De Cuyper M., Bulte J.W.M. - Physics and chemistry basis of biotechnology (Vol. 7) (2002)(en)

L. Henry Bryant, Jr. and Jeff W.M. Bulte

Attachment of a tripeptide growth factor (GHK) to a dendrimer periphery resulted in an enhancement on the GHK ability to facilitate the growth of hepatoma cells in solidsupport systems which may be useful in bioartificial liver support systems for liver transplantation [65]. The biodistribution of a G2 dendrimer coupled to indium or yttrium chelates has been evaluated in mice [66]. The biodistribution in mice was first in the kidneys followed by uptake in the liver, spleen and bone at most time points, Conjugation of the antibody did not change the biodistribution patterns. However, it has been noted that a greater understanding of whole body and cellular pharmacokinetics of naked and modified dendrimers is needed for optimal design of organand cell-specific structures [67].

The attachment of quaternary dimethyldodecylamine to the surface of G3 dendrimers yields dimethyl dodecylammonium chloride derivatised dendrimers which inhibited the growth of gram negative E. coli and gram positive S. aureus as detected by a bioluminescence method. The reduced luminescence quantitatively verified their use as dendrimer-based antibacterial agents [68]. The most widely used functionalities for the attachment of drug molecules include amide, carbonate, carbamate and ester bonds which are hydrolytically labile. A PEG-dendrimer allows for the water-soluble conjugation of cholesterol and two amino acids [69].

The synthesis of dendrimers which contain reactive terminal groups by the convergent method for attachment of a biomolecule has been reported [70]. However, there were difficulties in preparing large quantities of high-generation dendrimers with reactive terminal groups, so terminally reactive dendrimers prepared from the divergent method were employed.

Generalised methods for the functionalisation of methyl carboxylate and primary amino groups on the surface of dendrimers for coupling of biomolecules have been reported [71]. The surface groups were converted to either electrophiles such as the iodoacteamido, epoxy or N-hydroxysuccinimidyl groups or the nucleophiles such as the sulfhydryl group. These reactive groups allowed the covalent attachment of alkaline phosphatase. These protein-conjugated dendrimers could then be further conjugated with other antibodies such as the Fab' fragment of anti-CKMB antibody resulting in dendrimer-based multifunctional activities for use as immunoassay reagents. The appropriate selection of activating conditions for a dendrimer allows for the conjugation of two similar or dissimilar proteins. The presence of one protein does not affect the biological activity of the other protein on the same dendrimer [72]. Biotin, a molecule with high binding specificity for streptavidin, has been conjugated to dendrimers in efforts to pretarget radionuclides for cancer therapy [73]. A new biotinylation reagent was coupled to dendrimers, radioactively labelled with streptavidin and the in-vivo biodistribution and pharmacokinetics evaluated in mice, The dendrimers were rapidly cleared from the blood via both renal and hepatobiliary excretion. Kidney concentration increased with increasing dendrimer generation up to G3, being almost 50 % ID/g.

Potentially, localisation can be achieved by binding with an antibody conjugate previously localised on tumour cells. Several specific antibodies have been coupled to PAMAM dendrimers without losing their stability and immunological binding, both in

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solution and when immobilised onto a solid support [74] which holds promise in the production of immunodiagnostic products. The solubility was not altered significantly when antibodies were attached thus allowing improved sensitivity and shorter assay times of solution-phase binding of antibody to analyte compared to commercial solidphase or microparticulate reagents used in many automated immunoassay systems. (The dendrimer-based system showed analytical sensitivity and speed that was equivalent to or greater than commercial-based systems.)

