Multilayer fluorescent nanoparticles and methods of making and using same

Patent metadata

Field	Value
Publication number	US-2016018404-A1
Application number	US-201414768890-A
Country	US
Kind code	A1
Filing date	Feb 20, 2014
Priority date	Feb 20, 2013
Publication date	Jan 21, 2016
Grant date	—

Abstract

A multilayer, fluorescently responsive material (FRM)-containing nanoparticle and compositions comprising such nano-particles. The nanoparticles can be made using a layer-by-layer deposition method. The nanoparticles can be used in imaging methods such as, for example, cellular imaging methods.

First claim

What is claimed is:

1 ) A nanoparticle comprising:
a) a silica core comprising a plurality of a fluorescently responsive material (FRM) covalently bound to the silica network of the core; b) 1 to 100 FRM-containing silica layers, each layer comprising a plurality of the FRM covalently bound to the silica network of the FRM-containing silica layer; c) one or more FRM-free silica layers, wherein one of the FRM-free silica layers separates the silica core from one of the FRM-containing silica layers and, if present, each adjacent pair of the FRM-containing silica layers is separated by one of the FRM-free silica layers; d) an outermost FRM-free silica layer disposed on the outermost FRM-containing silica layer; and e) a plurality of poly(ethylene glycol) molecules covalently bound to the outer surface of the outermost FRM-free silica layer.

2 ) The nanoparticle of claim 1 , further comprising one or more moieties covalently bound to the poly(ethylene glycol) molecules covalently bound to the outer surface of the outermost FRM-free silica layer.

3 ) The nanoparticle of claim 2 , wherein the one or more moieties is selected from proteins, peptides, nucleic acids, aptamers, antibodies, antibody fragments, polymers, organic small molecules, and combinations thereof.

4 ) The nanoparticle of claim 3 , wherein the nucleic acids are selected from single-stranded DNA molecules, double-stranded DNA molecules, single-stranded RNA molecules, double-stranded RNA molecules, branched DNA molecules, and combinations thereof.

5 ) The nanoparticle of claim 1 , the nanoparticle having a diameter of 5 nm to 500 nm.

6 ) The nanoparticle of claim 1 , the nanoparticle having a diameter of 5 nm to 100 nm.

7 ) The nanoparticle of claim 1 , wherein each FRM-free silica layer has a thickness such that there is 10% or less measurable energy transfer between the FRM in the core and in an adjacent FRM-containing silica layer or in adjacent FRM-containing silica layers.

8 ) The nanoparticle of claim 1 , wherein each dye-free silica layer has a thickness of 1 nm to 20 nm.

9 ) The nanoparticle of claim 1 , wherein the core and all FRM-containing layers have a different FRM.

10 ) The nanoparticle of claim 1 , wherein the FRM is an organic dye.

11 ) The nanoparticle of claim 1 , wherein the FRM is selected from N-(7-dimethylamino-4-methylcoumarin-3-yl) (DAC), tetramethylrhodamine-5-maleimide (TMR), Cy5, or a combination thereof.

12 ) A method of making the nanoparticle of claim 1 comprising the steps of:
a) contacting a silica precursor, a plurality of a single type of FRM conjugate precursor, a solvent, and base such that a silica core having a plurality of FRM conjugated to the silica network of the silica core is formed,
b) contacting the material from step a) with a silica precursor and a solvent such that a FRM-free silica layer is formed on the silica core;
c) contacting the material from b) with a silica precursor, a single type of FRM conjugate precursor, a solvent, and base such that a FRM-containing silica layer is formed;
d) optionally, contacting the material from step c) with a silica precursor and a solvent such that a FRM-free silica layer is formed on the silica core and contacting the resulting material with a silica precursor, a single type of FRM conjugate precursor, a solvent, and base such that a FRM-containing silica layer is formed;
e) optionally, repeating step d) a desired number of times, wherein the contacting is to the material from a previously carried out step d);
f) contacting the material from step c), d, or step e) with a silica precursor and a solvent such that an outermost FRM-free silica layer is formed on the outermost FRM-containing layer; and
g) contacting the material from step f) with functionalized PEG molecules such that a nanoparticle having a plurality of PEG molecules covalently bound to the outer surface of the outermost FRM-free silica layer of the nanoparticle is formed.

13 ) The method of claim 12 , wherein the PEG molecules are heterobifunctional PEG molecules.

14 ) The method of claim 12 , further comprising the step of isolating the nanoparticle.

15 ) An imaging method comprising the steps of:
a) contacting a cell with a plurality of nanoparticles of claim 1 ; and b) obtaining a plurality of images of the sample, each image obtained using a different excitation wavelength and a different emission wavelength, wherein each different excitation wavelength is in the absorption spectrum of a different type of FRM present in the nanoparticle and each different emission wavelength is in the emission spectrum of a different type of FRM present in the nanoparticle.

16 ) The imaging method of claim 15 , further comprising the step of combining the plurality of images to provide a single image.

17 ) The imaging method of claim 15 , wherein the image is obtained by confocal microscopy.

18 ) The imaging method of claim 15 , wherein the cell is present in a subject.

Assignees

• Univ Cornell

Inventors

• Iyer Srikant K
• Wiesner Ulrich B

Classifications

• C09B69/103

  containing a diaryl- or triarylmethane dye · CPC title

• C09B69/105

  containing a methine or polymethine dye · CPC title

• C09B69/10

  Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N33/552

  Glass or silica · CPC title