Hermanson G. Bioconjugate Techniques, Second Edition

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350 7. Dendrimers and Dendrons

Figure 7.4 The chemical structure of a G-4 PAMAM dendrimer.

is the ease at which reactants are added and the growing dendron is puriﬁ ed from reaction

byproducts. This method also can be used to add terminal peptides onto the dendron for afﬁ n-

ity targeting purposes. Monaghan et al. (2001) used this approach to make peptide–dendrimer

conjugates for the investigation of integrin binding.

One of the most important advances in synthesizing dendrimers is through the use of the

cycloaddition reaction between azides and alkynes, which has become known as “click

chemistry ” (Chapter 17, Section 4). The copper I-catalyzed conjugation reaction forms cyclic

1,2,3-triazoles in very high yield. Using the appropriate monomers, the click chemistry-mediated

assemblage of dendrimers has provided dramatic improvement to both the yield and the ease

of synthesis and thus has dramatically decreased the cost of making bulk amounts of dendritic

molecules (Rouhi, 2004; Wu et al., 2004, 2005; Joralemon et al., 2005; Lee and Kim, 2005)

(Figure 7.7 ). Even unsymmetrical dendrimers can be prepared using a convergent synthe-

sis approach between propargyl-functionalized PAMAM dendrons and azido-functionalized

PAMAM dendrons (Lee et al ., 2007).

Regardless of how they are made, the higher the dendrimer generation the greater the density

of its branching becomes. Dendrimers of small size have an internally open conﬁ guration that

freely permits the ﬂ ow of small molecules within their inner structure. As dendrimers increase in

diameter from G-0 through G-7, their appearance and size becomes more and more similar to

Figure 7.5 The G-3 and G-4 PAMAM structures shown in Figures 7.3 and 7.4 are illustrated here as space-ﬁ lling

molecular models. The G-4 level begins to show similarity to globular proteins in its three-dimensional structure.

Figure 7.6 The divergent and convergent synthesis methods of dendrimer formation.

1. Dendrimer Construction 351

352 7. Dendrimers and Dendrons

that of globular proteins of corresponding mass. For instance, a G-0 PAMAM dendrimer has a

molecular weight of 517 and a diameter of about 1.5 nm, whereas a G-7 dendrimer has a mass

of 116,493 and a diameter of 8.1 nm, which are very similar in mass and diameter to peptides

and proteins of comparable sizes (Eichman et al., 2001). Unlike proteins, however, the surface

of dendrimers becomes increasingly dense as their size increases. This is due to the doubling of

branches and pendent groups on the outer surface for each generation increase in size. As the

dendrimer generation and size increases, the molecules become more symmetrical and spherical

in shape due to the dense outer branch packing. At a certain point, surface crowding becomes

so great that no further access to the internal structure is possible and the dendrimer becomes a

rigid ball. As dendrimer size increases, it also becomes increasingly difﬁ cult to add another layer

of monomers to the surface due to steric hindrance.

In general, dendrimers of size G-0 through G-3 have open, asymmetric, and ﬂ exible struc-

tures with effectively no protected internal areas, due to a large freedom of motion in their

branches, and they can readily accommodate additional covalent attachments to their surfaces.

See Figures 7.3–7.5 for illustrations of the two-dimension and three-dimension structure of a

Figure 7.7 The synthesis of dendrimer molecules using click chemistry proceeds with high yield. Each step results

in the cycloaddition reaction between azide-containing molecules and alkyne molecules to form triazole linkages.

G-3 and G-4 dendrimer, in which G-3 dendrimers can be seen to have a great deal of internal

space. However, G-4 through G-6 dendrimers display a more globular structure and contain

internal void areas, which can hold guest molecules, such as delivery agents or drugs. Above

this size, G-7 and greater, dendrimers are more like solid spheres or particles with inaccessible

interiors and highly dense surfaces, and they appear upon imaging much like polymer nanopar-

ticles of similar size (Chapter 14).

