Hermanson G. Bioconjugate Techniques, Second Edition
Подождите немного. Документ загружается.
600 14. Microparticles and Nanoparticles
particles. Two strategies can be used with crosslinking agents: (1) use of a homobifunctional
reagent, which contains the same reactive groups on either end or (2) use of a heterobifunc-
tional compound, which contains different reactive groups on each end.
A homobifunctional amine-reactive compound can be used initially to modify the amine
groups on particles, while leaving the remaining amine-reactive groups available to couple
with ligands. This type of reaction must be done with the crosslinker in great excess to prevent
polymerization of the amine particles themselves. There must be enough crosslinker present
Figure 14.8 Amine-containing particles can be conjugated using alkylation or acylation reactions to result in
secondary or tertiary amine linkages or amide bonds.
during the activation stage to avert the free end from attaching to another particle before that
particle’s amines are modifi ed with other crosslinkers.
Once the amine particles are modifi ed with a homobifunctional crosslinker, the excess reagent
is removed so that when the ligand is added, it doesn ’t become polymerized, particularly if it
has more than one amine (e.g., proteins). Homobifunctional crosslinkers containing NHS esters,
imidoesters, or aldehyde groups on each end can be used for this type of coupling process.
The following protocol illustrates this method using glutaraldehyde.
4.6. Glutaraldehyde
In one of the simplest methods, a homobifunctional amine-reactive crosslinker may be used
to activate the surface for coupling with an amine-containing ligand ( Figure 14.9 ). This has
been done with success using glutaraldehyde or polyglutaraldehyde (Rembaum et al., 1976;
Rembaum et al., 1978; Margel et al., 1979; Kaplan et al., 1983). The following protocol
Figure 14.9 Glutaraldehyde conjugation to amine particles can proceed via two routes. Reaction of the alde-
hyde groups with the amines using sodium cyanoborohydride results in secondary (or tertiary) amine linkages
with modifi cations containing a terminal aldehyde for further coupling to ligands. Alternatively, glutaraldehyde
polymers can react with amine particles via addition to double bonds, resulting in a polymeric coating that con-
tains both aldehydes and additional double bonds for further coupling with amine-containing molecules.
4. Polymeric Microspheres and Nanospheres 601
602 14. Microparticles and Nanoparticles
describes a two-step coupling method using glutaraldehyde on amine particles based on the
procedures of Kaplan et al. (1983) and Bang ’s Laboratories.
Protocol
1. Wash 10 mg of amine particles 3 times with 10 mM sodium phosphate, pH 7.4 (coupling
buffer).
2. After the fi nal wash, suspend the particles in coupling buffer containing 10 percent glu-
taraldehyde and mix well to dissolve.
3. React with mixing for 1 hour at room temperature.
4. Wash the particles with coupling buffer at least several times using centrifugation to
remove excess glutaraldehyde. Resuspend in 1 ml of the same buffer.
5. Add the protein to be coupled to the particle suspension in an amount equal to 1–10
molar excess over the calculated monolayer for the protein type to be coupled. Mix thor-
oughly to dissolve. Low concentrations of protein may result in particle aggregation,
because a single protein molecule can react with more than one particle.
6. React with mixing for 2–4 hours.
7. Add to the particle suspension a fi nal concentration of 0.2 M glycine (or another
amine-containing quench molecule, such as ethanolamine or Tris) and 10 mM sodium
cyanoborohydride. The blocking agent will couple to any remaining glutaraldehyde-reac-
tive sites and the reducing agent will convert all the resultant Schiff bases into stable sec-
ondary amine linkages.
8. Remove excess protein and reactants by washing with coupling buffer at least 3 times
using centrifugation. Store particles in a suitable buffer containing a preservative.
4.7. SPDP Coupling to Amine Particles
Another crosslinker-based method that has been used for coupling proteins to amine particles
involves the use of N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP). This reagent contains
an amine-reactive NHS ester and a thiol-reactive pyridyl disulfi de group (Chapter 5, Section 1.1).
Amine-containing particles can be activated by reaction with SPDP to form thiol-reactive deriv-
atives. Thiol-containing proteins, such as partially disulfi de-reduced antibodies, can be coupled
to the activated particles in a two-step reaction. An alternative method was outlined by Illum
and Jones (1985) that was based on Barbet et al. (1981) to couple SPDP modifi ed amine par-
ticles to antibodies that were also modifi ed with SPDP and then the pyridyl disulfi de group
reduced to form thiols. Mixing the thiolated antibody with the SPDP-activated particles results
in covalent coupling via disulfi de linkages ( Figure 14.10 ).
