Hermanson G. Bioconjugate Techniques, Second Edition
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610 14. Microparticles and Nanoparticles
mono-layer for the protein type to be coupled. The optimal amount of protein to be
added should be determined experimentally.
6. React with mixing for at least 18 hours at 4°C. Longer reaction times (24–48 hours) may
be necessary when using the pH 8.2 coupling buffer. Room temperature reactions will
increase the reaction rate.
7. Add ethanolamine to the particle suspension at a fi nal concentration of 0.1 M to quench
any remaining active groups and react with mixing for several hours.
8. Centrifuge and wash the particles at least 3 times with buffer to remove unreacted pro-
tein and ethanolamine. Finally, suspend the particles in a suitable storage buffer contain-
ing a preservative.
Protocol Using DSC
This method is derived from that of Miron and Wilchek (1993) for the activation of hydroxyl
groups on PEG molecules and Wilchek and Miron (1985) for the activation of agarose chro-
matography beads. Many types of hydroxyl-containing particles may be used in this proce-
dure, provided that the solvent used for the activation step does not deleteriously affect particle
integrity ( Figure 14.16 ).
1. Solvent exchange pHEMA particles into anhydrous acetone, dioxane, acetonitrile, THF, or
DMF (having very low amine content) using centrifugation and resuspension with sequen-
tial exchange into greater percentages of solvent in water until the particles are suspended
Figure 14.15 CDI can be used to activate hydroxyl-particles in organic solvent and then the intermediate reac-
tive imidazole carbamate brought into aqueous solution for coupling amine-containing ligands.
in 100 percent organic solvent. Wash several times with anhydrous solvent to eliminate any
remaining traces of water, letting the particles agitate in solvent between each centrifuga-
tion step. After the fi nal solvent wash, centrifuge the particles and remove excess solvent.
2. Resuspend the particles as a 5 percent suspension in anhydrous solvent containing DSC
at a concentration of 50 mg/ml (0.2 M).
3. React with mixing for 2 hours at room temperature.
4. Wash the activated particles 3 times with anhydrous solvent to remove excess DSC 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.
5. Immediately resuspend the particles at a concentration of 10 mg/ml in 0.1 M sodium
phosphate, pH 7.2 (coupling buffer) containing 1–10 mg of a protein or antibody and
mix to dissolve. Alternatively, add the protein 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 level of protein to be added should be determined experimentally.
6. React with mixing for at least 2 hours at room temperature or twice as long at 4°C.
7. Add ethanolamine to the particle suspension at a fi nal concentration of 0.1 M to quench
any remaining active groups and react with mixing for 1 hour. Other amine-containing
quenchers may be used, too, such as Tris buffer. Note: DSC-activated sites on the parti-
cles that completely hydrolyze will revert back to the original hydroxyls.
8. Centrifuge and wash the particles at least 3 times with buffer to remove unreacted pro-
tein and quenching agent. Finally, suspend the particles in a suitable storage buffer con-
taining a preservative.
Figure 14.16 DSC can be used to activate hydroxyl-particles to a reactive NHS-carbonate derivative. The sub-
sequent coupling of amine-containing ligands can be done in either organic solvent or aqueous conditions.
4. Polymeric Microspheres and Nanospheres 611
612 14. Microparticles and Nanoparticles
Protocol Using Cyanogen Bromide
Cyanogen bromide can be used to activate hydroxyl groups on particles to create reactive
cyanate esters, which then can be coupled to amine-containing ligands to form an isourea bond
(Figure 14.17 ). CNBr activation also can produce cyclic imidocarbonate groups, which are less
reactive than the cyanate ester, but can form imidocarbonate bonds. The exact reactive species
formed by the reaction is dependent on the structure of the hydroxylic support being activated
(Kohn and Wilchek, 1982).
Unlike the previous methods for activation of hydroxyls on particles, cyanogen bromide is
used under aqueous conditions, thus eliminating the need for organic solvents. This method
originated in the activation and coupling of ligands to agarose supports for affi nity chroma-
tography (Axen et al., 1967), and it subsequently was used to couple antibodies to hydroxyl-
containing latex particles (Yen et al., 1979) and more recently to immobilize protein A onto
pHEMA particles (Denizli et al. , 1995).
