Patent Application
20200315980
This application claims priority to Korean Patent Application No. 10-2019-0040944, filed on Apr. 8, 2019, the disclosure of which is incorporated herein by reference in its entirety.
The present disclosure relates to a drug delivery system in which urate oxidase and metal-based nanoparticles for hydrogen peroxide degradation are loaded in a nanocarrier, a pharmaceutical composition for treating hyperuricemia comprising the drug delivery system, and a method for producing the drug delivery system.
Therapeutic proteins are used for clinical treatment of various human diseases due to their favorable properties such as biocompatibility, selectivity and efficacy compared to chemical drugs. However, therapeutic proteins can cause side effects such as headache, diarrhea, temporary rash, reduced blood cells, cardiotoxicity, hypertension, hypersensitivity, exfoliative dermatitis, immunogenicity and serum disease. Reducing the side effects of drugs is an important issue in developing therapeutic proteins, along with other existing issues such as improving therapeutic efficacy and stability. To date, alteration of amino acid composition or conjugation of biocompatible molecules (e.g., polyethylene glycol (PEG)) has been extensively applied to reduce the dose of therapeutic protein and thus reduce side effects. However, this approach can compromise the critical properties of therapeutic proteins, and hence alternative strategies need to be developed.
Urate oxidase (UOX) is an enzyme that catalyzes the reaction of converting insoluble uric acid (UA) into soluble 5-hydroxyisoacetate (5-HIU). UOX is used to treat acute or chronic gout, gouty redness, gouty arthritis, kidney disease, cardiovascular disease, and hyperuricemia associated with tumor lysis syndrome (TLS). Tumor lysis syndrome (TLS) can occur during cancer treatment when many tumor cells are lysed. Gout is a common inflammatory arthritis caused by the deposition of uric acid crystals in joints and soft tissues. About 0.1 to 10% of the population worldwide suffers from gout. The conversion of UA to 5-HIU, catalyzed by UOX, also generates H2O2, which is toxic and can adversely affect patients deficient in glucose-6-phosphate dehydrogenase.
Gold (Au) nanoparticles (AuNPs) are highly biocompatible metal nanoparticles and are known to degrade hydrogen peroxide (H2O2), like catalase. Such enzyme-mimicking nanoparticles are called nanozymes. Nanozymes are attracting attention as an alternative to enzymes because of their high stability and cost effectiveness compared to enzymes. However, the enzyme-nanozyme system has been mainly used in the biosensor field because of its limitations in in vivo applications. For example, the use of a catalase-mimicking AuNP with UOX reduced H2O2 levels in vitro, but the application thereof in vivo is difficult. When UOX and AuNP are administered to blood, they are diluted to very low concentrations. Hence, they cannot be located close enough to each other to achieve effective cascade reactions consisting of 1) the generation of H2O2 by UOX and 2) the removal of H2O2 by AuNP. In order to encounter H2O2, a by-product to be efficiently removed by AuNP, AuNP and UOX need to be loaded together into a single carrier.
The patent documents and references cited herein are incorporated herein by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference.
The present inventors have made extensive efforts to develop a drug delivery system capable of effectively treating hyperuricemia in blood. As a result, the present inventors have experimentally demonstrated that when urate oxidase and catalytic metal nanoparticles capable of degrading hydrogen peroxide are loaded together in a nanocarrier having temperature-sensitive properties and administered in vivo, blood uric acid levels can be effectively lowered, thereby completing the present disclosure.
Therefore, an object of the present disclosure is to provide a drug delivery system capable of effectively treating hyperuricemia.
Another object of the present disclosure is to provide a pharmaceutical composition for preventing or treating hyperuricemia or hyperuricemia-related disease.
Still another object of the present disclosure is to provide a method for producing the drug delivery system.
Other objects and technical features of the present disclosure will be more clearly understood from the following detailed description of the disclosure, the appended claims and the accompanying drawings.
In accordance with one aspect of the present disclosure, there is provided a drug delivery system comprising: (i) urate oxidase; (ii) metal-based nanoparticles for hydrogen peroxide degradation; and (iii) a temperature-sensitive nanocarrier loaded with (i) and (ii).
