KR101220989B1 - Intracellular Delivery of Cargo Using Nanosyringe Array - Google Patents

Abstract

The present invention relates to a method for intracellular delivery of cargo for delivery purposes comprising the step of contacting cells with an open top of a nanoinjector array comprising cargo for delivery purposes. The present invention can be used in biological research to understand a variety of applications and intracellular functions, including cell-based therapies, in vivo imaging and tracking.

Nano syringe array, nanotubes, carbon, intracellular transport

Description

Intracellular Delivery of Cargo Using Nanosyringe Array

The present invention relates to an intracellular delivery method for intracellular cargo and an intracellular cargo vehicle.

Mesenchymal stem cells (Asahara, T., et al., Gene Ther., 7: 451-457 (2000); Mazhari, R. and Hare, JM, Nat. Clin. Pract. Cardiovasc. Med. , 4: S21- S26 (2007); Dazzi, F. and Horwood, NJ, Curr. Opin.Oncol . , 19: 650-655 (2007); Au, P., et al. , Blood , 111: 4551-4558 (2008); Lee, KB, et al. , Stem Cells , 25: 2964-2971 (2007)) or immune cells (Ljunggren, H.-G. and Malmberg, K.-J., Nat. Rev. Immunol. , 7: 329 -339 (2007); Matter, M., et al. , Cancer Res. , 67: 7467-7476 (2007); Kwon, ED, et al. , Proc. Natl. Acad. Sci. USA , 94: 8099- 8103 (1997)), with increasing interest in cell therapies, carrying genetic material into cells (Cemazar, M., et al. , Cancer Gene Ther. , 9: 399-406 (2002); Pack, DW) , et al. , Nat. Rev. Drug Discov. , 4: 581-593 (2005); Dalby, B., et al. , Methods , 33: 95-103 (2004)) and fluorescence for in vivo tracking or Labeling cells with magnetic nanoparticles (LaVan DA, et al. , Nature Biotechnol. , 21: 1184-1191 (2003); Lei, Y., et al. , Bioconjugate Chem. , 1 9: 421-427 (2008); Jaiswal, JK, et al. , Nat. Biotechnol. , 21: 47-51 (2003); Rogers, WJ, et al. , Nat. Clin. Pract. Cardiovasc. Med. , 3: 554-562 (2006); Ahrens, ET, et al. , Nat. Biotechnol. 23: 983-987 (2005). There is an increasing need for efficient devices and methods. Such devices preferably simultaneously and efficiently promote the intracellular transport of various foreign macromolecules or nanomaterials without external force and preferably do not adversely affect cell viability.

Plasma membranes of cells are an insurmountable barrier to the transport of foreign polymers in cell engineering, labeling, and cell therapy. Many attempts have been made to break through this barrier, especially using mechanical methods. Delivery of the dielectric material is typically carried out by the micro-injector method (Zhang, Y. and Yu, LC, BioEssays , 30: 606-610 (2008); Tsulaia, TV, et al. , J. Biomed. Sci. , 10: 328-336 (2003); Leonetti, JP, et al. , Proc. Natl. Acad. Sci. USA , 88: 2702-2706 (2003)) The difficulty lies in the damage on the cell membranes by needles. Bertozzi recently reported that functionalized carbon nanotubes attached to atomic force microscope tips can carry quantum dots (QDs) into cells without affecting cell viability (Chen). , X., et al. , Proc. Natl. Acad. Sci. USA , 104: 8218-8222 (2007). Both the micro-injector method and the carbon nanotube nano-injector system are limited delivery (ie, genes or nanoparticles) of cargo (eg genes or nanoparticles) into one cell as a function of time. Low speed) and the need for complex microscopic tools to control the injection position. In recent years, simultaneous gene transfer into numerous cells has been reported in carbon nanofiber arrays (McKnight, TE et al. , Nanotechnology , 14: 551-556 (2003); McKnight, TE, et al. , Nano Lett., 4: 1213-1219 (2004); Mann, DGJ, et al. , ACSNANO , 2: 69-76 (2008)) or silicon nanowire arrays with high aspect ratios (Kim, W , et al. , J. Am. Chem. Soc. , 129: 7228-7229 (2007)), but there are limitations to practical applications for the following reasons. First, external gravity by centrifugation is necessary to penetrate the cells with nanofibers (of various thicknesses and lengths), which causes severe damage to the cells. Second, only a small amount of plasmid DNA that is physically adsorbed or covalently attached to the carbon fiber surface is useful for intracellular transport. Third, the transport of various mixtures, such as plasmid DNA, is limitedly possible because of the absence of compartments capable of loading various cargoes with different characteristics and forms. As a result, there is a need in the art for a device that does not need to exert external force and can simultaneously and effectively promote intracellular transport of various foreign polymers without detrimental effect on cell viability.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

We sought to improve the nanoinjector array for intracellular transport of cargo. As a result, the inventors have developed a new platform for simple and sophisticated intracellular delivery using an array of vertically aligned nanoinjectors with adjustable heights. The present invention has been accomplished by confirming that various substances such as plasmid DNA and quantum dots can be efficiently transported into the cytoplasm of various cells such as cancer cells and human mesenchymal stem cells.

