TECHNICAL FIELD OF THE INVENTION
[001] The present invention is in the technical field of nanoparticle based biosensor for cell detection and isolation system, using ligand- receptor chemistry. The present invention also relates to components useful in preparation of the nanoparticle conjugated ligands specific for different cell populations of clinical interests, methods, processes, and uses thereof.
BACKGROUND OF THE INVENTION
[002] Cells expressing specific cell surface receptors exist in a heterogeneous population where cells have either the receptor of interest in large numbers or very low in numbers or completely nil. The current systems available to detect cells with specific receptors for characterization of a disease are based on tricky and expensive methods using Fluorescence-Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS).
[003] However, viability of cells in a very small population of receptor specific cells is difficult, as these cells after FACS and MACS if required to be cultured and used in downstream assays.
[004] In addition, Fluorescence-Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS) techniques are not amenable to high throughput and resolution, and are also fairly time consuming. Furthermore, both FACS and MACS require sophisticated instrumentation, a high level of technical expertise and are also prohibitively costly. These issues are especially relevant in resource-limited labs in developing countries.
[005] The conjugation of nanoparticles (NP) to monoclonal antibodies with high affinity makes nanoparticles useful as biosensors. However, the number of antibodies and their orientation on the surface of the NPs are crucial for effective diagnostic response.
[006] Furthermore, due to the presence of multiple reactive functional groups on antibodies, most of the conjugation techniques may lead to heterogeneous antibody orientations on the NPs, leading to nonspecific interaction.
[007] In addition, the conformational stability of an antibody is low and they are also prone to proteolytic, chemical, and thermal degradation, which can limit their utility in non-laboratory diagnostic environments.
[008] At present, use of magnetic separation of cells using magnetic particles is one of the leading enrichment methods for Circulating Cancer Cells (CTC) from the blood of a patient for detection and characterization, has been an important area of research. Most techniques use NPs, such as magnetic, fluorescent, metallic, conductive NPs or nanostructured substrates, to enrich or detect CTCs in blood samples. These nanocarriers have demonstrated desirable characteristics such as prolonged systemic circulation lifetime, reduced non-specific cellular uptake, targeting abilities, while minimizing undesirable side effects.
[009] Currently, numerous colorimetric nanobiosensors with specially engineered nanoparticles have the potential to detect specific cell types for different disease diagnosis. Gold nanoparticles (Au NPs) are used widely in various biological applications due to their unique optical properties.
[010] However, the challenge that persists with conventional techniques is the process of tagging the labeling molecule, i.e., proper binding of any foreign ligand to the receptor of interest so as to increase detection of specific cell population and sensitivity. Functionalization of NPs is a widely used technique that allows its conjugation with ligands possessing inherent ability to direct selective binding to specific cell types. The conjugation of Au NPs to monoclonal antibodies with high affinity makes them useful as biosensors.
[011] However, the number of antibodies and their orientation on the surface of the NPs are crucial for effective diagnostic response. Further, due to the presence of multiple reactive functional groups on antibodies, most of the conjugation techniques may lead to heterogeneous antibody orientations on the NPs, leading to nonspecific interaction.
[012] In addition, the conformational stability of an antibody is low and they are also prone to proteolytic, chemical, and thermal degradation, which can limit their utility in non-laboratory diagnostic environments. Further, the relatively high cost of antibodies makes working with them an expensive proposition. Therefore, alternative ligands such as small molecules are getting increasing attention due to their stability, ease of conjugation with nanoparticles, and cost effectiveness, provided they can be chemically synthesized with a high yield.
[013] In summary, there is an urgent need for proteolytic, chemical, and thermal stable non-laboratory diagnostic nano-particle kit/system for an easy and reliable method of cell detection and isolation, wherein higher viability of recovered cells and cell purity can be achieved.
SUMMARY OF THE INVENTION
[014] According to exemplary aspect, the present invention relates to a nanoparticle based biosensor for cell detection and isolation system, more particularly to isolate selective cell types possessing specific surface receptors from a heterogeneous cell population consisting of varied cell types bearing wide array of receptors. Furthermore, invention is based on ligand-receptor binding for isolation of cells of interest, wherein gold coated nanoparticle is conjugated with hyaluronic acid (HA) that acts as a ligand for the CD44 surface receptors present in certain cancer cell types. The nanoparticulate system can be used for various combinations of cancer cell-specific receptors and ligands. The present invention also relates to components useful in preparation of the nanoparticulate conjugation to ligand and successfully targeting CD44 receptor bearing cells, optimization methods for cell isolation, easy and reliable cell detection, besides retaining cell viability, economical; processes, and applications thereof.
[015] According to an exemplary aspect, the present invention discloses a gold coated nanoparticulate system, in particular, a Hyaluronic Acid (HA) conjugated for targeted detection and isolation of selectively specific CD44 surface receptor cells from a heterogeneous cell population.
[016] Yet another exemplary aspect of the present invention, a Hyaluronic Acid (HA) conjugated nanocomplex, wherein HA is the ligand for the CD44 receptor expressed specifically by certain cancer cell populations. The gold nanoparticle is conjugated to Hyaluronic Acid (HA) mediated by Polyethylene glycol (PEG), which is a polymer of ethylene oxide.
[017] The present invention also relates to components useful in preparation of the nanoparticulate conjugated system, as well as the compositions, methods, processes, and uses thereof.
[018] As will be appreciated by a person skilled in the art the present invention provides a variety of following advantages. Listing of Claims,
[019] A nanoparticle based biosensor for cell detection and isolation system comprising: a) gold nanoparticle, b) polymer, c) and linker molecule that acts as a ligand for cell surface receptors, wherein the gold nanoparticle is conjugated to linker molecule that acts as a ligand for cell surface receptors, wherein the polymer augments the efficacy of ligand-receptor binding.
[020] The nanoparticle based biosensor for cell detection and isolation system of [020], wherein the system is a specific combination of a cell-specific receptor and a particular ligand, wherein specific receptors/ligands are functionalized with specific chemical groups to make this system specific and effective for a specific cell type.
[021] The nanoparticle based biosensor for cell detection and isolation system of [020], wherein linker molecule is HA or DUPA or antibodies, wherein specific receptor/ligand is a CD44 cell surface receptor or a receptor protein, wherein the polymer is PEG.
[022] A method for cell detection and isolation system comprising:
a. formulation of Au-PEG-HA NPs platform
b. exposing Au-PEG-HA NPs cell recognition agent capable of selectively binding to the target receptor biomarker, wherein binding of ligand to receptor leads to aggregation of NPs and changes in spectral properties; and
c. detecting/isolating NPs bound target cells by absorbance/ colorimetric/differential centrifugation/ Fluorescence spectral analysis.
[023] The method for cell detection and isolation system of [023], wherein the cell detection and/or isolation method involve colorimetric observation and/or UV-absorption spectrum analysis and/or differential centrifugation and/or Fluorescence spectral analysis.
