Polydopamine-based drug delivery system for tissue regeneration and manufacturing method therefor

Abstract

The present invention relates to a polydopamine-based drug delivery system for tissue regeneration and a manufacturing method therefor and, more particularly, to a novel drug delivery system in which a hydrophobic drug for regeneration is loaded on polydopamine nanoparticles having hydrophilicity to maximize bone regeneration efficacy.

Description

Polydopamine-based drug delivery system for tissue regeneration and method for manufacturing the same

[0001] The present invention relates to a drug delivery system, and more particularly, to a polydopamine-based drug delivery system for tissue regeneration.

[0002]

[0003] Tissue regeneration is essential for repairing damaged tissues and organs, healing diseased areas, and restoring organ function. Traditionally, tissue regeneration primarily relied on transplantation and biomaterials to replace damaged tissue. However, these approaches have resulted in limited outcomes, including infection, immune rejection, and the need for secondary surgery. To address these issues, drug-induced therapies that promote tissue regeneration in targeted areas are attracting attention.

[0004] 1,4-Dihydrophenontroline-4-one-3-carboxylic acid (1,4-DPCA) is a low-molecular-weight compound that acts as a proline hydroxylase (PHD) inhibitor and promotes tissue regeneration. The detailed mechanism is as follows: 1,4-DPCA inactivates PHD, inducing upregulation of Hif-1α instead of spontaneous degradation. This upregulation promotes erythropoiesis, metabolism, and angiogenesis, leading to tissue regeneration. In particular, several studies have reported that the regenerative effects of 1,4-DPCA, which are due to increased Hif-1α protein expression, are particularly effective in bone regeneration.

[0005] Furthermore, it has been revealed that these bone regeneration effects are accelerated by the unique properties of Hif-1α, including its inhibitory effect on inflammatory responses and its ability to regulate osteogenic differentiation. However, despite the effective regenerative properties of 1,4-DPCA, several issues remain to be addressed.

[0006] For example, its inherent hydrophobicity makes it difficult to use alone due to its low solubility in water. Furthermore, concerns about cytotoxicity arise when using high concentrations of 1,4-DPCA. To overcome this, hydrogel-based drug delivery systems (DDS) have been developed that chemically bind to 1,4-DPCA and release it through ester hydrolysis. However, controlled drug release is limited due to rapid degradation by in vivo enzymatic reactions. Therefore, the development of novel DDSs is essential to maximize the tissue regeneration effects of 1,4-DPCA.

[0007] Polydopamine (PDA), a representative synthetic polyphenol, is highly valued for its role as a versatile drug carrier. This ability stems from its unique chemical structure, which enables the efficient encapsulation of various chemical drugs. The catechol group of polydopamine facilitates various non-covalent interactions, including hydrogen bonding, electrostatic interactions, and π-π stacking. These interactions not only stabilize the encapsulated drug but also enhance the loading capacity of polydopamine, enabling the effective release of various therapeutic agents. This versatility makes polydopamine advantageous for the design of targeted drug delivery systems, offering potential improvements in drug solubility, stability, and controlled release profiles.

[0008] The efficacy of polydopamine extends beyond its role as a multipurpose drug carrier, and its ability to enhance tissue regeneration is also recognized. Similar to natural polyphenols, polydopamine can generate reactive oxygen species (ROS) and induce local oxidative stress. These ROS activate antioxidant gene expression by modulating the Nrf2 signaling pathway to maintain cellular homeostasis. Indeed, antioxidant gene expression contributes to alleviating inflammation and promoting cell proliferation. Polydopamine's activity plays a crucial role in cell regeneration. Therefore, maintaining adequate levels of hydrogen peroxide (H2O2) production may aid tissue regeneration. In this regard, polydopamine-based drug carriers that generate ROS could be particularly useful for tissue regeneration.

[0009]

[0010] The present invention has been devised to solve the above problems, and the purpose of the present invention is to provide a novel drug delivery system encapsulating 1,4-DPCA, a regenerative drug, in polydopamine, and a method for manufacturing the same.

[0011] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0012]

[0013] In order to achieve the above object, the present invention provides a drug delivery system comprising polydopamine nanoparticles; and a drug loaded inside the polydopamine nanoparticles, wherein the drug is a hydrophobic regenerative drug.

[0014] The above drug may be 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA).

[0015] The size of the above polydopamine nanoparticles may be 100 to 1000 nm.

[0016] In addition, the present invention provides a method for manufacturing a drug delivery system, comprising the steps of: preparing a drug mixture by dissolving a drug in a solvent mixed with ammonium hydroxide, ethanol, and deionized water; adding dopamine hydrochloride to the drug mixture; and stirring the mixture to which dopamine hydrochloride has been added to produce polydopamine nanoparticles loaded with the drug.

[0017] The above drug may be 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA).

[0018] 300 to 700 parts by weight of the dopamine hydrochloride may be added to 100 parts by weight of the drug.

