Formation of colloidal metal dispersions using aminodextrans as reductants and protective agents

• US 5,248,772 A
• Filed: 01/29/1992
• Issued: 09/28/1993
• Est. Priority Date: 01/29/1992
• Status: Expired due to Term

• Current Assignee: Coulter International Corp.
• Original Assignee: Coulter Corporation (Danaher Corporation)
• Inventors: Burshteyn, Alexander, Siiman, Olavi
• Primary Examiner(s): GRIFFIN, RONALD
• Application Number:
  US07/827,347
• Time in Patent Office: 608 Days
• Field of Search: 536/112, 536/121, 252/62.53, 252/62.54, 424/9, 424/7.1, 424/1.1, 424/460, 424/461, 424/491, 424/493, 424/85.8, 424/94.1, 424/94.3, 424/94.67, 424/617, 424/618, 424/646, 424/649
• US Class Current: 536/112
• 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: G01N 33/548 Carbohydrates, e.g. dextran
• CPC Class Codes: G01N 33/553 Metal or metal coated
• CPC Class Codes: Y10T 428/2989 Microcapsule with solid core [includes liposome]
• CPC Class Codes: Y10T 428/2991 Coated
• CPC Class Codes: Y10T 428/2998 including synthetic resin or polymer

Abstract

The invention related generally to the preparation of colloidal metal(O) particles having a crosslinked aminodextran coating with pendent amine groups attached thereto. The aminodextran acts as both a reductant for reducing metal ions to metal(O) particles and as the protective agent which coats the metal(O) particles thus formed. After stabilizing the aminodextran coating by use of a crosslinking agent, the coated particles can be used to covalently bind proteins. The resulting protein containing colloidal particles can be used as markers in optical and electron microscopy, in immunological and biological assays, and possibly as therapeutic agents.

Claims

1. A method for preparing colloidal metal(O) particles from a solution of a metal salt and coating said particles with a polymeric organic compound which also is used as a reductant for said metal salt, said method comprising:

(a) heating a solution containing an amine derivatized, oxidizable sugar containing polysaccharide and a metal salt capable of being reduced by said oxidizable sugar containing polysaccharide for a time and at a temperature sufficient to reduce said metal salt to colloidal metal particles and simultaneously coating said particles with said polysaccharide; and

(b) stabilizing the amine derivatized polysaccharide coating on said particles by means of a bifunctional crosslinking agent, a diamine and a reducing agent.

2. The method of claim 1 wherein said amine derivatized polysaccharide is an aminodextran.

3. The method of claim 1 wherein said metal salt has a reduction potential of +0.7 volts or higher.

4. The method of claim 3 wherein said metal salt is selected from the group consisting of gold, silver, platinum, palladium, rhodium and iridium salts capable of being reduced by said polysaccharide.

5. The method of claim 4 wherein said metal salt is a gold or silver salt.

13. A method of preparing colloidal metal(O) particles coated with an amine derivatized, oxidizable sugar containing polysaccharide and having a protein bound thereto, said method comprising:

(a) preparing coated colloidal metal particles according to claim 1; and

(b) reacting the particles of step (a) with a protein capable of forming a covalent bond with the amine groups present on said particles,

14. The method of to claim 13 wherein said protein is a monoclonal antibody.

15. The method according to claim 13 wherein said colloidal metal particles are selected from the group consisting of colloidal gold, silver, platinum, palladium, rhodium and iridium colloidal particles.

16. The method of claim 15 wherein said colloidal particles are colloidal gold or silver particles.

17. A method of preparing colloidal metal(O) particles coated with an amine-derivatized reducible sugar containing polysaccharide and having a protein bound thereto, said method comprising:

(a) preparing colloidal metal particles according to claim 1;

(b) converting the amine groups present on said particles to a group selected from the group consisting of sulhydryl and maleimidyl groups; and

(c) reacting the product of step (b) with;

(1) a protein having a functional group reactive with said sulfhydryl and maleimidyl groups or

(2) a derivatized protein having at least one functional group selected from the group consisting of sulfhydryl and maleimidyl groups, such that when said particles contain sulfhydryl groups said proteins contains maleimidyl groups and when said particles contain maleimidyl groups said protein contains sulfhydryl groups.

18. The method of claim 17 wherein said protein is selected from the group consisting of a monoclonal and a polyclonal antibody either with or with a detectable label selected from the group consisting of an enzyme, a dye, an electron dense element and a substance containing a radioactive element.

19. The method of claim 17 wherein said colloidal metal particles are selected from the group consisting of colloidal gold, silver, platinum, palladium, rhodium and iridium colloidal particles.