3.2. GLYCOBIOLOGY

Glycobiology is the study of carbohydrate-protein interactions [75] which are prevalent at cellular levels. Most cells are coated with carbohydrates to which lectins or carbohydrate-binding proteins can attach. Interest is due to the ubiquitous and essential roles which sugar moieties of complex carbohydrates play in living systems and high hopes for glycodendrimers in the prevention of pathogenic infections and other related diseases. The attachment allows the adherence of various pathogens which can then take over the host tissues. Carbohydrates have multiple functional groups on each monomer unit which are capable of forming a myriad of structures, each one capable of a different specific biological message. The glycoside cluster effect is the binding of many sugar residues by a lectin which has clustered sugar-binding sites. By using convergent methods glycodendrimers have been synthesised by build-up of a gallic acid trivalent core to which carbohydrate residues were attached [76]. The convergent synthesis allows for the incorporation of other carbohydrates. The attachment of alphathiosialosides to a dendrimer allows the cluster of the active groups which significantly increases the inhibitory capacities compared to the monosialoside. Boronic acid groups, which facilitate the complexation of a variety of saccharides, have been exo-attached to dendrimers. The binding can be monitored by the fluorescence intensity of the host. Various symmetrically tethered sialodendrimers have been synthesised to generate families of multivalent glycoconjugates which may be used as inhibitors of haemaglutination of human erythrocytes by influenza viruses [77]. The influenza virus has two envelope glycoproteins, HA (hemagglutinin) and NA (neuraminidase). HA binds to sialic acid receptors (sialosides; cell surface carbohydrates) on cells and thus the virus gets internalised by receptor-mediated endocytosis. Competition with sialosides to HA using sialic acid coupled to dendrimer (i.e., vaccine) may allow the potential interaction with many HA moieties on a single virus. Multiple sialic acid (SA) residues coupled to dendrimers inhibit influenza-induced agglutination of red blood cells. The linear polyacrylamide backbones are cytotoxic. The ability of the SA coupled dendrimers to inhibit virus haemaglutination (HA) and to block viral infection of mammalian cells in-vitro has been reported [78]. Dendrimer polymers were not cytotoxic to mammalian cells at therapeutic levels. Variation in inhibitory effects were observed with different viruses.

A series of alpha-D-mannopyranose-containing dendrimers were synthesised using the convergent reaction scheme and were investigated with respect to the efficiency of these dendrimers in inhibiting the binding of a lectin to a purified yeast mannan fraction which is found in the serum of patients with Crohn's disease and may cause

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allergic responses from overexposure by bakers and brewers [79]. The preparation of dendroclefts (dendritic cleft-type receptors) up to G2 allow the chiral molecular complexation of mannosaccharides by H-bonding. The H-bonding observed mimics bacterial and protein binding to sugars (80]. The study of the interaction of sugarbinding proteins such as human IgG fraction to lactose has been undertaken by attaching p-aminophenyl-b-D-lactoside onto the dendrimer surface. The attachment of the glycodendrimers to solid-phase supports such as microtiter plate wells provided binding studies to be carried out with the proteins. There was a potential for marked selectivity of certain glycodendrimers towards distinct classes of sugar receptors [81]. The binding properties of PAMAM dendrimers ending with mannopyranoside residues were determined. They form insoluble carbohydrate-lectin complexes and can selectively precipitate a carbohydrate-binding protein from a lectin mixture; thus, they constitute new biochromatography materials [82].

In microtiter wells, dendrimer-based oligosaccharides which are recognised by cholera toxin and the enterotoxin of E. coli inhibited their binding to the wells which were coated with just the oligosaccharides in a competitive inhibition study. The toxins were labelled with 1-125 and the activity measured in the wells. These toxins cause traveller’s diarrhoea and if left untreated will result in death. These in-vitro experiments imply that administering the dendrimer-based oligosaccharide in-vivo may remove these toxins [83-85]. The cluster effect is more evident in small glycodendrimers possibly because of steric interaction as the generation of the glycodendrimer increases. Spacer arms have been placed between the dendrimer and the saccharide which allows for not only flexibility (relief from steric strain) but also the potential to control hydrophobichydrophilic interactions [86]. The mannose-binding protein which is an acute phase protein of immune response may be competitively challenged with mannoside-based dendrimers although no data was presented [87]. The scaffolding of poly-L-lysine allowed the covalent attachment of alpha-D-mannopyranoside glycodendrimers. Inhibition of the binding of yeast mannan to concanvalin A was up to 2000-fold higher than for the mannopyranoside alone [88]. Rather than being attached to the periphery of the dendrimer, the chemoenzymatic syntheses of N- acetyllactosamine has been reported based on the scaffolding of L-lysine. These disaccharides cores are associated with tumours, thyroid disorders and a sexually transmitted disease of the H. ducreyi pathogen and their binding properties studied showing the variability in the binding interactions suggesting additional research [89].

