the synthesis of hypochlorite, Bioorganic Chemistry 2011;37(4):510-521).

Brief description of the drawings

The invention is illustrated by drawings, where in Fig. 1 shows a photograph of suspensions of c-SWNTs (100 μg/ml) after treatment with hypochlorite at a total concentration of 1 - 0 μM, 2 - 100 μM, 3 - 1.5 mM, Fig. 2 shows the corresponding absorption spectra of the samples. Sample 3 illustrates that c-SWNTs can be completely destroyed by adding high concentrations of hypochlorite. In FIG. Figure 3 shows the Raman spectra of c-SWNTs after treatment with hypochlorite in the total concentration: 1 - 0 µM, 2 - 250 µM, 3 - 1.5 mM G-band 1597 cm-1, D-band 1350 cm-1. The spectra are normalized by the amplitude of the G-band.

Implementation of the invention

The proposed method for cleaning single-walled carbon nanotubes is carried out as follows.

The authors experimentally established the main regime characteristics of the method.

Nanotubes are dispersed in deionized water using an ultrasonic sonicator (Sonicator Q55, Qsonica, USA) at a power of 10 W for 30 s several times with 15 s intervals (the tube is placed on ice) or in an ultrasonic bath (Elma Ultrasonic, Germany) at 4- 10°C for several hours, so that the total time is at least 10 hours. The use of ultrasonic sonication makes it possible to obtain a suspension of single or several SWCNT aggregates assembled into bundles. The option of using an ultrasonic bath is preferable, since the native structure of nanoparticles is better preserved. Then the SWCNT suspension is centrifuged at 5000 g for 30 min to precipitate large aggregates of nanoparticles, and the supernatant is used for further processing. Stock suspensions of CNTs in water (0.8–1.5 mg/mL) are stored in the dark at 4°C. For long-term storage (more than 1 month), it is necessary to re-centrifuge.

At the same time, the concentration of nanotubes is determined using the extinction coefficient in the near infrared region (1050-1080 nm), known from the manufacturer or measured according to the standard method (Liu Z, et al. Supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery. ACS Nano 2007;1(1):50-56 doi: 10.1021/nn700040t.).

Then SWNTs are dissolved in 50 mM Na-phosphate buffer, pH 7.2 to a concentration of 100 μg/mL for further treatment with hypochlorite.

NaOCl is stable as a solution of 10-15% chlorine in water containing NaOH. The concentration of NaOCl in the original solution is determined by measuring the optical density at 290 nm (ε=350 M ^-1 cm ^-1 ) after successive dilutions of the reagent in 5 mm NaOH. - The concentration of commercial NaOCl solutions is between 1 and 2 M.

Aliquots of NaOCl are added to SWCNTs resuspended in buffer to a reagent concentration of 50-250 μM. If less than 5 µL of the initial hypochlorite solution must be added to the SWCNT suspension to achieve the desired NaOCl concentration, an intermediate solution of NaOCl in buffer with mM concentration must be prepared immediately before addition. An equivalent amount of water is added to the control unmodified SWCNT samples.

When implementing a method for cleaning single-walled carbon nanotubes, the authors monitored the oxidation of SWCNTs by measuring their absorption spectra, which can be done using a spectrophotometer that allows measurements in the near infrared region (for example, a Cary-50 spectrophotometer, Varian, USA). Measurement of the optical spectra of the SWCNT suspension is carried out no earlier than 7-8 hours after each addition of NaOCl. This time is sufficient for hypochlorite to react with SWCNTs if they do not contain impurities. Incubation can be carried out longer (eg overnight incubation). Incubation is carried out in a dark place at room temperature. If nanotube aggregates are visible in the suspensions or the absorption in the control changed by more than 10%, before recording the spectra, the SWCNT suspensions are subjected to ultrasonic treatment for 15 s twice with an interval of 10 s using an ultrasonic homogenizer (Sonopuls HD 2070, Bandelin Electronic, Germany) at 10 W, 20 kHz. To measure optical spectra, SWNTs are diluted in water so that measurements are made in the optimal absorbance range recommended for the instrument. To register Raman spectra, a drop of the solution is dried on glass. The study of samples by Raman spectroscopy is carried out using a 514 nm laser (Ar, 20 mW) using ND (neutral density) filters with a variable power in the range of 0.1-1 mW.

