Porous silicon nanoparticle-based drug delivery system inducing reactive oxygen species and self-activation thereof, and method for preparing same

Abstract

Proposed is a porous silicon nanoparticle with isothiocyanate moiety conjugated to the surface. The porous silicon nanoparticle with isothiocyanate moiety conjugated to the surface has low side effects due to high biocompatibility, and can generate reactive oxygen species in cells without an external initiator, which can promote decomposition and release of supported drugs.

Claims

1. A drug delivery system comprising: a porous silicon nanoparticles; and an isothiocyanate moiety conjugated to the surface of the porous silicon nanoparticle.

2. The drug delivery system of claim 1, wherein the drug delivery system further comprises a drug or prodrug supported on the porous silicon nanoparticles.

3. The drug delivery system of claim 2, wherein the prodrug is converted into a drug by reactive oxygen species.

4. The drug delivery system of claim 1, wherein the drug delivery system further comprises a tumor homing peptide conjugated to the surface of the porous silicon nanoparticles.

5. The drug delivery system of claim 4, wherein the tumor homing peptide is at least one peptide selected from the group consisting of an RGR-containing peptide, a CGKRK-containing peptide, and a CREKA-containing peptide.

6. The drug delivery system of claim 1, wherein the drug delivery system is represented by the following Formula 1: ##STR00005## In Formula 1, pSiNP is a porous silicon nanoparticle, and the R.sub.1 is an alkyl group having 1 to 8 carbon atoms.

7. The drug delivery system of claim 4, wherein the drug delivery system is represented by the following Formula 2: ##STR00006## In Formula 2, pSiNP is a porous silicon nanoparticle, the R.sub.1 is an alkyl group having 1 to 8 carbon atoms, the THP is a tumor homing peptide, the L is a linker, and X is 0 or 1.

8. A pharmaceutical composition for treating cancer, the composition comprising a porous silicon nanoparticle and a drug delivery system comprising an isothiocyanate moiety conjugated to the surface of the porous silicon nanoparticle as an active ingredient.

9. A method for manufacturing a drug delivery system, the method comprising: preparing porous silicon nanoparticles; and conjugating an isothiocyanate moiety to the surface of the porous silicon nanoparticle.

10. The method of claim 9, wherein the method further comprises supporting a drug or prodrug on the porous silicon nanoparticles.

11. The method of claim 9, wherein the method further comprises conjugating a tumor homing peptide to the surface of the porous silicon nanoparticles.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 shows a process in which a drug delivery system, according to an embodiment, is self-activated in a cell;

[0052] FIG. 2 shows a drug delivery system prepared with TEPITC, pSiNP, JS-11, and CGKRK peptides;

[0053] FIG. 3 shows a process in which a drug delivery system, according to an embodiment, is activated in tumor cells without being activated in the blood to release the drug and shows a synergistic effect leading to an increase in ROS, the release of a prodrug, and activation of a prodrug in the tumor cells;

[0054] FIG. 4 shows the results of confirming the effect of TEPITC on the ROS level in pancreatic cancer cells (BxPC-3) by DCF fluorescence;

[0055] FIG. 5 shows the results of confirming the effect of TEPITC on the ROS level in pancreatic cancer cells (BxPC-3) with a CLSM image;

[0056] FIG. 6 is a result confirming the cytotoxicity of TEPITC;

[0057] FIG. 7A shows the results of confirming the effect of TEPITC or PEITC on Nrf2 expression in BxPC-3 cells by Western blot;

[0058] FIG. 7B shows the results of confirming the effect of TEPITC on Nrf2 expression in BxPC-3 cells by Western blot;

[0059] FIG. 8 shows the results of confirming the effect of TEPITC on Nrf2 expression in BxPC-3 cells by immunofluorescence;

[0060] FIG. 9 shows the results of confirming the effect of TEPITC on HO-1 expression in BxPC-3 cells by Western blot;

[0061] FIG. 10 shows a process for preparing a porous silicon nanoparticle drug delivery system, including an isothiocyanate moiety, a tumor homing peptide, and a prodrug;

[0062] FIG. 11A shows the diameter of pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11);

[0063] FIG. 11B shows the zeta potential of pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11);

[0064] FIG. 12 shows a transmission electron microscope (TEM) image of pSiNP and ROSG-pSiNP (JS-11);

[0065] FIG. 13 shows the ATR-FTIR spectral analysis results of pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11);

[0066] FIG. 14A shows the results of confirming the degree of decomposition of ROSG-pSiNP according to the concentration of hydrogen peroxide;

[0067] FIG. 14B shows the degree of conversion of JS-11 released from ROSG-pSiNP to SN-38 according to the concentration of hydrogen peroxide;

[0068] FIG. 15 is a result of confirming the decomposition and shape change of ROSG-pSiNP according to the environment of hydrogen peroxide by TEM;

[0069] FIG. 16 shows the results of confirming the effect of ROSG-pSiNP (JS-11) on the ROS level in pancreatic cancer cells (BxPC-3) by DCF fluorescence;

[0070] FIG. 17 shows the results of confirming the effect of ROSG-pSiNP (JS-11) on the ROS level in pancreatic cancer cells (BxPC-3) with a CLSM image;

[0071] FIG. 18 shows the results of confirming the effect of ROSG-pSiNP (JS-11) on Nrf2 expression in BxPC-3 cells by Western blot;

[0072] FIG. 19 shows the results of confirming the degree of uptake of pSiNP, pSiNP-NCS, and ROSG-pSiNP (JS-11) in BxPC-3 cells with a CLSM image;

[0073] FIG. 20 shows the results of confirming the cytotoxicity of pSiNP, JS-11, pSiNPs (JS-11), and ROSGpSiNP (JS-11) to the BxPC-3 cell lines;

[0074] FIG. 21 shows the experimental process of administering pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11) to pancreatic cancer xenograft mice;

[0075] FIG. 22 shows the results of intravenous injection of pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11) into pancreatic cancer xenograft mice and confirms the degree of organ and tumor distribution after 2 hours;

[0076] FIG. 23 shows the PL signals of organs and tumor distribution after intravenous injection of pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11) into pancreatic cancer xenograft mice;