In the present disclosure, “urate oxidase” is an enzyme that catalyzes the conversion of uric acid to a more soluble product, that is, 5-hydroxyisoacetate (5-HIU) which is more easily secreted out of the body.
As used herein, the term “metal-based nanoparticles for hydrogen peroxide degradation” refers to a metal-based catalytic capable of promoting a reaction in which hydrogen peroxide (H2O2) is degraded into water (H2O) and oxygen (O2).
According to an embodiment of the present disclosure, the metal-based nanoparticles for hydrogen peroxide degradation may comprise metal, metal ion with chelate or metal oxide. The metal may comprise for example, gold (Au), platinum (Pt), manganese (Mn), silver (Ag), or iron (Fe), but is not limited thereto. Preferably, the metal for hydrogen peroxide degradation may be gold (Au), platinum (Pt), or silver (Ag). The metal ion with chelate comprise for example, manganese ion (Mn2+), Prussian Blue (PB) or iron ion (Fe2+). The metal oxide comprise for example, manganese oxide (MnO2), iron oxide (Fe2O3) or ceria (CeO2).
According to an embodiment of the present disclosure, the metal for hydrogen peroxide degradation may comprise, for example, gold (Au), platinum (Pt), manganese (Mn), silver (Ag), or iron (Fe), but is not limited thereto. Preferably, the metal for hydrogen peroxide degradation may be gold (Au), platinum (Pt), or silver (Ag).
As used herein, the term “temperature-sensitive” refers to a property in which the size of the nanocarrier decreases with increasing temperature and the size of the nanocarrier increases with decreasing temperature.
In the present disclosure, the diameter of the temperature-sensitive nanocarrier increases with decreasing temperature, whereas the diameter of the temperature-sensitive nanocarrier decreases with increasing temperatures.
In the present disclosure, this increase or decrease in the diameter of the nanocarrier with increasing or decreasing temperature is reversible.
In the present disclosure, the size of pores formed in the nanocarrier changes as the diameter of the nanocarrier increases or decreases. For example, when a drug to be delivered is encapsulated into the nanocarrier with increased pore size at a low temperature (e.g., 4° C.), and then applied to the human body, the pore size decreases. Due to such temperature-sensitive properties, when the drug delivery system of the present disclosure is administered to the human body, the distance between the urate oxidase and the metal-based nanoparticles for hydrogen peroxide degradation, loaded into the nanocarrier, may be decreased and the urate oxidase and the metal-based nanoparticles for hydrogen peroxide degradation may be positioned closer to each other. This proximity between the urate oxidase and the metal-based nanoparticles allows the rapid and efficient degradation of hydrogen peroxide.
According to one embodiment of the present disclosure, the temperature-sensitive nanocarrier is a Pluronic-based nanocarrier.
In the present disclosure, the “Pluronic” comprises a triblock copolymer composed of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO).
According to another embodiment of the present disclosure, the triblock copolymer composed of PEO-PPO-PEO may further comprise a photo-crosslinkable functional group.
According to a specific embodiment of the present disclosure, the Pluronic may be diacrylated Pluronic F127.
In one embodiment of the present disclosure, the weight ratio between the urate oxidase and the metal-based nanoparticles for hydrogen peroxide degradation, loaded in the temperature-sensitive nanocarrier, is 1 (urate oxidase): 0.15 to 1.5 (metal-based nanoparticles for hydrogen peroxide degradation).
In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating hyperuricemia or hyperuricemia-related disease comprising: (i) a therapeutically effective amount of a drug delivery system in which urate oxidase and metal-based nanoparticles for hydrogen peroxide degradation are loaded in a temperature-sensitive nanocarrier; and (ii) a pharmaceutically acceptable carrier.
In the present disclosure, the term “therapeutically effective amount” means an amount sufficient and suitable to treat hyperuricemia or hyperuricemia-related disease in vivo by administering the drug delivery system to a patient.
The pharmaceutical composition of the present disclosure may comprise a pharmaceutically acceptable carrier in addition to the active ingredient “drug delivery system”. Such carriers are those that are commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, menthol and mineral oil.
The pharmaceutical composition of the present disclosure may further comprise a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above-described components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
The suitable dose of the pharmaceutical composition of the present disclosure may vary depending on factors such as formulation method, mode of administration, and patient's age, weight, sex, disease conditions, diet, duration of administration, route of administration, rate of excretion, and response sensitivity.