The total amount of added hypochlorite required to remove impurities and start mild oxidation of the CNT surface depends on the amount of impurities, but usually does not exceed 1 mM. The purification of nanotubes from impurities and the oxidation of their surface can be considered completed when the absorption of the suspension at 1060 nm decreased by 12–20%. A decrease in optical absorption means that the modification of the SWCNT carbon surface begins; further processing can lead to the modification of the SWCNT structure and their degradation.

The invention is illustrated by examples.

Example 1

Carboxylated single-walled CNTs (c-CNTs) were purchased from Carbon Solutions Inc. (USA). 5 mg of c-SWNTs were placed in an Eppendorf tube and 1 ml of mQ water was added. Nanotubes were sonicated using an ultrasonic sonicator Sonicator Q55 (Qsonica, USA), placing the probe of the device into a test tube, which was placed in a beaker with ice. Ultrasonic treatment was carried out at a power of 10 W for 30 s 5 times with 15 interruptions. The resulting suspension was centrifuged at 5000g for 30 min, the supernatant was taken, and it was centrifuged again under the same conditions. To determine the c-SWNT concentration, an extinction coefficient of 13.2 ml mg ^-1 cm ^-1 was used (Vlasova II, et al. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes. Toxicol Appl Pharmacol. 2012; 264(1): 131-142 doi: 10.1016/j.taap.2012.07.027). The concentration was (1.5±0.2) mg/ml. The nanotube suspension was added to 2 ml of 50 mM Na-phosphate buffer, pH 7.2, so that the final concentration of c-SWNTs was 100 μg/ml. The suspension was distributed in 1 ml tubes - control and sample for treatment with NaOCl. The commercial NaOCl solution had a concentration of 1.5 M hypochlorite and was diluted 150 times to prepare a working 10 mM NaOCl solution in buffer. 5 µl of this solution was added to the sample to a final concentration of 50 µM. 5 μl of water was added to the control sample. After the first addition, the suspensions were incubated for 8 h at room temperature in a dark place; after incubation, the absorbance at 1060 nm was measured. To do this, an aliquot of c-SWNT suspension was taken and distilled water was added to it in a ratio of 1:2. The measurements were repeated 3 times; for measurements, a Cary-50 spectrophotometer, Varian, USA was used. The absorbance of the hypochlorite-treated sample did not change significantly (the absorbance in the sample with hypochlorite decreased by less than 10% relative to the control). After that, another 5 μl of 10 mM NaOCl was added to the suspension and the suspension was incubated for 16 h (overnight), after which the absorbance was measured: absorbance changes were (18 ± 4)% of the values in the control suspension. Spectra were recorded to control the NT modification. A photo of SWNT suspensions is shown in Fig. 1, the corresponding absorption spectra are shown in FIG. 2. Further additions of the reagent lead to degradation (destruction) of nanostructures (Figs. 1, 2, sample 3). The degradation of nanoparticles is accompanied not only by a decrease in the optical absorption of solutions, but also by the disappearance of the characteristic absorption bands of SWCNTs - metallic (Ml) and semiconductor (S2). In the case of cleaning and weak oxidation of the SWCNT surface, the addition of hypochlorite is possible only if the Ml and S2 bands are completely preserved (Fig. 2, curve 2).

CNT degradation can be characterized by other methods, such as dynamic light scattering and Raman spectroscopy. However, these methods are more laborious than the measurement of absorption spectra, are less reliable and require special equipment. In FIG. Figure 3 shows the Raman spectra of nanotubes with different degrees of degradation obtained using a multifunctional Raman scattering spectroscopy system (Insight Solutions, Russia). The main difference between the spectra is that the degradation of nanotubes is accompanied by an increase in the amplitude ratio of the G and D peaks, which serves as a standard marker of SWCNT oxidation (Russier J. et al. Oxidative biodegradation of single-and multi-walled carbon nanotubes. Nanoscale. 2011; 3(3):893 -896. doi: 10.1039/c0nr00779j).

The obtained SWCNTs do not contain organic impurities and amorphous carbon, they can be further precipitated by ultracentrifugation (at least 200,000 g) and dried; in this form, nanotubes are stored in a refrigerator for several years, and can later be used for functionalization with target molecules.