Unlike linear or ordinary branched polymers, dendrimers display intrinsically low viscosity,

even at high mass. As standard polymeric molecules increase in mass and size, their viscos-

ity normally increases continually. With dendrimers, viscosity increases only up to about the

fourth generation, after which it actually begins to decline (Mourey, 1992; Fréchet, 1994). In

addition, with control over the type of pendent groups that adorn the surface, dendrimers can

maintain high solubility regardless of size.

Dendrimer molecules of the mid-size range have been found to be excellent carriers of guest

molecules for solubilization and drug delivery. In addition, certain groups added to the surface

of such dendrimers can aid in the entrapment of molecules within the dendrimer cores. Such a

construct, called a dendrimer box, can be designed to release the guest molecules upon certain

conditions being met, such as pH-facilitated hydrolysis or a photoreaction of the surface capping

groups. In this case, the capping groups are reversible and thus the dendrimer can be induced to

release its cargo by cleavage (Jansen et al., 1994; Jansen and Meijer, 1995). Miklis et al. (1997)

used molecular dynamics to investigate the encapsulation of rose bengal molecules in a dendrimer

box, which was formed by the coupling of tBOC-L-Phe cap molecules to the 64 terminal primary

amines of a G-5 poly(propyleneimine) dendrimer. It was discovered that without the capping

groups, the rose bengal molecules were in equilibrium between the solvent, surface, and inte-

rior of the dendrimer. However, forming a dendrimer box with the tBOC-L-Phe caps stably was

found to keep the guest molecules within the dendrimer interior without leakage.

In some cases, a dendrimer box can be designed to provide a slow release of a drug by cap-

ping the branched structure with hydrophilic PEG groups. The internal structure is designed

to effectively dissolve the organic drug and sequester it, while the PEG capping groups provide

extreme dendrimer solubility and inhibit the movement of the drug out of the interior den-

dritic space. Liu et al. (2000) used this design to entrap indomethacin within the hydropho-

bic branches of a dendrimer built from phenyl-group-containing monomers and provided an

mPEG

cap to allow a time-release effect for the drug in vivo .

In addition to the PAMAM variety, many different chemical constituents have been used

as monomer units to build a dendritic molecule. Some have used trifunctional aromatic units,

rigid monomers to keep the branches from bending, polyethers, polyhydroxyls, heterocyclic

compounds, etc. In some dendrimer types, the interior structure is hydrophobic and can be

used potentially to carry small organic molecules, while the surface groups are made to be

hydrophilic to promote overall solubility. The potential variety of dendrimer construction is

limited only by the imagination of organic or inorganic building blocks, which can be con-

ceived and linked together.

2. Conjugation to Dendrimers

Many of the applications of dendrimers involve the covalent coupling of other molecules to

the dendrimer surface or to points within the branched structure. These attached molecules

2. Conjugation to Dendrimers 353

354 7. Dendrimers and Dendrons

can function as detection agents, afﬁ nity ligands, targeting components, radio-ligands, imaging

agents, or pharmaceutically active compounds. The methods used for dendrimer conjugation are

similar to the procedures used with other macromolecules and particles. The essential elements

in designing a dendrimer conjugate are the functional groups that are present on the dendrimer

and the functional groups on the molecule to be coupled.

Dendrimers are commercially available containing a variety of dendron structures and pendent

groups on their surface (Dendritic Nanotechnologies; Dendritech). Some of the functionalities

available allow for bioconjugation reactions to be done, but others are designed to create certain

solubility properties and cannot be used directly for coupling other ligands. Figure 7.8 shows

some of the available surface groups, which for traditional PAMAM-based dendrimers, including

amine, carboxylate, hydroxyl, methyl ester, mPEG, and a hydrophobic C

chain. Selections of

different diamine cores also are available with these surface functionalities, including the chain

lengths C

, C

, and a disulﬁ de cleavable cystamine core. Most dendrimers of the

PAMAM type are commercially available in sizes up to G-6, but can be custom ordered in higher

generations, which is more difﬁ cult to synthesize and more expensive.

