Nanocomposites for temporary photoperforation, formed from gold nanoparticles and upconverting nanoparticles

Abstract

The present invention provides: nanocomposites for temporary photoperforation of a cell membrane, formed by combining (i) gold nanoparticle and (ii) upconverting nanoparticles comprising ytterbium (Yb3+) and erbium (Er3+); and a pharmaceutical composition comprising same. The nanocomposite according to the present invention enables the use of near-infrared region light, which can effectively penetrate tissue, and enables temporary photoperforation through the formation of vapor nanobubbles caused by a momentary increase in temperature, and thus can promote the penetration of molecules (for example, drugs) into the plasma membrane / cell membrane.

Description

Nanocomposites for temporary photoperforation formed from gold nanoparticles and redox nanoparticles

[0001] The present invention relates to a nanocomposite for temporary photoporation formed from gold nanoparticles (AuNPs) and urea-converting nanoparticles (UCNPs). More specifically, the present invention relates to a nanocomposite for temporary photoporation formed from (i) gold nanoparticles; and (ii) ytterbium (Yb). 3+ ) and erbium (Er 3+ ) for temporary photoperforation of cell membranes, formed by combining reversible nanoparticles. The present invention also relates to a pharmaceutical composition comprising the nanocomposite, for example, a pharmaceutical composition for treating osteoarthritis.

[0002] Intracellular delivery (ICD) is a technology for delivering therapeutic agents to target cells. This technology is closely related to cell therapy and regenerative medicine. To enter cells, small molecules such as peptides and nucleic acids must cross the plasma membrane through passive or active transport. Methods to increase the efficiency of intracellular delivery of agents have been developed in recent decades. Various techniques, including carrier-mediated and membrane disruption-mediated methods, have been developed. Membrane disruption-mediated methods, designed to generate mechanical fields in the plasma membrane, are used to create temporary pores. These include electroporation, sonoporation, and photoporation. Many studies have reported increased membrane disruption efficiency using plasmonic nanoparticles (L. Wang, et al., Acta Biomater. 2023, 157, 24).

[0003] Plasmonic nanoparticles, such as polydopamine nano-sensitizers (IONPs) and gold nanoparticles (AuNPs) irradiated with 561 nm laser light, have been used to effectively deliver plasmid DNA (pDNA) into HeLa and Jurkat cells [a) J.C. Fraire, et al., Nat. Commun.2022,13, 1996; b) L. Raes, et al., Nano-Micro Lett.2020,12, 185; c) L. Raes, et al., Crystals2019,9, 411; d) A. Harizaj, et al., Adv. Funct. Mater.2021,6, 631]. Other examples include the delivery of FITC-dextran and siRNA to HeLa cells and H1299 lung carcinoma cells via AuNPs (R. Xiong, et al., ACS Nano 2014, 8, 6288), and siRNA delivery to ARPE-19 retinal cells using gold nanostars (MH Kafshgari, et al., Small 2021, 17, 2007577). Similarly, the non-invasive dye calcein was delivered to human prostate carcinoma cells and rat cardiomyocytes using carbon nanoparticles (A. Sengupta, et al., ACS Nano 2014, 8, 2889).

[0004] AuNPs have been used in various photoporation studies due to their ability to promote plasmonic responses and generate localized electric fields, as well as their high biocompatibility and absorption efficiency. During photoporation, the vibration of AuNPs generates heat in a localized region. The high thermal conductivity of AuNPs causes a transient temperature increase, forming vapor nanobubbles. This process facilitates the formation of transient pores in the plasma membrane. A widely used form of AuNPs, called nanospheres, is less than 100 nm in size and absorbs light in the 500 nm wavelength range. However, light in the 500 nm range has a relatively short wavelength, which limits its clinical application because it does not effectively penetrate tissues. Near-infrared light (NIR) (750-2000 nm) has a long wavelength range and can penetrate tissues, making it ideal for clinical applications [a) C. Ash et al., Lasers Med. Sci.2017,32, 1909; b) MV Padalkara, et al.,Analyst2015,140, ​​2093; c) M. Arink, et al., Eng. Proc.2021,9.]

[0005] Upconversion nanoparticles (UCNPs) absorb light in the NIR wavelength range and emit visible light. These nanoparticles are made of ytterbium (Yb), a lanthanide element. 3+ ), erbium (Er 3+ ) and thulium (Tm 3+) are formed by doping sodium ytterbium tetrafluoride (NaYF4) nanocrystals with doping elements. The combination and arrangement of doping elements and the doping efficiency determine the wavelength and quantum field of light emitted from the NaYF4 nanocrystals. Unlike photoluminescence with a typical Stokes reaction (λex<λem), the anti-Stokes reaction (λex>λem) converts photons. UCNPs are utilized in bioimaging, biosensing, and nanomedicine due to their high photostability [a) K. Jeong, et al.,ACS Nano2021,15, 18394; b) G. Liang, et al.,J. Nanobiotechnology2020,18, 154; c) A.-R. Hong, et al.,ACS Appl. Nano Mater.2020,3, 8662; d) S. Kuk, et al., Adv. Sci.2017,13, 1603139; e) J. Liu, et al.,J. Am. Chem. Soc.2014,136, 9701].

[0006] The present inventors conducted various studies to develop a novel photoperforation-based ICD method. In particular, the present inventors fabricated nanocomposites (NCs) composed of gold nanoparticles (AuNPs) and upconverting nanoparticles (UCNPs) and evaluated the applicability of the photoperforation-based ICD method through upconverting-photon quenching-mediated perforation influx (UCPPin). The present inventors synthesized nanocomposites (posAuNP@UCNPs) by combining positively charged gold nanoparticles (posAuNPs) with UCNPs at various ratios and compared and analyzed their morphological and physical properties. Furthermore, the present inventors confirmed that upconverting photons were quenched by AuNPs, and transient perforation of the plasma membrane was observed. In particular, the inventors of the present invention found that the nanocomposite (posAuNP@UCNPs) can effectively deliver baricitinib, an osteoarthritis treatment drug, into cells via reciprocal transduction-photon quenching-mediated perforation influx (UCPPin) in a 3D osteoarthritis model.