[024] The method of [023], wherein the nanoparticle based biosensor for cell detection and isolation system is used for detection and/or isolation selective cell types possessing specific surface receptors from a heterogeneous cell population, wherein selective cell types are circulating tumor cells in blood of metastatic cancer.
[025] The method of [023], wherein the nanoparticle based biosensor is used for cell detection and/or isolation and/or bioassays.
[026] The method of [023], wherein the method is an in-vitro cell detection and isolation method.
[027] The method of [023], wherein the subject is suffering premalignant and/or malignant stage of cancer or early stage for chemoprevention of dysplastic progression and for Stage II, Stage III, or Stage IV cancers for second primary prevention, or late-stage cancer, or metastasized cancer, or solid tumors, or cancer that has relapsed after treatment with another therapeutic modality, or carcinoma or sarcoma or melanoma or lymphoma or or leukemia.
[028] A kit for cell detection and isolation in a biological sample from a subject, wherein the kit comprising of at least one reagent that specifically binds to a cell surface receptor or cancer biomarker.
[029] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
[030] BRIEF DESCRIPTION OF THE DRAWINGS
[031] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
[032] FIG. 1 illustrates the Physico-chemical properties of Au NPs, Au-PEG NPs, Au-PEG-HA NPs: a. A schematic representation of synthesis of Au-PEG-HA; b. ATR-FTIR spectrum of Au NPs & Au-PEG NPs depicting PEGylation of Au NPs (3347.17 cm-1- OH group, 1095 cm -1- C-O-C, 1637 cm-1- C=O stretching); c.1H NMR spectroscopy demonstrating the chemical structure of HA in Au-PEG-HA NPs with characteristics peak labelled (inset mentioned in FIG. 7a), d. UV absorption spectra with a characteristic SPR peak (519-521 nm) of Au NPs, Au-PEG NPs, Au-PEG-HA NPs; e & f. Average size analysis of synthesized NPs Au NPs, Au-PEG NPs, Au-PEG-HA NPs determined by Transmission Electron Microscopy (TEM) image analysis.
[033] FIG. 2 illustrates the optimization of Au and HA concentration of Au-PEG-HA NPs: a. Colorimetric changes (with A650/A521 ratio) of Au-PEG-HA NPs after incubation with MDA-MB-231 (50,000 cells) and BT-474 (50,000 cells) at varied Au concentration (100-3.125 nM) show pink-violet shift (521 nm-559 nm) in MDA-MB-231 and no shift in BT-474; b. UV/VIS absorbance spectrum analysis of Au-PEG-HA NPs after incubation with MDA-MB-231 (50,000 cells) and BT-474 (50,000 cells) at 25 nM Au concentration. c. Colorimetric changes (with A650/A521 ratio) of Au-PEG-HA NPs after incubation with MDA-MB-231 (50,000 cells) and BT-474 (50,000 cells) at varied HA concentration (400-1.56 µg/mL) and 25 nM Au. d. UV/VIS absorbance spectrum analysis of Au-PEG-HA NPs after incubation with MDA-MB-231 (50,000 cells) and BT-474 (50,000 cells) at 100 HA µg/mL concentration (25 nM Au).
[034] FIG. 3 illustrates the differential identification of cells using Au-PEG-HA NPs (colorimetric changes and UV/VIS absorbance spectrum analysis): a. Comparison of colorimetric changes (with A650/A521 ratio) of Au-PEG-HA NPs after interaction with different cell populations (MDA-MB-231, BT-474, NIH 3T3) at varied cell numbers (50,000-195); b. UV/VIS absorbance spectrum analysis of Au-PEG-HA NPs after interaction with different cell population (MDA-MB-231, BT-474, NIH 3T3) at 50,000 cell number; c. Relationship between A650/A521 ratio and cell number after incubating Au-PEG-HA NPs with MDA-MB-231, BT-474, NIH 3T3 cells for cell numbers ranging from 195 to 100000.
[035] FIG. 4 illustrates CD 44-HA interaction for detecting cells of interest (MDA-MB-231) from a heterogeneous population. Cell mixtures were prepared by mixing MDA-MB-231 (CD44+ve/high) and BT-474 cells (CD44 –ve/low) in varying ratios, but keeping the total number of cells constant (2000). 100 µL of Au-PEG-HA solution (25 nm Au and 100 µg/mL HA) was added to each well, followed by 10 min incubation at room temperature. The colorimetric changes, UV-Vis absorption spectra and A650/A521 ratio (effect of aggregation) were recorded and compared with control (Au-PEG-HA NPs) values. a. colorimetric changes (with A650/A521 ratio) of Au-PEG-HA NPs after interaction with heterogeneous population of cells; b. UV/VIS absorbance spectrum analysis of Au-PEG-HA NPs after incubation with cell mixture; c. Variation in A650/A521 ratio of Au-PEG-HA NPs after incubation with cell mixture with respect to target cell population (MDA-MB-231). R2 values denotes the linearity of the plotted graphs; d. Recovery of viable cells (%) from the individual cells i.e. MDA-MB-231-GFP /BT-474/NIH 3T3/Blood (5000 cells) followed by nanoparticle (Au-PEG-HA: 25 nM, Au: 100 µg/mL HA) treatment, separation by differential centrifugation (600 rpm/30 min) and evaluated from pellet and supernatant by FACS analysis.
[036] FIG. 5 illustrates the Au-PEG-HA NP-mediated recovery of viable cells of interest from a heterogeneous population: a. colorimetric changes (with A650/A521 ratio) of Au-PEG-HA NPs after interaction with a mixture of cells (MDA-MB-231+BT-474, MDA-MB-231+NIH 3T3, MDA-MB-231+Blood cells) with different MDA-MB-231 cell densities (50%, 5%, 1% of MDA-MB-231 cells); b. Recovery of viable MDA-MB-231 cells from a mixture of spiked MDA-MB-231 cells with BT-474/NIH 3T3/Blood cells at different cell densities (50%, 5%, 1% of MDA-MB-231 cells) followed by a 10 minute incubation with Au-PEG-HA NPs and separation by differential centrifugation (600 rpm/30 min). The cells were disassociated from NPs and checked for viability by the trypan blue live/dead cell counting assay. c. Recovery of viable MDA-MB-231 cells from a mixture of spiked MDA-MB-231-GFP cells with BT-474/NIH 3T3/Blood cells at different cell densities (50%, 5%, 1% of MDA-MB-231 cells) followed by a 10 minute incubation with Au-PEG-HA NPs and separation by differential centrifugation (600 rpm/30 min). The isolated cells were evaluated by FACS analysis; d. Representative merged bright field and fluorescent microscopy images of MDA-MB-231 GFP cells cultured from recovered viable cells after 24 hr incubation.