[0019] In the step of manufacturing the polydopamine nanoparticles loaded with the drug by stirring the mixture to which the dopamine hydrochloride is added, the stirring can be performed at 100 to 500 rpm for 10 minutes to 40 hours.

[0020]

[0021] By means of solving the above problem, the present invention can provide a polydopamine-based drug delivery system for tissue regeneration and a method for manufacturing the same.

[0022] In addition, the present invention can maximize bone regeneration efficacy by loading a hydrophobic regenerative drug into hydrophilic polydopamine nanoparticles.

[0023] In addition, the polydopamine-based drug delivery system for tissue regeneration manufactured according to the present invention can maximize regenerative efficacy through the synergistic effect of polydopamine's generation of reactive oxygen species and sustained release of the drug.

[0024] In addition, the polydopamine-based drug delivery system according to the present invention can be applied not only to bone tissue but also to other tissues, and can be utilized as a new drug delivery system for a wide range of hydrophobic drugs requiring controlled release kinetics.

[0025]

[0026] The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.

[0027]

[0028] Figure 1 shows (A, B) scanning electron microscopy and (C, D) transmission electron microscopy images of polydopamine nanoparticles containing 1,4-DPCA.

[0029] Figure 2 shows the XPS analysis results. High-resolution N1s and O1s X-ray photoelectron spectroscopy (XPS) spectra of (A, B) polydopamine nanoparticles (PDA NPs), (C, D) polydopamine nanoparticles containing 1,4-DPCA (PDA w / DPCA NPs), and (E, F) 1,4-DPCA.

[0030] Figure 3 shows (A) the results of confirming drug release of PDA using 1,4-DPCA NPs, (B) a standard curve for calculating drug release using various concentrations of 1,4-DPCA, and (C) an in vitro drug release profile of PDA w / DPCA NPs. (au represents an arbitrary unit)

[0031] Figure 4 (A) is a schematic diagram of hydrogen peroxide analysis, and (B) and (C) show the quantified amounts of hydrogen peroxide generated using PDA NPs and PDA w / DPCA NPs in pH 7.4 PBS solution (n=7). (D) shows the relative gene expression of Nrf2, Gsta1, and Hmox1 in MC3T3-E1 cells treated with PDA NPs, PDA w / DPCA NPs, and 1,4-DPCA (n=3).

[0032] Figure 5 shows the results of evaluating the relative Hif-1α mRNA expression and protein levels in MC3T3-E1 cells treated with PDA NPs, PDA w / DPCA NPs, and 1,4-DPCA using (A) RT-qPCR and (B) ELISA (n = 6).

[0033] Figure 6 shows the results of in vitro biocompatibility evaluations. (A) shows in vitro cytotoxicity evaluated by the CCK-8 assay, and the bar graph shows the cell viability of cells treated with polydopamine nanoparticles (PDA NPs) and PDA NPs treated with 1,4-DPCA (PDA w / DPCA NPs) (n = 5). (B) shows the results of the in vitro hemolysis test of PDA w / DPCA NPs (n = 4).

[0034] Figure 7 shows the results of alkaline phosphatase (ALP) activity analysis of MC3T3-E1 cells treated with PDA NPs (PDA w / DPCA NPs) treated with 1,4-DPCA.

[0035] Figure 8 shows the results of in vivo tests in a mouse calvarial defect model. (A) is an evaluation schedule for evaluating the in vivo bone regeneration efficacy using a mouse calvarial defect model, (B) shows the percentage of new bone matrix within the calvarial defect site (the reported values ​​were calculated using MT staining images), (C) shows the average diameter of the residual defect at the site 4 weeks after surgery, and (D) shows micro X-ray images of the 3D reconstructed mandibular defect site for the no-treatment group, PDA NPs treatment group, and PDA w / DPCA NPs treatment group 4 weeks after surgery.

[0036] Figure 9 shows MT (Masson's trichrome) staining images and H&E (hematoxylin and eosin) staining images of the skull defect area. (A) is a representative MT staining image of the skull defect area in each group, and (B) is a representative H&E staining image of the skull bone defect cross-section obtained from animals in the untreated group, PDA NP, and PDA w / DPCA NP treated groups. Arrows indicate the defect area, and "ob" indicates old bone and "nb" indicates new bone.

[0037]

[0038] The terms used in this invention have been selected from widely used, current terms, taking into account the functions of the invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, in which case their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the invention.

[0039] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0040] When a part of a specification is said to “include” a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0041] Below, with reference to the attached drawings, embodiments of the present invention are described in detail so that those skilled in the art can easily implement them. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0042]

[0043] Hereinafter, the present invention will be described in detail.

[0044]

[0045] The present invention provides a drug delivery system comprising polydopamine nanoparticles; and a drug loaded inside the polydopamine nanoparticles, wherein the drug is a hydrophobic regenerative drug.

