KR20230119456A - Self-propelled hybrid amorphous aano-motor, Manufacturing method thereof, and Photothermal agent for photothermal ablation of cancer cell containing the same - Google Patents

Abstract

The present invention relates to a self-propelled amorphous nanomotor and a method for preparing the same by a continuous flow synthesis method, and provides an amorphous nanomotor that has excellent biocompatibility and is suitable for use in photothermal cancer treatment as well as in vivo drug delivery. it's about

Description

Self-propelled hybrid amorphous aano-motor, Manufacturing method thereof, and Photothermal agent for photothermal ablation of cancer cell containing the same}

The present invention relates to a self-propelled amorphous nanomotor and a method for preparing the same by a continuous microfluidic laminar flow synthesis method (or a continuous flow laminar flow synthesis method), which has excellent biocompatibility and provides in vivo drug delivery as well as photothermal cancer treatment It is about an amorphous nanomotor suitable for use in

Advances in nanotechnology have made it possible to form nanoscale structures and have revolutionized the field of nanomedicine to address unmet medical needs. Furthermore, the emergence of micro- and nanomotors powered by chemical catalysts (eg H ₂ O ₂ fuel) has received considerable attention. However, its clinical use has been severely limited due to biocompatibility issues.

Recently, biocatalytic micro/nanomotors driven by enzymes (urease, GO _x , catalase, etc.) achieve successful drug delivery due to their biocompatibility and are applied to the treatment of specific organs (bladder, stomach, etc.), and topical drugs It is applied to diagnosis in delivery and out-of-reach places. However, since biocatalytic micro/nano motors can be denatured by harsh external environments (e.g., high or low pH and high ion concentration), they have limitations in their movement speed and long-term use. Therefore, there are still many challenges to be solved in order to be applied in the real biomedical field.

Metal-organic-frameworks (MOFs), a type of crystalline material with high porosity and large surface area, have been applied to encapsulate various biomacromolecules as well as living cells. Among them, the zeolite imidazolate framework (ZIF) provides a biocompatible and encapsulated protective film from external reactive media. However, the highly crystalline ZIF-8 (micro-pores; 1-2 nm) synthesized in batches has a problem in that it has lower enzymatic activity (5-20 times) than natural enzymes.

In addition, polydopamine (Pdop) is a promising material due to its biocompatibility, light- or pH-responsive drug release, and bioimaging for local cancer treatment via photothermal therapy (PTT). In addition, Pdop-based micro/nano motors have been reported to have strong tissue adhesion and tissue penetration ability. However, Pdop's insufficient stability under harsh conditions (high or low pH) is a limiting problem for biocatalysis and drug delivery for diagnostics or combination therapy.

Korean Patent Publication No. 10-2019-0040490 (published on 2019.04.18)

1. X. Wu, H. Yue, Y. Zhang, X. Gao, X. Li, L. Wang, Y. Cao, M. Hou, H. An, L. Zhang, S. Li, J. Ma, H. Lin, Y. Fu, H. Gu, W. Lou, W. Wei, R. N. Zare, J. Ge, Packaging and delivering enzymes by amorphous metal-organic frameworks. Nat. Commun. 10, 1-8 (2019). 2. R. Kumar, K. Mondal, P. K. Panda, A. Kaushik, R. Abolhassani, R. Ahuja, H. G. Rubahn, Y. K. Mishra, Core-shell nanostructures: perspectives towards drug delivery applications. J. Mater. Chem. B. 8, 8992-9027 (2020). 3. A. Jin, Y. Wang, K. Lin, L. Jiang, Nanoparticles modified by polydopamine: Working as "drug" carriers. Bioact. Mater. 5, 522-541 (2020).

The present invention has been made to solve the above problems, and the continuous flow synthesis method further promotes the specific growth of an amorphous organometallic framework in a functional matrix and easily characterizes the porosity and crystallinity of self-assembled nanomaterials. It has been found that reliable synthesis of multipurpose Janus and core@shell hybrid structures can be achieved by tuning, and a core-shell type self-propelled hybrid amorphous nanomotor through a continuous flow synthesis method. The present invention was completed by finding out the optimal synthesis conditions that can be produced. That is, the present invention is to provide a self-propelled hybrid amorphous nanomotor, a method for manufacturing the same, an in vivo drug delivery system using the same, and a photothermal agent for photothermal treatment using the same.

In order to solve the above problems, the present invention relates to a self-propelled hybrid amorphous nanomotor, which is a core-shell nanoparticle, comprising a core containing polydopamine; and a shell including an organometallic framework in which a plurality of mesopores are formed.

As a preferred embodiment of the present invention, at least some of the plurality of mesopores are coupled with a self-propelling material.

As a preferred embodiment of the present invention, the self-propelling body may include at least one selected from urease, glucose oxidase and catalase.

As a preferred embodiment of the present invention, the transfer target material may include at least one selected from enzymes, proteins, DNA, and chemical drugs.

As a preferred embodiment of the present invention, at least one of the core and the shell may carry at least one in vivo delivery material selected from enzymes, proteins, DNA, and chemical drugs.

As a preferred embodiment of the present invention, the enzyme in the in vivo delivery material includes at least one selected from proteinase K, trypsin, lysine, and chymotrypsin. can do.

As a preferred embodiment of the present invention, the organometallic framework is amorphous, and the organometallic framework is ZIF (Zeolitic imidazole framework)-7, ZIF-8, ZIF-67, ZIF-8-90, ZIF-11 , ZIF-12, HKUST-1 (Hong Kong University of Science and Technology-1), MIL-53 (Material Institute Lavoisier-53), and UIO-66 (Universitetet i Oslo-66). there is.

As a preferred embodiment of the present invention, the core-shell nanoparticles may have an average diameter of 100 to 400 nm, and the shell may have a thickness of 10 to 100 nm.

Another object of the present invention is a method for manufacturing the self-propelled hybrid amorphous nanomotor described above, which is a continuous microfluidic laminar flow synthesis method (or continuous It can be prepared by performing a flow-type laminar flow synthesis method).

As a preferred embodiment of the present invention, in the first reaction unit of the tandem capillary reactor, a core containing polydopamine; And a shell comprising an organometallic framework; an amorphous nanomotor comprising a can be synthesized and formed.

As a preferred embodiment of the present invention, in the second reaction unit of the tandem capillary reactor, the self-propellant may be captured and bound to at least a portion of the plurality of mesopores formed in the shell of the synthesized amorphous nanomotor.

As a preferred embodiment of the present invention, the inlet of the first reaction unit includes a confluence to which a plurality of first reactant inlets are coupled, and the confluence is formed so that substances introduced from each of the plurality of inlets are simultaneously joined, An inlet for the second reactant may be formed at the end of the first reaction unit or at the beginning of the second reaction unit.

As a preferred embodiment of the present invention, the plurality of first reactant inlets include a first inlet, a second inlet and a third inlet, a metal ion precursor solution is injected into the first inlet, and a second inlet is injected. A polydopamine nanoparticle solution is injected, and an organic ligand precursor solution may be injected through the third inlet.

As a preferred embodiment of the present invention, a solution containing a self-propellant or a precursor solution of a self-propellant may be injected into the second reactant inlet.

As a preferred embodiment of the present invention, the metal ion precursor of the metal ion precursor solution may include a zinc ion (Zn ²⁺ ) precursor.

As a preferred embodiment of the present invention, the metal ion precursor solution may have a metal ion precursor concentration of 8.0 to 15.0 mg/ml.

As a preferred embodiment of the present invention, the organic ligand precursor solution is 2-methylimidazole, ethanedioic acid, propanedioic acid, butanedioic acid , pentanedioic acid, o-phthalic acid, m-phthalic acid, p-phthalic acid, 2-hydroxy-1,2,3- 2-hydroxy-1,2,3-propanetricarboxylic acid, benzene-1,3,5-tricarboxylic acid, 1H-1,2 ,3-triazole (1H-1,2,3-triazole), 1H-1,2,4-triazole (1H-1,2,4-triazole) and 3,4-dihydroxy-3-cyclo An organic ligand precursor including at least one selected from butene-1,2-dione (3,4-dihydroxy-3-cyclobutene-1,2-dione) may be included.

As a preferred embodiment of the present invention, the organic ligand precursor solution may have an organic ligand precursor concentration of 70 to 140 mg/ml.