3.3. PEPTIDE DENDRIMERS

Antibodies are potentially of great value in targeted drug therapy because of the inherent specificity of the antibody-antigen interaction. The conventional approach to preparing antibodies is to conjugate a peptide to a known protein or synthetic polymer, in order to mimic the macromolecular structure of the native protein. However, this method generates macromolecular carriers that are ambiguous in structure and composition. To improve on this approach, multiple antigenic peptide (MAP) systems were developed as efficient and chemically defined systems to produce immunogens in the absence of protein carriers. The MAP system consists of an oligomeric branching

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lysine core of seven lysine units and eight arms of peptides that contain antigenic epitopes. The overall structure of the MAP system is a polymer with a high density of surface peptide antigens and a molecular weight greater than 10 K [90]. The almost mutually exclusive desire to attach a large number of drug molecules to an antibody while still retaining maximal antibody immunoreactivity may be overcome by dendrimers which act as intermediate linkers between the drugs (capable of covalent attachment to the dendrimer) and the antibody (which binds to the dendrimer by only one modified site on the antibody).

A MAP dendrimer with a lipophilic surface has been synthesised for possible drug delivery. These lipoamino acids while lipophilic also retain the solvation properties of amino acids and peptides [91]. MAP dendrimers are peptide dendrimers which amplify peptide immunogenicity. Unlike most vaccines, MAPS can be stored or shipped as powders. There is an excellent review on MAPS [92]. They have been used in-vitro as immunogens, vaccines, immunodiagnostics, serodiagnostics, ligands, inhibitors, artificial proteins, epitope mapping, affinity purification, presentation of T-cell epitopes and intracellular delivery. The synthesis of MAP dendrimers has been improved by the use of a N-acetylation capping step which allows the direct (stepwise) synthesis and purification of MAPS. The automated peptide synthesiser was programmed to provide a N-acetylation capping reaction following each amino acid coupling reaction thus serving as a protecting group for further reaction with unwanted amino acids in the crude mixture [93].

3.4. BORON NEUTRON CAPTURE THERAPY

Boron neutron capture therapy is based on the nuclear reaction that occurs when a stable B-10 isotope is irradiated with low-energy neutrons to yield high LET radiation consisting of alpha particles and recoiling Li-7 which are energetic and cytotoxic [94]. To deliver the approximately 109 number of B- 10 atoms needed to effectively eradicate a tumour cell, dendrimers have been conjugated with a polyhedral borane and subsequently attached to a monoclonal antibody. The number of boron atoms range from 250 to 1000 per dendrimer molecule. Unfortunately, in-vivo studies with mice revealed hepatic and splenic uptake over tumour localisation. Instead of attaching the polyhedral borane to the periphery or surface of the dendrimer, the borane cluster has been incorporated into the interior of the dendrimer [95]. Decaborane was reacted with the alkyne functionality located in the interior of the dendrimer to give 0-carboranes. The incorporation into the interior of the dendrimer increased their aqueous solubility.

An interesting application of boronated dendrimers is in electron spectroscopic imaging-based immunocytochemistry [9G]. The dendrimers contain a boron cluster on one side of the dendrimer and an antibody fragment on the other side. These antibody- dendrimer-boranes were used to allow the visual detection of BSA in epithelial cells of ileum which had been internalised by endocytotic vesicles of ileal enterocytes in newborn piglets after administration of BSA. As determined by electron spectroscopic imaging of boron, a G4 starburst dendrimer bearing an epidermal growth factor was bound to the cell membrane and endocytosed in-vitro of the human malignant glioma U-343MG cell line expressing EGF receptors [97].