Cyanogen bromide is an extremely toxic chemical and should be used only in a well-
ventilated fume hood using the appropriate personal protective gear. The following protocol is
based on the method of March et al. (1974), as recommended by Bang ’s Laboratories.
All operations should be performed in a fume hood:
1. Wash 100 mg of hydroxyl-containing particles 2 times using centrifugation with 0.1 M
sodium carbonate, pH 8.5 (coupling buffer). After the second wash, resuspend the particles
at 10 mg/ml in 2 M sodium carbonate (activation buffer; no pH adjustment necessary).
2. Dissolve an amount of protein in 10 ml of coupling buffer equal to 1–10 excess over
the calculated monolayer for the type of particles being used.
Figure 14.17 Cyanogen bromide can be used to activate a hydroxyl-particle to a reactive cyanate ester, which
then can be used to couple amine-containing ligands.
3. In a fume hood, dissolve 1 g of cyanogen bromide in 0.5 ml of acetonitrile (highly toxic!).
4. Add a drop of the cyanogen bromide solution at a time to the particle suspension with
constant mixing at room temperature. The entire solution should be added to the parti-
cles over the course of about 10 seconds.
5. Activate the particles with mixing for exactly 2 minutes at room temperature.
6. Quickly wash the particles with ice-cold deionized water and then with a volume of cold
coupling buffer. Resuspend the particles in the protein solution prepared in step 2.
7. React for 24 hours at 4°C with mixing.
8. Wash the particles with coupling buffer and block excess reactive groups by resuspending
in 50 mM ethanolamine, pH 9.0. React for 1 hour at room temperature with mixing.
9. Thoroughly wash the particles with storage buffer (e.g., PBS, pH 7.5, or other suitable
buffer) and resuspend them at 10 mg/ml in storage buffer containing a preservative.
4.10. Coupling to Hydrazide Particles
Hydrazide particles can be made from carboxylate particles by modifi cation with a bis-hydrazide
compound using the carbodiimide reaction with EDC. Suitable bifunctional hydrazides include
the small carbohydrazide compound or the longer adipic dihydrazide (Chapter 4, Section 8).
A bis-hydrazide compound is reacted with a carboxylate particle population in large excess to
prevent particle polymerization during the reaction. The resultant hydrazide particles may be
used to couple to carbonyl-containing ligands, such as carbohydrates or glycans at their reduc-
ing ends or after the formation of aldehydes on carbohydrates using oxidation with sodium
periodate (Chapter 1, Section 4.4).
Perhaps a better design for a bis-hydrazide compound to modify carboxylate particles would
include a short PEG spacer arm between the two hydrazide groups. This type of linker would
result in a hydrophilic surface due to the presence of the PEG spacers, while providing the ter-
minal hydrazide functionality necessary for coupling to carbonyl compounds. Unfortunately,
this type of compound is not currently available, so the aliphatic bis-hydrazides are the only
choice.
Another route to the formation of a hydrazide on a surface is to use an aldehyde-containing
particle (such as HEMA/acrolein copolymers) and subsequently modify the aldehydes to form
hydrazone linkages with bis-hydrazide compounds, which then can be stabilized by reduction
with sodium cyanoborohydride (Chapter 2, Section 5). The resulting derivative contains termi-
nal hydrazides for immobilization of carbonyl ligands (see Figure 14.18 ).
Hydrazide-containing particles provide functional groups for the coupling of aldehyde- or
ketone-containing ligands through a dehydration reaction to form hydrazone linkages (a type
of Schiff base). However, a single hydrazone bond between a ligand and the particle surface
may not provide enough stability to prevent leaching of ligand due to hydrolysis. There are two
routes to overcome this instability: (1) reduce the hydrazone linkage using sodium cyanoboro-
hydride or (2) create multiple hydrazone linkages between the ligand and the particle surface.