As a preferred embodiment of the present invention, the polydopamine nanoparticle solution may contain 0.05 to 1.0% by weight of polydopamine nanoparticles.

As a preferred embodiment of the present invention, the self-propellant-containing liquid may have a self-propellant concentration of 1 to 20 mg/ml, and the self-propellant precursor solution may have a self-propellant precursor concentration of 1 to 20 mg/ml.

As a preferred embodiment of the present invention, the self-propelling body may include at least one selected from urease, glucose oxidase and catalase.

As a preferred embodiment of the present invention, the manufacturing method of the present invention can synthesize a self-propelled hybrid amorphous nanomotor under conditions satisfying Equation 1 below.

[Equation 1]

20 ≤ B/A ≤ 60

In Equation 1, A is the flow rate of the self-propellant-containing liquid or the precursor solution of the self-propellant injected through the second reactant inlet, and B is the flow rate of the polydopamine nanoparticle solution injected through the second inlet.

As a preferred embodiment of the present invention, the manufacturing method of the present invention can synthesize a self-propelled hybrid amorphous nanomotor under conditions satisfying Equation 2 below.

[Equation 2]

10 ≤ (C+D)/A ≤ 20

In Equation 2, A is the flow rate of the self-propellant-containing liquid or the precursor solution of the self-propellant injected into the second reactant inlet, C is the flow rate of the metal ion precursor solution injected into the first inlet, and D is injected into the third inlet. It is the sum of the flow rates of the organic ligand precursor solution.

As a preferred embodiment of the present invention, the manufacturing method of the present invention may further perform a process of restricting the growth of the shell by membrane filtration of the self-propelled hybrid amorphous nanomotor synthesized and obtained by performing the continuous microfluidic laminar flow synthesis method. .

As a preferred embodiment of the present invention, the manufacturing method of the present invention may further perform a process of performing washing and drying after performing a process of limiting the growth of the shell.

As a preferred embodiment of the present invention, the series-type capillary reactor of the present invention may further include a third reactant inlet in the first reaction part and/or the second reaction part.

As a preferred embodiment of the present invention, at least one in vivo delivery material selected from among enzymes, proteins, DNA, and chemical drugs supported on the core and/or shell of the core-shell nanoparticle is injected into the third reactant inlet. can

Another object of the present invention is to provide the above-described hybrid amorphous nanomotor as a photothermal agent for photothermal treatment of cancer cells.

In addition, another object of the present invention is to provide an in vivo drug delivery system using the hybrid amorphous nanomotor described above.

The hybrid amorphous nanomotor of the present invention has excellent biocompatibility and durability, and has much improved mobility (>80%) than polydopamine nanoparticles (<30%) and crystalline nanomotors (<50%). Nanomotors are suitable for use as photothermal agents for photothermal treatment of cancer cells, etc., as in vivo carriers for enzymes, proteins, drugs, etc., and can be applied to various nano-biomedical applications.

Figure 1a is a schematic cross-sectional view of a tandem capillary reactor used in the continuous flow synthesis method of the present invention.
Figure 1b is a photograph and schematic design of the tandem capillary reactor prepared in Preparation Example 1.
Figure 1c A shows a hybrid amorphous nanomotor (Pdop@enzyme@amorphous ZIF-8) prepared in Example 1 by continuous flow synthesis using a series capillary reactor (glass capillary, internal diameter 0.125 cm, total length 20 cm) It is a schematic process diagram showing a schematic diagram of removing cancer cells through sequential photothermal therapy using the same, and B shows nanomotor synthesis conditions of Example 1.
FIG. 3 is a result of measuring surface activity in the synthesis of nanomotors in Example 1 (Condition C2), Comparative Example 1 (Condition C1), and Comparative Example 3 (Condition C3) conducted in Experimental Example 1. FIG.
2, a) is a photograph of the flow system used in Experimental Example 1, b) is a schematic diagram of the flow system, and C) is a microscope image of triple and quadruple laminar flow in the first and second mixing regions (Top-1 flow: blue dye, middle flow: FITC dye, bottom flow: red dye, and Top-2 flow: rhodamine B dye).
4 is XRD and BET pore measurement results for the nanomotors prepared in Example 1 and Comparative Examples 1 and 2 conducted in Experimental Example 2.
5 is a measurement result of confirming the crystal structure of the nanomotor according to the reaction termination conditions conducted in Experimental Example 3, in which a) is an XRD measurement image and b) is an SEM measurement image.
6 is a graph showing the relative activity of urease of the nanomotors of Example 1 and Comparative Examples 1 to 3 in Experimental Example 4;
7 is an image of enzyme co-capture measurement conducted in Experimental Example 5.
8 is a particle characteristic measurement image of a nanomotor performed in Experimental Example 6.
9 shows SEM images, DLS measurement, and shell thickness measurement results of Pdop nanoparticles, C-motor, and A-motor conducted in Experimental Example 6.
10 is a photothermal characteristic test result conducted in Experimental Example 7.
11 is an infrared thermal image for each concentration, each NIR intensity, and each NIR intensity of the A-motor.
12 is a result of long-term activity evaluation of Pdop nanoparticles (Pdop/native urease, non-core@shell), A-motor (Example 1), and C-motor (Comparative Example 3) conducted in Experimental Example 7.
13 is a biocatalytic motion measurement result of Pdop nanoparticles, A-motor (Example 1), and C-motor (Comparative Example 3) conducted in Experimental Example 7.
14 is a measurement of Pdop degradation inhibition rate according to pH change and urea concentration of Pdop nanoparticles, A-motor (Example 1), and C-motor (Comparative Example 3) conducted in Experimental Example 7, that is, durability measurement results.
15 is a result of measuring the biocompatibility of the A-motor performed in Experimental Example 8, and is a result of measuring the cytotoxicity of NIH3GT3 for Pdop nanoparticles and the A-motor.
16 is a confocal microscope measurement image for the internalization and uptake evaluation of the A-motor in cancer cells performed in Experimental Example 8.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a self-propelled hybrid amorphous nanomotor that can be used as a nano-biomedical application product such as a photothermal agent for photothermal therapy (PTT) using near infrared ray (NIR), etc., and a biocarrier. The self-propelled hybrid amorphous nanomotor of the present invention is in the form of core-shell nanoparticles, and includes a core containing polydopamine; and a shell including an organometallic framework in which a plurality of mesopores are formed.

In addition, at least some of the plurality of mesopores are coupled with self-propellants, and the self-propellants coupled to the shell are asymmetrically distributed in the shell to have durable biocatalytic activity as nanomotors.

The self-propelling body may include at least one selected from urease, glucose oxidase, and catalase.

When urease is applied as a self-propellant, strong driving force can be obtained through a chemical reaction with urea (urea, NH ₃ ). In this case, the amorphous nanomotor of the present invention may be suitable for application as a photothermal agent for PTT to bladder cancer cells. there is.

In addition, materials such as enzymes, proteins, DNA, and chemical drugs can be supported in the mesopores of the core and/or the shell, so that the nanomotor of the present invention can be used as an in vivo delivery system.

For a preferred example of the enzyme among the supported materials to be transferred, the enzyme is selected from proteinase K, pepsin, trypsin, lysine, and chymotrypsin. One or more may be included.

The organometallic framework is an amorphous (amorphous) organometallic framework, and the organometallic framework may be composed of ZIF (Zeolitic imidazole framework), preferably ZIF (Zeolitic imidazole framework)-7, ZIF-8, ZIF-67, ZIF-8-90, ZIF-11,ZIF-12, HKUST-1 (Hong Kong University of Science and Technology-1), MIL-53 (Material Institute Lavoisier-53) and UIO- 66 (Universitetet i Oslo-66), and more preferably a ZIF including at least one selected from ZIF-7 and ZIF-8.

The size of the mesopores formed in the shell may be 2 to 20 nm, preferably 2 to 15 nm, and more preferably 2 to 8 nm.

of the present invention The self-propelled hybrid amorphous nanomotor (or the core-shell nanoparticle) may have an average diameter of 100 to 400 nm, preferably 150 to 350 nm, and more preferably 160 to 320 nm.