[0077] FIG. 24 shows a change in tumor volume after administration of pSiNP, JS_11, pSiNP (JS-11), or ROSG_pSiNP (JS-11) to pancreatic cancer xenograft mice;

[0078] FIG. 25A shows tumor sizes after administration of pSiNP, JS_11, pSiNP (JS-11), or ROSG_pSiNP (JS-11) to pancreatic cancer xenograft mice;

[0079] FIG. 25B is a result of confirming the change in tumor size after administration of pSiNP, JS-11, pSiNP (JS-11), or ROSG-pSiNP (JS-11) to pancreatic cancer xenograft mice;

[0080] FIG. 26 is a result of confirming plasma AST/ALT levels and hepatotoxicity after administration of pSiNP, JS-11, pSiNP (JS-11), or ROSG-pSiNP (JS-11) to pancreatic cancer xenograft mice; and

[0081] FIG. 27 is a result of administering pSiNP, JS-11, pSiNP (JS-11), or ROSG-pSiNP (JS-11) to pancreatic cancer xenograft mice and observing the expression of Nrf2/HO-1 protein in tumor tissue through an immunohistochemistry experiment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present disclosure is not limited to these examples.

EXPERIMENTAL METHOD

[0083] 1-1. Cell Culture

[0084] BxPC-3, AsPC-1, and PANC-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Va., U.S.A.). Cells were cultured in RPMI-1640 supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and streptomycin at 5% CO.sub.2, 37° C. conditions.

[0085] 1-2. Analysis of PEITC/TEPITC-Induced Intracellular ROS Generation

[0086] The intracellular ROS levels of cells were analyzed using the 2′,7′-dichlorofluorosine diacetate (DCFDA/H.sub.2DCFDA) cell ROS detection analysis kit. BxPC-3, AsPC-1, and PANC-1 cells (1×10.sup.5 cells/mL, 100 O/well) were inoculated into 96-well black plates and incubated at 37° C. for 24 hours. The cells were then treated with PEITC or TEPITC at 1.25, 2.5, 5, 10, and 20 μM, respectively, and then incubated at 37° C. for 2 hours. As a positive control group, a set treated with hydrogen peroxide (H.sub.2O.sub.2) was prepared. The cells were then treated with 20 μM DCFDA (100 μl) at 37° C. for 45 minutes. Excitation/emission wavelengths at 485/535 nm were measured with a microplate reader (Fluorostar Omega, BMG Labtech, Germany). Intracellular ROS generation was assessed by CLSM (LSM800, Carl Zeiss, Oberkochen, Germany) imaging using BxPC-3 cells. BxPC-3 cells (1×10.sup.5 cells/mL, 2 mL) were inoculated into confocal plates. After 24 hours incubation, cells were treated with 20 μM PEITC and TEPITC for 2 hours. The cells were then stained with 20 μM DCFDA (200 μL) at 37° C. for 45 minutes, washed 3 times with PBS, and stained with DAPI for 10 minutes. After washing 3 times, the cells were fixed with 4% paraformaldehyde for 20 minutes. The excitation and emission channels are: DAPI (405 nm, 400-520 nm band filter), ROS (488 nm, 400-585 nm band filter).

[0087] 1-3. Porous Silicon Nanoparticles Preparation

[0088] An electrolyte solution in which 48% aqueous hydrofluoric acid (HF) and absolute ethanol were mixed in a volume ratio of 3:1 on a p.sup.++-type single crystal silicon wafer doped with boron at a high concentration was treated and electrochemically etched to generate a porous sacrificial layer. Then, the porous sacrificial layer was removed using 2 M potassium hydroxide solution (KOH).

[0089] Then, a perforated etching waveform was applied to form a porous silicon layer. 300 cycles of low current density pulses of 46 mA cm.sup.−2 for 1.8 seconds and high current density pulses of 334 mA cm.sup.−2 for 0.4 seconds were repeated. Then, the porous silicon layer was lifted off from the silicon wafer by applying a current density of 3 mA cm.sup.−2 in a solution composed of 7.5% aqueous HF in ethanol for 300 seconds to obtain a pSi film.

[0090] The pSi film was placed in a sealed glass vial (22.18 mL size, VWR, Catalog No. 66011-143, Radnor, Pa., USA) containing deionized water (DI H.sub.2O, 6 mL) in an ultrasonic bath (VWR, model number VWRA142-0307, Radnor, Pa., USA) for 24 hours to obtain porous silicon nanoparticles (pSiNPs).

[0091] Porous silicon nanoparticles (pSiNPs) were incubated in DI H.sub.2O (12 mL) at 25° C. for 10 days to form a silicon oxide layer on the surface of the porous silicon nanoparticles, followed by filtering with a 0.22 μm syringe filter (Millipore, Millex syringe filter unit, 220 nm model number SLGP033RS, Burlington, Mass., USA). The filtered pSiNPs were then collected by centrifugation (14,000 rpm, 30 minutes) and redispersed/washed with EtOH (3 times).

[0092] 1-4. Preparation of pSiNP (JS-11)

[0093] pSiNP (to 1 mg) and JS-11 stock solution (20 mg/mL DMSO, 50 μL) were dispersed in EtOH (1.45 mL) and was mixed at 25° C. for 24 hours with a vortex mixer (600 rpm, Scientific Industries, Inc., Vortex-Genie 2, model No. SI-0246, Bohemia, N.Y., USA). The pSiNP loaded with JS-11 was centrifuged (14,000 rpm, 15 minutes) and washed with EtOH (3 times) to remove unloaded free JS-11.

[0094] To analyze the JS-11 loading efficiency, the supernatant from each centrifugation step was analyzed by measuring UV/vis absorption spectra using a spectrophotometer (Agilent Technologies Cary 8454, U.S.A.).

[0095] pSiNP supported with JS-11 (pSiNP (JS-11), 1 mg/100 μl DI H.sub.2O) was mixed with a 2 M CaCl.sub.2 stock solution (900 μl) for 90 minutes to prevent the release of JS-11 during the surface modification step. pSiNP (JS-11) was purified by centrifugation (14,000 rpm, 15 min) using DI H.sub.2O, 30% EtOH, 70% EtOH, and 100% EtOH.

