WO2015051465A1

WO2015051465A1 - Nanoparticular antibodies and uses thereof as contrast agents

Info

Publication number: WO2015051465A1
Application number: PCT/CA2014/050983
Authority: WO
Inventors: Danny BROUARD; Olivier Ratelle; Denis Boudreau
Original assignee: Héma-Québec; UNIVERSITé LAVAL
Priority date: 2013-10-11
Filing date: 2014-10-10
Publication date: 2015-04-16

Abstract

Metal/silica core-shell nanoparticles, such as silver/silica core-shell nanoparticles, having low polydispersity, and a process for their preparation, are described. The nanoparticles may comprise one or more fluorophores embedded with the silica shell, as well as one or more targeting moieties, such as an antibody, attached to the silica shell. Use of such nanoparticles for various applications, including the detection of the presence of a biomolecule on the surface of a cell, is also described.

Description

NANOPARTICULAR ANTIBODIES AND USES THEREOF AS CONTRAST AGENTS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application serial No. 61/889,651 , filed on October 11 , 2013, which is incorporated herein by reference in its entirety. TECHNICAL FIELD

The present invention generally relates to bioimaging and cell tagging applications, and more particularly to nanomaterials for bioimaging and cell tagging applications dedicated to blood product characterization and blood bank operations.

BACKGROUND ART

For the last two decades, nanotechnology has experienced a phenomenal growth, and its scope of applications has been extended to medicine, giving birth to a new research area called nanomedicine.^{1 2} The interest for nanometer-scale materials comes from the observation of drastic variations in the physical and photophysical properties of nanomaterials.³ Noble metals, such as gold and silver, can be wisely exploited to design high performance biosensors and contrast agents for bioimaging and cell tagging experiments.^{4 5} Nowadays, the field of application for these nanomaterials is expanding rapidly, and there is a strong demand for nanoparticular architectures offering multifunctionalities allowing many parameters to be measured simultaneously in a single experiment.⁶

There is thus a need for the development of novel nanomaterials useful for various applications such as bioimaging and cell tagging experiments.

The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides the following items 1 to 29:

1. Metal/silica core-shell nanoparticles comprising:

(i) a spherical core comprising a metallic material; and

(ii) a shell surrounding said core, said shell comprising silica;

wherein the core of the nanoparticles has a mean diameter of about 20 nm to about 100 nm, and at least 80% of the core of the nanoparticles have a diameter within 12 nm of the mean diameter.

2. The nanoparticles of item 1 , wherein at least 90% of the core of the nanoparticles have a diameter within 12 nm of the mean diameter. 3. The nanoparticles of item 1 , wherein at least 80% of the core of the nanoparticles have a diameter within 10 nm of the mean diameter.

4. The nanoparticles of item 1 , wherein at least 90% of the core of the nanoparticles have a diameter within 10 nm of the mean diameter.

5. The nanoparticles of any one of items 1 to 4, further comprising a targeting moiety attached to said shell.

6. The nanoparticles of item 5, wherein said targeting moiety is an antibody.

7. The nanoparticles of any one of items 1 to 6, wherein said targeting moiety is attached to said shell through an amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends.

8. The nanoparticles of item 7, wherein said crosslinker is sulfosuccimidyl-4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (sulfo-SMCC) group.

9. The nanoparticles of any one of items 1 to 8, further comprising one or more fluorophores incorporated within said shell.

10. The nanoparticles of item 9, comprising a plurality of fluorophores incorporated within said shell.

11 . The nanoparticles of any one of items 1 to 10, wherein said metallic material comprises silver or gold, preferably silver.

12. A method for detecting the presence of a biomolecule on the surface of a cell, said method comprising contacting said cell with metal/silica core-shell nanoparticles comprising:

(i) a core comprising a metallic material;

(ii) a shell surrounding said core, said shell comprising silica; and

(iii) a targeting moiety binding to said biomolecule attached to said shell.

13. The method of item 12, wherein nanoparticles are the nanoparticles of any one of items 1 to 11 .

14. The method of item 12 or 13, wherein said cell is a red blood cell.

15. The method of item 14, wherein said biomolecule is a blood group antigen.

16. The method of item 15, wherein said blood group antigen is a rare blood-group antigen.

17. A process for the preparation of metal/silica core-shell nanoparticles comprising the steps of:

providing metal nanoparticles,

providing one or more fluorescent silica precursors,

directly coating the metal nanoparticles with a fluorescent silica shell made from the fluorescent silica precursors and another silica precursor, functionalizing the surface of the silica shell,

reacting the functionalized surface with a coupling agent, and

attaching a targeting moiety to the coupling agent.

18. The process of item 17, wherein the metal is silver and the nanoparticles are provided by: adding an aqueous solution of AgNC>3 to a boiling and stirred aqueous solution of sodium citrate tribasic dehydrate, and

while stirring, boiling the mixture to allow for nanoparticle growth.

19. The process of item 18, wherein the mixture is further cooled down to room temperature.

20. The process of any one of items 17 to 19, wherein the fluorescent silica precursors are provided by reacting a isothiocyanate modified fluorophore with a silica precursor bearing an amine group.

21 . The process of item 20, wherein the silica precursor bearing an amine group is (3- aminopropyl)ethoxysilicate.

22. The process of any one of items 17 to 21 , wherein the fluorescent silica shell is coated on the metal nanoparticles by mixing together and reacting the fluorescent silica precursor(s), the other silica precursor, and the metal nanoparticles.

23. The process of item 22, wherein the other silica precursor is tetraethyl orthosilicate.

24. The process of any one of items 17 to 23, wherein said functionalizing is thiolating the surface of the silica shell.

25. The process of any one of items 17 to 23, wherein the coupling agent is an amine-to- sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends.

26. The process of item 25, wherein the coupling agent is sulfosuccimidyl-4-(N- maleimidomethyl)cyclohexane-1 -carboxylate.

27. The process of any one of items 17 to 26, wherein the targeting moiety is an antibody.

28. The process any one of items 17 to 27, wherein the nanoparticles are nanoparticles according to any one of items 1 to 11 .

29. Nanoparticles prepared according to the process of any one of items 17 to 28.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

In the appended drawings: FIGs. 1A and 1 B show UV spectra of several batches of nanoparticles produced according to a published method¹² (FIG. 1A) compared with UV spectra of several batches of nanoparticles according to an embodiment of the present invention (FIG. 1 B).

FIGs. 2A and 2B show transmission electron microscopy (TEM) images of silver/silica core-shell nanoparticles (Ag@SiC>2) . FIG. 2A shows the size distribution of the Ag@SiC>2 silver core (several transmission electron microscopy (TEM) images were used). FIG. 2B shows the spacer shell thickness (measured from TEM images) variability as a function of the amount of fluorophore precursor used during Ag@SiC>2 synthesis.

FIG. 3 shows the fluorescence emission spectra of eosin doped nanoparticles. The fluorescence intensity of Ag@SiC>2 varies as a function of the fluorescent silica precursor concentration used during synthesis.

FIGs. 4A to 4I show UV-Vis and fluorescence spectra of three Ag@SiC>2 nanoparticles: eosin (FIGs. 4A-4C), fluorescein (FIGs. 4D-F), and rhodamine (FIGs. 4G-I). FIGs. 4A, 4D and 4G show the variation of the UV-Vis spectra as a function of the concentration of fluorescent silica precursor used during the synthesis. FIGs. 4B, 4E and 4H present the fluorescence excitation and emission spectra of the corresponding Ag@SiC>2 nanoparticles. FIGs. 4C, 4F and 4I display TEM photographs of the Ag@SiC>2 nanoparticles.