[037] FIG. 6 illustrates the aggregation pattern of Au NPs and Au-PEG NPs by: a-b. size distribution of Au NPs and Au-PEG NPs upon incubation at room temperature at 0 hour, 24 hours and 7 days as determined from dynamic light scattering (DLS) method; c-d. UV/Vis absorbance spectrum analysis of Au NPs and Au-PEG NPs upon incubation at room temperature for 0 hour, 24 hours and 7 days.
[038] FIG. 7 illustrates the physico-chemical properties of Au NPs, Au-PEG NPs, Au-PEG-HA NPs: a. Inset of FIG. 1b: 1H NMR spectroscopy showing characteristic peaks of HA; broad signal (3.0 -3.8 ppm): protons in the sugar rings and 4.6 ppm: two anomeric protons attached to the carbons adjacent to the two oxygen atoms (highlighted in oval); 2.02 ppm: methyl (-CH3) protons of the N-acetyl group of HA (highlighted in rectangular). b. Size distribution pattern of synthesized Au NPs, Au-PEG NPs, Au-PEG-HA NPs as determined from dynamic light scattering (DLS) method; c. Surface morphology of NPs characterized by TEM.
[039] FIG. 8 illustrates the optimization of Au and HA concentration of Au-PEG-HA NPs: a-b. UV/VIS absorbance spectrum analysis of Au-PEG-HA NPs after incubation with MDA-MB-231 (50,000 cells) and BT-474 (50,000 cells) at 12.5 nM (a) and 50 (nM) (b) Au concentration; c-d. UV/VIS absorbance spectrum analysis of Au-PEG-HA NPs after incubation with MDA-MB-231 (50,000 cells) and BT-474 (50,000 cells) at 50 µg/mL (c) and 200µg/mL (d) HA concentration (25 nM Au).
[040] FIG. 9 illustrates the recovery of viable cells of interest from a heterogeneous population by using Au-PEG-HA NPs: a-c. UV absorption spectra of Au-PEG-HA NPs after interaction with a heterogeneous population of cells [MDA-MB-231+BT-474 (a), MDA-MB-231+NIH 3T3 (b), MDA-MB-231+Blood cells (c) at different cell densities (50%, 5%, 1% of MDA-MB-231 cells).
[041] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
DETAILED DESCRIPTION OF THE INVENTION
[042] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
[043] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[044] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
[045] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
[046] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
[047] Example embodiments of the present invention are described with reference to the accompanying figures.
[048] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[049] Definitions
[050] The following terms are used as defined below throughout this application, unless otherwise indicated.
[051] The terms “tumour” or “tumour tissue” refer to an abnormal mass of tissue which results from uncontrolled cell division. A tumour or tumour tissue comprises “tumour cells” which are neoplastic cells with anomalous growth properties and no functional bodily function. Tumours, tumour cells and tumour tissue can be benign or malignant.
[052] The phrase "differentially present" refers to differences in the quantity of the marker present in a sample taken from patients as compared to a control subject. A biomarker can be differentially present in terms of frequency, quantity or both.
[053] "Diagnostic" means identifying a pathologic condition.
[054] The terms "detection", "detecting" and the like, may be used in the context of detecting markers or biomarkers.
[055] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., µg/ml) or a relative amount (e.g., relative intensity of signals).
[056] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
[057] "Detectable moiety" or a "label" refers to spectroscopic, photochemical, biochemical, immunochemical, or chemical means of detection of a composition. For example, labels may include 32P, 35S, fluorescent dyes, biotin-streptavidin, dioxigenin, haptens, electron-dense reagents and enzymes. The detectable moiety generates a measurable signal that can quantify the amount of bound detectable moiety in a sample. Quantitation of the signal is done by scintillation counting, densitometry, or flow cytometry.
[058] "Antibody” refers to a polypeptide ligand encoded by an immunoglobulin gene(s), which specifically binds and recognizes an epitope.
[059] The terms "subject", "patient" or "individual" generally refer to a human or mammals. "Sample" refers to a polynucleotide, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.
[060] DUPA refers to 2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid that can act as a ligand.
[061] EMBODIMENTS OF THE INVENTION
[062] Proposed nanoplatform: Au-PEG-HA NPs
[063] Hypothesis
[064] The formulated Au-PEG-HA NPs comes in contact with cells that express the receptor (CD44) for HA that will lead to color change (and the accompanying absorbance shift) in the solution due to NPs-cell interaction that leads to aggregation of NPs. Again, these NPs bound target cells can be isolated from a heterogeneous population by differential centrifugation with high specificity and viability.
[065] Objectives:
[066] Synthesis of Au NPs and functionalization (Au-PEG-HA NPs)
[067] Optimization of gold (Au) and hyaluronic acid (HA) concentration for specific cell detection
[068] Differential identification of cells using Au-PEG-HA NPs (colorimetric and absorbance spectrum analysis with varied cell numbers i.e. MDA-MB-231 (CD44+ve/high), BT-474 (CD44 –ve/low) and NIH 3T3 (CD44 -ve).
[069] Exploiting CD 44-HA interaction for specific cell (MDA-MB 231) detection in a heterogeneous population
[070] Au-PEG-HA NPs mediated recovery of viable cells (MDA-MB-231) from different cell mixture (MDA-MB-231 + BT-474, MDA-MB-231 + NIH 3T3, MDA-MB-231 + Blood cells)
[071] IMPORTANT ATTRIBUTES OF THE INVENTION: SIGNIFICANT RESULTS:
[072] The colorimetric change from pink to violet upon the binding of the cells of interest to the Au-PEG-HA NPs is the underlying principle of this platform. Au-PEG-HA NPs on interaction with MDA-MB-231 cells (50,000) showed specific colorimetric change with a narrow UV-Vis absorbance spectrum (accompanying SPR shift from 521 to 559 nm) at optimized Au (25nM) and HA (100µg/mL) concentrations and such colorimetric changes were not observed with BT-474 cells. This makes it ideally suited for diagnostic devices, which rely on easily assessing the presence/absence of the desired parameter.
[073] The absorbance values i.e. A650 and A521 are related to the aggregated and dispersed state of NPs respectively, and thus the A650/A521 ratio defines the degree of aggregation of NPs. In this study inventors found that a linear relationship exists between the numbers of MDA-MB-231 cells (high CD44 expression) bound to the Au-PEG-HA NPs and the A650/A521 ratio. This helps with a quantitative estimation of cells, another feature relevant to any diagnostic platform.