[0046] The drug may be 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA). 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA) is a low-molecular-weight compound that acts as a proline hydroxylase (PHD) inhibitor and can promote tissue regeneration. More specifically, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA) inactivates PHD, thereby inducing upregulation of Hif-1α instead of spontaneous degradation. This upregulation can induce tissue regeneration by promoting erythropoiesis, metabolism, and angiogenesis. The regenerative effect of 1,4-DPCA due to increased Hif-1α protein expression is particularly effective in bone regeneration.

[0047] The polydopamine described above acts as a versatile drug carrier, capable of efficiently encapsulating various chemical drugs. This is due to its unique chemical structure, where its catechol groups can promote various non-covalent interactions, including hydrogen bonding, electrostatic interactions, and π-π stacking. These interactions not only stabilize the encapsulated drugs but also enhance the loading capacity of polydopamine, enabling the effective release of various therapeutic agents. Furthermore, polydopamine can enhance tissue regeneration. Similar to natural polyphenols, polydopamine can generate reactive oxygen species (ROS) and induce local oxidative stress.

[0048] The drug delivery system according to the present invention can maximize bone regeneration efficacy by loading the hydrophobic regenerative drug into the polydopamine nanoparticles.

[0049] The size of the above polydopamine nanoparticles may be 100 to 1000 nm. Preferably, it may be 100 to 400 nm, but is not limited thereto.

[0050] In addition, the present invention provides a method for manufacturing a drug delivery system, comprising the steps of: preparing a drug mixture by dissolving a drug in a solvent mixed with ammonium hydroxide, ethanol, and deionized water; adding dopamine hydrochloride to the drug mixture; and stirring the mixture to which dopamine hydrochloride has been added to produce polydopamine nanoparticles loaded with the drug.

[0051] The above drug is a hydrophobic regenerative drug, preferably 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), but is not limited thereto.

[0052] For 1 part by volume of the ammonium hydroxide, 10 to 30 parts by volume of ethanol and 30 to 50 parts by volume of deionized water can be mixed. Preferably, 20 parts by volume of ethanol and 40 parts by volume of deionized water can be mixed for 1 part by volume of the ammonium hydroxide, but this is not limited thereto.

[0053] 300 to 700 parts by weight of the dopamine hydrochloride may be added relative to 100 parts by weight of the drug. Preferably, 400 to 600 parts by weight of the dopamine hydrochloride may be added relative to 100 parts by weight of the drug, and more preferably, 500 parts by weight of the dopamine hydrochloride may be added relative to 100 parts by weight of the drug, but is not limited thereto.

[0054] In the step of producing polydopamine nanoparticles loaded with the drug by stirring the mixture to which the dopamine hydrochloride is added, the stirring may be performed at 100 to 500 rpm for 10 minutes to 40 hours. Preferably, the stirring may be performed at 100 to 300 rpm for 10 minutes to 30 hours, but is not limited thereto.

[0055] After the step of manufacturing polydopamine nanoparticles loaded with the drug by stirring the mixture to which the above dopamine hydrochloride has been added, a step of centrifuging the polydopamine nanoparticles loaded with the drug can be additionally performed.

[0056]

[0057] Hereinafter, the present invention will be described in detail using examples. However, the examples according to the present invention may be modified in various ways, and the scope of the present invention is not limited to the examples described below. The examples in this specification are provided to more fully explain the present invention to those of ordinary skill in the art.

[0058]

[0059] Manufacturing example. Synthesis of 1,4-DPCA

[0060] 8-Aminoquinoline (1.6 g, 11.1 mmol) and diethyl ethoxymethylene malonate (2.36 mL, 11.7 mmol) were heated to 100 °C for 1 h. Diphenyl ether (33 mL) was then added, and the mixture was refluxed at 250 °C for 2 h and then cooled to room temperature. The precipitated product was separated by centrifugation, washed with 10 mL of diethyl ether, and then washed twice with 5 mL of diethyl ether.

[0061]

[0062] Example 1. Preparation of polydopamine nanoparticles loaded with 1,4-DPCA (PDA w / DPCA NPs)

[0063] The drug 1,4-DPCA (0.01 g) was dissolved in a cosolvent consisting of ethanol (4 mL) and deionized water (8 mL) to which 0.2 mL of 25% ammonium hydroxide (NH4OH) was added. Then, 0.05 g of dopamine hydrochloride was dissolved in 1 mL of deionized water and injected into the 1,4-DPCA mixture. 1,4-DPCA-loaded polydopamine nanoparticles (PDA w / DPCA NPs) with an average size of 172 nm were prepared by constant stirring at 200 rpm for 40 min. The nanoparticles were collected by centrifugation at 8000 rpm for 5 min. The process of washing with deionized water and centrifugation was repeated three times. The collected nanoparticles were lyophilized and stored frozen for future use.

[0064]

[0065] Example 2. Preparation of polydopamine nanoparticles loaded with 1,4-DPCA (PDA w / DPCA NP)

[0066] The same procedure as in Example 1 was followed, except that 1,4-DPCA-loaded polydopamine nanoparticles (PDA w / DPCA NPs) with an average size of 282 nm were prepared by stirring for 24 hours.