And, the shell thickness of the core-shell nanoparticles is 10 to 100 nm, preferably 10 to 60 nm, more preferably 10 to 35 nm, and even more preferably 12 to 25 nm. can have

Such, the present invention is prepared through a continuous microfluidic laminar flow synthesis method (or continuous flow laminar flow synthesis method), thereby forming an amorphous organometallic framework to greatly improve the activity of enzymes coupled to mesopores. .

The continuous microfluidic laminar flow synthesis method can be performed using a series-type capillary reactor configured by integrating a plurality of reaction units in series, and for example, as schematically shown in FIG. 1A, the first reaction unit 30 and The synthesis may be performed using a series-type capillary reactor 100 configured by integrating the second reaction unit 50 in series.

The tandem capillary reactor 100 includes a confluence part 10 to which a plurality of first reactant inlets are coupled to the inlet of the first reaction part 30, and the confluence part 10 is formed at each of the plurality of inlets. It may be formed so that the incoming material is joined at the same time.

In addition, the plurality of first reactant inlets may be formed in a trident shape including a first inlet 1, a second inlet 2, and a third inlet 3, and the shape is a plurality of first reactant inlets. The shape of the inlet and the confluence is not particularly limited as long as the reactants injected through are joined at the same time.

In addition, a second reactant inlet (or a fourth inlet, 4) may be formed at the end of the first reaction unit 30 or at the beginning of the second reaction unit.

The above-described core-shell type self-propelled hybrid amorphous nanomotor of the present invention includes a core containing polydopamine in the first reaction unit; and a shell including an organometallic framework; a core-shell type amorphous nanomotor is synthesized, and at least a plurality of mesopores formed in the shell of the synthesized amorphous nanomotor are synthesized in the second reaction unit. It can be manufactured by incorporating a self-propelling body into some of them.

In addition, a metal ion precursor solution is injected into the first inlet, a polydopamine nanoparticle solution is injected into the second inlet, an organic ligand precursor solution is injected into the third inlet, and a self-reactant is injected into the second reactant inlet. Continuous microfluidic synthesis may be performed by injecting a propellant-containing liquid or a precursor solution of a self-propellant.

Depending on the injection rate (or flow rate) of the reactants injected into the first to fourth inlets, the degree of amorphous nanomotor formation, the degree of self-propellant bonding, etc. have a great effect, and the reactants are injected to satisfy the following equations 1 and 2 it is desirable

[Equation 1]

20 ≤ B/A ≤ 60, preferably 20 ≤ B/A ≤ 45, more preferably 23 ≤ B/A ≤ 35

In Equation 1, A is the flow rate of the self-propellant-containing liquid or the precursor solution of the self-propellant injected through the second reactant inlet, and B is the flow rate of the polydopamine nanoparticle solution injected through the second inlet.

[Equation 2]

10 ≤ (C+D)/A ≤ 20, preferably 12 ≤ (C+D)/A ≤ 19, more preferably 14 ≤ (C+D)/A ≤ 18

In Equation 2, A is the flow rate of the self-propellant-containing liquid or the precursor solution of the self-propellant injected into the second reactant inlet, C is the flow rate of the metal ion precursor solution injected into the first inlet, and D is injected into the third inlet. It is the sum of the flow rates of the organic ligand precursor solution.

When Equation 1 and/or Equation 2 are not satisfied, the rate at which the organometallic framework forming the shell of the core-shell nanoparticle is crystalline increases significantly, resulting in too low synthesis yield of amorphous nanomotors. There may be, and the amount of self-propellant coupled to the core-shell nanoparticles is too small, resulting in poor in vivo mobility of the amorphous nanomotor and insufficient physical properties such as in vivo activity and durability of the amorphous nanoparticles. .

The metal ion precursor solution injected through the first inlet may include a metal ion precursor, preferably a zinc ion (Zn ²⁺ ) precursor, and the zinc ion precursor is one selected from zinc nitrate, zinc chloride, and zinc acetate. May include more than one species.

And, the metal ion precursor solution may have a metal ion precursor concentration of 8.0 to 15.0 mg/ml, preferably 8.0 to 13.0 mg/ml, and more preferably 8.5 to 12.5 mg/ml. At this time, if the concentration of the metal ion precursor is less than 8.0 mg/ml, the concentration is too low, and the organic metal framework may not be formed well. If the concentration of the metal ion precursor exceeds 15.0 mg/ml, the shell thickness is too thick to form the nanomotor. Since the size may be too large and mobility in vivo may be poor, it is preferable to inject the metal ion precursor solution at a concentration within the above range.

In addition, the polydopamine nanoparticle solution injected through the second injection port preferably has a polydopamine nanoparticle content of 0.05 to 1.0% by weight, preferably 0.05 to 0.8% by weight, more preferably 0.08 to 0.6% by weight, at this time, If the polydopamine nanoparticle content is less than 0.05% by weight or more than 1.0% by weight, there may be a problem in that the yield of core-shell nanoparticles is lowered.

In addition, the organic ligand precursor solution injected through the third inlet includes 2-methylimidazole, ethanedioic acid, propanedioic acid, butanedioic acid, Pentanedioic acid, o-phthalic acid, m-phthalic acid, p-phthalic acid, 2-hydroxy-1,2,3-propane 2-hydroxy-1,2,3-propanetricarboxylic acid, benzene-1,3,5-tricarboxylic acid, 1H-1,2, 3-triazole (1H-1,2,3-triazole), 1H-1,2,4-triazole (1H-1,2,4-triazole) and 3,4-dihydroxy-3-cyclobutene An organic ligand precursor containing at least one selected from -1,2-dione (3,4-dihydroxy-3-cyclobutene-1,2-dione) may be included.

In addition, the organic ligand precursor solution preferably has an organic ligand precursor concentration of 70 to 140 mg/ml, preferably 80 to 135 mg/ml, and more preferably 85 to 125 mg/ml. If it is less than 70 mg/ml, formation of an organometallic framework may not be successful, and if the organic ligand precursor concentration exceeds 140 mg/ml, a large amount of unreacted organic ligand precursor may be generated and it may be disadvantageous to mesopore formation. It is better to use it in the concentration inside.

In addition, the self-propellant-containing solution and/or the self-propellant precursor solution injected through the fourth inlet has a concentration of 1 to 20 mg/ml, preferably 2 to 15 mg/ml, More preferably, it may be 2.5 to 10.0 mg/ml. At this time, if the concentration is less than 1 mg/ml, there may be a problem in that the mobility of the nanomotor is not good in vivo due to the small number of self-propellants coupled to the nanomotor, and the photothermal treatment effect may be reduced, and if the concentration exceeds 20 mg/ml However, since a large amount of self-propellant that cannot be bonded to the shell of the nanoparticle is generated and thus uneconomical, it is recommended to use the nanoparticle at a concentration within the above range.

In addition, the solvent used in the metal ion precursor solution, the polydopamine nanoparticle solution, the organic ligand precursor solution and/or the self-propellant-containing liquid (the self-propellant precursor solution) is water (preferably deionized water) and/or ethanol. good to use

Further explaining the continuous microfluidic laminar flow synthesis method of the present invention, zinc ions (Zn ²⁺ ) injected through the first inlet interact with catechol groups on the surface of polydopamine nanoparticles injected through the second inlet for exclusive nucleation, but , it is possible to form ZIFs that are not completely self-assembled into an amorphous structure due to the limited supply and short reaction time.

In addition, the self-propellant injected through the fourth inlet is supplied in a short time to be captured and combined with the amorphous shell that is not completely self-assembled.

In addition, in the manufacturing method of the present invention, the self-propelled hybrid amorphous nanomotor synthesized and obtained by performing the continuous microfluidic laminar flow synthesis method is rapidly cooled for less than 30 seconds to terminate the reaction (quenching), and then membrane filtration is performed to limit the growth of the shell. Further steps may be performed. In this way, by limiting the growth of ZIF constituting the shell, crystallization of the amorphous form can be prevented. At this time, the membrane filtration may be performed with a membrane filter made of nylon (polyamide) material, and for example, a membrane filter having a pore size of 0.1 to 0.3 μm and a diameter (Τ) of 40 to 50 mm.

In addition, the amorphous nanomotor obtained by membrane filtration was put in deionized water, centrifuged at 2,000 to 35,000 rpm for 8 to 20 minutes, and vacuum dried at room temperature for 18 to 48 hours to prepare a self-propelled hybrid amorphous nanomotor. can

The self-propelled hybrid amorphous nanomotor of the present invention prepared in this way can be used in various fields such as biological fields, sensor fields, and environmental fields. It can be used as a drug delivery system and the like.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to aid understanding of the present invention, and should not be construed as limiting the scope of the present invention to the examples.

