Lithium-loaded nanocapsules and uses thereof

Abstract

The present invention relates to a lithium isotope ( 6 The present invention relates to carbon nanocapsules (CNCs) encapsulating Li and their use preferably in neutron capture therapy and cancer therapy. The CNCs are in particular carbon nanohorn aggregates (CNHs) or carbon nanotubes (CNTs) with closed ends.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to a lithium isotope ( 6 The present invention relates to carbon nanocapsules containing ions (Li) and their use primarily in neutron capture therapy. This therapy is useful in the treatment of cancer among other diseases. The present invention is therefore in the field of medicine.

[0002] [Background technology] Cancer is one of the major diseases worldwide, with over 37.5 million cases diagnosed in recent years, and is associated with high morbidity, prevalence, and mortality. Currently, there are several cancer treatments, including surgery, radiation therapy, and chemotherapy, to extend the life expectancy of cancer patients. However, ineffective treatment or significant toxicity remain as the main challenges of currently available cancer treatments. Neutron capture therapy (NCT) is a high linear energy transfer form of radiation therapy that utilizes the potential of certain isotopes in cancer treatment, based on the capture and release of neutrons, a short-range charged particle generated at low energy. When certain isotopes are irradiated with low-energy thermal neutrons, nuclear reactions occur, producing high linear energy transfer (LET) particles suitable for eradicating cancer cells. The limited path length (5-9 μm) of the LET particles generated in NCT can limit the destructive effect on isotopes localized inside the cells. Thus, it gives this type of radiation therapy a high therapeutic precision. Furthermore, for the treatment of deep-seated tumors, NCT currently uses epithermal neutron beams, which have higher tissue penetration.

[0003] There are many nuclides that have a high affinity for capturing thermal neutrons and are therefore potential candidates in NCT. Of particular interest is boron, which has proven to be one of the most promising elements for the development of effective compounds for NCT. 10 B captures thermal neutrons and initiates a nuclear reaction 10 B(n,α) 7The ability to induce Li has made boron neutron cancer therapy (BNCT) an option for many patients. By selectively delivering therapeutic doses of boron compounds to tumor cells, they can be destroyed after irradiation with a thermal neutron beam. This therapy has the advantage of a large dose gradient between tumor and normal cells, resulting in less damage to the latter. To date, BNCT has been clinically investigated in a variety of disease sites, including glioblastoma multiforme, meningioma, head and neck cancer, lung cancer, breast cancer, hepatocellular carcinoma, sarcoma, skin malignancies, extramammary Paget's disease, recurrent cancer, pediatric cancer, and metastatic disease (Malouff T. et al. Frontiers in Oncology 2021, 11: 601820). Although there is still much room for clinical improvement, evidence indicates the potential of BNCT in treating tumors that are otherwise incurable due to dissemination, invasion, recurrence, or proximity to radiosensitive tissue. Recently, clinical studies on patients with genital melanoma that are thought to be resistant to radiation therapy and chemotherapy have shown very impressive results (Hiratsuka J. et al:38). 10 All patients treated with B-enriched L-BPA (L-para-borophenylalanine) showed complete local tumor control within 6 months without any significant side effects.

[0004] Recent technological improvements have enabled medical institutions to install accelerators, bringing NCT closer to real clinical application (Dymova MA, et al. Cancer Communications 2020, 40: 406). However, for many years, the scientific community of BNCT has focused on the need for new carriers characterized by high tumor specificity of boron compounds used as capture agents. This leads to improved efficiency of treatment and reduced risk of toxicity. Since the dawn of NCT 60 years ago, many different delivery agents have been developed and studied (He H, et al. Radiation Oncology 2021, 16: 216). However, the drug adopted in clinical trials is sodium mercaptoundecahydro-closo-dodecaborate (BSH; Na2 10 B 12 H11 SH) and L-para-borophenylalanine (BPA; CH 12 10 There are only two types of derivatives:

[0005] In the last few years, several modern BNCT agents have emerged. Most of them consist of a stable boron moiety coupled to a tumor targeting unit. This group may include novel polyhedral boranes, boronated nucleosides, amino acids and peptides, sugars, phospholipids, tetrapyrroles, and monoclonal antibodies (Barth RF. et al. Cancer Communications 2018, 38(1): 35). However, significant issues such as biodistribution and low tumor delivery obtained so far limit the efficiency of the available molecular systems. Therefore, to overcome the limitations of classical delivery compounds, novel nanocarriers are currently being investigated as delivery platforms. Gold nanoparticles capped with mercaptocarboranyl clusters or PEGylated gold nanoparticle-carborane assemblies have been hypothesized as potential therapeutic agents. Other types of nanoparticles, such as boron phosphate, silica, and boron nitride nanotubes (Nakamura H. et al. Bioorganic and Medicinal Chemistry Letters 2015, 25(2):172-174), have also been tested. The main drawback of these nanocarriers with externally attached boron moieties is their lack of stability under physiological conditions, which is necessary for (pre)clinical applications of NCT. To improve stability under physiological conditions, a new approach was explored in 2012 by designing core-shell biodegradable nanoparticles with boron clusters at the core (Sumitani S, Nagasaki Y.Polymer Journal 2012, 44(6):522-530). The resulting core-polymerized and boron-conjugated micelles showed high stability under physiological conditions and high accumulation in tumors. In the case of liposomal formulations, incorporation of boron species into the internal cavity of the host material improves stability. For example, 10% distearoyl boron lipid liposomes encapsulating BSH have been developed (Koganei H. et al. Bioconjugate Chemistry 2013, 24(1): 124-132). The liposomes have a high boron content (B / P ratio of 2.6) and show excellent delivery efficacy. A theranostic nanoplatform based on carborane-containing cholesterol derivatives has been explored for BNCT treatment of mesothelioma monitored in real time by MRI (Alberti D. et al. Organic and Biomolecular Chemistry 2014, 12(15): 2457-2467). In vivo testing with this new therapeutic agent revealed that tumor mass was dramatically reduced by approximately 80-85% after thermal neutron irradiation.

[0006] As the efficiency of boron compounds as NCT activators has declined, other elements have been tried. 157 The high cross section value of Gd ( 10 >60 times that of B; 157 Gd 254000b 10 Due to its high molecular weight (B 3800b) and its paramagnetic behavior, it stands out as a candidate for the development of a multifunctional NCT agent (gadolinium neutron capture therapy - GdNCT) that can be tracked by MRI imaging (Enger SA. et al. Radiation Measurements 2013, 59: 233-240). However, 155 / 157 The therapeutic efficiency of Gd is 10 B is significantly lower than that of Pigment Cell Research 1989, 2(4): 330-332, because interaction with thermal neutrons results in the release of short-range Auger electrons in tissues, and therefore a cytotoxic effect can only be achieved if the Auger occurs in close proximity to DNA to induce double-strand breaks (Martin RF. et al. Pigment Cell Research 1989, 2(4): 330-332).