[0067]

[0068] The product (2.00 g, 7.46 mmol) was combined with 40 mL of 10% (w / v) KOH, refluxed (110 °C) for 1 h, cooled to room temperature, and then the residual diphenyl ether was extracted using 28 mL of petroleum ether (boiling point 80 °C - 110 °C). The product was precipitated with 40 mL of 10% (w / v) HCl, filtered, washed with deionized water, and dried overnight under high vacuum conditions.

[0069]

[0070] Comparative Example 1. Synthesis of polydopamine nanoparticles (PDA NPs)

[0071] A co-solvent system consisting of deionized water (8 mL), ethanol (4 mL), and 25% NH4OH 0.2 mL was used as the reaction solution. Dopamine hydrochloride (0.05 g) dissolved in deionized water (1 mL) was slowly added to the mixed solution. The mixture was reacted for 24 h with constant stirring (200 rpm), and then centrifuged at 8,000 rpm for 5 min to collect polydopamine nanoparticles (PDA NPs). The process of washing with deionized water and centrifuging at 8,000 rpm for 5 min was repeated three times. The prepared polydopamine nanoparticles (PDA NPs) were freeze-dried and stored frozen before use.

[0072]

[0073] Experimental Example 1. SEM and TEM Analysis

[0074] The morphology (i.e., surface and nanostructural characteristics) of DPCA-loaded polydopamine nanoparticles (PDA w / DPCA NPs) was observed using scanning electron microscopy (SEM) (SU8220 & SU8230, Hitachi, Tokyo, Japan) and transmission electron microscopy (TEM) (Tecnai 12, Phillips, Amsterdam, Netherlands).

[0075] Referring to Fig. 1, SEM and TEM analysis results confirmed that the DPCA-loaded polydopamine nanoparticles had a round shape.

[0076]

[0077] Experimental Example 2. XPS Analysis

[0078] The elemental composition of the synthesized nanoparticles was analyzed by X-ray photoelectron spectroscopy (XPS) (Thermo Fisher, Nexsa, MA, USA). For sample preparation, double-sided copper tape was attached to a silicon wafer, and the sample was firmly positioned and dried with nitrogen.

[0079] Since 1,4-DPCA has a different ratio of chemical bonds (N1 and O1) compared to PDA, the XPS spectrum of PDA NPs randomly incorporated with 1,4-DPCA is expected to show a distinct difference from the spectrum of pure PDA NPs. However, according to the XPS analysis results (Fig. 2 and Table 1), the ratios of each chemical bond of PDA NPs incorporated with 1,4-DPCA were similar to those of pure PDA NPs. For example, in the high-resolution N1s XPS spectra, the ratios (%) of the -N=C-, -NH-, and -NH2 peaks of PDA NPs and PDA w / DPCA NPs were 23%, 58%, and 20%, and 25%, 57%, and 18%, respectively. In contrast, the ratios (%) of the -N=C-, -NH-, and -NH2 peaks of 1,4-DPCA were 53%, 25%, and 22%, respectively. Similarly, the ratios (%) of O=C and OC peaks in the high-resolution O1s XPS spectra of PDA NPs and PDA w / DPCA NPs were 92% and 8%, and 93% and 7%, respectively. For 1,4-DPCA, the ratios (%) of O=C and OC peaks were 59% and 41%, respectively. Since XPS analysis typically probes tens of nanometers below the surface and cannot reach the core of PDA NPs, it can be inferred that 1,4-DPCA is mainly located within the core of the nanoparticles.

[0080]

[0081] N1sO1s-N=C--NH--NH2O=CO-CPDA NPs (%)235820928PDA w / DPCA NPs (%)2557189371,4-DPCA (%)5325225941

[0082]

[0083] Experimental Example 3. In vitro drug release test

[0084] To evaluate the in vitro drug release rate, 3 mg of lyophilized PDA w / DPCA NPs with a particle size of 282 nm were injected into a conical tube containing 3 mL of 1X phosphate-buffered saline (1X PBS), pH 7.4. The samples were allowed to release 1,4-DPCA over a period of 1 h to 31 days. After each release interval, centrifugation was performed to separate the supernatant and perform HPLC analysis. The HPLC analysis was performed using a mobile phase of acetonitrile and water (30 / 70; v / v) at a constant flow rate of 1 mL / min. The concentration of the released drug at each time point was quantified by comparison with a standard curve of 1,4-DPCA.

[0085] First, the successful loading of the drug into the synthesized drug delivery system (DDS) was confirmed by HPLC. 1,4-DPCA was released from PDA w / DPCA NPs at a concentration of 50 μg / mL in 1X PBS solution (pH 7.4) for 1 h, and the resulting supernatant was analyzed by HPLC. The peak appeared at the same retention time as 1,4-DPCA, confirming that the regenerated drug was successfully loaded into the PDA NPs (Fig. 3A).

