# US 9,518,921 B2 · Silica nanoparticles with aggregation induced emission characteristics as fluorescent bioprobe for intracellular imaging and protein carrier

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- Number: US 9,518,921 B2
- Document ID: US9518921B2
- Status: Granted
- Kind: B2
- Application number: 13/728,150
- Filed: 2012-12-27
- Published: 2013-08-15
- Granted: 2016-12-13
- Earliest claimed: 2011-12-28
- Assignees: The Hong Kong University of Science and Technology
- Applicants: The Hong Kong University of Science and Technology
- Inventors: Benzhong Tang; Wing Yip Lam; Jianzhao Liu; Faisal Mahtab; Yang Liu; Yong Yu
- Web page: https://patentgrep.com/patent/US9518921B2

## CPC

- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- C07D 207/448: with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
- C07D 207/46: with hetero atoms directly attached to the ring nitrogen atom
- C07F 7/0807: {comprising Si as a ring atom}
- C07F 7/1804: {Compounds having Si-O-C linkages (Si-O-acyl linkages C07F7/1896)}
- C09B 23/14: Styryl dyes
- C09B 23/148: {Stilbene dyes containing the moiety -C6H5-CH=CH-C6H5 (stilbene azo dyes C09B29/00)}
- C09B 69/008: {Dyes containing a substituent, which contains a silicium atom}
- G01N 33/533: with fluorescent label
- G01N 33/54326: {Magnetic particles}
- G01N 33/54346: {Nanoparticles}
- G01N 33/582: {with fluorescent label}
- G01N 33/587: {Nanoparticles}
- B82Y 15/00: Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
- Y10S 977/773: Nanoparticle, i.e. structure having three dimensions of 100 nm or less
- Y10S 977/92: Detection of biochemical
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Provided herein are magnetic silica fluorescent nanoparticles and fluorescent silica nanoparticles comprising an aggregation induced emission luminogen and magnetite nanoparticles and use of the same as a fluorescent bioprobe for intracellular imaging and a protein carrier. Also provided are processes for preparing and fabricating the same.

## Claims

### Claim 1

A fluorescent bioprobe for intracellular imaging comprising an aggregation induced emission luminogen and magnetite nanoparticles; wherein the luminogen has a backbone structure selected from the group consisting of:
wherein
R is selected from the group consisting of H, alkyl, unsaturated alkyl, aryl, vinyl, acetyl, heteroalkyl, cycloalkyl, heterocycloalkyl, and heteroaryl;
X is selected from the group consisting of (CH2)n, C6H5, (Ph)n, O(CH2)n, NH(CH2)n, N[(CH2)n]2, (OCH2CH2)n, OCOR′C2H, COO-dioxopyrrolidine, CONH(CH2)n, OR′NH(CH2)n, R′CH2CH2S(CH2)n, R′CHCHS(CH2)n, R′NH(CH2)n, and R′-triazole-(CH2)n, wherein R′ is independently selected from the group consisting of alkyl, unsaturated alkyl, aryl, vinyl, heteroalkyl, cycloalkyl, heterocycloalkyl, and heteroaryl; and
n=0 to 20;
wherein X is capable of conjugating with a siloxane;
wherein the fluorescent bioprobe comprises magnetic fluorescent silica nanoparticles; and
wherein the magnetic fluorescent silica nanoparticles are surface functionalized with one or more biomolecules selected from the group consisting of glucose, galactose, and folic acid.

### Claim 2 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the luminogen has a chemical structure selected from the group consisting of:
wherein R1 is a substituent selected from the group consisting of alkyl, unsaturated alkyl, aryl, vinyl, heteroalkyl, cycloalkyl, heterocycloalkyl, and heteroaryl, and wherein R2, R3, and R4 are substituents independently selected from the group consisting of H, alkyl, unsaturated alkyl, aryl, vinyl, acetyl, heteroalkyl, cycloalkyl, heterocycloalkyl, and heteroaryl.

### Claim 3 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles are also protein carriers.

### Claim 4 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles exhibit aggregation-induced emission.

### Claim 5 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles exhibit superparamagnetism.

### Claim 6 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles exhibit negligible remanence and coercivity, wherein negligible remanence and coercivity indicate high magnetization.

### Claim 7 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles emit light; and wherein the light emission increases with increased luminogen loading.

