KR102368479B1 - Nanoparticle structure and method for forming the same - Google Patents

Abstract

A nanoparticulate structure and a method of forming the same are provided. The nanoparticle structure includes a porous support and nanoparticles coupled to the porous support, wherein the nanoparticles include manganese ferrite nanoparticles and ceria nanoparticles. The method of forming the nanoparticulate structure includes forming a porous support and nanoparticles, and bonding the porous support and the nanoparticles, wherein the nanoparticles include manganese ferrite nanoparticles and ceria nanoparticles.

Description

NANOPARTICLE STRUCTURE AND METHOD FOR FORMING THE SAME

The present invention relates to a nanoparticulate structure and a method for forming the same.

Rheumatoid arthritis is an inflammatory autoimmune disease that causes long-term synovitis and joint disorders. Although disease modifying anti-rheumatic drugs (DMARDs) are the main treatment for rheumatoid arthritis, side effects do not provide long-term disease relief.

The present invention provides a nanoparticle structure for the treatment of diseases.

The present invention provides a method for forming the nanoparticle structure.

Other objects of the present invention will become apparent from the following detailed description and accompanying drawings.

Nanoparticle structures according to embodiments of the present invention include a porous support and nanoparticles coupled to the porous support, wherein the nanoparticles include manganese ferrite nanoparticles and ceria nanoparticles.

The porous support may include mesoporous silica nanoparticles.

The nanoparticulate structure may further include a drug loaded on the porous support. The drug may include an arthritis drug.

The nanoparticulate structure can be injected into the human body to remove pro-inflammatory M1 macrophages and induce anti-inflammatory M2 macrophages.

The nanoparticle structure may be injected into the human body to remove reactive oxygen species and generate oxygen.

A method of forming a nanoparticulate structure according to embodiments of the present invention includes forming a porous support and nanoparticles, and combining the porous support and the nanoparticles, wherein the nanoparticles are manganese ferrite nanoparticles and ceria including nanoparticles.

The porous support may include mesoporous silica nanoparticles.

The method of forming the nanoparticulate structure may further include loading a drug onto the porous support.

The porous support is amine-functionalized, the nanoparticles are BMPA (2-Bromo-2-methylpropionic acid)-capped, and the bonding of the porous support and the nanoparticles includes, the amine-functionalized porous support and mixing the BMPA-capped nanoparticles.

Nanoparticle structures according to embodiments of the present invention may be injected into a living body and effectively used to treat diseases.

1 shows the treatment mechanism of MFC-MSN for rheumatoid arthritis.
2 shows a TEM image of MFC-MSN.
3 shows STEM images and EDX mapping images of MFC-MSN showing the conjugation of manganese ferrite nanoparticles and ceria nanoparticles in MSN.
4 shows the N ₂ adsorption/desorption isotherm of MFC-MSN, and the inset shows the pore size distribution of MFC-MSN.
5 shows UV-Vis spectra of RITC-labeled MSN and MFC-MSN.
6 shows the hydrodynamic size distribution according to the number of MSNs and MFC-MSNs.
7 shows H ₂ O ₂ decomposition curves in the presence of MF-MSN, C-MSN and MFC-MSN at pH 7.4.
8 shows O ₂ production curves in 1M H ₂ O ₂ solution in the presence of MF-MSN, C-MSN and MFC-MSN under physiological conditions.
9 shows an image in which O ₂ is generated in the presence of MF-MSN, C-MSN, and MFC-MSN.
10 shows the SOD activity of MF-MSN, C-MSN and MFC-MSN.
11 shows time-dependent hydroxyl radical generation of MF-MSN, C-MSN and MFC-MSN in the presence of H ₂ O ₂ .
12 shows the mechanism of synergistic O ₂ production and reactive oxygen species removal of MFC-MSN.
13 shows CLSM images showing cellular internalization of RITC-labeled MFC-MSN in murine BMDM.
14 shows BMDM viability as determined by MTS assay after incubation with C-MSN, MF-MSN or MFC-MSN at various concentrations for 24 hours.
15 shows CLSM images in BMDM incubated with MF-MSN, C-MSN or MFC-MSN in the presence of H ₂ O ₂ in the medium and the corresponding fluorescence intensity of intracellular H ₂ O ₂ (green).
16 shows HIF-1α (green) of BMDMs pretreated with MF-MSN, C-MSN or MFC-MSN for 4 hours and then incubated for 4 hours under hypoxic (1% O ₂ ) and inflammatory (+LPS) conditions. indicates dyeing.
17 shows protein expression of HIF1-α, M1 (iNOS, IL-1β and Cox-2) and M2 (Arg-1) macrophage markers in BMDM under various conditions.
18 shows mRNA expression of M1 and M2 macrophage markers in BMDM under various conditions.
19 shows optoacoustic images showing sO ₂ (red) in the knee joint before and after intra-articular injection of MFC-MSN.
20 shows CLSM images showing the uptake of MFC-MSN by synovial macrophages labeled with macrophage-specific F4/80 antibody in the knee joint 3 days after injection.
Figure 21 shows the inhibition of HIF-1α (red) expression in the knee joint synovial membrane and femur 30 days after injection of MFC-MSN.
22 shows immunohistochemical analysis of M1 (Cox-2; red) and M2 (CD206; green) macrophage markers in the synovial membrane 30 days after injection.
23 shows the results of qRT-PCR analysis of HIF-1α, M1 and M2 macrophage markers in synovial tissue 30 days after injection.
24 shows thermal image images of the left hind paw at various time points after treatment in a rheumatoid arthritis induced rat model.
Figure 25 shows the paw temperature of the left hind paw at various time points after treatment in a rheumatoid arthritis induced rat model.
26 shows paw width at various time points after treatment in a rheumatoid arthritis induced rat model.
27 shows the time required to traverse a 70 cm beam at various time points after treatment in a rheumatoid arthritis induced rat model.
28 shows histological and immunohistochemical evaluation of rheumatoid arthritis knee joints after 30 days of treatment in a rheumatoid arthritis induced rat model.

