Hermanson G. Bioconjugate Techniques, Second Edition

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380 7. Dendrimers and Dendrons

Protocol

1. Dissolve 10 mg (0.7 mol) of a G-4 PAMAM dendrimer in 1 ml of 0.1 M sodium phos-

phate, pH 9.0.

2. Add a 3-fold molar excess of biotinylation reagent over the molar quantity of dendrimer

present. For the use of sulfo-NHS-LC-biotin (MW 556), this represents the addition of

2.1mol or 1.16 mg. This reaction ratio will result in a modiﬁ cation level of about 2.5

biotin groups per dendrimer. Other molar ratios also may be used, depending on the

desired level of modiﬁ cation and the intended use for the conjugate.

3. React for 30–60 minutes at room temperature with mixing.

4. Purify the biotin-dendrimer using size exclusion chromatography on a desalting matrix

or by use of ultraﬁ ltration (e.g., centrifugal concentrators).

Fluorescent Labeling of Amine-Dendrimers

The multivalent nature of dendrimers can be used to advantage for signal enhancement in many

ﬂ uorescence detection schemes. A broad selection of hydrophilic organic ﬂ uorescent probes is

available as amine-reactive derivatives (Chapter 9), many of which may be used to form conju-

gates with amine-containing dendrimers. Coupling multiple ﬂ uorescent labels to each dendrimer

molecule creates a complex with increased luminescence or brightness for use in ﬂ uorescence

assays (Tomalia et al., 1998). In addition, combining a ﬂ uorescently labeled dendrimer with

the ability to link it to targeting molecules, such as antibodies or streptavidin, provides a rea-

gent system having superior detection capability than the use of ﬂ uorescently labeled proteins

directly. Manduchi et al. (2002) demonstrated that a ﬂ uorescent dendrimer could be a more

sensitive detection reagent than the use of directly labeled primary antibodies or indirect immu-

noassay methods using labeled secondary antibodies for microarray assay methods.

For instance, a dendrimer easily can be coupled with a large number of ﬂ uorescent dyes and

still provide additional coupling sites for biotinylation. The only limitation to the number of

ﬂ uorescent modiﬁ cations is if ﬂ uorescence quenching starts to take place, in which case no fur-

ther modiﬁ cations will result in increased signal. A series of such conjugates using different lev-

els of ﬂ uorophore modiﬁ cation should be done to determine the optimal level of dye-to-dendrimer

before quenching occurs.

A ﬂ uorescent, biotinylated dendrimer of this type then can be used in a (strept)avidin–biotin

assay to detect analytes. Having more than one biotin group on such a conjugate allows it to inter-

act with multiple streptavidin molecules, thus forming a megameric complex with a biotinylated

primary antibody bound at the site of an analyte. In this way, many more ﬂ uorescent molecules are

recruited to the point of detection than is possible using directly labeled proteins ( Figure 7.21 ). In

fact, for detection of DNA targets, dendrimer-based ﬂ uorescence enhancement methods have been

identiﬁ ed as an important route to increasing signal and avoiding polymerase chain reaction (PCR)

or other ampliﬁ cation methods (Nilsen et al., 1997; Vogelbacker et al., 1997; Kricka, 1999).

Fluorescent labels on dendrimers also are convenient to track the interaction of functional-

ized dendrimers for cell targeting. After the conjugation of targeting groups such as antibodies

or folic acid on a dendrimer along with radiolabels or toxic components, there are still amine

groups available to add ﬂ uorescent labels for detection purposes (Minard-Basquin et al., 2003;

Patri et al., 2004; Majoros et al., 2005). Such complexes may be assessed as to their speciﬁ city

by monitoring the ﬂ uorescent signal as the dendrimers bind to target cells.

Figure 7.21 Dendrimers that are ﬂ uorescently labeled as well as biotinylated create enhanced detection reagents

for use in (strept)avidin–biotin-based assays. Large complexes containing multiple ﬂ uorescent dendrimers can

bind to antigens and form a highly sensitive detection system that exceeds the detection capability of ﬂ uores-

cently labeled antibodies.