[0007] For example, in cell studies with Gd-DTPA or Gd-DOTA agents, penetration and accumulation in the cell nucleus were 84% and 56%, respectively. However, in studies of Gd-DTPA performed in human glioblastoma multiforme (GBM) tumors, these values ​​were sharply reduced (6.1%) (De Stasio G. et al. Neurological Research 2005, 27(4): 387-398). Recently, Lee et al. explored functionalized Gd2O3 nanoparticles as potential NCT prognostic agents (Ho SL. et al. RSC Advances 2018, 8(23):12653-12665). In addition to improved T1 magnetic resonance imaging (MRI) performance of the contrast agent, in vitro studies showed low values ​​of U87MG tumor cell death (28.1%) after thermal neutron beam irradiation (1.75-fold lower than that obtained with commercially available Gadovist). The few preclinical studies carried out on the basis of gadolinium compounds showed very disappointing results, leading the scientific community to almost completely abandon this GdNCT approach.Low selective delivery, poor water solubility, and poor therapeutic efficacy of gadolinium agents were proposed as the main reasons for the failure of GdNCT.

[0008] Recently, accelerators have been technically improved as a suitable neutron source for NCT. However, a major challenge for the successful clinical application of NCT remains the low selectivity of the delivery of current drugs to cancer cells.

[0009] With the aim of resolving this inconvenience of the prior art, novel carbon nanocapsules loaded with neutron-capturing elements have been developed, which significantly improve the efficiency of NCT in medical applications. Nanocapsules with a diameter of a few nanometers densely seal the neutron-capturing elements and prevent direct interaction of the neutron-capturing elements with biological media. The presence of functional groups on the external aromatic structure of the nanocarbon shelter gives it good water dispersibility and improves its biocompatibility.

[0010] [Disclosure of the Invention] The present invention presents an innovative and previously unexplored approach for the design of novel nanotherapeutic NCT agents. 6 A new concept based on carbon nanocapsules to encapsulate Li-active NCT nuclides was developed. 6 The Li active species is located in the internal cavity of the carbon nanocarriers, so that it is completely protected from the biological environment, avoiding toxicity and degradation. After the encapsulation of the active cargo, the external surface of the nanocarriers is modified to improve biocompatibility. The developed nanocapsules can be used to deliver therapeutic doses to cancer cells, 6 It is now possible to use Li compounds as NCT activators. 6 It is envisioned that nanoencapsulation of Li will be an early step in the successful development and implementation of lithium neutron cancer therapy (LiNCT). Additionally, the NCT agents described herein can be used in other types of treatments, such as neutron capture enhanced particle therapy (NCEPT), which involves injecting a neutron capture agent into a patient immediately prior to delivery of proton or heavy ion therapy.

[0011] High density 6 Li nuclides can be encapsulated in inorganic compounds, and the resulting nuclides are 6 The nuclear reaction of Li is 10 The fact that it produces more energetic particles than those resulting from B can significantly improve the efficiency of NCT in eradicating cancer cells.

[0012] Thus, a first aspect of the present invention relates to a lithium isotope 6 The present invention relates to carbon nanocapsules (CNCs) encapsulating Li, said nanocapsules being selected from carbon nanohorn aggregates (CNHs) and closed-ended carbon nanotubes (CNTs).

[0013] The term "carbon nanotube (CNT)" refers to a type of nanotube characterized by a cylindrical elongated shape and a nanometer-sized diameter. 2 It refers to one of the hybridized carbon allotropes. CNTs consist of a rolled-up honeycomb carbon lattice of graphene. Depending on the number of graphene layers, carbon nanotubes are classified as single-walled nanotubes or multi-walled nanotubes (designated SWCNT and MWCNT).

[0014] For purposes of the present invention, the term carbon nanotubes (CNTs) includes single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs).

[0015] The term "carbon nanohorn aggregates (CNH)" refers to aggregates (spherical clusters or bundles) of small graphene sheets, rolled to form horn-like cones with half fullerene caps, typically 30-50 nm long and 2-5 nm average diameter. The aggregates can have various morphologies, such as "dahlia" flowers or buds, with a total diameter (i.e., the larger dimension, referring to the longest distance between the two tips of the CNH) in the range of 50-120 nm. The stated values ​​for length, diameter, and total diameter refer to the average values ​​measured by SEM. These CNHs are available from the supplier Carbonium srl, Padua-Italy.

[0016] In a preferred embodiment, the CNC is a dahlia-type assembly of CNHs.

[0017] The CNCs of the present invention are CNH assemblies as defined above, which have closed tips or closed-ended carbon nanotubes, i.e., the carbon nanomaterials have their ends or tips closed and are free of lithium isotopes. 6 The result is a sealed capsule with an internal cavity containing Li. This sealed carbon capsule isolates the encapsulated compound from the external medium, which is an essential aspect for the development of CNCs for biomedical purposes.

[0018] In a preferred embodiment, the length of the closed-ended carbon nanotubes is between 80 and 300 nm and the outer diameter of said tubes is in the range of 1 to 30 nm. Preferably, for MWCNTs the outer diameter is in the range of 5 to 30 nm and for SWCNTs the outer diameter is in the range of 1 to 2 nm, determined by electron microscopy (TEM or SEM), preferably by TEM (Transmission Electron Microscopy). The indicated length and diameter values ​​represent average values.

[0019] In a preferred embodiment, the diameter (i.e., the larger dimension) of the CNHs ranges from 50 to 120 nm, as determined by scanning electron microscopy (SEM). The values ​​given refer to the average value (mean diameter).

[0020] Although CNTs and CNHs are both carbon allotropes with graphene-based layers, they have different morphologies: CNTs are cylindrical with only two ends, whereas CNHs have a dahlia-like shape with multiple ends that are sensitive to their packing.

[0021] In a preferred embodiment, the lithium isotope in the carbon nanocapsule 6 The Li concentration, as determined by XPS analysis, is 0.7 at.% to 10 at.%, more preferably 0.7 at.% to 1.1 at.%.

[0022] In a preferred embodiment, the carbon nanocapsules are 6 Contains no neutron-capturing elements other than Li.

[0023] The term "neutron-capture element" means a chemical isotope that, when irradiated with neutrons, captures neutrons in its nucleus and releases electromagnetic energy and / or particles.

[0024] In a preferred embodiment, the lithium isotope 6 Li is in the form of a lithium salt, preferably 6 LiCl, 6 LiF, 6LiI, 6 LiNO3, 6 Li2SO4, 6 Li2CO3, more preferably 6 It's LiI.

[0025] The exterior surface of the carbon nanocapsules can be functionalized to improve their water dispersibility, biocompatibility, and cellular internalization.

[0026] In a preferred embodiment, the exterior surface of the carbon nanocapsules is functionalized with groups selected from amines, carboxylic acids, hydroxyls, dextran, pluronic acid, polyethylene glycol (PEG) and albumin.

[0027] More preferably, the exterior surface is functionalized with free amine (-NH2) groups.

[0028] The amount of these groups in the carbon nanocapsules is about 1 to 8 mmol / g.

[0029] Another aspect of the present invention refers to a method for preparing the carbon nanocapsules according to the first aspect of the present invention.

