Patent Application
20230305008
The present invention relates to a SERS-nanotag comprising gold nanoparticle, an encapsulating agent, a Raman reporter and an antibody. The present invention also relates to a diagnostic kit having the SERS-nanotag for simultaneous detection of multiple breast cancer biomarkers selected from the group consisting of Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki67 in a paraffin embedded breast tissue sample.
Development of diagnostic SERS-nanoprobes for early and accurate detection of a disease is a challenging task in biomedical research. In the field of bio imaging, diagnostics and drug delivery, many optical imaging technologies are flourished, out of which SERS has emerged as a promising technique for detection of biological and chemical molecules adsorbed on nano roughened metallic surfaces like gold, silver etc. SERS employs the principle of Raman spectroscopy which is based on the inelastic scattering of incident radiation. It allows capturing of unique signatures corresponding to vibrations of molecules and provides signal enhancement up to 108-1014 folds than the normal Raman spectroscopy which enabled for minute chemical changes in biological samples even in cells and tissues.
Breast cancer is the most common cancer among women. Hormone receptors including Estrogen receptor (ER) and Progesterone receptor (PR) status are key biomolecules in breast cancer. Over-expression of HER2/Neu gene is associated breast cancer patient's prognosis and therapy and Ki67 is a proliferative marker. ER, PR, HER2 and Ki67 panel is essential in an estimation process of breast cancer prognosis which plays a significant role in treatment choice for breast cancer worldwide.
Presence of different biomarkers needs different modes of treatment strategy. Hence, it is very useful to detect the biomarkers quickly in real time and simultaneously. There are few reports on biomarker detection in clinical samples using SERS platform, but no such SERS based biomarker detection kit has been formulated yet especially for HER-2 grading. Salehi et al., in 2014 [Salehi, M., Schneider, L., Strobel, P., Marx, A., Packeisen, J., Schlucker, S. 2014. Two-Color SERS Microscopy for Protein Co-localization in Prostate Tissue with Primary Antibody-Protein A/G-Gold Nanocluster Conjugates. Nanoscale,6(4), pp. 2361-7] reported a formulation in which silica coated gold nanoclusters were used as SERS substrate for the detection of PSA and p63 on non-neoplastic prostrate tissue samples. Whereas, Wang et al., in 2017 [Wang, Y. W., Reder, N. P., Kang, S et al., 2017. Raman-encoded molecular imaging (REMI) with topically applied SERS nanoparticles for intraoperative guidance of lumpectomy. Cancer Research, 77(16), pp. 4506-16] reported a Raman-encoded molecular imaging (REMI) technique where the targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a panel of cell surface biomarkers at surgical margin on clinical samples. Currently immune-histochemical analysis is followed by pathologists to determine the multiple breast cancer biomarkers. To surmount the disadvantages associated with conventional immunohistochemistry technique such as being highly subjective and time consuming, there is a need for a technique for fast detection of multiple breast cancer biomarkers.
The main objective of the present invention is to provide a SERS-nanotag comprising colloidal AuNPs, a Raman reporter molecule, a biocompatible polymer and an antibody raised against a biomarker selected from the group consisting of Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki67 for specific simultaneous detection.
Another objective of the present invention is to provide a diagnostic kit for simultaneous detection of multiple biomarkers selected from the group consisting of Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki67 in a breast tissue sample by surface enhanced Raman scattering (SERS) modality where each biomarkers is identified by Raman fingerprint of the respective SERS-nanotags of the kit.
Still another objective of the present invention is to provide a tissue processing step and an antigen retrieval step to remove paraffin wax and unmask the antigens from the paraffin embedded breast cancer tissue.
Another objective of the present invention is to provide a SERS analysis i.e. scanning, and imaging to gather information from maximum locations in order to know the abundance of the biomarkers.
Yet another objective of the present invention is to provide a SERS intensity based semi-quantitative system for HER-2 gradation, since an over expression of HER-2 (2+and above from immunohistochemistry grading) is considered by the clinicians to judge the samples as positive.