[Example]

Preparation Example 1: Fabrication of a capillary reactor connected to a 3D printing channel

The tandem capillary reactor was constructed by connecting a glass capillary tube (inner diameter: 1250 μm, outer diameter: 1650 μm) and a 3D printing device (ø and Y-shaped devices) (see Fig. 1b), and the 3D printing device creates a virtual object. To do this, it was designed with AutoCAD software (Autodesk Inventor 2020, see B in FIG. 1B). In addition, the tandem capillary reactor was printed with a DLP (Digital Light Processing) desktop printer (Asiga Pico 2, Australia) using a UV curable acrylate resin (Plas. CLEAR, ASIGA). Specifically, the multi-digital mask was irradiated with UV at a wavelength of 385 nm (intensity: 2 mW/cm ² ) for an exposure time of 5 seconds with a set of 40 μm-thick layers, and then washed repeatedly with isopropanol and ethanol to wash away uncured 3D printing resin. . Next, it was post-treated with a UV radiation lamp at an intensity of 2 mW/cm ² for 30 minutes to obtain a more robust shape.

The reactor is divided into two parts: a first reaction part (core-shell synthesis, part 1) and a second reaction part (self-propellant synthesis, part 2). Both part 1 and part 2 have the same width (21mm), length (12mm) and inner channel diameter (1.6mm). In addition, the connection angle between the trident (ø ) connection of the first to third inlets of component 1 and the Y connection of the fourth inlet of component 2 is 45°.

Example 1: Through Continuous Microfluidic Laminar Flow Synthesis Fabrication of self-propelled hybrid amorphous nanomotors (A-motors)

(1) Synthesis of polydopamine (Pdop, size: 200 nm or less) nanoparticles

Pdop nanoparticles were prepared by mixing ethanol (40mL), deionized water (DI water, 90mL) and NH ₄ OH (1mL) under gentle stirring at room temperature (25-28 °C) for 30 minutes.

Next, dopamine hydrochloride (0.5 g/mL) aqueous solution (1 mL) was slowly added dropwise to the mixture, followed by reaction for 30 hours.

Next, the reaction product was washed with ethanol and deionized water, and then water was added several times and finally vacuum dried at room temperature for 24 hours to prepare Pdop nanoparticles.

(2) Preparation of fluorescently labeled enzymes (urease and proteinase K)

Proteinase K and urease were separately prepared by labeling them with proteins with Cy5 and Cy5.5 fluorophores, respectively.

Specifically, enzyme (1 mg/mL) in PBS (1x; pH 7.4) was added to 4 μL of sulfo-fluorophore-NHS ester (1 mM; Lumiprobe) and incubated for 30 minutes, then the modified enzyme was Fluorescently labeled enzymes, urease and proteinase K, were respectively prepared by purification with PBS three times through a 100 kDa filter (Millipore) while centrifuging at 3,600 rpm for 10 minutes.

(3) Manufacture of A-motor (Pdop@enzyme@aZIF-8)

A self-propelled hybrid amorphous nanomotor (A-motor) was fabricated by continuous microfluidic laminar flow synthesis using the serial capillary reactor prepared in Preparation Example 1, and a schematic diagram of the fabrication process is shown in FIG. 1C.

Laminar flow easily occurs in the glass capillary part, which plays a very important role in maintaining laminar flow because it helps fix the fluid.

Zinc nitrate solution as a metal ion precursor is in the first inlet, polydopamine nanoparticle (Pdop) solution prepared in advance is in the second inlet, and 2-methylimidazole (2-MIM) solution as an organic ligand precursor is in the third inlet. injected into. At this time, the concentration and injection flow rate of the zinc nitrate solution, the Pdop solution, and the 2-MIM solution are shown in Table 1 below, and the solvent of these solutions is deionized water.

In addition, an enzyme solution containing urease was injected into the fourth inlet, the concentration and injection flow rate are shown in Table 1 below, and the solvent was deionized water.

The three solutes (Pdop, Zn ²⁺ and 2-MIM) dissolved in deionized water diffused between the solvent interfaces while maintaining a triple laminar flow. Zn ²⁺ and 2-MIM diffuse in the Pdop solvent (middle part of triple laminar flow) to give various ZIF-8 forms (amorphous ZIF-8, crystalline ZIF-8, non-ZIF-8). Pdop (core) and shell (ZIF-8) were formed.

In addition, the formed core-shell nanoparticles meet the flow of enzymes (urease and proteinase K, 4mg/mL) in the second reaction part, and the enzymes bind to the mesopores of the shell to form self-propelled bodies (urease ) is formed. At this time, among the enzymes, urease is a self-propelling component, and the proteinase K is a component that serves to remove cancer cells by protein destruction.

The urease-conjugated core Pdop@enzyme@aZIF-8 synthesized through a tandem capillary reactor was collected in a vial filled with deionized water, and the reaction was terminated within 30 seconds.

Next, core-shell nanoparticles are separated from unreacted reagents (2-MIM, Zn ²⁺ ), and membrane filtration (membrane filter, nylon (polyamide), pore size: 0.2) is used to inhibit the growth of ZIF-8. μm, Τ: 47 mm) to limit the growth of ZIF-8.

Next, the resulting product was placed in deionized water and centrifuged at 3000 rpm for 10 minutes, and finally vacuum dried at room temperature for 24 hours to obtain an A-motor.

The average diameter of the synthesized A-motor was about 210 nm, and the thickness of the shell was 20 nm.

division inlet infusion rate
(flux) density zinc nitrate solution 1st inlet 40 μL/min (C) 9.3 mg/mL Pdop solution 2nd inlet 130 μL/min (B) 0.1% by weight 2-MIM solution 3rd inlet 40 μL/min (D) 102.5 mg/mL enzyme solution 4th inlet 5 μL/min (A) 4 mg/mL Equation 1 B/A = 26 equation 2 (C+D)/A = 16

Comparative Example 2 to Comparative Example 3

An A-motor was manufactured in the same manner as in Example 1, but, as shown in Table 2 below, the injection speed (flow rate) of the solution injected into the first to fourth inlets was changed to prepare an A-motor, and Comparative Examples 2 to 3 was carried out.

division Example 1 (C2) Comparative Example 1 (C1) Comparative Example 2 (C3) Equation 1 (B/A) 26 104 8 equation 2
((C+D)/A) 16 16 16

Comparative Example 4: Preparation of self-propelled hybrid crystalline nanomotor (c-motor) by batch synthesis method

Pdop and fluorescently labeled enzymes (urease and proteinase K) synthesized in the same manner as in Example 1 were prepared.

A mixture was prepared by sonicating zinc nitrate (0.372 g) and Pdop (0.1% by weight) in 17 mL of deionized water at room temperature (23-25° C.) for 10 minutes. Then, a mixture of 2-MIM (4.1 g) in 40 mL of deionized water was slowly added dropwise to the mixture, and immediately after adding enzymes (urease and proteinase K, 16 mg) mixed with 4 mL of deionized water, It was stirred at 500 rpm for 30 minutes at room temperature (23-25 °C).

Next, the growth of ZIF-8 was restricted by membrane filtration (membrane filter, nylon (polyamide), pore size 0.2 μm, Τ 47 mm) within 30 seconds to inhibit the growth of ZIF-8.

Then, the resulting solution was centrifuged at 3000 rpm for 10 minutes and finally vacuum dried at room temperature for 24 hours to obtain a self-propelled hybrid crystalline nanomotor (c-motor, Pdop@enzyme@cZIF-8).

Experimental Example 1: Surface diffusivity measurement experiment for measuring parameters for nanomotor synthesis according to flow rate

Interfacial diffusivity measurements were calculated by slightly modifying the mixing efficiency calculation method.

The flow monitoring system was set up the same as the synthesis system of Example 1. Before the experiment, the injection solutions of the third inlet (Bottom), the first inlet (Top-1), the second inlet (middle), and the fourth inlet (Top-2) were red, blue, FITC, It was stained with Rhodamine B (see Fig. 2 a) to c)).