The pharmaceutical composition of the present disclosure may be administered orally or parenterally. For parenteral administration, the pharmaceutical composition may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.
Meanwhile, considering that the pharmaceutical composition of the present disclosure is applied for the treatment or prevention of hyperuricemia or hyperuricemia-related disease, the pharmaceutical composition of the present disclosure may be administered by various oral or parenteral routes.
The concentration of the drug delivery system, which is an active ingredient contained in the pharmaceutical composition of the present disclosure, may be determined in consideration of the purpose of treatment, the condition of a patient, the period of time required, etc., and is not limited to a specific range of concentration.
The pharmaceutical composition of the present disclosure may be prepared in a unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method which can be easily carried out by a person having ordinary skill in the technical field to which the present disclosure pertains, or may be prepared by filling into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets or capsules, and may additionally comprise dispersing or stabilizing agents.
In the present disclosure, the hyperuricemia-related disease may be selected from the group consisting of acute or chronic gout, gout redness, gouty arthritis, kidney disease, cardiovascular disease, and tumor lysis syndrome (TLS).
In still another aspect of the present disclosure, there is provided a method for producing a drug delivery system comprising steps of:
(a) preparing urate oxidase and metal-based nanoparticles for hydrogen peroxide degradation; and
(b) loading a temperature-sensitive nanocarrier with the prepared urate oxidase and metal-based nanoparticles for hydrogen peroxide degradation.
According to one embodiment of the present disclosure, step (b) in the method for producing the drug delivery system may be performed at a low temperature so that the size of the temperature-sensitive nanocarrier is increased.
According to a specific embodiment of the present disclosure, the low temperature at which the size of the temperature-sensitive nanocarrier is increased may be 1 to 10° C., preferably 2 to 8° C., more preferably 3 to 5° C., and even more preferably 4° C.
Specific embodiments described herein are intended to represent preferred embodiments or examples of the present disclosure, and the scope of the present disclosure is not limited thereto. It is obvious to those skilled in the art that modifications and other uses of the present disclosure do not depart from the scope of the present disclosure as defined in the appended claims.
Experimental Materials and Method
1. Materials
Gold nanoparticles (diameter: 5 nm) coated with poly(vinylpyrrolidone) were purchased from nanoComposix Inc. (San Diego, Calif.). Ni-nitrilotriacetic acid (Ni-NTA) resin was purchased from Qiagen (Valencia, Calif., USA). A Vivaspin centrifugal concentrator with a molecular weight cutoff (MWCO) of 50 kDa was purchased from Sartorius Corporation (Bohemia, N.Y., USA). A PD-10 desalting column was purchased from GE Health Care (Piscataway, N.J., USA). Yeast extract, tryptone and agar were purchased from DB Biosciences (San Jose, Calif., USA). Pluronic F127 of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) (PEO100-PPO65-PEO100, molecular weight: 12.6 kDa) was obtained from BASF Corporation (Seoul, Korea). Acryloyl chloride was purchased from Tokyo Chemical Industry (TCI, Tokyo, Japan). 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl) ketone (Irgacure 2959) was obtained from Ciba Specialty Chemicals Inc. (Basel, Switzerland). A Nanosep centrifugal device (MWCO 300 kDa) for spin filtration was purchased from Pall Life Sciences (Ann Arbor, Mich., USA). Unless otherwise indicated, all other reagents were purchased from Sigma-Aldrich (Saint Louis, Mo., USA). All reagents were of analytical grade and were used without further purification.
2. Purification of Urate Oxidase (UOX) and Production of Gold Nanoparticles (AuNP)
UOX was prepared as follows. Briefly, the plasmid-encoding UOX gene (pQE80-UOX) was transformed into TOP10 Escherichia coli cells. The transformed TOP10_pQE80-UOX cells were cultured for UOX expression. Cells expressing UOX were harvested and subjected to immobilized metal ion affinity chromatography using a hexahistidine affinity tag attached to UOX. The purity of UOX is equal to or more than 95%, as analyzed by SDS-PAGE analysis. The catalytic activity of the purified UOX was analyzed by uric acid degradation assay.