An aspect of the present invention provides a SERS-nanotag comprising:
Another aspect of the present invention provides a process for synthesis of the SERS-nanotag comprising the steps of:
Yet another aspect of the present invention provides a diagnostic kit for detection of breast cancer biomarker comprising:
Still another aspect of the present invention provides a method for detecting breast cancer biomarker in a tissue sample comprising the steps of:
The objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings.
The present invention is focused on simultaneous detection of multiple biomarkers in a breast tissue sample based on SERS-nanotags using Raman fingerprint analysis. A nanoparticle probe comprising gold or silver nanoparticles having size in the range of 40-50 nm anchored with a Raman reporter molecule and encapsulated with a biocompatible polymer is conjugated with a breast cancer specific antibody which is transformed into a SERS-nanotag. Further, these SERS-nanotag conjugated to target specific antibodies raised against a biomarker selected from the group consisting of Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki67are used to validate the simultaneous recognition capabilities in paraffin embedded breast cancer tissue samples.
A diagnostic kit comprising the SERS-nanotag enables simultaneous detection of multiple biomarkers in a breast tissue sample with a highly sensitive and specific Raman peak of the Raman reporter attached to the nanoparticle which corresponds to the presence of respective antibody attached to the same nanoparticle.
An embodiment of the present invention provides a SERS-nanotagcomprising:
In another embodiment of the present invention, there is provided a SERS-nanotag, wherein the encapsulating agent is selected from the group consisting of a polysaccharide, polyethylene glycol, and serum albumin.
In still another embodiment of the present invention, there is provided a SERS-nanotag, wherein the polysaccharide is selected from the group consisting of chitosan, and hyaluronic acid.
In yet another embodiment of the present invention, there is provided a SERS-nanotag, wherein the encapsulating agent is polyethylene glycol
In another embodiment of the present invention, there is provided a SERS-nanotag, wherein the Raman reporter molecule is selected from the group consisting of cyanine dilipoic acid (Cy7DLA), hemicyaninecarbaldehyde (HCC), Pyryliniumhexylamine (PHA), Squaraine di-lipoic acid (SDL), Pyrenelipidene ethyl quartanised (Py L Et), crystal violet (CV) and Mercapto benzoic acid (MBA).
In still another embodiment of the present invention, there is provided a SERS-nanotag, wherein the antibody is a monoclonal or a polyclonal antibody.
In yet another embodiment of the present invention, there is provided a SERS-nanotag, wherein the antibody is a monoclonal antibody.
In still another embodiment of the present invention, there is provided a SERS-nanotag, wherein the antibody is a polyclonal antibody.
In an embodiment of the present invention, there is provided a SERS-nanotag, wherein the antibody is raised against a biomarker selected from the group consisting of Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki67.
An embodiment of the present invention provides a process for synthesis of the SERS-nanotag comprising the steps of:
In an embodiment of the present invention, there is provided a process for synthesis of the SERS-nanotag, wherein the gold nanoparticles are in a concentration in the range of 7×109 to 4×1010 particles/mL
In another embodiment of the present invention, there is provided a process for synthesis of the SERS-nanotag, wherein the Raman reporter molecule is in a concentration in the range of 0.5 to 100 μM.
In yet another embodiment of the present invention, there is provided a process for synthesis of the SERS-nanotag, wherein the antibody is in a concentration in the range of 2 to 20 μg.
In an embodiment of the present invention, there is provided a process for synthesis of the SERS-nanotag, wherein the Raman reporter molecule is selected from the group consisting of cyanine dilipoic acid (Cy7DLA), hemicyaninecarbaldehyde (HCC), Pyryliniumhexylamine (PHA), Squaraine di-lipoic acid (SDL), Pyrenelipidene ethyl quartanised (Py L Et), crystal violet (CV) and Mercapto benzoic acid (MBA).
In another embodiment of the present invention, there is provided a process for synthesis of the SERS-nanotag, wherein the encapsulating agent is selected from the group consisting of a polysaccharide, polyethylene glycol, and serum albumin
In yet another embodiment of the present invention, there is provided a process for synthesis of the SERS-nanotag, wherein the buffer is selected from the group consisting of MES buffer, Phosphate buffer and Tris buffer.