Hereinafter, the present invention will be described in detail through examples. Objects, features, and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the embodiments described herein, and may be embodied in other forms. The embodiments introduced here are provided so that the disclosed content can be thorough and complete, and so that the spirit of the present invention can be sufficiently conveyed to those of ordinary skill in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

Insufficiency of O ₂ supply to infiltrating immune cells in the cavity synovial membrane of rheumatoid arthritis (RA) upregulates hypoxia-inducing factor (HIF-1α) expression and induces reactive oxygen species (ROS) production Thus, both exacerbate synovial inflammation. Synovial inflammation in rheumatoid arthritis can be addressed by eliminating pro-inflammatory M1 macrophages and inducing anti-inflammatory M2 macrophages.

Nanoparticle structures according to embodiments of the present invention include manganese ferrite and ceria nanoparticle-anchored mesoporous silica nanoparticles (MFC-MSN).

MFC-MSN can synergistically scavenge reactive oxygen species and generate O ₂ to reduce M1 macrophage levels and induce M2 macrophages for the treatment of rheumatoid arthritis.

MFC-MSN exhibits a synergistic effect on O ₂ production and removal of reactive oxygen species. This leads to efficient polarization of M1 vs. M2 macrophages both in vitro and in vivo, while eliminating the intermediate hydroxyl radicals generated by manganese ferrite nanoparticles during O ₂ generation during Fenton reaction of ceria nanoparticles. due to a complementary reaction.

Intra-articular administration of MFC-MSN to a murine rheumatoid arthritis model alleviated joint hypoxia, inflammation and pathological features. Mesoporous silica nanoparticles (MSN) were used as drug delivery vehicles to continuously release the rheumatoid drug methotrexate to enhance the therapeutic effect of MFC-MSN.

Although the pathogenesis of rheumatoid arthritis has not been clearly elucidated, the mechanism involves an unwanted immune response that attacks the joint. Inflammatory cells, including macrophages, dendritic cells, T and B lymphocytes, neutrophils and natural killer cells, infiltrate the rheumatoid arthritis joint synovial membrane and contribute to severe destruction of joint bone and cartilage. In particular, macrophages play a key role in the pathophysiological response in rheumatoid arthritis.

Macrophages exist in two different phenotypes: a pro-inflammatory (M1) phenotype and an anti-inflammatory (M2) phenotype. M1 macrophages dominate in the early stages of inflammation and induce inflammation by secreting pro-inflammatory cytokines such as IL-1β and TNF-α. In contrast, M2 macrophages secrete anti-inflammatory cytokines and reduce inflammation. The macrophages of the rheumatoid arthritis joint are mainly of the M1 phenotype and promote the progression of rheumatoid arthritis by releasing various inflammatory cytokines.

Hypoxia-inducing factor (HIF-1α) expression and reactive oxygen species (ROS) in rheumatoid arthritis joints induce synovial inflammation and affect the balance of macrophage subtypes. Hypoxia is mainly induced in the rheumatoid arthritis synovial membrane through fibroblast-like synovial hyperplasia and increased oxygen demand of infiltrated inflammatory cells. Hypoxia upregulates HIF-1α in rheumatoid arthritis, which induces polarization of macrophages to the M1 subtype through reactive oxygen species production or other pathways. Therefore, inhibition of HIF-1α can attenuate the M1 phenotype, enhance the M2 polarization of macrophages, and effectively ameliorate rheumatoid arthritis.

Ceria nanoparticles can induce M2 polarization of macrophages by eradicating reactive oxygen species. Simultaneous inhibition of HIF-1α and reactive oxygen species shifts the phenotypic transition of pro-inflammatory M1 macrophages to the anti-inflammatory M2 subtype for the treatment of rheumatoid arthritis. Manganese ferrite nanoparticles down-regulate hypoxic markers such as HIF-1α by alleviating hypoxic conditions through the Fenton reaction. Ceria nanoparticles can scavenge reactive oxygen species for various inflammatory diseases.

The catalytic properties of ceria nanoparticles are due to the presence of two reversible oxidation states (Ce ³⁺ or Ce ^{4 +} ) on the surface of the nanoparticles. However, high concentrations of nanoparticles can cause cytotoxicity. Excessive concentration of manganese ferrite nanoparticles can lead to a decrease in cytotoxicity and oxygen production efficiency because hydroxyl radicals are generated as intermediates of the Fenton reaction. Since ceria nanoparticles can interfere with clinical translation with regard to their potential cytotoxicity, there is a need in biomedical applications to reduce nanoparticle dose by increasing efficiency. Manganese ferrite nanoparticles and ceria nanoparticles exhibit a synergistic effect on O ₂ production through efficient removal of intermediate reactive oxygen species, thereby inducing M2 polarization of macrophages even at relatively low nanoparticle concentrations.

MFC-MSN efficiently scavenges reactive oxygen species and generates O2 for M2 polarization of macrophages in a rheumatoid arthritis model (FIG. 1). In an in vitro experiment using bone marrow-derived macrophages (BMDM), inhibition of HIF-1α expression and M2 polarization were observed after treatment with MFC-MSN. In addition, methotrexate (MTX), a DMARD, was loaded into MSN for sustained release and efficient treatment of rheumatoid arthritis. MTX-loaded MFC-MSN showed great therapeutic potential in an in vivo rheumatoid arthritis mouse model.

Preparation and Characterization of MFC-MSN

MFC-MSN is formed by loading mesoporous silica nanoparticles (MSN) with 3 nm-sized ceria nanoparticles and 6 nm-sized manganese ferrite nanoparticles by a nucleophilic substitution reaction. The sizes of ceria and manganese ferrite nanoparticles were confirmed by transmission electron microscopy (TEM).

Inductively coupled plasma atomic emission spectrometer (ICP-AES) analysis showed that the weight ratio of manganese and iron ions to cerium ions was about 1.0. 80 μg of ions are conjugated to the surface of 1 mg of mesoporous silica nanoparticles. Conjugation of manganese ferrite nanoparticles and ceria nanoparticles to mesoporous silica nanoparticles was confirmed by TEM of MFC-MSN (FIG. 2).