[0086] The drug release profile of PDA w / DPCA NPs was further verified by HPLC analysis using a standard curve of 1,4-DPCA (Fig. 3B). To this end, the experimental drug loading efficiency of PDA w / DPCA NPs was first measured, and it was found to be approximately 10 wt%. Based on this, the amount of drug released from the total drug loaded into PDA w / DPCA NPs was calculated. As a result, PDA w / DPCA NPs steadily released 9.8 wt% of the loaded drug over 7 days (168 h, Fig. 3C).

[0087]

[0088] Experimental Example 4. Evaluation of reactive oxygen species (ROS) production and cellular antioxidant response activation by DDS.

[0089] Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), can be generated as byproducts from the drug carrier polydopamine through the autooxidation of the catechol moiety. To demonstrate that polydopamine generates H2O2, a peroxide assay was performed to quantify the generated H2O2 (Fig. 4A).

[0090] The concentration of ROS (i.e., hydrogen peroxide) generated from 1,4-DPCA-loaded polydopamine nanoparticles (PDA w / DPCA NPs) and control polydopamine (PDA) was quantified according to the instructions of the quantitative peroxide assay kit (Thermo Fisher Scientific, MA, USA). Samples were prepared at various concentrations (10, 25, and 50 μg / mL) in 1X PBS solution (pH 7.4). Each solution mixture was incubated at room temperature for 20 min and the absorbance was measured at 595 nm. Measurements were performed using a microplate reader (Infinite 200 Pro, Tecan, Menedorff, Switzerland). A standard curve for calculation was generated by measuring the absorbance at the same wavelength (595 nm) with 0.001–1 mM H2O2 solutions.

[0091] The results of the analysis showed that polydopamine nanoparticles (PDA NPs) generated 9.3, 9.0, and 10.6 μM of H₂O₂ at 10, 25, and 50 μg / mL, respectively (Fig. 4B). In contrast, 1,4-DPCA-loaded polydopamine nanoparticles (PDA w / DPCA NPs) generated approximately twice as much H₂O₂ at the same concentrations, 21.1, 26.3, and 29.5 μM (Fig. 4C). In the present invention, we hypothesized that the electron-donating amine group of 1,4-DPCA promotes catechol oxidation during the oxidation of the catechol moiety in polydopamine, thereby increasing H₂O₂ production.

[0092] ROS generated from polydopamine activate the Nrf2 / HO-1 signaling pathway by inducing appropriate levels of oxidative stress at target sites. This leads to the dissociation of Nrf2 from Keap1, translocation to the nucleus, binding to sMaf protein, and activation of the ARE, thereby promoting the expression of antioxidant genes such as Hmox1 and Gsta1. To confirm this, the expression of Nrf2, Hmox1, and Gsta1 genes in each condition was analyzed by qPCR. There was no significant difference in Nrf2 mRNA levels when MC3T3-E1 cells were treated with PDA NPs, PDA w / DPCA NPs, or 1,4-DPCA (Fig. 4D), indicating that none of the components of DDS affected basal Nrf2 gene expression.

[0093] In contrast, the mRNA levels of Hmox1 and Gsta1, which are Nrf2 target genes, showed a significant increase in the groups treated with PDA NPs and PDA w / DPCA NPs. Specifically, the Hmox1 mRNA level increased 4.5-fold in the PDA NPs-treated group and 7.4-fold in the PDA w / DPCA NPs-treated group, compared to a 1.1-fold increase in the 1,4-DPCA-treated control group (Fig. 4D). Similarly, the Gsta1 mRNA level increased 1.5-fold in the PDA NPs-treated group and 5.2-fold in the PDA w / DPCA NPs-treated group, whereas it decreased 0.63-fold in the 1,4-DPCA-treated group (Fig. 4D). These results suggest that treatment with PDA NPs and PDA w / DPCA NPs successfully enhanced the expression of Hmox1 and Gsta1, Nrf2 target genes involved in antioxidant responses. This suggests that PDA NPs and PDA w / DPCA NPs induce ROS production, thereby activating the Nrf2 pathway and enhancing the cellular antioxidant defense mechanism. Considering the superior ROS production and subsequent upregulation of antioxidant gene expression observed with PDA w / DPCA NPs compared to PDA NPs, it suggests that the combination of PDA and DPCA more effectively activates the Nrf2 pathway and promotes tissue regeneration.

[0094]

[0095] Experimental Example 5. Real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA)

[0096] To investigate whether 1,4-DPCA released from polydopamine nanoparticles contributes to the upregulation of Hif-1α mRNA and protein levels, MC3T3-E1 cells were treated with PDA w / DPCA NPs for 3 days, and RT-qPCR and ELISA analyses were performed. These results were compared with the control group treated with PDA NPs and 1,4-DPCA.

