Unagglomerated core/shell nanocomposite particles

• US 8,071,132 B2
• Filed: 06/01/2005
• Issued: 12/06/2011
• Est. Priority Date: 06/01/2004
• Status: Active Grant

• Current Assignee: Penn State Research Foundation
• Original Assignee: Penn State Research Foundation
• Inventors: Adair, James H., Rouse, Sarah M., Wang, Jun, Kester, Mark, Siedlecki, Christopher, White, William B., Vogler, Erwin, Snyder, Alan, Pantano, Carlo G., Ruiz-Velasco, Victor, Sinoway, Lawrence
• Primary Examiner(s): Tran, Susan
• Assistant Examiner(s): Ahmed, Hasan
• Application Number:
  US11/142,913
• Publication Number:
  US 20050281884A1
• Time in Patent Office: 2,379 Days
• Field of Search: None
• US Class Current: 424/489
• CPC Class Codes: A61K 47/6923 the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
• CPC Class Codes: A61K 47/6929 the form being a nanoparticle, e.g. an immuno-nanoparticle
• CPC Class Codes: A61K 49/0041 Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal in vivo G01N
• CPC Class Codes: A61K 49/0093 Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
• CPC Class Codes: B01J 13/02 Making microcapsules or microballoons for medical preparations A61K9/50
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]
• CPC Class Codes: Y10T 428/2989 Microcapsule with solid core [includes liposome]
• CPC Class Codes: Y10T 428/2991 Coated
• CPC Class Codes: Y10T 428/2995 Silane, siloxane or silicone coating

Abstract

The present invention provides a method for the synthesis of unagglomerated, highly dispersed, stable core/shell nanocomposite particles comprised of preparing a reverse micelle microemulsion that contains nanocomposite particles, treating the microemulsion with a silane coupling agent, breaking the microemulsion to form a suspension of the nanocomposite particles by adding an acid/alcohol solution to the microemulsion that maintains the suspension of nanocomposite particles at a pH of between about 6 and 7, and simultaneously washing and dispersing the suspension of nanocomposite particles, preferably with a size exclusion HPLC system modified to ensure unagglomeration of the nanocomposite particles. The primary particle size of the nanocomposite particles can range in diameter from between about 1 to 100 nm, preferably from between about 10 to 50 nm, more preferably about 10 to 20 nm, and most preferably about 20 nm.

Claims

1. A method of preparing unagglomerated, dispersed core/shell nanocomposite particles in suspension, comprising:

preparing a reverse micelle microemulsion by forming a mixture comprised of a surfactant, a solvent and an aqueous core precursor at room temperature;

adding a shell precursor material;

treating the reverse micelle microemulsion with a dispersing agent;

breaking the microemulsion to form a suspension of nanocomposite particles by adding a breaking agent to the microemulsion; and

washing and dispersing the suspension of nanocomposite particles simultaneously to produce said unagglomerated, dispersed core/shell nanoparticles in suspension.

2. The method of claim 1, wherein the acid/alcohol solution maintains the suspension of nanocomposite particles at a pH of between about 5 and 9 and further wherein the breaking agent is an acidified alcohol solution.

3. The method of claim 1, wherein the breaking agent is an acetic acid/ethanol solution and wherein the dispersing agent is selected from the group consisting of citrate, oxalate, succinate and phosphonates.

4. The method of claim 1, further comprising the additional steps of:

stirring the mixture for about 2 minutes to 24 hours;

adding base to form a suspension;

stirring the suspension for about 2 minutes to 24 hours;

wherein the shell percursor material is a silica or titania material; and

maturing the microemulsion for about 24 hours, wherein said unagglomerated dispersed core/shell nanoparticles in suspension maintain the nanostructure throuuh said silica or shell precursor material additions.

5. The method of claim 4, wherein the surfactant is poly(oxyetheylene)nonylphenyl ether.

6. The method of claim 4, wherein the solvent is cyclohexane.

7. The method of claim 4, wherein the aqueous core precursor is selected from the group consisting of Au, Ag, Co, Ni, Cu, CdS;

Pt, organic pigments, organic dyes, organic fluorophores such as the sodium salt of fluorescein, rhodamine, rhodamine derivatives, fluorescein, fluorescein derivatives, luciferin and one or more drug agents.