[0007] Accordingly, the present invention aims to provide nanocomposites (NCs) that can be usefully applied to temporary photoperforation through reversal-photon quenching-mediated perforation inflow.

[0008] In addition, the present invention aims to provide a pharmaceutical composition for treating osteoarthritis comprising a combination of the nanocomposite and barasitinib.

[0009] In one aspect of the present invention, (i) gold nanoparticles; and (ii) ytterbium (Yb 3+ ) and erbium (Er 3+ ) is provided for temporary photoperforation of cell membranes, formed by combining reversible nanoparticles.

[0010] In another aspect of the present invention, a pharmaceutical composition for treating osteoarthritis is provided, comprising a combination of a first compartment comprising the nanocomposite and a second compartment comprising baricitinib as an active ingredient.

[0011] The nanocomposite of the present invention enables the use of light in the near-infrared region, which can effectively penetrate tissues, by quenching upconverting photons formed by irradiation with light in the near-infrared region by gold nanoparticles. In addition, the nanocomposite of the present invention causes temporary photoperforation of the plasma membrane / cell membrane through the formation of vapor nanobubbles due to an instantaneous temperature increase. Therefore, the nanocomposite of the present invention can be usefully applied to a photoperforation-based ICD method through upconverting-photon quenching-mediated perforation influx (UCPPin). In addition, the pharmaceutical composition according to the present invention can effectively treat osteoarthritis through upconverting-photon quenching-mediated perforation influx (UCPPin).

[0012] Figure 1 shows the results of characterization of UCNPs and posAuNP@UCNPs for photoperforation.

[0013] (A) Schematic diagram of UCNP's repayment conversion mechanism.

[0014] (B) Photoluminescence intensity of UCNPs at different concentrations.

[0015] (C) Size distributions of UCNPs, posAuNPs, and posAuNP@UCNPs.

[0016] (D) Zeta potentials of UCNPs, posAuNPs, and posAuNP@UCNPs.

[0017] (E) TEM images of posAuNP and posAuNP@UCNPs.

[0018] Figure 2 shows the results of analyzing the morphological and physical properties of posAuNP@UCNPs with different UCNP ratios.

[0019] (A) TEM images showing the morphological characteristics of posAuNPs at different UCNP ratios. The UCNP-to-posAuNP ratio was varied from 0 to 10 to analyze the effect of the ratio on the morphology and properties of the particles. Scale bar, 40 μm.

[0020] (B) Schematic illustrating the excitation and plasmonic emission of UCNPs and the plasmonic absorption of AuNPs. This shows how the 545 nm plasmonic photon emitted from UCNPs is absorbed by AuNPs, resulting in photoluminescence quenching.

[0021] (C) Photoluminescence intensity of posAuNP@UCNPs hybrid as a function of posAuNP ratio.

[0022] (D) FEM simulation showing the electronic field distribution of posAuNP@UCNPs hybrid as a function of UCNP ratio at wavelengths of 980 nm and 545 nm.

[0023] Figure 3 shows the results of analyzing the RHL and morphological changes of posAuNP@UCNPs under light irradiation.

[0024] (A) Schematic diagram of the experimental setup for photoirradiation of posAuNPs and posAuNP@UCNPs.

[0025] (B) Heat generation of posAuNP@UCNPs (1:5) compared to posAuNP under irradiated conditions for up to 30 min.

[0026] (C) Graph showing the results of measurements using COMSOL of RHL generated by posAuNP@UCNPs under light irradiation.

[0027] (D) TEM images of posAuNPs and posAuNP@UCNPs under light irradiation for different periods. The blue arrow indicates posAuNPs@UCNPs, and the pink arrow indicates posAuNPs separated from the UCNP cap.

[0028] Figure 4 shows the results of observing the localization of posAuNP@UCNPs and evaluating the perforation / resealing of the plasma membrane in C28 / I2 cells under light irradiation.

[0029] (A) Observation of posAuNP@UCNPs localization in NP-treated C28 / I2 cells (suspension state) after 10 min.

[0030] (B) Time-lapse, voxel-wise images, and diagrams of pore formation and resealing under light irradiation after irradiation of C28 / I2 cells (attached state) for up to 20 min. Images were captured every 10 s from T0 (0 min) to T180 (20 min). Filled white arrows indicate regions of pore formation in the plasma membrane, and empty white arrows indicate regions of resealing after pore formation. posAuNP@UCNPs at a ratio of 1:5 were used as the nanocomposite. Scale bar, 5 μm.

[0031] (C) Line profiling graph from B 159 s and pore diameter. Blue line represents mean value.

[0032] (D) Changes in intracellular calcium levels over time after treatment of C28 / I2 cells with posAuNP@UCNPs 1:5. The red arrow indicates posAuNP@UCNPs 1:5.

[0033] Figure 5 shows the results comparing the UCPPin efficiency of posAuNP@UCNPs under different light conditions in C28 / I2 cell suspension and spiroids.

[0034] (A) UCPPin efficiency under continuous and pulsed IR analyzed in suspension cells. The percentage of Calc+ cells indicates successful UCPPin, PI+ cells indicate dead cells, and Calc+ / PI+ cells indicate that UCPPin occurred but the cells were damaged.

[0035] (B) UCPPin efficiency under pulsed IR at different pulse intervals (PI) analyzed in suspension cells. Pulse width (PW) and pulse number (PN) were fixed at 10 min and 2, respectively.

[0036] (C) Comparison of UCPPin efficiency between posAuNP@UCNPs 1:0 and 1:5 as analyzed in spiroids under pulsed IR. Scale bar, 100 μm.

[0037] Figure 6. Comparison of therapeutic efficacy between UCPPin and PassT for intracellular delivery of baricitinib in an in vitro model of osteoarthritis.

[0038] (A) Schematic diagram of the experimental procedure.

[0039] (B) Inhibition of STAT1 and STAT3 phosphorylation by baricitinib as analyzed by Western blotting.

[0040] (C) Evaluation of IL-6 levels using different intracellular delivery methods on day 1.

[0041] (D) Extracellular matrix (ECM) expression levels analyzed by Western blotting (E) and immunofluorescence at 1 and 2 weeks using different intracellular delivery methods.

