Nanocarrier for biomaterial delivery, nanocarrier-biomaterial complex, manufacturing methods thereof and treatment method for living plant-based object using nanocarrier

Abstract

A nanocarrier for delivering biomaterials, a nanocarrier-biomaterial complex, a method for manufacturing the same, and a method for treating a living plant-based target using the nanocarrier are disclosed. The disclosed nanocarrier is a nanocarrier for delivering biomaterials into a living plant-based target and may comprise a nanotube and a functional compound having an imidazole that is coupled to the surface of the nanotube via at least a polycyclic aromatic substance. A nanocarrier according to one embodiment may be configured to deliver a biomaterial that promotes the regeneration of plant cells of the target into the target.

Description

Technology Field

[0001] The present invention relates to a material carrier, a method for manufacturing the same, and its application, and more specifically, to a nanocarrier, a composite comprising a nanocarrier, a method for manufacturing the same, and its application. Background Technology

[0002] Population growth and global warming are driving the demand for the development of new breeding varieties and increased crop yields. Plant regeneration is an essential technology for genetic engineering applications, such as crop genome editing and transformation; however, low regeneration efficiency is known to be a major hurdle in genome editing (engineering). To breed high-value crops using agro-biotechnologies, tissue dedifferentiation (i.e., callus formation) and above-ground regeneration (i.e., shoot regeneration) must occur sequentially following plant genome editing or transformation. However, the efficiency of the plant regeneration process is very low, severely limiting the improvement of traits in major crops. With the recent advancement of CRISPR technology, the demand and market for precision breeding are growing explosively, and technologies to enhance plant regeneration efficiency are becoming essential requirements in this market.

[0003] Conventionally, attempts have been made to enhance plant (crop) regeneration efficiency by incorporating various hormones and additives into regeneration induction media, but these methods are facing significant limitations. This low regeneration efficiency of plants (crops) is attributed to genetic barriers or genetic factors. Therefore, to improve regeneration efficiency, it may be critically necessary to control the expression of genes (genetic factors) that essentially act as regeneration inhibitors during the tissue culture process.

[0004] MicroRNA (hereinafter miRNA), known as a genetic factor with significant influence on the process of plant regeneration, is a short single-stranded RNA having an average of 22 nucleotides. It is known to be expressed within cells and bind complementarily to the nucleic sequence of messenger RNA (mRNA) of target genes, thereby inhibiting the process of mRNA being translated into protein by ribosomes or causing mRNA to be degraded, thereby inhibiting the expression of target genes.

[0005] Although information regarding various genes associated with the process of plant regeneration is being revealed, efficient methodologies for controlling this process remain very limited. Currently, methods such as transformation using Agrobacterium tumefaciens or gene guns are utilized to overcome genetic factors; however, these methods are difficult to apply to a wide variety of crops due to limitations in target crops and issues regarding genetic modification (GM). Therefore, there is a need to develop gene delivery materials that enable temporary control of gene expression at specific times without the need for transformation, and to develop technologies that utilize these materials to overcome the limitations of plant callus regeneration. In particular, there is a demand for the development of novel materials that are applicable regardless of plant species and can stably introduce appropriate biomaterials into the calluses of various crop species. The problem to be solved

[0006] The technical problem that the present invention aims to solve is to provide a nanocarrier capable of effectively delivering biomaterials into a living plant-based object and a method for manufacturing the same.

[0007] Furthermore, the technical problem that the present invention aims to solve is to provide a nanocarrier capable of having excellent pH sensitivity and releasing biomaterials within plant cells, and a method for manufacturing the same.

[0008] Furthermore, the technical problem that the present invention aims to solve is to provide a nanocarrier and a method for manufacturing the same that are applicable regardless of plant species and can stably introduce appropriate biomaterials into a target plant species or a related object without limitation.

[0009] In addition, the technical problem that the present invention aims to solve is to provide a nanocarrier capable of delivering a biomaterial capable of promoting the regeneration of plant cells into a living plant-based target, and a method for manufacturing the same.

[0010] In addition, the technical problem that the present invention aims to solve is to provide a nanocarrier-biomaterial complex comprising the above-mentioned nanocarrier and a method for manufacturing the same.

[0011] In addition, the technical problem that the present invention aims to solve is to provide a method for treating an object using the aforementioned nanocarrier.

[0012] In addition, the technical problem that the present invention aims to solve is to provide a method for promoting the redifferentiation of plant cells using the aforementioned nanocarrier.

[0013] The problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be understood by those skilled in the art from the description below. means of solving the problem

[0014] According to one embodiment of the present invention, a nanocarrier for delivering biomaterials is provided, comprising: a nanotube; and a functional compound having an imidazole that is coupled to the surface of the nanotube via at least a polycyclic aromatic substance.

[0015] The above polycyclic aromatic material may include polycyclic aromatic hydrocarbons.

[0016] The above polycyclic aromatic substance may include pyrenyl.

[0017] The above nanocarrier may further include maleimide bound to the polycyclic aromatic substance and cysteine ​​bound to the functional compound, and the polycyclic aromatic substance and the functional compound may be mutually bonded by the bonding of the maleimide and the cysteine.

[0018] The above functional compound may include histidine containing the above imidazole.

[0019] The above nanotube may include carbon nanotubes.

[0020] The above nanotube may include SWNT (single-walled carbon nanotube).

[0021] The above nanocarrier is Py-His n- It may include a SWNT structure, wherein Py represents the pyrenyl, His represents histidine including the imidazole, and the SWNT may correspond to the nanotube.

[0022] The above n may be greater than or equal to 2 and less than 100.

[0023] The above object may include a callus.

[0024] The above biomaterial may include RNA (ribonucleic acid).

[0025] According to another embodiment of the present invention, a nanocarrier for delivering biomaterial is provided, comprising a nanotube as a nanocarrier for delivering biomaterial into a living plant-based target; and a functional compound having imidazole that is bound to the surface of the nanotube, wherein the nanocarrier is configured to deliver RNA (ribonucleic acid) that promotes the regeneration of plant cells of the target as the biomaterial into the target.

[0026] According to another embodiment of the present invention, a nanocarrier-biomaterial complex for application within a living plant-based object is provided, comprising: the aforementioned nanocarrier; and a biomaterial coupled to the functional compound of the nanocarrier.

[0027] The above biomaterial may include RNA (ribonucleic acid).

[0028] The absolute value of the zeta potential of the above nanocarrier-biomaterial complex can be about 20 mV or more.

[0029] The diameter of the above nanocarrier-biomaterial complex may be about 10 nm or less.