It is therefore an object of the present invention to provide a method for intracellular delivery of cargo for transport purposes.

Another object of the present invention is to provide an intracellular cargo carrier.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention is a method for intracellular delivery of cargo for delivery purposes comprising the step of contacting cells with an open top of a nanoinjector array comprising cargo for transportation purposes.

According to another aspect of the present invention, the present invention provides a method for producing a nanotube comprising: (a) an array of nanotubes having a tubular structure of an open portal; and (b) a hydrophilic or amphiphilic coating on the surface of the nanotubes. Provided are intracellular cargo carriers comprising amphiphilic polymers.

The inventors have sought to improve the nanoinjector array for intracellular transport of genetic material and nanoparticles. As a result, the inventors used vertically aligned nanoscanner arrays (CNSAs) (Kim, Y.-S., et al., Nanotechnology , accepted (2008)) with adjustable heights for simple and sophisticated intracellular delivery. We have developed a new platform. The present invention has been accomplished by confirming that various substances such as plasmid DNA and quantum dots can be efficiently transported into the cytoplasm of various cells such as cancer cells and human mesenchymal stem cells.

As used herein, the term 'quantum dots' (QDs) generally refers to semiconductor or metal nanoparticles that absorb light in different colors and quickly re-emit depending on the size of the dots. Nanocrystals, which can be called nanoscale single crystals or nanoscale single crystals, are largely divided according to their dimensions.The point corresponding to the 0 dimension or a single sphere, 1 Dimensions include nanowires, nanorods, nanoneedles, and two-dimensional nanodisks. The dot, which corresponds to the 0-dimensional nanocrystal, is commonly called a quantum dot and can be seen as a pattern of hemispheres in the form of droplets ranging from several tens to several tens of nanometers in diameter. If the size of the quantum dot is made very small and limited to a shorter region than the steady state material wave, the energy level can be increased. Depending on the energy state, the color of light emitted when each material is excited changes the wavelength of light emitted by controlling the size of the quantum dots. Thus, if the tiny quantum dots themselves are luminescent sources, they can be used in biological applications to understand various applications and intracellular functions, including cell therapy, in vivo imaging and tracking.

As used herein, the term “aspect ratio” is obtained by dividing the length of the first axis of the nanostructure by the average of the lengths of the second axis and the third axis, and the lengths of the second axis and the third axis are substantially the same.

According to the method of the present invention, the AAO template having a well-ordered pore structure is transformed into a two-step anodization process. The AAO template obtained through this anodization process is used for vertically adjustable carbon nanosyringe arrays (CNSAs). The two-stage anodization process consists of ion milling and chemical etching. The ion milling process is carried out by anodizing for 12 hours at 14 ° C. in a 0.3 M oxalic acid solution, followed by 12 hours at 60 ° C. with a mixture of phosphoric acid, chromic acid and tertiary-deionized water (6: 1.8: 92.9 wt%). A chemical etching process is carried out to anodize.

Carbon deposition includes thermal chemical vapor deposition (thermal CVD) (Kaatz, FH, et al. , Materials Science and Engineering: C , 141-144 (2003)), liquid epitaxy phase epitaxy (Oh SJ, et al. , Appl. Phys. Lett. , 84: 3738-3740 (2004)), vapor phase epitaxy (Klein, PB, et al. , Appl. Phys. Lett. , 79: 3527-3529 (2001)) or molecular beam epitaxy (Chang, JH, et al. , Appl. Phys. Lett. , 79: 785-787 (2001)). Can be carried out. Carbon deposition of the present invention is carried out using thermal chemical vapor deposition (thermal CVD). Argon (Ar) gas is introduced into the CVD chamber during the heat treatment of the carbon film. A gas mixture (1: 9 flow rate ratio) of acetylene (C ₂ H ₂ ) and ammonia (NH ₃ ) was deposited at 600 ° C. to deposit a carbon film on the surface of AAO pores by pyrolysis of acetylene. It is introduced into the CVD chamber for a time (100 mL / min).

According to a preferred embodiment of the invention, the cargo for transport is a chemical, a nanoparticle, a peptide, a polypeptide, a nucleic acid, a carbohydrate or a lipid.