Unlike the use of homobifunctional crosslinkers, heterobifunctional compounds usually
don’t have to be used in large excess with amine particles to prevent aggregation. This is due to
the fact that only one of the ends of the crosslinker can react with the amines on the particles.
Protocol
1. Wash 10 mg of amine particles into 10 mM sodium phosphate, pH 7.2, using centrifuga-
tion. Resuspend the particles in 1 ml of the same buffer.
2. Dissolve SPDP in dimethylformamide (DMF) at a concentration of 6.2 mg/ml (makes a
20 mM stock solution). Add 50 l of the SPDP solution to the 1 ml particle suspension
and mix to dissolve. Note: The small quantity of DMF in a polymeric particle suspension
should not affect particle stability, even if the polymer type is susceptible to swelling in
pure DMF. Other particle types, such as metallic or silica based, usually are not affected
by organic solvent addition, unless their surfaces are non-covalently coated with a dis-
solvable polymer.
3. React for 30 minutes at room temperature with mixing.
4. Wash particles with coupling buffer at least 3 times using centrifugation and resuspend in
the same buffer using a sonic probe to disperse fully the particles.
5. Add 1–10 mg of a protein or antibody containing an available thiol group to the particle
suspension. Alternatively, add the protein to be coupled to the particle suspension in an
amount equal to 1–10 molar excess over the calculated monolayer for the protein type
to be coupled. The optimal amount of protein to be added should be determined experi-
mentally. Creating thiol groups from disulfi des in proteins may be done by using a reduc-
ing agent or through the use of a thiolation reagent (Chapter 1, Section 4.1).
Figure 14.10 The crosslinker SPDP can be reacted with amine particles to create thiol-reactive pyridyl disulfi de
groups on the surface. Thiol-containing proteins or other thiol molecules can be reacted with these activated
particles to result in disulfi de linkages, which are reversible by reduction.
4. Polymeric Microspheres and Nanospheres 603
604 14. Microparticles and Nanoparticles
6. React with mixing for 2 hours at room temperature. At the completion of the reaction,
cysteine may be added at 50 mM to block excess pyridyl disulfi de-reactive sites.
7. Remove excess protein and reactants by washing with coupling buffer at least 3 times.
Store particles in a suitable buffer containing a preservative.
4.8. NHS-PEG
n
-Maleimide Coupling to Amine Particles
An alternative method for coupling thiol-containing proteins or antibodies to amine particles is
to use a heterobifunctional crosslinker containing an amine-reactive NHS ester at one end and a
thiol-reactive maleimide group on the other end (Chapter 5, Section 1). Unlike the pyridyl dithiol
reaction with a sulfhydryl as in the SPDP protocol described previously, a maleimide group
forms a stable thioether linkage with a sulfhydryl-containing ligand, which is not cleavable by
reduction.
A common choice of crosslinker for this type of reaction is sulfo-SMCC (succinimidyl-4-( N -
maleimidomethyl)cyclohexane-1-carboxylate), which has been used extensively for antibody
conjugation (Chapter 5, Section 1.3). However, perhaps a better option for particle conjuga-
tion is to use a similar crosslinker design, but one in which contains a hydrophilic PEG spacer
arm to promote particle hydrophilicity after modifi cation. The modifi cation of an amine par-
ticle with a NHS-PEG
n
-maleimide reagent can create a surface that is essentially coated with
PEG spacers (Chapter 18, Section 2). This helps to mask any hydrophobic character that the
particle surface may have, while providing terminal thiol-reactive maleimides for coupling lig-
ands ( Figure 14.11 ).
If a NHS-PEG
n
-maleimide compound is used for this type of activation and coupling, the
intermediate maleimide-activated particle should be quickly washed free of excess crosslinker
and used to couple ligand immediately. This is due to the fact that the maleimide hydrolyzes
in aqueous solution at a higher rate than that of a maleimide on an SMCC-type crosslinker,
because of the extreme hydrophilicity of the PEG spacer arm compared to the cyclohexane
spacer of SMCC.