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3.5.MR IMAGING AGENTS

Dendrimer gadolinium poly-chelates are a new class of MR imaging agents with large proton relaxation enhancements and high molecular relaxivity (relaxation rates per mM metal ion). Wiener at al [98] first introduced that the covalent attachment of gadolinium chelates to dendrimers have the potential to be blood-pool MR T1 imaging agents for use in MR angiography. The synthesis involved the covalent attachment of the acyclic GdDTPA chelate to a G2 and G6 dendrimer utilising a stable thiourea linkage between the chelate and the dendrimer. These dendrimer-based MR imaging agents had a molar relaxivity that was up to six times higher than for clinically used gadolinium chelates because of the high molecular weight of the dendrimer. The authors demonstrated the potential usefulness of these agents for vascular imaging by being able to delineate the vascular system of a rat for at least up to one hour. Not necessarily confined to complexation of Gd for T1-weighted MRI, a dysprosium chelate has been attached to a dendrimer in a similar fashion and opens up the opportunity for T2 MR imaging agents to utilise the macromolecular characteristics that the dendrimer provides [99]. The incorporation of Dy provides an unique T2 relaxation agent which may be important for tissue perfusion studies using MRI.

Proton Larmor Frequency (MHz)

Figure 2. TI MRD relaxiviw profiles for the G5 (upper triangles), G7 (diamonds), G9 (circles), and G10 (blocks) PAMAM dendrimers with attached Gd(-SCN-Bz-DOTA) chelates. For comparison, the MRD profile of Gd(p-NO2-Bz-DOTA) is also shown (lower triangles).

The three main parameters that dictate achieving maximum TI relaxivity for dendrimer-based gadolinium chelates are the amount of time that the water molecule interacts with the gadolinium, how fast the gadolinium tumbles in solution and how fast the paramagnetic electron spin density relaxes back to the ground state [100]. A limitation in achieving the full expected relaxivity for dendrimer-based MR contrast agents was observed and verified by 0-17 NMR studies of the G3, G4, and G5

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dendrimer-based MR imaging agents [101]. It was concluded that modification of the chelating ligand may result in faster water exchange and therefore higher relaxation rates. A plateau in the relaxivity was observed as the generation of the dendrimer increases from G5 to G10, providing evidence of even more severe limitation of achieving full relaxivity as the generation of the dendrimer increases [102]. Since contrast depends on the co-ordinated water molecule interacting with the bulk water, a long residence time at the gadolinium limits the relaxivity and therefore would limit the observed contrast

Figure 3. 3D-TOF MR angiogram ofa rut 50 minutesfollowing injection of 0 05 mmol/kg of Gd(-p-SCN-Bz-DOTA) attached to the surface of a G9 dendrimer The nanomolecular size of the dendrimer construct permits an extended vascular visualisation compared to the clinically used MR imaging agents

The potential of these dendrimer-based gadolinium chelates as blood pool imaging agents has been explored in pigs [103]. The dendrimer-based MR imaging agent was found to have the same blood pool properties as Gd-DTPA-polylysine. No statistical differences in relative signal intensities were observed in various pig organs between the two imaging agents. The MR angiographic properties of these dendrimer-based gadolinium chelates has been explored. In rats they are able to provide strong tumour rim enhancement and detailed angiographic definition of peritumoural vessels [ 104]. In the MRI of canine breast tumours a delayed tumour clearance was observed compared to the clinically used gadopentate dimeglumine [ 105]. The minimum effective dose of

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0.02mmol/kg of dendrimer-based gadolinium chelate was observed for visualisation of the mediastinum, abdomen and lower limbs of rabbits on 3D time of flight magnetic resonance angiography of the body [ 106].

For modified PAMAM dendrimers coupled to Gd chelates, the pharmacokinetic and biodistribution are found to depend on dendrimer size as well as the type of terminal

groups [ 107]. Hepatic localisation decreased and blood half-life increase by the covalent attachment of polyethylene glycol (PEG).