According to an embodiment of the present invention, the nanoscanner array of the present invention consists of nanotubes having a tubular structure of an open portal. According to a preferred embodiment of the invention, the nanotubes are surrounded by a template for forming the nanotubes and the top end of the template is etched to expose the nanotubes. According to a preferred embodiment of the invention, the height of the exposed nanotubes is 40-200 nm, more preferably 70-130 nm.

According to a preferred embodiment of the invention, the nanotubes are made of carbon, silicon, precious metals, silicon dioxide or titanium dioxide.

According to a preferred embodiment of the invention, the nanoscanner has a diameter of 20-200 nm.

According to a preferred embodiment of the present invention, the nanoscanner array is located at one end on the substrate. According to a preferred embodiment of the present invention, the nanoinjector is infiltrated into the cell and the height of the infiltrated nanoinjector is 40-200 nm. According to a more preferred embodiment of the invention, the height of the penetrated nanoinjector is 70-130 nm.

According to a preferred embodiment of the invention, the surface of the nanoscanner is coated with a hydrophilic or amphiphilic polymer. According to a more preferred embodiment of the present invention, the nanoscanner is coated with an amphiphilic polymer.

According to a preferred embodiment of the present invention, the amphiphilic polymer (amphiphilic) polymer is characterized in that represented by the following formula (1):

Formula 1

In the above formula, R ₁ is a silylalkyl group, (alkoxysilyl) alkyl group, (hydroxysilyl) alkyl group, substituted or unsubstituted alkyl group, substituted or unsubstituted aryl group, substituted or unsubstituted aralkyl group, or substituted or unsubstituted Ring alkaryl group; R ₂ is polyethylene glycol (PEG), dextran or polyvinylpyrrolidone; R ₃ is an aldehyde, epoxy, haloalkyl, primary amine, thiol, maleimide, ester, carboxyl group or hydroxyl group; R ₄ , R ₅ and R ₆ are, independently from each other, H or a C ₁ -C ₅ alkyl group; X and Y are independently of each other an oxygen, sulfur or nitrogen atom; l, m and n are each independently an integer of 1-10,000.

The term silyl alkyl used in the definition of R ₁ in formula (I) means a group substituted by a silyl group (preferably a silyl group having a carbon number of 1-10). For example, silylalkyl includes silylmethyl, silylethyl, silylpropyl and silylbutyl. The term (alkoxysilyl) alkyl refers to an alkoxy-substituted silylalkyl group (preferably (alkoxysilyl) alkyl having 1 to 10 carbon atoms). For example, (alkoxysilyl) alkyl may be trimethoxysilylethyl, trimethoxysilylpropyl, trimethoxysilylbutyl, trimethoxysilylpentyl, triethoxysilylethyl, triethoxysilylpropyl, triethoxysilylbutyl, Trimethoxysilylpentyl, methyldimethoxysilylethyl, methyldimethoxysilylpropyl, dimethylmethoxysilylethyl and dimethylmethoxysilylpropyl. The term (hydroxysilyl) alkyl means hydroxyl-substituted silylalkyl (preferably (hydroxysilyl) alkyl having 1 to 10 carbon atoms), for example trihydroxysilylethyl, trihydroxysilylpropyl, tri Hydroxysilylbutyl and trihydroxysilylpentyl.

The term "aralkyl" refers to an aryl group bonded to the structure by one or more alkyl groups, such as benzyl and phenylpropyl groups. The term "alkaryl" refers to an alkyl group bonded to a structure consisting of one or more aryl groups.

Preferably, in Formula 1, R ₁ is a (alkoxysilyl) alkyl group, a substituted or unsubstituted C _6-30 alkyl group, a substituted or unsubstituted C _6-30 alkoxy group, a substituted or unsubstituted aryl group, a substituted or unsubstituted A substituted aralkyl group or a substituted or unsubstituted alkaryl group, more preferably a (C _1-6 alkoxysilyl) C _1-10 alkyl group, a substituted or unsubstituted C _6-30 alkyl group, a substituted or unsubstituted phenyl group , A substituted or unsubstituted aralkyl group bound to a C _1-5 alkyl group with a phenyl group, or a substituted or unsubstituted alkaryl group bound with a C _1-5 alkyl group, most preferably (C _{1- 3} alkoxysilyl) C _1-5 alkyl group, substituted or unsubstituted C _8-20 alkyl group, substituted or unsubstituted C _6-20 alkoxy group, substituted or unsubstituted phenyl group, C ₁ as substituted or unsubstituted aralkyl group It is coupled to the phenyl group in _-5 alkyl group, or a substituted or non-substituted phenyl ring as an alkaryl group A C _1-5 alkyl group is bonded to.