[0030] The carbon nanocapsule of the present invention can be prepared from CNT, preferably MWCNT, or CNH aggregates, which are commercially available.Furthermore, these starting materials can be prepared according to the methods described in the prior art (for CNT, Sinnott S. et al. Critical Reviews in Solid State and Materials Sciences 2001, 26: 145, and for CNH, Liu X. et al. Biosensors and Bioelectronics 2020, 167: 112495).

[0031] The method for preparing carbon nanocapsules (CNCs) according to the first aspect of the present invention comprises the steps of: preparing CNCs from CNCs with open ends / tips: 6Li salt weight ratio of CNCs is 1:5~1:20 6 The mixture with Li salt is annealed at a temperature of 700 to 1300 °C for 4 to 12 hours. 6 This involves encapsulating Li and closing its open end / tip.

[0032] In a preferred embodiment, the annealing step comprises the steps of: 6 It is carried out with a weight ratio of Li salts of 1:10.

[0033] After the encapsulation and closure steps, functionalization of the exterior surface of the CNCs can be performed. Depending on the type of functional group to be included, one reagent or the other is used. For example, encapsulation of CNCs to include amine (-NH2) functionalization on the surface can be performed using 6 Li is subjected to a diazonium-based arylation reaction as known in the prior art (Georgakilas V. et al. Journal of the American Chemical Society 2002, 124(5): 760-761).

[0034] The starting material for preparing the CNCs according to the present invention is preferably commercially available CNTs or CNHs, which usually have closed ends or tips and therefore need to be opened to carry out the encapsulation process.

[0035] In a preferred embodiment of the method of the present invention, the CNCs are CNTs and the method comprises, prior to the encapsulation and closure steps, the following steps: Opening up the ends of closed-ended CNTs by treating them with a strong oxidizing medium, nitric acid or a mixture of nitric acid and sulfuric acid, for 6 to 15 hours. The CNTs obtained in the previous step are reduced in vacuum at 500-800°C for 1-2 hours.

[0036] After thermal reduction of the CNTs, the encapsulation and closure steps described above are carried out.

[0037] The acid treatment (oxidation step) allows the CNTs to be cut, metal particles (the catalysts used for CNT growth) to be removed, and O-containing sites (sp 3 This is a simple and efficient approach to induce the formation of nanotube defects.

[0038] The thermal reduction step allows the removal of the functionality incorporated into the graphitic structure of the CNTs by the acid treatment, and is carried out to avoid secondary reactions with the molten lithium salt during the loading step.

[0039] In a preferred embodiment, the thermal reduction is carried out at 800° C. for 2 hours.

[0040] In a preferred embodiment, the annealing step is carried out at a temperature of 1000-1300°C, more preferably at 1100°C.

[0041] In a preferred embodiment of the method of the present invention, the CNC is a CNH and the method comprises the steps of: The CNH is annealed in air to 400–550 °C to oxidize it and open the tip.

[0042] After oxidation of the CNH, the encapsulation and closure steps described above are carried out. In a preferred embodiment, the annealing for encapsulation and closure of the open ends / tips is carried out at a temperature between 700-1100°C, more preferably at 1100°C.

[0043] Another aspect of the present invention refers to a CNC as defined in the first aspect of the present invention for use as a medicament.

[0044] In a preferred embodiment, the present invention refers to a CNC as defined in the first aspect for use in the diagnosis or treatment of cancer.

[0045] In a preferred embodiment, the present invention also refers to a CNC as defined in the first aspect for use in neutron capture therapy (NCT), more preferably in the treatment of cancer by NCT, said CNC acting as a neutron capture therapy agent.

[0046] Because iodine is a contrast agent, the CNCs of the present invention can be used for diagnosis using, for example, X-ray computed tomography (XCT).

[0047] Although the CNCs described in this invention have a primary application in NCT, this does not preclude their use in other types of therapy, such as Neutron capture enhanced particle therapy (NCEPT).

[0048] Therefore, the present invention also refers to a CNC as defined in the first aspect for use in NCEPT which involves injecting a neutron capture agent into a patient immediately prior to irradiation by treatment with protons or heavy ions.

[0049] The CNCs described in this invention exhibit significant advantages over NCT agents known in the art. These advantages are summarized below: A large number of neutron-capturing atoms ( 6 Li) to avoid toxicity and direct interaction with the biological environment. Facile functionalization of the external surface of nanocapsules thus improving water dispersibility, biocompatibility, and cellular internalization. to cancer cells, which can promote cell death of cancer cells after neutron irradiation. 6 Delivery of high doses of Li therapeutics. It presents several advantages for the use of molecular entities, including extended circulation time in the blood and the possibility of accumulating at disease sites by both active and passive targeting. 6 Use of nanocapsules for the delivery of Li.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of this invention. Throughout this specification and claims, the word "comprise" and variations thereof are not intended to exclude other technical features, additives, ingredients, or steps. Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of this specification or may be learned by practice of the present invention. The following examples and figures are provided for illustrative purposes and are not intended to limit the present invention.

[0051] [Brief description of the drawings] Figure 1. Electron microscopy characterization of nanocarriers. TEM images of (a) MWCNTs treated with acid / centrifuged (20000 rpm) and (c) CNHs heat-treated (500 °C) under oxidizing atmosphere. (b, d) Histograms showing the size distribution of each carbon nanostructure as determined by SEM. Arrows indicate CNTs with open ends.

[0052] Figure 2. (a, b, and c) prepared by melt-phase filling. 6 LiI@MWCNT and (d, e, and f) 6 Z-contrast STEM and HRTEM images of LiI@CNHs.

[0053] Figure 3.a) 6 Qualitative neutron autoradiography of a KBr pellet containing LiI@MWCNT. b) Quantitative distribution of lithium in a portion of the sample.

[0054] Figure 4. High-resolution spectra of C1s, N1s, and I3d of functionalized CNCs:NH2_ 6 LiI@MWCNT and NH 2_ 6 LiI@CNH. Peak fitting was performed using Gaussian curves using Origin software.

[0055] Figure 5. a) CNH and b) MWCNT-produced 6 Qualitative neutron autoradiography of cell pellets exposed to Li@CNCs.

[0056] Figure 6. SEM images of MWCNTs after acid treatment and subsequent centrifugation at a) 4000 rpm and c) 20000 rpm. b) and d) show the respective length distribution histograms obtained by measuring over 1000 individual MWCNTs.

[0057] Figure 7. AFM images of acid-treated and centrifuged a) MWCNTs (20,000 rpm) and b) CNHs after oxidation treatment (500 °C).

[0058] Figure 8. Thermogravimetric analysis of pristine MWCNTs under flowing air after acid oxidation / centrifugation treatment and after reduction treatment at 800 °C under vacuum for 2 h.

[0059] Figure 9. Specific surface area by BET analysis of pristine CNH and CNH heat-treated to 400 °C (CNH 400 °C) or 500 °C (CNH 500 °C) at a heating rate of 1 °C / min in air.