In an embodiment of the present invention, there is provided a process for synthesis of the SERS-nanotag, wherein the antibody is raised against a biomarker selected from the group consisting of Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki67.
Another embodiment of the present invention provides a diagnostic kit for detection of breast cancer biomarker comprising:
Yet another embodiment of the present invention provides a method for detecting breast cancer biomarker in a tissue sample comprising the steps of:
In still another embodiment of the present invention, there is provided a method for detecting breast cancer biomarker in a tissue sample, wherein the breast cancer biomarker is selected from the group consisting of Estrogen receptor (ER), Progesterone receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki67.
The aim of the present invention is to detect multiple biomarkers in a breast tissue sample by SERS based diagnostic platform in a simultaneous detection mode using SERS-nanotags and confirm the presence or absence of the biomarkers much faster with high precision level as compared to analysis with current gold standards. The present invention provides a SERS based simultaneous diagnostic kit having a SERS-nanotag comprising a nanoparticle, a biocompatible polymer, a Raman reporter (RR) molecule and an antibody raised against breast cancer biomarkers selected from the group consisting of Estrogen receptor (ER), Progesterone receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki67.
The nanoparticle can be colloidal gold nanoparticles (AuNPs), colloidal silver nanoparticles (AgNPs), gold coated silver nanoparticles (Au@AgNPs) or gold (Au)/silver (Ag) coated glass slide. In a preferred embodiment of the present invention, the nanoparticles are AuNPs having size in the range of 40-50 nm. These gold nanoparticles are then encapsulated in a biocompatible polymer which is selected from the group consisting of a polysaccharide, polyethylene glycol, and serum albumin Both, in-house synthesized and commercially available Raman reporters are used as a signature Raman peaks to obtain simultaneous detection. The in-house Raman reporter (RR) molecules used in the present invention are Cy7DLA (cyanine dilipoic acid), HCC (hemicyaninecarbaldehyde), PHA (Pyryliniumhexylamine), SDL (Squaraine di-lipoic acid), Py L Et (Pyrenelipidene ethyl quartanised), and commercially available Raman reporters include CV (crystal violet) and MBA (Mercapto benzoic acid). The Raman reporter molecules are coupled to the commercially purchased antibodies raised against biomarkers i.e. ER, PR, HER2, and Ki67, to formulate the SERS-nanotags for detection of breast cancer biomarkers i.e. ER, PR, HER2, and Ki67.
The present invention also co-relates and validates the Raman fingerprint from the Raman reporter molecule with-respect-to biomarker in a breast tissue sample in a simultaneous
Raman fingerprint. This programme was approved by the local Ethics Committee and prior to specimen collection; all patients had signed informed consent forms. Pathologically confirmed breast cancer tissues with different ER, PR, HER2, Ki67 status were collected from Regional Cancer Centre (RCC), Trivandrum, Kerala, India. A separate bit of tumor tissue from each subject was paraffin embedded and 4 micron sections were obtained. Sections were selected after confirming the presence of tumor in Haematoxylin and eosin stained samples. Paraffin wax was removed by rinsing in xylene followed by different grades of alcohol and antigen retrieval was performed prior to the incubation with SERS-nanotag. SERS spectral analysis was carried out using with diode laser of 633/785 nm laser excitation source with spectrograph grating 600 gr/mm using maximum 1-20 sec integration time and around 1-15 accumulations.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Colloidal gold nanoparticles (AuNPs, size around 40-50 nm) were prepared by citrate reduction method [Kim Ling, J., Maier, M., Okenve, B., Kotaidis, V., Ballot, H. and Plech, A., 2006. Turkevich method for gold nanoparticle synthesis revisited. The Journal of Physical Chemistry B, 110(32), pp. 15700-15707]. The characterization of the synthesized nanoparticles was done through UV-VIS spectroscopy, Dynamic Light Scattering (DLS) and High Resolution Transmission Electron Microscopy (HRTEM).