[0096] 1.5 Preparation of ROSG-pSiNP (JS-11)

[0097] pSiNP (JS-11) (to 1 mg) and 3-aminopropyldimethylethoxysilane (APDMES, 20 μl) were mixed in EtOH (1 mL) and was vortex-mixed at 25° C. for 4 hours using vortex mixer (600 rpm, Scientific Industries, Inc., Vortex-Genie 2, Model No. SI-0246, Bohemia, N.Y., U.S.A.).

[0098] Amine-terminated nanoparticles were separated by centrifugation (14,000 rpm, 15 minutes), washed with EtOH (3 times), and then mixed 100 μl of maleimide-PEG-N-hydroxysuccinimide (MAL-PEG-NHS, MW=5 kDa)(10 mg/mL in EtOH) and reacted at 25° C. for 2 hours. The resulting particles were centrifuged (14,000 rpm, 15 minutes) and washed with EtOH (3 times) to remove unreacted MAL-PEG-NHS. The resulting particles were resuspended in EtOH (1 mL), and 3-isothiocyanatopropyltriethoxysilane (TEPITC, 20 μl) was added. The mixture was vortex-mixed at 25° C. for 4 hours using vortex mixer (600 rpm, Scientific Industries, Inc., Vortex-Genie 2, model number SI-0246, Bohemia, N.Y., USA), then centrifuged (14 000 rpm, 15 minutes) and washed with EtOH 3 times.

[0099] For targeting peptide conjugation, nanoparticle-containing EtOH (100 μL) was added to a CGKRK peptide stock solution (1 mg/mL in DI H.sub.2O, 100 μL) and incubated at 4° C. for 4 hours. Finally, the resulting nanoparticles were purified by centrifugation with EtOH (3 times) and dispersed in DI H.sub.2O (10 μl, stored at 4° C. before use).

[0100] 1-6. Nanoparticle Characterization

[0101] The fluid-dynamic size and zeta potential of pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11) were analyzed with a Malvern Instruments Zetasizer Nano ZS90 (Worcestershire, UK). The shape of nanoparticles was observed with a transmission electron microscope (TEM, Tecnai, G2 F30ST, FEI Company, OR, U.S.A.) at the Korea Basic Science Center (Korea University, Seoul, Korea). Attenuated total reflection Fourier transform infrared spectroscopy (FT-IR spectroscopy) results were obtained with a Thermo Scientific Nicolet iS 5 FT-IR spectrometer instrument (64 scans, Waltham, Mass., U.S.A.).

[0102] 1-7. CLSM Analysis for Cellular Uptake

[0103] The intracellular distribution of nanoparticles and intracellular ROS analysis were evaluated using BxPC-3 cells and confocal laser scanning microscopy (CLSM). Cells were inoculated in confocal dishes containing 2 mL of RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin at a density of 1×10.sup.5 cells/mL. After incubation at 37° C. for 24 hours, the medium was replaced with RPMI-1640 containing 100 μg/mL fluorescein-loaded pSiNP, fluorescein-loaded pSiNP containing TEPITC functionalization, and fluorescein-loaded pSiNP containing TEPITC/CGKRK functionalization. After 24 hours, the cells were washed 3 times with PBS (pH 7.4), and dyes (DAPI and cell mask plasma membrane) were used for nuclear and membrane staining. After washing 3 times with PBS (pH 7.4), the cells were fixed with 4% paraformaldehyde and incubated for 20 minutes. The excitation and emission channels are: DAPI (405 nm, 400-520 nm band filter), fluorescein (488 nm, 400-585 nm band filter) and cell membrane (640/670±20 nm).

[0104] 1-8. Analysis of Intracellular ROS Generation Induced by Nanoparticles

[0105] Nanoparticle-induced intracellular ROS was measured using the DCFDA/H2DCFDA Cell ROS Detection Assay Kit according to the manufacturer's protocol. BxPC-3 cells (1×10.sup.5 cells per well) were inoculated in 96-well black plates and incubated at 37° C. for 24 hours. The cells were then treated with 6.25, 12.5, 25, 50, and 100 μg/mL of pSiNP, JS-11, pSiNP (JS-11), and ROSG-pSiNP (JS-11) and incubated for 5 hours (the amount of JS-11 was calculated using the loading efficiency of pSiNP (JS-11)). After incubation, 20 μM DCFDA reagent (100 l) was added to each well, followed by incubation at 37° C. for 45 minutes.

[0106] Fluorescence excitation/emission wavelengths (485/535 nm) were measured using a microplate reader (FLUOstar Omega, BMG LABTECH, Germany). Nanoparticle-induced intracellular ROS generation was monitored by CLSM imaging of BxPC-3 cells.

[0107] Cells (1×10.sup.5 cells/mL, 2 mL) were inoculated in confocal dishes. After incubation for 24 hours, cells were treated with 100 μg/mL pSiNP and ROSG-pSiNP (except JS-11) at 37° C. for 2 hours and then stained with 20 μM DCFDA (200 μL) at 37° C. for 45 minutes. Cells were washed 3 times with PBS and then stained with DAPI for 10 minutes. After washing 3 times, the cells were fixed with 4% paraformaldehyde for 20 minutes. Finally, intracellular ROS was observed. The excitation and emission channels were DAPI (405 nm, 400-520 nm band filter) and ROS (488 nm, 400-585 nm band filter), respectively.

[0108] 1-9. Laboratory Animal Preparation

[0109] BALB/c nude mice (females and males, 4 weeks and 5 weeks old) were commercially obtained from Nara Co., Ltd., Gyeonggi-do, Korea. BxPC-3 cells (5×10.sup.6 cells, suspended in 100 μl mixture of RPMI medium and Matrigel, 1:1) were injected subcutaneously behind the flank of each mouse. All animal experiments were performed according to the guidelines of the Korea Kyunghee University Animal Care Committee (IACUC).