[0097] For RT-qPCR analysis, MC3T3-E1 cells were cultured in Alpha MEM medium supplemented with 10% FBS and 1% penicillin-streptomycin antibiotics at 37°C in a 5% CO2 incubator. Total RNA was isolated from the cells using TRIzol™ reagent (Invitrogen, MA, USA) and prepared for complementary DNA synthesis using the PrimeScript 1-Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). Gene expression was measured by RT-qPCR analysis using AccuPower®2X GreenStar™ qPCR Master Mix (BIONEER, Daejeon, South Korea) on a CFX Duet Real-Time PCR System (Bio-rad, CA, USA). Gene expression levels were normalized using the beta-actin housekeeping gene.

[0098] For ELISA analysis, MC3T3-E1 cells were thawed and adapted by culturing in T-75 flasks for three cycles, then dissociated with 0.25% trypsin-EDTA (Thermo Fisher Scientific, MA, USA) and counted using 0.4% trypan blue (Sigma-Aldrich, USA). Cells were seeded at 2.2 × 106 cells per well in 100-mm dishes and cultured in α-MEM medium for 2 days. Afterwards, the medium was replaced with special medium containing 50 μg / mL L-ascorbic acid 2-phosphate and 10 mM β-glycerophosphate to induce osteogenic differentiation. Differentiated cells were lysed in RIPA buffer (Elpis Biotech, Daejeon, Korea), cellular proteins were extracted, and quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Quantification of Hif-1α in MC3T3-E1 cells was performed using the Human / Mouse Total HIF-1α / HIF1A Kit (R&D Systems, MN, USA) at 100 μg of total protein per well. Absorbance was measured at 450 nm with compensation at 540 nm using a microplate reader (Infinite 200 Pro, Tecan, Menedorff, Switzerland).

[0099] Our analysis revealed that treatment with PDA NPs or PDA w / DPCA NPs did not show a statistically significant difference in Hif-1α mRNA expression levels compared to the control group. However, treatment with 1,4-DPCA significantly increased Hif-1α mRNA expression by 1.6-fold (Fig. 5A). Treatment with PDA NPs and PDA w / DPCA NPs significantly increased Hif-1α protein levels by 1.6-fold and 1.8-fold, respectively (Fig. 5B), whereas 1,4-DPCA treatment only increased Hif-1α protein levels by 1.2-fold. These results suggest that PDA NPs and PDA w / DPCA NPs can initially promote a transient upregulation of Hif-1α mRNA, which may not be detected at the 3-day time point due to rapid mRNA degradation. Interestingly, despite the fact that DPCA loaded into PDA w / DPCA NPs theoretically reduced Hif-1α protein levels by approximately one-tenth compared to the control group (1,4-DPCA alone), the effect on Hif-1α protein levels was significantly greater. These results highlight the effective drug delivery capacity of DDS for Hif-1α expression.

[0100] It is also noteworthy that PDA itself significantly increased HIF-1α protein expression compared to the control group. This may be due to ROS (e.g., H2O2) generated by PDA, which induces HIF-1α stabilization by mediating the dimerization of proline hydroxylase (PHD) via disulfide bonds. This result suggests that the highest HIF-1α protein overexpression was observed in the PDA w / DPCA NP-treated sample due to the synergistic effect of ROS and PHD inhibition by 1,4-DPCA.

[0101]

[0102] Experimental Example 6. In vitro biocompatibility evaluation

[0103] Cytotoxicity test

[0104] To evaluate the biocompatibility of PDA NPs and PDA w / DPCA NPs, cytotoxicity assays were performed using the CCK-8 assay. The cytotoxicity of synthesized 1,4-DPCA, polydopamine nanoparticles (PDA NPs), and DPCA-loaded polydopamine nanoparticles (PDA w / DPCA NPs) with a particle size of 282 nm were evaluated using the CCK-8 assay. Mouse fibroblast L929 cells were seeded at a density of 1 × 10 per well in 96-well cell culture plates (Costar, USA). 4 Cells were seeded at a density of 100 μL per well. They were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS and 1% penicillin-streptomycin, incubated at 37°C in a 5% CO2 atmosphere. After 24 h of incubation, various compounds (10, 25, and 50 μg / mL) were added to the cells. Samples treated with DMEM and DMSO alone served as controls. After 24 h of incubation, 10 μL of CCK-8 solution was added to each well, incubated for an additional 3 h, and then the absorbance was measured at 450 nm using a microplate reader (Infinite 200 Pro, Tecan, Mannedorf, Switzerland). Cell viability was calculated according to the following formula:

[0105] Normalized cell viability (%) = Absorbance of cells with compound / Absorbance of cells without compound × 100

[0106]

[0107] All samples demonstrated 80% cell viability at concentrations up to 25 μg / mL and were confirmed to be non-toxic (Figure 6A). These results indicate that the drug carrier according to the present invention is biocompatible and meets the biocompatibility standards of ISO 10993-5.

[0108]

[0109] Hemolysis test

[0110] To verify the injectability of the developed DDS, the hemolytic activity of PDA w / DPCA NPs with a particle size of 282 nm was analyzed to determine the toxicity level at which hemoglobin is ruptured when it comes into contact with human blood.