Manganese, iron, and cerium ions are observed on the surface of the mesoporous silica nanoparticles through energy-dispersive X-ray spectroscopy (EDX) analysis (FIG. 3). According to the nitrogen adsorption-desorption experiment, MFC-MSN has a Bruneter-Emmett-Teller (BET) surface area of 320.19 m ² /g and a mesopore size of 3.80 nm (FIG. 4). Mesoporous silica nanoparticles having a large pore size were synthesized for effective drug absorption by adding a swelling agent, mesitylene, to the reaction mixture.

Rhodamine B isothiocyanate (RITC) was incorporated into MSN for fluorescent labeling, which was verified by UV-Vis (ultraviolet-visible) and PL (photoluminescence) spectroscopy (FIG. 5). For biomedical applications, the surface of nanoparticles was coated with polyethylene glycol (PEG) to avoid non-specific protein adsorption under in vivo conditions. The hydrodynamic size increased from 67.3±1.3 nm of MSN to 80.8±2.2 nm of PEGylated MFC-MSN after conjugation with manganese ferrite nanoparticles and ceria nanoparticles ( FIG. 6 ).

Synergistic O of MFC-MSN ₂ Creation and removal of reactive oxygen species

The synergistic effect of manganese ferrite nanoparticles and ceria nanoparticles was investigated by comparing the catalytic properties of MFC-MSN with those of manganese ferrite nanoparticle-immobilized MSN (MF-MSN) and ceria nanoparticle-immobilized MSN (C-MSN). In the time-dependent H ₂ O ₂ assay under physiological conditions (pH 7.4), 31.6% and 20.5% of H ₂ O ₂ were degraded by MF-MSN and C-MSN, respectively, after 3 hours ( FIG. 7 ). However, most of H ₂ O ₂ was decomposed by the same concentration of MFC-MSN after only 1.5 hours. As the dissolved oxygen value increased and the number of O ₂ bubbles increased, the O ₂ production function of MFC-MSN was significantly improved at pH 7.4 compared to MF-MSN and C-MSN ( FIGS. 8 and 9 ).

These results indicate that manganese ferrite nanoparticles and ceria nanoparticles have a synergistic effect on H ₂ O ₂ decomposition and O ₂ generation. In addition, the mixture of MF-MSN and C-MSN showed almost no synergistic effect on H ₂ O ₂ decomposition and O ₂ generation compared with MFC-MSN, which was the short distance between manganese ferrite nanoparticles and ceria nanoparticles. This means that it plays a key role in the synergistic effect.

To understand this synergistic effect, the concentration change of the hydroxyl radical and the superoxide anion, which are intermediates in the intracellular H ₂ O ₂ metabolic pathway, was investigated. First, the superoxide anion decomposition ability of nanoparticles was evaluated using superoxide dismutase (SOD) assay at pH 7.4. The SOD activities of MF-MSN, C-MSN and MFC-MSN were 64.9±1.0%, 19.4±13.4% and 79.2±2.2%, respectively. All nanoparticles had SOD activity, but no synergistic effect on SOD activity was observed for manganese ferrite nanoparticles and ceria nanoparticles ( FIG. 10 ).

Hydroxyl radical generation in the presence of nanoparticles after addition of H ₂ O ₂ at pH 7.4 was evaluated. MF-MSN generates a hydroxyl radical as an intermediate in the Fenton reaction (FIG. 11). In particular, MFC-MSN did not generate hydroxyl radicals, indicating that ceria nanoparticles scavenge these radicals generated by manganese ferrite nanoparticles.

Therefore, the synergistic effect on efficient H ₂ O ₂ decomposition and O ₂ generation is attributed to the conversion of hydroxyl radicals into O ₂ molecules by ceria nanoparticles during the Fenton reaction of manganese ferrite nanoparticles ( FIG. 12 ). In addition, O ₂ production by MFC-MSN occurs at a low H ₂ O ₂ concentration such as 100 μM, which is an intracellular concentration of H ₂ O ₂ under inflammatory conditions.

In addition, only negligible amounts of Mn, Fe and Ce ions were leached, as determined by ICP-MS analysis, which showed the stability of MFC-MSN under inflammatory physiological conditions after 24 h.

Ability of MFC-MSN to scavenge reactive oxygen species and attenuate hypoxia in vitro

In vitro experiments were performed using BMDM isolated from mice. After 24 hours incubation with RITC-labeled MFC-MSN, confocal laser scanning microscopy (CLSM) showed efficient cellular uptake of MFC-MSN by BMDM (FIG. 13). Flow cytometry analysis also showed that the fluorescence intensity of cells increased after incubation with MFC-MSN in a concentration-dependent manner.

In addition, MFC-MSN did not show cytotoxicity for 24 hours at a concentration of up to 55 μg/mL, as measured by MTS assay ( FIG. 14 ). Therefore, subsequent in vitro experiments were performed at MFC-MSN concentrations below 55 μg/mL.

In order to evaluate the effect of removing reactive oxygen species of MFC-MSN under in vitro conditions, the level of intracellular H ₂ O ₂ was first evaluated using an intracellular H ₂ O ₂ assay kit ( FIG. 15 ). BMDMs pretreated with MF-MSN, C-MSN and MFC-MSN were incubated with 100 μM H ₂ O ₂ for 1 hour, and then intracellular H ₂ O ₂ levels were measured through fluorescence intensity. MFC-MSN degraded intracellular H ₂ O ₂ more efficiently than the control, which was consistent with previous results, showing the synergistic H ₂ O ₂ resolution of MFC-MSN ( FIG. 7 ).

The intracellular concentration of hydroxyl radicals was evaluated using HPF (3'-(p-hydroxyphenyl) fluorescein), which is an indicator of reactive oxygen species sensitive to hydroxyl radicals. BMDM pretreated with MF-MSN, C-MSN and MFC-MSN was treated with 100 μM H ₂ O ₂ , and then the fluorescence intensity of HPF was evaluated. BMDM pretreated with C-MSN or MFC-MSN had significantly reduced fluorescence intensity compared to H ₂ O ₂ -treated control, confirming that ceria nanoparticles could effectively remove intracellular hydroxyl radicals.