Different injection rate ratio (flow ratio) conditions (C1, C2, C3 in Table 1) at each position (#1-6; residence time (distance)) to investigate interfacial diffusion mixing by laminar flow in a series capillary reactor. The laminar flow pattern at each location was confirmed with an optical microscope (3 seconds (0.8 cm), 17 seconds (5.6 cm), 35 seconds (10 cm), 37 seconds (10.5 cm), 47 seconds (10.4 cm), 66 seconds) (18.9 cm)).

The position (#1-6) is the same as b) of FIG.

And, the interfacial diffusion rate was calculated in the following way.

First, the red code (ex: 0-255) of 6 nodes (6 columns vertically in the position image) was imported from the power point (microsoft co., PPT) of the microscope color image (see Table 3).

In addition, the standard deviation of the red code at the initial position (Table 4) and the standard deviation for each position were calculated and compared with the following Equation 1 (Table 4, Equation 1).

[Equation 1]

Equation 1 is the standard deviation of the red code values at 6 nodes, N is the number of nodes in the crossover location, Cl is the average red color in the crossover sample, and Cm is the average ratio of each location.

[Equation 2]

Equation 2 is the interfacial diffusion rate of each position (#1-6), and σinitial and σ are the standard deviations of each position depending on the initial position and inflow flow rate ratio (C1 to C3).

Red code under flow ratio C2 condition (B:A:(C+D) = 26 :1:16) division # One # 2 #3 # 4 #5 #6 node 1 255 226 217 222 211 216 node 2 254 215 215 220 198 211 node 3 233 179 179 213 191 209 node 4 200 153 165 209 190 207 node 5 179 155 162 204 185 204 node 6 164 160 162 169 162 175

flux
condition node 1 node 2 node 3 node 4 node 5 node 6 C1 23.5 20.0 16.6 14.5 14.5 13.3 C2 38.8 31.8 26.0 19.4 16.2 14.6 C3 38.6 27.7 24.4 12.3 9.6 3.0 Node 2 of condition C2: η=(1-√(38.8^2/(31.8^2)))Х100 = 17.9 %

In addition, in Example 1 (Condition C2), Comparative Example 1 (Condition C1), and Comparative Example 3 (Condition C3), the interfacial diffusivity measurement results during nanomotor synthesis are shown in a) of FIG. 3 .

Interfacial diffusion was controlled by varying three different core flow rates (FITC) over a constant flow ratio of 16:1 for the (red+blue):RB dye solution. In general, the degree of interfacial diffusion gradually increases as the channel length determined at positions #1 to #6 increases, because the degree of longitudinal diffusion is simply proportional to the flow time.

And, the lower the Middle (second inlet) flow rate, the higher the degree of interfacial diffusion.

At location #6, flow condition C3 with the lowest Middle (second inlet) flow rate had the highest interfacial diffusivity up to 92%, confirming that laminar flow was almost lost, and flow condition C1 with the highest core flow rate was the lowest interface. showed diffusion. The interfacial diffusion was lowered to 43% while maintaining laminar flow.

And the flow condition C2 showed an interfacial diffusion of 62% (see b) in FIG. 3). Presumably, this behavior showed longitudinal diffusion inversely proportional to the Middle (second inlet) flow rate in the tandem capillary reactor.

Experimental Example 2: XRD and BET pore measurement of nanomotors

XRD and BET pores of the core-shell structure of the nanomotors prepared in Example 1 and Comparative Examples 1 and 2 were measured, and these are shown in a) and b) of FIG. 4 .

Looking at Figure 4 a), Comparative Example 2 synthesized under the flow condition C3, which generally has the highest interfacial diffusivity, has (011), (002), (112), (022) peaks as well as micropores (1-2 nm) It was confirmed that this well-developed highly crystalline structure was obtained.

In contrast, Comparative Example 1 synthesized under the flow condition C1 having the lowest diffusion rate was observed to have a non-porous structure with a single broad XRD peak.

And, in Example 1 synthesized under suitable flow conditions C2, two broad peaks at 14 ^o ~ 26 ^o were observed to have mesopores in a wide range of 2 ~ 8 nm, which is amorphous ZIF-8 (aZIF-8) Indicates that it is a characteristic of the structure.

This result is due to the temporary time difference (C1 condition: 0.4 min C2 condition: 1 min and C3 condition: 2 min) were found to effectively confine the enzyme in an amorphous ZIF-8 shell that did not fully self-assemble. In the end, the biocatalytic activity of the different Pdop@urease@ZIF-8 NPs was relatively determined by measuring the change in UV absorption due to the conversion of phenol red. In the C1 condition with a non-porous layer on Pdop, there was no activity (2%). In C3 with crystalline ZIF-8-based core-shell micropores, the urease activity was less than 35%, similar to Pdop@urease@cZIF-8 synthesized in batch.

Experimental Example 3: Confirmation of crystal structure of nanomotors according to quenching conditions

Nanomotors were prepared in the same manner as in Example 1, but the reaction quenching time of the nanomotors obtained from the tandem capillary reactor was changed, and the unreacted reagent ( Isolation of nanoparticles from 2-MIM and Zn ²⁺ ) restricted the formation of crystalline cDia(Zn) as isomorphic Zn-MOFs.

PXRD and SEM measurement images of the nanomotors obtained according to the reaction termination conditions are shown in a) and b) of FIG. 5 . At this time, the PXRD (powder x-ray diffraction) pattern was measured with a high-resolution powder X-ray diffractometer-SmartLab (Rigaku) using a radiation source of CuKα, a wavelength of 1.5405 Å and a HyPix3000 detector at 30 kV and 30 mA power.

Through this, it was confirmed that cDia (Zn), an isomer, as well as amorphous ZIF-8 on polydopamine could be produced in a glass tube reactor depending on the termination time. When the termination time was less than 30 seconds, a typical amorphous ZIF-8 was confirmed in the XRD peak (14 ^o ), and a typical crystalline form of Dia (Zn) was observed in the XRD peak (10 ~ 20 ^o ) when the termination time was more than 1 minute.

Experimental Example 4: Measurement of relative activity of urease

The urease activity of each of the nanomotors prepared in Example 1 and Comparative Examples 1 to 4 was measured as follows, and the results are shown in FIG. 6 .

A solution of urea (300 mM) and phenol red (30 μL, 1 mg/mL) in PBS buffer (2.91 mL, pH 7.4) was added to a 15 mL conical tube (Falcon, polypropylene tube). Then, the nanomotor suspensions of Examples and Comparative Examples (120 μL, 1 mg/mL) were added thereto, and the conical tube was sealed, and the relative activity was measured using the increase in absorbance at 560 nm versus time (0 to 24 hours).

Relative activity was calculated based on the UV-vis absorption value of Pdop (Pdop/native urease) conjugated with natural urease, which was compared using Equation 3 below.

[Equation 3]

Referring to FIG. 6, the A-motor (amorphous motor) of Example 1 having an amorphous ZIF-8-based core shell prepared under C2 conditions had the highest urease activity (about 100%) without loss of urease activity, which was due to mesopores. Demonstrate successful capture of my enzyme, urease.

In contrast, Comparative Example 1 prepared under C1 conditions had a very low urease activity (2%), which is because almost no non-porous layer was formed on the shell.

In addition, Comparative Example 2 (C-motor, crystalline motor) prepared under the C3 condition showed a relative activity (35%) almost similar to that of Comparative Example 3 (C-motor, crystalline motor) prepared in batch mode. This is considered to be a result of the fact that the amount of urease bound is relatively small because ZIF, the shell of the nanomotors of Comparative Examples 2 and 3, has crystallinity (crystalline ZIF, cZIF).

These results indicate that the amorphous ZIF-8 shell that is not fully self-assembled in core-shell nanoparticles synthesized under C2 conditions effectively entraps and binds urease.

Experimental Example 5: Measurement of co-entrapment

An A-motor (Pdop@urease@aZIF-8, amorphous motor) was prepared in the same manner as in Example 1, but an enzyme solution in which urease and proteinase K were mixed as enzyme solutions in the fourth inlet. was injected to measure co-capture of the A-motor enzyme, and the results are shown in FIG. 7 .

To this end, proteinase K and urease were labeled with different fluorescent dyes (red and green), respectively.

Dual-color images observed under an optical microscope confirmed the successful co-loading of both enzymes in aZIF-8 (amorphous ZIF-8).