[0042] Figure 7 is a schematic diagram of the formation process and function of posAuNP@UCNPs.

[0043] (A) Schematic diagram of fabricating posAuNP using DOPA-PEI on AuNP.

[0044] (B) A schematic diagram of forming posAuNP@UCNP by combining posAuNP with carboxyl-UCNP is shown, showing the absorption of UCNP at a wavelength of 980 nm and luminescence at a wavelength of 545 nm, followed by the process of posAuNP absorbing light at 545 nm.

[0045] (C) Schematic diagram of the UCPPin process that occurs in cells.

[0046] Figure 8 shows the FTIR spectra of PEI and DOPA-PEI.

[0047] Figure 9 shows the TEM image and FTIR spectrum of posAuNP compared to AuNP.

[0048] Figure 10 shows the absorption and luminescence profiling of posAuNP and UCNP, respectively.

[0049] Figure 11 shows the characteristics of a 980 nm LED irradiator (A) and the absorbance curve of the LED-irradiated posAuNP@UCNPs nanocomposite (B).

[0050] Figure 12 shows confocal images (A) and CellSEM images (B) of posAuNP@UCNPs-treated C28 / I2 cells.

[0051] Figure 13 shows a long-distance illumination system of LEDs for time-lapse photography.

[0052] Figure 14 shows time-lapse images to estimate intracellular calcium levels during PM resealing.

[0053] Figure 15 shows the results of measuring the AuNP:DOPA-PEI ratio and incubation time analyzed by UCPPin efficiency.

[0054] Figure 16 shows the measurement results of posAuNP capacity analyzed by UCPPin efficiency.

[0055] Figure 17 shows the results of evaluating UCPPin efficiency according to IR time and pulse type.

[0056] Figure 18 shows the results of testing the survival rate during the UCPPin process at each step. (A) Schematic diagram of the experimental procedure. (B) Cell survival rate.

[0057] Figure 19 shows the results of evaluating UCPPin efficiency in C28 / I2 spiroids.

[0058] Figure 20 shows the results of evaluating the survival rate of C28 / I2 cells treated with UCPPin or PassT for 24 hours in a 3D model of osteoarthritis.

[0059] Photoporation technology based on plasmonic nanoparticles, such as gold nanoparticles (AuNPs), has been extensively studied for intracellular delivery of substances through cell membrane disruption. However, clinical application of AuNPs is challenging due to their absorption in the 500 nm region of the optical spectrum. To overcome this limitation, we used ion-converting nanoparticles (UCNPs) to stimulate AuNPs at near-infrared wavelengths. Positively charged gold nanoparticles (posAuNPs), obtained by coating AuNPs with DOPA-PEI, were combined with UCNPs to create a posAuNP@UCNPs nanocomposite, facilitating intracellular delivery under 980 nm NIR light irradiation. TEM and DLS confirmed that posAuNPs and UCNPs combined to form a nanocomposite. Furthermore, using multiple simulations, we analyzed the electric field distribution of posAuNPs based on morphological differences that changed with increasing UCNP ratio. Next, effective LED irradiation conditions were established by applying upconverting-photon quenching-mediated perforation influx (UCPPin) to C28 / I2 cells in suspension or spheroid form. The posAuNP@UCNP nanocomposite was confirmed to be effective in delivering baricitinib, an osteoarthritis treatment drug, in a 3D osteoarthritis model. Finally, chondrocyte differentiation was induced through intracellular delivery of baricitinib using posAuNP@UCNPs. These findings suggest that posAuNP@UCNPs have great potential as a tool for noninvasive drug delivery via UCPPin.

[0060] The present invention comprises (i) gold nanoparticles; and (ii) ytterbium (Yb 3+ ) and erbium (Er 3+ ) is combined to form a nanocomposite for temporary photoperforation of a cell membrane.

[0061] In the nanocomposite of the present invention, the gold nanoparticles are commercially available and are usually in the form of a sphere, i.e., a nanosphere, but are not particularly limited thereto. The gold nanoparticles may be positively charged gold nanoparticles for bonding with the cation-converting nanoparticles, for example, electrostatic bonding. The average particle diameter of the gold nanoparticles, preferably the positively charged gold nanoparticles, may be 75 to 85 nm, preferably 75 to 80 nm, and more preferably about 80 nm.

[0062] The positively charged gold nanoparticles can be obtained by coating gold nanoparticles with 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine, for example, to a thickness of about 2.5 nm. The 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine can be obtained by reacting polyethyleneimine (PEI) with 3,4-dihydroxyphenylalanine (DOPA). The reaction can be performed by a carbodiimide reaction, for example, according to the method disclosed in Park, JS et al., Multi-functional nanotracers for image-guided stem cell gene therapyNanoscale2017,9(14), 4665-4676 reported by the present inventors. If necessary, a fluorescent dye such as RITC can be chemically conjugated to the amine group of PEI through an isocyanate-amine reaction. The catechol group of the 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine reacts with metal substances including gold. Therefore, by vortexing a mixture of gold nanoparticles and 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine in an aqueous medium, the gold nanoparticles can be coated with 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine. The stirring can be performed at room temperature for about 30 to 40 seconds, but is not limited thereto. For example, the coating may be performed so that the weight ratio of the positively charged gold nanoparticles to the 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine is 1:0.5 to 0.9, preferably about 1:0.7.

[0063] In the nanocomposite of the present invention, the conversion nanoparticles are ytterbium (Yb 3+ ) and erbium (Er 3+) and the average particle diameter may be 30 to 40 nm, preferably 30 to 35 nm, and more preferably about 35 nm.

[0064] In one embodiment, the rechargeable nanoparticles may have a molecular formula of NaYYbErF4 having the atomic weights and mass ratios shown in Table 1 below.

[0065] Atomic mass ratio Na (sodium) 22.989769284.3525 Y (yttrium) 88.9058516.8318 Yb (ytterbium) 173.05432.7628 Er (erbium) 167.25931.6657 F (fluorine) 18.998403214.3872

[0066] The above-mentioned redox-converting nanoparticles are preferably functionalized with polyethylene glycol-COOH for binding with gold nanoparticles, for example, electrostatic binding. The redox-converting nanoparticles functionalized with polyethylene glycol-COOH can be obtained by a known method and are also commercially available. The redox-converting nanoparticles can be bound at a ratio of preferably 1 to 10 μg, more preferably 3 to 8 μg, and even more preferably about 5 μg, relative to 10 μg of the gold nanoparticles.

