KR102391328B1 - Method for Manufacturing and Use of Urease-powered Polydopamine Nanomotors - Google Patents

Abstract

The present invention relates to a biocompatible polydopamine nanomotor that can penetrate through the bladder wall and remain in the bladder for a long time in vivo.

Description

Method for Manufacturing and Use of Urease-powered Polydopamine Nanomotors using urease-powered Polydopamine Nanomotors

The present invention relates to a polydopamine nanomotor using a urease, a method for producing the polydopamine nanomotor, and a use thereof.

Intravesical therapy is a therapeutic drug into the bladder through a catheter for the treatment of various bladder diseases, such as overactive bladder (Ref. 1), interstitial cystitis (Ref. 2-4), and bladder cancer (References 5-6). A direct injection treatment method. Although intravesical drug delivery maximizes the therapeutic effect and minimizes systemic side effects by directly exposing the bladder tissue to the therapeutic agent, it still has limitations. Due to periodic urination, intravesical therapeutic agents can be rapidly flushed away, which requires frequent repeated injections for desirable therapeutic efficacy (Ref. 7). In addition, frequent injection of a therapeutic agent may cause discomfort to the patient as well as infection (Reference 8). Therefore, the delivered drug should be able to rapidly attach to the bladder mucosa and remain intact even after urination without disturbing the function of normal cells in the bladder.

Propelled microparticles and nanoparticles, i.e. micromotors/nanomotors, are of great interest for potential applications in nanomedicine because of their advantages such as rapid drug transport and penetration of biological barriers (Refs. 9-11). Recently, several nanomotor studies have been developed for in vivo applications. A system has been developed in which Zn or Mg reacts with water to generate hydrogen gas and moves to the gastrointestinal tract to deliver drugs (References 12-17). In addition, a silica/Fe ₃ O ₄ based micromotor in which motion is induced by a magnetic field has been developed for in vivo applications (Ref. 18). A nanomotor system has been applied to wound closure, and a Ni-based magnetic propeller has been developed to deliver drugs through the vitreous to the retina (Ref. 19). Although these synthetic motors outperformed conventional passive target particles in organs and tissues, synthetic micromotors are made of inorganic materials or metals that are not suitable for biological systems, and have no in vivo application to other organs and tissues. the current situation.

In order to solve the above problems, an object of the present invention is to provide a biocompatible nanomotor that can penetrate through the bladder wall and remain in the bladder for a long time in vivo.

The present invention is a hollow core; and a shell comprising polydopamine;

The polydopamine is bound to a urease,

The urease provides a polydopamine nanomotor that generates gas in the presence of urea to induce self-propulsion of the nanomotor.

In addition, the present invention comprises the steps of forming a polydopamine shell on silica particles to prepare silica-polydopamine core-shell particles;

preparing hollow polydopamine particles by removing the silica particles from the silica-polydopamine core-shell particles; and

It provides a method for producing a polydopamine nanomotor comprising the step of binding a urease to the surface of the hollow polydopamine particle to prepare a hollow polydopamine particle to which the urease is bound.

In addition, the present invention provides a carrier for a drug delivery system comprising the above-described polydopamine nanomotor.

The polydopamine nanomotor according to the present invention can penetrate through the mucosal layer and remain in the body for a long time.

In particular, urease-driven polydopamine nanomotors move autonomously in the presence of urea, and after reaching the bladder wall, they attach to bladder cells due to polydopamine on the nanomotor surface. After injection into the bladder, the nanomotors can penetrate deep bladder tissue by self-propelling, and thus many nanomotors can be retained in the bladder even after urination. Through this, the efficiency of drug delivery in the bladder is excellent, and the therapeutic effect can be maximized.