Dendrimers bearing certain ﬂ uorescent molecules attached on their periphery have been

shown to be environmentally sensitive probes for the presence of certain metal ions or to

changes in pH (Balzani et al., 2000; Paola et al., 2005). In addition, PAMAM dendrimers mod-

iﬁ ed with the relatively hydrophobic dye Oregon Green 488 were shown to be a more effective

transfection agent for anti-sense oligonucleotides than the dendrimer alone–plus the complex

could be tracked within the cell due to the ﬂ uorescence of the dye (Yoo and Juliano, 2000).

2. Conjugation to Dendrimers 381

382 7. Dendrimers and Dendrons

Speciﬁ c procedures for the conjugation of ﬂ uorescent labels to amine-containing molecules

can be found in Chapter 9. The following protocol describes one such reaction for an amine-

dendrimer with the ﬂ uorescent cyanine reactive probe, DyLight 649 NHS ester. This dye has

spectral properties that are nearly identical to the original Cy5-type dye, but the DyLight one

has negatively charged sulfonate groups, which makes the resultant compound extremely

water-soluble. Coupling reactions with other ﬂ uorescent dyes may be done similarly, but in

each case, optimization of the molar ratio of dye-to-dendrimer should be done to produce the

best conjugate for its intended application. Note that higher levels of dye incorporation do not

necessarily correlate to brighter ﬂ uorescent conjugates, because ﬂ uorescence energy transfer and

quenching may occur between dye molecules as the density of dyes increases on the dendrimer

surface. However, it appears that ﬂ uorescent dye substitution on dendrimers can be done at a

higher density than on proteins before such quenching effects occurs. Figure 7.22 illustrates the

reaction between an amine-dendrimer and a disulfonated Cy5 NHS ester derivative. Although

the exact structure of the DyLight 649 NHS ester is not published, it is similar to the known

Cy5 structure, but containing additional sulfonate groups to increase water solubility.

Protocol

1. In a fume hood, dissolve the DyLight 649 NHS ester at a concentration of 10 mg/ml in

dry DMF.

2. Dissolve the amine-dendrimer to be modiﬁ ed in DMF or buffer (50 mM sodium borate,

pH 8.5) at a concentration of at least 10 mg/ml. Avoid the use of amine-containing

buffers for an aqueous reaction, such as Tris or imidazole, as these will react with the

Figure 7.22 Fluorescent dyes such as an amine-reactive Cy5 derivative can be coupled to amine-dendrimers at

relatively high substitution levels to create intensely ﬂ uorescent detection agents. If the dendrimer also is deriva-

tized to contain an afﬁ nity group or a targeting group then speciﬁ c ﬂ uorescent detection at high sensitivity can

be realized.

NHS ester on the dye and prevent conjugation to the dendrimer. For reactions done in

organic solvent, add triethylamine to a ﬁ nal concentration of 1.25 times greater than the

amount of reactive dye to be added to the solution.

3. Add a quantity of the DyLight 649 dye to the dendrimer solution to provide at least a

1.25-fold molar excess of dye over the amount of dendrimer present (for nonaqueous

reactions) or a 6–15-fold molar excess for aqueous reactions. Mix well to dissolve. The

optimal amount of dye added should be determined experimentally by preparing a series

of conjugates using different molar ratios of dye-to-dendrimer.

4. React for 1 hour at room temperature with mixing.

5. Remove unreacted dye and reaction by-products from the modiﬁ ed dendrimer by gel ﬁ l-

tration or dialysis, using a molecular weight cutoff suitable for the size of the dendrimer.

3. Dendrimer-Chelate Derivatives for Imaging Applications

Imaging agents consisting of metal chelating compounds have been used extensively for the

modiﬁ cation of targeting agents for the in vivo detection of speciﬁ c cells, organs, or vascular

systems. The proper chelating group can coordinate securely a radioactive element or a con-

trast-enhancing agent, which can be imaged using radio-imaging techniques or magnetic reso-

nance imaging (MRI), respectively. Many such poly-dentate chelating agents are described in

Chapter 10. These compounds are designed to have bifunctional characteristics in that one end

is reactive for coupling to another molecule and the other end contains the chelating group.