FIG. 5 shows darkfield (left) and fluorescence (right) microscopy images of Ag@SiC>2 nanoparticles onto which fluorescein (top panels) and rhodamine (bottom panels) was coupled to the shell.

FIG. 6A shows the determination of the fluorescence enhancement factor (EF) for three types of

Ag@SiC>2 nanoparticles.

FIG. 6B shows TEM photographs of core-less nanoparticles after peroxide treatment;

FIG. 6C shows UV-Vis spectra of Ag@SiC>2 nanoparticles before and after peroxide treatment. FIG. 7 shows darkfield microscopy images of Ag@SiC>2 + FITC nanoparticles functionalized with APC-conjugated anti-CD235a using (bottom row) or not using (top) sulfo-SMCC for coupling the conjugated antibody.

FIGs. 8A and 8B show the specific recognition of red blood cells (RBCs) by NanoAbs. FITC- NanoAbs conjugated with an APC-conjugated anti-CD235a were used to tag RBCs and samples were observed by darkfield and fluorescence microscopy (FIG. 8A). A similar experiment was performed with FITC-NanoAbs conjugated with an unlabeled anti-CD41 a antibody and, as expected, RBCs were not labeled by these FITC-NanoAb-anti-CD41 (FIG. 8B).

FIGs. 9A and 9B show NanoAbs as cell markers for flow cytometry experiments. Cells tagged with highly fluorescent NanoAbs can easily be distinguished (FIG. 9A). Furthermore, NanoAbs induce significant changes in the side scattering channel of tagged cells (FIG. 9B). Cells tagged with fluorescent NanoAbs are easier to recognize since they induce changes in two channels simultaneously.

FIGs. 10A and 10B show RBC tagged with NanoAbs conjugated with an anti-CD235a. FIG. 10A: RBCs were observed by transmission darkfield microscopy (left) and confocal microscopy in darkfield imaging mode (right). FIG. 10B: Extinction spectra of three NanoAbs were recorded. NanoAbs' extinction spectra can be acquired and used to probe the local environment or to measure aggregation.

FIG. 11 shows fluorophore-free RBC imaging using NanoAbs. Non-fluorescent NanoAbs conjugated with an anti-CD235a were used to tag RBCs. Red channel shows the light scattered by the RBCs, and the green channel represents the LSPR light scattered by the NanoAbs.

FIGs. 12A and 12B show fluorescent NanoAbs vs. fluorophore-conjugated molecular antibodies.

RBCs were tagged with NanoAbs conjugated with an anti-CD235a (FIG. 12A), or phycoerythrin (PE)- conjugated anti-CD235a monoclonal antibodies (FIG. 12B). NanoAbs show greater detection sensitivity and are efficient at localizing cellular antigens.

FIG. 13 shows low-abundance antigen localization using dual-mode optical microscopy and fluorescent NanoAb. Red channel presents RBC scattered light recorded by confocal microscopy in darkfield mode. The green channel was used to record NanoAbs fluorescence. In this case, NanoAbs anti- Do(b) (Dombrock Do(b)) were used to locate Dombrock antigens on the surface of Do(ab) RBCs.

FIG. 14A shows UV-Vis spectroscopy characterization of gold nanoparticles (Au NPs) and fluorescent gold-core silica-shell nanoparticles.

FIG. 14B shows fluorescence spectroscopy characterization of gold-core silica-shell fluorescein nanoparticles (Au@Si02-FITC) and cleared supernatant recovered after washing NPs.

FIG. 15 shows a characterization of the antibody functionalization reaction. Median fluorescence intensity (MFI) signals from anti-human CD45 and anti-human CD4 were compared to MFIs from anti- human CD45-PerCP-Cy5 (A_exc = 488 nm, A_exc = 705 ± 10 nm) and anti-human CD4-PE (A_exc = 488 nm, Aexc = 590 ± 25 nm). MFI increases were observed for both NanoAbs, demonstrating successful crosslinking reactions. The relative increase measured for anti-human CD4-PE is lower because of the spectral overlap with the fluorescence from Ag@Si02 + FITC NPs.

FIG. 16 shows a fluorescence spectral characterization of the fluorophores (FITC, PE, PerCP-

Cy5.5)

FIGs. 17A to 17D shows density plots recorded for the monitoring of crosslinking reactions. FIG.

17A: Side scattering (SSC) vs. forward scattering (FSC) for anti-human CD45 NanoAbs. FIG. 17B: SSC vs. FITC fluorescence for anti-human CD45 NanoAbs. FIG. 17C: PerCP-Cy5 vs. FITC fluorescence for anti- human CD45 NanoAbs compared to anti-human CD45-PerCP-Cy5 NanoAbs (FIG. 17D). FIGs. 18A and 18B show the results of Jurkat cell tagging experiments using three different NanoAbs. FIG. 18A: Median side scattering intensity values were recorded for three different NanoAbs and compared to untagged Jurkat cells and Jurkat cells incubated with NanoAbs. FIG. 18B: MFIs for the FITC fluorescence channel were recorded for three different NanoAbs and compared to untagged Jurkat cells and Jurkat cells incubated with NanoAbs.

FIGs. 19A and 19B show the results of Jurkat cell tagging experiments using NanoAbs. FIG. 19A: Density plots recorded for Jurkat cells tagged with anti-human CD235 NanoAbs, anti-human CD4 NanoAbs, and anti-human CD45 NanoAbs (from left to right, respectively). FIG. 19B: FITC fluorescence channel intensity distributions for each NanoAb + Jurkat cell sample (dark grey), compared to untagged Jurkat cells (light grey).

FIGs. 20A and 20B show the results of Jurkat cell tagging experiments using monoclonal antibodies. FIG. 20A: Density plots recorded for Jurkat cells tagged with anti-human CD235-APC, anti- human CD4-PE, and anti-human CD45-FITC (from left to right, respectively). FIG. 20B: Fluorescence channel intensity distributions for each fluorescent antibody + Jurkat cell combination samples (dark grey), compared to untagged Jurkat cells (light grey).

DISCLOSURE OF INVENTION

In the claims and throughout the specification, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein. It should be understood that all combinations and subcombinations of features/embodiments, including preferred embodiments, described herein are encompassed by the present invention. The term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

The development of nanoparticular antibodies (NanoAbs) based on metal/silica core-shell nanoparticles, such as silver/silica (Ag@SiC>2) or gold/silica (Au@SiC>2) core-shell nanoparticles, is described herein.

Accordingly, in a first aspect, the present invention provides metal/silica core-shell nanoparticles comprising, consisting essentially of, or consisting of: (i) a spherical core comprising a metallic material; and

(ii) a shell surrounding said core, said shell comprising silica;

wherein the core of the nanoparticles has a mean diameter of about 20 nm to about 100 nm, and at least 80%, preferably at least 85 or 90%, of the core of the nanoparticles have a diameter within 12 nm of the mean diameter.

In another aspect, the present invention provides metal/silica core-shell nanoparticles comprising, consisting essentially of, or consisting of:

(i) a spherical core comprising a metallic material;

(ii) a shell surrounding said core, said shell comprising silica; and

(iii) one or more fluorophores covalently attached to, or covalently

incorporated/embedded into, said shell.

In another aspect, the present invention provides silver/silica core-shell nanoparticles comprising, consisting essentially of, or consisting of:

(i) a spherical core comprising silver and/or gold, preferably silver;

(ii) a shell surrounding said core, said shell comprising silica; and

(iii) one or more fluorophores covalently attached to, or covalently

incorporated/embedded into, said shell.

(i) a spherical core comprising a metallic material;

(ii) a shell surrounding said core, said shell comprising silica; and

(iii) one or more targeting moieties, preferably antibodies, covalently attached to said shell.