[0067] As described above, the nanocomposite of the present invention can induce photoperforation by irradiating with light in the near-infrared region, for example, light of about 980 nm, which can effectively penetrate tissue. Accordingly, temporary photoperforation of the nanocomposite of the present invention can be achieved by irradiating with light of about 980 nm, and such light irradiation can be performed for 5 to 20 minutes, preferably 10 to 20 minutes.

[0068] The present invention also provides a pharmaceutical composition for treating osteoarthritis, comprising a combination of a first compartment comprising the nanocomposite and a second compartment comprising baricitinib as an active ingredient.

[0069] In the pharmaceutical composition of the present invention, the first compartment and the second compartment may be formulated as separate dosage forms. Accordingly, the second compartment may be formulated as a known dosage form containing baricitinib, for example, a single-dose or multiple-dose dosage form. In one embodiment, the second compartment may be administered once or multiple times daily at a known dosage of baricitinib or a pharmaceutically acceptable salt thereof, for example, a dosage of 0.5 μM to 1 μM. Of course, the dosage and frequency of administration may be appropriately adjusted depending on the patient's condition, age, severity, etc.

[0070] The first compartment may be in the form of a parenteral administration preparation such as an injection, for example, a form suitable for intramuscular, intraperitoneal, subcutaneous, or intravenous administration, and may be a single-dose or multiple-dose administration form. The injection is usually prepared as a sterile solution of the nanocomposite of the present invention, and the pH of the solution should be suitably adjusted and buffered. In the case of intravenous administration, the total concentration of the solute should be adjusted so as to impart isotonicity to the preparation. The first compartment may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier, such as saline having a pH of 7.4. In one embodiment, the first compartment may be administered once or multiple times daily at a dosage of 0.01 to 0.1 mg / kg of the nanocomposite of the present invention. Of course, the dosage and the frequency of administration may be appropriately adjusted depending on the patient's condition, age, severity, etc.

[0071] Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention and the present invention is not limited to them.

[0072] Example

[0073] 1. Materials and test methods

[0074] (1) Materials

[0075] Polyethylene glycol (PEG)-COOH-modified cyclic peptide nanoparticles (UCNP-PEG-COOH) (QUN91W11) were purchased from ACS Material (California, USA). Gold nanoparticles (AuNP) (GC80) were purchased from ACS Material BBI solution (UK). Polyethyleneimine (25k), propidium iodide (P4170), and calcein (C0875) were purchased from Sigma Aldrich (St. Louis, USA). Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM F12), fetal bovine serum (FBS), and Dulbecco's phosphate-buffered saline (DPBS) were purchased from HyClone (Utah, USA). Hoechst 33342 (H3570) and Calcein-AM (65-0853-78) were purchased from Invitrogen (CA, USA). STAT1 and STAT3 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). P-STAT1 and P-STAT3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

[0076] (2) Preparation and characterization of posAuNP@UCNPs

[0077] For posAuNP, DOPA-conjugated PEI (DOPA-PEI) was prepared by carbodiimide reaction according to the method disclosed in Park, JS et al., Multi-functional nanotracers for image-guided stem cell gene therapy Nanoscale 2017, 9(14), 4665-4676. In addition, the fluorescent dye RITC (λ) was prepared by isocyanate-amine reaction. ex : 562 nm; λ em: 583 nm) was chemically conjugated to the amine group of PEI to prepare RITC-DOPA-PEI.

[0078] 10 μg of AuNP solution was transferred to an E-tube, 7 μg of DOPA-PEI was added, and the solution was vortexed at maximum speed to prepare posAuNPs. If fluorescence observation was required, posAuNPs were prepared using RITC-DOPA-PEI. 10 μg of posAuNP was fixed at 1, and UCNPs were added at different ratios (0, 1, 2, 5, and 10 μg) and vortexed at maximum speed to prepare posAuNP@UCNPs.

[0079] Each nanocomposite solution (10 μL) was added dropwise to the TEM grid formvar surface and allowed to adhere for 10 min. The remaining solution was completely removed using 3M paper, and the TEM grid was dried. The remaining NP solution was measured using DLS.

[0080] (3) IR effect on posAuNP@UCNPs

[0081] posAuNP and posAuNP@UCNPs (1:5) were prepared in glass vials, and the two nanocomposites were simultaneously irradiated with LED emitting light at 980 nm. Subsequently, the temperature at each time point was measured using an IR thermometer (TG267, FLIR, USA).

[0082] TEM images were taken to determine the morphology of NPs with and without LED irradiation. Samples for TEM observation were prepared in the same manner as described above.

[0083] The electric field and RHL formed by NPs were calculated and presented using the Wave Optics module of COMSOL Multiphysics software V. 6.1. The NP morphology according to the UCNP ratio was investigated using TEM images to generate a three-dimensional geometric structure. In addition, a PML was established by cutting out a quarter of the sphere [(a) S. Manrique-Bedoya, et al., J. Phys. Chem. C2020, 124, 17172; b) K. Zhang, et al., Lasers Med. Sci. 2019, 34, 1849; c) J. Shin, et al., Org. Electron. 2019, 66, 94]. The electric field was implemented by inputting the wave equation, assuming that plane waves of 980 nm (UCNP λex) and 545 nm (UCNP λem) were applied in the X-axis direction while the particles were arranged in the XZ plane. RHL was also calculated under these conditions.

[0084] (4) Localization of posAuNP@UCNPs

[0085] C28 / I2 cells were harvested with trypsin / EDTA, resuspended, and each group was divided into 1.0x10 5 cells mL -1 These were transferred to E-tubes. Cells were treated with each nanocomposite, incubated at 4°C for 20 min, fixed with 4% PFA, and subjected to cell TEM analysis at Eulji University (Seongnam, Korea).