The method of the present invention is carried out without artificial force. According to a preferred embodiment of the invention, the transport of cargo into the cells takes place in a spontaneous manner by gravity acting on the cells themselves.

According to a preferred embodiment of the present invention, the cells are normal cells, cancer cells, stem cells or immune cells. According to a more preferred embodiment of the invention, the cells are cancer cells or human mesenchymal stem cells.

According to a specific embodiment of the present invention, the amphiphilic polymer coating is performed on the exposed carbon nanoinjector, which is represented by Chemical Formula 2 of the amphiphilic polymer.

(2)

In the formula, l, m and n are polymers having a molar ratio of 2: 2: 1. Amphiphilic polymer (1 wt%) and CNSA used in the present invention are emulsified for 24 hours to convert the hydrophobic surface of the carbon nanoinjector into a hydrophilic surface. This surface processing step is necessary for the cargo to be loaded (by capillary activity) into the hollow core (compartment space) of the coronary CNSA syringe, which is typically an aqueous solution of targeted cargo for intracellular delivery. This is because it is dissolved or scattered on. According to an embodiment of the present invention, the exposed carbon nanoinjector has a diameter of 50 nm to 200 nm as a tubular structure of an open portal. According to a preferred embodiment of the invention, the carbon nanoinjector has a diameter of 50 nm to 150 nm, more preferably 50 nm to 100 nm, even more preferably 50 nm to 60 nm, most preferably 50 nm. .

As a final step, cells seeded in CNSA are penetrated in a spontaneous fashion by the cell's own gravity (Kim, W., et al. , J. Am. Chem. Soc. , 129: 7228-7229 (2007)). Cargo's intracellular transport follows.

According to an embodiment of the invention, the cargo is a genetic material, peptide, protein, lipid, polysaccharide, organic molecule and quantum dots. According to a preferred embodiment of the invention, the cargo is plasmid DNA and protons. According to a preferred embodiment of the invention, the genetic material is a non-viral gene vector, plasmid DNA, RNA, RNAi, siRNA and viral particles. According to a more preferred embodiment of the invention, the genetic material is plasmid DNA.

According to an embodiment of the invention, the label of cargo is a green fluorescence protein, a radioisotope (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), a chemical (eg, biotin), fluorescence Substances [e.g. fluorescein, fluoresein Isothiocyanate (FITC), rhodamine 6G (rhodamine 6G), rhodamine B (rhodamine B), 6-carboxy-tetramethyl-rhodamine (TAMRA), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole) and Coumarin], luminescent material, chemiluminescent and fluorescence resonance energy transfer (FRET). According to a preferred embodiment of the invention, the label of the cargo is a green fluorescence protein.

According to a preferred embodiment of the invention, the cells are cancer cells, HEK 293 cells, NIH3T3 cells, mouse melanoma cells, HaCat cells, B16F10 cells and human mesenchymal stem cells (MSCs). According to a more preferred embodiment of the invention, the cells are cancer cells and human mesenchymal stem cells.

The features and advantages of the present invention are summarized as follows:

(i) The present invention is to create a new platform for intracellular transport based on vertically aligned carbon nanoinjector arrays.

(ii) According to the present invention, plasmid DNA and quantum dots can be effectively transported into the cytoplasm of cancer cells and human mesenchymal stem cells.

(iii) According to the present invention, various cargoes can be loaded into the hollow coronary compartments of CNSA and can be transported to the cytoplasm of cells naïve without modification and intracellular transport is spontaneous (ie, no external force required) and simultaneously It occurs in cells (ie high-throughput).

(iv) In accordance with the present invention, the CNSA platform can be used for biological applications to understand various applications and intracellular functions, including cell-based therapy, in vivo imaging and tracking.

As described in detail above, the present invention provides a method of carbon nanosyringe arrays (CNSAs) for intracellular transport of genetic material and nanoparticles. The present invention also provides a delivery system for effectively delivering plasmid DNA and quantum dots to the cytoplasm of cancer cells and human mesenchymal stem cells. According to the method of the present invention, various cargoes can be spontaneously delivered to the cytoplasm of the cells naïve without modification.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

material

All organic solvents were used intact without further purification. PEGFPC1 vector DNA (4.7 kbp; Novagen, Madison, Wis.) Was amplified in Escherichia coli DH5α and Endofree plasmid groups were purified with the Frap Kit (Qiagen, Valencia, CA, USA). Lipofectamine ^™ 2000 and viability / cytotoxicity kit were purchased from Invitrogen (Carlsbad, CA).