More specifically, the plasmid encoding a recombinant urate oxidase (UOX) gene derived from Aspergillus flavus under the control of T5 promoter (pQE80-UOX) was transformed into TOP10 E. coli cells. The pre-cultured transformed cells were inoculated into a fresh 2×YT medium containing 100 μg/mL of ampicillin, and shake-cultured at 220 rpm and at 37° C. When the optical density at 600 nm (OD600 nm) reached 0.5, 1 mM isopropyl β-D-1-thiogalactopyranoside was added to the culture for induction of UOX expression and incubated for 5 hours. Then, the cells were pelleted by centrifugation at 6,000 rpm for 10 minutes. The cell pellets were stored at −80° C. until use, resuspended in lysis buffer (50 mM sodium phosphate, 0.3 M sodium chloride, 10 mM imidazole, pH 7.4), and incubated with 100 μg/mL of lysozyme on ice for 10 minutes. The resuspended cell pellets were sonicated on ice for 15 min (10-sec pulse on and 20-sec pulse off). The cell lysate was centrifuged at 12,000 rpm at 4° C. for 40 minutes, and then Ni-NTA resin was added to the supernatant which was then incubated at 4° C. for 30 minutes. The supernatant incubated with the Ni-NTA resin was loaded onto a polypropylene column and washed with washing buffer (50 mM sodium phosphate, 0.3 M NaCl, 20 mM imidazole, pH 7.4). After washing, UOX was eluted with elution buffer (50 mM sodium phosphate, 0.3 M sodium chloride, 250 mM imidazole, pH 7.4). The eluted UOX solution was buffer-exchanged with PBS buffer (pH 7.4) using a PD-10 column. The UOX concentration was determined by absorbance measurement at 280 nm using Synergy™ multimode microplate reader (BioTek, Winooski, Vt., USA) according to Beer-Lambert's law. The extinction coefficient of UOX was 53,520 M−1cm−1.
Before encapsulation into nanocarriers (NC), the catalase-mimic catalytic activities of AuNPs were measured by H2O2 degradation assay.
3. Determination of Degradation Rate of Uric Acid by UOX (Urate Oxidase)
In order to optimize the ratio of UOX to AuNPs, various concentrations of AuNPs (1, 2.5, 5, 10, 15, 20 and 25 μg/mL) were mixed with 500 nM UOX. To determine the degradation rate of UA, 200 μM UA was mixed with UOX in PBS buffer (pH 7.4), and the absorbance at 293 nm was monitored using Synergy™ multi-mode microplate reader. The extinction coefficient of UA at 293 nm was 12,300 M−1cm−1.
4. Production of UOX-AuNP@NCs
Pluronic-based nanocarriers (NCs) were synthesized by photo-crosslinking the micelle state of diacrylated pluronic F127 (DA-F127).
Briefly, a 10 wt % DA-F127 solution was prepared in deionized water (DIW). Then, 0.154 mL of the DA-F127 solution was added to 1.846 mL of DIW containing 0.057 wt % of irgacure 2959 to induce micelle formation of DA-F127. Using an ultraviolet (UV) lamp (VL-4.LC, 8 W, Vilber Lourmat, France), a photocrosslinking process for NC synthesis was performed by irradiation at 1.3 mW/cm2 for 15 minutes. The produced NCs were purified by dialysis for 1 day and freeze-dried. UOX and AuNPs were co-encapsulated into the nanocarriers (NCs) using the temperature-dependent size change of the NCs (UOX-AuNP@NCs).
Briefly, 1 mg of NCs was dissolved in 1 mL of a 5 μM UOX solution containing 50 μg of AuNPs. The mixture was incubated overnight at 4° C. to induce the size expansion of the NCs. Then, the temperature of the mixed solution was increased to 37° C. and maintained for 10 minutes to reduce the size of the NCs for encapsulation. The final solution was centrifuged using a Nanosep centrifugal device to remove unloaded UOX and AuNPs (11,000 rpm, 37° C., 10 min). Then, the amount of UOX in UOX-AuNP@NCs was measured by bicinchoninic acid protein assay (Micro BCA protein analysis kit, Thermo Fisher Scientific, Waltham, Mass., USA). The loaded amount of AuNPs in UOX-AuNP@NCs was calculated by absorbance of AuNPs at 525 nm using a spectrometer (Molecular Devices, Spectra-MaxM2e, Trenton, N.J., USA).