NHS-PEG
n
-maleimide crosslinkers are available in a number of spacer lengths depending
on the size of the polymer chain in the PEG component (Chapter 18, Section 2). Long-chain
crosslinkers of this type use PEG polymers of molecular weight approximately 2,000–5,000 Da,
containing from about 45 to over 100 repeating polyethylene oxide units. These full-length
polymers are polydisperse and actually consist of a broad range of polymer lengths, which
makes reproducibility of the crosslinker size nearly impossible to achieve. Modifying a particle
with this type of polymer diversity causes variability in the length of the crosslinkers displayed
on the surface, which may be detrimental for coupling some ligands.
However, shorter, discrete crosslinkers containing PEG spacers of known chain length are
now available (Thermo Fisher, Quanta BioDesign). These reagents are designed to contain an
exact number of PEG units, typically from between 2 repeating units to 24 repeating units. The
following protocol may be used with any of these compounds with the appropriate adjustments
in the quantity of crosslinker added to the reaction to take into account differences in molecular
weight due to the PEG length. The longer of these discrete NHS-PEG
n
-maleimide crosslinkers
will provide the greatest degree of hydrophilicity after modifi cation of an amine particle surface
(see Chapter 18 for additional details on discrete PEG compounds).
Protocol
1. Wash 10 mg of amine particles into 10 mM sodium phosphate, pH 7.2 (coupling buffer)
using centrifugation. Resuspend the particles in 1 ml of the same buffer.
2. Dissolve NHS-PEG
6
-maleimide (MW 601.6) into dimethyl sulfoxide (DMSO) at a con-
centration of 20 mM. PEG-type crosslinkers often exist as a thick oily mass, and prepar-
ing the solution may involve dissolving an entire vial of the compound into DMSO to
determine accurately the required concentration.
3. Add 50 l of the NHS-PEG
6
-maleimide solution to the 1 ml particle suspension and mix
thoroughly to dissolve.
4. React for 1 hour at room temperature with mixing.
5. Quickly wash the particles with coupling buffer at least twice using centrifugation.
Figure 14.11 The modifi cation of amine-containing particles with NHS-PEG
4
-maleimide produces hydrophilic
PEG spacers containing terminal thiol-reactive groups. Coupling of thiol-containing proteins then results in the
formation of thioether linkages.
4. Polymeric Microspheres and Nanospheres 605
606 14. Microparticles and Nanoparticles
6. Add 1–10 mg of a protein or antibody containing an available thiol group to the particle
suspension. Alternatively, add the protein to be coupled to the particle suspension in an
amount equal to 1–10 molar excess over the calculated monolayer for the protein type
to be coupled. The optimal amount of protein to be added should be determined experi-
mentally. Creating thiol groups on proteins or peptides may be done from disulfi des by
reduction. Alternatively, a thiolation reagent may be used to add thiols to the protein
surface for coupling (see the protocols in Chapter 1, Section 4.1).
7. React with mixing for 2 hours at room temperature. At the completion of the reaction,
cysteine may be added at 50 mM to block excess maleimide-reactive sites.
8. Remove excess protein and reactants by washing with coupling buffer at least 3 times
using centrifugation. Store particles in a suitable buffer containing a preservative.
4.9. Coupling to Hydroxyl Particles
Polymeric particles containing hydroxyl groups often are created from copolymers or composites
of pHEMA, frequently with other more rigid polymer cores, such as polystyrene ( Figure 14.12 ).
pHEMA particles have surfaces that contain an abundance of primary hydroxyls, which tend
to produce favorable hydrophilic surface characteristics (Tauer et al., 2005). The hydroxyls can
hydrogen bond with a layer of water molecules in aqueous solution, which forms an interface
between individual particles and stabilizes them against aggregation, even in the presence of
relatively high salt concentrations. The interaction of biomolecules with pHEMA particles typi-
cally lowers nonspecifi c binding potential compared to particles of more hydrophobic polymer
construction. This enhanced hydrophilicity of pHEMA particles translates into a high degree of
biocompatibility, which is important for decreasing background in particle-based assays and in
preventing denaturation of immobilized proteins on the particle surface.
Although hydroxyls are not spontaneously reactive toward functional groups on biomole-
cules, they can be activated for covalent coupling by a number of known reaction mechanisms.
Most of the reactions that can result in covalent attachment of ligands to hydroxylic or pHEMA
particles originated in the development of immobilization technology for affi nity chromatogra-
phy using larger hydroxyl-containing porous beads (for a review, see Hermanson et al., 1992).