[0086] (5) Live imaging for transient perforation and re-sealing

[0087] For confocal laser scanning microscopy (CLSM), 1.0 x 10 5C28 / I2 cells were seeded in 30 mm dishes. The following day, the cell membrane was stained with PlasMem Bright Green (Dojindo) for 20 min. After treatment with posAuNP@UCNPs at a ratio of 1:5, a long-distance LED irradiation system was installed to perform time-lapse imaging using LED IR. To confirm cell membrane resealing, intracellular calcium levels were measured with Fluo-4.

[0088] (6) UCPPin efficiency

[0089] The efficacy of UCPPin was analyzed in cell suspensions and spheroids. C28 / I2 cells were harvested with trypsin / EDTA, resuspended, and each group was divided into 1.0 × 10 5 cells mL -1 These were transferred to E-tubes. In addition, 5 μg of calcein, 2 μg of propium iodide (PI), and 2 μg of each nanocomplex were added to the cells and incubated at 4°C for 10 minutes, after which LED IR was initiated. After IR, the remaining dye and nanocomplexes were washed with DPBS to remove them, and the cells were analyzed by FACS.

[0090] For spheroid cell analysis, 1.0 x 10 harvested C28 / I2 cells 5 cells mL -1 The cells were separated into E-tubes at a concentration of 10 μg / ml. The cells were treated with each nanocomplex, incubated at 4°C for 10 minutes, and then washed with DPBS. The cells treated with each nanocomplex were resuspended in DMEM F12 medium, divided into 10 wells in a u-bottom 96-well plate, and cultured for 1 day. The following day, spheroid formation was examined, and after the addition of calcein and PI, LED irradiation was performed. The cells were then washed with DPBS, and CLSM analysis was performed.

[0091] (7) 3D modeling and analysis of osteoarthritis in vitro

[0092] Nanocomposite, osteoarthritis inducer (LPS 1 μg mL -1 and IL-6 20 ng mL -1 ) and 0.5 μM baricitinib (BRN) were added to C28 / I2 cells suspended in DMEM F12 serum- and antibiotic-free medium. Cells were incubated at 4°C for 10 min and illuminated with LED for 20 min. After washing with DPBS, cells were lysed with RIPA buffer (containing protease / phosphatase inhibitors), and STAT induction was confirmed by Western blotting.

[0093] In another assay, cells were washed after IR, resuspended in DMEM F12 medium, divided into six wells in a U-bottom 96-well plate, and cultured for up to 2 weeks. On day 1 of 3D culture, cells were diluted threefold, and IL-6 cytokine levels were measured using an IL-6 ELSA kit (430501, BioLegend, USA) according to the manufacturer's protocol.

[0094] During the first week of 3D culture, Western blotting was performed using cells lysed in RIPA buffer. In the second week, spheroids were transferred to 35 mm dishes for CLSM, allowed to adhere to the bottom of the dish, and IF was performed the following day.

[0095] (8) Statistical analysis

[0096] Statistical analysis was performed using Student's t-test with SigmaPlot software 10.0. *P<0.05, **P<0.01, ***P<0.001 were considered statistically significant.

[0097] 2. Test Results and Discussion

[0098] 2.1. Characterization of CNPs and posAuNP@UCNPs

[0099] The UCNP (NaYYbErF4@PEG-COOH) used in this study was ytterbium (Yb) as a sensitizer. 3+ ) and the activator erbium (Er) 3+ ) is a nanoparticle composed of Yb of UCNP 3+ The ions absorb light at 980 nm, triggering an anti-Stokes reaction, resulting in the emission of light at 545 nm (green light) (Figure 1A). This phenomenon, known as upconversion, allows UCNPs to convert low-energy near-infrared (NIR) light into high-energy visible light, making them a useful tool for a variety of applications, including imaging and therapy.

[0100] Using TEM, it was observed that UCNPs had a uniform size of approximately 30 nm and emitted light at 545 nm when irradiated with 980 nm light. The photoluminescence intensity increased proportionally to the amount of UCNPs (Fig. 1B).

[0101] The sizes and zeta potentials of AuNPs, UCNPs, posAuNPs, and posAuNP@UCNPs were measured (Fig. 1C and Fig. 1D). Determining the zeta potential of particles is an important factor in the photoporation efficiency. We found that photoporation was more effective in cationic particles than in anionic particles. Therefore, we modified the surface of AuNPs with 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine (DOPA-PEI) (Fig. 8) to prepare posAuNPs, which were positively charged AuNPs. Despite their small size, UCNP particles tend to aggregate in aqueous solutions; When posAuNPs were combined with UCNPs, the size of posAuNP@UCNPs increased to approximately 100 nm, and the zeta potential became positive (approximately +30 mV).

[0102] TEM observations of AuNPs and posAuNPs revealed that the positive surface charge of posAuNPs was due to the DOPA-PEI layer (Fig. 9). This result was confirmed using FTIR spectroscopy, which revealed the characteristic NH stretching of PEI. When UCNPs were bound to posAuNPs, they tended to bind only to one side of the posAuNPs, resulting in a "hat-like" appearance (Fig. 1E).

[0103] 2.2. Morphological and physical characterization of posAuNP@UCNPs using different UCNP ratios

[0104] To determine the optimal ratio of UCNPs to posAuNPs, the mass ratio of posAuNPs was fixed at 1, and the UCNP ratio was varied from 0 to 10. Using TEM, it was observed that the morphology of posAuNPs changed depending on the UCNP ratio (Fig. 2A). When the UCNP ratio increased to 1, 2, and 5, only a quarter of the posAuNP cross-section was bound to the surface with UCNPs. In the 1:10 group, a form in which more than half of the NP cross-section was bound to UCNPs was observed, and a form well bound to NPs was confirmed even when the UCNP ratio increased. The diameters corresponding to the major and minor axes, which were not evenly bound to all surfaces, were measured (Fig. 2A, lower panel). As the UCNP ratio increased, the number of UCNPs bound to posAuNPs also increased.

