Patent Application
20220054660
This application claims priority to and the benefit of Korean Patent Application No. 10-2020-0103976 filed in the Korean Intellectual Property Office on Aug. 19, 2020, the entire contents of which are incorporated herein by reference.
The present invention relates to a nanoparticle technology for biological imaging based on X-ray attenuation.
The incidence and mortality of cancer are steadily growing, and cancer is one of the major barriers to increasing life expectancy worldwide. Early diagnosis of cancer leads to increased chances for curative treatment and improved survival against most cancers, especially those that are aggressive or have no early symptoms. Although non-invasive anatomical imaging technologies such as magnetic resonance imaging (MRI), computed tomography (CT) and ultrasound (US) imaging have become indispensable to cancer diagnosis, they have their respective drawbacks such as long acquisition time, high radiation exposure and low resolution at increased depth. Furthermore, the contrast issues of these tomographical techniques significantly limit their use if the morphology of tumor is similar to that of healthy tissues; thus they are less able to detect early or subtle changes that occur in cancers such as pancreatic cancer and colon cancer.
Alternatively, the technology of using non-invasive optical imaging with fluorescent molecules or nanoparticles (NPs) designed to specifically bind to cancer has steadily advanced. Researches on the fluorescence imaging using near-infrared (NIR), especially second-NIR (NIR-II, 900-1700 nm), light have rapidly increased since it provides longer tissue penetration and better temporal and spatial resolution compared to visible or ultra-violet (UV) light due to its reduced tissue absorption, scattering and autofluorescence. Nevertheless, intrinsic problems of the fluorescence-based approaches, such as limited penetration depth of the absorption and emission light and a trade-off between the depth and resolution, are unavoidable. The issues on bio-toxicity, low quantum yield and insufficient detection systems also restrict their clinical adoption.
In contrast, X-ray-based imaging is fast, easy to use and has almost no limitation in penetration depth. Specifically, compared to other X-ray techniques such as CT, plain X-ray imaging such as those used in chest X-rays has additional advantages including lower cost, much less exposure to radiation and easier accessibility.
Therefore, there is a need to develop nanoparticles capable of real-time in vivo imaging including early diagnosis of tumors by non-invasive plain X-ray examination and research into in vivo imaging technology using the same.
1. Nat. Biomed. Eng. 1, 697-713 (2017) (published on Sep 12, 2017)
An object of the present invention is to provide nanoparticles for biological imaging based on X-ray attenuation, which are capable of non-invasive in vivo imaging through plain X-ray imaging and a method of preparing the same.
Also, another object of the present invention is to provide a composition for biological imaging based on X-ray attenuation comprising the nanoparticles.
In addition, another object of the present invention is to provide a method of an X-ray attenuation-based biological imaging comprising the nanoparticles.
In order to achieve the above object, the present invention provides nanoparticles for biological imaging based on X-ray attenuation comprising: a core structure comprising an X-ray attenuating material; and a shell layer formed on the core structure and made of a material having biocompatibility and non-reactivity in vivo.
Also, the present invention provides a composition for biological imaging based on X-ray attenuation comprising the nanoparticles for biological imaging based on X-ray attenuation as an active ingredient.
In addition, the present invention provides a method of biological imaging based on X-ray attenuation comprising the steps of reacting the nanoparticles for biological imaging based on X-ray attenuation with a biological sample; and observing an image by X-ray imaging.
Furthermore, the present invention provides a method of preparing nanoparticles for biological imaging based on X-ray attenuation comprising the steps of preparing an X-ray attenuating material precursor solution (Step 1); preparing a core structure by adding the solution of the step 1 to the core precursor solution (Step 2); annealing the core structure of the step 2 (Step 3); and preparing nanoparticles having a core precursor-shell layer by adding annealed core structure of the step 3 to a shell precursor solution (Step 4).
Hereinafter, the present invention will be described in detail.