[0060] Figure 10. Thermogravimetric analysis of pristine CNHs and CNHs heat-treated at 400 °C and 500 °C (under air flow).

[0061] Figure 11. a) SEM image of CNHs after oxidation treatment (500 °C) and b) respective length distribution histograms obtained by measuring over 500 individual CNHs.

[0062] Figure 12. Encapsulated in MWCNT 6 HRTEM image of LiI. Annealing promotes simultaneous filling and end closure of CNTs (carbon nanocapsules).

[0063] Figure 13. 6 Energy dispersive X-ray analysis of LiI@MWCNT. The inset corresponds to the area of ​​the sample where the data was acquired.

[0064] Figure 14. 6 Energy dispersive X-ray analysis of the LiI@CNH sample. The inset corresponds to the sample for which the data was acquired.

[0065] Figure 15. Sample a) 6 LiI@MWCNT and b) 6 High-resolution XPS spectrum of I3d for LiI@CNH.

[0066] Figure 16. Schematic of surface functionalization by cycloaddition of CNCs prepared on CNH.

[0067] [Example] The present invention will now be illustrated by some examples carried out by the inventors to demonstrate the effectiveness of the products of the present invention.

[0068] Example 1: Preparation of CNCs from MWCNTs and CNHs:

[0069] 1.1. Purification, shortening, and end-opening of MWCNTs The raw MWCNTs used in this study were obtained from NanoAmor (Short MWNT (95+%, OD20-30nm), product ID 1237YJS). Oxidative cutting of MWCNTs was performed by a combination of acid treatment and ultrasonic treatment as previously reported (Kierkowicz M. et al. Carbon 2018, 139: 922-932). Briefly, 150mg of MWCNTs were dispersed in 150mL of a mixture of H2SO4 / HNO3 (3 / 1) and ultrasonicated for 24 hours at a temperature of approximately 40°C. The resulting suspension was washed with distilled water and vacuum filtered using a 0.2μm PTFE membrane (Omnipore) until the filtrate had a neutral pH. The MWCNTs were then ultrasonically dispersed again in ultrapure water (200mL) and centrifuged at either 4000rpm or 20000rpm for 30 minutes. The precipitate (bundles or long nanotubes) was discarded and this process was repeated twice. The MWCNT suspension was collected by vacuum filtration and dried overnight in an oven at 100 °C. Finally, the prepared short MWCNTs were reduced by heat treatment in order to remove the oxygen functional groups introduced during the shortening process. For this purpose, short MWCNTs (50 mg) were placed in a silica tube and the sample was left under vacuum for 2 h. Afterwards, the tube was placed in a furnace and annealed at 800 °C in vacuum for 2 h (heating rate 200 °C / h).

[0070] 1.2. Oxidative heat treatment of CNH to open the tip CNHs were obtained from Carbonium (SWCNH dahlia type). CNHs were heat treated at two different temperatures under oxygen atmosphere (O2) to oxidize the carbon tips and allow full access to the internal cavity with this method, following the protocol described by Utsumi et al. (Utsumi S, et al. The Journal of Physical Chemistry B 2005, 109(30):14319-14324). Briefly, 50 mg of CNHs were placed in a silica boat, which was then placed in a tubular furnace. The heat treatment was carried out under artificial dry air, reaching a maximum temperature of 400 °C or 500 °C (heating rate 1 °C / min). After reaching the maximum temperature, the samples were immediately cooled.

[0071] 1.3. 6 Synthesis of Li@CNC The loading of MWCNTs was attempted with different lithium compounds by molten capillarity wetting, following a modified protocol reported by Sloan et al. for the encapsulation of LiI in single-walled CNTs (Brown G. et al. Applied Physics A 2003, 76(4): 457-462). Briefly, 10 mg of shortened MWCNTs were mixed with 100 mg of the selected lithium compound in an Ar-filled glove box (using an agate mortar and pestle) until the sample had a homogenous color. The powder was then sealed under vacuum in a silica tube approximately 10 cm long. The silica ampoules were placed in a furnace and the mixture was annealed at 5 °C / min until the first dwell step was reached (50 °C above the melting points of the corresponding salts: LiI, LiCl, and LiF, >99% pure, obtained from Sigma-Aldrich. After 4 h, the temperature was increased (5 °C / min) to 1100 °C, where the system was allowed to dwell for 10 min. Finally, the samples were cooled (1 °C / min) to 50 °C above the melting points of the inorganic salts, followed by a cooling rate of 5 °C / min to room temperature. This heat treatment was designed to allow the formation of filled, closed-end CNTs in a single step (Martincic M. et al. Carbon 2019, 141: 782-793). For the loading of CNH with lithium salts, a slight modification of the annealing procedure was applied. Silica ampoules (sealed under vacuum) containing the corresponding mixtures (lithium salts / CNH) were placed in a tubular furnace and then annealed at 950 °C for 12 h. The system was subsequently cooled down to 600 °C at 1 °C / h and finally to room temperature at 5 °C / h. The inorganic salts that showed high loading of CNCs (MWCNT as well as CNH) were investigated for their enhanced morphology, 6 LiI (Sigma-Aldrich, >99% purity) was also probed.

[0072] After the loading process, the CNCs were purified by washing excess inorganic material from the external surface. The mixture was first dispersed in water (ultrasonicated for 15 min) and then refluxed at 110 °C for 4 h. This purification cycle was repeated three times to ensure complete removal of excess unencapsulated lithium compounds. The purified CNCs were collected by vacuum filtration using a 0.2 μm PTFE membrane (Omnipore). The samples were then dried at 100 °C overnight.

[0073] 1.4. 6 External surface functionalization of Li@CNC External surface functionalization of CNCs with free amine groups was carried out using a diazonium-based arylation reaction (Georgakilas V. et al. Journal of the American Chemical Society 2002, 124(5): 760-76). First, CNCs (20 mg) were ultrasonically dispersed in DMF (20 mL) for 30 min. Then, 200 mg of 4-[(N-Boc)aminomethyl]aniline was added to the dispersion and dissolved by mechanical stirring. After the mixture was cooled to 0 °C, 200 μL of isopentyl nitrite was added. The reaction temperature was increased to 80 °C and stirring was continued for 12 h. Purification of the sample was carried out by vacuum filtration using the following solvent order: DMF / water / methanol / diethyl ether. Finally, the sample was dried under vacuum overnight. Deprotection of the N-Boc functional groups on the CNC surface was performed by treatment with 4 M dioxane solution (in HCl). The obtained CNCs were first well dispersed in 20 mL of dioxane solution by sonication for 15 min, and then the dispersion was stirred at room temperature overnight. Purification of the samples was performed by vacuum filtration using the following solvent order: water / methanol / diethyl ether. Finally, the samples were dried under vacuum overnight.