The polyvalent nature of dendrimers has been investigated as vehicles for carrying multi-

ple chelator groups to enhance signals in various imaging applications (Barthand Soloway,

1994; Yoo et al., 1999; Kobayashi et al., 2000, 2001; Sato et al., 2001). In addition, in certain

chelate-dendrimer constructs, excess amines on the dendrimer surface can aid in the cellular

uptake process through charge-mediated endocytosis.

Roberts et al. (1990) conjugated porphyrins to PAMAM dendrimers as carriers to link them

to antibody molecules for in vivo targeting. The porphyrin derivative was a N -(4-nitrobenzyl)-

5-(4-carboxylphenyl)-10,15,20-tris(4-sulfophenyl)porphine containing one carboxylate group,

which could be coupled to the amine groups on the dendrimer using the EDC/NHS method to

form an amide bond. This porphyrin compound was effective at chelating copper ions, espe-

cially

Cu, which has nuclear decay properties ideal for potential use as a chemotherapeutic

agent.

One of the more common chelating groups used for imaging applications is DTPA. This

compound is available in amine-reactive form as the anhydride or as a isothiocyanatobenzyl

derivative [e.g., 2-( p-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaaceticacid chelate

(1B4M; MW 555) (Kobayashi et al., 1999). There are various isothiocyanatobenzyl-DTPA

derivatives available that avoid conjugation of one of the chelator ’s carboxylate groups, which

would be necessary using the anhydride form (Mirzadeh et al., 1990; Brechbiel and Gansow,

1991; Michel et al., 2002; Behr et al., 1999; Brouwers et al., 2003; Macrocyclics, Inc.). Any

of these derivatives can be used to modify an amine-dendrimer to contain multiple chelat-

ing groups on its surface ( Figure 7.23 ). Mamede et al. (2003) used the SCN-Bzl-DTPA com-

pound 1B4M to modify a biotinylated, G-4 PAMAM dendrimer to contain approximately 52

chelating group substitutions. The conjugate then was loaded with

111

In and complexed with

avidin for investigation of its use for radiotherapy applications.

3. Dendrimer-Chelate Derivatives for Imaging Applications 383

384 7. Dendrimers and Dendrons

Kobayashi et al. (2003) similarly prepared a DTPA modiﬁ ed PAMAM dendrimer using the

same isothiocyanate derivative for use in MRI imaging. A G-8 amine-dendrimer was reacted

with a 1,024-fold excess of chelating compound, resulting in a heavily loaded complex. In this

case, the resultant conjugate was charged with

153

Gd to create a high-intensity contrast agent

for in vivo imaging use in animals.

The following protocol for the modiﬁ cation of an amine-dendrimer with an SCN-Bzl-DTPA

chelator is based on the literature references previously cited. Dendrimers of other generations

will work well in this procedure provided that the molar ratios of reactants are adjusted for the

size of dendrimer being used and the substitution level desired.

Protocol

1. Dissolve 10 mg of a G-4 amine-dendrimer in 1 ml of 0.1 M sodium carbonate buffer,

pH 9.0.

2. Add a quantity of the SCN-Bzl-DTPA bifunctional chelating agent to obtain the desired

molar excess of label over the amount of dendrimer present. The optimal ratio may be

determined experimentally by preparing a series of dendrimer-chelate conjugates using dif-

ferent molar ratios and choosing the one that works the best in the intended application.

Figure 7.23 Chelating groups such as the isothiocyanate derivative of DTPA can be used to create multiva-

lent chelating complexes with amine-dendrimers. Such complexes are able to coordinate multiple metal ions for

detection, imaging, or radioimmunotherapy purposes.

3. React for at least 1 hour at room temperature with mixing. Some reactions have been

done for up to 24 hours at elevated temperatures (e.g., 40°C).

4. Purify the DTPA-dendrimer using dialysis, size exclusion chromatography, or spin-tube

concentrators having a molecular weight cutoff of 5,000 Daltons.

Other chelate-dendrimer constructs may be prepared using alternative bifunctional chelating

agents according to Chapter 10.