[074] Furthermore, the fundamental principle of this platform is the exploitation of ligand-receptor binding chemistry for cell detection/isolation. Specifically, the invention suggests that in a heterogeneous-cell population, the NPs will selectively bind only to cells expressing CD44 that was proved by two separate experiments. It was observed that in the cell mixture (MDA-MB-231+ BT-474 cells), when MDA-MB-231 cells were increased from 0 to 2000 (simultaneous decrease in BT-474 cells from 2000 to 0), inventors observed a clear colorimetric change (pink to violet) accompanied by a SPR shift (521 nm to 559 nm). So it can be concluded that the specificity with which Au-PEG-HA NPs bind to MDA-MB-231 cells is driven by a strong HA-CD44 interaction, resulting in specific colorimetric changes as well as SPR peak shift. In a separate experiment, upon Au-PEG-HA NPs treatment with 5000 MDA-MB-231-GFP cells carrying CD44 receptor and their separation by differential centrifugation (600 rpm, 30 minutes), 96.87±2.80 % cells were found in pellet. Interestingly, in the case of BT-474/NIH 3T3/Blood cells which do not carry or very less expression of CD44 receptor, the percentage of cells in the supernatant was found to be 98.33±1.67, 96.67±3.33 and 95.77±4.23 respectively and significantly very less number of cells were found in pellet. Therefore, inventors concluded that Au-PEG-HA NPs selectively and specifically bind to MDA-MB-231 cells through CD44-HA interaction and helps in pelleting down the cells.
[075] Upon spiking MDA-MB-231 cells (high CD44 expression) with BT-474/ NIH 3T3/blood cells (low/negative expression of CD44) at different cell density (50%-1%), MDA-MB-231 cells were isolated with 60-94% efficiency and these viable cells can be cultured for further downstream experiments. Particularly, when the blood cells (low/negative expression of CD44) were spiked with 1% MDA-MB-231 cells (high CD44 expression). Inventors were able to isolate MDA-MB-231 with 74% efficiency that opened the window for circulatory tumor cells detection and isolation in blood upon further improvement.
[076] The harvested cells were cultured for up to 24hrs in DMEM+10% FBS. MDA-MB-231-GFP cells were observed at under fluorescent microscope, thus proving that the cells isolated from Au-PEG-HA NPs are viable and can be used in further downstream assays.
[077] IMPORTANT EMBODIMENTS AND EXPERIMENTAL PROCEDURES OF THE INVENTION:
[078] Objective 1: synthesis of gold nanoparticles and functionalization
[079] Citrate-capped Au NPs were prepared according to a modified Frens method (Huang et al. 2014; Ji et al. 2007). Typically, an aqueous solution of HAuCl4 (0.25 mM, 50 mL) was heated under reflux and then 1.3 mL solution of sodium citrate (1%) with or without PEG 400 (25µg/mL) was added under vigorous stirring. The solution was kept for boiling for 15 minutes, resulting in PEGylated Au NPs. The PEGylated Au NPs were next conjugated to HA by carbodiimide reaction with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalyst. The coupling chemistry used was EDC/NHS chemistry, which provides a covalent bond without the addition of a spacer (Fischer 2010). Briefly, 50 mg of the desalted HA was dissolved in 5 mL of NaHCO3/acetic acid buffer solution (pH 4.7), stirred for 2 hrs (400 rpm) and then activated with a mixture of NHS and EDC (molar ratio of NHS/EDC is 0.25), reacting for 3h with constant stirring (600 rpm) at room temperature. Subsequently, Au-PEG NPs were added into reaction mixture to obtain HA/NH2-PEG molar ratios of 1.0. The reaction was performed for 48 hrs with constant stirring (600 rpm) at room temperature. The resulting product (Au-PEG-HA NPs) was purified using a dialysis tube (12,000 MWCO) against distilled water for 24 hr.
[080] Methods: The conjugation of PEG and HA with Au NPs was confirmed by FTIR spectroscopy and NMR spectroscopy respectively. The colloidal Au NPs, Au-PEG NPs, Au-PEG-HA NPs were also characterized by using ultraviolet-visible spectroscopy (UV-VIS), transmission electron microscopy (TEM), dynamic light scattering (DLS) measurements.
[081] Results: The synthesis of Au-PEG-HA NPs was carried out by conjugating HA to PEGylated Au NPs as per the schematic mentioned in the FIG. 1a. Citrate-capped Au NPs were prepared according to a modified Frens method [See Methods]. The Au NPs were functionalized with PEG (PEGylation). The addition of PEG increases the hydrophilicity as well as the stability of the Au NPs (by preventing aggregation), and provides active functional groups (-NH2) required for HA conjugation. The Au-PEG NPs, in contrast to Au NPs, are stable at room temperature without any change in their properties, indicating that PEG is an efficient stabilizer for Au NPs as measured by DLS (FIG. 6a-b) and it’s SPR peaks (FIG. 6c-d). FTIR analysis confirmed polyethylene glycol capping on gold nanoparticles (Au-PEG NPs) by showing the signature peaks of PEG at 3444.17 cm-1 (O-H), 1095 cm-1 (C-O-C stretching) and 1637 cm-1 (C=O stretching) (FIG. 1b). Subsequent surface modification of Au-PEG NPs with HA was confirmed by 1H-NMR spectra demonstrating its chemical structure with characteristic peaks labeled i.e. broad signal (3.0 -3.8 ppm): protons in the sugar rings; 4.6 ppm: two anomeric protons attached to the carbons adjacent to the two oxygen atoms; 2.02 ppm: methyl (-CH3) protons of the N-acetyl group of HA (FIG. 1c and inset in FIG. 7a). In homogenous samples, all the three groups of NPs exhibit the same optical response upon illumination. All the three NP groups were pinkish in color with a characteristic surface plasmon resonance (SPR) peak located at 519-521 nm in UV-Vis spectra (FIG. 1d). Upon conjugation of HA to Au-PEG NPs, the SPR peak showed a slight shift from 519 to 521 nm and there was no change in color of the solution, indicating unaltered optical properties of Au NPs (FIG. 1d). The size of Au NPs, Au-PEG NPs and Au-PEG-HA NPs was characterized by using TEM and DLS studies (FIG. 1e and FIG. 7b-c). The average size of Au NPs, Au-PEG NPs, Au-PEG-HA NPs, assessed by TEM, was found to be 21.95±6.4 nm, 47.21±15.54 nm and 67.98 ±20.4 nm respectively (FIG. 1e). The increase in size of Au nanoparticles upon PEGylation, followed by HA conjugation was observed and further supported via DLS analysis. The hydrodynamic diameter of Au NPs, Au-PEG NPs and Au-PEG-HA NPs was 16.53 nm, 37.18 nm and 67.95 nm evaluated through dynamic light scattering (DLS) analysis (FIG. 7b). The range of size distribution of Au NPs, Au-PEG NPs and Au-PEG-HA NPs was 10-35 nm, 18-82 nm and 22-110 nm respectively (FIG. 1f). So, considering the lower margin, Inventors infer that ~18% particles are present in 1-30 nm range in case of Au-PEG NPs. Similarly in case of Au-PEG-HA NPs, ~3% particles are present in the range 1-30 nm and ~18% particles are present in the range of 30-50 nm and ~79% of the particles were present in the 50-110 nm range (FIG. 1f). The varied size of nanoparticles present in the Au-PEG-HA NPs alluded to the inefficiency of the conjugation process. Inventors believe that optimization of this process will lead to a narrower particle size distribution due to uniform distribution of HA on the surface of PEGylated Au NPs.