[0110] 1-10. Biodistribution Analysis

[0111] IVIS imaging was performed to confirm the biodistribution of nanoparticles in xenograft mice. Mice with xenograft tumors were randomly assigned to 4 groups (females, n=3 per group). IVIS imaging experiments were performed when the longest diameter of the subcutaneous tumor reached to 10 mm. Mice in each group were divided into the following four groups. (1) control group (PBS intravenous injection (i.v.)), (2) pSiNP group (pSiNP intravenous injection), (3) pSiNP (JS-11) group (pSiNP (JS-11) intravenous injection), and (4) ROSG-pSiNP group (JS-11) (ROSG-pSiNP (JS-11) intravenous injection). Particles (20 mg/kg in 100 μl PBS suspension) were injected intravenously (i.v.) into mice and mice were sacrificed 2 hours later. The fluorescence signals of the resected tumor/major organs (lung, heart, liver, spleen, kidney) were measured using the VISQUE In Vivo Smart LF luminescence and fluorescence animal imaging system (Vieworks Co., Ltd., Korea), and GFP excitation (λ.sub.ex=390 to 490 nm) and an ICG emission filter (λ.sub.em=810 to 860 nm) were used.

[0112] 1-11. Therapeutic Efficacy Assay

[0113] Tumor-bearing mice were randomly divided into 5 groups (n=5 per group). (1) PBS only, (2) pSiNP only, (3) JS-11, (4) pSiNP (JS-11), and (5) ROSG-pSiNP (JS-11) were administered by intravenous injection (i.v.) 4 times at intervals of 3 days at a dose of 20 mg/kg, 100 μl each.

[0114] The mouse tumor size was recorded every 3 days until the end of the experiment, and the tumor volume was calculated by the formula V=[(tumor length)×(tumor width).sup.2]/2.44. At the end of the experiment (day 22), the body of each mouse was photographed with a digital camera, and body weight was measured.

[0115] The experiment was terminated considering the conditions of the control group according to the Guidelines for Endpoint Monitoring and Humane Termination of the IACUC. Tumors of each mouse were collected after whole blood perfusion using ethylenediaminetetraacetic acid (EDTA) coated tubes after anesthesia.

[0116] Tumor growth inhibition T/C ratio (ratio of mean tumor weight of treatment group to control group) was used to calculate treatment efficacy. The tumor growth inhibition T/C ratio equation is as follows. T/C ratio=(T.sub.t/T.sub.0)/(C.sub.t/C.sub.0), where T.sub.t=median tumor volume treated at time t, T.sub.0=median tumor volume treated at time 0, C.sub.t=median tumor in control group at time t volume, and C.sub.0=median tumor volume of the control group at time 0).

[0117] For histological analysis, dissected organs (liver, kidney, spleen, heart, and lung) and tumors were immersed in 10% formalin solution. After blood coagulation, serum was separated by centrifugation at 4,000×g for 30 minutes immediately for hematological analysis. All experiments using animals were performed according to the guidelines and protocols approved by IACUC, Kyunghee University, Seoul, Korea.

<Example 1> Nanoparticle Design for Self-Activation Therapy

[0118] Based on ROS generation by ITC, ROSG-pSiNP-(JS-11), a self-activating therapeutic nanoparticles (SATN) system consisting of the following four main components was prepared. (See FIG. 2)

[0119] (i) 3-isothiocyanatopropyltriethoxysilane (TEPITC): it is a ROS generating moiety. The TEPITC moiety can be functionalized on the surface of pSiNPs via a condensation reaction in an ethanol solvent. Specifically, the pSiNP surface can be functionalized through the condensation reaction of triethoxysilane ((C.sub.2H.sub.5O).sub.3Si) of the TEPITC moiety and silanol (Si—OH) of the pSiNP surface in an ethanol solvent. According to one embodiment, the surface-functionalized pSiNP may be represented by Formula 3 below:

##STR00004##

[0120] (ii) pSiNP (porous Si Nanoparticle): it is a drug delivery platform that is degraded in response to ROS. pSiNP can be decomposed in a physiological environment and can accelerate decomposition when exposed to ROS.

[0121] (iii) JS-11: it is a ROS-reactive prodrug conjugated with mefenamic acid and SN-38. The mefenamic acid ester moiety of JS-11 can be cleaved by ROS to release the anticancer drug SN-38. Double-shielded formulations by prodrug formulation and nanoparticle encapsulation can increase the therapeutic effect of SN-38 and reduce side effects.

[0122] (iv) CGKRK: it is a cancer-targeting peptide. CGKRK has a high binding affinity for neovascular endothelial cells, so it can strengthen the intravascular penetration of nanoparticles and also enhance cell penetration of nanoparticles.

[0123] The nanoparticles combined with (i) to (iv) were expected to cause an ensemble action of cancer-targeting, in situ ROS self-generation for ROS-mediated treatment, and ROS-mediated self-activation (decomposition of nanoparticles, release of prodrugs, and conversion to drugs of released prodrugs) (refer to FIG. 3). A drug delivery system capable of self-activation and turn on-type drug release within a target can solve the problems of drug release due to non-specific degradation and non-cancer targeting of the existing DDS platform.

<Example 2> Intracellular ROS Upregulation and Cytotoxicity of the Isothiocyanate Moiety

[0124] 2-1. Upregulation of Intracellular ROS of Isothiocyanates

[0125] Cells were contacted with ITC, and intracellular ROS generation was assessed. Human pancreatic cell lines BxPC-3, AsPC-1, and PANC-1 were treated with TEPITC, and ROS levels were measured using the DCFDA/DCF kit. When 2′,7′-dichlorofluorescein diacetate (DCFDA) diffuses into cells, it is deacetylated to H.sub.2DCF by cellular esterase, and when H.sub.2DCF comes into contact with ROS, it is converted to green fluorescent DCF Phenethyl isothiocyanate (PEITC) and H.sub.2O.sub.2 were used as positive control groups. Each chemical substance (TEPITC, PEITC, and H.sub.2O.sub.2, concentration=0 to 20 μM) were treated on the cell line and incubated for 2 hours.