5. Size and Surface Charge of UOX-AuNP@NC
The sizes and surface charges of NCs and UOX-AuNP@NCs were analyzed at 37° C. using an electrophoretic light scattering system (ELS-8000, Otsuka Electronics Osaka, Japan). Then, the stability of UOX-AuNP@NCs was determined by measuring the size change of UOX-AuNP@NCs at 1 mg/mL in a cell culture medium (RPMI 1640, Gibco, N.Y., USA) containing 10% fetal bovine serum (FBS) at 37° C. and 100 rpm. The size of UOX-AuNP@NCs was observed at predetermined time points for 7 days.
6. Determination of In Vitro UA Degradation Rate in NCs
In order to compare the UA degradation efficiencies, four enzyme systems were prepared as follows: (1) UOX, (2) UOX in NCs (UOX@NC), (3) UOX with AuNPs (UOX-AuNP), and (4) UOX-AuNP in NCs (UOX-AuNP@NC). Enzyme samples were incubated at 37° C. for 15 minutes and enzymatic assays of UOX at 10 nM were performed. 200 μL of assay buffer (50 mM sodium borate buffer, pH 8.0, 100 μM UA) was used and the UA degradation rates were calculated by measuring absorbance change at 293 nm.
7. Determination of H2O2 Levels In Vitro
Concentrations of H2O2 generated by UOX systems (UOX, UOX@NC, UOX-AuNP, or UOX-AuNP@NC) when adding UA to the samples were estimated using degradation of N,N-dimethyl-4-nitroaniline (RNO). 0.25 M of RNO and 0.03 M of histidine were prepared in DIW and mixed together. Then, UOX, UOX@NC, UOX-AuNP or UOX-AuNP@NC solution was added to the RNO solution. The concentrations of UOX and UA were fixed at 200 nM and 1 mM, respectively. Then, H2O2 generation from each sample was measured by incubating the sample at room temperature for 10 minutes and observing N,N-dimethyl-4-nitroaniline (RNO) concentration by measuring absorbance at 440 nm.
8. In Vitro Cytotoxicity of UOX-AuNP@NC
Cytotoxicity of UOX-AuNP@NCs without UA was examined using squamous cell carcinoma-7 (SCC7) cells obtained from American Type Culture Collection (Rockville, Md., USA). First, SCC7 cells were seeded at 5×103 cells/well in a 96-well tissue culture plate with a cell culture medium (RPMI 1640, Gibco, N.Y., USA) containing 10% FBS and 1% penicillin-streptomycin. The seeded cells were incubated for 24 hours at 37° C. under a 5% CO2 condition. Then, the cells were washed with PBS buffer (pH 7.4) and treated with the prepared UOX-AuNP@ NC samples at different concentrations (from 0 to 1 mg/mL on the basis of NC concentration). The sample-treated cells were incubated overnight at 37° C. Then, the cells were washed with PBS buffer, and the cytotoxicity of the UOX-AuNP@NCs without UA was calculated using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Kumamoto, Japan).
9. In Vitro Cytotoxicity Determination of H2O2 Generated by the UOX Systems
To examine the cytotoxicity of H2O2 generated by the UOX systems, the cytotoxicity of UOX-AuNP@NC in the presence of UA was compared to that of UOX, UOX@NC and UOX-AuNP. SCC7 cells were seeded at 5×103 cells/well in a 96-well tissue culture plate and cultured overnight at 37° C. under 5% CO2. The cultured cells were washed with PBS and treated with 100 μL of each of samples such as UOX, UOX@NC, UOX-AuNP and UOX-AuNP@NC. Each sample was processed to a final concentration of 200 nM UOX and 1 mM UA. Among them, UOX@NC was a negative control. The sample-treated cells were incubated for 3 hours at 37° C. and washed with PBS. The cytotoxicity of sample groups in the presence of UA was determined using a Cell Counting Kit-8.