Measure

Fluorescence images were observed using a Leica DMRBE microscope (Leica Microsystem AG, Wetzlar, Germany) equipped with magnifications × 100, × 200, × 400 and × 630 (1.4 aperture) oil objective. Images were obtained using a CoolSNAP ^™ fix CCD camera powered by Metamorph Imaging Software (Universal Imaging Co., Downingtown, PA, USA). After gene expression, cells were measured using a Coulter EPICS XL ^™ cytometer (Beckman Coulter, Inc., Fullerton, Calif.) Run with an Expo32 ADC analysis program. The cell morphology on carbon nanoinjectors was examined by scanning electron microscopy using S4700 electron microscope (Hitachi Ltd., Tokyo, Japan).

Manufacturing of the CNSA Platform

Preparation of AAO Nano Templates :

Highly ordered porous AAO templates were prepared via a two-step anodization process. To prepare AAO with 50 nm pore diameter, high purity (99.9995) aluminum to form a mirror at 5 ° C. with a mixture of perchloric acid and ethanol (1: 5, v / v) under an applied potential of 15 V. Goodfellow was degrease and electropolish for 5 minutes. The electro-polished aluminum foil was anodized for 12 hours at 14 ° C. in a 0.3 M oxalic acid solution under a constant voltage of 40 V. The pre-anodized oxide film with irregular pupil pattern was removed by chemical etching at 60 ° C. for 12 hours with a mixture of phosphoric acid, chromic acid and tertiary-deionized water (6: 1.8: 92.9 wt%). The second anodization was carried out for 3 hours under the same conditions as the first anodization step (0.3 M oxalic acid solution, 14 ° C., 40 V). The AAO nanotemplate was a hexagonal pattern and had a very constant array of appropriate 50 nm diameter and 6 μm long pupils.

Carbon Nanotube Growth :

The carbon film was incubated in the cavity of an AAO template prepared using a thermal chemical vapor deposition (thermal CVD) method without a catalyst. Argon (Ar) gas was introduced into the CVD chamber during the heat treatment process. In order to deposit the carbon film on the surface of AAO pupils by pyrolysis of acetylene, a gas mixture (1: 9 flow rate ratio) of acetylene (C ₂ H ₂ ) and ammonia (NH ₃ ) was added at 600 ° C. Was introduced into the CVD chamber for 1 hour (100 mL / min).

Partial Exposure of Carbon Nanoinjectors :

Removing the first precipitated carbon surface film by Ar ion milling until the AAO surface is exposed and etching the AAO film at 60 ° C. with a mixture of phosphoric acid, chromic acid and tertiary-deionized water (6: 1.8: 92.9 wt%) The carbon nanoscanner was partially exposed through the step of partially etching by emulsifying the solution. The length of the exposed carbon nanoinjector can be controlled very systematically by adjusting the emulsion time.

CNSA was sterilized by continuous soaking in acetone and ethanol to avoid infection during the intracellular transport process before the amphiphilic polymer coating. Upon coating, CNSA was soaked in amphiphilic polymer solution (1 wt% in Millipore water) for 24 hours, then washed three times with Millipore water and dried in 100 ° C. oven.

Cell Culture and Reagents

NIH3T3 cells (American Type Culture Collection, Manassas, VA) were maintained in adhesive culture and added with 10% (v / v) fetal bovine serum (FBS, GIBCO) and 100 IU / mL streptomycin (GIBCO). Incubated in DMEM medium (Dulbecco's Modified Eagle's Medium; GIBCO-Invitrogen, Grand Island, NY) with a single membrane using a 37 ° C humidity cabinet. Human MSCs were maintained in low-glucose DMEM medium containing supplements such as NIH3T3 cell cultures.

Cargo Loading and CNSA-mediated Intracellular Transport

For intracellular delivery of cargo, a drop of water containing either plasmid DNA (400 μg / mL) or quantum dots (1 nM) was placed in CNSA and allowed to react for 12 hours. The cargo-loaded CNSA was then washed with Millipore water using a micropipette to remove unloaded cargo. Cells were dispensed into CNSA in wells (1 × 10 ⁵ cells / well) of 24-well cell culture plates and incubated at 37 ° C. under 5% CO ₂ /95% air for 12 hours. Treated cells were isolated from CNSA using trypsin / EDTA and gentle pipetting. The isolated cells were dispensed back into 24-well cell culture plates and allowed to adhere for 12 hours before intracellular transport of cargo was examined.

On the other hand, the CNSA-mediated intracellular transport experiment using the force was performed. CNSA was prepared as above and 5 nM Qdot605 (Quantum Dot Corporation) was loaded on CNSA surface for 24 hours. Next, active lymphoside cells (activator PMA (phorbol 12-myristate 13-acetate) and ionomycin treated cells) were seeded on CNSA loaded with Qdot605. Cells were then moved downward by centrifugal force (800 g or 1000 g), incubated for 24 hours, and intracellular delivery of Qdot605 was investigated.

