Beads for capturing target cells from bodily fluid

• US 7,439,062 B2
• Filed: 12/23/2004
• Issued: 10/21/2008
• Est. Priority Date: 12/23/2004
• Status: Active Grant

• Current Assignee: Biocept, Inc.
• Original Assignee: Biocept, Inc.
• Inventors: Bhatt, Ram S., Tsinberg, Pavel
• Primary Examiner(s): Naff; David M
• Application Number:
  US11/021,304
• Publication Number:
  US 20060141045A1
• Time in Patent Office: 1,398 Days
• Field of Search: None
• US Class Current: 435/325
• CPC Class Codes: A61K 9/1641 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
• CPC Class Codes: C12N 2533/30 Synthetic polymers

Abstract

Spheroidal beads present an exterior surface of a hydrophilic hydrogel, which is an isocyanate-functional polymer that is polymerized by urethane bonds and cross-linked by urethane and urea bonds. Sequestering agents present at the surface are covalently bound to isocyanate groups or to intermediate linkers that are so bound. These beads allow sequestering agents to retain their native three-dimensional configuration, and as a result of such surface characteristics and hydrophilicity, they achieve highly effective capture of very small subpopulations of rare cells from bodily fluids or the like and very effectively deter nonspecific binding of other biomaterials present in such bodily fluid. They may be all-hydrogel spheroids or hydrogel-coated substrates.

Claims

1. Beads for capturing target cells from a bodily fluid, which beads comprise:

spheroids of a size between about 5 μ

m and about 500 μ

m, said spheroids having an exterior hydrophilic hydrogel surface that is constituted of an isocyanate- or a thiocyanate-functional polymer containing residues of PEG, PPG, or a copolymer thereof that are polymerized by urethane bonds, and

agents which are directly or indirectly bound to said hydrogel via isocyanate or thiocyanate groups of said polymer, wherein said agents can contact and bind ligands on the surfaces of said target cells.

2. Beads according to claim 1, wherein said PEG, PPG or copolymer thereof has an average molecular weight of about 2000-7000 daltons, and wherein said hydrogel contains crosslinking.

3. Beads according to claim 2, wherein said hydrogel is obtained by reacting polyoxyalkylene diols or polyols with difunctional or polyfunctional isocyanate or thiocyanate compounds.

4. Beads according to claim 1, wherein said hydrogel is a reaction product of reactant (a), which is an isocyanate containing at least a major portion of diisocyanates, with reactant (b), which is said PEG, PPG, or copolymer thereof, wherein the molecules of reactant (b) each contain two or more hydroxyl groups, and wherein the reactants are reacted at a ratio of isocyanate of reactant (a) to hydroxyl of reactant (b) of about 1.2 to about 2.2.

5. Beads according to claim 1, wherein said spheroids are solid polymeric substrates coated by said hydrogel, and wherein said hydrogel coating is at least about 1 micron thick in the dry state and swells to at least about 3 times its thickness in an aqueous environment.

6. Beads according to claim 1, wherein said spheroids are substantially entirely hydrogel and contain said agents dispersed substantially throughout.

7. Beads according to claim 1, wherein said agents are selected from the group consisting of antibodies, lectins, receptor ligands, carbohydrates, and hormones.

8. Beads according to claim 1, wherein said agents are selected from the group consisting of antibodies, lectins, receptor ligands, carbohydrates, and hormones, and wherein said agents retain their native 3-dimensional configuration.

9. Beads according to claim 1, wherein said agents are antibodies and said antibodies retain their native 3-dimensional configuration.

10. Beads according to claim 1, wherein said agents are indirectly bound to said hydrogel.

11. Beads according to claim 1, wherein said polymer is an isocyanate-functional polymer, and wherein said agents are directly or indirectly bound to isocyanate groups of said polymer.

12. A method for capturing target cells from a bodily fluid, which method comprises the steps of:

(a) providing beads according to claim 1, and

(b) bringing said beads into contact with said bodily fluid, so that said target cells are captured by binding to said agents that are specific to said target cells.

13. The method according to claim 12, wherein said spheroids are washed following step (b) to remove nonspecifically bound biomaterial so that substantially only the captured target cells adhere to said beads.

14. The method according to claim 12, wherein said PEG, PPG, or copolymer thereof has a molecular weight of about 6000-7000 daltons, and wherein said hydrogel contains crosslinking.

15. The method according to claim 12, wherein said hydrogel is a reaction product of reactant (a), which is an isocyanate and/or polyisocyanate, with reactant (b), which is said PEG, PPG, or copolymer thereof, wherein the molecules of reactant (b) each contain two or more hydroxyl groups, and wherein the reactants are reacted at a ratio of isocyanate of reactant (a) to hydroxyl of reactant (b) of about 1.2 to about 2.2.

16. The method according to claim 12, wherein said spheroids are solid polymeric substrates coated by said hydrogel, and wherein said hydrogel coating is at least about 1 micron thick when dry and swells to at least 3 times its thickness in an aqueous environment.

17. The method according to claim 12, wherein said hydrogel is obtained by reacting polyoxyalkylene diols or polyols with difunctional or polyfunctional isocyanate or thiocyanate compounds.

18. The method according to claim 12, wherein said spheroids are substantially entirely hydrogel and contain said agents dispersed substantially throughout.

19. The method according to claim 12, wherein said agents are selected from the group consisting of antibodies, lectins, receptor ligands, carbohydrates, and hormones.

20. The method according to claim 12, wherein said agents are selected from the group consisting of antibodies, lectins, receptor ligands, carbohydrates, and hormones, and wherein said agents retain their native 3-dimensional configuration.

21. The method according to claim 12, wherein said agents are antibodies and said antibodies retain their native 3-dimensional configuration.

22. The method according to claim 12, wherein said agents are indirectly bound to said hydrogel.

23. The method according to claim 12, wherein said polymer is an isocyanate-functional polymer, and wherein said agents are directly or indirectly bound to isocyanate groups of said polymer.