Example 2

As an example of the contamination of nanotubes with organic compounds, we used lipopolysaccharides (LPS), which were added to a suspension of c-SWNTs. c-SWNTs were prepared as described in Example 1. Next, the nanotubes were diluted in 1 ml of 50 mM Na-phosphate buffer, pH 7.2, containing 1 mg/ml LPS (about 200 μM). The solution was distributed into test tubes of 0.5 ml - control and sample for processing. A working solution of 10 mM NaOCl in buffer was prepared. Hypochlorite interacts rapidly with LPS, as with any organic molecule, it can be expected that part of the added hypochlorite will go to interact with LPS. Therefore, additives increased the concentration of hypochlorite in the samples. The addition of 75 μM NaOCl followed by an incubation of 7 hours did not change the absorbance of the sample relative to the control (sample to which water was added). After the second addition of 75 μM and subsequent incubation for 16 hours, no significant (more than 10%) differences were observed in the absorption of the sample and control. After the third addition of 75 µl, the absorption of the suspension decreased by (15±2)%. At the same time, the shape of the spectra did not change; the specific absorption bands M1 and S2 had the same shape as in the control sample. The total concentration of added hypochlorite was 225 μM, which is higher than in example 1, which indicates the interaction of NaOCl with added LPS.

Example 3

Amidated SWCNTs (am SWCNTs) contain amide groups covalently bonded to the surface of nanotubes. Not more than 1% of the carbon atoms of CNTs are associated with amide groups. 3 mg of c-SWNTs were placed in an Eppendorf tube and 500 µl of mQ water was added. In order not to disturb the functionalization of SWCNTs, in this case, softer ultrasonic treatment was used. Sonication of nanotubes was carried out in an ultrasonic bath (Elma Ultrasonic, Germany) at 4–10°C for 2 hours 8 times with interruptions for several hours. The resulting suspension was centrifuged at 5000g for 30 min, the supernatant was taken and centrifuged again under the same conditions. An extinction coefficient of 15 ml mg ^-1 cm ^-1 was used to determine the PEG-SWNT concentration. The absorption spectra of these CNTs also look like the spectra of c-SWNTs (Fig. 2). Hypochlorite reacts with amide (peptide) groups to form chloramides (Hawkins CL et al. Hypochlorite-induced oxidation of amino acids, peptides and proteins. Amino acids 2003; 25:259-274. DOI 10.1007/s00726-003-0016-x) , so the concentration of hypochlorite in the suspension after each addition was increased to 100 μm. After the first and second additions of 100 μM NaOCl and sample incubation for 8 and 16 hours, respectively, no difference was observed in absorbance at 1060 nm between the control sample and the sample treated with hypochlorite. The third addition of 100 μM NaOCl resulted in a decrease in sample uptake by (10±2)% relative to the control. After the fourth addition, the absorption of control and sample differed by (19±2)%. The spectra of the control and samples treated with hypochlorite did not differ in shape. Thus, the processing of am-SWNTs was verified after the addition of 400 μM NaOCl.

Example 4

PEGylated SWCNTs (600 Da PEGs) were obtained from Carbon Solutions Inc. (USA). The type of PEG-CNT used is a copolymer in which not more than 1% of the CNT carbon atoms are bonded to PEG molecules and the PEG content is 10-25 wt. %. Analogously to example 3, an ultrasonic bath was used to disperse SWNTs. To determine the concentration of PEG-SWNTs, an extinction coefficient of 20.2 ml mg ^-1 cm ^-1 was used. The absorption spectra of these CNTs also look like the spectra of c-SWNTs (Fig. 2). To purify PEG-SWNTs, NaOCl was added to them so that the concentration of the reagent after each addition was 250 μM. Hypochlorite does not react with PEG. To reduce the amplitude of the spectrum of the suspension of PEG-SWNTs in phosphate buffer pH 7.2 in the region of 1060 nm by (20 ± 2)%, it was necessary to make 2 additions of hypochlorite, with a total concentration of 500 μM NaOCl.

Thus, the proposed method achieves complete removal of possible impurities from the SWCNT suspension, such as organic molecules and amorphous carbon, followed by partial oxidation of the SWCNT surface (a decrease in absorption by 12–20%), which improves the biocompatibility and biodegradability of nanoparticles.

Claims (1)

A method for cleaning single-walled carbon nanotubes (SWCNTs), which consists in dispersing SWCNTs in water, then diluting them in a buffer at neutral pH to a concentration of 100 µg/ml, after which the treatment of SWCNTs with hypochlorite is started, adding it to a concentration of 50-250 µM , while monitoring oxidation by instrumental measurement of optical absorption spectra not earlier than 7-8 hours after each addition of hypochlorite, which is carried out until the amplitude of the suspension spectrum in the near infrared region decreases by 12-20% relative to the control sample, to which instead of hypochlorite is added equivalent volume of water.