The newer Priostar dendrimers from Dendritic Nanotechnology, which are created using a new

manufacturing process, provide greater ﬂ exibility in dendrimer design and much better stability

in the ﬁ nal product. These dendrimers are stable at room temperature, unlike PAMAM dendrim-

ers, and can withstand extremes in pH or temperature without hydrolyzing or decomposing. In

addition, the dendritic structures can be formed to have internal functional groups along their

branches for conjugation, including secondary amines and hydroxyls. Therefore, ﬂ uorescent mol-

ecules or other organic molecules can be attached at internal locations, leaving the surface groups

available for additional conjugation sites.

Figure 7.8 The pendent groups available on the surface of dendrimer molecules are highly varied. Some groups

provide functional or reactive groups for bioconjugation, while other groups create unique solubility character-

istics for the dendrimer.

Priostar dendrimers are available with amine, hydroxyl, carboxylate, or epoxy functionali-

ties on their surface for bioconjugation reactions. Also, due to the monomers used for synthesis,

each generation of Priostar dendrimer contains more surface functionalities than the corre-

sponding PAMAM dendrimer. For example, PAMAM dendrimers with ethylenediamine cores

have 4, 8, 16, and 32 pendent groups present for generations G-0, G-1, G-2, and G-3, respec-

tively. By contrast, Priostar dendrimers have 4, 12, 30, and 100 pendent functionalities on their

surface for G-0, G-1, G-2, and G-3, respectively. The result is much greater surface functionality

at much lower generation number and size; therefore, surface functionality can be maximized

without growing the dendrimer so large that it no longer can accommodate guest molecules

within its structure.

Other than the epoxy groups available on one Priostar dendrimer type and a methyl ester

available on a PAMAM dendrimer, the commercial suppliers generally don ’t offer a selection

of spontaneously reactive dendrimers for bioconjugation purposes. For this reason, most of the

applications published for coupling biomolecules to dendrimers have used various modiﬁ cation or

activation steps to create the appropriate reactive groups for conjugation (e.g., Leon et al., 1996).

Due to the multivalent nature of dendrimers, the ﬁ rst consideration for conjugating molecules

to them is to decide how many modiﬁ cations should occur on its surface. For some molecules,

maximizing the ligand:dendrimer modiﬁ cation ratio may be desirable. An example is in creat-

ing sugar-dendrimer derivatives to interact with carbohydrate binding proteins on cell surfaces.

Since many sugar-lectin associations are of low afﬁ nity, creating a dendrimer conjugate having

numerous sugar molecules attached to its surface is advantageous to form multiple interaction

points. This approach results in the sugar-dendrimer complex binding to the cell surface with

higher avidity than a single sugar derivative would be able to achieve.

However, for other bioconjugation applications, the optimal number of molecules attached

to a dendrimer may have to be determined by experimentation. Too many modiﬁ cations may

result in decreased activity of the ﬁ nal conjugate as compared to a similar conjugate made

without the use of a dendrimer. For instance, numerous ﬂ uorescent molecules can be attached

to a G-3 amine-containing dendrimer to provide an enhanced ﬂ uorescent conjugate, which is

brighter than a single ﬂ uorescent molecule for labeling proteins. However, if too many ﬂ uores-

cent molecules are attached, ﬂ uorescence quenching may take place and obviate any beneﬁ t the

use of a multivalent dendrimer may provide toward signal enhancement. Therefore, for any

given dendrimer conjugate preparation, some thought must be given to optimizing the number

of modiﬁ cations (or the ligand:dendrimer ratio) to obtain the best possible conjugate activity in

the intended application.

In addition, if a dendrimer is to be labeled with one molecule and then ultimately attached to

another molecule to form the complete complex, then the second conjugation step also must be

planned from the beginning. Are some surface groups going to be used for coupling the ﬁ rst mol-

ecule and then the remaining groups used for coupling to the second one or will a disulﬁ de den-

dritic core be used for coupling to the second molecule after cleavage of the modiﬁ ed dendrimer?

Such decisions will affect the conjugation strategies used with dendrimers and often govern the

usefulness of the resultant conjugate.