The ability for these dendrimer-based MR imaging agents to be site-specific has been shown [108]. The attachment of folate to dendrimer-based chelates targets these particles to folate binding proteins which exist in the serum as well as on the surface of many cancer cells.

A kinetic theory for describing the dynamic properties of an intermediate-sized MR imaging agent, cascade-Gd-DTPA-24 polymer, (Schering AG, Berlin, Germany, MW<30 kDA) has been developed [109]. The dendrimer-based MR imaging agent was considered intermediate in size relative to Gd-DTPA and albumin-Gd-DTPA-30, The method has clinical applications based on its potential for pixel-by-pixel mapping. The first covalent attachment of tetraaza macrocycles to the terminal phosphorous group of a G1 and G3 P-S containing dendrimer has been achieved [ 110]. The co-ordination of Gd to the grafted macrocycle still needs to be explored, but if possible, it would open up the possibility of having two nuclei, Gd157 and P-3 1, which are detectable by MRI and may be useful in multitiuclear MRS.

3.6. METAL ENCAPSULATION

The complexation of divalent cations such as Cu, Zn, Ni and Au on the periphery of dendrimers has been reported [111, 112]. However, recently cooper nanoclusters have been trapped within dendrimers [113]. Ten weeks later Tomalia published his article on copper-dendrimer nanocomposites [1 14]. The trapping involves zerovalent copper nanoclusters within dendrimers as well as other elemental metals or metal sulphides such as Ag(I), Pt(II), Pd(II), Ru(III) and Ni(II) [115]. These nanocomposites could conceptually incorporate any metal ion which could be chemically reduced once inside the dendrimer shell thus trapping the metal ions as clusters giving rise to intradendrimer metal nanoparticles. Instead of discrete metal atoms within dendrimers, 2-3 nm gold colloids have been stabilised by multiple amine-terminated dendrimers, thus giving rise to inter-dendrimer nanoparticles [116]. The potential applications of these intraor inter-dendrimer nanoparticles could be in catalysis and as use as nanodevices, although one could also envision the delivery of metal ions for therapeutic or diagnostic applications, including MR imaging [117].

3.7. TRANSFECTION AGENTS

Dendrimers have been shown to form physiologically stable complexes with DNA and to mediate transfection of the DNA into a wide variety of cells in culture [118]. It was found that the transfection efficiency was both a function of the particular dendrimer

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generation and the type of cell. By using twenty different types of polyamidoamine dendrimers with a broad range of cell lines it was found that the size, shape and number of surface groups on the dendrimer affects the degree of transfection. The use of heatactivated dendrimers has increased the efficiency of cellular transfection of DNA by more than 50-fold [119]. Heating in a solvolytic solvent such as water for various times degraded the dendrimers. The highest molecular weight component of the degraded products, termed a 'fractured dendrimer, mediated transfection. The degradation supposedly occurs by cleavage of the amide bonds within the dendrimer. The results suggest that other physical properties such as dendrimer flexibility is important in transfection ability. These heat-activated dendrimers are commercially available as SuperFectTM (Qiagen). SuperFect-DNA complexes possess a net positive charge which allows them to bind to negatively charged receptors, such as sialylated glycoproteins on the surface of eukaryotic cells. It has been proposed that once inside the cell, SuperFectTM buffers the lysosome after it has fused with the endosome, leading to pH inhibition of lysosomal nucleases. This ensures stability of SuperFect-DNA complexes and the transport of intact DNA to the nucleus. In general, significantly higher transfection efficiencies were found than for widely used liposomal reagents. SuperFectTM is suitable for both adherent and suspension cells. These include primary cells such as pig endothelial, human smooth muscle, and HUVEC or sensitive cell lines such as HaCaT and HT-1080.