[0030] The above nanocarrier-biomaterial complex may have the characteristic of releasing the biomaterial within the plant cells of the target.

[0031] According to another embodiment of the present invention, a method for treating an object using a nanocarrier is provided, comprising the steps of: preparing a complex solution containing the aforementioned nanocarrier-biomaterial complex; and bringing the complex solution into contact with a living plant-based object to introduce the nanocarrier-biomaterial complex into the interior of the object.

[0032] The above biomaterial may include RNA (ribonucleic acid), and the regeneration of the plant cells of the subject may be promoted by the RNA.

[0033] The above object may include plant tissue or a plant body.

[0034] The above object may include a callus.

[0035] The absolute value of the zeta potential of the above nanocarrier-biomaterial complex can be about 20 mV or more.

[0036] The diameter of the above nanocarrier-biomaterial complex may be about 10 nm or less.

[0037] According to another embodiment of the present invention, a method for treating an object using a nanocarrier is provided, comprising the steps of: preparing a complex solution containing a nanocarrier-biomaterial complex in which a nanocarrier comprising a functional compound having imidazole and a biomaterial comprising RNA (ribonucleic acid) are combined; and contacting the complex solution with a living plant-based object to introduce the nanocarrier-biomaterial complex into the interior of the object and promoting the regeneration of the plant cells of the object using the RNA. Effects of the invention

[0038] According to embodiments of the present invention, a nanocarrier capable of effectively delivering biomaterials into a living plant-based target can be realized. Furthermore, according to embodiments, a nanocarrier capable of releasing biomaterials within plant cells with excellent pH sensitivity can be realized. Additionally, according to embodiments, a nanocarrier applicable regardless of plant species can be realized that a biomaterial suitable for a target plant species or a related target can be stably introduced without limitation. Furthermore, according to embodiments, a nanocarrier capable of delivering biomaterials capable of promoting plant cell regeneration into a living plant-based target can be realized.

[0039] According to embodiments of the present invention, a nanocarrier-biomaterial complex comprising the above-described nanocarrier can be realized. Additionally, according to embodiments, a method for treating a target using the above-described nanocarrier can be provided. Additionally, according to embodiments, a method for promoting the redifferentiation of plant cells using the above-described nanocarrier can be provided.

[0040] According to one embodiment, a nanocarrier can be implemented that can easily deliver external genetic material (biomaterial) into plant cells, which can be applied to enhance the regeneration efficiency of plants. In addition, unlike gene delivery technologies using a gene gun, which has low delivery efficiency and involves physicochemical shock from the outside, or Agrobacterium tumefaciens, which has limitations in applicable plant species, the nanocarrier according to one embodiment can provide a simple, non-destructive method of delivering genetic material that is easy to apply to various plant species.

[0041] In addition, according to one embodiment, unlike conventional carriers that have difficulty delivering genetic materials such as RNA due to limitations in the shape or size of bindable biomaterials, a method can be provided to efficiently deliver biomaterials such as RNA into a living plant-based target, such as a callus, by using a nanocarrier having excellent pH sensitivity. The technology according to one embodiment can effectively enhance / promote plant regeneration by controlling genes that inhibit regeneration of the callus.

[0042] However, the effects of the present invention are not limited to the above effects and can be extended in various ways without departing from the technical concept and scope of the present invention. Brief explanation of the drawing

[0043] FIG. 1 is a diagram illustrating a nanocarrier for delivering biomaterials into a living plant-based object according to one embodiment of the present invention. FIGS. 2a and 2b are drawings for exemplarily illustrating a method for manufacturing a nanocarrier according to one embodiment of the present invention. FIG. 3 is a diagram showing a nanocarrier-biomaterial complex for application within a living plant-based object according to one embodiment of the present invention. FIG. 4 is a diagram illustrating an exemplary method for treating an object using a nanocarrier according to one embodiment of the present invention. FIG. 5 is a photographic image showing a solution containing Py-His6-SWNT synthesized according to one embodiment of the present invention. Figure 6 is a graph showing the results of measuring the zeta potential of Py-His6-SWNT synthesized according to one embodiment of the present invention. Figure 7 is a graph showing the results of measuring the size distribution of Py-His6-SWNT particles synthesized according to one embodiment of the present invention. FIG. 8 shows Py-His6-SWNT synthesized according to one embodiment of the present invention and dCS synthesized according to a comparative example. cov This is a graph showing the change in zeta potential according to the change in pH of SWNT. FIG. 9 shows (GT) on Py-His6-SWNT synthesized according to one embodiment of the present invention 15 This is a graph showing the results of evaluating the fluorescence characteristics of Cy3 before and after the binding of -Cy3. Figure 10 is a graph showing the results of evaluating the degree of biomaterial (ssDNA) release according to pH change of Py-His6-SWNT synthesized according to one embodiment of the present invention. Figure 11 is an image showing the results of observing the phenotype of the cleavage activity of a callus into which Py-His6-SWNT synthesized according to an embodiment of the present invention was introduced. FIG. 12 is a microscopic image showing a callus treated with Py-His6-SWNT according to an embodiment of the present invention. Figure 13 is an image showing the penetration characteristics of Py-His6-SWNT into a callus treated with Py-His6-SWNT according to an embodiment of the present invention, analyzed by confocal Raman microscopy. Figure 14 is a graph showing the change in zeta potential according to the mixing ratio of a complex containing Py-His6-SWNT and RNA (STTM396) according to an embodiment of the present invention. FIG. 15 is a graph showing the change in size according to the mixing ratio of a complex containing Py-His6-SWNT and RNA (STTM396) according to an embodiment of the present invention. FIG. 16 is a microscopic image showing a callus treated with a complex of Py-His6-SWNT and RNA (STTM396) according to an embodiment of the present invention. Figure 17 is an image showing the penetration characteristics of the complex into a callus treated with a complex of Py-His6-SWNT and RNA (STTM396) according to an embodiment of the present invention, analyzed by confocal Raman microscopy. FIG. 18 is a drawing showing an image in which the regeneration of the callus is evaluated as a phenotype after treating the callus according to an embodiment and a comparative example of the present invention. FIG. 19 is a graph showing the results of quantitatively evaluating the regeneration of callus after treating callus according to the embodiments and comparative examples of the present invention. Specific details for implementing the invention

[0044] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings.

[0045] The embodiments of the present invention described below are provided to more clearly explain the present invention to those skilled in the art, and the scope of the present invention is not limited by the following embodiments, and the following embodiments may be modified in various other forms.