In Figure 1 (A) is a schematic diagram showing the penetration (penetration) and retention (retention) in the bladder wall of polydopamine nanomotors, (B) is polydopamine using silica nanoparticles (SiNP) and polydopamine (PDA) It is a schematic diagram showing the manufacturing process of a nanomotor.
In FIG. 2, (A) is an HR-TEM photograph of Ur-PDA NC, and (B) is an EDS map (scale bar = 200 nm) corresponding to green C, purple N, yellow O, and red S. .
In FIG. 3, (A) is a hydrodynamic size distribution for each manufacturing step of polydopamine nanomotors, (B) is a zeta potential of SiNP, PDA@SiNP, PDA NC and Ur-PDA NC, (C ) shows the FT-IR spectra of SiNP, PDA@SiNP and Ur-PDA NC, and (D) shows the activity of urease and free urease conjugated to polydopamine nanomotors.
In FIG. 4 (A) is trajectories of polydopamine nanomotors for 30 seconds in the presence of urea (0, 50 and 100 mM) in simulated urine and real urine, (B) is the diffusion coefficient value calculated from MSD (**P ≤ 0.01, urea vs no urea), (C) is x of 20 particles (polydopamine nanomotors) in each condition (0, 50, 100 mM and real urine) and mean square displacement (MSD) with increasing time interval, analyzed by y-coordinate tracking, and (D) represent the rate at which the concentration of the element increases.
5 shows the cytotoxicity test results of polydopamine nanomotors.
Figure 6 shows two-photon microscopy images of the bladder at depths (0, 12, 27, 42 and 57 μm) after injection of FITC-labeled (A) SiNPs, (B) PDA NCs and (C) Ur-PDA NCs ( two-photon microscopic images). Fluorescence images of the first and second columns were measured at 0 and 12 hours after injection, respectively (scale bar = 50 μm).
Figure 7 shows the mean fluorescence intensity of the bladder at the depth after (A) intravesical injection and (B) 12 hours after injection of the FITC-labeled group (**P ≤ 0.01, Ur-PDA NC vs. PDA NC).
8 is a photographic image of an intact bladder 12 hours after injection of (A) PBS, FITC-labeled (B) SiNP, (C) PDA NC and (D) Ur-PDA NC (first row). and ex vivo fluorescence images (second row) (scale bar = 1 cm).
Maximum projection intensity of the bladder 12 hours after injection of FITC-labeled (E) PDA NCs and (F) Ur-PDA NCs (scale bar = 50 μm).
9(A) shows the fluorescence intensity of the intact bladder 12 hours after injection of the FITC-labeled group. (B) shows that the FITC-labeled group reduced the fluorescence intensity of the bladder 12 hours after injection and after 0 hours (100%) compared to (**P ≤ 0.01, *P ≤ 0.05, PDA NC vs. SiNP and Ur-PDA NC vs PDA NC).
10 shows the H&E staining results of bladder tissue 12 hours after injection of (A) PBS and (B) polydopamine nanomotors (scale bar = 100 μm).

The present invention is a hollow core; and a shell comprising polydopamine;

The polydopamine is bound to a urease,

The urease relates to a polydopamine nanomotor that generates gas in the presence of urea to induce self-propulsion of the nanomotor.

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

In the present invention, the term “nanomotor” is defined as a nanoparticle that can be propelled with force by various external stimuli, and is defined as a microscopic device that has its own driving force by a chemical reaction of a catalyst in a liquid. These nanomotors could contribute to solving complex and esoteric problems while being tasked with maintaining self-propelled forces in liquids.

Polydopamine nanomotors according to the present invention include a hollow core; and a shell comprising polydopamine.

In the present invention, the polydopamine nanomotor has a structure of hollow nanoparticles or nanocapsules. That is, the core of the nanomotor is hollow. By forming the core hollow, the weight of the polydopamine nanocore can be minimized and the speed of the nanomotor can be maximized. And, through this, it is possible to maximize the penetration effect of the nanomotors in the mucosa.

In the present invention, the shell includes polydopamine. Dopamine, a monomer form of polydopamine, is a mimic molecule of the 3,4-Dihydroxy-Lphenylalanine (L-DOPA) molecule found in sea mussels. The polydopamine has a covalent bond, catechol and imine functional groups, so it has a very strong bond not only on organic materials such as biomaterials and synthetic polymers, but also on solid surfaces such as electrodes or separators of batteries. can be formed Accordingly, it is possible to form a surface reforming, a surface modification, a self-assembled multilayer, a nanocomposite thin film, and the like. The catechol functional group of dopamine is easily oxidized in the presence of oxygen and can form polydopamine shells of various thicknesses by self-polymerization.

In the present invention, the polydopamine may exhibit high adhesion to a tissue by forming a covalent/non-covalent bond with a target tissue, that is, a mucosal tissue.

In one embodiment, the polydopamine may be an autopolymer of dopamine.