[0074] 1.5. Characterization Samples for microscopy were prepared by dropping onto a holey carbon grid. A suspension was prepared by sonicating a small amount of CNCs in absolute ethanol. Transmission electron microscopy (TEM) images were acquired using a JEOL1210 microscope operated at 120 kV. HRTEM, HAADF and STEM were performed on a FEI, Tecnai G2 F20 microscope operated at 200 kV. Scanning electron microscopy (SEM) images were acquired using a Quanta FEI 200 ESEM FEG microscope operated at 5 kV. An atomic force microscope (NanoScope IV SPM Controller, Veeco) in tapping mode was used to observe the surface morphology of various carbon nanostructures deposited on a mica substrate. Brunauer-Emmett-Teller (BET) analysis was performed on 15–30 mg of raw CNH after annealing in dry air at 400 °C or 500 °C. The samples were subjected to an adsorption outgassing treatment at 150 °C for 2 h, followed by nitrogen adsorption / desorption.

[0075] Thermogravimetric analysis was carried out on a Netzsch instrument, model STA449F1 Jupiter®, under air flow at a heating rate of 10° C. / min. Samples for XPS were prepared by dropping a dispersion prepared by sonication in dry ethanol onto a silicon wafer (5 mm x 5 mm, previously washed with water / acetone). XPS measurements were performed using a SPECS PHOIBOS 150 hemispherical analyzer (SPECS GmbH, Berlin, Germany) with monochromatic Al Kalpha radiation (1486.74 eV) as excitation source at 5 x 10 -10 The experiment was carried out at room temperature with a base pressure of 1000 mbar. High-resolution spectra in the C1s, O1s, N1s and I3d regions were also registered.

[0076] Example 2: Neutron irradiation experiment 2.1. Sample preparation for neutron irradiation 6 Neutron irradiation experiments with LiI@CNC were carried out using KBr pellets. The pellets consisted of 200 mg of KBr in 2 mg of6 The nanomaterials were prepared by grinding the LiI@CNCs with an agate mortar and pestle. The resulting mixture was pressed (5 ton) using a 1.0 cm diameter metal pellet press die. These pellets were prepared for qualitative autoradiography (see below) to examine the presence and uniformity of lithium in the nanomaterials.

[0077] 2.2. Cell preparation and 6 Processing by Li@CNC To assess uptake, we employed the rat osteosarcoma UMR-106 cell line because many BNCT experiments have been performed using this model (Bortolussi S. et al. Radiation Oncology 2017, 12(1): 130). The cells were cultured in a medium consisting of DMEM high glucose, 10% FCS, and 1% gentamicin. Afterwards, the cells were cultured in a 75 cm 2 2 flasks of 3x10 6 After 48 hours, the culture medium was replaced and the cells were grown in medium enriched with lithium-containing carbon nanomaterials for 4 hours. 6 The Li@CNCs were diluted in 20 ml of culture medium and stirred until dissolved, followed by sonication for 30 min. Before adding this enriched cell culture medium to the cell culture flask, the enriched medium was UV-irradiated for 15 min to avoid cell contamination. After the contact time was over, the medium was removed, and the cells were washed five times with PBS, trypsinized, and counted. Finally, 4 million cells were sorted and centrifuged to form a condensed cell pellet. The pellet was attached to a Mylar disc and allowed to dry.

[0078] 2.3 Neutron autoradiography Neutron autoradiography is based on the use of passive detectors sensitive to charged radiation. A sample containing elements that capture neutrons and emit charged particles is irradiated in contact with the detector. After irradiation, the detector is etched in a chemical solution that expands the latent image locus and makes it visible for microscopic analysis. It is employed to obtain quantitative information about the element concentration in the sample, or a qualitative image of the spatial distribution, depending on the calibration of the technique (neutron fluence, etching parameters). Using quantitative neutron autoradiography, 6 Thermal neutron capture reaction on Li:

number

[0079] Dry cell samples on Mylar disks were irradiated twice in the terminal neutron column of the TRIGA MARK II reactor at the Laboratori Energia Nucleare Applicata (LENA) of the University of Pavia. In the first irradiation, the samples were placed on a CR39 and irradiated for 30 min with a reactor power of 2 kW, resulting in a total thermal neutron fluence of 1.97 ± 0.01 × 10 10 cm -2 This neutron autoradiography setup is sensitive to alpha particles, allowing quantitative autoradiographic analysis by counting the tracks generated by alpha particles (Postuma I. et al. Reports of Practical Oncology & Radiotherapy 2016, 21(2): 123-128). The results are presented as a composition of 40 (8x5) sequential photographs. Each image has an area of ​​0.3 mm. 2 (0.632mmx0.474mm) and the total measured area is 12mm 2 , which is a representative portion of the entire sample.

[0080] Qualitative images were obtained from the second irradiation for both the cell sample and the KBr pellet. The sample was placed on another CR-39 and irradiated in the same position at 250 kW for 2 hours. A higher neutron fluence leads to a higher track density on the detector, allowing the formation of a Li distribution map in the sample (Altieri S. et al. Applied Radiation and Isotopes 2008, 66(12):1850-1855). Qualitative autoradiography results in a black and white image, where the grey halos represent areas with lithium uptake. The relatively white parts of the image are areas of high lithium concentration.

[0081] 2.4. In vitro neutron irradiation experiments The NCT effect was tested using the cell lines mentioned above. To evaluate the effect of lithium due to neutron irradiation, four flasks were prepared: two were used as controls, without any drug treatment (non-enhanced cells) and two were used to treat the cells. 6 The rat osteosarcoma UMR-106 cells were cultured in a medium consisting of DMEM high glucose, 10% FCS and 1% gentamicin. Then, 75 cm 2 3x10 in 4 flasks 6 After 48 h, the medium was replaced for two flasks and the cells were grown for 4 h in the medium enriched with lithium-containing carbon nanomaterials, following the same procedure described in section 2.2. After the end of the contact time, the cells were washed and new medium was added to the flask. The other two control flasks were subjected to the same procedure. Finally, the non-enriched and enriched cell flasks were irradiated for 11 min at 250 kW in the thermal column of a TRIGA Mark II reactor. The irradiation position features a 250 kW thermal field of approximately 100 Hz. 10 cm -2 The thermal neutron flux was 100 ng / s. Cell viability was assessed by a cloning assay measuring plating efficiency.

[0082] Results and Observations from Examples 1 and 2 Above Preparation of CNCs from MWCNTs and CNHs As shown above, two different types of carbon nanostructures, namely MWCNTs and CNHs, were used to enhance the binding of cancer cells. 6 We have explored MWCNTs as nanocarriers for the delivery of Li. We realized that the preparation of viable carbon nanomaterials for melt-filling depends on the carbon nanostructure employed. In the case of MWCNTs, the preparation of nanocapsules typically required a two-step process. The first step consists in an oxidation protocol combining a mixture of H2SO4 / HNO3 and ultrasonic treatment. The acid treatment allows the cutting of the MWCNTs, removing metal particles (the catalysts used in the growth of CNTs) and creating O-containing sites (sp 3 This is a simple and efficient approach to induce the formation of defects (defects) in the CNTs. With the aim of discarding the long CNTs or bundles that may still be present after oxidative scission, we took advantage of the aqueous dispersibility of the acid-treated MWCNTs and fractionated them by length by centrifugation (at 4000 rpm and 20000 rpm). The material that settled at the bottom of the centrifuge tube was discarded and the supernatant was collected. In this way, purified, very short MWCNTs with narrow length distribution and open ends were obtained (Figure 1a).