The present inventors have prepared nanoparticles comprising an X-ray attenuating material such as lead bromide (CsPbBr3, CPB) perovskite quantum dots (QD) having excellent biosafety and stability and completed the present invention by confirming that cancer can be imaged by the X-ray attenuation reaction of quantum dots only with plain X-ray imaging to be useful for early diagnosis of cancer, as well as in vivo imaging.
The present invention provides nanoparticles for biological imaging based on X-ray attenuation comprising: a core structure comprising an X-ray attenuating material; and a shell layer formed on the core structure and made of a material having biocompatibility and non-reactivity in vivo.
At this time, the X-ray attenuating material may comprise an ABX3 perovskite structure material, which is a quantum dot material having an average diameter of 5 to 15 nm, and the shell layer may comprise at least one selected from the group SiO2, TiO2, ZnO, ZrO2 and Al2O3.
(The A is selected from the group consisting of Ti, Sr, Ca, Cs, Ba, Y, Gd, La, Fe and Mn, the B is selected from the group consisting of Pb, Sn, Cu, Ni, Bi, Co, Fe, Mn, Cr, Cd, Ge and Yb, and the X is selected from the group consisting of lyBr(1−y), lyCl(1−y) and BryCl(1−y) (0≤y≤1)).
According to an embodiment of the present invention, the nanoparticles according to the present invention can be completely prevented from being decomposed or released by encapsulating an X-ray attenuating quantum dot material with SiO2.
In addition, the shell layer further may comprise at least one targeting agent selected from the group consisting of enzyme substrates, ligands, amino acids, peptides, proteins, nucleic acid, lipids, cofactors, carbohydrates and antibodies on the surface, but it is not limited thereto, and the targeting agent may increase the absorption rate of the nanoparticles, and specifically target and bind to a biological sample such as cells or tissues in vivo. Furthermore, any antibody may be used as long as the antibody specifically targets only cancer cells and binds to cancer cells.
In addition, the cancer cells may be selected from the group consisting of colon cancer, liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or cervical cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal cancer near the anus, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, and brain tumor. According to the invention, cancer can be diagnosed by a change caused by X-ray attenuation by plain X-ray imaging, so all cancer cells known to a person skilled in the art can be diagnosed.
In addition, the present invention provides a method of biological imaging based on X-ray attenuation comprising the steps of reacting the nanoparticles for biological imaging based on X-ray attenuation with a biological sample; and observing an image by X-ray imaging.
According to an embodiment of the present invention, the antibody, which is a cancer-specific targeting agent bound to the nanoparticles, attaches the nanoparticles to cancer cells, and when X-rays are irradiated thereto, lead cesium bromide quantum dot scintillator significantly reduces the amount of X-ray photons that transmit cancer cells by emitting bright or fluorescing at the tumor site due to the X-ray attenuation or fluorescence characteristics of the lead cesium bromide quantum dot scintillator so as to diagnose cancer.
In addition, the present invention provides a composition for biological imaging based on X-ray attenuation comprising the nanoparticles for biological imaging based on X-ray attenuation as an active ingredient.
The composition may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include physiological saline, polyethylene glycol, ethanol, vegetable oil and isopropyl myristate, but it is not limited thereto.
In addition, the present invention provides a method of preparing nanoparticles for biological imaging based on X-ray attenuation comprising the steps of preparing an X-ray attenuating material precursor solution (Step 1); preparing a core structure by adding the solution of the step 1 to the core precursor solution (Step 2); annealing the core structure of the step 2 (Step 3); and preparing nanoparticles having a core precursor-shell layer by adding annealed core structure of the step 3 to a shell precursor solution (Step 4).
According to an embodiment of the present invention, a method of preparing the nanoparticle is a new and effective synthesis method for rapid co-synthesis of CPB QD, that is, QD and NP inside SiO2 NP, and this rapid co-synthesis can lead to CPB QD being trapped inside SiO2 NPs before its decomposition.