4. Dendrimer Derivatives as Surface Modiﬁ cation Agents

The extremely branched nature of dendrimers provides multiple functionalities for creat-

ing high-capacity surfaces for coupling afﬁ nity molecules. The presence of numerous pendent

groups on dendrimers can be used to advantage both in facilitating attachment to the surface

and in providing a biocompatible coating for protein coupling. For instance, dendrimer modi-

ﬁ cation of gold surfaces, both planer and nanoparticle, have the potential to create a matrix

for high-density ligand coupling and low nonspeciﬁ city in assays. However, the dendrimer type

and the characteristics of the pendent functionalities come into play in forming the optimal sur-

face environment. PAMAM or amine-dendrimers of other types could result in relatively high

nonspeciﬁ c binding, particularly if unmasked terminal amines provide sites for charge interac-

tions with other proteins (Yoon et al ., 2002; Hong et al ., 2003a, 2004;).

Amine-dendrimers also can be used to pattern biological molecules on gold surfaces (Hong

et al., 2003b). In this application, PAMAM dendrimers were printed onto a SAM surface pre-

pared using 11-mercaptoundecanoic acid to create sites for linking biomolecules. The carbox-

ylate groups on the termini of the decanoic acid groups were activated with carbodiimide

(EDC) and petaﬂ uorophenol to create reactive PFP esters (Chapter 2, Section 1.13). A PAMAM

dendrimer solution in methanol (0.7 nM) was printed onto this activated SAM surface by con-

tact printing. Reaction between the PFP esters on the SAM molecules and the amines on the

PAMAM molecules covalently coupled the dendrimers to the surface via amide bond linkages.

The dendrimers then were biotinylated with sulfo-NHS-biotin, washed, and allowed to interact

with ﬂ uorescently labeled avidin, which was bound to the surface through interaction with the

biotin groups. The ﬂ uorescent label was used to detect the bound protein and assess the efﬁ -

ciency of dendrimer surface functionalization.

Alternatively, PAMAM dendrimers can be linked to glass surfaces containing aldehyde function-

alities through Schiff base formation and reductive amination using sodium cyanoborohydride.

Hong et al. (2003b) patterned dendrimers on aldehyde slides and then blocked excess aldehyde

groups using a 2 hour incubation with 1 M 2-(2-aminoethoxy)ethanol. The result was cova-

lently linked dendrimers on the slides containing an abundance of dendritic amines for further

conjugation.

Hong et al. (2004) also found that modiﬁ cation of PAMAM dendrimers with a short PEG

linker arm could act to reduce nonspeciﬁ city caused by the amines on the dendrimer-modiﬁ ed

surface. An azido-PEG

-amine spacer was activated with nitrophenyl carbamate to yield an

activated intermediate that could be used to modify the amines on the dendrimer ( Figure 7.24 ).

Reaction at high molar ratio resulted in about 61 PEG-azido spacers on the dendrimer. Reduction

of the azido group to an amine using triphenylphosphine in THF provided the dendrimer-PEG-

amine derivative for surface modiﬁ cation. The added presence of the PEG spacer arm reduced

4. Dendrimer Derivatives as Surface Modiﬁ cation Agents 385

386 7. Dendrimers and Dendrons

nonspeciﬁ c binding to the dendrimer-surface despite the fact that both the PAMAM and PEG-

modiﬁ ed dendrimer both had terminal amino groups present. Another bioconjugation route to

using this azido-PEG-dendrimer is to make an alkyne-containing protein or ligand and couple it

to the modiﬁ ed dendrimer using click chemistry. The reaction of an azide with an alkyne in the

presence of Cu(I) results in a cycloaddition reaction, which forms a triazole ring. See Chapter 17,

Section 4 for more details on the click reaction.

Figure 7.24 Azido-PEG-amine linkers can be coupled to amine-dendrimers by formation of an intermediate

reactive nitrophenyl carbamate group. This compound then can be reacted with the amines on a dendrimer

to form an azido-dendrimer derivative that can be used in chemoselective ligation strategies. The azide groups

can be reacted with alkyne derivatives in a click chemistry reaction to couple ligands through a triazole link-

age. Alternatively, the azido groups can undergo a Staudinger reaction with a phosphine compound to give the

amino-PEG-dendrimer derivative. In addition, a Staudinger ligation reaction can be done using a phosphine

derivative that would result in a covalent amide bond linkage (see Chapter 17, Section 4).