In one embodiment, polydopamine on the surface of the shell may be bound to a bioenzyme. The polydopamine may form a bond with the bioenzyme through a covalent bond or a hydrogen bond, and specifically, the catechol group of the polydopamine may form a bond with the amine group of the bioenzyme. In this case, the bond may be formed by a micelle addition reaction and/or a Schiff base reaction.

The bioenzyme is a bioavailable enzyme, and is a protein catalyst that mediates a chemical reaction in a living body. As such a bioenzyme, a urease (urease, urease) may be used.

The urease is an enzyme that hydrolyzes urea. The urease may serve as an engine to move the nanomotor while decomposing urea present in a high concentration in the bladder, and also has biocompatibility. Urea may be decomposed into ammonia and carbon dioxide by the urea decomposition furnace.

In one embodiment, the polydopamine nanomotor can induce self-propelling by generating gas by the action of a biological enzyme.

When the bioenzyme is a urease, the urease decomposes the urea in a urea environment to generate carbon dioxide, and the nanomotor may be propelled and driven through the generated carbon dioxide. Through this, the nanomotors are attached to mucous membranes such as the bladder wall, and can also penetrate into the mucosa. Therefore, when using urease, polydopamine nanomotors can be expressed as urease-conjugated polydopamine nanomotors or urease-powered polydopamine nanomotors.

In one embodiment, the thickness of the shell may be between 10 and 30 nm or between 15 and 25 nm. Excellent biocompatibility can be provided in the thickness range, and the speed of the nanomotor can be maximized by minimizing the weight of the nanomotor.

In addition, the size of the polydopamine nanomotor may be 800 to 1,500 nm or 1,000 to 1,200 nm. In the above size range, it has the advantage of being easy to adhere to and penetrate into the body. If the size is too small, it is not possible to obtain a driving force as a nanomotor, and if the size is too large, there is a fear that the bio-penetration force is reduced.

In one embodiment, the polydopamine nanomotor may further include a drug. At this time, the drug may form a bond with polydopamine of the shell. The drug can form a bond with polydopamine by π-ð stacking and hydrophobic bonding.

The type of the drug is not particularly limited, and an anticancer agent may be used in the present invention. The anticancer agents include paclitaxel, texotere, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carbopletin, doxorubicin, daunorubicin, idarubicin, 5-fluorouracil and methotrexate, At least one selected from the group consisting of actinomycin-D may be used.

In one embodiment, polydopamine nanomotors can be used for the treatment of bladder diseases. In this case, the type of bladder disease is not particularly limited, and, for example, may be selected from the group consisting of overactive bladder, interstitial cystitis, and bladder cancer.

In general, the treatment of bladder disease uses a method of injecting a drug using a catheter into the bladder. At this time, the drug does not adhere well to the bladder wall and is washed away by frequent urination. In the present invention, when the polydopamine nanomotor is injected into the bladder using the polydopamine nanomotor, the nanomotor is propelled by a high urea concentration in the bladder, and the polydopamine nanomotor can efficiently penetrate the mucosal layer of the bladder wall. It may also be present longer after urination within the lesion wall. The improved penetration and penetration of these nanomotors suggests that the nanomotors can be used as a novel method for the treatment of various bladder diseases.

In addition, the present invention relates to a method for producing the above-described polydopamine nanomotor.

The polydopamine nanomotor according to the present invention comprises the steps of forming a polydopamine shell on silica particles to prepare silica-polydopamine core-shell particles;

preparing hollow polydopamine particles by removing the silica particles from the silica-polydopamine core-shell particles; and

By binding a urease to the surface of the hollow polydopamine particles can be prepared through the step of preparing the urease-coupled hollow polydopamine particles.

In the present invention, the step of preparing the silica-polydopamine core-shell particles is a step of forming a polydopamine shell on the silica particles. The silica-polydopamine core-shell particles may be expressed as PDA@SiNP.

In the above step, silica particles may be suspended in a dopamine precursor solution, and the dopamine precursor may be self-polymerized on silica particles to prepare polydopamine.

Specifically, dopamine undergoes self-polymerization (self-polymerization) in a buffer solution of about pH 8.5, and polydopamine formed through this process is highly reactive and can easily make new bonds on the surface. .

In one embodiment, the average particle diameter of the silica particles may be 800 to 2,000 nm or 1,000 to 1,500 nm.

In one embodiment, the dopamine precursor may be dopamine hydrochloride.

In one embodiment, the content of the dopamine precursor may be 60 to 90 or 70 to 80 parts by weight based on 100 parts by weight of the silica particles.