Cytotoxic Assays in vitro )

NIH3T3 cells incubated in CNSA for 12 hours were re-divided (5 × 10 ³ cells / well) into 96-well plates containing 100 μL medium. After 24 hours the medium was aspirated and washed twice with PBS. Fresh culture (100 μL) was added to each well, after which 20 μL of MTT solution (2.5 mg / mL in PBS) was added. The cells were further incubated at 37 ° C. for 4 hours. The medium was carefully aspirated. After lysing the cells in 200 μL dimethylsulfoxide (DMSO), the absorbance of each well was measured at 570 nm using a FL600 microplate reader (Bio-Tek Inc., Winooski, VT). Data is expressed as percent of live cells compared to control.

Scanning electron microscope

After incubation in CNSA for 12 hours, NIH3T3 cells were fixed in 3% glutaaldehyde solution, washed with PBS buffer, dehydrated with grade ethanol series and lyophilized. Cells were observed using S4700 SEM (Hitachi Ltd., Tokyo, Japan).

Experiment result

1A illustrates the overall process for intracellular delivery of cargo using the CNSA platform of the present invention. Prepare a CNSA platform, including the following successive chemical and physical steps: i) preparing nanoporous anodized aluminum oxide (AAO); ii) carbon deposition in the vacant cavity of the AAO; And iii) performing ion milling and chemical etching to expose to an array of carbon nanoinjectors of adjustable height. 1B shows a typical scanning electron microscope image of CNSA with an exposure length of about 80 nm to 120 nm from an AAO film. As shown in transmission electron microscopy images of one carbon nanoscanner (see FIG. 5), each nanoscanner is a tubular structure with an outer diameter of about 50 nm and an open portal. In the next step, an amphiphilic polymer (at 1 wt% in water) dispersed in water to form a stable coat carbon nanotube (Park, S., et al. , Chem. Comm. , 2876- 2878 (2008)) to convert the hydrophobic surface of the nanoinjector into a hydrophilic surface. 1C shows the chemical structure of an amphipathic polymer. This surface processing step is necessary for the cargo to be loaded (by capillary activity) into the hollow core (compartment space) of the coronary CNSA syringe, in which the targeted cargoes for intracellular transport are usually dissolved or dissolved in aqueous solution. It is scattered. As a final step, cells seeded in CNSA are penetrated in a spontaneous fashion by the cell's own gravity (Kim, W., et al. , J. Am. Chem. Soc. , 129: 7228-7229 (2007)). Intense intracellular transport follows.

We investigated the possibility of CNSA as an intracellular delivery platform for genetic material using increased green fluorescent protein (EGFP) -encoded plasmid DNA (pEGFP) as a model cargo. A series of CNSAs of varying heights (40, 80, 120 and 160 nm) were used to investigate the best height for intracellular delivery. In order to load the cargo, CNSA pieces (˜1 cm × 1 cm) were treated with a drop of water contained in pEGFP (100 μg / mL) and unloaded pEGFP was removed by a light wash. NIH3T3 cells were cultured by aliquoting to pEGFP loaded CNSA. Lipofectamine ^™ 2000, one of the most useful non-viral gene transport vectors, was used as a positive control. 2 (a and b) show typical fluorescence microscopy images and flow cytometry histograms of NIH3T3 cells after treatment with CNSA (with 80 nm and 120 nm height) or Lipofectamine 2000, respectively. CNSA at 40, 80, and 160 nm height also resulted in high gene expression (-24-30% according to cell flow analysis), but CNSA at 120 nm height showed the highest gene expression efficiency (-34%). On the other hand, lower gene transfer efficiency was seen when using CNSA without an amphiphilic polymer coating (see FIG. 6), which means that the amphiphilic polymer coating is important for cargo loading. Relatively low gene expression of the CNSA platform compared to lipofectamine 2000 may be beneficial for direct delivery of empty plasmid DNA into the cytoplasm but not to the nucleus; However, the gene expression of the CNSA platform is much higher than other nonviral vectors (Nishikawa, M. and Huang, L., Hum. Gene Ther. , 12: 861-870 (2001); Kodama, K., et. al. , Curr. Med. Chem., 13: 2155-2161 (2006). MTT cell proliferation assays performed on NIH3T3 cells after incubation on CNSA formed at 80 nm and 120 nm showed that all had a viability of at least 85% and no toxicity to the cells compared to the control (FIG. 1C). This survival rate is due to the minimal damage to the plasma membrane during infiltration by a ~ 50 nm diameter nanoscanner. Since lipofectamine 2000 is only used in specific applications such as gene delivery but is toxic to cells (FIG. 1C) (Bell, H., et al. , Neuroreport, 30 : 793-798 (1998); Filion, MC and Phillips) , NC, Int. J. Pharm ., 162: 159-170 (1998), the CNSA platform of the present invention provides a more effective approach. We observed that although CNSA set at 160 nm exhibited a similar degree of gene expression (˜30%), cell viability was significantly reduced after adhesion from the transport platform in that case compared to shorter CNSA. For this reason we used a 120 nm high CNSA in subsequent experiments.