20. The method of claim 19 wherein said colloidal particles are gold or silver colloidal particles.

6. A method of preparing colloidal metal(O) particles coated with an amine derivatized, oxidizable sugar containing polysaccharide, said method comprising:

(a) heating an aqueous solution of a sugar reducible metal salt and adding to said solution an aqueous solution of an amine derivatized polysaccharide having at least one reducing sugar component;

(b) heating and mixing the solution resulting from step (a) for about 10-30 minutes at a temperature in the range of about 90°

-100°

C. to reduce said metal salt by said polysaccharide to colloidal metal(O) particles suspended in said polysaccharide solution and coated with said polysaccharide;

(c) cooling the solution resulting from step (b) to room temperature and adding to the cooled solution an aqueous first bifunctional reagent capable of reacting with amine groups on said amine derivatized polysaccharide;

(d) adding an organic diamine to the product of step (c);

(e) reducing the reducible groups formed in step (d);

(f) dialyzing the solution of step (e) against water; and

(g) centrifuging the product of step (f) to obtain amine derivatized polysaccharide coated particles capable of being dispersed as colloids.

7. The method of claim 6 wherein said derivatized polysaccharide is an aminodextran.

8. The method of claim 6 wherein said metal salt has a reduction potential of +0.7 volts or higher.

9. The method of claim 8 wherein said metal salt is selected from the group consisting of gold, silver, platinum, palladium, rhodium and iridium salts capable of being reduced by said polysaccharide.

10. The method of claim 9 wherein said metal salt is selected from the group consisting a gold or silver salt.

11. The method of claim 6 wherein said first bifunctional reagent is glutaraldehyde.

12. The method of claim 6 wherein said diamine is selected from the group consisting of H₂ NCH₂ --(CH₂)_x --CH_y (CH₃)_z --NH₂ and C₆ H_4+a (NH₂)₂, where x=0-3, y=1 or 2, and Z=1 when y=1 or Z=0 when y=2;

and a=0 or 6.

21. Colloidal metal(O) particles having a polymeric organic coating thereon of a substance used to form said metal(O) particles by reduction of a metal salt with said substance, said coating comprising a stabilized amine derivatized polysaccharide and said coating being stabilized by means of a bifunctional crosslinking agent, a diamine and a reducing agent.

22. The particles of claim 21 wherein said amine derivatized polysaccharide is an aminodextran.

23. The particles of claim 21 wherein said bifunctional crosslinking agent is glutaraldehyde.

24. The particles of claim 21 wherein said diamine is selected from the group consisting of H₂ NCH₂ --(CH₂)_x --CH_y (CH₃)_z --NH₂ and C₆ H_4+a (NH₂)₂, where x=0-3, y=1 or 2, and Z=1 when y=1 or Z=0 when y=2;

and a=0 or 6.

25. The particles of claim 21 wherein said metal(O) is selected from the group consisting of gold, silver, platinum, palladium, rhodium and iridium.

26. The particle of claim 25 wherein said metal(O) is silver or gold.

27. Coated colloidal metal particles having a protein bound thereto, said particles comprising colloidal metal(O) particles coated with a stabilized amine derivatized polysaccharide and a protein bound to said coating, said coating being stabilized by means of a bifunctional crosslinking agent, a diamine and a reducing agent, and said metal(O) particles being formed by reduction of a metal salt with said amine derivatized polysaccharide.

28. The particles of claim 27 wherein said amine derivatized polysaccharide is an aminodextran.

29. The particles of claim 27 wherein said bifunctional crosslinking reagent is glutaraldehyde.

30. The particles of claim 27 wherein said metal(O) is selected from the group consisting of gold, silver, platinum, palladium, rhodium and iridium.

31. The particles of claim 30 wherein said metal(O) is gold or silver.

32. The particle of claim 27 wherein said diamine is selected from the group consisting of H₂ NCH₂ --(CH₂)_x --CH_y (CH₃)_z --NH₂ and C₆ H_4+a (NH₂)₂, where x=0-3, y=1 or 2, and Z=1 when y=1 or Z=0 when y=2;

and a=0 or 6.

33. The particles according to claim 27 wherein said protein is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, enzymes and lectins.

34. The particles according to claim 27 wherein said protein is a monoclonal or polyclonal antibody.

35. The particles according to claim 27 wherein said protein has a detectible enzyme or a substance containing a radioactive element or a dye attached thereto.

36. The particles according to claim 27 wherein said stabilized amine derivatized polysaccharide coated particles coated are reacted with selected reagents to produce particles having either reactive maleimidyl or sulfhydryl groups attached to said polysaccharide coating and said protein is covalently bound to the sulfhydryl or maleimidyl group on said coated particles.

37. Particles according to claim 36 wherein said protein is derivatized to contain either reactive sulfhydryl or maleimidyl groups prior to binding said protein to said maleimidyl or sulfhydryl containing polysaccharide coated particles, provided that when said particles contain sulfhydryl groups said protein contains maleimidyl groups and when said particles contain maleimidyl groups said protein contains sulfhydryl groups.