Multi-site attachment provides suffi cient ligand stability, because not all the hydrazones will
hydrolyze simultaneously to release ligand, and when one hydrazone bond breaks, it will have
enough time to reform before the other hydrazones hydrolyze. Thus, glycosylated proteins cou-
pled after oxidation to hydrazide particles most likely will be stable due to the presence of
4. Polymeric Microspheres and Nanospheres 613
614 14. Microparticles and Nanoparticles
more than one aldehyde group, but small ligands containing only a single carbonyl group prob-
ably should be treated with cyanoborohydride to stabilize the hydrazone bond.
The reactions involved with coupling carbonyl-containing ligands to hydrazide particles
originated with the activation and coupling chemistry associated with the preparation of affi n-
ity chromatography supports (O ’Shannessy and Wilchek, 1990). The application of this strat-
egy to hydrazide-containing microparticles or nanoparticles is straightforward and will work
well so long as accommodation is given to particle stability during the process. Using this
method, a broad range of carbonyl-containing ligands can be coupled, such as reducing sugars
or carbohydrates containing a reducing end, glycans after release from proteins, glycoproteins
and other glycoconjugates, and small organic compounds containing an aldehyde or ketone
group ( Figure 14.19 ). Horak et al. (1999) used hydrazide-pHEMA particles to couple oxidized
horseradish peroxidase (HRP) in good yield and retention of enzymatic activity.
The following protocol describes the oxidation of carbohydrate (glycans) on antibody mol-
ecules to form aldehydes and the subsequent coupling to hydrazide particles.
Protocol
1. Dissolve the antibody to be coupled in 10 mM sodium phosphate, 0.15 M NaCl, pH
7.5, at a concentration of 10 mg/ml. The antibody must be glycosylated to work in this
procedure.
2. Prepare a solution of 0.1 M sodium periodate in water. Protect from light.
3. With mixing, add 0.1 ml of the periodate solution to each ml of the antibody solution.
4. React for 30 minutes at room temperature, protected from light.
5. The reaction may be quenched by the addition of 0.1 ml of glycerol per ml of reaction
or by the addition of sodium bisulfi te to a fi nal concentration of 10 mM.
Figure 14.18 Carboxylate-particles or aldehyde-particles can be modifi ed with the carbohydrazide in excess to
create a hydrazide-particle that can be used to couple with aldehyde-containing molecules.
6. Remove excess reactants from the reaction mixture using size exclusion chromatogra-
phy on a column of Sephadex G25 (or equivalent).
7. Wash 100 mg of hydrazide particles 2 times with 10 ml of 10 mM sodium phosphate,
0.15 M NaCl, pH 7.5 (coupling buffer) using centrifugation.
8. After the fi nal wash, resuspend the particles at a concentration of 10 mg/ml in coupling
buffer and add an appropriate amount of the solution from step 6, which contains the
purifi ed, oxidized antibody. The amount of oxidized antibody to add to the particles
should be about 1–10 over the amount of the calculated monolayer for the particle
type used. ( Note: For 100 mg of 1 m hydrazide particles, a monolayer equivalent of
antibody will be about 1.5 mg, so the total amount added should be in the range of
1.5–15 mg for a 1–10 excess).
9. React with mixing for at least 6 hours at room temperature or overnight at 4°C.
10. Wash the beads at least several times with coupling buffer and resuspend in the same
buffer containing 0.05–0.1 percent of a blocking molecule (such as BSA, gelatin, non-
fat dried milk, PEG, PVP, etc.). The best blocking agent may be found by experimenta-
tion and testing of the antibody-coupled particles in the intended application.
11. Wash the particles several times with coupling buffer and store in an appropriate buffer
containing a preservative at 4°C.