To confirm the hypoxia attenuation ability of MFC-MSN, the HIF-1α expression level was evaluated. After pretreatment with MF-MSN, C-MSN and MFC-MSN, BMDMs were incubated for 4 hours under hypoxic and inflammatory conditions, which were hypoxic (1% O ₂ , 5% CO ₂ and 94% N ₂ ) and 0.2 This was achieved by the addition of μg/mL lipopolysaccharide (LPS). Hypoxia and LPS upregulated HIF-1α expression, which was attenuated by treatment with MF-MSN, C-MSN and MFC-MSN ( FIG. 16 ). The decrease in HIF-1α expression level was most pronounced in MFC-MSN, indicating synergistic generation of O ₂ by MFC-MSN.

M1 to M2 phenotypic transition in vitro of BMDM induced by MFC-MSN

M1 to M2 phenotypic transfer of BMDM in vitro after incubation with MFC-MSN was evaluated. Western blot analysis showed that protein expression of HIF-1α and pro-inflammatory M1 markers including iNOS, IL-1β and Cox-2 was significantly increased after hypoxia and LPS treatment ( FIG. 17 ). The expression levels of HIF-1α and M1 markers showed similar trends, indicating that HIF-1α plays an important role in the inflammatory behavior of activated macrophages.

In particular, treatment with MFC-MSN significantly reduced the expression levels of HIF-1α and pro-inflammatory M1 markers. The expression level of arginase-1 (Arg-1), a representative M2 marker, was significantly increased in BMDM treated with MFC-MSN compared to that of other groups. qRT-PCR analysis of BMDM showed that hypoxia and LPS treatment increased mRNA expression levels of M1 markers including iNOS, IL-6 and TNF-α ( FIG. 18 ).

Similarly, MFC-MSN treatment significantly reduced the mRNA expression level of the M1 marker. In contrast, anti-inflammatory M2 markers including Arg-1, CD206 and IL-10 were significantly increased after MFC-MSN treatment. Manganese ferrite nanoparticles and ceria nanoparticles also showed a macrophage polarizing effect, but less than that of MFC-MSN. Successful M1 to M2 phenotypic transition of macrophages is confirmed by MFC-MSN synergistically scavenging reactive oxygen species and generating O ₂ .

Oxygen production and M2 polarization of synovial macrophages in a murine rheumatoid arthritis model

To investigate the in vivo effect of MFC-MSN for the treatment of rheumatoid arthritis, a rat adjuvant-induced arthritis (AIA) model was established through intradermal injection of complete Freund's adjuvant. Important pathological features of human rheumatoid arthritis, including chronic inflammation and cartilage and bone destruction, are shared in the AIA model.

Using the murine AIA model, hemoglobin oxygen saturation (sO ₂ ) in the blood of the hypoxic knee joint was first visualized using photoacoustic imaging ( FIG. 19 ). Intra-articular injection of MFC-MSN immediately increased sO2 levels from 17.0±0.7% to 22.9±1.2%. This result shows that MFC-MSN can generate O ₂ immediately and efficiently in a hypoxic environment.

One day after injection, the synovial tissue was excised to trace the intracellular pathway of MFC-MSN ( FIG. 20 ). CLSM images of synovial tissue stained with macrophage-specific F4/80 antibody show intracellular accumulation of MFC-MSN in macrophages, likely due to their strong phagocytosis ability on foreign matter compared to other immune cells .

It was confirmed whether O ₂ generated by MFC-MSN relieves hypoxia of rheumatoid arthritis knee joint. Immunohistochemical staining for HIF-1α in synovial tissues and femurs indicates that injection of MFC-MSN successfully attenuated HIF-1α expression in both synovial and bone tissues ( FIG. 21 ). In addition, MFC-MSN acting as a reactive oxygen species scavenger and O ₂ generator induced a phenotypic change of synovial macrophages from M1 to M2 phenotype in rheumatoid arthritis knee joints ( FIGS. 22 and 23 ).

M1 macrophage-specific biomarkers such as Cox-2, TNF-α and iNOS were increased after induction of rheumatoid arthritis, whereas MFC-MSN injection significantly reduced their levels. Rheumatoid arthritis also stimulated expression of M2 markers due to macrophage infiltration, but injection of MFC-MSN further up-regulated anti-inflammatory M2 markers. Consequently, MFC-MSN immediately increased hemoglobin sO ₂ levels to alleviate hypoxia around rheumatoid arthritis synovial joints, and changed the macrophage phenotype from pro-inflammatory M1 to anti-inflammatory M2 subtype.

In vivo therapeutic effect of MTX-loaded MFC-MSN on rheumatoid arthritis

Based on the unique properties of MSN to enable sustained integrated drug release, we investigated whether MFC-MSN could deliver the anti-rheumatic drug MTX to enhance the therapeutic effect. To prevent rapid clearance and severe cytotoxicity due to repeated infusion of high-concentration MTX, sustained release of low-dose MTX is very beneficial for the treatment of rheumatoid arthritis. The loading capacity and loading efficiency of MTX were 10.4 μg/mg and 20.8%, respectively. As can be seen from the release profile of MTX, about 60% of MTX was released slowly over 7 days, showing sustained release.

As inflammation and hypoxia in rheumatoid arthritis cause hyperthermia and swelling of the feet, thermal imaging measurements were used to evaluate foot temperature and edema following administration of MFC-MSN or MTX-loaded MFC-MSN. After 30 days, the paw temperature and width increased by about 37% and about 120%, respectively, in the PBS-treated group ( FIG. 24 ). In contrast, hyperthermia and paw edema were dramatically reduced in mice treated with MFC-MSN and further decreased after treatment with MTX-loaded MFC-MSN ( FIGS. 25 and 26 ). However, injection of free MTX at the same dose as loaded in MFC-MSN did not show a therapeutic effect on rheumatoid arthritis due to the rapid clearance of MTX. In addition, behavioral tests were performed on rheumatoid arthritis mice receiving different treatments by determining the time required to traverse a 70 cm beam. The mice injected with MFC-MSN or MTX-loaded MFC-MSN showed significantly reduced time compared to the PBS or MTX-injected group ( FIG. 27 ).