(i) a spherical core comprising silver and/or gold;

(ii) a shell surrounding said core, said shell comprising silica; and

(i) a spherical core comprising a metallic material;

(ii) a shell surrounding said core, said shell comprising silica; (iii) one or more fluorophores covalently attached to, or covalently incorporated/embedded into, said shell; and

(iv) one or more targeting moieties, preferably antibodies, covalently attached to said shell.

(i) a spherical core comprising silver and/or gold;

(ii) a shell surrounding said core, said shell comprising silica;

(iii) one or more fluorophores covalently attached to said shell; and

The metallic material may be any metal (or combination of metals) amenable to nanoparticle formation. Preferred metals are those providing metal-enhanced fluorescence (MEF). In an embodiment, the metallic material comprises/is gold, silver, platinum, aluminum, palladium, copper, cobalt, iron, indium, nickel or any combination thereof. In a further embodiment, the metal metallic material comprises/is silver or gold. In an embodiment, the metallic material comprises/is silver. In another embodiment, the metallic material comprises/is gold.

In an embodiment, the core of the nanoparticles has a mean diameter of about 20 nm to about 100 nm, of about 30 to about 90 nm, of about 30 to about 80 nm, of about 30 to about 70 nm, of about 40 to about 65 nm, or of about 45 to about 60 nm, such as for example about 40, 45, 50, 55 or 60 nm, preferably about 50, 51 , 52, 53, 54 or 55 nm. In these and other embodiments, at least 80%, 85% or 90% of the nanoparticles have a core with a diameter within about 12 nm, preferably within about 10, 9, 8, 7, 6 or 5 nm of the mean diameter.

In embodiments, the thickness of the silica shell is of about 5 to about 20 nm, of about 6 to about 19 nm, of about 7 to about 18 nm, of about 8 to about 17 nm, of about 9 to about 16 nm or of about 10 to about 15 nm, such as for example about 8, 9, 10, 11 , 12, 13, 14 or 15 nm, more preferably about 10 nm.

As used herein, the term "fluorophore" refers to a molecule, label or moiety that has the ability to absorb energy from light, transfer this energy internally, and emit this energy as light of a characteristic wavelength. Any fluorescent label or fluorophore may be used without limitation with the nanoparticles, methods and compositions provided herein. Examples of fluorophores that may be used include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), 5-carboxyfluorescein (FAM), 2'7'-dimethoxy- 4'5'-dichloro-6-carboxyfluorescein (JOE), Oregon green, eosin, rhodamine, TRITC, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'- dimethylaminophenylazo) benzoic acid (DABCYL), 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), Texas Red®, coumarin (and derivatives thereof such as hydroxycoumarin, aminocoumarin and methoxycoumarin), cyanine (and derivatives thereof such as indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole), pyrene derivatives (e.g., Cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, malachite green), tetrapyrrole derivatives (e.g., porphin, phthalocyanine, bilirubin), BODIPY® (Invitrogen®), Alexa Fluor® (Invitrogen®), R-Phycoerythrin (PE), Cy2, Cy3, Cy5, Cy7, allophycocyanin (APC), and PerCP. Preferred fluorophores include fluorescein, eosin and rhodamine. In an embodiment, the nanoparticles comprise one fluorophore. . In another embodiment, the nanoparticles comprise a plurality of fluorophores. In a further embodiment, the nanoparticles comprise two fluorophores. In another embodiment, the nanoparticles comprise there, four or five fluorophores.

In an embodiment, the nanoparticles further comprise one or more targeting moieties attached to said shell. The term "targeting moiety" as used herein refers to a molecule that is capable of binding the nanoparticles of the present invention to a binding site on a target cell. Thus, the targeting moiety is a ligand through which nanoparticles of the present invention binds to a selected target cell. Such targeting moiety may be, for example, a natural or synthetic ligand of a molecule expressed by a cell, for example a nucleic acid, a peptide, a polypeptide, a sugar moiety (carbohydrate), a lectin, a small molecule, a lipid, and the like. The targeting moiety may bind to a receptor, substrate, antigenic determinant, or other binding site on a target cell. In an embodiment, the targeting moiety is a polypeptide, preferably an antibody (or an antigen-binding fragment thereof), for example an antibody capable of binding to a protein (e.g., a receptor) expressed at the surface of a cell. The target cell may be any target cell (any cell type). In an embodiment, the target cell is a blood cell, such as a peripheral blood mononuclear cell (PBMC, e.g., lymphocytes, monocytes/macrophages), a thrombocyte/platelet or a red blood cell. In an embodiment, the targeting moiety is not tagged with a label, such as a fluorophore. In another embodiment, the targeting moiety is tagged with a label, such as a fluorophore.

The term "antibody" is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies, multispecific antibodies, and antibody fragments, as long as they exhibit the desired activity (e.g., binding to a target cell). Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. In an embodiment, the antibody is a monoclonal antibody. In an embodiment, the antibody is not labelled, e.g., with a fluorophore. In another embodiment, the antibody is detectably labelled, e.g., with a fluorophore.

In embodiments, the antibody is anti-glycophorin A (anti-CD235a), which is a molecular antibody specific to red blood cells, anti-platelet glycoprotein lib (anti-CD41 a), which is a molecular antibody specific to thrombocytes, anti-Dombrock Do(b) (anti-Do(b)), specific to the b allele of the Dombrock antigen, anti- CD4 which is a molecular antibody specific to CD4-expressing cells such as subsets of T lymphocytes (T- helper lymphocytes), monocytes, macrophages and dendritic cells, or an anti-CD45, which is a molecular antibody specific to CD45-expressing cells, such as most leukocytes.

In an embodiment, the nanoparticles do not comprise an intervening layer between the metal nanoparticle surface and the fluorescent silica shell. In particular, there is no "pure" silica layer (i.e. silica without fluorophores) between the metal nanoparticle surface and the fluorescent silica shell. Accordingly, in other aspects, the present invention provides the metal/silica core-shell nanoparticles as defined above, wherein the nanoparticles do not comprise an intervening layer (e.g., a pure silica layer) between the metal nanoparticle surface and the fluorescent silica shell.

In another aspect, the present invention provides a detection mixture comprising the metal/silica core-shell nanoparticles as defined above and a cell. In an embodiment, the detection mixture further comprises a physiologically acceptable buffer, e.g. phosphate buffer saline (PBS).

In another aspect of the invention, there is provided a method of producing the above metal/silica core-shell nanoparticles. This method comprises the steps of:

providing metal nanoparticles,

providing one or more fluorescent silica precursors,

directly coating the metal nanoparticles with a fluorescent silica shell made from the fluorescent silica precursors and another silica precursor,

functionalizing the surface of the silica shell,

reacting the functionalized surface with a coupling agent, and

attaching a targeting moiety to the coupling agent.

In embodiments, the metal is silver and the nanoparticles are provided by:

adding an aqueous solution of AgNC>3 to a boiling and stirred aqueous solution of sodium citrate tribasic dehydrate, and

while stirring, boiling the mixture to allow for nanoparticle growth.

The addition of the AgNC>3 aqueous solution to the boiling sodium citrate tribasic dehydrate solution is advantageous compared to the addition of sodium citrate tribasic dehydrate solution to a boiling solution of AgNC>3. Indeed, it surprisingly greatly increases the reproducibility of the method. The nanoparticles produced consistently have repeatable features, such as UV spectra and polydispersity [i.e., measure of the size heterogeneity of the nanoparticles in the mixture). FIG. 1A shows the UV spectra of several batches of nanoparticles produced according to a published method¹² compared with several batches of nanoparticles according to the presently claimed invention (FIG. 1 B) (see Example 1 below for details). Clearly, the UV spectra of the nanoparticles of the invention are more reproducible that those of the method disclosed in reference 12. Furthermore, since the width of the UV extinction band varies as a function of nanoparticle polydispersity, it is also clear that nanoparticles of the invention consistently have a good polydispersity, which is not the case with the method disclosed in reference 12. Without being bound by theory, it is believed that this is due to a stabilizing effect of sodium citrate tribasic dehydrate on the pH of the reaction medium, which in turn impacts on the nucleation and nanoparticle growth rate.