[082] Objective 2: optimization of au and ha concentration in au-peg-ha nps for specific cell detection
[083] Invention suggests that when the NPs comes in contact with cells that express the receptor for HA (CD44), it will lead to color change (and an accompanying absorbance shift) in the solution due to NPs-cell interaction-mediated aggregation of Au-PEG-HA NPs. Therefore, the next step was to optimize the concentration of both Au and HA in Au-PEG-HA NPs group to allow for clear visualization of the colorimetric changes induced by its binding to cells of our interest. For this, inventors utilized two cell lines that express varying levels of CD44: MDA-MB-231 (CD44+ve/high) and BT-474 (CD44 –ve/low).
[084] Methods: Au-PEG-HA NPs were serially diluted in a 96 well ELISA plate (concentration Au: 100-3.125 nM). Cells (MDA-MB-231 and BT-474) were trypsinized at 80% confluence and suspended in PBS (catalogue no: 10010023, GIBCO, Invitrogen, USA). 50,000 cells were added to each well and incubated for 10 min at room temperature. Optimal Au concentration was evaluated based on variation in colorimetric changes as well as absorbance spectrum for the detection of specific cells.
[085] Similarly, optimal concentration for hyaluronic acid (HA) for specific colorimetric detection was carried out by varying HA concentration (HA: 400-1.56 µg/mL, optimized Au concertation: 25 nM). 50,000 cells (MDA-MB-231 and BT-474) were added to each well and incubated for 10 min at room temperature. The colorimetric changes and absorbance spectrums were recorded (UV-VIS spectra) and analyzed for optimal HA concentration to detect specific cells. The aggregation effect of Au-PEG-HA NPs due NPs-cell interaction was monitored by observing A650/A521 ratio.
[086] Results: Inventors first varied the concentration of Au (100- 3.125 nM) and found that when 50,000 MDA-MB-231 (CD44+ve/high) cells are added to the NPs solution, the color of the solution changes from pink to violet (FIG. 2a), and this was accompanied by and a shift of the SPR peak from 521 to 559 nm (FIG. 2b). There was a simultaneous increase of absorbance at 650 nm as well as a corresponding increase in A650/A521 ratio, which confirmed the aggregation of NPs due to the interaction of Au-PEG-HA NPs with MDA-MB-231 cells (FIG. 2b). They also observed that the colorimetric change from pink to violet was extremely saturated at the highest Au gold concentration tested (100 nM), and the intensity of the color change showed a gradual reduction with lower Au concentrations (FIG 2a).
[087] Next, they added the same number (50,000) of BT-474 cells to the NPs solution and inventors observed a slight decrease in the absorbance peak value (521 nm) and a minor increase in the absorbance at 650 nm indicating some level of aggregation due to nonspecific NP-cell interaction (also inferred from the increase in A650/A521 ratio). Inventors observed an extremely saturated violet color at the highest Au concentration, which gradually reduced with decreasing Au concentrations (FIG. 2a). Since our goal was to develop a platform to detect the specific binding of cells of our interest as a function of colorimetric changes, visualization of the color change was a key parameter for evaluating the efficacy of this platform. Therefore, inventors chose the optimized Au concentration of 25 nM as this allowed for a clear observation of colorimetric changes with narrow and defined UV-Vis absorbance spectrum (with violet shift). Moving forward, the higher concentrations of Au (100 nM and 50 nM) were not considered to avoid color saturation as well broad absorbance spectrum (FIG. 2a & FIG. 8b). Similarly, the lower concentrations of Au (12.5 nM was excluded due to the broad absorbance spectrum (FIG. 8a).
[088] Next, they varied the concentration of HA (400-1.56 µg/mL) and kept a constant concentration of Au (optimized at 25 nM). Data showed the Au absorbance peak value decreased when MDA-MB-231 cells (50,000) were added, and this was accompanied by a shift of the SPR peak from 521 to 559 nm (FIG. 2c). Inventors also observed a change in the color of the NPs solution from pink to violet upon adding MDA-MB-231 cells (FIG. 2c) at all concentration of HA tested. No colorimetric change was observed as well as A650/A521 ratio variations (FIG. 2c-d). For all subsequent experiments in this study, the optimized HA concentration was selected to be 100 µg/mL based on the clear observation of colorimetric changes with narrow UV-Vis absorbance spectrum (with violet shift). The higher concentrations of HA (400, 200 µg/mL) were not considered due to the slightly viscous nature of HA at higher concentration, which causes improper optical absorbance (FIG. 2c and FIG. 8c-d). To sum up, Au-PEG-HA NPs on interaction with MDA-MB-231 cells showed specific colorimetric change with a narrow UV-Vis absorbance spectrum (accompanying SPR shift from 521 to 559 nm) at optimized Au (25nM) and HA (100µg/mL) concentrations and such colorimetric changes were not observed with BT-474 cells (FIG. 2c).
[089] Objective 3: differential identification of cells using au-peg-ha nps
[090] Methods: In a typical test, the interaction of Au-PEG-HA NPs with different cells (at varying number) was studied based on colorimetric changes as well SPR peak shift. The cells (MDA-MB-231, BT-474, NIH 3T3) were harvested by trypsiniaztion at 80% confluence and suspended in PBS. The cells were serially diluted (two fold) in 200 µL PBS from 50,000 to 195. 100 µL of Au-PEG-HA NPs (25 nM Au and 100 µg/mL HA) solution was added to each well (final volume 300 µL) followed by 10 min incubation at room temperature. The observed colorimetric change and UV-Vis absorption spectra were recorded and compared with the control (Au-PEG-HA NPs) values. The influence of cell numbers on the aggregation of the Au-PEG-HA NPs was investigated by monitoring the changes in the absorbance ratio at 650 and 521 nm (A650/A521) to develop a quantitative estimation of cell numbers bound to NPs.
[091] Results: Next, they added varying numbers (50,000-195) of MDA-MB-231 cells (CD44+ve/high) to Au-PEG-HA NPs and, as shown in FIG. 3a, they observed a gradual reduction of violet color with decreasing cell numbers, indicating an inverse relationship between colorimetric change and cell number which is dependent on degree of NPs-cell interactions. They also observed a shift of the SPR peak from 521 to 559 nm and a simultaneous increase in absorbance at 650 nm (higher A650/A521 ratio), which confirmed the aggregation of NPs due to a competition between cells and PBS (salt) that interact with Au-PEG-HA NPs (FIG. 3b). In case of BT-474 cells (CD44 –ve/low) and NIH 3T3 cells (CD44 -ve), inventors observed a decrease in the absorbance peak value (521 nm) and slight increase in A650 that indicate some level of nonspecific binding, but they did not see any colorimetric change (violet shift) as well as any meaningful variation in the A650/A521 ratio (FIG. 3a).