The therapeutic effects of MFC-MSN and MTX-loaded MFC-MSN were further evaluated by histological and immunohistochemical analysis ( FIG. 28 ). These rheumatoid arthritis-induced pathological features were evaluated as severe rheumatoid arthritis cartilage destruction, synovial inflammation and stimulated angiogenesis in response to hypoxia. Safranin-O staining of the knee joint showed complete destruction of cartilage in the PBS-injected group. In contrast, injection of MFC-MSN or MTX-loaded MFC-MSN showed efficient preservation of cartilage structures. Mankin's score was applied to the quantification of cartilage structures. In addition, synovitis was significantly reduced in the MFC-MSN and/or MTX-loaded MFC-MSN infusion groups as assessed by H&E staining. In addition, the number of inflammatory cells was significantly reduced after treatment with MFC-MSN or MTX-loaded MFC-MSN.

Finally, angiogenesis, a typical response induced by hypoxia to increase oxygen supply, was analyzed by immunohistochemical staining of blood vessels with von Willebrand factor (vWF). CLSM and quantification of vWF-positive vessels indicate that injection of either MFC-MSN or MTX-loaded MFC-MSN significantly reduced angiogenesis in the knee joint.

During treatment, MTX-loaded MFC-MSN was monitored by monitoring key liver function markers and body weight in the blood, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine. It was evaluated whether it had a negative effect on the rat's health. No significant changes in body weight and blood levels were observed compared to healthy mice, and the toxicity of MTX-loaded MFC-MSN was negligible.

Overall, manganese ferrite, ceria and MTX in MTX-loaded MFC-MSN are synergistic and show optimal therapeutic effects for rheumatoid arthritis.

As described above, MFC-MSN can relieve inflammation by removing reactive oxygen species and generating O ₂ to induce polarization of macrophages from M1 to M2 in the rheumatoid arthritis knee joint. The synergistic effect of manganese ferrite and ceria nanoparticles is due to the hydroxyl radical scavenging effect of ceria nanoparticles, whereby intermediate radicals generated by manganese ferrite nanoparticles can generate O ₂ . MFC-MSN can induce polarization of M1 to M2 macrophages under hypoxic and inflammatory conditions both in vitro and in vivo.

Intra-articular injection of MFC-MSN into rheumatoid arthritis mice oxygenates inflamed synovial joints, downregulating HIF-1α and attenuating inflammation through polarization of M1 to M2 macrophages. Anti-rheumatic drug delivery using MFC-MSN further alleviates rheumatoid arthritis. MFC-MSN can be applied as an anti-inflammatory agent and M2 macrophage stimulator for the treatment of other hypoxia-related inflammatory diseases such as myocardial infarction.

[ Example and Analysis example ]

Synthesis of 6nm-sized manganese ferrite nanoparticles

Manganese ferrite nanoparticles with a size of 6 nm were synthesized through thermal decomposition of manganese oleate and iron oleate complexes. The iron oleate complex was prepared by dissolving 40 mmol of iron chloride and 120 mmol of sodium oleate in 60 mL of deionized water, 80 mL of ethanol and 140 mL of n-hexane. Manganese oleate complex was prepared by substituting manganese chloride for iron chloride during the synthesis of iron oleate complex.

The prepared solution was heated to 70° C. and stirred for 4 hours. The upper organic layer was separated, and the solvent was evaporated using a rotary evaporator to obtain a metal oleate complex. For the synthesis of manganese ferrite nanoparticles, 0.67 mmol of manganese oleate, 1.33 mmol of iron oleate, and 4 mmol of oleic acid were dissolved in 10 g of 1-octadecene. After degassing under vacuum, the mixture was heated to 320° C. at a constant heating rate of 3.3° C./min and aged for 1 hour. The resulting nanoparticles were washed several times with ethanol and dispersed in chloroform.

Synthesis of 3nm Ceria Nanoparticles

1 mmol of cerium(III) acetate and 12 mmol of oleyl amine were dissolved in 15 mL of xylene. The mixture was stirred at room temperature for 2 h and heated to 90 °C. After adding 1 mL of deionized water to the solution, the resulting solution was aged at 90° C. for 3 hours and cooled to room temperature. The ceria nanoparticles were washed several times with ethanol and redispersed in chloroform.

BMPA - capped manganese ferrite nanoparticles and Seria Synthesis of nanoparticles

For the synthesis of BMPA-capped manganese ferrite nanoparticles, 0.5 g of 2-Bromo-2-methylpropionic acid (BMPA), 0.05 g of citric acid and 15 mg of oleic acid capped manganese ferrite nanoparticles were dissolved in a mixture of chloroform and DMF. (50/50 v/v, 15 mL). Similarly, BMPA-capped ceria nanoparticles were synthesized by mixing 0.25 g of BMPA, 0.025 g of citric acid, and 15 mg of ceria nanoparticles in a mixture of chloroform and DMF. The mixture was stirred overnight and then centrifuged to remove excess ligand. BMPA-capped nanoparticles were dispersed in ethanol for conjugation to mesoporous silica nanoparticles.

RITC and amine Large pore size meso containing functional groups Porus Synthesis of silica nanoparticles (MSN)

2 g of cetyltrimethylammonium chloride (CTAC) and 0.08 g of triethanolamine were dissolved in 20 mL of deionized water and heated at 95° C. with vigorous stirring. After 1 hour, 1.5 mL of mesitylene, 1.5 mL of TEOS, and 0.15 mL of the prepared APTES ((3-Aminopropyl) triethoxysilane)-RITC were added to the mixture, and the mixture was further aged for 1 hour. The product was collected by centrifugation and washed several times with ethanol. CTAC was removed three times by treatment with ammonium nitrate (60 mg/mL in methanol). To synthesize amine-functionalized MSN, 0.15 mL of APTES was added to MSN, heated to 70° C. and aged for 3 hours. The final product was obtained by washing several times with ethanol.