The reaction medium is advantageously cooled down to room temperature before further use. The nanoparticles can then be isolated by known means, for example, centrifugation.

Fluorescent silica precursors are silica precursors having attached thereto a fluorophore as described above. Preferred fluorescent silica precursors are those produced by reacting an isothiocyanate modified fluorophore with a silica precursor bearing an amine group, such as (3-aminopropyl)ethoxysilicate (APTES, a silica precursor). Such silica precursors and their method of making are known in the art (see reference 13, incorporated herein by reference). They can be produced, for example, in dimethylformamide (DMF) in the presence of a base such as triethylamine. Other fluorescent silica precursors are well-known in the art; see, for example: Hermanson G (2013), "Bioconjugate Techniques, 3^rd Edition" (Academic Press), and references cited therein, incorporated herein by reference.

The produced fluorescent silica precursor solution can generally be kept cold (-20°C) in the dark for about 2 weeks without modification to its fluorescence properties.

In the next step, metal nanoparticles are directly coated with a fluorescent silica shell comprising fluorescent silica precursors. Herein, "directly coated" means that there is no intervening layer between the metal nanoparticle surface and the fluorescent silica shell. In particular, there is no "pure" silica layer (i.e., silica without fluorophores) between the metal nanoparticle surface and the fluorescent silica shell. It was indeed surprisingly found that good fluorescence enhancement factors (EF) were obtained even in the absence of such an intervening "pure" silica layer. It is known in the art that fluorophores located too close to a metal core are subject to fluorescence extinction, which reduces the measured EF. However, it is shown in the examples below that the EFs measured in metal nanoparticles directly coated with a fluorescent silica shell according to the invention are nevertheless surprisingly high and thus constitute a good compromise, considering the simplicity of synthesis, easier large-scale production and low level of polydispersity. The fluorescent silica shell can be produced through a variant of the well-known Stober method (see, for example, reference 12). This variant involves mixing together and reacting the fluorescent silica precursor(s), as well as a silica precursor (non-fluorescent, for example tetraethyl orthosilicate (TEOS)), with the metal nanoparticles. This would usually be carried out in ethanol (e.g., anhydrous ethanol), at a basic pH (for example pH 10, adjusted using NH4OH), stirring, for about 20 hours at room temperature in the dark.

The nanoparticles can then be isolated by known means, for example, centrifugation.

It was observed that the fluorescence of the nanoparticles increased as a function of the fluorescent silica precursor(s) concentration up to a plateau. On the other hand, the thickness of the shell was not much affected by that concentration .

More than one fluorescent silica precursor may be successfully incorporated into the silica shell, thereby allowing multiplex applications, the simultaneous detection of a plurality of markers in a single assay.

To attach a targeting moiety on the surface of the metal/silica core-shell nanoparticles, the surface of the silica shell should be functionalized. Different methods to do so have been described in the art (Hermanson G (2013), "Bioconjugate Techniques, 3^rd Edition" (Academic Press), and references cited therein). One preferred method is to thiolate the surface (add thiol groups on the surface), which is described in the art. This can done by reacting the nanoparticles with a thiolating agent; refer to: Hermanson G (2013), "Bioconjugate Techniques, 3^rd Edition" (Academic Press), and references cited therein, for a thorough list of thiolating agents. A preferred thiolating agent is 3-mercaptopropyl-trimethoxy- silane (MPT MS). Suitable reaction conditions are, for example, in ethanol at room temperature and/or at a higher temperature (for example, about 45-55°C, or about 50°C).

Advantageously, the nanoparticles are then washed, for example by centrifugation.

Then, the nanoparticles are reacted with a coupling agent that will allow attaching a targeting moiety to the nanoparticles. Several coupling agents are known in the art; refer to Hermanson G (2013), "Bioconjugate Techniques, 3^rd Edition" (Academic Press), and references cited therein, for a thorough list of souch coupling agents. A preferred coupling agent is sulfosuccimidyl-4-(N-maleimidomethyl)cyclohexane-1 - carboxylate (sulfo-SMCC), an amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane spacer arm. It was observed that this coupling agent advantageously reduces undesirable aggregation of the nanoparticles. Suitable reaction conditions for use of sulfo-SMCC are, for example, in DMF at room temperature. Other examples of amine- to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends of spacer arms (of different lengths) are commercially available from Thermo Scientific, and include Pierce® AMAS, Pierce® BMPS, Pierce® EMCS, Pierce® GMBS, Pierce® LC-SMCC, Pierce® MBS, Pierce™ SM(PEG)n crosslinkers, Pierce™ SMCC, Pierce® SMPB and Pierce® SMPH, Pierce® Sulfo-EMCS, Pierce® Sulfo- GMBS, Pierce® Sulfo-KMUS, Pierce® Sulfo-MBS and Pierce® Sulfo-SMPB.

Finally, a targeting moiety is attached to the nanoparticles. As stated above, preferred targeting moieties include antibodies or antigen-binding fragments thereof. Suitable reaction conditions for attachment of antibodies on nanoparticles with sulfo-SMCC are, for example, in phosphate buffer at room temperature.

Advantageously, the nanoparticles are then washed, for example by centrifugation. They are then ready for use.

In another aspect, the present invention relates to a method for detecting the presence of a biomolecule on the surface of a cell, said method comprising contacting said cell with the metal/silica core- shell nanoparticles described herein, wherein the formation of a complex between the biomolecule and the metal/silica core-shell nanoparticles (through the targeting moiety) is detected by a detectable change. In an embodiment, the above method comprises measuring said detectable change.

In an embodiment, the metal/silica core-shell nanoparticles comprise a detectable moiety such as a fluorophore, a dye or a radioactive label, and the method comprises detecting the fluorophore, dye or radioactive label on the surface of the cell. Methods for visualizing the detectable change resulting from biomolecule complex formation include any fluorescent detection method, including without limitation fluorescence microscopy, a microtiter plate reader or flow cytometry.

As exemplified below, a simple, fast and cost effective method for the synthesis of NanoAbs was developed. The preparation steps appear to be scalable (at least up to 10x), and the observed extended Ag@SiC>2 shelf-life (see the Examples below) should allow them to be stored (e.g. in anhydrous ethanol in the dark) for months until use.

NanoAbs core made of a spherical silver particle, characterized by a narrow size distribution (i.e., low polydispersity) with a mean size of about 54 nm were developed. The size of the silver core could be varied.

The use of a silver core confers some of the special NanoAb photophysical properties. First, the core can interact with excitation light to improve the luminescence properties of fluorophores located in the silica shell, a phenomenon known as metal-enhanced fluorescence (MEF).^7"9 Secondly, the silver core can generate localized surface plasmon resonance (LSPR). This resonance is responsible for the color of Ag@SiC>2 nanoparticles under darkfield optical microscopy.¹⁰ Such NanoAbs were indeed observed as diffraction-limited bluish spots by darkfield microscopy. The nanoparticles' outer silica shell has many functions. It acts as a steric and electrostatic barrier preventing the silver core from chemical attacks and confers to the NanoAb their long-term stability in most common biological media.

Different types of fluorophores can be embedded inside the silica shell during its synthesis (by modifying the silica precursor), and their respective concentrations can be modulated to make the NanoAb spectrally distinguishable and amenable to multiplex analysis.

Sulfosuccimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (sulfo-SMCC), a bifunctional molecule, was used to couple antibodies onto the surface of Ag@SiC>2 or Au@SiC>2 nanoparticles through a simple and efficient cross-linking reaction allowing the antibodies to retain their detection specificity.

Furthermore, the maximum extinction wavelength of NanoAbs can be modified by their local environment or red-shifted with the interparticular distance. The latter phenomenon can be exploited as a ruler to evaluate distances between cell receptors or as a visual observation of analyte-triggered nanoparticle aggregation. The color of NanoAbs, as observed by dark field microscopy, can thus be used to characterize their immediate chemical/biological environment. When two or more particles get close to one another, plasmonic coupling may occur, resulting in a spectral shift towards more reddish hues. This phenomenon can be measured, calibrated, and used to detect the presence of an analyte that causes aggregation of the particles.

NanoAbs can be used to specifically label some cells in a population in order to differentiate them later by flow cytometry. Highly fluorescent NanoAbs could easily distinguish labeled cells from those that were not. The NanoAbs can thus be used both in cell imaging and cell labeling for flow cytometry applications.

Because of their plasmonic properties and to localized surface plasmon resonance (LSPR), NanoAbs can be observed by dark field microscopy. By filtering scattered light, it is possible to isolate NanoAbs within the sample. Thus, imaging applications are possible without using complex equipment.

The NanoAbs extinction spectra can be acquired and used to probe the local environment or as an index of aggregation.

Nanoparticles can be used to locate low density cellular receptors. The fluorescence intensity of a single nanoparticle could be easily detected. The detection of a labeled receptor by NanoAbs was possible. NanoAbs could contribute to significant improvements in the performance of phenotyping tests and reduce the need to rely on genotyping tests and costs related thereto.

Finally, fluorophore-free NanoAbs can be prepared and used as cheap contrast agents for cell imaging experiments requiring low excitation power or extended exposure times (no photobleaching). MODE(S) FOR CARRYING OUT THE INVENTION

The present invention is illustrated in further details by the following non-limiting examples.

Example 1 : Materials and Methods

Silver core synthesis

Silver nanoparticles were prepared by sodium citrate reduction of silver nitrate. More specifically, in a 1 L Erlenmeyer flask, 500 ml_ of a 0.74 mM sodium citrate tribasic dehydrate solution in deionized water was bought to a rolling boil and kept under vigorous stirring. 500 μΙ_ of a 1.06 M AgNC>3 solution was added using a volumetric pipet. The solution was left to boil for 50 minutes and cooled down under constant stirring until the mixture reached room temperature. Finally, the total volume of the solution was adjusted to 350 ml_ with deionized water. The nanoparticle concentration was calculated at 1 ^E11 NPs/mL by ICPAES elemental determination of silver, assuming an average nanoparticle diameter of 50 nm.

Core-shell nanoparticle preparation

Prior to the nanoparticle shell synthesis, fluorescent silica precursors for each fluorophore to be incorporated were prepared. Fluorescein, eosin and rhodamine silica precursors were synthetized as follows.

First, a total of 5.7 μιτιοΙ of isothiocyanate-modified fluorophores are dissolved in 1 14 μΙ_ of dimethyl formamide. 11 .5 μιτιοΙ of triethylamine and 6.8 μιτιοΙ of (3-aminopropyl)triethoxysilane were added and the solution was left to react under vigorous stirring for 2 hours at room temperature. The volume of the solution was adjusted to 13.5 ml_ with anhydrous ethanol to obtain a final concentration of ~ 0.4 mM of fluorescent silica precursor.

The fluorescent silica coating was made using a modified Stober method. 5 μΙ_ of previously prepared nanoparticles were mixed to 30 ml_ of anhydrous ethanol in a 50 ml_ conical plastic flask. 700 μΙ_ of a 9.9 mM TEOS/ethanol solution with 400 μΙ_ of NH4OH and 270 μΙ_ of the fluorophore precursor solution were added subsequently. The nanoparticle mixture was allowed to react under continuous stirring for 20 hours at room temperature in the dark.

The highly luminescent nanoparticles were then purified by centrifugation (3X, 9700 RCF, t = 15 minutes) and resuspended in a final volume of 12 mL anhydrous ethanol. The nanoparticle concentration was calculated at ~ 6^E10 NPs/mL by ICPAES elemental determination of silver using an average nanoparticle diameter of 54 nm after sample digestion in an aqueous 10% HNO3 solution.

The core-shell nanoparticle synthesis could be scaled up 10 times without any modification in the reactive molar ratios and stored for months in anhydrous ethanol, away from room illumination (in the dark).

A transmission electron microscopy (TEM) image of the obtained core-shell nanoparticles is presented in FIG. 2. Analysis of the TEM image showed that the silver core had a mean diameter of about 54 ± 12 nm and bore a silver shell about 10 ± 1 nm thick.

This figure also shows that the nanoparticles have a low polydispersity. The top histogram shows the size distribution of the Ag@SiC>2 silver core. The bottom histogram indicates that the spacer shell thickness varies as a function of the amount of fluorophore precursor used during the Ag@SiC>2 synthesis.

During development, it was also observed that the concentration of the silica precursor used for the synthesis does not significantly alter the thickness of the silica layer. Also, the size of the silver core could be varied.

Very inexpensive and quickly prepared, the nanoparticles presented a bluish hue when observed by dark-field microscopy, and were observed to be stable in biological media.

Antibody functionalization (NanoAb preparation)

The surface of the nanoparticles was first thiolated. In a 50-mL conical plastic tube, 100 μΙ_ of 3- mercaptopropyl-trimethoxy-silane (MPTMS) were mixed to 10 mL of core-shell silver nanoparticles resuspended in ethanol. The solution was left to react for a first 2-hour period at room temperature. To maximize the covalent reaction yield, the temperature was raised to 50°C for 1 hour.¹⁴ Thiol-modified nanoparticles were washed by centrifugation (3X, 9700 RCF, 15 min) and resuspended in a final volume of 10 mL anhydrous ethanol.

Depending on the amount of nanoparticular antibodies required for the analysis, 150 μί of a 0.075 M sulfo-SMCC solution in DMF per 1 mL of thiolated NPs resuspended in DMF were mixed and left to react under continuous stirring for 1 hour at room temperature. Then, nanoparticles were washed (3X, 9700 RCF, 15 min) and resuspended in phosphate buffer (PB, 0.1 M, pH 8.43).

Finally, 20 μί of a 0.5 mg/ml {- 160 kDa) antibody solution are added to 1 ml of NPs in PB and the mixture was left to react for 2 hours. The antibody-functionalized core-shell NPs are washed (3X, 9700 RCF, 15 minutes) and resuspended in PB.

Red blood cell extraction from blood samples

Venous blood samples were obtained by phlebotomy from voluntary donors after informed consent. After reception, blood samples (V = 7 mL) were split into six aliquots of 1 mL and centrifuged (510 RCF, 7 minutes). Supernatants were eliminated from packed red blood cells (RBCs) and adjusted to 1 mL by adding Additive Solution Formula 3 (AS-3). This process was repeated two more times, and washed RBCs were stored at 4°C for a maximum of 40 days.

NanoAb-RBC tagging Anti-glycophorin A (anti-CD235a) and anti-platelet glycoprotein lib (anti-CD41 a) molecular antibodies were functionalized at the surface of core-shell nanoparticles (NanoAb anti-CD235a and NanoAb anti-CD41 a).

Anti-CD235a and anti-CD41 a molecular antibodies are specific to RBCs and to thrombocytes, respectively. The latter were used as a negative control to evaluate the detection specificity of NanoAbs.