[0126] According to FIG. 4, cells treated with TEPITC showed a significant increase in fluorescence within the cytoplasm, indicating that the in situ ROS level was upregulated. Compared with before chemical treatment, the fluorescence intensity-based enhancement factor was 6.34 times higher in the TEPITC (20 μM) treatment group and 3.67 times higher in the PEITC (20 μM) treatment group. According to fluorescence analysis and confocal laser scanning microscopy (CLSM) images, TEPITC upregulates the intracellular ROS level, and the effect was superior to PEITC, which is known for its anticancer effect. (See FIG. 5).

[0127] 2-2. Cytotoxicity of Isothiocyanates

[0128] Next, the cytotoxicity of TEPITC was analyzed. BxPC-3 cells were treated with 0 to 10 μM of TEPITC or PEITC and incubated for 24 hours, and cytotoxicity and viability were analyzed by CCK-8 assay. According to FIG. 6, both TEPITC and PEITC showed similar cytotoxicity results in a dose-dependent manner, and the cytotoxicity of BxPC-3 cells treated with 10 μM TEPITC was significantly increased up to 80% compared to the untreated group cells. Similar results were observed in other cell lines including AsPC-1 and PANC-1.

[0129] 2-3. Relationship of Isothiocyanates with Nrf2/HO-1 Oxidative Stress Signaling

[0130] Oxidative stress-related signaling pathways were analyzed to confirm the association between TEPITC treatment and intracellular ROS upregulation. PEITC was used as a positive control group. PEITC is known to induce Nrf2 accumulation in human prostate cancer PC-3 cells and increase the expression of endogenous heme oxygenase-1 (HO-1) protein.

[0131] According to FIG. 7A, BxPC-3 cells were treated with TEPITC 20 μM or PEITC 20 μM and incubated for 0 to 4 hours, as a result of incubation, the expression of a regulator of cell resistance to oxidative stress, nuclear factor erythroid 2-related factor 2 (Nrf2), was significantly increased. Both the TEPITC-treated group and the PEITC-treated group showed the greatest increase in Nrf2 expression when 30 minutes had elapsed after incubation. It is noteworthy that the expression of Nrf2 in the TEPITC treatment group was maximum 30 minutes after incubation began but decreased from 1 hour later. This indicates that the stimulation of TEPITC on the oxidative stress signaling pathway of cells may be subject to time constraints. According to FIG. 7B, as a result of treatment with TEPITC at a concentration of 0 to 20 μM, an increase in Nrf2 level by TEPITC was observed to be concentration-dependent in the range of 2.5 to 10 μM.

[0132] CLSM imaging was used to verify that TEPITC induces nuclear translocation of cytoplasmic Nrf2. BxPC-3 cells were treated with TEPITC (0 to 10 μM), incubated for 30 minutes, and visualized by treatment with Nrf2 antibody and DAPI.

[0133] According to FIG. 8, the immunofluorescence results were consistent with the Western blot results. An increase in Nrf2 expression in the cytoplasm was observed in the TEPITC (2.5 μM) treatment group, and in the nucleus Nrf2 expression was observed in the TEPITC (5 μM) treatment group at a concentration twice as high. The Nrf2 signal in the nucleus was weakened in the TEPITC (10 μM) treatment group at a concentration four times higher than this, and the Nrf2 signal appeared mainly in the cytoplasm.

[0134] The expression level of HO-1, an antioxidant enzyme essential for cell protection against oxidative stress, was also analyzed. Cells were treated with TEPITC (5 μM) and incubated for 0 to 6 hours. According to FIG. 9, the expression level of intracellular HO-1 was significantly increased by TEPITC. This is thought to be because Nrf2 is a transcription factor that regulates the expression of HO-1. The level of Nrf2 was maximum at 0.5 to 1 hour points, whereas the expression amount of HO-1 was the highest at 2 hours and 4 hours, but decreased from 6 hours.

[0135] In summary, isothiocyanate may affect the Nrf2/HO-1 signaling system by increasing the intracellular ROS level and may have a cancer cell killing effect by exhibiting cytotoxicity.

<Example 3> Preparation and Characterization of ROSG-pSiNP (JS-11)

[0136] Based on the results of intracellular ROS generation by TEPITC, a SATN formulation was prepared in which TEPITC was functionalized on the surface of pSiNP and containing the ROS-responsive prodrug JS-11 in the pores of pSiNP.

[0137] A preparing process will be described with reference to FIG. 10. A porous silicon film was prepared by electrochemically etching a p.sup.++-type silicon wafer doped with boron at a high concentration, and pSiNPs were prepared by ultrasonic fragmentation. The prodrug JS-11 was mixed with pSiNPs together with calcium ions (Ca.sup.2+) and vortex-mixed to load JS-11 into the pores of pSiNP. (3-aminopropyl)-dimethylethoxysilane (APDMES) was treated on pSiNP (JS-11) to modify the surface of pSiNP to generate a primary amine group. A polyethylene glycol (PEG) linker, maleimide-PEG-N-hydroxysuccinimide (MAL-PEG-NHS) (5 kDa), was conjugated to the primary amine on the surface of pSiNP (JS-11) through amide coupling. Next, TEPITC was grafted onto the nanoparticle surface through hydrolytic condensation. Finally, the CGKRK tumor-targeting peptide was conjugated by a thiolene coupling reaction between the maleimide terminus of PEG and the cysteine terminus of the peptide. The prepared nanoparticles were named ROSG-pSiNP (JS-11).

[0138] When ROSG-pSiNP (JS-11) is administered, ROSG-pSiNP (JS-11) may be accumulated in the tumor site due to the homing property of CGKRK, and penetrates into the cancer cell to generate in situ ROS. By inducing degradation of pSiNP by ROS, a prodrug supported thereon is released, and finally, since the prodrug can be converted into a drug by ROS, a cancer cell-specific and high therapeutic effect and reduced side effects can be expected.

[0139] JS-11 was synthesized by a carbodiimide coupling reaction between mefenamic acid and 7-ethyl-10-hydroxycamptothecin (SN-38). (yield 95.5%). Mefenamic acid is a class of nonsteroidal anti-inflammatory drugs (NSAIDs) used to treat pain. JS-11 where mefenamic acid and SN-38 are ester-bonded can be cut by H.sub.2O.sub.2 treatment and converted to SN-38, and hence can be used as a prodrug reacting to ROS. The purity of JS-11 was confirmed by .sup.1H/.sup.13C nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS).