In one embodiment, the reaction may be performed at 20 to 30° C. or room temperature for 8 to 15 hours or 10 to 13 hours. Since polydopamine can be self-polymerized at room temperature, it has the advantage that it can be coated without additional reagents or equipment, so the manufacturing process cost and process efficiency are excellent.

In one embodiment, after performing the above steps, centrifugation and/or washing may be performed to remove unreacted dopamine precursors.

In the present invention, the step of preparing the hollow polydopamine particles is a step of removing the silica particles from the silica-polydopamine core-shell particles. The hollow polydopamine particles may be expressed as PDA NC.

The above step may be performed by supporting the silica-polydopamine core-shell particles prepared in the above-mentioned step in an acid solution. Silica particles, which are cores, may be removed through the above step.

In one embodiment, the acid solution may include HF and NH ₄ F. Also, the pH of the acid solution may be 3 to 6.

In one embodiment, the step may be performed for 3 to 10 minutes or 5 to 7 minutes.

In the present invention, the step of preparing the hollow polydopamine particles to which urease is coupled is a step of binding the urease to the surface of the hollow polydopamine particles. The hollow polydopamine particle to which the urease is bound is a polydopamine nanomotor, and may be expressed as a urease junction (or binding) nanocapsule (or nanomotor) or Ur-PDA NC.

The above step can be performed by adding an aqueous solution containing PDA NC to the urease solution, and through the above step, the catechol group of polydopamine of PDA NC is micelle addition reaction with the amine group of urease (Michael addition and / Alternatively, a bond may be formed through a Schiff base reaction.

In one embodiment, the reaction may be performed at 20 to 30° C. or room temperature for 8 to 15 hours or 10 to 13 hours.

In the method for producing a polydopamine nanomotor of the present invention, a step of binding a drug to the surface of the hollow polydopamine particle may be additionally performed. In this case, the binding of the drug to polydopamine may be performed after the urease is bound, but the drug may be bound first before binding the urease.

In one embodiment, the drug may form a bond with polydopamine by π-π stacking and hydrophobic bonding.

In one embodiment, the drug may be of any of the aforementioned types.

The present invention also relates to the use of the aforementioned polydopamine nanomotors.

The polydopamine nanomotor according to the present invention can be used as a carrier for a drug delivery system. In addition, the polydopamine nanomotor may be coated on a medical device such as a catheter and injected into a living body.

The polydopamine nanomotor may include a drug, and the drug may be delivered into the living body through the mucosa by the propulsion of the nanomotor. In this case, the above-mentioned kind may be used as the kind of drug.

The polydopamine nanomotor of the present invention can be used for the treatment of various diseases depending on the type of drug, and specifically can be used for the treatment of bladder diseases. The bladder disease may be selected from the group consisting of overactive bladder, interstitial cystitis and bladder cancer.

In an embodiment of the present invention, hollow nanoparticles (nanomotors) were prepared using polydopamine. Polydopamine exhibited high adhesion to tissues by forming covalent/non-covalent bonds with target tissues. In addition, in order to propel the nanomotor, an enzyme urease (urease) was conjugated to the surface of the polydopamine nanomotor. Nanomotors driven by urease were propelled by decomposing urea (~300 mM) present in high concentrations in the bladder into carbon dioxide and ammonia. It was confirmed that after being injected into the bladder through a catheter, it penetrated into the bladder wall through propulsion and that a large amount of nanomotors remained even after urination.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example.

<Reference> Statistical analysis

Statistical analysis was performed through t-test using software of SigmaPlot10.0. ** Values of P<0.01 were considered statistically significant. Data are presented as mean ± standard deviation (SD) from several individual experiments. All experiments were performed in triplicate and 20 nanomotors were measured for each group.

Example 1. Polydopamine nanomotor synthesis

(1) Polydopamine nanocapsule (PDA NC) synthesis

5 mg of silica particles (5 wt%) were pre-washed 3 times with 10 mM TRIS buffer (pH 8.5) and centrifuged to form a pellet. The resulting pellet was resuspended in 2 mL of dopamine hydrochloride solution (2 mg mL ^-1 ). The suspension darkened within 10 minutes and the reaction allowed to proceed for 12 hours. Thereafter, the yellowish-brown particles were centrifuged (1000 g, 30 sec) and washed with fresh TRIS buffer to remove excess unreacted dopamine. PDA NC was manufactured through a centrifugation/redispersion process (4500 g for 5 minutes, 3 cycles) after removing the silica core at 20° C. for 5 minutes with 2M HF/8 M NH ₄ F solution (pH 5).