In another interesting route to creating dendrimer-functionalized surfaces, Pathak et al .

(2004) activated the silanol groups on glass slides using CDI (Chapter 3, Section 3). The result-

ant reactive imidazole carbamate groups could be coupled to amine-containing dendrimers to

form covalent carbamate linkages directly to the surface. Studying the effect of surface modi-

ﬁ cation with generations 1–5 of a series of poly(propyleneimine) dendrimers resulted in the

conclusion that dendrimers of G-3 size and above created surfaces of very high-binding activity

for coupling afﬁ nity ligands, with a G-4 dendrimer giving the greatest binding potential. This

method may provide an excellent modiﬁ cation strategy for immobilizing afﬁ nity ligands onto

planer and spherical silica surfaces. Once the amine-dendrimer surface was created, coupling

proteins to it was done using the heterobifunctional crosslinker, sulfo-GMBS, which formed a

thiol-reactive surface.

Benters et al. (2002) used three different coupling strategies to coat glass slides with amine-

dendrimers for subsequent immobilization of oligonucleotides for preparing DNA microarrays.

Cleaned slides were initially modiﬁ ed with 3-aminopropyltriethoxysilane (APTS) or glycidy-

loxypropyltrimethoxysilane (GOPTS) to create functional groups for further modiﬁ cation.

The APTS modiﬁ ed slides subsequently were reacted either with glutaric anhydride to form

terminal carboxylate groups or with 1,4-phenylenediisothiocyanate (PDITC) to create reactive

isothiocyanate groups on the surface. The carboxylate-containing slides were activated using

the carbodiimide DCC along with NHS in DMF to form reactive NHS ester groups for cou-

pling the amine-dendrimers.

G-4 PAMAM dendrimers were attached to each of the three activated slides using a

100 mg/ml solution in methanol. The result formed covalently attached dendrimer layers on the

slide surfaces, which then were again activated for the coupling of oligonucleotides. The den-

drimer activation process was similar to that of the APTS surface to form reactive NHS esters.

The dendrimers ﬁ rst were modiﬁ ed with glutaric anhydride and then activated using DCC and

NHS in DMF. This process formed amine reactive NHS esters on the dendrimers, which then

could be used to immobilize directly 5  -amine modiﬁ ed oligos ( Figure 7.25 ).

Dendrimer coated slides prepared using the methods of Benters et al. (2002) were found to

provide signiﬁ cantly greater signal in ﬂ uorescent DNA hybridization assays than conventional

slides prepared by coupling DNA directly to silane or poly-

L-lysine modiﬁ ed surfaces. Clearly,

dendrimers provide a three-dimensional surface with greater functionality and biocompatibility

for producing high-activity arrays.

Le Berre et al. (2003) also investigated the use of dendrimers to provide enhanced cou-

pling capability and increased signal for DNA microarrays. In this application, the dendrim-

ers consisted of a core of hexachlorocyclo-triphosphazene (N

), which terminated in

aryl aldehyde groups on the surface (Launay et al., 1994, 1997; Slomkowski et al., 1999).

A G-4 aldehyde dendrimer containing 96 pendent aldehydes was coupled to a slide surface

after modiﬁ cation with APTS to contain amines. Multiple Schiff base interactions with the

amine groups on the derivatized slide effectively immobilized the dendrimers. Next, the spot-

ting of amine-modiﬁ ed DNA to the aldehyde-dendrimer surface with overnight incubation

followed by reduction with sodium cyanoborohydride resulted in covalently linking both the

dendrimers to the surface and the oligos to the aldehyde groups through secondary amine

bonds. A comparison of the dendrimer-coupled DNA slides to 11 commercially available acti-

vated glass slides indicated that the dendrimer slide provided among the highest ﬂ uorescence

intensities in hybridization assays and 10- to 100-fold higher detection sensitivity than conven-

tional slides.