The following methods of linking molecules to dendrimers present options for deciding the

best reactions to exploit to create a conjugate. In all cases, the ratio of reactants and the nature

of the ﬁ nal conjugate should be carefully considered. In the end, running a series of trial conju-

gations to optimize the ﬁ nal conjugate will result in a method that is appropriate to the intended

application and consistent in performance from batch to batch.

2. Conjugation to Dendrimers 355

356 7. Dendrimers and Dendrons

2.1. Coupling to Amine-Dendrimers

With PAMAM (or Priostar) amine-containing dendrimers, the surface is adorned with many pri-

mary amine groups, the number of which is dependent on the generational size of the dendritic

structure. For instance, a G-3 PAMAM dendrimer has 32 amine groups on the outer ends of

its branches, a G-4 has 64 amines, and a G-5 has 128 amines. As long as the dendrimer size is

within the mid-range where crowding of surface components is not severe, then the reactivity of

these pendent amines is much greater than the amines on a globular protein. This is due to the

fact that with protein molecules solvent accessibility of amines is dependent on the relative expo-

sure of the side chain lysine amines to the environment. For many globular proteins, a percentage

of amines are highly accessible and react quickly, but for another population of amines, they are

somewhat buried below the surface polypeptide structure and don ’t react as readily (Chapter 1,

Section 1.1).

With dendrimers, the pendent amine groups are all approximately equally accessible and very

reactive for bioconjugation or modiﬁ cation reactions (Fréchet, 1994). The only potential limi-

tation for coupling to a dendrimer surface would be steric crowding, which would restrict the

number of large molecules from coupling to every functional group on the dendrimer. For the

coupling of small molecules such as sugars or biotin derivatives, the total number of possible

modiﬁ cations will approach the total number of amine groups on the surface, providing the

dendrimer is not so large that there is already severe surface crowding of the branches. For glob-

ular protein coupling, however, the number of potential coupling points may be much less than

the total number of functional groups available, because each protein molecule will overlap and

block some functional groups as it is coupled to the dendrimer surface.

Modiﬁ cation of Amine-Dendrimers with Sulfo-NHS-LC-SPDP

The amines of a PAMAM dendrimer may be reacted with any heterobifunctional crosslinker,

which contains an amine-reactive group on one side and another end having different reactivity.

The result will form a reactive dendrimer that is useful for coupling proteins and other ligands,

which have functional groups able to form a covalent linkage with the second reactive group on

the crosslinker. Sulfo-NHS-LC-SPDP contains a sulfo-NHS ester that will couple with the pen-

dent amine groups on the dendrimer and a pyridyl disulﬁ de at its other end to couple with thiol-

containing ligands (Chapter 5, Section 1.1). Reaction of this reagent with an amine-containing

dendrimer yields a reactive intermediate containing a long aliphatic spacer arm and terminating

in reactive pyridyl disulﬁ de groups ( Figure 7.9 ). A disulﬁ de linkage can be formed by reaction

of the pyridyl disulﬁ de end with a thiol-containing ligand, which is reversible by reduction with

DTT or TCEP.

Another strategy using this crosslinker is purposely to reduce the pyridyl disulﬁ de end after

dendrimer modiﬁ cation to create a free thiol on the dendrimer. Singh (1998) used this process to

thiolate dendrimers for subsequent coupling with protein molecules containing a group reactive

to thiols, such as sulfo-SIAB (or sulfo-SMCC) activated antibodies. After the initial activation,

SPDP-PAMAM dendrimers may be stored indeﬁ nitely either lyophilized or frozen, because the

thiol-reactive group is stable to hydrolysis or degradation. The modiﬁ ed dendrimers also may

be treated with DTT to release pyridine-2-thione and create free sulfhydryls on the dendrimer

surface. A thiolated dendrimer should be kept in the presence of at least 10 mM EDTA and used

immediately for bioconjugation to prevent oxidation of the thiols, which could form oligomers

over time if disulﬁ des are formed between dendrimer molecules.

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM den-

drimers may be done along with the use of a secondary conjugation reaction to couple a detectable

label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation

method along with amine-reactive ﬂ uorescent labels (FITC or 6-carboxytetramethylrhodamine

succinimidyl ester) to create an antibody conjugate, which also was detectable by ﬂ uorescent

imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate den-

drimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were

labeled with FITC at a level of 5 ﬂ uorescent molecules per G-5 PAMAM molecule.