To investigate the usefulness of the CNSA platform for different cargoes, we investigated the intracellular transport of quantum dots using CNSA 120 nm high. 2D shows a typical fluorescence image of NIH3T3 cells cultured in CNSA containing quantum dots (QDs; 3 nm size, fluorescence wavelength [λ _em ] = 525 nm). As expected, many high fluorescence dots were found in the cytoplasm and not in the nucleus, indicating that the quantum dots were transported directly into the cytoplasm. The 120 nm high CNSA appears small enough not to infiltrate the nucleus at the center of the cell. We could not observe any fluorescent dots in cells treated with quantum dots in the absence of CNSA platform. These results clearly demonstrate the usefulness of the CNSA platform for the intracellular transport of various cargoes dispersed or dissolved in aqueous solutions. There have been many interests in using quantum dots as fluorescent labels to track target proteins in the cytoplasm of living cells. However, it was very difficult to transport the quantum dot labeled protein into the cytoplasm in its original form, since it is typically wrapped in an endozomal vesicle after endocytosis and fails to track. The CNSA platform of the present invention makes it possible to overcome this problem by delivering naked quantum dot-protein conjugates directly into the cytoplasm.

In order to further investigate the application of the CNSA platform of the present invention, the inventors investigated the intracellular transport of pEGFP and quantum dots in human mesenchymal stem cells (MSCs), which carry genetic material into cells or in vivo tracking. This is because there is a broad need for an effective device or method for labeling cells with fluorescent or magnetic nanoparticles. As described in the experiments of NIH3T3 cells, human MSCs were dispensed and cultured in CNSA containing either pEGFP or quantum dots. 3 (a and b) are the resulting fluorescence images and flow cytometry results. Most cells showed green fluorescence density and expression efficiency similar to that seen in NIH3T3 cells (˜30% for CNSA versus 42% for lipofectamine 2000), indicating effective gene transport and expression. The transport of quantum dots was very successful by having clear fluorescent quantum dots very high only in the cytoplasm (FIG. 3C). We then conducted a live / dead assay to investigate whether 120 nm high CNSA affects the permeability of human MSC membranes. Most cells were alive (FIG. 3D), indicating little effect on intracellular permeability after treatment with CNSA. Thus, the CNSA platform of the present invention is an effective device for the delivery of both genetic material and quantum dots to human MSCs in applications involving cell therapy and in vivo tracking. In order to investigate how cells are matched with the CNSA (120 nm) of the present invention, NIH3T3 cells were cultured on an array after being fixed for scanning electron microscopy. 4A (low magnification) clearly shows healthy cells with morphology spread across the surface of CNSA. 4B shows the cells subjected to the nanoinjector at high magnification. As indicated by the arrows, each nanoinjector appears in an embossed-like shape that penetrates the cell. Although scanning electron microscopy images do not provide direct evidence of the CNSA-mediated intracellular transport mechanism, it is found that the cells are initially penetrated by gravity-induced carbon nanoinjectors and cargo diffuses into the cytoplasm of the cells in the nanoinjectors. Can be. On the other hand, the effect of applying additional force was confirmed. 7 is a confocal (100 ×) micrograph when 800 g of centrifugal force is applied, indicating that the quantum dots are transported into the cells.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

1 (a) is a schematic diagram of the structure and use of the carbon nanoinjector array in the intracellular transport of cargo in the present invention: (i) Carbon film deposition in a well-ordered pore structured porous AAO template. Thermal CVD methods for; (ii) chemical etching to remove the top layer carbon surface from the AAO template by ion milling and to expose the carbon nanoinjectors; (iii) surface coating of carbon nanoinjectors with amphiphilic polymers and loading of cargo for transport; And (iv) intracellular delivery using a carbon nanoinjector platform. 1 (b) is a scanning electron microscope image of CNSA exposed to a height of 80-120 nm. Figure 1 (c) is the chemical structure of the amphipathic polymer used in the coated carbon nanoinjector.