50 μΙ_ of a freshly prepared anti-CD235a NanoAb solution were mixed with 200 μΙ_ of a 1/10000 dilution of packed red blood cells (pRBCs) in 1X phosphate-buffered saline (PBS). The mixture was gently mixed by rotation in a 500 μΙ_ Eppendorf® tube for 20 minutes at 4°C. After the incubation time, the sample was washed three times by centrifugation (510 RCF, 10 minutes) to eliminate unbound NanoAbs from the RBC mixture. The final volume was adjusted to 500 μΙ_ using 1X PBS and kept refrigerated (4°C) until analysis.

The very same procedure was performed with the anti-CD41 a NanoAb. Fluorescence confocal microscopy measurements.

The fluorescence confocal apparatus used for this study was custom-made and built around the IX-71 inverted microscope from Olympus®. The excitation source was a pulsed supercontinuum white laser from Fianium®. The repetition rate was set to 20 MHz and the excitation wavelength fixed to 488 nm using a Fianium® white laser and a versachrome 490 nm (TBP01-490/15-25x36, Semrock®). The maximum excitation power used was measured at 50 W and found to give the best overall signal-to-background noise ratio. A UIS2 series 100X oil immersion objective (Olympus®, UPLFLN 100x012) was used for all confocal experiments presented in this study. Fluorescence was filtered using bandpass filters, focused on a 75 μιτι diameter pinhole and detected with an avalanche photodiode. The instrumental setup was completely automated and computer-controlled from sample motion to image acquisition (Symphtime®, PicoQuant®). Image observations and 3D reconstructions were performed post-analysis using the Imaris® software from Bitplane®.

Epifluorescence and darkfield measurements.

The very same instrumental setup used for confocal analysis was employed for epifluorescence and darkfield experiments. The excitation source was switched to a halogen lamp and fluorescence filter cubes were used for wavelength selection. Three objectives (40X, 60X, 100X, Olympus®) were available, and images captured with a color charge-coupled device (CCD) camera. For the darkfield microscopy analysis, a cardioid condenser was positioned in the illumination pathway. Diffused light from the sample was collected using the same 60X and 100X objectives, adjusting their numerical aperture (N.A.) to a slightly lower value than the cardioid condenser (N.A. = 0.92), and imaged on the same color CCD camera used for the epifluorescence analyses.

Gold (Au@.SiO?) NanoAb Preparation

For the experiment with gold NanoAbs, commercial gold nanoparticles were used as starting material (40 nm gold nanospheres, NanoXact® 0.05 mg/ml, citrate surface, 25 ml; nanoComposix®, Inc., San Diego, CA, USA).

A. Conditioning of the Au nanoparticle surface and fluorescent silica coating synthesis:

1. 2.5 ml Au NPs were diluted to 15 ml using phosphate buffer, pH 8.5, at room temperature.

2. Solution was under continuous magnetic stirring throughout the synthesis.

3. 20 μΙ (3-aminopropyl)trimethoxysilane 97% (Sigma-Aldrich®, cat. #281778) was added to the solution. Final concentration of (3-aminopropyl)trimethoxysilane was « 7.5 mM.

4. Reaction time: 3 hours; reaction temperature: 25°C, under continuous magnetic stirring.

5. NPs were washed by three cycles of centrifugation (10 000 RCF, t = 10 minutes).

6. NPs were resuspended in a final volume of 15 ml ethanol.

7. Starting with a 15-ml solution of conditioned Au NPs under continuous magnetic stirring, 10 μΙ of TEOS (Sigma-Aldrich®, cat. #131903) were added. Final concentration of

TEOS ^« 3 mM.

8. 50 μΙ of FITC fluorescent silica precursors (APS-FITC) were added. The preparation of the fluorescent silica precursor was as described for silver core NPs. Final concentration of APS-FITC ^« 15 μΜ.

9. 625 μΙ NH₄OH 28.0-30.0% (Sigma-Aldrich®, cat. #320145) was added to the nanoparticle solution. Final concentration of NH4OH ~ 0.3M.

10. Reaction time: 24 hours; reaction temperature: 25°C, under continuous magnetic stirring.

11 . Nanoparticles were washed by three cycles of centrifugation (10 000 RCF, t = 10 minutes).

12. Nanoparticles were resuspended in a final volume of 5 ml ethanol.

B. Sulfhydryl (SH) functionalization: 1. Starting with a 5-ml solution of Au@SiC>2-FITC under continuous magnetic stirring, 10 μΙ (3-mercaptopropyl)trimethoxysilane (Sigma Aldrich®, cat. #175617) was added. Final concentration of (3-mercaptopropyl)trimethoxysilane ~ 10 mM.

2. Reaction step 1 : reaction time: 2 hours, reaction temperature: 25°C, under continuous magnetic stirring.

3. Reaction step 2: reaction time: one hour, reaction temperature: 50°C, under continuous magnetic stirring.

4. Nanoparticles were washed by three cycles of centrifugation (10 000 RCF, t = 10 minutes).

5. Nanoparticles were resuspended in a final volume of 5 ml ethanol.

C. Antibody functionalization:

Monoclonal antibody functionalization of the surface of Au@SiC>2 NPs was performed as described for Ag@SiC>2 NPs above.

D. Spectral characterization of Au@SiC>2-FITC nanoparticles (FIG. 14):

1. UV-Vis spectral characterization was performed using a NanoDrop® 2000 spectrophotometer (Thermo Scientific®). Two-μΙ aliquots were directly loaded onto the sample holder.

2. Fluorophore incorporation into the silica shell:

i. 200 μΙ NP suspension in a 96-well plate.

ii. Centrifugation (10 000 RCF, t = 10 minutes); 200 μΙ cleared supernatant recovered and put aside NP pellets.

3. Comparison of fluorescence spectra of NP pellets and cleared supernatants.

E. Cell tagging:

1. Cell tagging protocol was as described above for silver core NPs. An excess of NanoAbs was incubated with target cells. After incubation (t = 20 minutes, T = 4°C), unbound NanoAbs were removed by centrifugation (510 RCF, t = 10 minutes).

2. Final volume was adjusted to 500 μΙ using 1X PBS and kept refrigerated (4°C) until analysis.

F. Cell analyses by flow cytometry:

1. Depending on the cell concentration, a 10- to 50-μί aliquot of NanoAb-tagged cells were added to one ml 1 χ PBS in a sample injection tube.

2. Sample elution was performed at count rates below 20 events/second.

3. Cells were gated using the side scattering channel (SSC) vs forward scattering channel (FSC) panel. 4. Specific vs. non-specific binding was evaluated by comparing FITC fluorescence distributions from Jurkat cells (blank) and NanoAb-incubated Jurkat cells (FIGs. 18A and 18B).

Table 1 : Antibodies used for Jurkat cell tagging experiments

Antibody identification Short name Manufacturer Catalog number

Anti-Human CD45 PerCP-Cy5 (HI30) Anti-CD45 PerCP-Cy5 Tonbo Biosciences 65-0459 T025

Anti-Human CD45 FiTC (HI30) Anti-CD45 FiTC Tonbo Biosciences 35-0459-T025

Anti-Human CD45 (Hi30) Anti-CD45 Tonbo Biosciences 70-0459 U100

Anti-Human CD4 PE (OKT4) Anti-CD4 PE Tonbo Biosciences 50-0048 T025

Anti-Human CD4 (OKT4) Anti-CD4 Tonbo Biosciences 70-0048 U100

Anti-Human CD235 APC Anti-CD235 APC BD Biosciences 551336

Anti-Human CD235 Anti-CD235 BD Biosciences 555569

Example 2: Results

FIG. 3 shows the fluorescence emission spectra of eosin-doped nanoparticles. The fluorescence intensity of Ag@SiC>2 varies as a function of the fluorescent silica precursor concentration used during the synthesis (0 μΜ produces a flat line, then curves from bottom to top are 0.9 μΜ, 2.1 μΜ, 3.3 μΜ, and 4,5 μΜ). The fluorescence increases until it reaches a peak/maximum. At that level, fluorophores become too close to each other and there is fluorescence quenching by non-radiative energy transfer (homotransfer).