### Claim 8 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the aggregation induced emission luminogen is in solid form.

### Claim 9 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles comprise magnetic cores covered by a silica shell.

### Claim 10 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles are spherical with substantially uniform sizes and narrow particle distributions.

### Claim 11 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles possess high surface charges, measured by zeta potential, and good colloidal stabilities, which result from high surface charges at a workable pH.

### Claim 12 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the aggregation induced emission luminogen is covalently bonded to a silica network through amine and amide functional groups.

### Claim 13 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the aggregation induced emission luminogen is covalently bonded to silica nanoparticles via alkyne-azide cycloaddition.

### Claim 14 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the aggregation induced emission luminogen is grafted onto the surface of silica nanoparticles.

### Claim 15 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles are surface functionalized with one or more functional groups selected from the group consisting of amino, azido, carboxylic acid, and thiol functional groups.

### Claim 16 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles are conjugated with the one or more biomolecules via an esterification reaction.

### Claim 17 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles are nontoxic to living cells.

### Claim 18 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles are effectively taken up by cancer cells.

### Claim 19 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles can image cytoplasm of cancer cells.

### Claim 20 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles can target specific cancer cells.

### Claim 21 (depends on 1)

The fluorescent bioprobe of claim 1, wherein the magnetic fluorescent silica nanoparticles can adsorb protein molecules selected from the group consisting of bovine serum albumin (BSA) and lysozyme.

### Claim 22 (depends on 2)

A process for preparing the bioprobe of claim 2 comprising a sol-gel reaction of silole-APS conjugate and tetraethoxysilane in a magnetic fluid of magnetite.

### Claim 23 (depends on 2)

A process for preparing the bioprobe of claim 2 comprising a first sol-gel reaction of tetraphenylethene-containing siloxane, followed by a second sol-gel reaction with tetraethoxysilane.

### Claim 24 (depends on 1)

A process for preparing the bioprobe of claim 1 comprising:
(a) preparation of tetraphenylethene-containing siloxane and silole-containing siloxane;
(b) sol-gel reactions of the tetraphenylethene-containing siloxane and the silole-containing siloxane; and
(c) reactions of the tetraphenylethene-containing siloxane and silole-containing siloxane with tetraethoxysilane.

### Claim 25 (depends on 2)

A process for preparing the bioprobe of claim 2 comprising:
(a) preparation of siloxane 1,2-Bis(4-{1-[2-(triethoxysilyl)ethyl]-4-triazolyl}phenyl)-1,2-diphenylethene (32) and 2,5-Bis(4-{1-[2-(triethoxysilyl)ethyl]-4-triazolyl}phenyl)-3,4-diphenyl-1,1-dimethylsilole (33);
(b) sol-gel reactions of siloxane 1,2-Bis(4-{1-[2-(triethoxysilyl)ethyl]-4-triazolyl}phenyl)-1,2-diphenylethene (32) and 2,5-Bis(4-{1-[2-(triethoxysilyl)ethyl]-4-triazolyl}phenyl)-3,4-diphenyl-1,1-dimethylsilole (33); and
(c) reactions of siloxane 1,2-Bis(4-{1-[2-(triethoxysilyl)ethyl]-4-triazolyl}phenyl)-1,2-diphenylethene (32) and 2,5-Bis(4-{1-[2-(triethoxysilyl)ethyl]-4-triazolyl}phenyl)-3,4-diphenyl-1,1-dimethylsilole (33) with tetraethoxysilane.

### Claim 26 (depends on 1)

A process for preparing the bioprobe of claim 1 comprising surface grafting of the aggregation induced emission luminogen onto the magnetite nanoparticles.

### Claim 27 (depends on 1)

A process for surface functionalization of the bioprobe of claim 1 comprising bioconjugation with glucose molecules using alkyne-azide cycloaddition.

### Claim 28 (depends on 1)

A process for surface functionalization of the fluorescent bioprobe of claim 1 comprising a click reaction of sugar-bearing phenylacetylene with
respectively.

### Claim 29 (depends on 1)

A process for surface functionalization of the bioprobe of claim 1, comprising a reaction of folic acid with
respectively.

### Claim 30

A process for preparing lysozyme-decorated fluorescent silica nanoparticles comprising adsorption of lysozyme by