[0046] The terms used herein are for describing specific embodiments and are not intended to limit the invention. Terms used herein in the singular form may include plural forms unless the context clearly indicates otherwise. Additionally, the terms “comprise” and / or “comprising” used herein specify the presence of the mentioned features, steps, numbers, actions, components, elements, and / or groups thereof, and do not exclude the presence or addition of one or more other features, steps, numbers, actions, components, elements, and / or groups thereof. Furthermore, the term “connected” used herein means not only that components are directly connected, but also includes the concept of indirectly connecting components through the interposition of additional components between them.

[0047] Furthermore, when a component is described in this specification as being located "on" another component, this includes not only cases where a component is in contact with another component, but also cases where another component exists between the two components. The term "and / or" as used in this specification includes any one of the listed items and all combinations of one or more thereof. Additionally, terms of degree such as "about" and "substantially" as used in this specification are used to mean a range of numerical values ​​or degrees or approximate values, taking into account inherent manufacturing and material tolerances, and are used to prevent an infringer from unfairly exploiting the disclosures in which precise or absolute figures provided to aid in understanding this specification are mentioned.

[0048] Embodiments of the present invention will be described in detail below with reference to the attached drawings. The sizes or thicknesses of the areas or parts depicted in the attached drawings may be slightly exaggerated for the clarity of the specification and convenience of explanation. Throughout the detailed description, the same reference numerals indicate the same components.

[0049] FIG. 1 is a drawing illustrating a nanocarrier (100) for delivering biomaterials within a living plant-based object according to one embodiment of the present invention.

[0050] Referring to FIG. 1, a nanocarrier (100) according to an embodiment of the present invention may comprise a nanotube (10) and a functional compound (20) having imidazole that is bonded to the surface of the nanotube (10). The functional compound (20) may be bonded to the surface of the nanotube (10) via at least a polycyclic aromatic substance. The polycyclic aromatic substance may be, for example, a polycyclic aromatic hydrocarbon or may include the same. The polycyclic aromatic substance may be, for example, pyrenyl (12) or may include the same. The specific type of the polycyclic aromatic substance is not limited to pyrenyl (12) and may vary depending on the case. Additionally, according to another embodiment, the nanotube (10) and the functional compound (20) may be bonded to each other by covalent bonding without an intermediate bonding substance such as the polycyclic aromatic substance. In addition, the method of bonding between the nanotube (10) and the functional compound (20) can be varied.

[0051] The nanotube (10) may be a carbon nanotube. For example, the nanotube (10) may be a single-walled carbon nanotube (SWNT). The length of the nanotube (10) may be, for example, tens of nm to hundreds of nm. The diameter of the nanotube (10) may be, for example, about 1 nm to several nm. The diameter of the nanotube (10) may be, for example, about 10 nm or less. The nanotube (10) may be a type of nanostructure. Also, the nanotube (10) may be a type of nanoparticle.

[0052] Carbon nanotubes can serve as useful biomaterial carriers that overcome the limitations of existing carriers to deliver biomaterials (e.g., genetic material) into plant tissues or the plant body. Carbon nanotubes can be classified into single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) based on the number of walls. Carbon nanotubes possess advantages such as a high aspect ratio, stiffness, and a large surface area, making them suitable for the delivery of biomaterials (biomolecules). In particular, SWNTs exhibit a characteristic Raman peak at approximately 1590 cm⁻¹. -1Since this is demonstrated, it can be utilized to easily observe the delivery characteristics of SWNTs into the target (biological organism). However, despite the utility of carbon nanotubes, their use has been limited due to their low solubility and dispersibility. Furthermore, in the case of cationic carbon nanotubes used as gene carriers, there are limitations in the size or type of gene that can be delivered, as the gene remains attached to the carbon nanotube (a type of nanoparticle) without detaching, thereby inducing the expression of the effect. In the case of SWNTs modified by the positively charged SWNT modification method (e.g., PEI-SWNT, Chitosan-SWNT), the pattern of biomolecule release from the SWNT within the cell is limited, thus limiting the size and type of material that can be delivered.

[0053] However, in an embodiment of the present invention, by modifying the nanotube (10) with a functional compound (20) containing imidazole, the nanotube (10) can be configured to have the characteristic of releasing biomaterial within the cell after entering the cell with biomaterial. Here, the imidazole may be a type of functional group. The nanotube (10) may be modified with imidazole or modified with a functional compound (20) containing imidazole. The imidazole may have a lower pKa value than the amine group. For example, the imidazole may have a pKa value of about 6.0. In this regard, the nanotube (10) modified with imidazole may have high pH sensitivity. The nanotube (10) modified with imidazole may have higher pH sensitivity at a neutral pH compared to the positively charged SWNT based on the amine group. Considering that the pH inside a plant cell is about 7 to 8, it may be desirable to modify the nanotube (10) with an imidazole having a pKa of about 6.0. The nanotube (10) modified with imidazole may have the characteristic of transporting biomaterial (biomolecule) into the cell and then releasing the biomaterial more efficiently at a location with a neutral pH, for example, cytoplasm.

[0054] The functional compound (20) can be bonded to the surface of the nanotube (10) via at least pyrenyl (12). The pyrenyl (12) can be bonded to the surface of the nanotube (10) by van der Waals forces through π-π interactions. The pyrenyl (12) can provide a bonding force to the functional compound (20) without damaging the properties of the nanotube (10). Even if the pyrenyl (12) is bonded to the surface of the nanotube (10), characteristic Raman peak properties by the nanotube (10) may appear.

[0055] According to one embodiment, the functional compound (20) may be histidine containing the imidazole or may contain histidine. According to one example, the nanocarrier (100) may further include maleimide (14) bound to pyrenyl (12) and cysteine ​​(22) bound to the functional compound (20), and the pyrenyl (12) and the functional compound (20) may be mutually bound by the bonding of maleimide (14) and cysteine ​​(22). Maleimide (14) may be bound to pyrenyl (12), cysteine ​​(22) may be bound to maleimide (14), and the functional compound (20) may be bound to cysteine ​​(22). Accordingly, the functional compound (20) can be bound to the nanotube (10) via pyrenyl (12), maleimide (14), and cysteine ​​(22). Maleimide (14) and cysteine ​​(22) can provide excellent binding properties between pyrenyl (12) and the functional compound (20). Additionally, maleimide (14) and cysteine ​​(22) may not impair the biomaterial transport properties and dispersibility of the nanocarrier (100).