[0105] Next, the physical properties of posAuNPs conjugated with UCNPs were investigated. UCNPs are excited by 980 nm light and emit resonant photons at 545 nm, whereas AuNPs are plasmonic nanoparticles that absorb light and form localized electric fields (Fig. 2B). Since the wavelength band of 545 nm is similar to the maximum absorption wavelength of 80 nm-sized AuNPs (i.e., 550 nm) (Fig. 10), we hypothesized that the 545 nm resonant photons emitted from UCNPs would be absorbed by AuNPs, resulting in photon quenching. To verify this hypothesis, the photoluminescence intensity was measured by fixing the UCNP ratio to 1 and increasing the posAuNP ratio (Fig. 2C). It was found that the photoluminescence intensity gradually weakened as the posAuNP ratio increased, which confirms that the photon quenching occurred due to the reabsorption of the photons emitted by the UCNPs by the posAuNPs.

[0106] The influence of UCNP binding on the electric field generated by posAuNPs was investigated by theoretical simulations using the finite element method (FEM)-based Wave Optics module of COMSOL Multiphysics software. Based on the TEM images shown in Fig. 2A, three-dimensional geometries were generated at each UCNP ratio. Then, each wavelength range of 980 nm and 545 nm was applied to the X-axis of the XZ plane, which is the perfectly matched layer (PML), and the obtained electric fields are shown in Fig. 2D. For ratios of 1:0, 1:1, 1:2, and 1:5, the maximum electric field value at 980 nm was approximately 3.23 V / m, indicating that UCNPs at these ratios had little effect on the electric field. However, at a ratio of 1:10, which is sufficient to cover half of the posAuNP cross-section, a slight increase was observed in a localized region near the UCNP. A similar pattern was also observed at 545 nm, but the overall increase in localized areas was 50% higher than that observed at 980 nm. These results suggest that UCNPs have little effect on the formation of local electric fields by posAuNPs, and that posAuNP@UCNPs retain the properties of plasmonic NPs without affecting their function in the reversal-photon quenching-mediated perforation inflow process.

[0107] 2.3. Evaluation of the effect of 980 nm light irradiation on posAuNP@UCNPs

[0108] To evaluate the effect of 980 nm light irradiation on posAuNP@UCNPs, an experiment was conducted (Fig. 3A). The temperature change that occurred when posAuNP@UCNPs (1:5) was irradiated with 980 nm light was measured for 30 min and compared with the temperature change of the irradiated posAuNPs (Fig. 3B). From 2 min after irradiation, posAuNP@UCNPs exhibited greater heat generation than posAuNPs, with a temperature increase of approximately 20-35%.

[0109] When posAuNP@UCNPs are irradiated with electromagnetic waves, the temperature rises due to the generated resistance, resulting in a physical phenomenon known as resistive heating loss (RHL). To confirm this, a simulation was performed using the same structure and conditions as those used in the previous electric field analysis using COMSOL Multiphysics software (Fig. 3C). The simulation results showed that the surface of posAuNP@UCNPs in the 1:0 ratio group had an electric field of approximately 762 W / cm. 2 While the RHL of the group was evenly generated, the local regions of all other groups were 1460 to 1860 W / cm 2 The RHL of UCNPs was generated. These values ​​are approximately 1.9 to 2.4 times higher than those of the 1:0 ratio group, respectively, indicating that a large resistance occurs at the local site where UCNPs are bound, leading to heat generation. The generation of heat is an important factor for AuNP photoporation because it leads to the formation of vapor nanobubbles (VNBs) that burst and perforate cell membranes. Therefore, these results confirm that the binding of AuNPs and UCNPs increases the RHL of AuNPs, which increases the formation of VNBs, and ultimately leads to higher UCPPin efficiency.

[0110] To investigate how the above results are related to the morphological characteristics, posAuNP@UCNPs were observed before and after IR irradiation using TEM (Fig. 3D). In the IR 0 min group, posAuNPs and UCNPs were well combined (blue arrows). However, in the IR 20 min group, UCNPs were separated from posAuNP@UCNPs and existed in a free state (pink arrows). The change in size was measured by DLS (Fig. 3D, right panel), which showed that the nanoparticles separated from each other and remained dispersed in a free form up to 60 min of IR. In addition, the power (mW / cm) as a function of the distance from the light source 2 ) and Fluence (J / cm) which indicates the intensity of energy applied to the area according to the investigation time. 2 ) was measured and shown in Fig. 11A. The particle size distribution according to the irradiation time was estimated by performing an absorbance scan as described in Fig. 4D (Fig. 11B).

[0111] 2.4. Effect of electromagnetic wave irradiation on chondrocyte membrane perforation using posAuNP@UCNPs

[0112] The effect of PosAuNP@UCNPs on the cell membrane of the human-derived chondrocyte cell line C28 / I2 (adherent cells) was observed after electromagnetic irradiation at 980 nm. After treatment with each nanocomposite in the resuspended culture medium, the cells were fixed for 20 min, and TEM images were taken. TEM images showed that all treated NPs were attached to the cell membrane surface (Fig. 4A). The same results were confirmed by SEM when the cells were in an adherent state (Fig. 12). To further confirm the location of the NPs, posAuNPs were conjugated with RITC-DOPA-PEI, which confirmed that the treated NPs were attached to the cell surface. Furthermore, SEM confirmed the attachment of NPs to the surface of C28 / I2 cells treated with posAuNP@UCNPs at a posAuNP:UCNP ratio of 1:5. TEM and SEM confirmed that all NPs were bound to the cell membrane.

[0113] Next, time-lapse real-time imaging was performed to determine whether UCPPin was generated when cells were treated with posAuNP@UCNPs (ratio 1:5). After C28 / I2 cells were attached to a dish for confocal laser scanning microscopy (CLSM), the plasma membrane (PM) was stained, and the sample was irradiated with a long-range irradiator at 980 nm (Figure 13). Images were captured every 6.5 s for up to 180 cycles (Figure 4B). Irradiation of UCNPs with 980 nm light transfers light energy to AuNPs in solution. The heat energy generated during this process creates vapor nanobubbles (VNBs), which create small pores in the biomembrane as they pass through it. This transfer of material into the PM by temporary perforation using light is referred to as photoporation. In this study, we used a modified method using UCNP-conjugated AuNPs, termed upconverting-photon quenching-mediated perforation influx (UCPPin). At 0 s, posAuNP@UCNPs were bound to the cell surface. At 139 s, a loss of PM integrity and pore formation were observed in the area indicated by the white arrow. The pores were stable for approximately 20 s, and PM integrity was restored at 159 s. The voxel view images on the right are real-time captured and three-dimensionally displayed as time-sequential accumulations. The attachment of posAuNP@UCNPs (red) to the PM (green) resulted in pore formation (filled arrow), followed by re-sealing of the PM (hollow arrow). Line profiling of the PM in the white arrow area was performed at 152 seconds, and the diameter of the generated pores was measured and shown in the graph of Fig. 4C.These results confirmed that posAuNP@UCNPs were attached to the cell membrane surface and formed pores on the membrane surface under NIR irradiation.