[0140] To confirm whether JS-11 is converted to SN-38 by ROS, the change in the emission spike of JS-11 using H.sub.2O.sub.2 at 37° C. was measured. Since JS-11 has no electron push-pull capability, there is no emission in aqueous media but decomposes upon exposure to H.sub.2O.sub.2 and fluoresces at 560 nm. JS-11 reacted only with H.sub.2O.sub.2 and did not show the conversion to SN-38 under various pH conditions (pH 3 to 10), metal ions, and biomolecules.

[0141] The diameter and zeta potential of the pSiNP nanoparticles were measured. According to FIG. 11, it was seen that the average fluid-dynamic diameter of pSiNPs was uniform (172.2±56.7 nm, polydispersity index (PDI)=0.105). As a result of measuring the zeta potential by dynamic light scattering (DLS), it showed a negative zeta potential value of −35.4±7.75 mV. JS-11-loaded nanoparticles, pSiNPs (JS-11), slightly increased in size (194.3±86.7 nm, PDI=0.229) and slightly decreased in negative surface charge (−26.7±7.66 mV). The APDMES-modified pSiNP (JS-11) had a significant change in zeta potential (25.9±9.22 mV) due to the positive primary amine functional group of APDMES. MAL-PEG-NHS-conjugated pSiNPs (JS-11) showed a slightly reduced zeta potential (13.7±5.7 mV). Nanoparticles to which TEPITC was attached did not show a significant change in surface charge (9.4±4.2 mV). The final formulation, ROSG-pSiNP (JS-11), in which both TEPITC and CGKRK peptides were conjugated, showed an increased particle size (303.2±84.2 nm, PDI=0.318) with a slight surface charge (0.61±3.31 mV).

[0142] In the environment of phosphate buffer saline (PBS, pH 7.4) and cell culture medium (DMEM), ROSG-pSiNP (JS-11) has a similar size distribution (PBS: 337.7±81.4 nm, DMEM: 350.8±90.9 nm), showing a negative surface charge (PBS:4.69±7.2 mV, DMEM:5.64±6.2 mV).

[0143] ROSG-pSiNP (JS-11) showed no particle aggregation or rapid decomposition in DI H.sub.2O, PBS, and DMEM. This indicates the high colloidal stability of ROSG-pSiNP (JS-11).

[0144] FIG. 12 shows a transmission electron microscope (TEM) image of pSiNP and ROSG-pSiNP (JS-11). According to FIG. 12, the uniformity of the shape and size of pSiNP was maintained even after drug loading and ROS generating moiety/peptide conjugation.

[0145] According to FIG. 13, the attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectrum of pSiNPs showed two bands of 3550 to 3200 cm.sup.−1 corresponding to ν(OH) stretching and 1065 cm.sup.−1 corresponding to ν(Si—O) stretching. After drug loading, the pSiNP (JS-11) formulations show the bands of 2950 to 2860 cm.sup.−1 corresponding to ν(C—H) stretching vibration of the aliphatic chain, 1710 to 1680 cm.sup.−1 corresponding to ν(C═O) stretching, and 1310 to 1250 cm.sup.−1 corresponding to the stretching vibration for ν(C—O) of the aromatic ester. This suggests that JS-11 was successfully loaded onto pSiNP. ROSG-pSiNP (JS-11) nanoparticles showed characteristic bands at 2140 and 1650 cm.sup.−1. This is due to the bending mode of isothiocyanate and the stretching vibration of the amide bond, indicating successful conjugation of the TEPITC and CGKRK peptides.

[0146] ROSG-pSiNP (JS-11) showed a loading efficiency of JS-11 at to 26.7±2.3%. The loading efficiency of JS-11 was calculated by Equation 1 below.

[00001]$\begin{array}{cc}\mathrm{loading}\mathrm{efficiency}\left(\%\right)=\frac{W^{\mathrm{totalJS}-11}-W^{\mathrm{unloaded}-\mathrm{loadedJS}-11}}{W^{\mathrm{loadedJS}-11}+W^{\mathrm{NPs}}}\times 100& \left[\mathrm{Equation}1\right]\end{array}$

[0147] The concentration of unloaded JS-11 was calculated from the standard absorption curve. To determine whether JS-11 loaded on nanoparticles was not released during the surface modification step of pSiNP (JS-11), each surface modification step [(1) calcium treatment, (2) APDMES modification, (3) NHS-PEG-MAL conjugation, (4) TEPITC modify, (5) CGKRK peptide conjugation] were analyzed by measuring UV/vis absorption spectra.

[0148] Since the characteristic absorption peak of JS-11 was not observed in each surface modification step, it was confirmed that JS-11 loaded on pSiNP was not released during the surface modification step.

[0149] After the characterization of nanoparticles, ROSG-pSiNP were incubated with a small amount of H.sub.2O.sub.2 at 37° C. to determine whether particle degradation was induced by ROS and whether JS-11 was converted to SN-38 by ROS. pSiNP exhibit absorption at 405 nm by the Si backbone, and the transparency of pSiNP increases as it decomposes.

[0150] According to FIG. 14A, the absorption intensity of pSiNPs decreased over time (0 to 60 minutes) due to decomposition, and the decrease was increased as the concentration of H.sub.2O.sub.2 (0 to 1000 PM) increased.

[0151] The JS-11 release and SN-38 conversion profiles of ROSG-pSiNP (JS-11) were analyzed. ROSG-pSiNP (JS-11) was incubated in PBS (pH 7.4) containing H.sub.2O.sub.2 (1 mM), and the fluorescence signal of SN-38 was measured at regular time intervals (0 to 24 hours).

[0152] According to FIG. 14B, in the experimental group with H.sub.2O.sub.2, 90% or more of SN-38 was converted after 24 hours, whereas in the experimental group without H.sub.2O.sub.2, less than 20% of SN-38 was converted even after 24 hours.