[0056] According to one embodiment, the nanocarrier (100) is Py-His n - It may include a SWNT structure. Here, Py represents pyrenyl (12), His represents histidine including imidazole, and the SWNT may correspond to a nanotube (10). nIt may correspond to a functional compound (20). According to one example, the above n may be greater than or equal to 2 and less than 100. The above n may be 2 or greater and less than 100, or 2 or greater and less than 50. If the above n is 1, the binding characteristics of the biomaterial to the nanocarrier (100) may be reduced. Meanwhile, if the above n is excessively large at the polymer level, the dispersibility of the nanocarrier (100) or the transport characteristics of the biomaterial may be reduced. According to an example, by selecting an appropriate range of n, the binding characteristics of the biomaterial, the dispersibility of the nanocarrier (100), and the transport characteristics of the biomaterial can be secured.

[0057] According to one embodiment, the living plant-based object may include plant tissue or a plant body. For example, the object may include a callus. A callus is a type of plant tissue and may be an undifferentiated, irregular mass of cells. A plant body may be expressed as plant cells undergo redifferentiation from the callus. However, in the embodiments of the present invention, the type of object is not limited to a callus. In some cases, the object may be a plant body such as a seedling or a bud.

[0058] According to one embodiment, the biomaterial or biological material may be a genetic material. The biomaterial may be a biomolecule. According to one example, the biomaterial may include ribonucleic acid (RNA). The biomaterial may include synthetic RNA. For example, the biomaterial may include a short tandem target mimic (STTM).

[0059] RNA complementary to miRNA can be used to inhibit the activity of miRNA that inhibits the redifferentiation of plant cells (tissues), and RNA can be used to promote the redifferentiation of plant cells (tissues). Existing technologies related to this have been dependent on transformation technology, so their scope of application has been limited. However, according to an embodiment of the present invention, biomaterials prone to degradation, such as RNA, can be easily and conveniently delivered into a target without separate chemical treatment or physical shock, and the efficiency of plant cell redifferentiation within the target can be enhanced. In this case, the nanocarrier (100) can be configured to deliver RNA that promotes the redifferentiation of plant cells in the target as the biomaterial into the target.

[0060] STTM (short tandem target mimic) is a short single-stranded RNA created to artificially inhibit short-length RNA, such as miRNA, and may be a nucleotide inhibitor that inhibits the function of miRNA. STTM may have sequences complementary to the target RNA capable of recognizing the target at both ends, and may have a sequence in the middle capable of forming a stem-loop for the structural stability of STTM within the cell. STTM may have a specific and efficient inhibitory function against target RNA and may be used to inhibit the activity of short-length RNA. However, in the embodiments of the present invention, the biomaterial is not limited to RNA / STTM, and in some cases, said biomaterial may include DNA (deoxyribonucleic acid). Furthermore, said biomaterial may include both RNA and DNA.

[0061] FIGS. 2a and 2b are drawings for exemplarily illustrating a method for manufacturing a nanocarrier according to one embodiment of the present invention.

[0062] Referring to FIG. 2a, a nanocarrier according to an embodiment of the present invention can be prepared, for example, using a functional compound (20) containing histidine containing imidazole. More specifically, an Ac-CH₄ with cysteine ​​(22) applied to n histidine ends. n After preparing the -NH2 group, it can be reacted with pyrenyl maleimide, and as a result, Py-His n A structure can be obtained. Here, Py represents pyrenyl (12), and His represents histidine containing the imidazole. His n It may correspond to a functional compound (20). According to one example, n may be greater than or equal to 2 and less than 100. Pyrenyl (12) and the functional compound (20) may be mutually bonded by the combination of maleimide (14) and cysteine ​​(22).

[0063] Referring to FIG. 2b, the Py-His n By inducing a π-π interaction between the pyrenyl (12) and the nanotube (10), a nanotube (10) modified with a functional compound (20) can be obtained. The nanocarrier (100) obtained in FIG. 2b can correspond to the nanocarrier (100) described in FIG. 1.

[0064] The method for manufacturing a nanocarrier according to one experimental example may be as follows.

[0065] (1) Py-His6 can be produced by reacting 150 µl of 10 mg / ml of N-(1-pyrenyl)maleimide with 150 µl of 20 mg / ml of Ac-CH6-NH2 peptide in a thermo mixer for 18 hours (1200 rpm, 25℃).

[0066] (2) 3 mL of diluted Py-His6 can be obtained by adding 2700 µL of distilled water to Py-His6.

[0067] (3) A SWNT suspension can be prepared by placing 3 mL of 0.5 mg / ml SWNT and mL of Py-His63 into a container and performing tip sonication (1 hr, 40% amplitude, in an ice bath). Through the sonication, the distance between the SWNTs is increased so that pyrenyl can bind better to the SWNTs.

[0068] (4) To remove Py-His6 that has not reacted (bound) with SWNT, the SWNT suspension can be dialyzed with distilled water for 48 hours (using a 100 kD dialysis membrane).

[0069] (5) Py-His6-SWNT can be obtained by centrifuging the solution that underwent the above dialysis at 17,500 rpm for 4 hours and collecting about 80% of the supernatant. Through the above centrifugation, large nanostructures (nanoparticles) can be removed.

[0070] However, the manufacturing conditions and specific methods of the nanocarriers disclosed in the above-described experimental examples are merely exemplary and may be varied.

[0071] FIG. 3 is a drawing showing a nanocarrier-biomaterial complex (200) for application within a living plant-based object according to one embodiment of the present invention.

[0072] Referring to FIG. 3, a nanocarrier-biomaterial complex (200) according to an embodiment of the present invention may include a nanocarrier (100) and a biomaterial (50) bound to a functional compound (20) of the nanocarrier (100). The nanocarrier (100) may correspond to the nanocarrier (100) according to the embodiment described with reference to FIG. 1. The biomaterial (50) may be bound to the functional compound (20) by electrostatic interaction. The biomaterial (50) may include, for example, RNA. In this case, the biomaterial (50) may include a short tandem target mimic (STTM). Redifferentiation of plant cells (tissues) can be promoted using RNA capable of inhibiting the activity of miRNA. The nanocarrier (100) may be configured to deliver RNA that promotes the redifferentiation of plant cells of the target into the target as the biomaterial (50). However, in the embodiments of the present invention, the biomaterial (50) is not limited to RNA / STTM. In some cases, the biomaterial (50) may include DNA. Additionally, the biomaterial (50) may include both RNA and DNA.

[0073] According to one embodiment, a nanocarrier-biomaterial complex (200) can be prepared by first preparing a nanocarrier solution containing a nanocarrier (100) and then mixing a biomaterial (50) into the nanocarrier solution. That is, the method for preparing a nanocarrier-biomaterial complex according to the embodiment may include the step of preparing a nanocarrier solution containing a nanocarrier (100) and the step of mixing a biomaterial (50) into the nanocarrier solution. The step of preparing the nanocarrier solution may include, for example, the method described with reference to FIGS. 2a and 2b.