10. Determination of In Vivo Uric Acid Level
For in vivo experiments, 8-week old C57BL/6 mice (Orient Bio Inc., Seongnam, South Korea) were used. To induce a hyperuricemia state in the mice, the mice were treated with hypoxanthine and potassium oxonate. Before the induction of hyperuricemia, all the mice were fasted overnight. Hypoxanthine was dissolved in 3% soluble starch solution and injected intragastrically into the mice at a dose of 475 μg/g. Then, potassium oxonate was dissolved in a co-solvent composed of a mixture of lanolin and liquid paraffin (3:2, v/v) and injected subcutaneously into the hypoxanthine-treated mice at a dose of 95 μg/g.
Five groups of mice were prepared: (1) normal mice (control), (2) untreated hyperuricemia mice, (3) hyperuricemia mice treated with UOX, (4) hyperuricemia mice treated with UOX-AuNP, and (5) hyperuricemia mice treated with UOX-AuNP@NC. 100 μL of sample solution (UOX concentration was fixed at 1 μM) of either PBS, UOX, UOX-AuNP or UOX-AuNP@NC was injected into the tail vein of the hyperuricemia mice. At predetermined time points (pre-induction and 1, 3, 6, 12, and 18 hours after injection), blood was collected from the sample-treated mice via retro-orbital bleeding. At 18 hours after injection of samples, blood was collected by cardiac puncture. The collected blood was centrifuged at 4° C. and 3000 rpm for 5 minutes to obtain serum. The UA concentration in the serum was analyzed after sample injection to compare the effect of UA degradation by sample treatment. For measuring the serum level of uric acid, samples were analyzed by the previously reported method with little modification. Serum samples were mixed with 0.3 M acetate buffer (pH 3.6) and then stored at 4° C. until analysis. Reagent mixtures for determination of serum UA were prepared in 0.3 M acetate buffer (pH 3.6) at a concentration of 0.83 mM 2,4,6-tripyridyl-s-triazine (TPTZ) and 1.66 mM FeCl3.H2O. The absorbance at 593 nm was measured to determine the UA concentration. In addition, in the presence or absence of NCs, the UA degradation rates of serum samples at 18 hours after sample injection were measured to evaluate the residual activity of UOX.
Experimental Results
1. Physiochemical Properties and Optimal Ratio of UOX and AuNPs Encapsulated in NCs
In the present disclosure, Pluronic-based NCs were used to deliver UOX and AuNPs simultaneously. UOX and AuNPs were co-encapsulated in NCs through temperature-dependent size changes. In order to achieve efficient UA degradation by UOX in the presence of AuNPs, the optimal ratio of UOX to AuNPs was examined. Various concentrations of AuNPs (1, 2.5, 5, 10, 15, 20, and 25 μg/mL) were mixed with 500 nM UOX, and then encapsulated in NCs. In all cases, the loading efficiencies were 90% or higher.
The enzymatic activity assay of samples showed that 5 μg/mL was the optimal concentration of AuNPs for efficient UA degradation (see
The size and surface charge of the produced NCs were analyzed using an electrophoretic light scattering system at 37° C. to predict the size of NCs in the body. When UOX and AuNPs were not loaded, the size and surface charge of NCs were 67±15 nm and −4.2±0.8 mV, respectively (see
2. Enhanced Catalytic Activities of UOX Encapsulated Together with AuNPs in NCs
The present inventors analyzed the change in catalytic efficiency of UOX upon co-encapsulation with AuNPs in NCs. It was hypothesized that the efficiency of catalytic activity will be enhanced by two factors. One is that the addition of AuNPs will degrade the side-product H2O2, accelerating the UA degradation reaction. The other is the proximity effect of multiple catalysts in cascade reactions. Co-encapsulation of UOX and AuNPs in NCs will greatly reduce the distance between UOX and AuNPs, accelerating H2O2 degradation in situ. In order to prove these hypotheses, four groups of enzyme systems were prepared, including UOX alone (UOX), UOX encapsulated in NC (UOX@NC), a free mixture of UOX and AuNPs (UOX-AuNP), and UOX and AuNPs encapsulated in NCs (UOX-AuNP@NC). The degraded amounts of UA and H2O2 were measured. As shown in
Next, the present inventors examined whether different enzyme systems led to different H2O2 levels during UA degradation in vitro. H2O2 levels were estimated by measuring RNO degradation (