Synthesis of MFC-MSN, MF-MSN and C-MSN

20 mg of amine-functionalized MSN was added to 15 mg of BMPA-capped manganese ferrite nanoparticles and 15 mg of BMPA-capped ceria nanoparticles and stirred overnight at room temperature. MF-MSN and C-MSN were synthesized using 15 mg of manganese ferrite nanoparticles and 60 mg of ceria nanoparticles, respectively. The product was washed several times with ethanol and water and then coated with PEG using methoxy poly(ethylene glycol) succinimidylglutarate (mPEG-SG) and methoxy poly(ethylene glycol) amine (mPEG-AM). The resulting MFC-MSN, MF-MSN and C-MSN were redispersed in 5 mL of deionized water.

MTX loading and emission testing

1 mL of MFC-MSN and 1 mL of 0.2 mg/mL MTX were mixed in deionized water and stirred overnight at room temperature to load MTX. Excess MTX was removed by washing several times with deionized water. After centrifugation, the absorbance at 305 nm of the supernatant was measured to obtain the release profile of MTX up to 7 days.

Characteristics of MFC-MSN

TEM analysis was performed using a JEOL EM-2010 microscope operated at 200 kV. Metal ion concentrations including manganese, iron and ceria were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using an ICPS-7500 spectrometer (Shimadzu). The hydrodynamic size of the nanoparticles was measured using a dynamic light scattering instrument (Malvern). The Brunauer-Emmett-Teller (BET) surface area and pore size of MFC-MSN were measured by nitrogen adsorption at 77 K using a 3Flex surface characterization analyzer (Micrometritics). RITC labeling of MSN was confirmed using a UV-Vis spectrophotometer (Agilent Technologies). MTX loading was also demonstrated by UV-Vis spectrophotometry by measuring the intrinsic absorbance of MTX at 305 nm. Successful labeling of RITC in MSN was investigated using PL spectrophotometry (Agilent Technologies).

H ₂ O ₂ decomposition analysis

MF-MSN, C-MSN and MFC-MSN containing 2.5 mM H ₂ O ₂ and 60 μg/mL manganese ferrite and ceria were mixed in PBS at room temperature. 50 μL of the above solution was added to 100 μL of a Ti(SO ₄ ) ₂ solution containing 1.33 mL of 24% Ti(SO ₄ ) ₂ 8.33 mL of H ₂ SO ₄ in 50 mL of deionized water, and added every 30 minutes until 3 hours. . After the reaction was completed, the absorbance of the solution was measured at 405 nm to evaluate the H ₂ O ₂ concentration.

O ₂ generative analysis

MF-MSN, C-MSN and MFC-MSN containing 1M H ₂ O ₂ and 150 μg/mL of manganese ferrite nanoparticles and ceria nanoparticles were mixed in 20 mL of PBS at room temperature, and dissolved oximetry (HI9136, HANNA) instrument, Korea) to measure the O ₂ concentration every 5 minutes. In order to observe O ₂ bubbles in the Eppendorf Tube, MF-MSN, C-MSN, MFC-MSN containing 1M H ₂ O ₂ and 75 μg/mL manganese ferrite nanoparticles and ceria nanoparticles Mix and monitor for up to 1 hour. The O ₂ generating ability of MFC-MSN was also evaluated under hypoxic and inflammatory conditions achieved by uptake of Ar in PBS and addition of 100 μM H ₂ O ₂ .

SOD mimic catalytic activity

The SOD mimicking catalytic activity of MF-MSN, C-MSN and MFC-MSN was investigated using the SOD assay kit (Sigma Aldrich) according to the provided protocol. WST (aqueous tetrazolium salt) working solution was prepared by diluting 1 mL of WST solution with 19 mL of buffer, and 15 μL of enzyme solution was diluted with 2.5 mL of dilution buffer to prepare enzyme working solution. The SOD activity was calculated using the unique property of WST to generate a water-soluble formazan dye upon reduction with a peroxide anion. WST working solution and enzyme working solution were added to MF-MSN, C-MSN and MFC-MSN at a final concentration of 300 μg/mL, followed by thorough mixing and incubation at room temperature for 20 minutes. The absorbance at 450 nm was measured using a microplate reader, and the slope of the inhibition rate compared to the blank was measured to calculate the SOD activity (%).

Hydroxyl radical production

The production of hydroxyl radicals by MF-MSN, C-MSN and MFC-MSN was assessed using the hydroxyl radical antioxidant capacity (HORAC) assay (Cell Biolabs, Inc.) with a slightly modified protocol. A 1X fluorescein solution and 1X hydroxyl radical initiator were prepared by diluting a fluorescein probe (100X) and a hydroxyl radical initiator (5X) with an analytical diluent and deionized water, respectively. 40 μL of MF-MSN, C-MSN and MFC-MSN containing 20 μg/mL of manganese ferrite and ceria were mixed with 140 μL of 1X fluorescein solution and incubated for 30 minutes.

20 μL of 1X hydroxyl radical initiator was added to the solution for nanoparticle and Fenton reaction. Fluorescence intensity of the solution was measured every 30 minutes for up to 2 hours using a microplate reader (ex/em = 480/530). The hydroxyl radical generation rate was calculated by evaluating the oxidation rate of the fluorescent probe by hydroxyl radicals through a hydrogen atom transfer (HAT) process.