[0074] According to one embodiment, the absolute value of the zeta potential of the nanocarrier-biomaterial complex (200) may be about 20 mV or more. The diameter (outer diameter) of the nanocarrier-biomaterial complex (200) may be about 10 nm or less. When these conditions are satisfied, the nanocarrier-biomaterial complex (200) can be well dispersed in a solution and can penetrate well into the cytosol. If the particle size of the nanocarrier-biomaterial complex (200) is too large or the particles aggregate, the dispersibility and penetration characteristics may be degraded. Additionally, when the absolute value of the zeta potential of the nanocarrier-biomaterial complex (200) is about 20 mV or more, the penetration characteristics into the cytosol may be improved.

[0075] According to one embodiment, the nanocarrier-biomaterial complex (200) may have the characteristic of releasing biomaterial (50) within the plant cell of the subject. The nanocarrier (100) may have the characteristic of releasing biomaterial (50) within the cell after carrying the biomaterial (50) into the cell by including a functional compound (20) containing imidazole. The nanocarrier (100) containing imidazole may have higher pH sensitivity at a neutral pH. The nanocarrier (100) containing imidazole may have the characteristic of releasing the biomaterial (50) more efficiently at a location with a neutral pH, for example, cytoplasm, after transporting the biomaterial (50) into the cell.

[0076] A method for treating an object using a nanocarrier according to an embodiment of the present invention may include the steps of preparing a composite solution containing a nanocarrier-biomaterial composite and contacting the composite solution with a living plant-based object to introduce the nanocarrier-biomaterial composite into the interior of the object. The step of contacting the composite solution with the object may be a simple process of immersing or contacting the object with the composite solution. Through this, the biomaterial loaded on the nanocarrier-biomaterial composite can be easily delivered into the interior of the object. The biomaterial may include, for example, RNA, and the regeneration of the plant cells of the object may be promoted by the RNA. The object may include plant tissue or a plant body. For example, the object may include a callus. The absolute value of the zeta potential of the nanocarrier-biomaterial composite may be about 20 mV or more. The diameter of the nanocarrier-biomaterial complex may be about 10 nm or less. The nanocarrier-biomaterial complex may have the characteristic of releasing the biomaterial within the plant cells of the target. The complex solution may be an aqueous solution. Therefore, water may be used as a buffer when introducing the nanocarrier-biomaterial complex into the interior of the target.

[0077] According to one embodiment, a method for treating a target using a nanocarrier may include the steps of: preparing a complex solution containing a nanocarrier-biomaterial complex in which a nanocarrier comprising a functional compound having imidazole and a biomaterial comprising RNA are combined; and contacting the complex solution with a living plant-based target to introduce the nanocarrier-biomaterial complex into the interior of the target and promoting the regeneration of the target's plant cells using the RNA.

[0078] In addition, all features described with reference to FIGS. 1 to 3 may be applied to the method for treating an object using a nanocarrier according to an embodiment of the present invention.

[0079] FIG. 4 is a diagram illustrating an exemplary method for treating an object using a nanocarrier according to one embodiment of the present invention.

[0080] Referring to FIG. 4, a composite solution (300) containing a nanocarrier-biomaterial complex may be prepared, and the nanocarrier-biomaterial complex may be introduced into the interior of a living plant-based object (500) by bringing the composite solution (300) into contact with the object (500). As a non-limiting example, the object (500) may be immersed in or brought into contact with the composite solution (300) while the composite solution (300) is contained in a predetermined container (CT1). The type and size of the container (CT1) may vary.

[0081] FIG. 5 is a photographic image showing a solution containing Py-His6-SWNT synthesized according to one embodiment of the present invention. The solution may be a dispersion in which Py-His6-SWNT particles are dispersed.

[0082] FIG. 6 is a graph showing the results of measuring the zeta potential of Py-His6-SWNT synthesized according to one embodiment of the present invention. The zeta potential was obtained by dynamic light scattering (DLS) (Malvern Panalytical) measurement.

[0083] Referring to Fig. 6, it can be confirmed that the Py-His6-SWNT synthesized according to the example has a positive (+) zeta potential. Therefore, the Py-His6-SWNT, which corresponds to a nanocarrier, can have a positive (+) charge and can be used as a carrier for biomaterials having a negative (-) charge. Biomaterials having a negative (-) charge can bind well to the Py-His6-SWNT.

[0084] Figure 7 is a graph showing the results of measuring the size distribution of Py-His6-SWNT particles synthesized according to one embodiment of the present invention. The size distribution was obtained by measuring with Nanosight NM10 (Malvern Panalytical) using the nanoparticle tracking analysis (NTA) method.

[0085] Referring to Fig. 7, it can be seen that the synthesized Py-His6-SWNT particles have a relatively small size and a relatively uniform size distribution. The size measured in Fig. 7 is the hydrodynamic radius, which may differ from the actual size of the Py-His6-SWNT particles.

[0086] FIG. 8 shows Py-His6-SWNT synthesized according to one embodiment of the present invention and dCS synthesized according to a comparative example. cov This is a graph showing the change in zeta potential according to the change in pH of SWNT. Here, dCS according to the comparative example cov -SWNT is Chitosan-SWNT made by covalently bonding deacetylated chitosan to COOH-SWNT.

[0087] Referring to Fig. 8, to verify the pH sensitivity of Py-His6-SWNT, the change in zeta potential of Py-His6-SWNT was examined in water with various pH values ​​(pH 5–8). For comparison, among existing cationic polymers, Chitosan-SWNT, specifically dCS, in which deacetylated chitosan is covalently bonded to COOH-SWNT, was compared. cov -SWNT and pH sensitivity were compared. As a result, Py-His6-SWNT dCS cov - It was observed that charge neutralization proceeded relatively faster with increasing pH compared to SWNT.

[0088] To confirm the biomaterial release characteristics of the nanocarrier according to an embodiment of the present invention, Cy3-bound (GT) 15 Experiments were performed using [the substance]. Cy3 is a fluorescent dye, and (GT) 15 is the full sequence name of ssDNA (single strand DNA).

[0089] FIG. 9 shows (GT) on Py-His6-SWNT synthesized according to one embodiment of the present invention 15 This is a graph showing the results of evaluating the fluorescence characteristics of Cy3 before and after the binding of -Cy3.

[0090] Figure 10 is a graph showing the results of evaluating the degree of biomaterial (ssDNA) release according to pH change of Py-His6-SWNT synthesized according to one embodiment of the present invention.