8. The method of claim 7, wherein the drug agent is a genetic therapeutic agent which delivers nucleic acids to cells in a transcriptionally active form.

9. The method of claim 7, wherein the fluorescein and fluorescein derivatives are selected from the group consisting of BDCECF;

BCECF-AM;

Calcien-AM;

5,(6)-carboxy-2′

,7′

-dichlorofuorescein;

5,(6)-carboxy-2 ′

7′

-dichlorofuorescein diacetate Nsuccinimidylester;

5,(6)-carboxyeosin;

5,(6)-carboxycosin diacetate;

5,(6)-carboxyfluorescein;

5-carboxyfluorescein;

6-carboxyfluorescein;

5,(6)-carboxyfluoresceinacetate;

5,(6)-carboxyfluorescein acetate N-succinimidyl ester;

5,(6)-carboxyfluorescein Nsuccinimidylester;

5(6)-carboxyfluorescein octadecyl ester;

5,(6)-carboxynaphthofluoresceindiacetate;

eosin-5-isothiocyanate;

eosin-5-isothiocyanate diacetate;

fluorescein-5(6)-carboxamidocaproic acid;

fluorescein-5(6)-carboxamidocaproic acid N-succinimidyl ester;

fluorescein isothiocyanate;

fluorescein isothiocyanate isomer 1;

fluorescein isothiocyanate isomer 2;

fluorescein isothocyanate diacetate;

fluorescein octadecyl ester;

fluorescein sodium salt;

napthofluorescein;

napthofluorescein diacetate;

or N-octadecyl-N7-(5 fluoresceinyl) thiourea (F18).

10. The method of claim 7, wherein the rhodamine and rhodamine derivatives are selected from the group consisting of 5,(6)carboxytetramethylrhodamine;

5-carboxytetramethylrhodamine N-succinimidyl ester;

6-carboxytetramethylrhodamine Nsuccinimidylester;

5,(6)-carboxytetramethylrhodamine N-succinimidyl ester;

5,(6)-carboxy-X-rhodamine;

dihydrorhodamine 123;

dihydrorhodamine 6G;

lissaninc rhodamine;

rhodamine 110 chloride;

rhodamine 123, rhodamine B hydrazide;

rhodamine,B; and

rhodamine WT.

11. The method of claim 7, wherein the organic pigments and dyes are selected from the group consisting of hematoporphyrin dyes, such as 7,12-bis(1-hydroxyethyl)-3,8,13,17-tetramethyl-2 1 H,23H-porphine-2 and 18-dipropanoic acid, and cyanine dyes and derivatives, such as indocyanine green;

indoine blue;

R-phycoerythnn (PE), PE-Cy 5;

PE-Cy 5.5;

PE-Texas Red;

PE-Cy 7;

Cy 3 NHS ester;

Cy 3 maleimide and hydrazide;

Cy 3B NHS ester;

Cy 3.5 NHS ester;

Cy 3 amidite;

Cy 5 NHS ester;

Cy-5;

Cy 5 amidite;

Cy 5.5;

Cy-5.5 NHS ester;

Cy 5.5 annex in V;

Cy 7;

Cy 7 NHS ester;

Cy 7Q NHS ester;

allophycocyanin (APC);

APC-Cy 7;

APC Cy 5.5;

propidium iodide (PI);

crystal violet lactone;

patent blue VP;

brilliant blue G;

or cascade blue acetyl azide.

12. The method of claim 1, wherein the shell precursor material is selected from the group consisting of SiO₂, TiO₂, ZnO, Fe₂O₃, Zr₂, NiO and GeO₂, Sn, Pb, Ag and Au, CaPO_x, CaCO₃, tetraethoxysilane (TEOS), titanium (IV) isopropoxide, and a calcium phosphosilicate having the general formula Ca_x(PO₄)_y(OH),(SiO₂)_a.