(2) Synthesis of urease-conjugated polydopamine nanomotors (Ur-PDA NC)

The aqueous solution containing the PDA NC synthesized in (1) was suspended in a PBS solution containing urease (3 mg mL ^-1 ). The reaction was allowed to proceed at room temperature for 12 hours. Thereafter, the urease-conjugated (binding) polydopamine nanomotor (Ur-PDA NC) solution was washed 3 times with PBS and then centrifuged (4500 g for 5 minutes).

In the present invention, Figure 1A is a schematic diagram of the bladder wall penetration and retention of polydopamine nanomotors in the bladder.

A urease-driven polydopamine nanomotor can be designed and fabricated by a three-step process as shown in Figure 1B. In the first step, silica particles (SiNP) are added to a dopamine solution in Tris-HCl buffer (pH 8.5) to polymerize dopamine on the SiNP (PDA@SiNP) surface. It can be seen that the polydopamine film completely covers the silica nanoparticles with a thickness of 20 nm. In the second step, HF solution is added to remove the silica core and hollow nanocapsules are fabricated while maintaining the structural shape. It can be seen that the particle size of the nanocapsules is approximately 1 μm. Thereafter, the urease is conjugated to the surface of the nanocapsule by the reaction of the amine group of the enzyme with the catechol group of polydopamine through the Schiff base reaction.

Experimental Example 1. Characterization of urease conjugated nanomotors (Ur-PDA NC)

(1) method

First, the characteristic morphology of the urease-conjugated nanomotor (Ur-PDA NC) was confirmed by high-resolution transmission electron microscopy (HR-TEM), and the components were confirmed through energy dispersive spectrum (EDS).

In addition, the size and zeta potential of the nanocapsules were measured for each synthesis step, and the deformation of the surface properties was measured through the FT-IR spectrum.

(2) Results

The HR-TEM image of the urease-conjugated nanomotor (Ur-PDA NC) is shown in FIG. 2A.

As shown in FIG. 2A , it can be seen that the nanomotor maintains a capsule shape having a hollow structure even after binding of the urease, and the size of the nanomotor is 1 μm.

The energy dispersion spectrum (EDS) results are shown in FIG. 2B.

As shown in FIG. 2B, it can be confirmed that the urease conjugated nanomotor (Ur-PDA NC) includes carbon, nitrogen, oxygen and sulfur components. In particular, sulfur is detected by the thiol group of the urease, and through this, it can be confirmed that the urease is conjugated to the nanomotor. The detected compositions of carbon, nitrogen and oxygen and sulfur were 68.02, 13.93, 17.03 and 1.01%, respectively.

Meanwhile, the size of the nanocapsules for each synthesis step is shown in FIG. 3A.

As shown in Fig. 3A, it can be seen that the size of the nanocapsules is slightly changed for each synthesis step. It can be seen that the size of PDA@SiNP was larger than that of SiNP due to the polydopamine coating, and the size of PDA NC was slightly reduced after removing the silica core. In addition, in the last step, the size of Ur-PDA NCs increased slightly again, which indirectly confirms successful conjugation of urease.

In addition, the zeta potential analysis results of the nanocapsules for each synthesis step are shown in FIG. 3B. The zeta potential shows the charge characteristics of the nanocapsules generated in each manufacturing step, and the silica core showed a negative zeta potential (-43 ± 0.87 mV), whereas the PDA@SiNP showed 20.6 ± due to the amine group on the polydopamine surface. It can be seen that a positive value of 0.89 mV is indicated. However, after conjugation of the urease, the zeta potential value of the nanocapsule was negative due to the introduction of the urease molecule (-23.5 ± 0.70 mV).