[092] To estimate the number of cells bound to the NPs as a function of the observed A650/A521 ratio, inventors plotted a standard curve between cell number and A650/A521 ratio. In MDA-MB-231 cells, a logarithmic relationship was observed between cell number and A650/A521 ratio (R2 value = 0.988), confirming that CD44-HA binding governs the interaction between NPs and cells (FIG. 3c). Such relationships were not observed for BT-474 cells and NIH 3T3 cells. This helps with a quantitative estimation of cells based on an easily measured parameter, another feature relevant to any diagnostic platform.
[093] Objective 4: exploiting cd44-ha interaction for specific cell detection in a heterogeneous population
[094] The underlying principle of this platform is the exploitation of ligand-receptor binding chemistry for cell detection/isolation. Specifically, inventors postulate that in a heterogeneous-cell population, the NPs will selectively bind only to cells expressing CD44.
[095] Method: To evaluate the efficacy of ligand-receptor chemistry in Au-PEG-HA NPs for specific cell detection, cell mixture was prepared by mixing MDA-MB-231 (CD44+ve/high) and BT-474 cells (CD44 –ve/low) in varying ratios, but keeping the total number of cells constant (2000) in each well (FIG. 4). Next, 100 µL of Au-PEG-HA NPs solution (25 nM Au and 100 µg/mL HA) was added to each well followed by 10 min incubation at room temperature. The colorimetric changes, UV-Vis absorption spectra and A650/A521 ratio (effect of aggregation) were recorded and compared with control (Au-PEG-HA NPs) values.
[096] In another experiment, recovery of cells isolated with Au-PEG-HA NPs was evaluated for all the different cells used in this study (MDA-MB-231/BT-474/NIH 3T3/Blood). So, individual cells i.e. MDA-MB-231 GFP /BT-474/NIH 3T3/Blood (5000 cells) were mixed with Au-PEG-HA NPs for 10 min, then separated by differential centrifugation (600 rpm/30 min). Cell viability (%) was evaluated in both the pellet and the supernatant by FACS analysis. BD Canto II FACS instrument was used for determining the percentage of MDA-MB-231 GFP cells captured in both the supernatant and the pellet. Analysis of the data was performed using Flowing software.
[097] Results: In order to evaluate the efficacy of the Au-PEG-HA NPs platform for specific cell detection, cell mixtures were prepared by mixing MDA-MB-231 (CD44+ve/high) and BT-474 cells (CD44 –ve/low) in varying ratios, but keeping the total number of cells constant (2000) (FIG. 4a). 100 µL of Au-PEG-HA solution (25 nm Au and 100 µg/mL HA) was added to each well, followed by 10 min incubation at room temperature.
[098] Inventors observed that in the cell mixture, when MDA-MB-231 cells were increased from 0 to 2000 (simultaneous decrease in BT-474 cells from 2000 to 0), inventors observed a clear colorimetric change (pink to violet) accompanied by a SPR shift (521 nm to 559 nm) (FIG. 4a-b). Also, the presence of MDA-MB-231 cells in the cell mixture, contributes to a slight decrease in absorbance at 650 nm (as reflected from decreased A650/A521). Here, inventors also observed a linear relationship between MDA-MB-231 cell number and A650/A521 ratio (R2 value= 0.97) (FIG. 4c). Inventors conclude that the specificity with which Au-PEG-HA NPs bind to MDA-MB-231 cells is driven by a strong HA-CD44 interaction, resulting in specific colorimetric changes as well as SPR peak shift.
[099] In a separate experiment, inventors added 5000 MDA-MB-231-GFP cells to a solution containing Au-PEG-HA NPs and allowed the cells to incubate with the NPs for 10 minutes. After this period, the NP-bound cells were separated as a pellet by differential centrifugation (600 rpm, 30 minutes). Inventors reasoned that if our hypothesis was correct, allowing for some non-specific binding, the vast majority of the MDA-MB-231-GFP would be contained in the NP-containing pellet and not in the supernatant. Additionally, if cells that have low to negative expression of CD44 (BT-474, NIH 3T3 and blood) were to be added to the NP solution. The analyzed cells present in both the pellet and the supernatant by FACS inventors found that in the case of MDA-MB-231, 96.87±2.80 % cells were found in pellet. Interestingly, in the case of BT-474/NIH 3T3/Blood, the percentage of cells in the supernatant was found to be 98.33±1.67, 96.67±3.33 and 95.77±4.23 respectively and significantly very less number of cells were found in pellet (FIG. 4d). Therefore, they concluded that Au-PEG-HA NPs selectively and specifically bind to MDA-MB-231 cells through CD44-HA interaction.
[0100] Objective 5: au-peg-ha nps mediated recovery of high percentage of viable cells from target population
[0101] Methods: The cells (MDA-MB-231, BT-474, NIH 3T3) were harvested by trypsiniaztion at 80% confluence and suspended in PBS. The blood samples (10 ml) were collected in 10% (w/w) sodium citrate by venipuncture from healthy volunteers at Mazumdar Shaw Medical Center, Bangalore, India. For lysed blood, 1 ml of whole blood was mixed with 10 mL of ACK lysing buffer (ThermoFisher Scientific A1049201) for 5 min at room temperature. After red blood cells were lysed, the white blood cells were collected by centrifuging at 1200 rpm for 5 min. The supernatant was aspirated and white blood cells were resuspended in 1 mL PBS to generate the lysed blood. To evaluate the cell capture efficiency and viability, MDA-MB-231 cells were mixed with BT-474/NIH 3T3/Blood at varying densities (50%, 5%, 1%). 100 µL of Au-PEG-HA NPs solution was added followed by 10 min incubation at room temperature. The colorimetric changes, UV-Vis absorption spectra and aggregation effect (A650/A521) were measured and compared with the control (Au-PEG-HA NPs) values. Then, the cells attached to the Au-PEG-HA NPs were harvested by centrifuging at 600 rpm for 30 min (Kowalczyk et al., 2011). The viability (Live/Dead) of recovered cells in the pellet was evaluated by using the trypan blue assay. The harvested cells were cultured for up to 48hrs in DMEM+10% FBS and observed at 24hrs and 48hrs under fluorescent microscope for viability and growth pattern.
