Dendrimer–drug interactions

Advanced Drug Delivery Reviews 57 (2005) 2147 – 2162 www.elsevier.com/locate/addr

Dendrimer–drug interactionsB Antony D’Emanuele*, David Attwood School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PL, UK Received 29 March 2005; accepted 13 September 2005 Available online 28 November 2005

Abstract The interaction between drugs and dendrimers is reviewed with particular reference to the entrapment of drugs within the dendrimer architecture and the electrostatic and covalent complexation of drugs to the dendrimer surface. The application of dendrimer–drug complexation in the enhancement of drug solubility and bioavailability and the use of the complexes as vehicles for the controlled release of drugs and drug targeting is discussed. D 2005 Elsevier B.V. All rights reserved. Keywords: Dendrimers; Encapsulation; Solubility Enhancement; PEGylation; Electrostatic Interaction; Conjugation; Block Copolymers

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Encapsulation of drugs within the dendritic architecture . . 2.1. Unimolecular micelles . . . . . . . . . . . . . . . . 2.2. PEGylated dendrimers . . . . . . . . . . . . . . . . 2.3. Dendritic box . . . . . . . . . . . . . . . . . . . . 2.4. Cored dendrimers . . . . . . . . . . . . . . . . . . 2.5. Dendrimer-based block copolymers . . . . . . . . . 3. Surface interactions between drugs and dendrimer . . . . . 3.1. Electrostatic interaction between drug and dendrimer 3.2. Conjugation of drug to dendrimer . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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This review is part of the Advanced Drug Delivery Reviews theme issue on "Dendrimers: a Versatile Targeting Platform", Vol. 57/15, 2005. * Corresponding author. Tel.: +44 161 275 2333; fax: +44 709 203 0763. E-mail address: [email protected] (A. D’Emanuele).

0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2005.09.012

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1. Introduction Despite the fact that the term dendrimer was coined in the early 1980s [1], dendritic architectures can be traced back to 1978 [2]. These unique hyperbranched materials are considered as a new class of polymer and indeed they possess many interesting and unique properties. Dendrimers differ from many classical polymers, such as linear and branched polymers, in that they are well-defined, with a high degree of molecular uniformity and monodispersity, as well as a highly functional surface [1,3–5]. In general, low generation dendrimers have an open structure, but as the generation increases the structure becomes more globular and dense. Dendrimers are similar in size to a number of biological structures, for example, fifthgeneration polyamidoamine (PAMAM) dendrimers are approximately the same size and shape as haemoglobin (5.5 nm diameter) [6]. Several classes of dendrimers have been synthesised with a variety of core materials, branching units and surface modifications. Although dendritic structures were first proposed in the late 1970s, it was only in the early 1990s when a significant number of publications started to appear in the literature. This delay may be accounted for by the time taken to develop efficient synthetic methods, high costs, and the lack of commercial availability of dendrimers. Dendrimers have found numerous and diverse applications including those in the fields of biomedicine (e.g. drug delivery, DNA delivery and cancer diagnostics) [6–8], catalysis [9–11] and electronics (e.g. light harvesting devices, optical sensors and displays) [12–16]. An area that has attracted great interest is the interaction between drugs and dendrimers. Several types of interactions have been explored, which can be broadly subdivided into the entrapment of drugs within the dendritic architecture (involving electrostatic, hydrophobic and hydrogen bond interactions) and the interaction between a drug and the surface of a dendrimer (electrostatic and covalent interactions). The applications of such systems have been severalfold, including the use of dendrimers to enhance drug solubility and bioavailability, and to act as release modifiers and platforms for drug targeting. In this overview a range of drug/model compound–dendrimer interactions will be considered together with their proposed applications.

2. Encapsulation of drugs within the dendritic architecture The open nature of the dendritic architecture has led several groups to investigate the possibility of encapsulating drug molecules within the branches of a dendrimer. This offers the potential of dendrimers to interact with labile or poorly soluble drugs. Such systems may enhance drug stability and bioavailability. Encapsulation of a drug within a dendrimer may also be used to provide a means of controlling its release. Maciejewski indeed suggested the use of egg shell-like architectures for the encapsulation of guest molecules in polymers in 1982 [17]. Several types of dendrimer have been investigated for the encapsulation of drugs, including systems designed for triggered release. The nature of drug encapsulation within a dendrimer may be simple physical entrapment, or can involve non-bonding interactions with specific structures within the dendrimer. Much of the work in this area has been based on the assumption that dendrimers possess a hollow core and a dense shell [7], and indeed much of the literature based on drug encapsulation supports this hypothesis. However, several theoretical studies since 1990 have shown that a dendritic structure made up from flexible bonds should exhibit maximum density at the centre of the molecule with a decrease in density towards the periphery [18,19]. These theoretical models have been supported by small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). The actual internal geometry of a dendrimer is likely to be determined by the type of molecule used to form the branches, the nature of the bonds (flexible vs rigid) and nature of the surface groups (bulkiness). Dendrimer size will also be relevant to the three dimensional shape; lower generation dendrimers tend to be open and amorphous structures whereas higher generations can adopt a spherical conformation capable of incorporating drug molecules. 2.1. Unimolecular micelles Dendrimers consisting of an apolar core and polar shell have been referred to as bunimolecular micellesQ. Unlike conventional micelles, however, the dendritic structure is independent of dendrimer concentration [20–24]. The first such structure was a