In addition, the FT-IR spectrum measurement result is shown in FIG. 3C. The FT-IR spectrum can show evidence of modification of surface properties and successful conjugation of urease. The strong absorption peak at 1020-1110 cm ^-1 corresponds to the Si-O-Si asymmetric stretching oscillations, and the peak at 960 cm ^-1 is due to the asymmetric bending and stretching oscillations of Si-OH. After coating with polydopamine, shear oscillations of NH in the amide group and novel absorption picks at 1520 and 1615 cm ⁻¹ corresponding to the aromatic ring of dopamine were observed. After removing the silica core and bonding the urease, the peaks of Si-O-Si and Si-OH disappear, and some characteristic absorption peaks of the urease with weak strength are observed in the range of 1000-1800 cm ^-1 became

Experimental Example 2. Quantification of urease and analysis of urease activity

(1) method

The concentration of urease present on the surface of the polydopamine nanomotor was measured using a BCA protein analysis kit. The BCA protein assay kit quantifies the amount of protein by correlating the amount of the protein with the reduction of copper by peptide binding.

In addition, the enzymatic activity of urease bound to PDA NC was evaluated using a commercial kit for determining the concentration of ammonia produced by Berthelot's method.

At this time, the concentration of the nanomotor was 0.5 mg mL ^-1 , and the experiment was performed according to the manufacturer's instructions.

(2) Results

The amount of urease on the surface of the polydopamine nanomotor was quantified using a BCA protein analysis kit, and the results of comparing the activities of the urease conjugated to the nanomotor and the free urease at the same concentration are shown in FIG. 3D .

As shown in FIG. 3D , it can be confirmed that the activity of the conjugated urease was similar (86%) to that of the free urease without a significant decrease. Through this, it can be confirmed that urease can be used for propulsion of nanomotors.

Experimental Example 3. Video recording and nanomotor motility analysis.

(1) method

An optical microscope was used to observe and record video of the movement of polydopamine nanomotors. Aqueous samples of nanomotors were placed on glass slides and mixed well with various urea concentrations (0, 50 and 100 mM) and real urine. For actual urine experiments, urine was extracted from rats and collected in 1.5 mL e-tubes. Urine was centrifuged (8000 rpm for 5 min) and syringe filtered using a 0.22 μm filter. The movement of the nanomotor containing urine was recorded for 30 s at a frame rate of 8 fps. More than 20 nanomotors per condition were analyzed and the tracking path, mean square displacement (MSD) and velocity of the nanomotors were analyzed with ImageJ. Thereafter, the diffusion coefficient was obtained by matching the MSD data to the following equation.

MSD (Δt) = 4De Δt

where De denotes the effective diffusion coefficient and Δt denotes the time interval.

(2) Results

The nanomotor driven by the urease converts urea into ammonia and carbon dioxide as shown in Equation 1 below.

(NH ₂ ) ₂ CO + H ₂ O → CO ₂ + 2NH ₃

Although the geometrical asymmetry of synthetic motors has been regarded as an important requirement for generating propulsion, recent studies have shown that the unbalanced distribution of molecules conjugated to the surface of nanomotors is sufficient for propulsion of synthetic motors driven through biocatalytic transformation of enzymes. it turned out that The kinetic profiles of urease-driven nanomotors were evaluated at urea concentrations of 0, 50 and 100 mM and real urine.

The tracking trajectory of the nanomotor is shown in Fig. 4A. The tracking trajectory was recorded for 30 seconds at 8 frames per second. In addition, the velocity and mean square displacement (MSD) were calculated through the tracked trajectory, which is shown in FIGS. 4D and 4C.

In the absence of the element, the nanomotors were randomly moved at a speed of 0.77 μm s ⁻¹ due to Brownian motion and exhibited no directionality. However, after urea was added (50 and 100 mM), the nanomotors exhibited enhanced diffusion and directionality at velocities of 1.49 and 2.23 μm s ⁻¹ , respectively. MSD increased linearly with time as a typical pattern of diffusion kinetics, and further increased with increasing urea concentration.

In particular, the MSD and speed of nanomotors in actual urine were higher than those of other groups, confirming that nanomotors using urease can be effectively promoted for application to the treatment of bladder diseases.

In addition, the effective diffusion coefficient was obtained using MSD, which is shown in FIG. 4B. was obtained according to the following equation.

As shown in FIG. 4B , it can be seen that diffusion increases as the urea concentration increases, and the diffusion rate becomes saturated at a high urea concentration. It can be seen that this is a behavior similar to the behavior assumed by the Michaelis-Menten mechanics.

Experimental Example 4. Cytotoxicity test

(1) method

Before in vivo testing, human bladder cells (RT4) and nanomotors were cultured for 24 hours and then the cell compatibility of the nanomotors was analyzed.