4.11. Coupling to Epoxy Particles
Polymeric particles containing epoxide groups can be used to couple thiol-, amino-, or
hydroxyl-containing ligands via a ring-opening reaction facilitated under basic conditions. This
reactive group can be used to couple proteins, nucleic acids, sugars and carbohydrates, and
other organic molecules containing these functionalities. Epoxide groups can be introduced
into polymeric particles through free-radical copolymerization with oxirane-containing vinyl
monomers, such as allyl glycidyl ether, or they may be introduced by surface modifi cation using
a bis-epoxide compound, such as 1,4-butanediol diglycidyl ether (Sundberg and Porath, 1974).
Epoxy activation has been used extensively to immobilize affi nity ligands onto porous beaded
chromatography supports, and it can be used with equal success to couple ligands to micropar-
ticles and nanoparticles.
Epoxide-containing particles can be used to couple thiol-containing ligands at slightly basic
pH (pH 7.5–8.5), amine-containing ligands at higher pH values (pH 9–11), and hydroxyl-
containing ligands at very high alkaline conditions (pH 11). The following protocol can be
Figure 14.19 Aldehyde-containing molecules, such as periodate-oxidized carbohydrates or glycoproteins, can
be coupled to hydrazide-particles to form a hydrazone bond. This bond can be further stabilized by reduction
with sodium cyanoborohydride.
4. Polymeric Microspheres and Nanospheres 615
616 14. Microparticles and Nanoparticles
used to couple thiol-, amine-, or hydroxyl-containing ligands with the proper pH adjustment of
the carbonate coupling buffer ( Figure 14.20 ).
Protocol
1. Wash 100 mg of epoxy particles with coupling buffer (i.e., 0.1 M sodium carbonate, pH
10 for coupling amine-containing ligands). Use higher pH conditions if coupling hydrox-
ylic molecules and lower pH for coupling thiol-containing ligands. Suspend the particles
at a 5 percent solution in coupling buffer.
2. With mixing, add to the particle suspension a quantity of ligand dissolved in coupling
buffer in an amount that represents a 1–10 excess over the molar quantity of epoxide
groups present on the particles.
3. React at room temperature (for sensitive ligands) or at 45–60°C (for more stable ligands)
for at least 20 hours with mixing.
4. Block excess epoxy groups by the addition of cysteine to a fi nal concentration of 50 mM.
Other small molecules can be used, provided they will effi ciently react with the excess
epoxides and not result in a modifi cation that could interfere with the subsequent use of
the particles. Continue the reaction with mixing for at least 2 hours.
5. Wash the particles thoroughly with coupling buffer and then into a more moderate pH
storage buffer containing a preservative.
Figure 14.20 Particles containing reactive epoxy groups can be coupled with amine-, thiol-, or hydroxyl-
containing molecules.
4.12. Coupling to Aldehyde Particles
Polymeric particles containing aldehydes are produced by two general routes: copolymers
containing an aldehyde monomer (e.g., acrolein) or through periodate oxidation of diols
incorporated onto the surface of particles. An example of copolymer aldehyde particles are
HEMA/acrolein derivatives (Kumakura and Kaetsu, 1984; Chang et al., 1986; Colvin et al. ,
1988), which are hydrophilic due to the large number of hydroxyl groups and provide alde-
hydes for covalent attachment of amine-containing ligands. Epoxy particles also can be used
to create surface aldehydes by opening up the epoxide ring by acid hydrolysis and oxidation of
the resultant diols by sodium periodate (Schiel et al. , 2006).
Aldehyde particles are spontaneously reactive with hydrazine or hydrazide derivatives,
forming hydrazone linkages upon Schiff base formation. Reactions with amine-containing
molecules, such as proteins, can be done through a reductive amination process using sodium
cyanoborohydride ( Figure 14.21 ).
Protocol
1. Wash 10 mg of aldehyde particles 3 times with 10 mM sodium phosphate, pH 7.4 (cou-
pling buffer). Buffers of higher pH value (i.e., carbonate buffer at pH 10) will result in
more effi cient Schiff base formation with amine-containing molecules than neutral pH
conditions.
2. After the fi nal wash, suspend the particles at 5–10 mg/ml in coupling buffer and add a
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. ( Note: It takes
about 18 mg of BSA or 15 mg of IgG to saturate 1 g of 1 m particles, and more protein
if the particles are smaller.) Mix thoroughly to dissolve. Low concentrations of protein
may result in particle aggregation, because a single protein molecule can react and bridge
more than one particle.