Through this, it was confirmed that the rapid flow production of core-shell nanoparticles under low stress on enzymes enables scalable and efficient co-administration of multiple drugs through a single system, which has high potential for combination therapy of drug delivery. .

Experimental Example 6: Measurement of particle characteristics of A-motor and C-motor

The particle characteristics of the Pdop nanoparticles, the A-motor of Example 1, and the C-motor of Comparative Examples 2 and 3 were measured, and the results are shown in FIG. 8 .

FE-SEM and energy dispersive X-ray spectroscopy (EDX) were performed using JSM 7401F. And, HR-TEM was measured using JEOL JEM-2100F. Then, selective area electron diffraction (SAED) patterns, electron energy loss spectroscopy (EELS), and fast Fourier transform (FFT) were performed. X-ray photoelectron spectroscopy (XPS) spectra were performed on an ESCALAB 250 (Thermo Scientific., energy resolution 0.45 eV, ion pore performance ≤5 μA).

8a, a) are pre-synthesized Pdop nanoparticles and b) are FE-SEM (scale: 400 nm, inset scale: 50 nm) images of the A-motor.

And, c) of FIG. 8b is an HR-TEM measurement image (scale: 50 nm) and elemental mapping image (EELS, electron energy loss spectroscopy) of the A-motor.

In addition, d) of FIG. 8C is a SAED (Selected Area Electron Diffraction Patterns) TEM image of the A-motor and e) of the C-motor synthesized in Comparative Example 2.

8D and 8E are HR-TEM and FFT images of the A-motor of Example 1 and the C-motor of Comparative Example 3, respectively.

8f is an X-ray photoelectron spectrum measurement image of Zn 2p for the C-motor (blue line) of Comparative Example 3 and the A-motor of Example 1 synthesized in a batchwise manner.

Looking at a) of FIG. 8a, the Pdop nanoparticles showed a uniform size of 200 nm with a relatively smooth surface. In contrast, in b), when the urease was entrapped in the shell, aZIF-8, under flow condition C2 (Pdop:enzyme:ZIF-8 = 26:1:16), the A-motor became a rough surface, and the shell The thickness was about 15 to 25 nm.

In contrast, the shell of the C-motor of Comparative Example 3 synthesized in a batchwise manner had a thickness of 40 to 50 nm.

Referring to Figure 8b, the presence of aZIF-8 and urease in Pdop was confirmed by EELS, indicating that the composition of the A-motor (Pdop@urease@aZIF-8), that is, carbon, oxygen, sulfur (thiol group of urea enzyme) , zinc (Zn ²⁺ of aZIF-8) and nitrogen.

Referring to FIGS. 8c, 8d, and 8e, SAED and TEM-FFT of the A-motor of Example 1 show amorphous ZIF-8 that is significantly different from the clear crystal structure of the C-motor synthesized in the batch method.

In addition, looking at Figure 8f, looking at the results of XPS analysis examining the molecular situation of the A-motor (Pdop@urease@aZIF-8), the Zn 2P spectrum of the A-motor (Pdop@urease@cZIF) and C-motor (Pdop@urease@cZIF), respectively. It exhibited two strong chemical shifts at 1044.9 eV and 1022.0 eV that were well assigned to the same Zn 2P ^1/2 and Zn 2P ^3/2 states, respectively, identical to the C-motors obtained batchwise. This result means that the amorphous layer of the hybrid nanoparticles has a typical ZIF-8 structure as a coordination compound between Zn ²⁺ and 2-MIM.

In addition, SEM images of the Pdop nanoparticles, the A-motor of Example 1 and the C-motor of Comparative Example 3 are shown in a) of FIG. 9, and the DLS measurement results are shown in b) of FIG. 9, respectively. The results of measuring the shell thickness of the particles are shown in c) of FIG. 9 .

Looking at a) and c) of FIG. 9, the Pdop nanoparticles do not have a shell, the C-motor has a shell with a thickness of about 45 nm, and the A-motor of Example 1 has a shell of 25 nm or less, preferably 10 ~ It was confirmed that a shell having a thickness of 20 nm was formed.

Experimental Example 7: Measurement of Pdop degradation inhibition rate in photothermal performance, biocatalytic movement and alkaline conditions

To confirm the potential bioapplication of the A-motor, photothermal properties and biocatalytic kinetic properties were tested. Although asymmetric micro/nano motors are known to generate enhanced motion at low Reynolds numbers, it has recently been reported that symmetric micro/nano motors can induce enhanced propulsion by enzyme-based asymmetric distribution.

(1) Measurement of photothermal properties

The trajectory, the mean displacement, and the diffusion coefficient were obtained from Pdop nanoparticles (Pdop/native urease), C motor of Comparative Example 3, and A motor of Example 1. Urea (300 mM) under NIR-on/off conditions. was used, and the measurement results are shown in a) of FIG. 9, and the temperature change per hour (minutes) is shown in b).

Figure 10a) is Pdop, C-motor (NIR-off), A-motor (NIR-off) according to the presence or absence of irradiation of low-intensity NIR (808 nm, 0.8 W/cm ² ) under urea conditions (urea, 300mM) ) and diffusion coefficients measured under A-motor (NIR-on) conditions.

In addition, FIG. 10 b) and FIG. 11 a) to c) are comparison results of photothermal performance according to NIR laser irradiation time, and FIG. Nanoparticles (Pdop/native urease), C motor (Comparative Example 3) and A motor (Example 1) were each put into an EP tube and irradiated with an 808 nm NIR laser for 17 minutes (intensity: 2 W/cm ² ). Next, the temperatures of various samples were recorded for each minute, and FIG. 11 a) and A motor (Example 1) by A-motor concentration (NIR laser intensity = 2 W/cm ² ), FIG. 11 b) By NIR laser intensity (A-motor concentration = 400 μg/ml), c) is an infrared thermal image by NIR intensity.

Looking at b) of FIG. 10, the photothermal performance of the A-motor (60°C) and the Pdop nanoparticles (62°C) was superior to that of the C-motor (54°C) by 10% or more.

Referring to FIG. 11, as the A-motor concentration increased, the photothermal effect tended to increase as the NIR intensity increased. A significant increase was observed at intensities exceeding 0.8 W/cm ² .

In addition, it was confirmed that the A-motor of the present invention can also be used for photothermal imaging through the measurement of the infrared thermal image of FIG. 11 c).

(2) Evaluation of long-term activity

The diffusion coefficient test and mobility were monitored for 5 days, and long-term activity evaluation results are shown in a) and b) of FIG. 12 .

12 a) is the diffusion coefficient (Pdop/native urease and A-motor) calculated by tracking 10 particles for 5 days to analyze biocatalyst protection reuse, and b) is 5 days to confirm substrate protection. ΔT (temperature change) of Pdop/native urease (light gray and dark gray lines) and A-motors (black and gray lines) at various urea concentrations (0 and 300 mM) and NIR intensities (2 W/cm ² ) during nanomotor diffusion. The coefficient was determined by fitting a parabola of the mean square displacement (<r2>) according to the equation <r2>= 4DΔT, where D is the diffusion coefficient t represents the time interval.

Looking at a) of FIG. 12, the diffusion coefficient of Pdop nanoparticles (Pdop/native urease) was initially 5.8 μm ² /sec and significantly decreased to 2.1 μm ² /sec after 5 days, while the A-motor initially had a diffusion coefficient of 5.4 μm 2 /sec. It can be confirmed that it slightly changed to μm ² /sec and 4.3 μm ² /sec after 5 days, and through this, it was confirmed that the long-term active stability (durability) was excellent.

Looking at b) of FIG. 12, the long-term photothermal effect was performed by raising the temperature with or without urea (300 mM) for 5 days, and the Pdop / native urease photothermal ability was obtained after 5 days, as can be seen in the degree of protection. almost disappeared (0%, ΔT: 30.9 -> 5.5 °C). However, the A-motor showed little change in the photothermal effect after 5 days (protection level: 85%, ΔT: 29.3 -> 25.1 °C).

(3) Measurement of biocatalytic kinetics

Under urea conditions (urea, 300 mM), low-intensity NIR (808 nm, 0.8 W/cm ² ) Pdop nanoparticles (Pdop/native urease) with or without irradiation, C-motor (Comparative Example 3) and A-motor (Example The biocatalytic movement of Example 1) was measured, and the results are shown in a) and b) of FIG. 12 . a) Swimming trajectories (Y), and b) Mean Square Displacement (MSD).