Human bladder cancer cells (RT4) were cultured at 37°C in a humidified incubator containing 5% carbon dioxide. Bladder cancer cells were cultured in a medium containing 10% fetal bovine serum, 100 IU mL ^-1 penicillin and 100 mg mL ^-1 streptomycin. RT4 cells were incubated with nanomotors (5, 10, 50, 100, 500 and 1000 μg mL ⁻¹ ) for 24 hours, and then washed with serum-free medium.

Cell viability was assessed by MTT assay. MTT solution was prepared at a concentration of 0.5 mg mL ^-1 and then sterilized with a 0.2 μm filter. Then, 0.5 mL of the solution was added to each well. The wells were incubated at 37 °C for 30 min and 200 μL of DMSO was added. After incubation for 2 h, absorbance was measured at a wavelength of 595 nm.

(2) Results

The results of the cytotoxicity test are shown in FIG. 5 .

As shown in FIG. 5 , it can be confirmed that the cytotoxicity of the nanomotors is almost nonexistent up to a concentration of 1,000 μg mL ⁻¹ .

Example 2. Labeling with FITC

To label PDA NCs and Ur-PDA NCs with FITC fluorescent dye, 100 μL of FITC solution (1 mM) was added to 2 mL of PDA NCs and Ur-PDA NC aqueous solutions, respectively. The mixture was incubated at room temperature for 12 hours to proceed with the reaction. Then, a solution of labeled PDA NCs and Ur-PDA NCs was dialyzed (3.5 kDa) for 24 hours to remove excess unreacted FITC molecules.

As a control, SiO ₂ was labeled with FITC as previously reported elsewhere. 50 μL of APTES was added to 2 mL of SiO ₂ nanoparticle solution (5 mg mL ⁻¹ of ethanol). After stirring at room temperature for 12 hours, the modified silica nanoparticles were separated and excess APTES was removed by centrifugation/redispersion process (1000 g, 30 sec, 3 cycle). Then, 1 mL of the resulting solution was mixed with 0.5 mL of FITC ethanol solution (1 mM). After stirring for 4 hours, the particles were collected by centrifugation/redispersion process (1000 g, 30 sec, 3 cycle).

Experimental Example 5. Two-photon fluorescence imaging of nanomotors in the bladder

(1) method

To investigate the ability of nanomotors to penetrate the bladder wall and remain in the bladder, FITC fluorescent materials were labeled on the motor surface. In addition, SiNP and PDA NC groups as controls were also labeled with FITC on the surface.

SD female rats were randomly divided into three groups (SiNP, PDA NC, Ur-PDA NC) (n=3), and the rats were anesthetized by inhalation anesthesia. 50 μL suspensions of FITC-labeled SiNPs, PDA NCs and Ur-PDA NCs were administered via catheter into the bladder. After 0 and 12 hours of dosing, mice were sacrificed and the bladder was dissected and excised intact to examine the bladder wall. Tissues were then rinsed with PBS, leveled, and visualized using two-photon fluorescence microscopy.

The two-photon fluorescence signal of FITC and the second harmonic generation (SHG) of the collagen structure were observed at a wavelength of 950 nm. Images were collected as Z-stacks (xyz, 400 Hz) at 512 × 512 pixels and analyzed with Leica's LAS AF Lite 2.6.1.

(2) Results

6 and 7 show the results of visualization and quantification of bladder fluorescence intensity 0 and 12 hours after injection of the FITC-labeled group. All rats urinated several times during 12 h and were used for sample retention analysis.

In the case of the SiNP group, it was confirmed that the intensity of green fluorescence in the bladder wall was very low (FIG. 5A), but in the case of the PDA NC and Ur-PDA NC groups, strong green fluorescence was observed in the bladder wall (FIG. 6B and C). Through this, it can be confirmed that polydopamine improves adhesion to bladder tissue.

In particular, Ur-PDA NCs were observed up to a depth of 60 μm in the bladder wall (Fig. 7A). Propulsion of nanomotors could enable penetration through the mucosal layer within the bladder. Despite the decrease in fluorescence intensity, the fluorescence trend was similar even after 12 h after injection. It can be seen that the fluorescence intensity of all groups decreased to 27 μm in the bladder wall due to urination, whereas the fluorescence deeper than 27 μm was preserved even after 12 hours ( FIG. 7B ). Through this, it can be confirmed that in order to remain in the bladder for a long time, the penetration of the nanomotor into the bladder wall is a necessary requirement.