[0083] Figure 6 shows the length distribution of acid-cut MWCNTs after applying different centrifugation speeds of 4000 rpm and 20000 rpm. The length distribution was determined by measuring more than 1000 specimens from SEM images. Statistical analysis showed that the average length distribution of MWCNTs centrifuged at 4000 rpm (Figure 6a) and b)) and 20000 rpm (Figure 6c) and d)) was 186.2 ± 81.9 nm and 144.3 ± 53.9 nm, respectively. Therefore, MWCNTs obtained at the highest centrifugation speed were adopted for the following studies due to their small average particle size and narrow distribution. It is important to note that the length distribution obtained is within the range where the biocompatibility of CNTs is improved (Serpell CJ. et al. Nature Communications 2016, 7: 13118). AFM imaging confirms the very short length of MWCNTs observed by SEM and provides a further overview of the morphology of the samples (Figure 7). Subsequently, the short MWCNTs were thermally reduced to remove the functional groups introduced into the graphitic structure by the acid treatment. This step is performed to avoid secondary reactions with the molten lithium salt during the loading step. The removal of the aliphatic fraction was confirmed by thermogravimetric analysis (TGA, Figure 8). While the TGA curve of the acid-treated MWCNTs shows a continuous weight loss due to the removal of adsorbed moisture and functional groups (red line), the vacuum-annealed sample becomes thermally stable until complete combustion at about 550 °C (green line).

[0084] The preparation of CNHs suitable for the synthesis of CNCs is very simple and requires only a single oxidation step for controlled tip opening of the carbon structure. This controlled thermal oxidation can be applied because the destruction of CNHs occurs preferentially through the tip, where a high density of structural defects or high deformation is commonly observed. The raw CNHs were treated individually at 400 and 500 °C in air. The recovered samples were analyzed by BET and the extent of tip opening at each treatment temperature was estimated by measuring the specific surface area (SA). Raw CNHs (CNH = 419 m 2 / g) and 400°C (CNH 400℃ =1317m 2 / g) and 500 °C (CNH500℃ =1857m 2 A comparison of the specific surface area of ​​the CNHs after annealing at 1000 nm (0.1 μm / g) with that of the CNHs reveals the effect of temperature on the degree of tip opening (Figure 9). The highest surface area is achieved at the highest temperature employed. 500℃ The specific surface area value achieved is 1450 m, which was previously reported by Fan et al. 2 / g (J. Phys. Chem. B 2006, 110, 4, 1587-1591). This nearly reaches the value achieved for graphene (Wang JT-W. et al. Carbon 2020, 162: 410-422), indicating that both sides of the single carbon layer of the CNH are fully accessible (Martincic M. Tobias G. Expert Opinion on Drug Delivery 2015, 12(4):563-581). Further characterization using TGA under air flow reveals that all heat-treated CNHs exhibit similar burning curves as the pristine CNHs (Figure 10). This indicates that the controlled oxidation at 400 and 500 °C occurs only at the tips of the CNHs, preserving the structural integrity of the CNHs. Therefore, the CNHs with the highest SA were selected for the development of nanocapsules. The star-shaped carbon structure of CNH makes it possible to obtain a large number of open tips, which is expected to maximize the loading rate of lithium compounds.

[0085] Figure 1b is a TEM image of the CNHs after oxidative heat treatment at 500 °C. Statistical analysis reveals that the median diameter, determined by measuring more than 500 specimens from the SEM images, is 59.3 ± 16.9 nm. AFM and SEM images of the CNHs are shown in Figures 7 and 11.

[0086] 6 Li inclusion in CNCs ( 6 LiI@CNC) In order to maximize the lithium loading in short MWCNTs, via capillary wetting of the molten phase, several lithium salts were attempted, namely lithium iodide, lithium chloride, and lithium fluoride. These salts were individually ground together with a given amount of MWCNTs and sealed under vacuum in silica ampoules. The mixture was then heated above the melting temperature of the salt, allowing capillary wetting of the internal cavity of the open-ended MWCNTs by the molten halide. The results obtained showed that the high reactivity of lithium chloride and lithium fluoride with silica ampoules at the temperatures employed in the loading process limits their use in this method. Therefore, for the preparation of CNCs, lithium iodide ( 6 LiI) was used. Generally, bulk loading of carbon nanomaterials leads to the presence of a large amount of non-encapsulated compounds (Kierkowicz M. et al. ACS Sustainable Chemistry & Engineering 2017, 5(3): 2501-2508). Therefore, the amount of cations remaining on the outside of MWCNTs or CNHs after loading experiments was considered to be small. 6 LiI was removed from the sample by washing several times with water (LiI solubility = 1.67 g / g, 25 °C).

[0087] MWCNT and CNH 6 The successful loading of LiI was first determined by Z-contrast imaging. Due to the large difference in atomic numbers of the carbon and iodine from the loaded salts, a significant contrast difference between the components was detected by high-angle annular dark-field (HAADF) imaging in a high-resolution scanning transmission electron microscope (STEM). Thus, the internal cavities of MWCNTs and CNHs were filled with LiI. 6 The presence of LiI was confirmed (Figures 2a and 2d, respectively). Indeed, the homogeneous distribution of bright linear small fragments within the darker carbon shell confirmed the efficiency of the method employed for the preparation of CNCs. Moreover, no trace of bright particles was observed outside the carbon nanostructures, indicating that they were not encapsulated. 6It was confirmed that LiI was completely removed. The free LiI was removed while retaining the encapsulated material of the same nature. 6 The selective dissolution of LiI indicates that the ends / tips of carbon nanomaterials were closed during the filling experiments. This sealed carbon system isolates the encapsulated compounds from the external medium, which is an essential aspect for the development of CNCs for biomedical purposes. It has been previously reported that the ends of MWCNTs can be closed by thermal annealing, provided that the system is subjected to a sufficiently high temperature (Martincic M. et al. Carbon 2019, 141: 782-793). However, we also provide evidence herein that the tips of CNHs can also be regenerated by appropriate heat treatment.

[0088] The thermal regeneration of the ends of carbon nanostructures depends on their diameter. For example, higher temperatures are required for sealing of multi-walled CNTs compared to single-walled CNTs (Martincic M. et al. Carbon 2019, 141: 782-793). For short MWCNTs, 1100 °C was adopted, while for CNHs, 950 °C was set as the temperature to simultaneously promote the filling and end-closure processes.