[0111] For hemolysis assays, EDTA-stabilized single-donor human whole blood was purchased from Innovative Research (November, MI) and centrifuged at 500 × g for 5 min. The pellet was washed with 150 mM NaCl solution and 1 × PBS buffer (pH 7.4) and then resuspended in a series of buffers representing the pH range from physiological (pH 7.4) to late endolysosomal (pH 6.2). Each well of a 96-well plate was seeded with 190 μL of red blood cell (RBC) suspension. Subsequently, 10 μL of various concentrations of DDS prepared in DPBS was added to each well. The plates were then incubated at 37°C for 1 h. The 96-well plate was then centrifuged at 500 × g for 5 min to pellet the RBCs. Hemoglobin absorbance of the supernatant was measured at 590 nm using a microplate reader (Infinite 200 Pro M Plex, Tecan, Menedorff, Switzerland). Considering that other cytoplasmic components extracted from red blood cells, such as proteins and carbohydrates, in addition to hemoglobin, may contain small amounts of signal that interferes with spectrophotometric measurements, 100% hemolysis was corrected for with red blood cell lysates treated with Triton X-100. The percentage of red blood cell hemolysis was calculated using the following formula:

[0112]

[0113] Hemolysis(%)={(absorbance of sample)-(absorbance of negtive control)}÷{(absorbance of positive control)-(absorbance of negtive control)}×100

[0114]

[0115] (A hemolysis percentage value of less than 2% indicates that the test sample is not hemolytic; between 2% and 5% indicates that the test sample is slightly hemolytic; and greater than 5% indicates that the test sample is hemolytic.)

[0116]

[0117] Hemolysis test results showed that the hemolysis rate increased with increasing concentration of PDA w / DPCA NPs, and the hemolysis level also increased slightly in the pH range (pH 6.2). The highest concentration of 50 μg / mL caused approximately ~1.9% hemolysis across the entire pH range. The remaining concentrations of 10 and 20 μg / mL showed ~1% hemolysis across the entire pH range (Figure 6B). According to the ASTM E2524-08 standard, a hemolysis rate exceeding 5% indicates that the test substance causes damage to red blood cells. The hemolysis test results showed that the hemolysis rate was less than 5% across the entire concentration range, demonstrating the safety and injectability of the substance.

[0118]

[0119] Experimental Example 7. In vitro and in vivo studies of bone regeneration using DDS.

[0120] Alkaline phosphatase activity (ALP) assessment

[0121] To demonstrate the in vitro bone regeneration effect of the developed DDS, ALP activity was assessed in MC3T3-E1 cells treated with PDA w / DPCA NPs. ALP activity is a key indicator of early osteoblast differentiation and mineralization for bone development. Therefore, ALP activity can confirm active bone formation in osteoblasts and / or osteoblasts.

[0122] MC3T3-E1 cells were seeded in 96-well cell culture plates at a density of 1 × 104 cells / well. After 24 h, the culture medium containing 10% FBS and 1% penicillin-streptomycin antibiotics was replaced with osteogenic medium (OSM). OSM was prepared by adding 10 mM β-glycerol phosphate and 50 μg / mL L-ascorbic acid 2-phosphate to the culture medium. PDA w / DPCA NPs at a concentration of 10 μg / mL were added to the medium and mixed with OSM. The cells were cultured for 1, 3, and 7 days, with the medium replaced with OSM (control) or OSM containing PDA w / DPCA NPs. After 1, 3, and 7 days, the cells were washed with DPBS and lysed with lysis buffer. Absorbance was measured at a wavelength of 405 nm using a microplate reader (Infinite 200 Pro M Plex, Tecan, Menedorff, Switzerland) and an ALP kit (Sigma-Aldrich, St. Louis, MO, USA) according to the following formula:

[0123]

[0124] ALP(IU / L or umol / (L·min))={(0D T50 - OD T0 )×RxnVol×35.3}÷{(0D cal - 0D Blank )×SmplVol×T}

[0125]

[0126] (OD here T50 = OD value at 405 nm of 50-minute sample, OD T0 = OD value at 405 nm of 0 minute sample, OD Cal = OD value at 405 nm of the calibrator solution included in the kit, OD Blank = OD value at 405 nm in deionized water, RxnVol = total reaction volume (200 μL), T = reaction time (50 min), SmplVol = amount of sample used in the reaction (50 μL))

[0127]

[0128] ALP activity was monitored for 1, 3, and 7 days in MC3T3-E1 cells treated with PDA w / DPCA NPs at a concentration of 10 μg / mL, and the results are shown in Fig. 7. Referring to Fig. 7, changes in ALP activity in MC3T3-E1 cells treated with PDA w / DPCA NPs and then differentiated were evaluated, and it was observed that bone activity was enhanced in all groups compared to the untreated control group according to the differentiation period. Specifically, ALP activity increased 1.2-fold on day 1, 1.8-fold on day 3, and 1.1-fold on day 7 compared to the control group. These results suggest that the designed drug carrier promotes bone formation and osteoblast differentiation in MC3T3-E1 cells.