[0091] As shown in Fig. 9, (GT) 15 When Cy3 is attached to a modified SWNT (i.e., Py-His6-SWNT) rather than when it exists alone, the fluorescence of Cy3 is quenched and its intensity decreases, and (GT) 15When Cy3 moves away from the surface of the modified SWNT, the fluorescence intensity of Cy3 increases again. Referring to Figure 10, it can be seen that Py-His6-SWNT releases a large amount of ssDNA at a pH of about 7–8, which is the pH inside a plant cell.

[0092] The plant material (subject) and its growth conditions used in the embodiments of the present invention are described as follows.

[0093] In the embodiments of the present invention, the Columbia-0 ecotype Arabidopsis thaliana (Col-0) was used as the wild type. After sterilizing the surface of the Arabidopsis thaliana seeds, they were planted and cultured on 0.7% agar plates containing 1 / 2 MS (Murashige and Skoog). The plant material was cultured under a white fluorescent lamp (120 μmol photons·m²) at a temperature of approximately 22–23°C. -2 ㆍs -1 ) was cultured under long-day conditions (16 hours light / 8 hours dark cycle) using ). For plant callus formation, hypocotyl explants of 7-day-old plants were cultured in the dark for 7 days at a temperature of 22°C in a callus-inducing medium (MS medium supplemented with 0.5 μg / ml 2,4-dichlorophenoxyacetic acid and 0.05 μg / ml kinetin). However, these plant materials (subjects) and growth conditions are merely exemplary and may vary.

[0094] The method of introducing and analyzing a nanocarrier (a type of nanoparticle) into a callus used in an embodiment of the present invention is described as follows.

[0095] Calluses were soaked in 200 µl of aqueous Py-His6-SWNT solutions of various concentrations and cultured for 18 hours. Culture was conducted under dark conditions at room temperature. After the culture period ended, residual SWNTs were removed by transferring the calluses three times to 200 µl of distilled water, and the calluses were subsequently transferred to solid media for observation. To determine the optimal Py-His6-SWNT culture time for the calluses, callus activity after SWNT delivery was observed through the phenotype 14 days after the completion of SWNT treatment (Fig. 11). Additionally, the characteristic Raman peak of SWNT, 1590 cm⁻¹, was observed. -1 By observing Raman bands on both the surface and inside the callus, the penetration efficiency of SWNTs into the callus was observed through Raman analysis (Fig. 13). As a result, it was confirmed that Py-His6-SWNT did not affect (negatively affect) the cleavage activity of the callus under all Py-His6-SWNT culture conditions, and it was confirmed that Py-His6-SWNT could penetrate the entire callus when 2 mg / L Py-His6-SWNT (i.e., Py-His6-SWNT with a SWNT concentration of 2 mg / L) was treated to the callus for 18 hours. However, the aforementioned method of introducing the nanocarrier is merely exemplary and can be varied.

[0096] Figure 11 is an image showing the results of observing the phenotype of the cleavage activity of a callus into which Py-His6-SWNT synthesized according to an embodiment of the present invention was introduced. Figure 11 also includes results for a negative control and a callus treated with DIW (deionized water) for comparison.

[0097] Referring to Figure 11, it can be confirmed that there are no problems with the cleavage activity of the callus to which Py-His6-SWNT was introduced, that is, the callus treated with Py-His6-SWNT. It was confirmed that Py-His6-SWNT does not have a negative effect on the cleavage activity of the callus under all Py-His6-SWNT culture conditions.

[0098] FIG. 12 is a microscopic image showing a callus treated with Py-His6-SWNT according to an embodiment of the present invention.

[0099] Figure 13 is an image showing the penetration characteristics of Py-His6-SWNT into a callus treated with Py-His6-SWNT according to an embodiment of the present invention, analyzed by confocal Raman microscopy. Figures 13a, b, and c correspond to the results at points a, b, and c in Figure 12. Figure 13 is the result of an experiment using Py-His6-SWNT with 2 mg / L of SWNT applied.

[0100] Referring to Fig. 13, the area marked in red in the confocal Raman microscopy analysis image may indicate the region where SWNTs are present. From these results, it can be confirmed that when a callus is treated with Py-His6-SWNT for a predetermined time, Py-His6-SWNT can penetrate the entire interior of the callus.

[0101] The method for the in vitro synthesis (i.e., in vitro transcription) of STTM396 used in the embodiments of the present invention is described as follows. Here, STTM396 is an example of a biomaterial used in the embodiments of the present invention.

[0102] In an embodiment of the present invention, for the in vitro synthesis of STTM396 that inhibits the activity of miRNA396, a template plasmid DNA inserted with the sequence of STTM396 (Sequence No. 1) was cloned using a 2-fragment Gibson assembly method. Through nucleotide sequencing analysis, it was confirmed that the sequence was that of a template plasmid with the inserted STTM396 sequence. The template plasmid DNA was linearized by cutting the 3' end of the STTM396 sequence using the Spe I restriction enzyme, and the template DNA was purified using a "Gel & PCR purification kit." In vitro synthesis was carried out using T7 RNA polymerase, and subsequently, DNA in the tube was removed through Dnase I treatment. The synthesized STTM396 was purified using an RNA cleanup kit. However, the types of biomaterials and synthesis methods applied in the embodiments of the present invention are not limited to those described above.

[0103] In an embodiment of the present invention, Py-His6-SWNT and RNA (i.e., STTM396) can be reacted at various ratios for about 10 minutes to form a complex of Py-His6-SWNT and RNA. The reaction ratio (mixing ratio) for forming the complex can be determined based on zeta potential and particle size data analyzed based on dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) measurement results.

[0104] FIG. 14 is a graph showing the change in zeta potential according to the mixing ratio of a complex containing Py-His6-SWNT and RNA (STTM396) according to an embodiment of the present invention. In FIG. 14, "SWNT only" is the case where only Py-His6-SWNT is present without the binding of RNA.

[0105] Referring to FIG. 14, changes in the zeta potential of the complex according to the mixing ratio of Py-His6-SWNT and RNA (STTM396) can be observed. According to one embodiment, an absolute value of the zeta potential of the complex of Py-His6-SWNT and RNA of about 20 mV or higher may be advantageous for penetration into the cytoplasm of the target. In this regard, in the embodiment of FIG. 14, it may be preferable for the mixing ratio of Py-His6-SWNT and RNA to be 10:1, 1:1, and 1:5. However, these results are merely exemplary, and embodiments of the present invention are not limited thereto.