[0089] Inside MWCNTs and CNHs 6The successful formation of sealed nanostructures with LiI nanometric crystals was confirmed by transmission electron microscopy (Figure 2). Furthermore, it is possible to confirm by visual inspection that the graphitic ends of the MWCNTs and CNHs (indicated by arrows) are reconstructed, thus forming the desired sealed CNCs (see additional images in Figure 12). The intensity profile of the crystals observed in Figure 2c reveals a lattice spacing of 3.47 Å, which is in good agreement with the (111) plane of LiI (Fm-3m(225), ICSD). It is interesting to notice that a tubular contrast is observed in the internal cavities of some MWCNTs. This has been previously reported for other metal halides leading to the formation of tubular van der Waals heterostructures (Sandoval S. et al. ACS Nano 2018, 12(7):6648-6656.Sandoval S. et al. Carbon 2017, 123:129-134). Figure 2f shows an HRTEM image of the crystals trapped within the CNH. The intensity profile (inset) reveals an interplanar spacing of 3.01 Å, which is consistent with the (200) planes of the LiI cubic structure.

[0090] Energy dispersive X-ray analysis (EDX) was then employed to confirm the presence of I and to semi-quantitatively determine the atomic composition of the synthesized materials. 6 LiI@MWCNT and 6 For LiI@CNH, estimated concentrations of 0.25 at.% I and 0.63 at.% I were measured, respectively (Figures 13-14).

[0091] X-ray photoelectron spectroscopy (XPS) measurements show a signal at approximately 620 eV corresponding to I3d, consistent with the presence of I (from LiI) filled into the cavities of both the MWCNTs and CNHs (Figure 15). Because the atomic weight of lithium is very small, the signal intensity was too low to be measured by this technique. Therefore, the concentration of enriched lithium was estimated using the I3d 5 / 2 The determination was based on the quantification of iodine in the high-resolution spectrum of (Table 1). Considering that I and Li are equimolar (1:1 ratio), 6The estimated atomic percentage of Li is 6 1.4 at.% for LiI@MWCNT, and 6 For LiI@CNH, it is 3.5 at.% (Table 1). 6 Li concentration ( 6 LiI@MWCNT and 6 Although the concentrations of LiI@CNHs (0.25 at.% and 0.63 at.%; also obtained by indirect measurement using iodine concentration) were much lower than those evaluated by XPS, it was revealed that the cavities of CNHs contained more Li than those of MWCNTs.

[0092] [Table 1]

[0093] Quantitative neutron autoradiography Developed 6 To test the potential of Li@CNCs for LiNCT, they were dispersed in KBr pellets. 6 An initial evaluation was performed using LiI@MWCNT by neutron autoradiography. 6 LiI@MWCNT is 6 Better than LiI@CNH 6 It was chosen for these initial neutron irradiation tests because it contains a low amount of Li, and therefore reacts with neutrons. 6 If the Li nucleus has already been detected, 6 The same is expected for LiI@CNH. Figure 3 shows an image of the lithium distribution in the KBr pellet. The grey level map shows that alpha particle tracks from the lithium neutron capture reaction are visible. It is important to note that the tracks are distributed throughout the pellet. Since MWCNTs tend to aggregate in bundles and the KBr pellets were prepared by simple grinding, the track distribution is inhomogeneous. This inhomogeneity is indicated by white dots, which are track clusters consisting of agglomerates of lithium atoms. Quantitative neutron autoradiography analysis reveals a 44±8 track mm -2 This was a 12mm2 The track counts in the region show a 20% scatter due to low heterogeneity. Surprisingly, this analysis confirmed the presence of active lithium species on the sample.

[0094] External surface functionalization of CNCs (NH2- 6 Li@CNC) CNC@ 6 The external surface functionalization of LiI was carried out using a diazonium-based arylation reaction. Figure 16 shows a schematic diagram of the functional groups involved. The presence of free amine groups on the external aromatic structure of the nanocarbon shelter is expected to significantly improve its water dispersibility and thus its biocompatibility.

[0095] After chemical modification 6 The composition of LiI@CNCs was determined by XPS analysis (Figure 4). High-resolution XPS spectra of C1s, N1s and I3d were used to quantitatively evaluate the concentrations of C, N and I in the amine-functionalized samples. After considering the presence and stoichiometry of Li (from LiI) and H (present in the aminophenyl groups), 6 The relative atomic content of N1s was found to be comparable in both LiI@CNCs (1.9 at.%). The slight changes in C and I (the latter is NH2_ 6 In the case of LiI@CNH (1.1 at.%), NH 2_ 6 LiI@MWCNT (higher than 0.7 at.% of I), the presence of H (six H atoms per amino group-containing moiety), and I and 6 Considering equimolar concentrations of Li, the amino loading was calculated, which was 1.61 mmol and 1.67 mmol of -NH2 per gram of CNH and MWCNT, respectively, leading to surface chemical modification. These results indicate that the yield of the employed cycloaddition reaction for covalent surface modification of carbon nanostructures is hardly affected by their morphology.

[0096] Qualitative neutron autoradiography Figure 6 shows the results of the ZnO / ...6 Qualitative neutron autoradiography of Li@CNCs is shown. The image in Fig. 5a) shows that cells exposed to CNH-produced nanoparticles homogeneously absorb lithium. This is confirmed by quantitative analysis, resulting in an average track density of 23 ± 5 tracks mm -2 where the uncertainty reflects the distribution in the sample. This low uncertainty is due to the fact that the track density is very regular and is consistent over a given sampling area (0.3 mm 2 This means that there is little variation between different parts of the sample (Figure 5b). On the other hand, the lithium-enhanced nanoparticles produced by MWCNTs are taken up into the cells with poor uniformity (Figure 5b). This is also confirmed by quantitative analysis, which shows an average track density of 20 ± 20 tracks mm -2 The 100% uncertainty is the area where the track density is sampled (0.3 mm 2 This heterogeneity may be due to the poor dispersibility of the compound in the cell culture medium, resulting in limited or no supply of lithium to the cells.

[0097] Biocompatibility and neutron irradiation experiments For this study, promising CNH-produced 6 Only the Li@CNC compound was used for neutron irradiation of the cells to measure the viability. Table 2 shows the results obtained, evaluated by plate efficiency. When the cells were not irradiated but enriched with lithium, the viability of the cells was 92%, a slight but tolerable effect of the cytotoxicity of the nanocapsules. When the cells were irradiated without lithium treatment, the viability dropped to 4.2%, due to the interaction of the neutrons with the hydrogen and nitrogen in the cells (background dose). When the cells were irradiated after treatment with the lithiated compound, the viability was halved (1.8%), due to the effect of the interaction of the neutrons with lithium. [Table 2]

[0098] As this is an initial test, a high dose was used to increase the possibility of a therapeutic effect. The use of such a dose results in high cell death in the absence of lithium, but surprisingly, in the presence of the nanocapsules, a 50% decrease in cell viability was observed. In this approach, the encapsulated cargo remains biologically inert until activated by external neutron irradiation. This allows the nanocapsules to be internalized by cells or delivered to diseased tissues in vivo without biological action. Only when the nanocapsules are positioned at the target site are they activated by external neutron flux, becoming lethal and behaving as carbon nuclear nanobombs (CNBs). This is in contrast to previous studies using radionuclides, where nanocapsules delivering radionuclides have already been administered in vivo in radioactive form.