[0102] Inventors showed that the viability of the isolated cells of interest (MDA-MB-231) from a heterogeneous cell population (BT-474/NIH 3T3/blood) to be between 76-88% depending upon various experimental conditions (spiking density of 50%, 5% and 1% of MDA-MB-231 cells) (FIG. 5b). To further validate the trypan blue assay results, a separate experiment was designed to determine the specific cell capture efficiency where GFP-expressing MDA-MB-231 cells were spiked into BT-474/NIH 3T3/Blood cell suspension at varying spiking densities (50%, 5%, 1%), the cells bound to Au-PEG-HA NPs were isolated using differential centrifugation (600 rpm for 30 min). The supernatant and the pellet were analyzed at green fluorescence range to determine the percentage of captured MDA-MB-231-GFP cells by BD Canto II FACS instrument. Analysis of the data was performed using Flowing software to calculate the percentage of MDA-MB-231-GFP cells in the pellet (FIG. 5c). Finally, in order to assess whether the cells recovered from the NP platform retain their morphology and phenotype in culture, we cultured them for up to 24 hours in DMEM+10% FBS. Our data showed that MDA-MB-231-GFP cells retain their morphology in culture, leading us to conclude that they can be used in further downstream assays (Fig. 5d).
[0103] Results: In any real-world usage scenario, the relevance of this platform would be measured by two attributes: (a) how effectively can it ‘sort’ the cells of interest from a heterogeneous population; and, (b) are the ‘sorted’ cells viable for further downstream processing? To answer these questions we mixed MDA-MB-231 cells with BT-474/NIH 3T3/Blood at varying densities (50%, 5%, 1%). The intention of including blood cells here was to increase the application scenario of our synthesized NPs (i.e., can it be employed for the detection of specific cells, like circulating tumor cells, present in the blood?). In each scenario, the absorbance peak value decreased and was accompanied by a violet shift of the SPR peak from 521 to 559 nm and a simultaneous increase in absorbance at 650 nm, which confirmed the aggregation of NPs due to NPs-cell (MDA-MB-231) interaction (also observed from the increase in A650/A521 ratio) (FIG 5 a and FIG. 9a-c). The isolation and recovery of target cells was evaluated in a heterogeneous population of cells where MDA-MB-231 cells were spiked into BT-474/NIH 3T3/Blood cells at different cell densities (50%, 5%, 1%). The cell mixtures were then incubated with Au-PEG-HA NPs (25 nM Au, 100 µg/mL HA) for 10 minutes. Subsequently, the cells bound to the NPs were separated by differential centrifugation (600 rpm/30 min). The resulting pellet, which contains NP-bound cells, was disassociated and cells were checked for viability by the trypan blue live/dead cell counting assay. Inventors observed 76-88% recovery of viable cells from the heterogeneous populations (FIG. 5b). Specifically, in the mixture of MDA-MB-231 and BT-474 cells, the cell viability was found to be 83.24±0.45 %, 74±1.73% and 79.70±3.15% at 50%, 5% and 1% spiking density respectively (FIG. 5b). Similarly, in the MDA-MB-231+NIH 3T3 mixture, the cell viability was 88.50±1.78%, 88.46±1.19% and 78.63±10.96% at 50%, 5% and 1% spiking density respectively. Again, in the mixture of MDA-MB-231 and blood cells, the cell viability was found to be 84.85±1.62 %, 79.39±2.23% and 78.52±3.06% at 50%, 5% and 1% spiking density respectively. The harvested cells were cultured for up to 24hrs in DMEM+10% FBS. MDA-MB-231-GFP cells were observed at 24hrs and 48hrs under fluorescent microscope, thus proving that the cells isolated from Au-PEG-HA NPs are viable and can be used in further downstream assays (FIG. 5d).
[0104] It can be argued that the trypan blue live/dead cell count assay provides information about total cell viability; this data likely includes viability of the negative cell population captured due to nonspecific interactions with the NPs. So, to specifically evaluate the viability of cells of our interest, MDA-MB-231-GFP cells were spiked in to BT-474/NIH 3T3/Blood cell suspension at varying densities (50%, 5% and 1%) and the specific-cell (MDA-MB-231-GFP) capture efficiency of Au-PEG-HA NPs was evaluated by counting GFP +ve cells (cells-of-interest) corresponding to GFP expression through FACS analysis. FACS analysis (FIG. 5 c) showed that more than 90% of live MDA-MB-231/GFP +ve cells were recovered at 50% cell density (5000:5000). With a decrease of target cells in the heterogeneous population, the percentage of GFP +ve cells decreased. Specifically, at 50% of MDA-MB-231-GFP (5000:5000), the cell recovery was 92.73±4.43% (with BT-474), 92.54±3.51% (with NIH 3T3) and 94.45±5.25% (with blood cells). When the MDA-MB-231-GFP cells were reduced to 5% (5000:95000), the percentage of GFP +ve cells was further decreased to 70.87± 13.10% (with BT-474), 78.03±12.24% (with NIH 3T3) and 81.90± 6.49% (with blood cells). Again, on further reduction of MDA-MB-231-GFP cells to 1% (1000:99000), the percentage of GFP +ve cells was decreased to 62.39± 21.03% (with BT-474), 59.74±11.95% (with NIH 3T3) and 73.60± 5.79% (with blood cells). Here inventors concluded that upon spiking MDA-MB-231 cells (high CD44 expression) with BT-474/ NIH 3T3/blood cells (low/negative expression of CD44) at different cell density (50%-1%), inventors are able to isolate MDA-MB-231 cells with 60-94% efficiency and these viable cells can be cultured for further downstream experiments.
[0105] IMPORTANT ATTRIBUTES OF Au-PEG-HA NPs SYSTEM
[0106] Exploiting the unique spectral properties of Au NPs and the high affinity of ligand-receptor binding features in Au-PEG-NPs, CD44 expressing cells can be easily detected by the clear observation of colorimetric change (SPR shift) due to NPs-cell interaction leading the aggregation of NPs.
[0107] By exploiting receptor-ligand specificity, the target cells from a heterogeneous population can be separated upon interaction with Au-PEG-HA NPs followed by differential centrifugation method (even at 1% target cell density) and it compares favorably, both in terms of specificity as well as cell viability, with current platforms described in the literature
[0108] Importantly, cell viability with this platform is as high as 76–88%, which is beneficial for successive exploitation and manipulation of cells including culturing them and performing further downstream experiments.
[0109] IMPORTANT EMBODIMENTS OF THE PRESENT INVENTION:
[0110] In the present research Inventors have shown that our platform can be used easily and reliably to detect (colorimetric observation) and isolate (differential centrifugation) cells at low cost in resource-limited conditions (which preclude expensive instrumentation), with high efficiency, specificity and cell recovery by exploiting substrate-ligand binding using Au NPs. These NPs based platform can also be very useful for single cell isolation even at lower number of cells (below 10,000) unlike FACS that proves its efficacy. Again, this research could be a viable alternative to antibody-mediated targeting of cells by minimizing issues like high cost and the possibility of nonspecific chemical interactions.
[0111] Au-PEG-HA NPs can be simple and effective platform for cell detection based only on colorimetric observation and UV-absorption spectrum analysis that does not require sophisticated instrumentation, a high level of technical expertise.