FIGs. 4A to 4I show the spectral characterization (UV-Vis and fluorescence) of three Ag@SiC>2 nanoparticles, eosin (FIGs. 4A-4C), fluorescein (FIGs. 4D-F), and rhodamine (FIGs. 4G-I). FIGs. 4A, 4D and 4G show the variation of the UV-Vis spectra as a function of the concentration of fluorescent silica precursor used during the synthesis. FIGs. 4B, 4E and 4H present the fluorescence excitation and emission spectra of the corresponding Ag@SiC>2. FIGs. 4C, 4F and 4I display TEM photographs of each Ag@SiC>2 nanoparticle. These figures demonstrate that it is possible to integrate different fluorophores in the silica layer of the nanoparticles. Also, the architecture of the nanoparticles is shown to be independent of the composition of the silica layer.

FIG. 5 shows darkfield (left) and fluorescence (right) microscopy images of Ag@SiC>2 nanoparticles onto which fluorescein (top row) and rhodamine (bottom row) was incorporated within the shell.

Ag@SiC>2 nanoparticles. FIG. 6B shows TEM photographs of core-depleted nanoparticles after peroxide treatment. FIG. 6C shows UV-Vis spectra of Ag@SiC>2 nanoparticles before and after peroxide treatment. These figures show that the nanoparticles, irrespective of the fluorophore incorporated into the silica layer, exhibit advantageous plasmon enhancement of their fluorescence (MEF).

The amplification factor (EF) is slightly lower than what is reported in the literature for nanoparticles that have a more complex architecture, such as spacing between the metal core and the silica shell (EF = 10). This decrease is caused by close proximity (3-5 nm) between the silver core and the fluorescent silica layer. Fluorophores located too close to the core are indeed subject to fluorescence extinction, which reduces the measured EF. However, the measured EFs are nevertheless surprisingly high and thus constitute a good compromise, considering the simplicity of synthesis, easier large-scale production and low level of polydispersity of the nanoparticles prepared according to the invention.

To demonstrate antibody functionalization at the surface of the nanoparticles, Ag@SiC>2 + FITC nanoparticles were coupled with APC-conjugated anti-CD235a in the absence or presence of the bifunctional molecule (sulfo-SMCC).

The crosslinking reaction with monoclonal antibodies is efficient with, but not possible without, sulfo-SMCC. FIG. 7, top row, shows the absence of fluorescence in the APC channel for the nanoparticles functionalized without sulfo-SMCC, whereas the bottom row demonstrates that fluorescence is observed when sulfo-SMCC is used for crosslinking. Of note, little non-specific adsorption was observed when no sulfo-SMCC was used.

Two types of fluorescent (FiTC) NanoAbs were prepared. The first was coupled with a fluorescent antibody (ACP) specific to a membrane receptor for red blood cells, glycophorin A (anti-CD235a). The second NanoAb was coupled with an antibody against an antigen (anti-CD41 a) that is absent from erythrocytes.

FIGs. 8A and 8B show the specific recognition of red blood cells (RBCs) by NanoAbs. FiTC- NanoAbs conjugated with an APC-anti-CD235a were used to tag RBCs and samples were observed by darkfield and fluorescence microscopy (FIG. 8A). A similar experiment was performed with FITC-NanoAbs conjugated with an unlabeled anti-CD41 a antibody and, as expected, RBCs were not labeled by these FITC-NanoAbs-anti-CD41 (FIG. 8B). This experiment demonstrates that monoclonal antibodies do not lose their specificity of detection when coupled to the surface of silver-core / silica-shell nanoparticles.

FIGs. 9A and 9B show NanoAbs as cell markers for flow cytometry experiments. Cells tagged with highly fluorescent NanoAbs can be easily distinguished (middle row). Furthermore, NanoAbs induce significant changes in the side scattering channel of tagged cells (top and bottom rows). Therefore, cells tagged with fluorescent NanoAbs are easier to recognize, since they induce changes in two channels simultaneously. This shows that NanoAbs can be used to specifically label some cells in a population, so as to differentiate them later by flow cytometry. The highly fluorescent NanoAbs could easily distinguish labeled cells from those that were unlabeled. The NanoAbs can thus be used both in cell imaging and cell labeling for flow cytometry applications.

FIGs. 10A and 10B show a RBC tagged with NanoAbs anti-CD235a. The RBC was observed by darkfield microscopy (left) and darkfield confocal microscopy (right). The extinction spectra of the three NanoAbs (circled in right micrograph) were recorded and are shown in the bottom row. The NanoAbs extinction spectra can be acquired and used to probe the local environment, or else to monitor aggregation. The color of the NanoAbs observed by dark field microscopy can be measured and used to characterize their relative proximity. When particles are relatively close to each other, plasmonic coupling can occur, resulting in a spectral shift towards more reddish hues. This phenomenon can be measured, calibrated and used to measure the density and relative distance between, e.g., antigens on a cell surface.

FIG. 11 shows fluorophore-free RBC imaging using NanoAbs. Non-fluorescent NanoAbs anti- CD235a was used to tag RBCs. Red channel (dimmer grey halos) shows light scattered by the RBCs, and the green channel (brighter grey spots) represents the localized surface plasmon resonance (LSPR) light scattered by the NanoAbs. Due to their plasmonic properties and to LSPR, NanoAbs can be observed by dark field microscopy. Filtering the scattered light, it is possible to observe isolated NanoAbs within the sample. Thus, imaging applications are possible without the need for complex equipment.

FIGs. 12A and 12B show fluorescent NanoAbs vs. fluorophore-conjugated molecular antibodies. RBCs were tagged with NanoAbs anti-CD235a (top) and phycoerythrin (PE)-conjugated molecular antibody anti-CD235a (bottom). Nanoparticles show greater detection sensitivity, and are better suited to localize cellular antigens. Both strategies are complementary. Nanoparticles exhibit higher fluorescence intensity and excellent resistance to photodegradation. Nanoparticle fluorescence is discrete, which allows to localize antigens, and to perform statistical research related to antigen density, distance between antigens, and the like.

FIG. 13 shows the localization of rare antigens using dual-mode optical microscopy and fluorescent NanoAbs. The red channel (darker grey ring) presents RBC-scattered light recorded by darkfield microscopy. The green channel (brighter grey spots) was used to record NanoAb fluorescence. In this case, anti-Do(b) (Dombrock Do(b)) NanoAbs were used to locate Dombrock antigens on the surface of Do(ab) RBCs. This shows that nanoparticles can be used to detect and locate low-density cellular receptors. The fluorescence intensity of a single nanoparticle could be easily detected. The detection of a labeled receptor by NanoAbs was possible. NanoAbs could help to significantly improve the performance of phenotyping tests and reduce the occurrence of genotyping tests and costs related thereto.

FIG. 14 shows fluorophore incorporation into the silica shell of Au@Si02 NPs. A red shift in the extinction spectrum of Au nanoparticles indicates a variation in the dielectric environment at the nanoparticle-solvent interface caused by the presence of the silica shell (FIG. 14A). Assessment of successful fluorescein functionalization on gold-core silica-shell nanoparticles. The fluorescence signal from Au@SiC>2 + FITC NPs (light grey line) was compared to that of the cleared supernatant (dark grey line) after separation by centrifugation (FIG. 14B).