Most activation strategies for hydroxylic particles are done under nonaqueous conditions,
because the activating agent and the intermediate reactive group typically are susceptible to
hydrolysis. A convenient method of activation is to form a reactive carbonyl group on the
hydroxyl particle using compounds such as carbonyldiimidazole (CDI; Bethell et al., 1979)
or disuccinimidyl carbonate (DSC; Miron and Wilchek, 1993). These activating agents create
imidazole carbamates (using CDI) or NHS-carbonates (using DSC) on the particle surface, which
then are spontaneously reactive toward amines (see Figures 14.13 and 14.14 ). After washing away
excess activating agent in organic solvent, the particles are centrifuged to remove most solvent
and then resuspended in aqueous buffer containing the amine ligand to be coupled (e.g., a protein).
The imidazole carbamate group is more stable to hydrolysis in aqueous buffer than the NHS-
carbonate group, which is similar in reactivity to an NHS ester. However, this means that the
imidazole carbamate also is slower to react and couple with amines. NHS-carbonate reactions
usually go to completion within 1–2 hours at room temperature, whereas imidazole carbamates
typically require higher pH conditions and overnight incubations to get maximal yield of ligand
coupling.
The following protocols involve the activation of hydroxylic pHEMA particles in organic
solvent using either CDI or DSC and the subsequent coupling of amine-containing molecules
using aqueous buffer conditions. Various solvents may be used for the activation step as long
as the activation agents are soluble in them and the particles do not get damaged due to sol-
vent exposure. Only water miscible solvents should be used to facilitate exchange into and out
of aqueous conditions. However, the solvents should be anhydrous to prevent hydrolysis of the
activation agent or the subsequent reactive group formed on the particles.
Protocol Using CDI
This method is derived from that of Bethell et al. (1979) and Colvin et al. (1988):
1. Solvent exchange pHEMA particles into anhydrous THF using centrifugation and resuspen-
sion with sequential exchange into greater percentages of THF in water until the particles
Figure 14.12 A core/shell polymeric particle made of a hydrophobic polystyrene core that is capped with a
hydrophilic polyHEMA shell, which contains numerous hydroxyl groups.
4. Polymeric Microspheres and Nanospheres 607
608 14. Microparticles and Nanoparticles
are suspended in 100 percent THF. Wash several times with anhydrous THF to elimi-
nate any remaining traces of water, letting the particles agitate in solvent between each
centrifugation step. After the fi nal THF wash centrifuge the particles and remove excess
solvent ( Figure 14.15 ).
2. Resuspend the particles as a 5 percent suspension in anhydrous THF containing CDI at a
concentration of 50 mg/ml (0.3 M).
Figure 14.13 Hydroxyl-containing particles can be activated for coupling ligands using a number of strategies,
which involve either aqueous or nonaqueous reactions. Epoxy and vinyl sulfone activation procedures provide
reactive groups able to couple with amine-, thiol-, or hydroxyl-containing ligands. Cyanogen bromide activa-
tion and the CDI and DSC methods provide reactive groups for amine coupling.
3. React with mixing for 2 hours at room temperature.
4. Wash the activated particles 3 times with anhydrous THF to remove excess CDI and
reaction by-products. After the fi nal wash, remove the solvent and perform a quick
wash with ice-cold water to remove most traces of solvent in the particle pellet. Finally,
resuspend the particles at 10 mg/ml in cold 0.1 M sodium phosphate, pH 8.2, or 0.1 M
sodium carbonate, pH 9.5 (coupling buffer). The higher pH coupling buffer will result in
greater reactivity of the imidazole carbamate and greater coupling yields for proteins.
5. Add 1–10 mg of a protein or antibody containing an available thiol group to the parti-
cle suspension in coupling buffer and mix to dissolve. Alternatively, add the protein to
the particle suspension in an amount equal to 1–10 molar excess over the calculated
Figure 14.14 Additional hydroxyl-particle activation methods include bis-epoxide modifi cation, tosyl activa-
tion, and tresyl activation methods. The tosyl chloride and tresyl chloride activation procedures must be done
in dry organic solvent, but the coupling of an amine-containing ligand can be done in either organic solvent or
aqueous buffer.
4. Polymeric Microspheres and Nanospheres 609