[0129]

[0130] In vivo testing using a mouse calvarial defect model

[0131] To evaluate the in vivo bone regeneration efficacy of the optimized DDS, PDA w / DPCA NPs were applied to a mouse calvarial defect model and the results were compared with the control group.

[0132] The experimental mice were housed in separate cages and allowed to acclimate to the animal environment for 7 days before surgery. The mice were anesthetized with an intraperitoneal injection of ketamine (75 mg / kg body weight). A sagittal skin incision was made on the scalp from the frontal to the occipital bone, and the periosteum was included in the skin flap. A full-thickness circular defect (5 mm in diameter) was created in the left parietal bone using a 5-mm diamond-coated trephine bur (Cogoogon, China) with a slow dental handpiece (Marathon-N3, SMT, China). An untreated empty skull defect served as a negative control. The experimental groups included groups treated with PDA NPs or PDA w / DPCA NPs with a particle size of 282 nm. The drug loading concentration was 10 μg / mL. To minimize the risk of drug diffusion to other sites, the solution was applied dropwise to the skull defect side to allow accumulation at the damaged area. Considering that PDA NPs readily aggregate, the solution was sonicated in a water bath before use. The incision was closed with 5-0 Catgood suture (Ethicon), and the rats were kept warm using a heat lamp until they fully recovered. The animals were returned to their cages and monitored daily for complications or abnormal behavior.

[0133] The experimental timeline for evaluating the in vivo bone regeneration efficacy is shown in Figure 8A using a mouse calvarial defect model. Overall, treatment with PDA and DPCA NPs resulted in complete filling of the defect site with new bone, which became denser, more uniform in shape, and more stable, closely resembling the surrounding old bone. In contrast, the control group exhibited disorganized fibrous connective tissue in the defect site. Quantitative results (Figures 8B and 8C) calculated from the Masson's trichrome (MT) stained image (Figure 8A) showed that the untreated group had minimal bone tissue and collagen. However, the group treated with PDA NPs showed enhanced bone regeneration, as evidenced by increased expression of antioxidant genes that induce anti-inflammatory effects. This was further enhanced in the group treated with PDA w / DPCA NPs, which exhibited the highest rate of new bone formation in the calvarial defect site, as confirmed by Masson's trichrome (MT) and hematoxylin and eosin (H&E) staining (Figures 9A and 9B). The PDA w / DPCA NPs treatment group showed an approximately 6-fold increase in bone formation compared to the untreated group, indicating that the synergistic effect of the regenerative drug (1,4-DPCA) and the ROS-generating drug carrier (PDA NPs) significantly enhanced bone formation.

[0134] Furthermore, using 3D-reconstructed micro-X-ray images, we analyzed the whole skull 4 weeks after surgery to confirm the induction of new bone formation in the skull defect area (Fig. 8D). No bone formation was observed in the untreated control group. In contrast, new bone formation was detected in the center of the defect in the group treated with PDA NPs. The group treated with PDA w / DPCA NPs showed noticeably more extensive bone formation in a wider area in the center of the defect compared to the group treated with PDA NPs. This suggests that newly formed bone cells are gradually assembling to close the defect area. This observation is consistent with the MT and H&E staining results (Figs. 9A and 9B). The above results clearly demonstrate the efficacy of DDS designed for bone tissue regeneration.

[0135]

[0136] We have discussed specific embodiments of the present invention. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. Polydopamine nanoparticles; and Contains a drug loaded inside the above polydopamine nanoparticles, A drug delivery system characterized in that the above drug is a hydrophobic regenerative drug.

2. In paragraph 1, A drug delivery system characterized in that the drug is 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA).

3. In paragraph 1, A drug delivery system characterized in that the size of the above polydopamine nanoparticles is 100 to 1000 nm.

4. A step of preparing a drug mixture by dissolving the drug in a solvent mixed with ammonium hydroxide, ethanol, and deionized water; A step of adding dopamine hydrochloride to the above drug mixture; and A method for manufacturing a drug delivery system, characterized in that it comprises a step of manufacturing polydopamine nanoparticles loaded with the drug by stirring a mixture to which the dopamine hydrochloride is added.

5. In paragraph 4, A method for manufacturing a drug delivery system, characterized in that the drug is 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA).

6. In paragraph 4, A method for manufacturing a drug delivery system characterized by adding 300 to 700 parts by weight of the dopamine hydrochloride to 100 parts by weight of the drug.

7. In paragraph 4, A method for manufacturing a drug delivery system, characterized in that in the step of manufacturing polydopamine nanoparticles loaded with the drug by stirring the mixture to which the dopamine hydrochloride is added, the stirring is performed at 100 to 500 rpm for 10 minutes to 40 hours.

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