At this time, the X-ray attenuating material precursor is selected from the group consisting of lead bromide (PbBr2), cesium bromide (CsBr), cesium iodide (Csl), cesium chloride (CsCl), and lead chloride (PbCl2), and the X-ray attenuating material precursor solution may include a basic catalyst, specifically an ammonia catalyst, but it is not limited thereto.
In addition, the core precursor or the shell precursor may be selected from the group consisting of tetramethyl orthosilicate and tetraethyl orthosilicate, but it is not limited thereto.
In addition, the annealing may be performed for 1 to 3 hours at 100 to 200° C., and stability is improved by reducing the hydroxyl groups on the surface of the nanoparticles by this annealing process.
In addition, it may further include the step of binding a targeting agent to the shell layer of the nanoparticles having the core precursor-shell layer, and the binding the targeting agent to the shell layer of the nanoparticles having the core precursor-shell layer may comprise the steps of modifying the shell layer by reacting the nanoparticles having the core precursor-shell layer with 3-aminopropyl triethoxysilane and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt; and conjugating modified nanoparticles with a targeting agent.
The absorption rate of the nanoparticles can be increased by the process of binding such a targeting agent to the shell layer of the nanoparticles, and it can be specifically targeted and bound to a biological sample such as cells or tissues in vivo.
In addition, the present invention provides a novel and effective X-ray attenuation-based in vivo imaging method that can be detected anywhere, such as cells or tissues in vivo, using a lead cesium bromide (CsPbBr3, CPB) perovskite quantum dot (QD) scintillator. The CPB perovskite QD has excellent ability to convert incident X-ray photons into visible light emission with excellent spatial resolution. However, its use was limited because of its poor stability against moisture and the possibility of releasing toxic Pb compounds.
According to an embodiment of the present invention, the stability problem was solved by introducing silicon dioxide (SiO2) core/shell nanoparticles (NP) embedded with CPB QD. The core portion was formed by rapid co-synthesis of CPB QD and SiO2 NP; the shell portion was formed by growing an additional SiO2 layer outside the synthesized core NP to completely prevent QDs from being released or decomposed. Subsequently, an anti-CD44 antibody (Ab) targeting a cancer cell surface-attached receptor was conjugated to the surface of CPB-SiO2@SiO2 NP. A small amount of CPB-SiO2@SiO2-Ab NP (2.8 μg based on QD) was injected intravenously into mice with pancreatic tumors of approximately 5 mm in size; Thereafter, a plain X-ray image was taken. The bright white spots from the strong X-ray attenuation of CPB QD at the tumor site gradually became intense and reached the highest intensity in 2 hours after injection. In addition, the stability, X-ray attenuation properties of CPB QD, and bio-distribution and toxicity of QD-containing SiO2 NPs were evaluated for clinical application.
The basic strategy for in vivo imaging of cancer by plain X-ray examination was shown in
Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.
Lead bromide (PbBr2, 99.999%), cesium bromide (CsBr, 99.999%), oleic acid (OA, 90%, technical grade), oleyl amine (OLA, 70%, technical grade), tetramethyl orthosilicate (TMOS, 98%), tetraethyl orthosilicate (TEOS, 98%, reagent grade), 3-aminopropyl triethoxysilane (APTES, 99%) and ammonium hydroxide (NH4OH, 28%), anti-CD44 (antibody produced in rabbit), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) and 2-mercapto-ethyl-amine hydrochloride (2-MEA, 98%) were purchased from Sigma-Aldrich and used as received. N,N-dimethylformamide (DMF, 99.5%), toluene (99.7%) and anhydrous ethanol (99.8%) were purchased from DAEJUNG and used without further purification.