The gold nanoparticles of size 40-45 nm were concentrated from 25 mL to 3.6 mL by centrifugation at 6000 rpm, for 30 minutes. To this, 0.05% of TWEEN 20 was added for stabilizing the gold nanoparticles and vortexed for few minutes. Then, ˜400 μL of 80 μM Raman reporter1 (crystal violet (CV) in dimethyl sulfoxide (DMSO)) was added and incubated for half an hour. For making the tag biocompatible, 45 μL SH-PEG-COOH was added and incubated for 10 minutes. To this solution, 275 μL SH-PEG-OCH3 was added and further incubated for 3 hrs. Then, the solution was concentrated to 1 mL by centrifugation at 10,000 rpm for 10 minutes. Excess PEG was removed by centrifuging the solution again at 10,000 rpm for 10 minutes. Finally, the solution was re-suspended in milliQ water to obtain AuNP@PEG@CV.
Anti-HER2 (Rabbit Monoclonal antibody; ABCAM) for the biomarker HER2 was purified using 3 KDa centrifugal filters. PEG encapsulated nanoparticles obtained in example 2 (1-1.5 mL) were centrifuged at 8000 rpm for 15 minutes and re-suspended in −500 μL MES buffer (HIMEDIA) (50 mM, pH 6.1). 5 μL of freshly prepared EDC (SIGMA-ALDRICH) (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide, 250 mM) was added and after a few minutes gap, 6 μL of freshly prepared sulfo-NHS (ALDRICH CHEMISTRY) (N-hydroxysuccinimide, 250 mM) was also added. After incubating the reaction mixture for 30 minutes, the reaction mixture was centrifuged at 10000 rpm for 10 minutes and re-suspended in 500 μL MES buffer. Then, 4 μg antibody was added to that and incubated in a shaker incubator for 2 hrs at room temperature and kept for overnight incubation at 4° C. After that, the mixture was centrifuged at 10000 rpm for 10 minutes and finally re-suspended in fresh 500 μLMES to obtain the antibody conjugated SER-nanotag AuNP@PEG@CV@Anti HER2.
The gold nanoparticles of size 40-45 nm were concentrated from 7.2 mL to 1 mL by centrifugation at 6000 rpm, for 30 minutes). 0.05% of TWEEN 20 was added for stabilizing the gold nanoparticles and vortexed for few minutes. Then, 37 μL of 100 μM Raman reporter 2 (Squaraine di-lipoic acid (SDL) in dimethyl sulfoxide) and 262.5 μL milliQ water was added and incubated for 10 minutes. For making the tag biocompatible, 62 μL SH-PEG-COOH was added and incubated for 10 minutes. To this solution, 368 μL SH-PEG-OCH3 was added and further incubated for 3 hrs. Then, the solution was concentrated to 1 mL by centrifugation at 10,000 rpm for 10 minutes. Excess PEG was removed by centrifuging the solution again at 10,000 rpm for 10 minutes. Finally, the solution was re-suspended in milliQ water to obtain AuNP@PEG@SDL.
Anti-ER (Rabbit Monoclonal antibody; ABCAM) for the biomarker ER was purified and conjugated by EDC-NHS coupling as described in example 3 (procedure for HER2 conjugation). Here also, 4 μg antibody was added to the PEGylated AuNPs obtained in example 4 to obtain antibody conjugated SERS-nanotag: AuNP@PEG@SDL@Anti ER.
The gold nanoparticles of size 40-45 nm were concentrated from 25 mL to 3.6 mL by centrifugation at 6000 rpm, for 30 minutes. To this, 0.05% of TWEEN 20 was added for stabilizing the gold nanoparticles and vortexed for few minutes. Then, 400 μL of 200 μM Raman reporter 3 Mercapto benzoic acid (MBA) was added and incubated for half an hour. For making the tag biocompatible, 45 μL SH-PEG-COOH was added and incubated for 10 minutes. To this solution, 275 μL SH-PEG-OCH3 was added and further incubated for 3 hrs. Then, the solution was concentrated to 1 mL by centrifugation at 10,000 rpm for 10 minutes. Excess PEG was removed by centrifuging the solution again at 10,000 rpm for 10 minutes. Finally, the solution was re-suspended in milliQ water to obtain SERS-nanotag: AuNP@PEG@MBA.