[0153] Additionally, according to the TEM analysis result of FIG. 15, after 1 hour has elapsed after treatment of pSiNP with PBS or H.sub.2O.sub.2, there was no change in the shape of the nanoparticles in PBS, but decomposition proceeded in H.sub.2O.sub.2, and the shape was significantly changed. As a result, it can be seen that the degradation of pSiNP is accelerated when exposed to ROS. This is related to the formation of silanol (Si—OH) on the surface by ROS and conversion to orthosilicic acid (Si(OH).sub.4).

<Example 4> In Vitro Cytotoxicity Evaluation of ROSG-pSiNP (JS-11)

[0154] According to the in vitro analysis of the Example 3, a theoretical basis was prepared that ROSG-pSiNP (JS-11) could release the drug after self-activation by ROS within the tumor.

[0155] DCFDA/DCF analysis was performed to confirm the intracellular ROS generating ability of ROSG-pSiNP (JS-11) on BxPC-3 cells. BxPC-3 cells were treated with pSiNP, JS-11, pSiNP (JS-11), and ROSG-pSiNP (JS-11) (nanoparticle concentration of 0 to 100 μg/mL), respectively, and incubated for 5 hours.

[0156] According to FIG. 16, in the ROSG-pSiNPs (JS-11) treatment group, the intensity of DCF fluorescence signal increased in a dose-dependent manner. The ROS level of cells treated with 100 μg/mL of ROSG-pSiNP (JS-11) was 8.3 times higher than that of the PBS control group, and the value was consistent with the ROS level of cells treated with 325 μM H.sub.2O.sub.2. In addition, it was confirmed that the ROSG-pSiNP formulation without JS-11 had ROS generating properties.

[0157] According to the CLSM cell imaging of FIG. 17, in the experimental group treated with ROSG-pSiNP, it was observed that the fluorescence signal of DCF was higher than that of the experimental group treated with PBS and pSiNP. This was consistent with the results of cytotoxicity analysis and fluorescence signal analysis.

[0158] To confirm the hypothesis that ITC surface-modified nanoparticles increase intracellular ROS levels, BxPC-3 cells were incubated at 37° C. for 6 hours with ROSG-pSiNP (JS-11) (concentration 0 to 100 g/mL), and GSH levels and glutathione peroxidase (GPx) enzyme activity were analyzed. In cells treated with ROSG-pSiNP (JS-11), GSH was depleted up to 75% in a dose-dependent manner, and intracellular GPx activity was also significantly reduced. These results indicate that ITC functionalized nanoparticles (ROSG-pSiNP (JS-11)) not only deplete intracellular GSH levels but also inhibit GPx activity, leading to severe ROS accumulation in BxPC-3 cells.

[0159] According to FIG. 18, in the ROSG-pSiNP treated group, Nrf2 expression appeared 2 hours after the start of incubation, and the results were consistent with the DCF analysis results. Analysis of CLSM imaging and Nrf2 expression levels using pSiNP without JS-11 loading confirmed that intracellular oxidative stress and ROS generation were not induced by JS-11.

[0160] The cancer-targeting ability of ROSG-pSiNP (JS-11) was further investigated. BxPC-3 cells were incubated with (i) fluorescein loaded pSiNP, (ii) fluorescein loaded TEPITC functionalized pSiNP, and (iii) fluorescein loaded TEPITC/CGKRK functionalized pSiNP at 37° C. for 24 hours. DAPI staining and cell mask plasma membrane staining were performed, and the fluorescence signal of fluorescein was analyzed using confocal laser scanning microscopy (CLSM) imaging, and the cellular uptake and intracellular localization of nanoparticles were confirmed.

[0161] According to FIG. 19, targeted nanoparticles, ROSG-pSiNP (fluorescein loaded pSiNP with TEPITC/CGKRK functionalization), were found to have higher absorption in BxPC-3 cells than non-targeted nanoparticles (fluorescein loaded pSiNP and fluorescein loaded TEPITC functionalized pSiNP). In images containing DAPI and cell mask plasma membrane dye, the fluorescein signal (green) was mainly observed in the cytoplasm, which means that particles penetrated the cell efficiently.

[0162] Next, the cytotoxicity of pSiNP, JS-11, pSiNPs (JS-11), and ROSG-pSiNP (JS-11) was evaluated for BxPC-3 cell lines. The throughput of pSiNP (JS-11) and ROSG-pSiNP (JS-11) was corrected with the concentration of JS-11 (0 to 10 μM).

[0163] According to FIG. 20, the negative control pSiNP had very low cytotoxicity, but the rest showed significant toxicity in a dose-dependent manner. The group treated with ROSG-pSiNP (JS-11) showed the highest toxicity. The results were as follows: (i) at low concentration (0.625 μM JS-11): JS-11 was 21.6%; pSiNP (JS-11) was 31.11%; and ROSG-pSiNP (JS-11) is 42.5%, (ii) at high concentration (10 μM JS-11): JS-11 was 42.4%; pSiNP (JS-11) was 54.3%; and ROSG-pSiNP (JS-11) was 79.1%.

[0164] The cytotoxicity of two nano-formulations, pSiNP-NCS (without JS-11 and TEPITC-modifying) and pSiNP-NCS/CGKRK (without JS-11 and CGKRK-modifying), was further analyzed to confirm the synergistic cytotoxic effect of JS-11 and TEPITC. At low concentration (0.625 μM JS-11, 12.5 μg/mL nanoparticles), pSiNP-NCS and pSiNP-NCS/CGKRK showed lower toxicity than nanoparticles containing JS-11 nano-formulation. pSiNP-NCS was 16.7% and pSiNP-NCS/CGKRK was 33.1%, while pSiNP (JS-11) was 31.11% and ROSG-pSiNP (JS-11) was 42.5%. At high concentration (10 μM JS-11, 100 μg/mL nanoparticles), pSiNP-NCS (53.1%) and pSiNP-NCS/CGKRK (67.1%) showed lower toxicity than that of ROSG-pSiNP (JS-11) (79.1%).

[0165] The higher toxicity of pSiNPs (JS-11) than JS-11 was due to the improved cell penetration, and the higher toxicity of ROSG-pSiNPs (JS-11) is due to their higher toxicity than pSiNPs (JS-11) and nanoparticles without JS-11 (pSiNP-NCS and pSiNP NCS/CGKRK). This is thought to cause a synergistic effect by the combination of cell penetration improvement by CGKRK peptide, in situ ROS generation by TEPITC moiety, and conversion from JS-11 to SN-38 in cells.