Experimental Example 6. In vivo fluorescence imaging

(1) method

To further investigate the retention of the sample in the intact bladder after urination, the fluorescence intensity of the entire bladder was visualized and quantified 12 h after intravesical injection.

SD female rats were randomly divided into 4 groups (control, SiNP, PDA NC, Ur-PDA NC) (n=3), the rats were anesthetized by inhalation, and then FITC-labeled samples were injected intravesically (50 µL). Injected via catheter. 12 hours after injection, fluorescence images were obtained in the wavelength range of 500 to 550 nm using an in vivo fluorescence imaging system. Bladder was extracted from sacrificed mice, excised, and observed with an in vivo fluorescence imaging system.

(2) Results

The results are shown in FIGS. 8 and 9 .

As shown in FIGS. 8A-E and 9A , the fluorescence intensity of FITC-labeled PDA NCs and Ur-PDA NCs was stronger than that of other groups (control and SiNP). In particular, Ur-PDA NCs showed the strongest fluorescence intensity.

In addition, as a result of comparing the fluorescence intensity of each group before and after urination, as shown in FIG. 9B, the bladder fluorescence intensity of the SiNP, PDA NC and Ur-PDA NC groups after 12 hours was 4.5%, 35.8%, and 49.2%, respectively. decreased, and it can be seen that a significant amount of Ur-PDA NC remains after urination compared with other groups. This suggests that the urease-driven polydopamine nanomotor system may be an effective drug delivery system for bladder disease.

Experimental Example 7. Tissue analysis

(1) method

After confirming the presence of nanomotors in the bladder, histological analysis of the bladder tissue was performed by H&E staining.

PBS and urease-propelled nanomotors were injected into the bladder for 24 hours, and after 12 hours, the bladder was extracted from the sacrificed mice, excised, and fixed in 4% paraformaldehyde. The fixed bladder was embedded in paraffin blocks, and 4 μm-thick sections were made for H&E staining. The stained area was observed under an optical microscope.

(2) Results

The staining results are shown in FIG. 10 .

As shown in Figure 10, it can be confirmed that there is no significant difference in the bladder tissue injected with PBS and nanomotors. This shows that the nanomotors are not histotoxic.

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Claims (12)

hollow core; and a shell comprising polydopamine;
The polydopamine is bound to a urease,
The urease induces self-propelling of the nanomotor by generating gas in the presence of urea,
A polydopamine nanomotor having a size of 800 to 1,500 nm.
The method of claim 1,
A polydopamine nanomotor that forms a bond between the catechol group of polydopamine and the amine group of urease.
The method of claim 1,
The thickness of the shell is 10 to 30 nm polydopamine nanomotors.
delete The method of claim 1,
A polydopamine nanomotor in which a drug is bound to a polydopamine nanomotor.
6. The method of claim 5,
The drug is an anticancer agent, and the anticancer agent is paclitaxel, texotere, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carboplatin, doxorubicin, daunorubicin, idarubicin, 5-fluoro A polydopamine nanomotor that is at least one selected from the group consisting of uracil, methotrexate, and actinomycin-D.
The method of claim 1,
A polydopamine nanomotor for use in the treatment of one or more bladder diseases selected from the group consisting of overactive bladder, interstitial cystitis and bladder cancer.
Forming a polydopamine shell on the silica particles to prepare a silica-polydopamine core-shell particles;
preparing hollow polydopamine particles by removing the silica particles from the silica-polydopamine core-shell particles; and
A method for producing polydopamine nanomotors having a size of 800 to 1,500 nm, comprising the step of binding a urease to the surface of the hollow polydopamine particles to prepare hollow polydopamine particles to which a urease is bound.
9. The method of claim 8,
The method for producing a polydopamine nanomotor wherein the removal of the silica particles in the step of preparing the hollow polydopamine particles is performed under acidic conditions.
9. The method of claim 8,
In the step of preparing the hollow polydopamine particles to which the urease is coupled, the binding of the urease is a micelle addition reaction or a Schiff base reaction. Preparation of polydopamine nanomotors Way.
9. The method of claim 8,
Method for producing polydopamine nanomotors further comprising the step of binding a drug to the surface of the hollow polydopamine particles.
A carrier for a drug delivery system comprising the polydopamine nanomotor according to claim 1 .

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