13. The method of claim 12, wherein the precursor is tetraethoxysilane (TEOS).

14. The method of claim 12, wherein the precursor is titanium (IV) isopropoxide (TIPO).

15. The method of claim 1, wherein the dispersing agent is a silane coupling agent is selected from the group consisting of 3-aminopropyltrimethoxysilane, 3-aminopropylsilsesquioxanc, 3-glycidoxypropyltrimethoxysilane, trimethoxysi lylpropyldiethylenetriamine, 3-trimethoxysi lylpropylsuccinic anhydride and amide-linked carboxyl groups.

16. The method of claim 15, wherein the silane coupling agent is 3-aminopropyltrimethoxysilane.

18. The method of claim 1, further comprising adding a surface modifier to said dispersing agent in order to use the nanocomposite particles in vivo in an animal or human.

19. The method of claim 18, further wherein said surface modifier is a binder.

20. The method of claim 19, wherein the binders are antibodies.

21. The method of claim 4, further comprising attaching moieties selected from the group consisting of organic groups, metals, enzymes, macromolecules and plasmids to the shell precursor material.

22. The method of claim 4, wherein the diameter of the nanocomposite particles is modified by manipulating the molar ratio of aqueous phase to surfactant, the molar ratio of aqueous phase to the shell precursor material, and the molar ratio of base to the shell precursor material.

23. The method of claim 1, further comprising:

using a size-exclusion HPLC system to wash and disperse the nanocomposite articles comprised of an HPLC column packed with spherical silica beads of about I μ

m to about 100 μ

m in diameter;

pumping breaking agent through the HPLC column;

limping the nanoparticle suspension into the HPLC column through a stationary phase at a flow rate from of about 1 ml/min to about 100 ml/min;

measuring changes in UV absorbance or fluorescence with detectors connected to an out-flow of the HPLC packed column in order to determine when the column is fully saturated with nanoparticles; and

eluting and redispersing the nanoparticles with a water miscible organic solvent and water solution of up to about 250 volume percent solution.

24. The method of claim 23, wherein the spherical silica beads are about 20 μ

m in diameter.

25. The method of claim 23, wherein the water miscible organic solvent and water solution is up to about 70 volume percent solution.

26. The method of claim 23, wherein the HPLC column is about 5×

50 mm in length.

27. The method of claim 23, wherein the stationary phase is comprised of microspheres which have been treated with the dispersing agent.

28. The method of claim 1, wherein the nanocomposite particles have a primary particle size of between about 1 to 100 nm in diameter.

29. The method of claim 1, wherein the nanocomposite particles have a primary particle size of between about 10 to 20 nm in diameter.

30. The method of claim 1, wherein the nanocomposite particles have a primary particle size of about 20 nm in diameter.

32. The method.of claim 1 further comprising the additional steps of forming a first mixture containing a surfactant, a solvent, a quantity of aqueous core precursors pH adjusted to pH 6-8, and calcium in the form of Ca⁺⁺;

forming a second mixture containing a surfactant, a solvent, a quantity of aqueous core precursors pH adjusted to pH 6-8, and phosphorus in the form of PO₄<sup>−

−

−</sup>

and optional SiO₃<sup>−

−</sup>

; and

mixing together and maturing said first mixture and said second mixture for about 2 minutes.

33. The method of claim 32 wherein the aqueous core precursors are pH adjusted to pH 7.4.

34. The method of claim 32 wherein the surfactant is poly(oxyethyl ene) nonyiphenyl ether.

35. The method of claim 32 wherein the solvent is cyclohexane.

36. The method of claim 32 wherein the aqueous core precursor is selected from the group consisting of Au, Ag, Co, Ni, Cu, CdS;

Pt, organic pigments, organic dyes, organic fluorophores such as sodium salt of fluorescein, rhodamine, rhodamine derivatives, fluorescein, fluorescein derivatives, luciferin and one or more drug agents.

37. The method of claim 36 wherein the drug agent is a genetic therapeutic agent which delivers nucleic acids to cells in a transcriptionally active form.