[0114] To further confirm pore formation, intracellular calcium concentrations were measured in C28 / I2 cells (Fig. 4D). While the intracellular calcium concentration in the control group (CONT) remained stable, it decreased rapidly at 600 s and then decreased again at 800 s in the posAuNP@UCNPs 1:5 group. Calcium-free medium maintained low calcium concentrations in both groups (Fig. 14). Based on these results, we concluded that treatment of C28 / I2 cells with posAuNP@UCNPs 1:5 resulted in transient cell perforation followed by calcium-dependent PM resealing.

[0115] 2.5. Comparison of UCPPin efficiency of posAuNP@UCNPs at different IR times in C28 / I2 cell suspension and spheroids.

[0116] Several screenings were performed to determine the conditions for optimal UCPPin efficiency. First, the optimal ratio of AuNPs to DOPA-PEI (DP) for forming posAuNPs was determined using staining with propidium iodide (PI), a non-invasive fluorescent dye, and calcein (Figure 15A). The tested AuNP to DP ratios were 1:0.5, 1:0.7, and 1:0.9, respectively. The efficiency of posAuNPs without UCNPs (1:0) and posAuNPs with UCNPs (1:1) was compared. The results showed that the higher the DP ratio, the greater the number of cells stained with PI, leading to an increase in the number of dead cells. In contrast, calcein staining, an indicator of live cells, was barely detectable. These results imply that cells stained with PI but not with calcein require weaker stimulation, as strong stimulation may cause hyperperforation, leading to PM resealability and death. The UCPPin efficiency was compared with and without UCNPs at the same DP ratio. Calcein-positive cells were observed in the UCNP (1:1) group, and the number of PI-positive cells was slightly less than that in the UCNP (1:0) group. A DP ratio of 0.5 was considered too low because few PI-stained cells were detected, and a DP ratio of 0.9 resulted in too many dead cells. Therefore, the DP ratio in this study was set to 0.7.

[0117] Second, a screening step was performed to determine the optimal NP incubation time prior to LED irradiation (Figure 15B). Comparing the results obtained with a UNCP ratio of 1 between 0 and 20 minutes, the number of dead cells increased with increasing incubation time. Therefore, the optimal incubation time was determined to be up to 10 minutes.

[0118] Third, a screening step was performed to determine the optimal dose of posAuNP (Fig. 16). It was confirmed that the number of dead cells increased as the amount of posAuNP increased, with 11.6 μg of posAuNP showing 48% of dead cells. However, because the number of dead cells slightly decreased when UCNPs and posAuNPs were combined in a previous experiment, it was decided not to keep the posAuNP dose too low, and the optimal dose was determined to be 10 μg.

[0119] An analysis was performed to determine the optimal UCNP ratio for UCPPin using posAuNP@UCNPs. First, the efficiency of UCPPin was confirmed by continuous wave irradiation at 980 nm for 0, 10, 15, and 20 min with an intensity of approximately 16 mW (Fig. 5A and Fig. 17). As the irradiation time increased, the number of calcein-positive and PI-positive cells increased. However, the number of PI-positive cells decreased as the UCNP ratio increased. Higher UCPPin efficiencies were observed at posAuNP to UCNP ratios of 1:1, 1:2, 1:5, and 1:10 than at 1:0. The efficiency was highest at 20 min of irradiation time (19.2 J / cm 2 ) was particularly high at a ratio of 1:5 or higher. Under these conditions, a high efficiency of approximately 63% was confirmed. The viabilities at each ratio and stage are shown in Figure 18.

[0120] Next, the optimal pulse interval (PI) was investigated (Fig. 5B). The pulse width (PW) and pulse number (PN) were fixed at 10 min and 2, respectively. The PI was set to 2, 5, and 10 min for comparison. The 2-min PI group showed a high UCPPin efficiency similar to that of the 20-min continuous irradiation time, but the UCPPin efficiency decreased as the PI increased. At AuNP to UCNP ratios of 1:5 and 1:10, the UCPPin efficiency was high without a significant difference in efficiency between the two ratios. However, since treating a small amount is more suitable than treating a large amount, the 1:5 ratio was used. As the irradiation time increased, the electromagnetic energy applied to the cells accumulated, resulting in membrane destruction, dead cells, and higher UCPPin efficiency. However, as the PI lengthened, energy accumulation by cells weakened, and thus the number of dead cells and UCPPin efficiency showed a decreasing pattern.

[0121] The longer the exposure time, the greater the accumulation of electromagnetic energy applied to the cells, leading to membrane destruction, cell death, and increased UCPPin efficiency. Conversely, the longer the PI, the less energy is applied to the cells, leading to a decrease in the number of dead cells and UCPPin.

[0122] Finally, an experiment was conducted to determine whether pulsed waves could be applied to cell spheroids (Fig. 5C). Cell spheroids with a diameter of 150 μm were generated and pulsed at 24 J / cm 2 (PW=20 min, PN=2, PI=2 min, PW=5 min) pulsed. Images were captured using CLSM. The CONT and 1:0 groups showed UCPPin efficiencies of less than 5%. However, the 1:5 group showed an efficiency of approximately 30%, indicating high UCPPin efficiency. Similar results were confirmed using larger spiroids (Fig. 19).

[0123] 2.6. Comparison of the efficacy of UCPPin and PassT in delivering baricitinib for the treatment of osteoarthritis in a three-dimensional model in vitro.