Preparation of BMDM

In order to isolate murine bone marrow-derived macrophages (BMDM), bone marrow was collected from the femurs of 5-week-old male Sprague Dawley (SD) mice (Coatech, Gyeonggi-do, Korea) as described above. First, the hind legs of the animals were collected and muscle tissue was removed to expose the femurs. The bone was then flushed with PBS using a syringe to isolate bone marrow cells. After centrifugation, the collected bone marrow cells were harvested in 30% (v/v) L929 cell conditioned medium, 15% (v/v) FBS, 5% (v/v) horse serum and 1% (v/v) penicillin/streptate. They were differentiated into macrophages for 4 days in differentiation medium consisting of high-glucose DMEM supplemented with mycin. Conditioned medium was prepared from L929 cells by growing the cells in high-glucose DMEM containing 10% (v/v) FBS, and 1% (v/v) penicillin/streptomycin. After 4 days of incubation, the macrophage differentiation medium was discarded and adherent BMDMs were obtained using a cell scraper.

Cell uptake and cell viability testing

To confirm cellular uptake of RITC-labeled MFC-MSN, BMDM was incubated with MFC-MSN for 24 h, followed by flow cytometry using FACS AriaII in National Center for Inter-University Research Facilities (NCIRF) and CLSM analysis. Analysis was performed. For CLSM analysis, cell nuclei were co-stained using Hoechst 33342 (Thermo Fisher Scientific). Cell viability was assessed using the MTS cell proliferation assay (Promega). After BMDM was incubated with MF-MSN, C-MSN and MFC-MSN for 24 hours, MTS solution was added to the medium and absorbance was measured at 490 nm using a microplate reader.

intracellular H ₂ O ₂ analyze

To evaluate the intracellular H ₂ O ₂ concentration, an intracellular H ₂ O ₂ assay (Sigma Aldrich) was used. 100 μM of H ₂ O ₂ was treated with BMDM, and pre-incubated with 40 μg/mL of MF-MSN, C-MSN and MFC-MSN for 24 hours. After 1 hour, the cell medium was replaced with an assay buffer and the intracellular H ₂ O ₂ concentration was measured using CLSM (ex/em = 490/520).

Intracellular OH radical generation

The ability to generate hydroxyl radicals in cells was investigated using 3'-(p-hydroxyphenyl) fluorescein (HPF, Thermo Fisher Scientific), a reactive oxygen species indicator sensitive to hydroxyl radicals. BMDMs were incubated with 40 μg/mL of MF-MSN, C-MSN and MFC-MSN for 24 hours and then incubated with 100 μM of H ₂ O ₂ for an additional 1 hour. The H ₂ O ₂ containing medium was removed and replaced with 5 μM HPF working solution. After 30 min, the green fluorescence intensity of the wells was investigated using CLSM assay.

HIF - 1α immunostaining

HIF-1α immunostaining was performed on BMDMs cultured in lipopolysaccharide (LPS)-containing medium under hypoxic conditions after treatment with MF-MSN, C-MSN and MFC-MSN. Hypoxia was achieved by incubation in a hypoxic chamber containing 1% O ₂ , 5% CO ₂ and 94% N ₂ gas for 4 hours. BMDM cells pre-incubated with 40 μg/mL of MF-MSN, C-MSN, and MFC-MSN for 4 hours were cultured in a medium containing 0.2 μg/mL of LPS under hypoxic conditions for another 4 hours, followed by HIF-1α was stained with a primary antibody against (Abcam) and Alexa Fluor 488-labeled secondary antibody (Abcam). F-actin was co-stained using rhodamine paloid (Thermo Fisher Scientific).

qRT-PCR and western blot analysis

Rat BMDM was pretreated with MSN, C-MSN, MF-MSN and MFC-MSN for 4 hours, followed by extensive PBS washing. Fresh DMEM supplemented with LPS (0.2 μg/mL) was added and cells were incubated in hypoxic conditions (1% O ₂ , 5% CO ₂ and 94% N ₂ ). After 4 hours, total RNA and protein from BMDM were extracted with TRIzol ^® (Life Technologies, CA) and Cell Lysis Buffer ^® (Cell Signaling Technology, MA), respectively. The concentration of total RNA was measured using a NanoDrop spectrometer (ND-2000, NanoDrop Technologies, Wilmington, DE). cDNA was then synthesized with 600 ng of total RNA.

For qRT-PCR, TOPOne ^TM SYBR Green premix (Enzynomics, Daejeon, Korea) was added to cDNA in the StepOnePlus real-time PCR system (Applied Biosystems, CA) at 94°C for 10 seconds, at 60°C for 15 seconds, at 72°C. Amplification was carried out in 50 cycles for 30 seconds. To perform Western blots, Bradford assays were performed for determination of protein concentrations.

Proteins were then mixed with NuPAGE ^® LDS sample buffer (Life Technologies, CA), boiled at 95° C. for 3 minutes for denaturation, and loaded onto a 12% (w/v) SDS-polyacrylamide gel. After 100 min of electrophoresis at 80 V, proteins were transferred to a nitrocellulose membrane (Bio-Rad, CA) using a Trans-Blot ^® SD semi-dry electrophoretic transfer cell (Bio-Rad) at 20 V for 50 min. For specific binding, the membranes were blocked with 5% skim milk for 2 hours. Membranes were probed with primary antibodies, HIF-1α (Novus Biologicals, CO), iNOS, IL-1β, Cox-2, Arg-1 (Abcam, UK) overnight at 4°C. The next day, the membrane was washed 3 times with TBS-T and reacted with HRP-conjugated secondary antibody for 50 min. After washing, the membrane was developed using a chemiluminescence detection system (GE Healthcare, UK). Blotted membranes were visualized using a Gel Logic 2200 PRO imaging system (Carestream Health, NY).

Adjuvant-induced arthritis (AIA) model and intra-articular injection of nanoparticles

All animal studies were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. For establishment of the AIA model, 10-week-old SD mice were purchased from Coretech and acclimated for 1 week. Arthritis was induced by tail-based intradermal injection of 10 mg/mL complete Prowin's adjuvant (Chondrex, WA) as described above. Animals were randomly assigned after the first signs of inflammation were observed on day 8. AIA-induced animals were randomly assigned to 8 groups (n = 6).