38. The method of claim 32 wherein the diameter of the nanocomposite particles is modified by manipulating the molar ratio of aqueous phase to surfactant, the molar ratio of aqueous phase to the shell precursor material, and the molar ratio of base to the shell precursor material.

40. The method of claim 4, wherein the shell precursor material is selected from the group consisting of SiO₂, TiO₂, ZnO, Fe₂O₃, Zr₂, NiO and GeO₂, Sn, Pb, Ag and Au, CaPO_x, CaCO₃, tetraethoxysilane (TEOS), titanium (IV) isopropoxide, and a calcium phosphosilicate having the general formula Ca_x(PO₄)_y(OH)z(SiO₂)_a, and wherein the reverse micelle microemulsion is synthesized by the addition of the first and second microemulsions to one another.

41. The method of claim 40, wherein the precursor is tetraethoxysilane (TEOS).

42. The method of claim 40, wherein the precursor is titanium (IV) isopropoxide (TIPO).

17. The method oftlaim 1, wherein the core of the nanocomposite particle contains hydrogel materials selected from the group consisting of polyvinyl alcohol, polymethyl methacrylate and 2-hydroxylethyl methacrylate.

31. A method of synthesizing unagglomerated, dispersed core/shell nanocomposite particles, comprising:

preparing a reverse micelle microemulsion, comprising;

(i) forming a mixture comprised of poly(oxyetheylene)-nonylphenyl ether, cyclohexane and an aqueous active-medical-agent precursor at room temperature;

(ii) stimng the mixture for about 30 minutes;

(iii) adding NH₄OH to form a suspension;

(iv) stirring the suspension for about 15 minutes;

(v) adding a shell precursor; and

(vi) maturing the microemulsion for about 24 hours;

treating the reverse micelle microemulsion with 3-aminopropyltrimethoxysilane;

breaking the microemulsion to form a suspension of the nanocomposite particles by adding to the micro emulsion an acetic acidethanol solution which maintains the suspension at a pH of between about 6 and 7; and

washing and dispersing the suspension of nanocomposite particles using a size-exclusion HPLC system comprising;

(i) packing a HPLC column with spherical silica beads of about 20 μ

m in diameter;

(ii) pumping ethanol through the HPLC column;

(iii) pumping the nanoparticle suspension into the HPLC column through a stationary phase at a flow rate of about 1 ml/min;

(iv) measuring changes in UV absorbance or fluorescence with detectors connected to an out-flow of the HPLC packed column in order to determine when the column is fully saturated with nanoparticles; and

(v) eluting and redispersing the nanoparticles with an ethanol/water solution of up to about 70 v/o water.

39. A method of synthesizing unagglomerated, dispersed corelshell nanocomposite particles, comprising:

preparing a reverse micelle microemulsion, comprising;

(i) forming a first mixture comprised of poly(oxyetheylene)-nonylphenyl ether, cyclohexane, a drug agent precursor and Ca⁺⁺ at room temperature;

(ii) forming a second mixture comprised of poly(oxyetheylene)-nonylphenyl ether, cyclohexane, a drug agent precursor and PO4<sup>−

−

−</sup>

with optional SO₂⁻

; and

(iii) mixing and maturing the first and second mixtures for about two minutes;

treating the reverse micelle microemulsion with 3-aminopropyltrimethoxysilane;

breaking the micro emulsion to form a suspension of the nanocomposite particles by adding to the microemulsion an acetic acid/ethanol solution which maintains the suspension at a pH of between about 6 and 7; and

washing and dispersing the suspension of nanocomposite particles using a size-exclusion HPLC system comprising;

(i) packing a HPLC column with spherical silica beads of about 20 μ

m in diameter;

(ii) pumping ethanol through the HPLC column;

(iii) pumping the nanoparticle suspension into the HPLC column through a stationary phase at a flow rate of about 1 ml/min;

(iv) measuring changes in UV absorbance or fluorescence with detectors connected to an out-flow of the HPLC packed column in order to determine when the column is fully saturated with nanoparticles; and

(v) eluting and redispersing the nanoparticles with an ethanovwater solution of up to about 70 v/o water.