[0099] conclusion Using both MWCNTs and CNHs, 6 We developed a nanocapsule to confine Li. 6 It was shown that Li enters UMR cells. 6 The accumulation of Li was greater in the CNHs than in the MWCNTs. 6 The Li@CNCs are more uniform. In fact, in the first case, the Li@CNCs present in the cells 6 The concentration of Li was 23±5 mm in CR39. -2 in the second case, this resulted in an average spatial density of 20±20 track mm -2 was equivalent to 6 Initial preliminary studies of thermal neutron irradiation of UMR cells treated with Li@CNH showed that at this concentration, 6 It was shown that the cell viability was reduced by 50% compared to irradiation without Li. Furthermore, this compound showed low toxicity at the cellular level. These results clearly demonstrate the possibility of using this compound for Lithium Neutron Capture Therapy (LiNCT), and to generate a complete cell survival curve to evaluate the therapeutic potential of this developed nanocapsule, we performed a series of experiments:

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[0100] [Figure 1] Electron microscopy characterization of nanocarriers. TEM images of (a) acid-treated / centrifuged (20000 rpm) MWCNTs and (c) CNHs heat-treated (500 °C) under oxidizing atmosphere. (b, d) Histograms showing the size distribution of each carbon nanostructure as determined by SEM. Arrows indicate CNTs with open ends. [Diagram 2] Z-contrast STEM and HRTEM images of (a, b, and c) 6LiI@MWCNTs and (d, e, and f) 6LiI@CNHs prepared by melt-phase filling. [Diagram 3] a) Qualitative neutron autoradiography of a KBr pellet containing 6LiI@MWCNT. b) Quantitative distribution of lithium in a portion of the sample. [Figure 4] High-resolution spectra of C1s, N1s, and I3d of functionalized CNCs: NH2_6LiI@MWCNT and NH2_ 6LiI@CNH. Peak fitting was performed with Gaussian curves using Origin software. [Diagram 5] Qualitative neutron autoradiography of cell pellets exposed to 6Li@CNCs produced by a) CNHs and b) MWCNTs. [Figure 6] SEM images of MWCNTs after acid treatment and subsequent centrifugation at a) 4000 rpm and c) 20000 rpm. b) and d) show the respective length distribution histograms obtained by measuring over 1000 individual MWCNTs. [Figure 7]AFM images of acid-treated and centrifuged a) MWCNTs (20,000 rpm) and b) CNHs after oxidation treatment (500 °C). [Figure 8] Thermogravimetric analysis of pristine MWCNTs under flowing air after acid oxidation / centrifugation treatment and after reduction treatment at 800 °C under vacuum for 2 h. [Figure 9] Specific surface area by BET analysis of raw CNH and CNH heat-treated to 400 °C (CNH 400 °C) or 500 °C (CNH 500 °C) at a heating rate of 1 °C / min in air. [Figure 10] Thermogravimetric analysis (under air flow) of raw CNH and CNH heat-treated at 400 °C and 500 °C. [Figure 11] a) SEM image of CNHs after oxidation treatment (500 °C) and b) length distribution histograms obtained by measuring over 500 individual CNHs. [Figure 12] HRTEM image of 6LiI encapsulated in MWCNTs. Annealing promotes simultaneous filling and end closure of the CNTs. [Figure 13] Energy dispersive X-ray analysis of 6LiI@MWCNT. The inset corresponds to the area of ​​the sample where the data was acquired. [Figure 14] Energy dispersive X-ray analysis of the 6LiI@CNH sample. The inset corresponds to the sample for which the data was acquired. [Figure 15] High-resolution XPS spectra of I3d for samples a) 6LiI@MWCNT and b) 6LiI@CNH. [Figure 16] Schematic of surface functionalization by cycloaddition of CNCs prepared on CNH.

Claims

1. Lithium isotopes ６ A carbon nanocapsule (CNC) containing Li, wherein the nanocapsule is selected from carbon nanohorn aggregates (CNHs) and carbon nanotubes (CNTs) with closed ends.

2. The carbon nanocapsule according to claim 1, wherein the CNC is a closed-end CNT selected from single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs), preferably having a length of 80 to 300 nm, and the outer diameter of the tube being in the range of 5 to 30 nm in the case of MWCNTs, and in the range of 1 to 2 nm in the case of SWCNTs.

3. The carbon nanocapsule according to claim 1, wherein the CNC is an aggregate of dahlia-like carbon nanohorns, and preferably the diameter of the CNH is in the range of 50 to 120 nm.

4. Lithium isotopes in the aforementioned CNC ６ The carbon nanocapsule according to claim 1, wherein the concentration of Li is in the range of 0.7 at.% to 10 at.%.

5. CNC, ６ The carbon nanocapsule according to claim 1, which does not contain neutron-trapping elements other than Li.

6. Lithium isotope ６ Li is ６ LiCl, ６ LiF, ６ LiI, ６ LiNO ３ , ６ Li ２ SO ４ and ６ Li ２ CO ３ The carbon nanocapsule according to claim 1, which is in the form of a lithium salt selected from

7. The carbon nanocapsule according to claim 1, wherein the outer surface of the CNC is functionalized with a group selected from amine, carboxylic acid, hydroxyl, dextran, pluronic acid, polyethylene glycol (PEG), and albumin, and the outer surface of the CNC is functionalized with a free amine group -NH₂.

8. The carbon nanocapsule according to claim 6, wherein the group functionalizing the surface is in the range of 1 to 8 mmol / g.

9. A method for preparing a CNC according to claim 1, comprising: a CNC having an open end / tip; ６ Li salt weight ratio of 1:5 to 1:20, CNC ６ By annealing the mixture with Li salt at a temperature of 700-1300°C for 4-12 hours, ６ A method comprising encapsulating Li and closing the open end / tip, wherein the annealing in the step is carried out at a weight ratio of CNC:6 Li salt of 1:

10.

10. The method according to claim 9, wherein the CNC is a CNT, and before the encapsulation and closing process, the following steps are included: To open the ends of CNTs, oxidize CNTs with closed ends by treating them with a strong oxidizing medium, such as nitric acid or a mixture of nitric acid and sulfuric acid, for 6 to 15 hours. The CNTs obtained in the previous process are thermally reduced under vacuum at 500-800°C for 1-2 hours.

11. The method according to claim 9, wherein the CNC is CNH, and before the encapsulation and closing process, the following steps are included: The CNH is annealed in air to 400-550°C to oxidize the CNH and open the tip of the CNH.

12. After the aforementioned encapsulation and closing steps, a diazonium-based arylation reaction is performed to remove the free amine group -NH ２ The method according to claim 9, further comprising the step of functionalizing the external surface of the CNC.

13. A carbon nanocapsule according to claim 1, for use as a pharmaceutical product.

14. Carbon nanocapsules according to claim 1, for use in the diagnosis or treatment of cancer.

15. The carbon nanocapsule according to claim 1, for use in neutron capture therapy (NCT) or neutron capture-enhanced particle therapy (NCEPT).

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