On the 10th day after adjuvant administration, 50 μL of PBS, MTX, MSN, MSN+MTX, C-MSN, MF-MSN, MFC-MSN, MFC-MSN+MTX was administered to the knee joint through intra-articular injection. As determined by ICP-AES analysis, C-MSN, MF-MSN and MFC-MSN were injected with 10 μg dose of manganese ferrite and ceria. MSN was injected at the same concentration as silica in MFC-MSN. Free MTX was injected in equal amounts into MSN and MFC-MSN at a dose of 2 μg.

Thermal imaging and evaluation of physical function in arthritis

After intra-articular injection, temperature changes and photothermal images of the hind paw were recorded up to 30 days after adjuvant administration using an infrared thermal imaging system (FLIR i2, FLIR Systems Inc., OR). The change in foot width was measured by caliper. Both hind paws of the animals were counted in cumulative measurements of paw temperature and width. The physical function of the AIA animals was assessed by recording the mean time through the 70 cm beam. Animals were acclimated to the beam on days 3 and 6 prior to the first signs of inflammation.

Optoacoustic imaging of oxygen levels in the knee joint

Vascular O ₂ saturation (sO2) around synovial joints was measured using a Vevo LAZR optoacoustic imaging system (VisualSonics Inc., Canada) with LZ550 transducer differential optics of peroxidized and deoxygenated hemoglobin at different wavelengths of 850 nm and 750 nm, respectively. was measured by absorption. In the rat AIA model, sO2 around the synovial joint was measured before and after administration of 50 μL of MFC-MSN to the synovial joint through intra-articular injection.

Immunohistochemical analysis

Histopathology was evaluated to determine the characteristics of rheumatoid arthritis, including hypoxia, M1/M2 equilibrium, angiogenesis, synovitis (synovitis) and cartilage destruction. On day 30, the animals were sacrificed and the knee joint was recovered. Knee joints were fixed in 4% paraformaldehyde for 3 days at 4°C, then transferred to descaling solution (Sigma, MO) and placed on an orbital shaker. Tissues were transcribed at room temperature for 14 days. The transcribed knee joint was placed in paraffin and cut with a microtome at 10 μm. For immunohistochemistry, tissue sections were dewaxed and stained with primary antibodies against HIF-1α (Novus Biologicals), Cox-2, CD206 (Abcam), F4/80 and vWF (Santa Cruz Biotechnology, TX). . The secondary antibodies used were goat anti-mouse Alexa Fluor ^® 568 (Abcam) against HIF-1α, goat anti-rabbit Alexa Fluor ^® 488 (Abcam) against F4/80 and CD206 and goat against Cox-2 and vWF. anti-rabbit Alexa Fluor ^® 568 (Abcam). Tissue sections were then stained with DAPI mounting solution and observed using a confocal fluorescence microscope (LSM780, Carl Zeiss, Germany). For quantification of vWF-positive vessel density, vWF-positive areas relative to DAPI-positive areas were measured using ImageJ software.

qRT-PCR analysis of M1/M2 expression in collected synovial joints

On day 30 after adjuvant administration, the knee joints of all animals were retrieved and placed in RNAse/DNAse-free conical tubes. Synovial tissue of the knee joint was collected using a sterile surgical blade. The obtained tissue was minced with a surgical blade and then homogenized in the presence of TRIzol ^® (D1000, Benchmark Scientific, NJ), and total RNA was isolated. Synthesis of cDNA and qRT-PCR was performed as described above for in vitro assays.

Safranin-O and H&E staining

For the evaluation of cartilage destruction and synovial inflammation, safranin-O and H&E staining were performed, respectively. Safranin-O staining for proteoglycans in cartilage was performed according to the method described above. Briefly, dewaxed tissue sections were stained with Weigert's hematoxylin working solution consisting of 1% hematoxylin and 30% iron chloride (anhydrous) for 10 min.

After extensive washing, sections were counter-stained with fast green (FCF) solution for 5 min. After washing with 1% acetic acid, sections were stained with 0.1% safranin O solution for an additional 5 min. For H&E staining, dewaxed tissue sections were initially stained with hematoxylin working solution for 10 min. After extensive washing with running tap water and 95% alcohol, slides were stained with 1% eosin Y solution for 3 min.

After dehydration, safranin-O and H&E stained tissue sections were fixed with Canadian balsam (Sigma). Knee joint and synovial tissues were visualized under light microscopy for evaluation of cartilage destruction and synovial inflammation, respectively. Cartilage destruction was scored using Mankin's method as described above, with 7 investigators unaware of the experimental animal group.

So far, specific embodiments of the present invention have been described. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (10)

nanoparticles including manganese ferrite nanoparticles and ceria nanoparticles; and
Nanoparticle structure comprising a porous support for supporting the nanoparticles by binding to the nanoparticles. The method of claim 1,
The porous support is a nanoparticle structure, characterized in that it comprises mesoporous silica nanoparticles. The method of claim 1,
Nanoparticle structure, characterized in that it further comprises a drug loaded on the porous support. 4. The method of claim 3,
The drug is a nanoparticulate structure, characterized in that it comprises an arthritis drug. The method of claim 1,
The nanoparticulate structure is injected into the human body to remove pro-inflammatory M1 macrophages and to induce anti-inflammatory M2 macrophages. The method of claim 1,
The nanoparticulate structure is injected into the human body to remove reactive oxygen species and generate oxygen. forming a porous support and nanoparticles; and
Comprising the step of binding the porous support and the nanoparticles,
The nanoparticles are a method of forming a nanoparticle structure, characterized in that it comprises manganese ferrite nanoparticles and ceria nanoparticles. 8. The method of claim 7,
The porous support is a method of forming a nanoparticle structure, characterized in that it comprises mesoporous silica nanoparticles. 8. The method of claim 7,
Method of forming a nanoparticulate structure further comprising the step of loading a drug on the porous support. 8. The method of claim 7,
wherein the porous support is amine-functionalized,
The nanoparticles are BMPA-capped,
The step of binding the porous support and the nanoparticles,
Method of forming a nanoparticulate structure comprising the step of mixing the amine-functionalized porous support and the BMPA (2-Bromo-2-methylpropionic acid)-capped nanoparticles.

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