The swimming trajectories were measured by 2D motion tracking using an optical video (pco.edge sCMOS camera with NIS-Elements BR 4.500.00 64-bit) and an inverted microscope equipped with a 60x microscope. The movement of the nanomotor (10 particles) was captured as an image at room temperature using a Nikon Ti-DH, 20 fps). The trajectory was analyzed using the video analysis and modeling tool Tracker 5.1.3 program, and the mean square displacement (MSD) and mean velocity were calculated using synthesized nanoparticle samples (concentration 400 μg/mL). Then, the biocatalytic magnetophoretic movement of the nanomotor was monitored by transferring the sample to an aqueous urea solution (300 mM) for 20 seconds, and the thermal catalytic movement was measured under urea (300 mM) and NIR-on (0.8 W/cm2) conditions. . And, the MSD was calculated based on Equation 3 below.

[Equation 3]

In Equation 3, N is the number of particles, r _i (t)-r _i (t+Δt) is the vector distance traveled by the particle during an interval of time (Δt), and the velocity of the particle is the number of individual trajectories (up to 2 seconds) It was calculated by fitting the MSD to the quadratic equation of MSD(Δt)=4Dt+v2t2 and taking the average among all particles.

Referring to FIG. 13 , it was confirmed that the biocatalytic movement of the A-motor was high before NIR irradiation, and in particular, the A-motor biocatalytic kinetic activity significantly increased during NIR irradiation.

(4) Measurement of Pdop degradation inhibitory effect

The degree of protection and the degree of Pdop degradation (or degradation inhibition rate) according to the pH conditions were measured, and the results are shown in a) and b) of FIG. 14, and the measurement method is as follows.

Pdop degradation inhibition rate was evaluated in alkaline conditions of Pdop/native urease (non-core@shell), A-motor (Example 1), and C-motor (Comparative Example 3), and urea (300 mM) and urease before degradation test (400μg/mL) pH was measured after 24 hours of incubation at room temperature in order to confirm the pH change by the reaction. The change in pH was 9.5.

Urea (0 and 300 mM) or different pH (pH 7.4 and 9.5) was measured by applying NIR power 2 W/cm ² for 5 minutes, and to test the protective ability for each condition, the nanomotor (400 μg/ mL) was dispersed in an alkali or urea solution, incubated for 5 days, and recorded every 1 day.

Looking at a) of FIG. 14, there was no decomposition of Pdop in Pdop/native urease and A-motor for 5 days in a neutral solution, pH 7.4, and the temperature increase was not affected.

In a solution with high pH (pH: 9.5), Pdop/native urease was decomposed, and the temperature of Pdop was also increased. On the other hand, A-motor's aZIF-8 minimized the effect of temperature rise by preventing deterioration.

Ammonia generated in the reaction of urea + urease -> CO ₂ + 2NH ₄ induces the basic pH condition to pH 9.5 when measured, induces denaturation of Pdop and urease, and significantly reduces photoconversion and biocatalytic performance. It is known to be able to.

However, the aZIF-8 shell prevented denaturation of Pdop and the enzyme by maintaining the catecholic bond of Pdop and maintaining the conformational change and active site of the enzyme. activity was maintained, and the 2-MIM ligand and surface structure of the aZIF-8 shell protected the core from -OH to maintain the original function of the hybrid nanoparticles.

Experimental Example 8: Biocompatibility and photothermal therapy experiment of urease-driven A-motor with enhanced metastasis to bladder cancer cells under simulated conditions

(1) Measurement of the biocompatibility of the A-motor

The biocompatibility of the synthesized nanomotor was analyzed with fibroblasts (NIH3T3).

Different concentrations (100 μg/mL, 200 μg/mL, 400 μg/mL) of Pdop nanoparticles (control) and A-motors, respectively, in Dulbecco's Modified Eagle Medium (DMEM) cell culture medium supplemented with 10% fetal bovine serum (FBS). , and separately added to a 96-well plate containing NIH3T3 cells at a cell concentration of 2x10 ⁴ CFU/ml per well.

After culturing for 24 hours in a CO ₂ incubator at 37° C., MTS assay was performed to quantitatively measure cell viability. All experiments were performed in triplicate.

A graph of cell viability per concentration is shown in FIG. 15 .

The survival rate by concentration of Pdop nanoparticles was 100 μg/mL (92±2%), 200 μg/mL (93±1%), and 400 μg/mL (90±1%), and the survival rate by concentration of the A-motor was 100 μg/mL (83±6%), 200 μg/mL (85±3%), and 400 μg/mL (93±3%).

Through this, it was confirmed that the A-motor had low cytotoxicity and excellent biocompatibility.

(2) Internalization and uptake of A-motor into cancer cells

The internalization and uptake of the A-motor on cancer cells was further investigated. For better visualization, rhodamine B (RB)-conjugated A motors were prepared according to the reported method.

Specifically, rhodamine B (5 mg) and nanoparticles (Pdop nanoparticles, A-motor) were stirred in deionized water (10 mL) for 12 h at 360 rpm.

First, rhodamine B (5 mg) and Pdop nanoparticles (4 mg) were stirred in a brown vial with 10 ml of 10 mL deionized water for 12 hours at room temperature. Then, the resulting solution was centrifuged at 3000 rpm for 10 minutes and finally vacuum dried at room temperature for 24 hours.

Fluorescently labeled RB-Pdop nanoparticles (Pdop-rhodamine B) and RB-A-nanomotors were prepared separately in McCoy's 5a medium at a concentration of 400 μg/mL.

T-24 cells cultured at a concentration of 4x10 ⁵ CFU/mL in a 24-well plate were co-cultured with Pdop nanoparticles or A-motors for 24 hours. To better visualize the localization of Pdop nanoparticles (control) and A-motors, cell cytoplasm and cell nuclei were stained with Calcein AM and DAPI.

Cellular uptake of Pdop nanoparticles or A-motors was investigated by confocal microscopy, and the measured images are shown in FIG. 16 .

In FIG. 16, green is the cytoplasm stained with Calcein AM, blue is the cell nucleus stained with Hoechst, and red is the A-motor stained with Rhodaman B (RB).

Referring to Figure 16, it can be seen that the A-motor (red) was successfully internalized into the cytoplasm (green).

(3) nanomotor transmigration and sequential photothermal therapy

To investigate the translocation behavior of the A-motor across blood endothelial tissue and to confirm its applicability to photothermal therapy (PTT) in cancer tissue, we further developed an in vitro blood bladder cancer model using transwell inserts. More specifically, HUVEC cells were seeded with 2x10 ⁴ CFU dispersed in EGM (endothelial cell growth medium) and co-cultured with bladder cancer cells (T-24) on a transwell membrane (pore size 8 μm) ( see Fig. 17 schematic).

When a confluent monolayer of HUVEC tissue was formed, the HUVEC layer was cultured on top of a 24-well plate containing 2x10 ⁴ CFU of T-24 cells. Then, 400 μg/ml of Pdop nanoparticles, C-motors (Comparative Example 3) or A-motors (Example 1) were separately introduced into HUVEC cells so that the nanoparticles or nanomotors could cross the HUVEC layer to the T-24 It was allowed to be transferred to cells (transmigration). Then, after culturing for 24 hours in a CO ₂ incubator at 37° C., the T-24 cells were irradiated with a NIR laser (2 W/cm ² ) for 5 minutes. Cells were stained with Calcein AM and Hoechst. T-24 cells not treated with Pdop nanoparticles or nanomotors were used as a control.

Normal adult blood as well as the bladder contains urea, but the amount varies from person to person. In order to mimic the similar conditions in vivo, urea was added to the in vitro blood-bladder cancer model as a medium when a confluent monolayer of HUVEC cells was formed, and the concentration difference of nanoparticles (Pdop, C-motor, and A-motor) was used to form the HUVEC layer. has been transferred to.

After 24 hours of co-cultivation, the HUVECs containing the transwell were removed, and the supernatant of the T-24 cells was collected to analyze the amount of nanoparticles delivered to the bladder tissue using UV-Vis spectroscopy. 18 and 19 are shown. In this case, FIG. 18 shows UV-vis absorbance measurement data of the upper membrane before and after the transfer and fluorescence images of the lower membrane after the transfer. At this time, the UV-vis adsorption spectrum was measured with a UV-vis spectrometer (Thermo Scientific, NanoDrop 2000) of quartz cuvettes.

