Isolation of cells or the like from bodily fluids

• US 20060141045A1
• Filed: 12/23/2004
• Published: 06/29/2006
• Est. Priority Date: 12/23/2004
• Status: Active Grant

• Current Assignee: Biocept, Inc.
• Original Assignee: Biocept, Inc.
• Inventors: Bhatt, Ram S., Tsinberg, Pavel
• Granted Patent: US 7,439,062 B2
• Time in Patent Office: Days
• US Class Current: 424/489
• 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. Generally spherical beads for sequestering biological cells from a bodily fluid or the like, 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-functional polymer containing residues of PEG, PPG or a copolymer thereof that are polymerized by urethane bonds, and

sequestering agents which are directly or indirectly bound to said hydrogel via said functional isocyanate groups, which sequestering agents retain their native 3-dimensional configuration and are present at least at or near the exterior surface boundaries of said spheroids where they can contact and bind with ligands on the surfaces of target cells of interest.

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

3. Beads according to claim 2 wherein said hydrogel polymer is a reaction product of some trifunctional or greater hydroxyl molecules or isocyanate molecules.

4. Beads according to claim 1 wherein said hydrogel is a reaction product of (a) an isocyanate containing at least a major portion of diisocyanates and (b) said PEG, PPG or copolymer thereof, and wherein the reactants were reacted in an isocyanate to hydroxyl ratio of about 1.2 to about 2.2.

5. Beads according to claim 1 wherein said spheroids are solid polymeric substrates which are covered by a hydrogel coating that 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 sequestering agents and/or intermediate linkers dispersed substantially therethroughout.

7. A method for separating target biological cells from a bodily fluid or the like employing beads according to claim 1, which method comprises the steps of:

(a) providing beads according to claim 1, and

(b) bringing said beads into contact with bodily fluid or the like, so that target biological cells are captured at or near the exterior boundaries thereof by binding to said sequestering agents that are specific to said target cells.

8. The method according to claim 7 wherein said spheroids are washed following step (b) to remove nonspecifically bound biomaterial so that substantially only the captured cells adhere to said beads.

9. The method according to claim 7 wherein said PEG, PPG or copolymer has a molecular weight of about 6000-7000 daltons and wherein said hydrogel prepolymer is a reaction product containing residues of diisocyanates and/or polyisocyanates and residues of trifunctional or greater polyols.

10. The method according to claim 9 wherein said hydrogel is a reaction product of a reaction that was carried out at an isocyanate to hydroxyl ratio of about 1.2 to about 2.2 to form the prepolymer, solution of which prepolymer is used in providing said beads.

11. The method according to claim 7 wherein said beads are spheroidal substrates coated with a hydrogel layer that is at least about 1 micron thick when dry and that constitutes a hydrogel which swells so that its thickness becomes at least about 3 times greater in an aqueous environment.

12. A method of providing beads adapted to the efficient isolation of cells from a bodily fluid or the like, which method comprises:

providing spheroidal substrates having a size between about 5 μ

m and about 1000 μ

m,

covering the entire outer surfaces of said substrates with a hydrophilic hydrogel prepolymer coating, which is an isocyanate-functional polymer containing residues of polyoxyalkylene diol and/or polyol reactants that are prepolymerized by urethane bonding to diisocyante and/or polyisocyanate molecules,

binding sequestering agents directly or indirectly to said hydrogel via the isocyanate groups so that said sequestering agents retain their native 3-dimensional configuration and are present at least at or near exterior boundaries of said hydrogel coatings where they can contact and bind with ligands on the surfaces of target cells of interest, and

completing polymerization of said coated prepolymer.

13. The method according to claim 12 wherein said polyoxyalkylene diols include PEG, PPG or copolymers thereof and have an average molecular weight of about 2000-7000 daltons and wherein said hydrogel polymer is a reaction product that includes some crosslinking via polyol molecules.

14. The method according to claim 13 wherein said reaction product is one where the reactants were reacted in an isocyanate to hydroxyl ratio of about 1.2 to about 2.2 to form the prepolymer and wherein a solution of the prepolymer is coated onto said substrates and polymerized thereupon.

15. The method according to claim 14 wherein said coating is applied so as to be at least about 1 micron thick when dry and constitutes a hydrogel which swells so that its thickness becomes at least about 3 times greater in an aqueous environment.

16. The method according to claim 12 wherein said sequestering agents are mixed with a solution of the prepolymer and become covalently bonded to isocyanate groups of said polymer, and wherein such mixture is coated onto said substrates and polymerized thereupon.

17. A method of providing beads adapted to the efficient isolation of cells from a bodily fluid or the like, which method comprises:

providing a prepolymer in the form of an isocyanate-functional polymer that contains residues of PEG, PPG or a copolymer thereof having a MW between about 2,000 and 7,000 which residues are linked by urethane bonds to isocyanates, and that contains at least about 0.2 meq. of active isocyanate per gram,

mixing sequestering agents or intermediate linkers with said prepolymer under conditions which cause said agents or linkers to covalently bind with isocyanate groups,

forming said prepolymer mixture into spheroidal beads having a size between about 5 μ

m and about 500 μ

m, and

optionally linking sequestering agents with said linkers when intermediate linkers are used,

whereby the entire outer surface of said beads is a hydrophilic hydrogel where sequestering agents are present that retain their native 3-dimensional configuration so that they can contact and bind with ligands on the surfaces of target cells of interest.

18. The method according to claim 17 wherein said PEG, PPG or copolymer has an average molecular weight of about 6000-7000 daltons, wherein said prepolymer is a reaction product formed from isocyanates and some tri-functional or greater polyols and wherein said isocyanates, said PEG, PPG or copolymer and said polyols were reacted in an isocyanate to hydroxyl ratio of about 1.2 to about 2.2.

19. The method according to claim 17 wherein said sequestering agents in aqueous solution are mixed with the prepolymer and such mixture is injected in droplet form into a moving body of substantially immiscible liquid where gelling into spheroidal beads occurs.

20. The method according to claim 19 wherein said droplets are injected into mineral oil and contain sufficient Abs to provide an Abs density on said bead surfaces of at least about 3.6 micromole per mg of beads.