Reaction of a G-3 PAMAM dendrimer with only a 4- to 10-fold molar excess of sulfo-NHS-

LC-SPDP was found to yield a partially modiﬁ ed surface containing only a few pyridyl disulﬁ de

groups and leaving the rest of the amines available for further conjugation (Singh, 1998). To

create 7–10 thiols per dendrimer, the reaction can be carried out using a 20-fold excess of sulfo-

NHS-LC-SPDP. The following protocol should be optimized to incorporate the level of thiolation

best suitable for the application of the ﬁ nal conjugate.

Figure 7.9 Amine-containing dendrimers can be activated with SPDP to create thiol-reactive derivatives.

Alternatively, the pyridyl dithiol group may be reduced to create free thiols on the dendrimer surface for subse-

quent conjugation.

2. Conjugation to Dendrimers 357

358 7. Dendrimers and Dendrons

Protocol

1. Dissolve the amine-containing PAMAM dendrimer in methanol or a buffered aqueous

medium at a pH of 7–9 (e.g., 50 mM sodium phosphate, pH 7.5) and at a concentration of

at least 10 mg/ml. Note that Singh (1998) used a concentration of 110 mg/ml in methanol,

but other dendrimer concentrations should work equally well. For nonaqueous reactions,

the addition of a proton acceptor may aid in driving the reaction to maximal yields (i.e.,

triethylamine or dimethylaminopyridine).

2. Dissolve sulfo-NHS-LC-SPDP at a concentration of 20 mM (5.2 mg/ml) in DMSO or

water. If water is used, the solution must be used immediately to prevent hydrolysis of

the sulfo-NHS ester.

3. With mixing, add an aliquot of the crosslinker to the dendrimer solution to provide the

desired molar excess of reagent. For many applications, less than 10 pyridyl disulﬁ de groups

are needed per dendrimer molecule; therefore, molar ratios in the range of 5–20  excess

of crosslinker over the amount of dendrimer present typically are used.

4. React with mixing for at least 30 minutes at room temperature. Longer reactions may be

used without problems.

5. Purify the derivatized dendrimer using gel ﬁ ltration (size exclusion chromatography) on

a desalting column or through use of ultraﬁ ltration spin-tubes (for G-4 and above). For

smaller dendrimers, the derivatives may be puriﬁ ed by repeated precipitation from a meth-

anolic solution by addition of ethyl acetate, dioxane, or benzene. The SPDP-dendrimer

may be dried by lyophilization (if in water or buffer) or by solvent evaporation in vacuo

(if the precipitation method was used).

The SPDP-modiﬁ ed dendrimer of step 5 may be further derivatized with an amine-reactive

ﬂ uorescent molecule for use in ﬂ uorescence detection applications. The ﬂ uorophore mod-

iﬁ cation may be done prior to coupling an antibody or other molecules to the dendrimer

for targeting purposes. The following steps illustrate the procedure used to obtain the ﬂ u-

orescently labeled dendrimer and then to use the SPDP-modiﬁ ed dendrimer to form thiols

on the surface through reduction or to link to another molecule containing a thiol. Step 6

is optional for adding ﬂ uorescence detection capabilities, and the protocol of either step

7 or 8 may be used to conjugate the SPDP-reactive group to another protein.

6. Dissolve the puriﬁ ed SPDP-modiﬁ ed dendrimer of step 5 in 50 mM sodium phosphate,

0.15 M NaCl, pH 7.5, or in DMSO at a concentration of at least 10 mg/ml. Add a

10–20  molar excess of an amine-reactive ﬂ uorescent molecule (i.e., NHS-rhodamine or

a hydrophilic NHS-Cy5 derivative see section on ﬂ uorescent probes). React with mixing

for 1 hour at room temperature. Purify the ﬂ uorescently labeled SPDP-modiﬁ ed den-

drimer using gel ﬁ ltration or ultraﬁ ltration. Follow the method of either step 7 or 8 to

conjugate the dendrimer to another protein or molecule.