Anti-PR (Rabbit Monoclonal antibody; ABCAM) for the biomarker PR was purified and conjugated to the AuNP@PEG@MBA obtained in example 6 by the procedure described in example 3 to obtain antibody conjugated SERS-nanotag: AuNP@PEG@MBA@Anti-PR.
The gold nanoparticles of size 40-45 nm were concentrated from 25 mL to 3.6 mL by centrifugation at 6000 rpm, for 30 minutes. To this, 0.05% of TWEEN 20 was added for stabilizing the gold nanoparticles and vortexed for few minutes. Then, 400 μL of 100 μM Raman reporter 4 Pyrenelipidene ethyl quartanised (Py L Et) was added and incubated for half an hour. For making the tag biocompatible, 45 μL SH-PEG-COOH was added and incubated for 10 minutes. To this solution, 275 μL SH-PEG-OCH3 was added and further incubated for 3 hrs. Then, the solution was concentrated to 1 mL by centrifugation at 10,000 rpm for 10 minutes. Excess PEG was removed by centrifuging the solution again at 10,000 rpm for 10 minutes. Finally, the solution was re-suspended in milliQ water to obtain SERS nanotag: AuNP@PEG@Py L Et.
Anti-Ki67(ABCAM, Mouse monoclonal antibody) for the biomarker Ki67 was purified and conjugated to the AuNP@PEG@Py L Et (4 μg) obtained in example 8 by the procedure described in example 3 to obtain antibody conjugated SERS-nanotag AuNP@PEG@Py L Et@Anti-Ki67.
Breast cancer tissue samples having various biomarker expression status were collected from Regional Cancer Centre, Trivandrum, Kerala, India for SERS analysis. Ethical approval for the same was obtained from the consigned authorities prior to the experiments.
Prior to spectral analysis, tissue processing was carried out by the following standardized steps for the paraffin embedded formalin fixed tissues.
The present diagnostic strategy is able to detect single, duel and triple biomarkers in breast cancer tissue sample accurately.
Breast cancer tissue samples with different levels of HER-2 expression were collected from Regional Cancer Centre (RCC), Trivandrum, Kerala, India. Tissues were analyzed by immunohistochemistry to confirm the grading by a pathologist. Samples were processed as mentioned in above examples. For HER-2 grading, AuNP@CV@HER-2 was added to the tissue samples and incubated for 30 min and washed thoroughly to take the signature peaks from SERS analysis. Spectra were collected by spectral accumulation and also through image scanning. All the tissue processing procedure was as early described and the SERS analysis was performed with 633 nm laser, under 10× objective of confocal Raman microscope. For single spectral analysis, integration time used was 3-5 seconds with 2-5 accumulations using 10 mW power. Image scanning was performed for 150×150 nm area with 0.01 seconds integration time. Average CCD counts were taken for comparison of different HER-2 graded samples.
HER-2 grading is an important aspect during the selection of treatment regimens for breast cancer patients as HER-2 over expression, i.e., 3+ and above are considered as HER-2 positive whereas 2+ expression is considered as borderline. Here, tissue samples with varying HER-2 expression levels (1+ to 4+) were analyzed by single spectral obtained from the Raman mapping providing the average spectra. Both the analysis showed an increase in spectral intensity of Raman reporter CV in accordance with the Her-2 expression levels. Table 1 denotes the CCD counts obtained from the Raman image analysis corresponding to the 440 and 1615 cm−1 peaks of CV where both the peak intensities were found to be increasing from 3 to 40 w.r.t to peak at 440 cm−1 and 18 to 150 w.r.t. peak at 1615 cm−1 respectively as the HER-2 expression levels increases from 1+ to 4+. Thus, the present Her-2 grading by Raman spectral analysis can be utilized as a complementary technique to IHC to confirm the HER-2 grading.
Immunohistochemistry (IHC) is the existing gold standard method for detection of breast cancer biomarkers in formalin fixed paraffin embedded tissue samples. Table 2 provided below compares both the techniques in terms of specificity, easiness and time required for sample processing and analysis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011034768 | Aug 2020 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2021/050577 | 6/14/2021 | WO |