First, the CPB precursor solution was prepared by adding 0.3 g PbBr2, 0.17 g CsBr, 1.2 ml OLA and 3.6 ml OA to 20 ml DMF and stirring the mixture at 90° C. until it became transparent. The catalytic amount of 28% aqueous NH4OH solution was slowly added to the CPB precursor solution. Two millilitres of this ammonia-containing CPB precursor solution was rapidly injected into a 400 μl/100 ml TMOS/toluene solution, followed by stirring for 2 h at room temperature. The synthesized CPB-SiO2 NPs were collected using centrifugation and were washed with ethanol three times. The obtained powder was annealed at 150° C. for 2 h to remove surface hydroxyl groups. With the addition of NPs to the 1.2 ml/40 ml TEOS/ethanol solution, stirring at room temperature for 1 h, and then injection of 4 ml 28% NH4OH solution, additional SiO2 layers were formed on the annealed-CPB-SiO2 NPs. After 20 h of stirring at room temperature, synthesized CPB-SiO2@SiO2 NPs were collected by centrifugation and washed with ethanol three times.
The precursor solution was prepared by adding 0.2212 g Pbl2, 0.08376 g PbBr2, Csl 0.1248 g, CsBr 0.068 g, 1.2 ml OLA and 3.6 ml OA to 20 ml DMF and stirring the mixture at 90° C. until it became transparent. The catalytic amount of 28% aqueous NH4OH solution was slowly added to the CsPbl2Br1 precursor solution. Two millilitres of this ammonia-containing CsPbl2Br1 precursor solution was rapidly injected into a 1600 μl/100 ml TMOS/toluene solution, followed by stirring for 2 h at room temperature. The synthesized CsPbl2Br1-SiO2 NPs were collected using centrifugation and were washed with ethanol three times. The obtained powder was annealed at 150° C. for 2 h to remove surface hydroxyl groups. With the addition of NPs to the 1.2 ml/40 ml TEOS/ethanol solution, stirring at room temperature for 1 h, and then injection of 4 ml 28% NH4OH solution, additional SiO2 layers were formed on the annealed-CsPbl2Br1-SiO2 NPs. After 20 h of stirring at room temperature, synthesized CsPbl2Br1-SiO2@SiO2 NPs were collected by centrifugation and washed with ethanol three times.
The precursor solution was prepared by adding 0.0134 g CsCl, 0.2936 g PbBr2, 1.2 ml OLA and 3.6 ml OA to 20 ml DMF and stirring the mixture at 90° C. until it became transparent. The catalytic amount of 28% aqueous NH4OH solution was slowly added to the CsPbBr2Cl1 precursor solution. Two millilitres of this ammonia-containing CsPbBr2Cl1 precursor solution was rapidly injected into a 400 μl/100 ml TMOS/toluene solution, followed by stirring for 2 h at room temperature. The synthesized CsPbBr2Cl1-SiO2 NPs were collected using centrifugation and were washed with ethanol three times. The obtained powder was annealed at 150° C. for 2 h to remove surface hydroxyl groups. With the addition of NPs to the 1.2 ml/40 ml TEOS/ethanol solution, stirring at room temperature for 1 h, and then injection of 4 ml 28% NH4OH solution, additional SiO2 layers were formed on the annealed-CsPbBr2Cl1-SiO2 NPs. After 20 h of stirring at room temperature, synthesized CsPbBr2Cl1-SiO2@SiO2 NPs were collected by centrifugation and washed with ethanol three times.
The precursor solution was prepared by adding 0.0117 g CsBr, 0.2225 g PbCl2, 1.2 ml OLA and 3.6 ml OA to 20 ml DMF and stirring the mixture at 90° C. until it became transparent. The catalytic amount of 28% aqueous NH4OH solution was slowly added to the CsPbBr1Cl2 precursor solution. Two millilitres of this ammonia-containing CsPbBr1Cl2 precursor solution was rapidly injected into a 400 μl/100 ml TMOS/toluene solution, followed by stirring for 2 h at room temperature. The synthesized CsPbBr1Cl2-SiO2 NPs were collected using centrifugation and were washed with ethanol three times. The obtained powder was annealed at 150° C. for 2 h to remove surface hydroxyl groups. With the addition of NPs to the 1.2 ml/40 ml TEOS/ethanol solution, stirring at room temperature for 1 h, and then injection of 4 ml 28% NH4OH solution, additional SiO2 layers were formed on the annealed-CsPbBr1Cl2-SiO2 NPs. After 20 h of stirring at room temperature, synthesized CsPbBr1Cl2-SiO2@SiO2 NPs were collected by centrifugation and washed with ethanol three times.