[0106] FIG. 15 is a graph showing the change in size according to the mixing ratio of a complex containing Py-His6-SWNT and RNA (STTM396) according to an embodiment of the present invention. In FIG. 15, "SWNT only" is the case where only Py-His6-SWNT is present without the binding of RNA.

[0107] Referring to Fig. 15, it can be seen that the size of the complex changes depending on the mixing ratio of Py-His6-SWNT and RNA (STTM396). However, the size measured in Fig. 15 is the hydrodynamic radius and may differ from the actual size of the complex. As a non-limiting example, it may be desirable for the diameter (outer diameter) of the nanocarrier-biomaterial complex to be about 10 nm or less.

[0108] According to one embodiment, the absolute value of the zeta potential of the nanocarrier-biomaterial complex may be about 20 mV or more, and the diameter (outer diameter) of the nanocarrier-biomaterial complex may be about 10 nm or less. When these conditions are satisfied, the nanocarrier-biomaterial complex can be well dispersed in solution and can penetrate well into the cytosol.

[0109] The method for introducing a complex of Py-His6-SWNT and RNA (STTM396) into a callus and the method for analyzing the introduced complex used in the embodiments of the present invention are described as follows.

[0110] The above-mentioned Py-His6-SWNT and RNA (STTM396) complex can be cultured in the dark on callus at a concentration of 2 mg / L based on SWNT for 18 hours. The method of introducing the Py-His6-SWNT and RNA complex into the callus was carried out in the same manner as previously described. Subsequently, the penetration characteristics and efficiency of the complex into the callus were observed through Raman analysis.

[0111] FIG. 16 is a microscopic image showing a callus treated with a complex of Py-His6-SWNT and RNA (STTM396) according to an embodiment of the present invention.

[0112] Figure 17 is an image showing the penetration characteristics of a complex into a callus treated with a complex of Py-His6-SWNT and RNA (STTM396) according to an embodiment of the present invention, analyzed by confocal Raman microscopy. Figures 17a, b, and c correspond to the results at points a, b, and c in Figure 16. Figure 17 shows the experimental results using Py-His6-SWNT to which 2 mg / L of SWNT was applied. Additionally, in Figure 17, the mass ratio of Py-His6-SWNT to RNA was 1:1.

[0113] Referring to Fig. 17, the area marked in red in the confocal Raman microscopy analysis image may indicate the region where SWNT is present. From these results, it can be confirmed that when a callus is treated with a complex of Py-His6-SWNT and RNA for a predetermined time, the complex can penetrate the entire surface and interior of the callus.

[0114] The technologies according to the embodiments of the present invention can provide conditions for forming a complex that do not lose the dispersibility of the nanocarrier even after loading a biomaterial (e.g., RNA) onto the nanocarrier, and can provide culture conditions that can effectively deliver the biomaterial without losing the activity of the callus even after the complex is inserted into the callus. In addition, the amount of biomaterial released by the complex according to the embodiments of the present invention may vary depending on the pH, which can facilitate the delivery of the biomaterial into the plant cell. Therefore, the technology according to the embodiments can be utilized as a technology capable of selective gene expression control at a specific time during the callus tissue culture process.

[0115] Embodiments of the present invention allow for the convenient delivery of biomaterials to plant tissues or plants through a simple process of forming a complex by mixing biomaterials with SWNTs modified with imidazole and immersing a plant in a solution containing the complex, and can be used for the development of new breeding varieties without separate physicochemical shock. Embodiments of the present invention may be applicable to new breeding development technologies, the production of plant biotechnology-based pharmaceuticals, etc.

[0116] The method for analyzing the redifferentiation phenotype of a callus treated with a complex of Py-His6-SWNT and RNA (STTM396) used in the embodiments of the present invention is as follows.

[0117] Calluses were treated for 18 hours under dark conditions at concentrations of 2 mg / L and 5 mg / L relative to SWNT using a complex prepared by reacting Py-His6-SWNT and RNA (STTM396) in a 1:1 mass ratio. The method of introducing the Py-His6-SWNT and RNA complex into the calluses was the same as previously described. For callus redifferentiation, the treated calluses were cultured for 21 days in shoot-inducing medium (MS medium supplemented with 0.9 μmol / L 3-indoleacetic acid and 2.5 μmol / L 2-isopentenyl adenine) at 25°C under continuous light conditions. As a result, it was confirmed that the cell redifferentiation rate of the calluses increased when the calluses were treated with the Py-His6-SWNT and RNA complex. However, the method for analyzing cell redifferentiation using a complex is not limited to what has been described above and can be varied in many ways.

[0118] FIG. 18 is a drawing showing an image in which the regeneration of the callus is evaluated as a phenotype after treating the callus according to an embodiment and a comparative example of the present invention.

[0119] FIG. 19 is a graph showing the results of quantitatively evaluating the regeneration of callus after treating callus according to the embodiments and comparative examples of the present invention.

[0120] In FIGS. 18 and 19, (A) through (D) correspond to comparative examples, and (E) and (F) correspond to examples. (A) corresponds to a mock case as a control group. (B) is the case where the callus was treated with DIW. (C) is the case where the callus was treated with STTM396. (D) is SWNT modified with imidazole (i.e., Py-His nThis is a case where the callus was treated with -SWNT. (E) and (F) are cases where the callus was treated with a complex of Py-His6-SWNT and RNA (STTM396) according to the example. (E) is a case where the callus was treated with a concentration of 2 mg / L based on SWNT, and (F) is a case where the callus was treated with a concentration of 5 mg / L based on SWNT.

[0121] Referring to Figures 18 and 19, it can be seen that the cell redifferentiation rate of the callus was increased when the callus was treated with a complex of Py-His6-SWNT and RNA (STTM396) in the examples rather than in the comparative examples.

[0122] According to the embodiments of the present invention described above, a nanocarrier capable of effectively delivering biomaterials into a living plant-based target can be realized. Furthermore, according to the embodiments, a nanocarrier capable of releasing biomaterials within plant cells with excellent pH sensitivity can be realized. Additionally, according to the embodiments, a nanocarrier applicable regardless of plant species can be realized that a biomaterial suitable for a target plant species or a related target can be stably introduced without limitation. Furthermore, according to the embodiments, a nanocarrier capable of delivering biomaterials capable of promoting plant cell regeneration into a living plant-based target can be realized.

[0123] According to embodiments of the present invention, a nanocarrier-biomaterial complex comprising the above-described nanocarrier can be realized. Additionally, according to embodiments, a method for treating a target using the above-described nanocarrier can be provided. Additionally, according to embodiments, a method for promoting the redifferentiation of plant cells using the above-described nanocarrier can be provided.