Figure 4. Illustration of dendrimer-DNA complex DNA wraps around the dendrimers which condense the DNA and allows cellular incorporation (transfection)

Dendrimers have also been found to transfect cells with antisense oligonucleotides and plasmid expression vectors coding antisense mRNA (antisense nucleic acids) through energy-dependent endocytosis which allowed higher transfection efficiency compared to DNA alone or lipid-mediated transfection [120]. The cells were assayed for transfection based on the inhibition of luciferase expression. The unmodified oligonucleotides were found to form stable complexes with dendrimers and to function as native oligonucleotides, thus requiring no chemical modification of the oligonucleotide to prevent degradation or intracellular destruction. The transfection of plasmid DNA by dendrimers in-vivo has been demonstrated [121]. The G5 ethylenediamine-core dendrimer enhanced the transfer of plasmid DNA which encodes for viral interleukin- 10, a gene which regulates immune responses, into transplanted

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mouse cardiac isografts in-vivo. Compared to the DNA alone, the transfection of higher amounts of the DNA for longer periods of time by the dendrimer resulted in longer graft survival times. By varying the charge ratio of DNA to dendrimer the survival time was extended to 39 days compared to 14 days for the control. Interestingly, the use of the G9 dendrimer showed a decrease in survival time from 39 days to 27 days suggesting that many factors including the dendrimer generation must also be taken into account when used in-vivo. The possibility of using dendrimers to overcome corneal allograft rejection or to treat disorders of the corneal endothelium appears promising [122]. The immune response to transplanted corneal allografts produces high levels of tumour necrosis factor (TNF). Blocking the action of TNF may prolong some graft survival times. A dendrimer was used to deliver the gene encoding for tumour necrosis factor receptor immunoglobulin (TNFR-lg), a TNF blocker, to rabbit corneal endothelium ex-vivo. The gene was expressed for up to 9 days in the transfected corneas.

Dendrimers allow for the transfection of mammalian cells with various genes. The interactions have been explored by EPR and the idea is to be able to extend the work to the mechanism of gene transfer [ 123]. Using nitroxide-labelled dendrimers in the presence of various oligonucleotides, the G2 dendrimer at a pH of 5.5 showed significant interaction with polynucleotides which decreased with an increase in dendrimer concentration presumably because of the aggregation of the dendrimers. The interaction of the G6 dendrimer with the polynucleotides increased with dendrimer concentration until the interacting sites were saturated. It has been proposed that coulombic interaction between the negatively charged phosphate groups of the nucleic acid and positively charged amino groups of the dendrimer (at physiological pH) allow ionic complex formation and that the current polymeric polyelectrolyte theory does not allow a reliable theoretical calculation of the compositions [124]. It has been noted that an excess of the cationic dendrimer increases transfection efficiency. However, further studies need to be conducted in vivo to assess the cellular localisation and fate of transfected nucleotide and dendrimer [ 125].

3.8. DENDRITIC BOX

Meijer stumbled on the idea of the dendritic box in his quest for a chiral dendrimer encapsulating amino acids by reaction of the terminal amines with BOC-protected amino acids. [126]. The dendritic box is made up of PPI dendrimers which have diameters of 5 nm as revealed by EPR and NMR spectroscopy [127]. The synthesis of a rigid, dense outer shell around the dendrimer in the presence of guest molecules results in a dendritic box which encapsulates the guest molecules. Hydrolysis (i.e., enzymatic) of the outer shell allows the release of the trapped guest molecule. A second class of dendritic host involves an alkyl chain-propagating dendrimer which behaves as an inverted micelle which transfers the guest from the aqueous to the organic phase using pH as the control parameter. The use of low MW surfactants solubilises the dendrimerguest in water. The lowering of the pH organises the globular inverted dendritic micelles into cylindrical amphoteric vesicles which can then assemble into unimolecular micelles with the subsequent release of the guest—thus providing a novel

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drug-delivery system, especially the encapsulation and Solubilisation of hydrophobic drugs. One industrial application is the enhancement of the dyeability of polyolefins and to improve the dispersion of silica in rubber formulations [128].

4. Concluding remarks

Although there are almost unlimited potential applications of dendrimers, it is interesting that the only commercial application of dendrimers to date are as transfection agents. With research in the 1990's directed toward applications, it is envisioned that other viable commercial applications will be announced in the new millennium. The interest in dendrimers has been so profound that a web-site has been set up at www.dendrimers.com.

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