The synthesized CPB-SiO2@SiO2 NPs were dissolved in anhydrous ethanol at a concentration of 5 mg/ml. Excess amount of APTES was added to the solution and then kept overnight at 60° C. CPB-SiO2@SiO2 NP surface-amine-functionalized NPs were collected by centrifugation and reacted with sulfo-SMCC for 2 h at room temperature. Separately, anti-CD44 antibodies were incubated with 50 mM 2-MEA for 1.5 h at 37° C. and then, by passing the mixture through a desalting column, separated from excess 2-MEA. Finally, antibody conjugation on the CPB-SiO2@SiO2 NPs was performed by mixing at 4° C. for 2 h the maleimide-activated NPs and sulfhydryl groups containing antibodies.
Panc-1 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen-Strep) at 37° C. under 5% CO2 overnight. To estimate the uptake efficiency of the NPs, the cells were seeded in 60-mm dishes (1×106 cells) and incubated for 24 h at 37° C. in the CPB-SiO2@SiO2-Ab NPs-containing phosphate-buffered saline (PBS) solution at various NP concentrations (0.1, 0.2, 0.5 and 1.0 mg/ml). The cells were fixed with formaldehyde and fluorescence emission was measured by PL spectroscopy.
For the cell viability test, Panc-1 cells were seeded in 96-well plates and treated with CPB-SiO2@SiO2-Ab NP solutions of various concentrations (0.1, 0.2, 0.5 and 1.0 mg/ml in PBS) for various durations (24, 48 and 72 h). Cell viabilities were analyzed using the water-soluble tetrazolium salt (WST-1) assay (Dogen) according to the manufacturer's protocol. WST-1 solution was added to each well and the cells were incubated at 37° C. for 30 min. After the incubation, absorbance at 450 nm was measured using a microplate reader (BioTek).
Panc-1 cells were seeded in a 4-well chamber slide (1.5×104 cells per well) and treated with CPB-SiO2@SiO2-Ab NP solution (0.5 mg/mL) for various durations. The cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 15 min and then blocked in 2% BSA at room temperature for 1 h. The cells were stained with DAPI (Abcam) and examined using confocal laser scanning microscopy (CLSM).
All animal experiments were performed under the guidelines of the Seoul St. Mary's Hospital animal care and use committee. BALB/c nude mice (Orient Bio) were implanted subcutaneously with Panc-1 (3×106 cells) in matrigel. Tumor growth was monitored until it reached an acceptable size of about 150±30 mm3. 200 μl of CPB-SiO2@SiO2-Ab NP solution (1 mg/ml) was injected into the tail vein. Prior to obtaining real-time X-ray images, all mice were anesthetized with isoflurane and medical grade oxygen. To investigate the biodistribution of the CPB-SiO2@SiO2-Ab NPs, mice were sacrificed and dissected 2 h or 10 days after NP injection.
The size and shape of the synthesized CPB-SiO2 and CPB-SiO2@SiO2 NPs were characterized by high-resolution transmission electron microscopy (HRTEM, JEM-2100F, JEOL Ltd.) and field emission scanning electron microscopy (FE-SEM, JSM-7610F, JEOL Ltd.). The variation of the hydroxyl groups on the surface of CPB-SiO2 NPs was characterized by Fourier-transform infrared (FT-IR) spectrometry (Nicolet™ iS™ 50 FTIR Spectrometer, Thermo Fisher) and X-ray photoelectron spectroscopy (XPS, PHI 5000 VersaProbe, ULVAC PHI). The PL spectra of CPB-SiO2 and CPB-SiO2@SiO2 NPs were recorded using a fluorescence spectrophotometer (SM245, Korea Spectral Products and Darsa Pro-5200, PSI Co. Ltd.) with excitation of 405 nm. The release of elemental lead from CPB-SiO2@SiO2 NPs was detected by inductivity coupled plasma mass spectroscopy (ICP-MS, NexION 350D, Perkin-Elmer SCIEX). In vivo fluorescence and X-ray images were acquired on an Optical in vivo Imaging System-IVIS Lumina XRMS (PerkinElmer Inc.). Tumor and major organs were dissected and immediately fixed in 10% formalin for 24 h, and then their images were taken with the same IVIS Lumina XRMS. For microscopic assessment, the tissues were embedded in paraffin and sectioned (thickness: 5-15 μm), and the sections were stained with hematoxylin and eosin (H&E).