[0124] According to one embodiment, a nanocarrier can be implemented that can easily deliver external genetic material (biomaterial) into plant cells, which can be applied to enhance the regeneration efficiency of plants. In addition, unlike gene delivery technologies using a gene gun, which has low delivery efficiency and involves physicochemical shock from the outside, or Agrobacterium tumefaciens, which has limitations in applicable plant species, the nanocarrier according to one embodiment can provide a simple, non-destructive method of delivering genetic material that is easy to apply to various plant species.

[0125] In addition, according to one embodiment, unlike conventional carriers that have difficulty delivering genetic materials such as RNA due to limitations in the shape or size of bindable biomaterials, a method can be provided to efficiently deliver biomaterials such as RNA into a living plant-based target, such as a callus, by using a nanocarrier having excellent pH sensitivity. The technology according to one embodiment can effectively enhance / promote plant regeneration by controlling genes that inhibit regeneration of the callus.

[0126] This specification discloses preferred embodiments of the present invention. Although specific terms have been used, they are used merely in a general sense to facilitate the explanation of the technical content of the invention and to aid in understanding the invention, and are not intended to limit the scope of the invention. It is obvious to those skilled in the art that, in addition to the embodiments disclosed herein, other variations based on the technical concept of the present invention are possible. Those skilled in the art will understand that the nanocarrier for biomaterial delivery, the nanocarrier-biomaterial complex, the method for manufacturing the same, and the method for treating a living plant-based target using the nanocarrier, according to the embodiments described with reference to FIGS. 1 to 19, can be variously substituted, changed, and modified within the scope of the technical concept of the present invention. Therefore, the scope of the invention should not be determined by the described embodiments but by the technical concept described in the claims. Explanation of the symbols

[0127] * Explanation of symbols for major parts of the drawing * 10 : Nanotube 12 : Pirenil 14 : Malaimide 20: Functional Compounds 22: Cysteine 50 : Biomaterial 100 : Nanocarrier 200 : Nanocarrier-biomaterial complex 300: Complex solution 500 : Object

Claims

Claim 1 A nanocarrier for delivering biomaterials, comprising: a nanotube as a nanocarrier for delivering biomaterials into a living plant-based object; and a functional compound comprising an imidazole that is bonded to the polycyclic aromatic substance, wherein at least a polycyclic aromatic substance is non-covalently bonded to the surface of the nanotube by π-π interactions rather than covalent bonds, and the nanocarrier is introduced into the object to deliver the biomaterials. Claim 2 In claim 1, the polycyclic aromatic material is a nanocarrier containing pyrenyl. Claim 3 A nanocarrier according to claim 1, further comprising maleimide bonded to the polycyclic aromatic substance and cysteine ​​bonded to the functional compound, wherein the polycyclic aromatic substance and the functional compound are mutually bonded by the bonding of the maleimide and the cysteine. Claim 4 delete Claim 5 In claim 1, the nanotube is a nanocarrier comprising a carbon nanotube. Claim 6 In claim 5, the nanotube is a nanocarrier comprising a single-walled carbon nanotube (SWNT). Claim 7 In claim 2, the nanocarrier is Py-His n - A nanocarrier comprising a SWNT structure, wherein Py represents the pyrenyl, His represents histidine including the imidazole, and the SWNT is a nanocarrier corresponding to the nanotube. Claim 8 In claim 7, the nanocarrier is greater than or equal to 2 and less than 100. Claim 9 In claim 1, the object is a nanocarrier comprising a callus. Claim 10 In claim 1, the biomaterial is a nanocarrier comprising RNA (ribonucleic acid). Claim 11 A nanocarrier for delivering biomaterials, comprising: a nanotube as a nanocarrier for delivering biomaterials within a living plant-based object; and a functional compound comprising an imidazole that is bonded to the polycyclic aromatic substance, wherein the polycyclic aromatic substance is non-covalently bonded to the surface of the nanotube by π-π interactions rather than covalent bonds, and the nanocarrier is configured to deliver the biomaterial that promotes the regeneration of plant cells of the object into the object. Claim 12 A nanocarrier-biomaterial complex for application within a living plant-based object, comprising: a nanocarrier as described in any one of claims 1 to 3 and 5 to 11; and a biomaterial coupled to the functional compound of the nanocarrier. Claim 13 In claim 12, the biomaterial is a nanocarrier-biomaterial complex comprising RNA (ribonucleic acid). Claim 14 A nanocarrier-biomaterial complex according to claim 12, wherein the absolute value of the zeta potential of the nanocarrier-biomaterial complex is 20 mV or more, and the diameter of the nanocarrier-biomaterial complex is 10 nm or less. Claim 15 In claim 12, the nanocarrier-biomaterial complex is a nanocarrier-biomaterial complex having the characteristic of releasing the biomaterial within the plant cells of the target. Claim 16 A method for treating an object using a nanocarrier, comprising: a step of preparing a complex solution containing a nanocarrier-biomaterial complex described in claim 12; and a step of bringing the complex solution into contact with a living plant-based object to introduce the nanocarrier-biomaterial complex into the interior of the object. Claim 17 In claim 16, the biomaterial comprises RNA (ribonucleic acid), and a method for treating a target using a nanocarrier in which the regeneration of plant cells of the target is promoted by the RNA. Claim 18 In claim 16, the object is a method for treating an object using a nanocarrier comprising plant tissue or a plant body. Claim 19 In claim 16, the object is a method for treating an object using a nanocarrier comprising a callus. Claim 20 A method for treating a target using a nanocarrier, wherein, in claim 16, the absolute value of the zeta potential of the nanocarrier-biomaterial complex is 20 mV or more, and the diameter of the nanocarrier-biomaterial complex is 10 nm or less. Claim 21 A method for treating a target using a nanocarrier comprising a nanocarrier-biomaterial complex in which a nanocarrier comprising a functional compound having imidazole and a biomaterial comprising RNA (ribonucleic acid) are combined; and a step of contacting the complex solution with a living plant-based target to introduce the nanocarrier-biomaterial complex into the interior of the target and promoting the regeneration of the target's plant cells using the RNA, wherein the nanocarrier has at least a polycyclic aromatic substance non-covalently bonded to the surface of a nanotube by π-π interactions rather than covalent bonds, and the nanocarrier has the imidazole bonded to the polycyclic aromatic substance. Claim 22 In claim 1, the object is a nanocarrier for the delivery of biomaterials including plant cells. Claim 23 In claim 11, the subject comprises a plant cell, and the biomaterial is a nanocarrier for biomaterial delivery comprising RNA (ribonucleic acid).

Images