Referring to FIG. 18, both the A-motor and the C-motor showed improved transfer efficiency after 24 hours of incubation due to exercise, similar to the Pdop nanoparticles. Referring to FIG. At over 80%, the transfer rate was twice as good as that of the C-motor.

This is because the nanoparticles containing urease generate an asymmetric gradient along with urea decomposition, resulting in increased movement, and the mesopores (2-8 nm) of aZIF-8 are micropores (<2 nm). This is because it increases the reactivity of urea and urease more than cZIF-8.

(4) Measurement of photothermal therapy (PTT) potential for cancer cells

To measure the photothermal therapeutic potential of synthesized Pdop and nanomotors, photothermal therapy to treat cancer cells was additionally evaluated by irradiating T-24 cells that had absorbed Pdop or nanomotors with NIR laser (2W/cm ² ) for 5 minutes. is shown in Figure 20. At the time of measurement, nanoparticles remaining in the culture medium were removed by washing the T-24 cells before NIR irradiation.

T-24 cells not treated with Pdop nanoparticles or A-motor were used as a control. And the control group was also investigated by NIR. After 5 minutes of NIR laser irradiation, live cells were stained with Calcein AM, and all experiments were performed three times.

Referring to FIG. 20, bare Pdop nanoparticles with excellent PTT effect showed lower photothermal therapy performance than A-motor due to low mobility due to high adhesion to venous endothelium. In contrast, significant cell death was investigated in both the C- and A-motors, whereas complete cancer cell death was observed in the A-motors.

The improved movement efficiency of the A-motor leads to high temperatures through excellent NIR absorption, which can be used as an effective photothermal agent for photothermal therapy.

Through the above examples and experimental examples, the core-shell type hybrid nanomotor prepared by the continuous microfluidic laminar flow synthesis method proposed by the present invention has an amorphous shell and an amorphous shell that is not crystalline, so that a large amount of urea body ( enzymes) are effectively combined, and the resulting hybrid amorphous nanomotor has excellent in vivo mobility, excellent biocompatibility due to no biotoxicity, and is suitable for use as a photothermal agent for photothermal treatment for cancer cell removal. In addition, the self-propelled hybrid amorphous nanomotor of the present invention can be used as an in vivo drug delivery system by carrying additional enzymes, proteins, drugs, etc. in the core and / or shell, and can also be used for photothermal imaging due to its excellent photothermal properties was able to confirm

1: 1st inlet 2: 2nd inlet 3: 3rd inlet
4: 4th inlet 10: confluence 30: first reaction unit
50: second reaction unit 100: serial capillary reactor

Claims (16)

As a core-shell nanoparticle,
a core containing polydopamine; and
A shell including an organometallic framework in which a plurality of mesopores are formed;
Self-propelled hybrid amorphous nanomotor, characterized in that at least some of the plurality of mesopores are coupled with a self-propellant.
The self-propelled hybrid amorphous nanomotor according to claim 1, wherein the self-propelled body includes at least one selected from urease, glucose oxidase and catalase.
The self-propelled hybrid amorphous nanomotor according to claim 1, wherein at least one of the core and the shell carries one or more in vivo delivery materials selected from among enzymes, proteins, DNA, and chemical drugs.
The method of claim 3, wherein the enzyme in the in vivo delivery material is at least one selected from proteinase K, pepsin, trypsin, lysine, and chymotrypsin. A self-propelled hybrid amorphous nanomotor comprising a.
The method of claim 1, wherein the organometallic framework is amorphous,
The organometallic framework is selected from Zeolitic imidazole framework (ZIF)-7, ZIF-8, ZIF-67, ZIF-8-90, ZIF-11, ZIF-12, HKUST-1, MIL-53 and UIO-66. A self-propelled hybrid amorphous nanomotor comprising at least one.
The method of claim 1, wherein the core-shell nanoparticles have an average diameter of 100 to 400 nm,
The self-propelled hybrid amorphous nanomotor, characterized in that the shell has a thickness of 10 ~ 100 nm.
It is prepared by performing a continuous microfluidic laminar flow synthesis method using a series-type capillary reactor configured by integrating the first reaction part and the second reaction part in series,
In the first reaction unit, a core containing polydopamine; And a shell comprising an organometallic framework; an amorphous nanomotor comprising a is synthesized,
In the second reaction unit, a self-propelled hybrid amorphous nanomotor manufacturing method, characterized in that a self-propelled material is coupled to at least some of the plurality of mesopores formed in the shell of the synthesized amorphous nanomotor.
The method of claim 7, wherein the inlet of the first reaction unit includes a confluence to which a plurality of first reactant inlets are coupled,
The joining portion is formed so that materials introduced from each of the plurality of inlets are simultaneously joined,
A method for manufacturing a self-propelled hybrid amorphous nanomotor, characterized in that a second reactant inlet is formed at the end of the first reaction unit to the beginning of the second reaction unit.
The method of claim 8, wherein the plurality of first reactant inlets include a first inlet, a second inlet and a third inlet,
A metal ion precursor solution is injected into the first inlet, a polydopamine nanoparticle solution is injected into the second inlet, and an organic ligand precursor solution is injected into the third inlet,
The method of manufacturing a self-propelled hybrid amorphous nanomotor, characterized in that the self-propellant-containing solution or the precursor solution of the self-propellant is injected into the second reactant inlet.
10. The method of claim 9, wherein the metal ion precursor comprises a zinc ion (Zn ²⁺ ) precursor,
The organic ligand precursor solution is 2-methylimidazole, ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, o-phthalic acid, m-phthalic acid, p-phthalic acid, 2-hydroxy-1,2,3-propanetricarboxylic acid (2-hydroxy -1,2,3-propanetricarboxylic acid), benzene-1,3,5-tricarboxylic acid, 1H-1,2,3-triazole (1H-1 ,2,3-triazole), 1H-1,2,4-triazole (1H-1,2,4-triazole) and 3,4-dihydroxy-3-cyclobutene-1,2-dione (3 , 4-dihydroxy-3-cyclobutene-1,2-dione) method for manufacturing a self-propelled hybrid amorphous nanomotor, characterized in that it comprises an organic ligand precursor containing at least one selected from.
The method of manufacturing a self-propelled hybrid amorphous nanomotor according to claim 9, characterized in that it satisfies Equation 1 below;
[Equation 1]
20 ≤ B/A ≤ 60
In Equation 1, A is the flow rate of the self-propellant-containing liquid or the precursor solution of the self-propellant injected through the second reactant inlet, and B is the flow rate of the polydopamine nanoparticle solution injected through the second inlet.
The method of manufacturing a self-propelled hybrid amorphous nanomotor according to claim 9, characterized in that it satisfies Equation 2 below;
[Equation 2]
10 ≤ (C+D)/A ≤ 20
In Equation 2, A is the flow rate of the self-propellant-containing liquid or the precursor solution of the self-propellant injected into the second reactant inlet, C is the flow rate of the metal ion precursor solution injected into the first inlet, and D is injected into the third inlet. It is the sum of the flow rates of the organic ligand precursor solution.
The method of claim 9, wherein the metal ion precursor solution has a metal ion precursor concentration of 8.0 ~ 15.0 mg / ml,
The organic ligand precursor solution has an organic ligand precursor concentration of 70 to 140 mg/ml,
The polydopamine nanoparticle solution has a polydopamine nanoparticle content of 0.05 to 1.0% by weight,
Preparation of a self-propelled hybrid amorphous nanomotor, characterized in that the self-propellant-containing liquid has a self-propellant concentration of 1 to 20 mg/ml, and the self-propellant precursor solution has a self-propellant precursor concentration of 1 to 20 mg/ml. method.
[Claim 8] The method of claim 7, wherein the self-propelled hybrid amorphous nanomotor obtained by performing the continuous microfluidic laminar flow synthesis method is subjected to membrane filtration to limit shell growth. .
An in vivo drug delivery system comprising the self-propelled hybrid amorphous nanomotor of any one of claims 1 to 6.
A photothermal agent for photothermal treatment of cancer cells, characterized by using the self-propelled hybrid amorphous nanomotor of any one of claims 1 to 5.