7. To create a thiolated dendrimer, the pyridyl disulﬁ de groups may be reduced by addi-

tion of 50 mM DTT or TCEP in 50 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA,

pH 7.5, which will release pyridine-2-thione groups and leave sulfhydryls on the dendrimer

surface. Remove excess reducing agent by gel ﬁ ltration on a column of Sephadex G-25 or the

equivalent. The thiolated dendrimer should be used immediately to conjugate to a protein or

another molecule containing a thiol-reactive group, such as a maleimide or iodoacetyl group.

8. Alternatively, a thiol-containing protein may be directly conjugated to the SPDP-

modiﬁ ed dendrimer to create a disulﬁ de linkage. Add a sulfhydryl-containing protein

or other molecule to the puriﬁ ed SPDP-modiﬁ ed dendrimer in 50 mM sodium phos-

phate, 0.15 M NaCl, 10 mM EDTA, pH 7.5. The amount of the thiol-containing protein

to be added to the dendrimer should be determined experimentally to be optimal for

the intended application of the conjugate. Typically, an excess of thiol-protein over the

number of SPDP groups per dendrimer is added to assure that the number of proteins

coupled will efﬁ ciently utilize the number of modiﬁ cations initially made using sulfo-

NHS-LC-SPDP. Thus, controlling the molar ratio used in the initial crosslinker reaction

will control the molar ratio of protein-to-dendrimer in the ﬁ nal conjugate.

NHS-PEG-Maleimide Coupling to Amine-Dendrimers

An alternative method to the use of sulfo-NHS-LC-SPDP for coupling thiol-containing proteins

or antibodies to PAMAM dendrimers is to use a heterobifunctional crosslinker containing an

amine-reactive NHS ester and a thiol-reactive maleimide group (Chapter 5, Section 1). Unlike the

pyridyl disulﬁ de reaction with a sulfhydryl as in the SPDP protocol described previously, a male-

imide 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, which has been

used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for den-

drimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic

PEG spacer arm to promote dendrimer hydrophilicity after modiﬁ cation. Derivatization of an

amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with

water-soluble PEG spacers. This modiﬁ cation helps to mask any potential for nonspeciﬁ c inter-

actions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides

for coupling ligands ( Figure 7.10 ).

If a NHS-PEG-maleimide compound is used for this type of activation and coupling, the

intermediate maleimide-activated dendrimer should be quickly puriﬁ ed of excess crosslinker

and reaction by-products and immediately used to couple ligand. This is due to the fact that

the maleimide hydrolyzes in aqueous solution at a higher rate than an SMCC-type crosslinker,

because of the extreme hydrophilicity of the PEG spacer arm compared to the cyclohexane

spacer of SMCC.

NHS-PEG-maleimide crosslinkers are available in a number of spacer lengths depending on

the size of the polymer chain in the PEG arm. Long chain crosslinkers of this type use PEG

polymers of molecular weight approximately 2,000 to 5,000, containing from about 45 to

over 100 repeating polyethylene oxide units. Shukla et al. (2003) used PEG modiﬁ cations to

increase the half-life of folate receptor-targeted dendrimers for boron neutron capture therapy.

However, a major deﬁ ciency of such full-length PEG polymers is that they are extremely poly-

disperse and consist of a broad range of polymer lengths, which makes reproducibility of their

size difﬁ cult to achieve. Modifying a dendrimer with this type of polymeric crosslinker results

in variability of the length of the PEG chains displayed on its surface, which may be detrimen-

tal for coupling some ligands. In addition, the use of full-length PEG polymers perhaps should

be avoided if the resultant hydrodynamic volume of the dendrimer derivative becomes unac-

ceptably large for some applications.

Alternatively, shorter, discrete crosslinkers containing PEG spacers of known chain length

are now available (Thermo Fisher, Quanta BioDesign; see Chapter 18). These reagents are

designed to contain an exact number of PEG units, typically from between 2 repeating units

2. Conjugation to Dendrimers 359