<Example 5> In Vivo Anti-Cancer Effect of ROSG-pSiNP (JS-11)

[0166] The cancer-specific therapeutic efficacy of ROSG-pSiNP (JS-11) has been demonstrated in the xenograft mice model for pancreatic cancer.

[0167] First, the hemolytic properties of ROSG-pSiNP (JS-11) were investigated to confirm safety during intravenous injection. Red blood cells were incubated at 37° C. for 1 hour in a solution containing 0.1 mg/mL of ROSG-pSiNP (JS-11). PBS and Triton X-100 were used as negative and positive control groups, respectively. ROSG-pSiNP (JS-11) showed a negligible hemolysis rate of <1% in red blood cells (p<0.001), thus confirming excellent safety for intravenous injection.

[0168] Next, the biodistribution of ROSG-pSiNP (JS-11), pSiNP (empty), and JS-11-loaded pSiNP was confirmed. The nanoparticles were administered intravenously (i.v.) at a dose of 20 mg/kg to pancreatic cancer model mice. (See FIG. 21). 1 hour and 2 hours after nanoparticle injection, internal organs (heart, lung, liver, kidney, and spleen) and tumors were obtained and analyzed for pSiNP intrinsic photoluminescence (PL).

[0169] For organs and tumors 1 hour after injection, the control group (PBS injected) did not show tissue autofluorescence signals, but all nano-formulations (pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11)) showed a strong signals in at the heart, lung, liver and kidney compared to the tumor site.

[0170] However, 2 hours after injection, accumulation in the liver and kidneys decreased dramatically. In addition, according to FIGS. 22 and 23, the non-targeted nano-formulations (pSiNP and pSiNP (JS-11)) showed stronger PL signals in the heart, lung, kidney, and liver than in tumor, but the targeted nano-formulation ROSG-pSiNP (JS-11) showed a decreased signal in organs and a stronger signal within the tumor site.

[0171] The process of ROSG-pSiNP (JS-11) removal in vivo was analyzed. ROSG-pSiNP (JS-11) was intravenously injected into BALB/c mice at a dose of 20 mg/kg, and the mice were sacrificed after 2 hours and 24 hours circulation. Major organs (lung, heart, liver, spleen, and kidney) were obtained and the PL signal of pSiNP was measured. At the 2-hour cycle time point, ROSG-pSiNP (JS-11) was mainly accumulated in the liver and kidney. However, at the time of 24-hour circulation, ROSG-pSiNP (JS-11) accumulated in the organ was significantly removed. Since the removal mechanism of pSiNP is known to be excreted after decomposition into soluble silicic acid (Si(OH).sub.4), it is thought that ROSG-pSiNP (JS-11) will also be removed from the body by the same mechanism.

[0172] Finally, the cancer treatment efficacy of ROSG-pSiNP (JS-11) was evaluated in pancreatic cancer xenograft model mice. To induce pancreatic tumors, BxPC-3 cells were injected subcutaneously behind the flank of mice on day 0. The tumor size after 4 weeks of injection of BxPC-3 cells was about 150 mm.sup.3. On days 34, 37, 40, and 43 after tumor injection, PBS, JS-11, pSiNP (JS-11), and ROSG-pSiNP (JS-11) were administered to mice with pancreatic tumors at doses of 20 mg/kg via tail veins. Changes in tumor volume and body weight in mice were monitored, and toxic effects were investigated for practical applications.

[0173] According to FIG. 24, the experimental group administered with ROSG-pSiNP (JS-11) showed a significant decrease in tumor volume as of day 51 compared to the experimental group administered with empty pSiNP, JS-11, and pSiNP (JS-11).

[0174] According to FIG. 25, pSiNP and pSiNP (JS-11) slightly reduced tumor size, whereas ROSG-pSiNP (JS-11) dramatically reduced tumor size. The mean tumor volume of the ROSG-pSiNP (JS-11) treated group was 93.5±22.7 mm.sup.3, whereas the mean tumor volume of the pSiNP, JS-11, and pSiNP (JS-11) treated groups was 259.6±57.8 mm.sup.3, 183.1±40.8 mm.sup.3, and 179.0±37.6 mm.sup.3, respectively. No significant body weight change occurred in all mice injected with nanoparticles.

[0175] The hepatotoxicity of pSiNP, JS-11, pSiNP (JS-11), and ROSG-pSiNP (JS-11) was analyzed by measuring plasma levels of alanine transferase (ALT) and aspartate transaminase (AST), which are hepatotoxicity markers. 20 mg/kg of nanoparticles (pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11)) and corresponding concentration of JS-11 (5.2 mg/kg) were injected into the tail vein of pancreatic cancer xenograft model mice. On day 51, after BxPC-3 cell inoculation, plasma AST/ALT levels were analyzed.

[0176] According to FIG. 26, hepatotoxicity was not shown in the PBS, pSiNP, pSiNP (JS-11), and ROSG-pSiNP (JS-11) treatment groups (AST<200 U/L, ALT<60 U/L), but the JS-11 treatment groups had 2.8 and 3.5 times higher AST levels than the PBS control groups, respectively. According to the experimental results, since pSiNP (JS-11) and ROSG-pSiNP (JS-11) do not release drugs during liver metabolism, it suggests that tumor-specific delivery of JS-11, a highly hepatotoxic anticancer drug, can lower side effects and increase therapeutic effects.

[0177] Nrf2/HO-1 protein expression related to oxidative stress in tumor tissues was observed by immunohistochemistry experiments.

[0178] According to FIG. 27, it was confirmed that the expression of Nrf2 and HO-1 proteins increased in the tumor tissue administered with the ROSG-pSiNP formulation, which was consistent with the in vitro results. Summarizing the experimental results, the ROSG-pSiNP (JS-11) formulation can selectively and significantly reduce the size of pancreatic cancer without weight loss and hepatotoxicity by inducing tumor-specific ROS generation and oxidative stress.