2A-2D show the transport of various cargoes into NIH3T3 cells using CNSAs of the invention. Fluorescence images of NIH3T3 cells expressing GFP after treatment with pEGFP-loaded CNSAs (80 nm and 120 nm height) and pEGFP / Lipofectamine ^™ 2000 complex as positive control vectors, respectively (FIG. 2 (a)) And flow-cytometry histogram profiles; FIG. 2 (b)). Scale bars represent 20 μm. The dashed line and solid line in FIG. 2 (b) represent flow cytometry of NIH3T3 cells before and after gene delivery, respectively. FIG. 2 (c) is the viability of NIH3T3 cells incubated in CNSA for 12 hours or treated with lipofectamine 2000 for 6 hours. 2 (d) is a typical fluorescence image of NIH3T3 cells treated on CNSA loaded with quantum dots (QDs, fluorescence wavelength [λ _em ] = 525 nm). Scale bars represent 20 μm.

FIG. 3 shows fluorescent images of human MSCs expressing GFP obtained after treatment of pEGFP-loaded CNSAs and pEGFP / lipopeptamine 2000 complexes as positive control vectors, respectively, at 120 nm height (FIG. 3A) and Flow-cytometry histogram profiles (FIG. 3 (b)). Scale bars represent 20 μm. 3 (c) is a typical fluorescence image of human MSC treated on CNSA loaded with quantum dots (QDs, fluorescence wavelength [λ _em ] = 525 nm). Scale bars represent 20 μm (× 630). 3 (d) is a live / dead assay of human MSCs performed after incubation in CNSA for 24 hours (× 400).

4 is a scanning electron microscope image of NIH3T3 cells incubated in CNSA at 80 nm height for 24 hours in the present invention: (a) x 500 and (b) x 100 K. The arrow in FIG. 4 (b) clearly shows the nanoinjector infiltrated area in the cell.

5 is a transmission electron microscope image of a hollow carbon nanoinjector of about 50 nm diameter with an outer wall of about 7 nm thick in the present invention.

FIG. 6 is a graph showing the gene transfer effect of pEGFP-loaded CNSAs without an amphiphilic polymer coating. The exposure lengths of CNSA are 40 nm (a), 80 nm (b), 120 nm (c) and 160 nm (d).

7 is a confocal photograph showing intracellular transport results of quantum dots when additional force is applied using amphiphilic polymer coated CNSAs of the present invention.

Claims (30)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete intracellular cargo comprising (a) an array of nanotubes having a tubular structure of an open portal, and (b) an amphiphilic polymer coated on the surface of the nanotubes. With carrier, The hollow core in the nanotube is the loading position of the cargo and the surface of the hollow core is coated with the amphiphilic polymer; The transport of cargo by the carrier into the cell occurs in a spontaneous manner by gravity acting on the cell itself; The amphiphilic polymer is a carrier, characterized in that represented by the following formula (1): Formula 1

In the above formula, R ₁ is a silylalkyl group, (alkoxysilyl) alkyl group, (hydroxysilyl) alkyl group, substituted or unsubstituted alkyl group, substituted or unsubstituted alkoxy group, substituted or unsubstituted aryl group, substituted or unsubstituted Aralkyl group or a substituted or unsubstituted alkaryl group; R ₂ is polyethylene glycol (PEG), dextran or polyvinylpyrrolidone; R ₃ is an aldehyde, epoxy, haloalkyl, primary amine, thiol, maleimide, ester, carboxyl group or hydroxyl group; R ₄ , R ₅ and R ₆ are, independently from each other, H or a C ₁ -C ₅ alkyl group; X and Y are independently of each other an oxygen, sulfur or nitrogen atom; l, m and n are each independently an integer of 1-10,000. 19. The carrier of claim 18, wherein the carrier further comprises a chemical, nanoparticle, peptide, polypeptide, nucleic acid, carbohydrate or lipid as a cargo for transport within the nanotube. 19. The carrier of claim 18, wherein said nanotubes are made of carbon, silicon, precious metals, silicon dioxide or titanium dioxide. 19. The carrier of claim 18, wherein said nanotubes have a diameter of 20-200 nm. 19. The carrier of claim 18, wherein said carrier further comprises a substrate and said nanotubes are located at one end on said substrate. 19. The carrier of claim 18, wherein the nanotubes penetrate into the cell and the height of the nanotubes penetrated is 40-200 nm. The carrier of claim 23, wherein the height of the nanotubes to penetrate is 70-130 nm. 19. The carrier of claim 18, wherein the nanotubes are surrounded by a template for forming nanotubes and the top of the template is etched to expose the nanotubes. 27. The carrier of claim 25, wherein the height of the exposed nanotubes is 40-200 nm. 27. The carrier of claim 26, wherein the height of the exposed nanotubes is 70-130 nm. delete delete delete

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