3. Incubate with mixing for 2–4 hours at room temperature.
Figure 14.21 Aldehyde-particles can be reacted with amine-containing proteins or other molecules to form
intermediate Schiff bases, which can be stabilized by reduction with sodium cyanoborohydride.
4. Polymeric Microspheres and Nanospheres 617
618 14. Microparticles and Nanoparticles
4. Add to the particle suspension a quantity of sodium cyanoborohydride in water to bring
the fi nal concentration up to 10 mM. If a high pH buffer was used for the initial incuba-
tion between the particles and the protein, perform a quick wash with 10 mM sodium
phosphate, pH 7.4, to bring the pH down to a point in which the reducing agent is
active. Mix for 30 minutes at room temperature. The reducing agent will convert all the
resultant Schiff bases into stable secondary amine linkages.
5. Add to the particle suspension a quenching molecule (such as glycine, ethanolamine,
or Tris) to give a fi nal concentration of 0.2 M. The blocking agent will couple to any
remaining aldehyde-reactive sites.
6. 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.
5. Silica Particles
The use of silica particles in bioapplications began with the publication by Stöber et al. in 1968
on the preparation of monodisperse nanoparticles and microparticles from a silica alkoxide
monomer (e.g., tetraethyl orthosilicate or TEOS). Subsequently, in the 1970s, silane modifi -
cation techniques provided silica surface treatments that eliminated the nonspecifi c binding
potential of raw silica for biomolecules (Regnier and Noel, 1976). Derivatization of silica with
hydrophilic, hydroxylic silane compounds thoroughly passivated the surface and made possi-
ble the use of both porous and nonporous silica particles in all areas of bioapplications (Schiel
et al. , 2006).
The modifi cation of silica particles to provide sites for coupling affi nity ligands can be done
similarly through covalent derivatization of the surface with a functional silane containing
a side-chain-reactive group or functional group (see Chapter 13). For instance, reaction of a
silica particle with 3-aminopropyltriethoxysilane (APTS) under the appropriate condi-
tions coats the surface with primary amino groups for conjugation with electrophilic groups.
Reagents containing alkoxy silane groups can condense, particularly after hydrolysis to cre-
ate reactive silanols, with the silanol ⎯ OH groups on standard silica particles to form stable
siloxane linkages (or oxane bond; Figure 14.22 ). This reaction typically is catalyzed by heat or
through the use of at least a partially aqueous environment, which forms the requisite silanols
from the alkoxy groups on the silane compound. Unlike other alkoxy silanes, trimethoxy
silane compounds can react directly with particle silanols without the need for high-temperature
conditions or hydrolysis to form siloxane linkages. Choice of the appropriate silane func-
tional group for surface modifi cation can provide a broad range of silica particle properties for
subsequent coupling of biomolecules or other affi nity ligands (for review see VanDerVoort
et al. , 1996).
Silica particles have some advantages over polymer particles. They have a higher density
than polymeric particles (1.96 g/cm
3
versus 1.05 g/cm
3
) and thus they can be washed using
centrifugation, even when working with nanometer-sized particles. Typically, silica particles as
small as 30–40 nm still can be separated from suspension using a benchtop microfuge, making
handling and processing of silica particles potentially much simpler than polymeric particles of
the same size.
Another advantage of using inorganic silica particles over polymer particles is that they
don’t shrink or swell when exposed to aqueous or nonaqueous environments. In the case of
silica, nonaqueous solvents may be used for activation reactions without concern that soften-
ing or dissolving of polymeric structures will damage the particles, as silica does not dissolve in
organic solvent environments.
A potential disadvantage of silica-based particles, however, is the tendency for the siloxane
linkages between silicon atoms within the particle to dissolve hydrolytically, especially under
Figure 14.22 Silica particles can be functionalized to contain amine group by reaction with APTS.
5. Silica Particles 619