4. Dendrimer Derivatives as Surface Modiﬁ cation Agents 387

388 7. Dendrimers and Dendrons

Figure 7.25 The multivalent nature of dendrimers can be used to add increased functionality to surfaces.

Aminopropyl silane surfaces can be activated with either PDITC or through use of a cyclic anhydride plus DCC/

NHS to give amine-reactive surfaces. These reactive surfaces can be used to couple amine-dendrimers to provide

a high density of amine groups on the surface for further bioconjugation.

5. Dendrimer Fluorescent Quantum Dots

Dendrimers can be used to effectively coat and passivate ﬂ uorescent quantum dots to make

biocompatible surfaces for coupling proteins or other biomolecules. In addition, the ability of

dendrimers to contain guest molecules within their three-dimensional structure also has led to

the creation of dendrimer-metal nanoclusters having ﬂ uorescent properties. In both applica-

tions, dendrimers are used to envelop metal or semiconductor nanoparticles that possess ﬂ uo-

rescent properties useful for biological detection.

Huang and Tomalia (2005) used PAMAM dendrimers to coat gold nanoparticles or CdSe/

CdS core/shell ﬂ uorescent quantum dots by preparing a disulﬁ de-core dendrimer (using

cystamine). The dendrimer then was succinylated to create terminal carboxylate groups, its

core reduced with DTT, and the thiol-dendron used to modify quantum dots by dative bonding

to the particle surface. The result was an organized polymeric coating on the gold particles or

quantum dots that terminated in multiple carboxylate groups for conjugation of biomolecules

(Figure 7.26 ). The negative charges on the dendron terminals provided charge repulsion to

maintain colloidal stability of the small nanoparticles in solution, while the polyvalent nature

of the dendrons made available an abundance of coupling sites for conjugation.

Testing of G-1, G-2, and G-3 dendrimers in this application provided insight into the density

of surface modiﬁ cation needed to passivate completely the particles and prevent aggregation.

The G-1 dendron was insufﬁ cient in this regard, but both the G-2 and G-3 dendron were big

enough to create a surface barrier, which resulted in excellent colloidal stability of the particles

in solution.

Zheng and Dickson (2002) created a new type of ﬂ uorescent dendrimer construct by seques-

tering small nanoclusters of silver within hydroxyl-terminated G-2 or G-4 PAMAM dendrim-

ers (16 hydroxyls on G-2 and 64 hydroxyls on G-4). The internal structure of a dendrimer is

known to interact with charged silver ions in solution (Varnavski et al., 2001). Without adding

a reducing agent, such as sodium borohydride, which is typically used to form silver nano-

particles, it was discovered that the silver ion dendrimer complex could be photoactivated to

cause silver reduction to elemental silver within the internal structure of the dendrimers. The

resulting very small dendrimer/silver nanoclusters displayed strong ﬂ uorescence with absorp-

tion bands at 345 nm and 430 nm and a broad emission curve extending from slightly less than

500 nm to nearly 700 nm. By contrast, if borohydride reduction was used, larger silver nano-

particles were formed (3–7 nm) within the dendrimers, which displayed no ﬂ uorescence charac-

teristics, but only strong plasmon absorption at 398 nm. Thus, small ﬂ uorescent nanoclusters

containing only up to about 8 atoms of silver were formed by photoactivation and stably

sequestered within the G-2 or G-4 interior. These properties were in agreement with studies on

silver clusters of 2–8 atoms, which have been shown to have size-dependent ﬂ uorescence char-

acteristics (Tani and Murofushi, 1994).

The broad emission band displayed by these silver/dendrimer constructs actually was found

to consist of 5 overlapping ﬂ uorescent peaks caused by individual silver/dendrimer complexes.

Each of these complexes evidently contained a uniquely sized silver nanocluster, which resulted

in an individual emission peak. Therefore, all the silver/dendrimer complexes together in solution

presented a combined average of these 5 discrete emission peaks, and thus displayed the broad

emission band covering nearly 200 nm in width across the spectrum.

Lesniak et al. (2005) also describe the preparation and use of similar dendrimer/silver

nanoclusters using G-5 PAMAM dendrimers terminated with either amino, hydroxyl, or

5. Dendrimer Fluorescent Quantum Dots 389