FIG. 15 shows an assessment of successful antibody functionalization. Anti-human CD45, anti- human CD45-PerCP-Cy5, anti-human CD4 and anti-human CD4-PE were covalently linked to the surface of Au@SiC>2-FITC NPs. After crosslinking, fluorescence from luminescent antibodies was compared to that of non-luminescent antibodies. An increase in median fluorescence intensity (MFI) was recorded for NPs functionalized with fluorescent antibodies.

FIG. 16 shows a spectral characterization of the fluorophores (FITC, PE, PerCP-Cy5.5).

FIGs. 17A to 17D show cytometry density plots recorded for scattering (FIG. 17A) and fluorescence (FIG. 17B) profiles of Au@Si0₂-FITC NPs. Cytometry density plots for Au@Si0₂-FITC NPs functionalized with anti-human CD45 (FIG. 17C) was compared to Au@Si02-FITC NPs functionalized with anti-human CD45-PerCP-Cy5 (FIG. 17D). The increase in fluorescence recorded for anti-human CD45-PerCP-Cy5 confirms the covalent binding of antibodies on the surface of Au@Si02-FITC NPs.

FIGs. 18A and 18B show an assessment of successful cell tagging applications. Three different

NanoAbs were prepared with distinct detection specificities towards Jurkat cells: a (black bars) anti-human CD45 NanoAbs (high level of expression on Jurkat cells); b (light grey bars): anti-human CD235 NanoAbs (negative control - no expression on Jurkat cells); c (dark grey bars): anti-human CD4 NanoAb (low to moderate level of expression on Jurkat cells).

FIG. 18A shows NanoAb median side scattering intensity (MSSI) compared to median fluorescence intensities (MFIs) from Jurkat cells (blank) and Jurkat cells incubated with NanoAbs. A slight increase in the MSSI was recorded for Jurkat cells incubated with a. MSSI variations of NanoAb-tagged Jurkat cells is lower than that reported for silver-core silica-shell NanoAbs and red blood cells (RBCs) as targets. The extinction maximum of Au@Si02 NPs is located at 530 nm, while that of Ag@Si02 NPs is around 420 nm. The latter yields greater overlap with the SSC laser source (488 nm). Moreover, Ag@Si02 NPs present better overall plasmonics properties, and Jurkat cells are significantly smaller than RBCs, which leads to a lower absolute number of nanoparticles per cell with Jurkat cells compared to RBCs.

FIG. 18B shows NanoAb MFIs compared to MFIs from Jurkat cells (blank) and Jurkat cells incubated with NanoAbs. An increase in the MFI was recorded for Jurkat cells incubated with a. No significant increase was recorded for b, and a slight increase in MFI was recorded for c. The crosslinking reaction used to functionalize monoclonal antibodies at the surface of Au@Si02- FITC NPs did not alter the specificity of the Abs.

FIGs. 19A and 19B show cell tagging experiments using fluorescent monoclonal antibodies (see Table 1 for the list of antibodies used). FIG. 19A shows density plots recorded for Jurkat cells incubated with NanoAbs b, c and a described above for FIGs. 18A and 18B. FIG. 19B depicts histogram plots showing the fluorescence distribution of Jurkat cells (blank) compared to the corresponding NanoAb-tagged Jurkat cells. This experiment confirms the detection specificity of NanoAbs.

FIGs. 20A and 20B show cell tagging experiments using NanoAbs. FIG. 20A shows density plots recorded for Jurkat cells incubated with anti-human CD235-APC (negative control), anti-human CD4-PE (low-density antigen, positive control) and anti-human CD45-PerCP-Cy5 (high-density antigen, positive control). FIG. 20B depicts histogram plots showing the fluorescence distribution of Jurkat cells (blank) compared to the corresponding fluorescent monoclonal antibody-tagged Jurkat cells. Results can be compared to the ones obtained with NanoAbs in FIGs. 19A and 19B.

Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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Claims

WHAT IS CLAIMED IS:

1. Metal/silica core-shell nanoparticles comprising:

(i) a spherical core comprising a metallic material; and

(ii) a shell surrounding said core, said shell comprising silica;

2. The nanoparticles of claim 1 , wherein at least 90% of the core of the nanoparticles have a diameter within 12 nm of the mean diameter.

3. The nanoparticles of claim 1 , wherein at least 80% of the core of the nanoparticles have a diameter within 10 nm of the mean diameter.

4. The nanoparticles of claim 1 , wherein at least 90% of the core of the nanoparticles have a diameter within 10 nm of the mean diameter.

5. The nanoparticles of any one of claims 1 to 4, further comprising a targeting moiety attached to said shell.

6. The nanoparticles of claim 5, wherein said targeting moiety is an antibody.

7. The nanoparticles of any one of claims 1 to 6, wherein said targeting moiety is attached to said shell through an amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends.

8. The nanoparticles of claim 7, wherein said crosslinker is sulfosuccimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) group.

9. The nanoparticles of any one of claims 1 to 8, further comprising one or more fluorophores incorporated within said shell.

10. The nanoparticles of claim 9, comprising a plurality of fluorophores incorporated within said shell.

11 . The nanoparticles of claim 9 or 10, wherein said shell with the fluorophores directly surrounds the core, the nanoparticles being free of an intervening layer between the core and said shell.

12. The nanoparticles of any one of claims 1 to 11 , wherein said metallic material is silver or gold.

13. The nanoparticles of claim 12, wherein said metallic material is silver.

14. A method for detecting the presence of a biomolecule on the surface of a cell, said method comprising contacting said cell with metal/silica core-shell nanoparticles comprising

(i) a core comprising a metallic material;

(ii) a shell surrounding said core, said shell comprising silica; and

(iii) a targeting moiety binding to said biomolecule attached to said shell.

15. The method of claim 14, wherein nanoparticles are the nanoparticles of any one of claims 1 to 13.

16. The method of claim 14 or 15, wherein said cell is a red blood cell.

17. The method of claim 16, wherein said biomolecule is a blood group antigen.

18. The method of claim 17, wherein said blood group antigen is a rare blood group antigen.

19. A process for the preparation of metal/silica core-shell nanoparticles comprising the steps of: providing metal nanoparticles,

providing one or more fluorescent silica precursors,

functionalizing the surface of the silica shell,

reacting the functionalized surface with a coupling agent, and

attaching a targeting moiety to the coupling agent.

20. The process of claim 19, wherein the metal is silver and the nanoparticles are provided by: adding an aqueous solution of AgNC>3 to a boiling and stirred aqueous solution of sodium citrate tribasic dehydrate, and

while stirring, boiling the mixture to allow for nanoparticle growth.

21 . The process of claim 20, wherein the mixture is further cooled down to room temperature.

22. The process of any one of claims 19 to 21 , wherein the fluorescent silica precursors are provided by reacting a isothiocyanate modified fluorophore with a silica precursor bearing an amine group

23. The process of claim 22, wherein the silica precursor bearing an amine group is (3- aminopropyl)ethoxysilicate.

24. The process of any one of claims 19 to 23, wherein the fluorescent silica shell is coated on the metal nanoparticles by mixing together and reacting the fluorescent silica precursor(s), the other silica precursor, and the metal nanoparticles.

25. The process of claim 24, wherein the other silica precursor is tetraethyl orthosilicate.

26. The process of any one of claims 19 to 25, wherein said functionalizing is thiolating the surface of the silica shell.

27. The process of any one of claims 19 to 26, wherein the coupling agent is an amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends.

28. The process of claim 27, wherein the coupling agent is sulfosuccimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate.

29. The process of any one of claims 19 to 28, wherein the targeting moiety is an antibody.

30. The process of any one of claims 19 to 29, wherein the nanoparticles are nanoparticles according to any one of claims 1 to 13.

31 . Nanoparticles prepared according to the process of any one of claims 19 to 30.