A new and effective synthetic method was developed to embed CsPbX3 QDs inside SiO2 NPs, namely the rapid co-synthesis of QDs and NPs. After rapidly injecting a QD precursor solution containing the base catalyst for SiO2 synthesis into a Si precursor solution, base-catalyzed synthesis of SiO2 NPs and ligand-assisted re-precipitation (LARP) of CsPbX3 QDs occurred simultaneously in a very short time. This rapid co-synthesis resulted in the CsPbX3 QDs being trapped inside the SiO2 NPs before their decomposition occurred.
The embedded amounts of CPB QDs in the CPB-SiO2@SiO2 NPs was estimated by depth-profile XPS measurements of the spin-coated NP thin films (
The X-ray attenuation by the CPB-SiO2@SiO2 NPs was evaluated using clinical X-ray equipment (EVA-HF520, COMED). Radiographic images of the NPs-containing plastic cylinder with a thickness of 0.5, 1.0 and 2.0 cm were acquired at various X-ray tube potentials from 40, 50, 60 kVp (peak kilovoltage) (
To target the CD44 surface adhesion receptor of the pancreatic cells, the surface of the synthesized CPB-SiO2@SiO2 NPs was modified with anti-CD44 antibodies. Because its expression is generally associated with a poor prognosis, CD44 is an important prognostic marker and therapeutic target of pancreatic cancer. The anti-CD44 antibodies were conjugated on the surface of the CPB-SiO2@SiO2 NPs by reacting the maleimide-activated NP surface with sulfhydryl groups on the antibodies. The uptake of CPB-SiO2@SiO2-Ab NPs in Panc-1 cells was evaluated by confocal laser-scanning microscopy (CLSM) (
To verify the uptake efficiency of the CPB-SiO2@SiO2-Ab NPs, Panc-1 cells were seeded at a density of 1×106 on a 60-mm cell culture plate and held for 24 h; this was followed by treatment with various concentrations of NPs (0, 0.1, 0.2, 0.5 and 1 mg/ml) for another 24 h, after which cells were fixed with formaldehyde. The PL intensity of the Panc-1 cells, indicative of CPB QD uptake, increased steadily as the concentration of NPs increased to 0.5 mg/ml, but showed no apparent increase thereafter (
When the transplanted Panc-1 cells had grown to a sufficient volume in the xenograft mouse, 200 μl of 1 mg/ml CPB-SiO2@SiO2-Ab NP solution, equaling an NP dose of 10 mg/kg body weight, was injected intravenously.
Real-time biodistribution of injected CPB-SiO2@SiO2-Ab NPs was monitored through X-ray and fluorescence imaging, with results shown in
Therefore, it was confirmed that cancer can be efficiently and in real time detected by a non-invasive plain X-ray examination without cytotoxicity using CPB QD stably confined in SiO2 NP.
The nanoparticles for real-time biological imaging based on X-ray attenuation according to the present invention can be rapidly prepared by co-synthesis, and have excellent safety and stability in vivo.
In addition, it has an effect of enabling real-time biometric imaging such as simple and rapid early diagnosis of cancer by plain X-ray imaging, such as those used for non-invasive chest X-ray.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0103976 | Aug 2020 | KR | national |