Similar UA degradation and H2O2 production by UOX alone and UOX@NC indicate that NCs themselves did not substantially affect the enzymatic activity of UOX. More importantly, these results imply that NCs do not hinder the transport of UA as well as H2O2 across NCs or inside NCs. Thus, NCs not only provided the efficient loading of enzyme and nanoparticles, but also allowed the transport of small molecules including substrate and product for enzymatic reaction across or inside the carrier, which is a key requirement for proper action of delivered enzymes against target molecules present inside the body. A significant increase in the UA degradation and a significant decrease in H2O2 level of UOX-AuNP@NC compared to UOX-AuNP imply that NCs did not hinder the cascade reactions between them. In addition, these results demonstrate the hypothesis of the proximity effect made by co-localizing UOX and AuNPs in NCs on H2O2 removal and UA degradation. The present inventors speculated that the distances between UOX and AuNPs were closer in NCs than those between a free mixture of UOX and AuNPs. Considering that the blood volume is much greater than the sample injection volume, samples are expected to be substantially diluted in vivo upon administration. Therefore, the more pronounced effect of co-encapsulation of UOX and AuNPs in NCs on H2O2 removal and UA degradation would be expected in vivo. Through the above-described experimental results, the present inventors confirmed the favorable features of the Pluronic-based delivery platform having enhanced efficacy.
3. In Vitro Cytotoxicity of UOX-AuNP@NC with or without UA
Before the in vivo experiment using UOX-AuNP @NC, the cytotoxicity of UOX-AuNP@NC without UA was measured to verify the biosafety of the nanosystem using SCC7 cancer cells (
In order to evaluate H2O2-related cytotoxicity, the cytotoxicity of UOX-AuNP @ NC system was compared to those of other samples in the presence of UA (
4. Evaluation of In Vivo Activities of NC Loaded with UOX and AuNP
To evaluate the therapeutic effect of UOX-AuNP @NC in vivo, a hyperuricemia mouse model was used, as shown in
First, among the experimental groups, the hyperuricemia group showed a much higher (over 3-fold) UA concentration in serum at 1 hour compared to the pre-induction (0 hour) or the normal control (no hyperuricemia) group (
In addition, at 6 hours or later, the UOX and UOX-AuNP groups showed similar UA concentrations compared to the normal or hyperuricemia group in contrast to the maintenance of lower concentration of UA for the UOXAuNP@NC group, indicating that only the UOX-AuNP@NC group was effective up to 18 hours. Considering the biodistribution results of Pluronic-based NCs, which showed that when administered intravenously, the administered NC stayed in the main organ (predominantly in the liver) for at least 1 day in the previous experiment, the residual UA degradation effect at a later time point appeared to be because the in vivo half-life of UOX and AuNP was extended by encapsulation into NCs. At 18 hours, the residual UOX activity in serum by UOX-AuNP@NC was confirmed (
Clearance of the toxic intermediate H2O2 can facilitate UA degradation. Also, the effective removal of H2O2 by UOX-AuNP@NC can lower the toxicity issue associated with H2O2. Thus, the UA degradation effect was significantly enhanced by co-delivering UOX and AuNP using NC (UOX-AuNP@NC) in vivo, and the delivery platform of the present disclosure has an obvious clinical potential for hyperuricemia treatment through synergistic effect of UOX and AuNPs.
The effects and advantages of the present disclosure are summarized as follows.
(i) The present disclosure is directed to a drug delivery system in which urate oxidase and metal-based nanoparticles for hydrogen peroxide degradation are loaded in a temperature-sensitive nanocarrier, and a pharmaceutical composition for treating hyperuricemia-related disease comprising the drug delivery system.
(ii) In the drug delivery system of the present disclosure, urate oxidase and metal-based nanoparticles for hydrogen peroxide degradation are positioned close to each other, thereby effectively removing the toxic substance hydrogen peroxide (H2O2) generated during uric acid degradation.
(iii) The drug delivery system of the present disclosure may be developed as an active ingredient of a drug for preventing or treating hyperuricemia-related disease.
Although the present disclosure has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present disclosure. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereto.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0040944 | Apr 2019 | KR | national |