[0112] Similarly, this nano-biosensor can be made widely useful by using different receptors/ligands specific to different target cells.
[0113] By exploiting receptor-ligand specificity, the target cells from a heterogeneous population can be separated by differential centrifugation method with high specificity and viability (even at 1% target cell density).
[0114] Also, the continual improvements in small molecule screening will allow for incorporation of multiple ligand-receptor combinations on Au NPs, thereby improving the detection limit of a platform beyond 0.01%, making it useful for detecting circulatory tumor cells in blood.
[0115] Importantly, cell viability with this platform is as high as 76–88%, which is conducive for subsequent manipulation of cells including culturing them and performing molecular biological diagnosis.
[0116] Finally, our platform provides a convenient and cost-efficient alternative for cell sorting in laboratories and for isolation of rare populations of cells that cannot feasibly be done using existing technologies such as FACS or MACS.
[0117] Also, this NPs system with high affinity of ligand-receptor binding chemistry can be a viable alternative to antibody-mediated targeting of cells by minimizing issues like high cost and the possibility of nonspecific chemical interactions.
[0118] IMPORTANT ATTRIBUTES OF THE PRESENT INVENTION:
[0119] 1. Effective conjugation: Effective conjugation of the ligand Hyaluronic acid(HA) to the cell specific CD44 receptor is achieved, so that higher percentage of receptor bearing cells are easily detected and reliable.
[0120] 2. Specific targeting: This NPs system can used for specific cell-detection that works by exploiting the high affinity of ligand-receptor binding
For example, Inventors have used HA (Hyaluronic Acid) to specifically target CD44 expressing cells, where, HA can bind to CD44 cell surface receptor. So, Au-PEG-HA NPs selectively bind to the cells expressing the CD44 receptor, demonstrating CD44-HA receptor-ligand specificity.
[0121] Similarly, this NP based platform can be made widely useful by using different receptors/ligands specific to other cells. Also, the continual improvements in small molecule screening will allow for incorporation of multiple ligand-receptor combinations on Au NPs, thereby improving the detection limit of a platform beyond 0.01%, making it useful for detecting circulatory tumor cells in blood.
[0122] 3. Applicable for various receptor-ligand combination: This nanoparticle delivery system is made more specific by targeting cancer cell-specific receptor by using particular ligands. HA (Hyaluronic acid) has been used to specifically target breast carcinoma where, HA can bind to CD44 receptor, highly expressed in cancer cells. Furthermore, this nanoparticulate system can be made widely useful by using different receptors/ligands specific to different cancer.
[0123] 4. Effective cell isolation: Subsequently, the target cells bound to the NPs can be effectively separated by differential centrifugation (600 rpm/30 min) with a high percentage recovery of cells of interest (60-94%), demonstrating the high specificity and robustness of the developed NPs.
[0124] 5. Cell viability: Importantly, the recovered cell viability with this platform is as high as 76-88%, which is conducive for subsequent manipulation of cells including culturing them and performing molecular biological diagnosis.
[0125] 6. Cost effectiveness: Our platform provides a convenient and cost-efficient alternative for cell sorting in laboratories and for isolation of rare populations of cells that cannot feasibly be done using existing technologies such as FACS or MACS.
[0126] Alternative to antibody-mediated cell targeting: Also, this NPs system with high affinity of ligand-receptor binding chemistry can be a viable alternative to antibody-mediated targeting of cells by minimizing issues like high cost and the possibility of nonspecific chemical interactions.
a. Objectives achieved,
b. Formulation of Functionalized ligand conjugated Nanocomplex (AuNP-PEG-HA)
c. In vitro Efficiency (Cell Specific)
d. Identify specific receptor bearing cells
e. Isolation of selective cell population of cells bearing the specific receptor
f. High percentage cell viability
g. Cost-effectiveness.
h. Alternative to antibody-mediated cell targeting.
[0127] Merely for illustration, only representative number/type of graph, chart, block and sub- block diagrams were shown. Many environments often contain many more block and sub- block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[0128] According to a non-limiting exemplary aspect of the present invention, the markers can be used for the development of kits that enable sample collection, processing and marker detection. These diagnostic kits developed can then be utilized by hospitals/private clinics/dental doctors or the public as such to screen/diagnose oral cancer.
[0129] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0130] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0131] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
[000] References:
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,CLAIMS:CLAIMS

I/We Claim,

1) A nanoparticle based biosensor for cell detection and isolation system comprising:
a) gold nanoparticle,
b) polymer, and
c) linker molecule that acts as a ligand for cell surface receptors,
wherein the gold nanoparticle is conjugated to linker molecule that acts as a ligand for cell surface receptors, wherein the polymer augments the efficacy of ligand-receptor binding.
2) The nanoparticle based biosensor for cell detection and isolation system of claim 1, wherein the system is a specific combination of a cell-specific receptor and a particular ligand, wherein specific ligands are functionalized with specific chemical groups to make this system specific and effective for a specific cell type.
3) The nanoparticle based biosensor for cell detection and isolation system of claim 1, wherein linker molecule is HA or DUPA or antibodies, wherein specific receptor is a CD44 cell surface receptor or a receptor protein, wherein the polymer is PEG.
4) A method for cell detection and isolation in a biological sample from a subject, the method comprising the steps of:
a) formulation of Au-PEG-HA NPs platform
b) exposing Au-PEG-HA NPs cell recognition agent capable of selectively binding to the target receptor biomarker, wherein binding of ligand to receptor leads to aggregation of NPs and changes in spectral properties; and
c) detecting/isolating NPs bound target cells by absorbance/ colorimetric/differential centrifugation/ Fluorescence spectral analysis.
5) The method for cell detection and isolation system of claim 4, wherein the cell detection and/or isolation method involve colorimetric observation and/or UV-absorption spectrum analysis and/or differential centrifugation and/or Fluorescence spectral analysis.
6) The method of claim 4, wherein the nanoparticle based biosensor for cell detection and isolation system is used for detection and/or isolation of selective cell types possessing specific surface receptors from a heterogeneous cell population, wherein selective cell types can be circulating tumor cells in blood resulting from primary/metastatic cancer.
7) The method of claim 4, wherein the nanoparticle based biosensor is used for cell detection and/or isolation and/or bioassays.
8) The method of claim 4, wherein the method is an in-vitro cell detection and isolation method.
9) The method of claim 4, wherein the subject is suffering premalignant and/or malignant stage of cancer or early stage for chemoprevention of dysplastic progression and for Stage II, Stage III, or Stage IV cancers for second primary prevention, or late-stage cancer, or metastasized cancer, or solid tumors, or cancer that has relapsed after treatment with another therapeutic modality, or carcinoma or sarcoma or melanoma or lymphoma or leukemia.
10) A kit for cell detection and isolation in a biological sample from a subject, wherein the kit comprising of at least one reagent that specifically binds to a cell surface receptor or cancer biomarker.