[0124] The therapeutic efficacy of baricitinib (BRN), a STAT1 and STAT3 phosphorylation inhibitor, was confirmed as a treatment for osteoarthritis (OA). To facilitate intracellular drug delivery via posAuNP@UCNPs, an in vitro 3D model was treated with posAuNP@UCNPs (1:5), and passive transport (PassT), another mechanism for drug delivery across the cell membrane, was tested. Suspended C28 / I2 cells were treated with posAuNP@UCNPs, OA inducers (LPS and IL-6), and BRN. The solutions were incubated at 4°C for 10 min, irradiated with a 980 nm LED for 20 min, cultured in 3D, and analyzed for STAT inhibition, IL-6 concentration, and ECM expression levels (Fig. 6A). The level of inhibition of STAT phosphorylation by BRN was confirmed by Western blot (Fig. 6B) and used to determine whether BRN was delivered to cells via UCPPin and PassT. As shown by the inhibition of STAT phosphorylation, BRN was effectively delivered to cells by both UCPPin and PassT, regardless of the presence or absence of IR. The survival rate was measured on day 1 (Fig. 20), and no difference in survival rate was observed between the groups. However, the expression of IL-6, an inflammatory cytokine that acts downstream of STAT, differed between cells delivered with BRN via UCPPin and cells delivered with BRN via PassT. IL-6 expression in PassT-BRN-transduced cells was similar to that in control cells (CONT), but was lower (approximately 40-45%) in UCPPin-BRN-transduced cells (Fig. 6C). Therefore, as evidenced by the level of inhibition of IL-6 expression, UCPPin delivers BRN to cells more efficiently than PassT.

[0125] ECM expression levels were analyzed by Western blot and immunofluorescence staining at 1 and 2 weeks, respectively. At week 1, the expression levels of COL II, SOX9, and MMP13 were analyzed in duplicate for each experimental group. SOX9 is a key transcription factor in chondrogenesis, COLII is a representative ECM, and MMP13 is an enzyme that degrades ECM such as COL II, so it was necessary to confirm the protein expression of these markers. COL II expression was significantly higher in the UCPPin group than in the CONT and PassT groups, especially in the UCPPin / IR+ group (Fig. 6D). MMP13 expression was similar in CONT, PassT, and UCPPin / IR- cells, but decreased in UCPPin / IR+ cells. SOX9 showed the highest expression in the UCPPin / IR- group, but it is a transcription factor that acts at all stages of chondrogenesis and functions to initiate differentiation, so it is more appropriate as a measure of differentiation potential rather than a measure of the degree of differentiation. Therefore, the higher expression of SOX9 in the UCPPin / IR- group may be more significant in terms of differentiation potential than in the CONT group. Furthermore, the UCPPin / IR+ group, like the IR- group, also has significance because it has higher expression than the other groups.

[0126] Figure 6E shows that COL II and aggrecan (AGG) expression increased in the UCPPin group at week 2. COL II is expressed inward from the center of the spheroids in a three-dimensional environment, while AGG is expressed on the outside of the spheroids. Taken together, these results demonstrate that UCPPin / IR-mediated BRN delivery is more effective in promoting ECM expression and tissue formation than PassT-mediated BRN delivery, and that IR further promotes these effects of UCPPin / IR-mediated BRN delivery.

[0127] 3. Conclusion

[0128] In this study, we characterized the morphological and physical properties of posAuNP@UCNPs, and evaluated the changes induced by 980 nm light irradiation of posAuNP@UCNPs. Temporary perforation and resealing of PM were observed after treatment with posAuNP@UCNPs. Furthermore, we compared the efficacy of posAuNP@UCNPs for UCPPin according to IR time and type, and analyzed its effects on C28 / I2 cell suspension and spheroids. Finally, we compared the therapeutic efficacy of UCPPin and PassT for intracellular delivery of baricitinib in an in vitro 3D osteoarthritis model. UCPPin via posAuNP@UCNP nanocomposites demonstrated superior mass delivery than PassT. Although we delivered a small molecule, BRN, this study suggests that UCPPin is not only effective for mass delivery but also has potential for the treatment of osteoarthritis. In the future, UCPPin may be used to deliver RNAs and other macromolecules, further expanding its scope of clinical therapeutic applications.

Claims

1. (i) gold nanoparticles; and (ii) ytterbium (Yb) 3+ ) and erbium (Er 3+ ) for temporary photoperforation of cell membranes, formed by combining reversible nanoparticles.

2. A nanocomposite according to claim 1, characterized in that the gold nanoparticles are positively charged gold nanoparticles.

3. A nanocomposite according to claim 2, characterized in that the average particle diameter of the positively charged gold nanoparticles is 75 to 85 nm.

4. A nanocomposite according to claim 2, characterized in that the positively charged gold nanoparticles are obtained by coating the gold nanoparticles with 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine.

5. A nanocomposite according to claim 4, characterized in that the 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine is obtained by reacting 3,4-dihydroxyphenylalanine with polyethyleneimine through a carbodiimide reaction.

6. A nanocomposite characterized in that the weight ratio of the positively charged gold nanoparticles to the 3,4-dihydroxy-L-phenylalanine-conjugated polyethyleneimine in the fourth paragraph is 1:0.5 to 0.

9.

7. A nanocomposite according to claim 1, characterized in that the average particle diameter of the rechargeable nanoparticles is 30 to 40 nm.

8. A nanocomposite according to claim 1, characterized in that the rechargeable nanoparticle has a molecular formula of NaYYbErF4.

9. A nanocomposite according to claim 1, characterized in that the rechargeable nanoparticles are functionalized with polyethylene glycol-COOH.

10. A nanocomposite characterized in that, in the first paragraph, the rechargeable nanoparticles are formed by combining with 1 to 10 μg of the gold nanoparticles.

11. A nanocomposite characterized in that, in the 10th paragraph, the rechargeable nanoparticles are formed by combining them at a ratio of about 5 μg with respect to 10 μg of the gold nanoparticles.

12. A nanocomposite according to claim 1, characterized in that the temporary photoperforation is formed through light irradiation of about 980 nm.

13. A nanocomposite according to claim 12, characterized in that the light irradiation is performed for 5 to 20 minutes.

14. A pharmaceutical composition for treating osteoarthritis, comprising a combination of a first compartment comprising a nanocomposite according to any one of claims 1 to 13 and a second compartment comprising baricitinib as an active ingredient.

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