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[27]-arborol reported by Newkome et al. in 1985 [20]. The same group also synthesised a symmetrical, four directional saturated hydrocarbon cascade polymer containing 36 carboxylic acid moieties with a neopentyl core [22]. It was shown that lipophilic probes were located within the lipophilic infrastructure of the dendritic structures and it was concluded that the polymers exist as single molecules capable of molecular inclusion and therefore act as unimolecular micelles. Hawker et al. [24] described the synthesis of dendritic polyether unimolecular micelles based on an electron-rich 3,5-dihydroxybenzyl alcohol building block with carboxylate surface groups. The dendrimers were able to solubilize a range of polycyclic compounds in water due to k–k interactions, and a relationship was found between the solubilizing power of the dendrimer and the electron density of the polycyclic aromatic. The solubilizing power of the dendrimers was found to be similar in magnitude to that of sodium dodecyl sulfate, but unlike conventional micelles, increased with increase in the concentration of dendrimer at very low concentration, i.e. the dendrimers did not have a critical micelle concentration. Globular dendritic amphiphile structures were also synthesised in which hydrophobic and hydrophilic chain ends were segregated at distinct ends of the structure, thus allowing for preferential orientation at the interface between an organic solvent and water. One of the limitations in the solubilization capacity of unimolecular micelles is a consequence of conformational collapse of the hydrophobic core in water. Smith et al. [25] have synthesised polyphenylene dendrimers which contain no flexible linkages. The rigid structure in these dendrimers makes them less likely to collapse in an aqueous environment and thus the cavities within the dendrimer are maintained when in an aqueous environment. 2.2. PEGylated dendrimers Poly(ethylene glycol) (PEG) has been used to modify dendrimers in the design of solubilizing and drug delivery systems [26–34]. PEG is typically conjugated to the surface of a dendrimer to provide a hydrophilic shell around a hydrophobic dendritic core to form a unimolecular micelle. PEG is of particular interest in the design of dendrimer systems

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for pharmaceutical applications because of its high water solubility, biocompatibility and ability to modify the biodistribution of carriers [35]. Liu et al. synthesized water-soluble dendritic unimolecular micelles based on 4,4-bis(4V-hydroxyphenyl)pentanol building blocks and a surface shell of PEG chains (Fig. 1) [26,27]. The pentanol-based monomer was used to increase the flexibility and cavity size of the dendritic architecture. A single dendritic molecule of G1, G2 and G3 was found to dissolve 0.39, 0.97 and 2.9 molecules of pyrene in water, respectively. A model drug (indomethacin) was loaded to a level of 11% w / w in a G3 dendrimer, a value that corresponds to approximately nine drug molecules per dendrimer. The drug-loaded dendrimer provided sustained release of indomethacin (I) over a period of approximately 30 h. O

Cl

N O O OH

I. Indomethacin

The influence of dendrimer generation (G3 and G4) and PEG molecular weight (550 or 2000) on the ability of PEG grafted dendrimers to encapsulate the hydrophobic drugs adriamycin (II) and methotrexate (III) was examined by Kojima et al. [28]. NMR data indicated that essentially every terminal amino group on the dendrimer was successfully reacted with a PEG chain. It was found that drug loading increased with dendrimer size and increasing chain length of PEG grafts, with up to 6.5 adriamycin or 26 methotrexate molecules incorporated per dendrimer (G4) molecule. The higher encapsulation of methotrexate was attributed to the fact that it is an acidic drug and can therefore interact with the basic interior of the dendrimer. While there was evidence of the sustained release of methotrexate from a dendrimer carrier in an aqueous solution of low ionic strength, no control could be achieved in isotonic solutions. In

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Fig. 1. Structure of G2 water-soluble dendritic unimolecular micelles based on 4,4-bis(4V-hydroxyphenyl)pentanol building blocks and a surface shell of polyethylene glycol (PEG) chains [27].

order to overcome this problem and improve the retention of guest molecules, the same group introduced a shell structure on the dendrimer surface [33]. G4 PAMAM dendrimers were prepared with PEG 2000 conjugated to the surface and a methacryloyl group at every chain end of the dendrimer through an l-lysine residue. The methacryloyl groups were polymerized using a free radical initiator to form a nanocapsule that could retain small molecules within the PAMAM environment. When polymerization of the methacryloyl groups took place in the presence of Bengal Rose (IV) the guest molecules were found to be tightly associated with the dendrimer. However, the encapsulation efficiency was low, with an average of only 0.4 Bengal Rose molecules associated per dendrimer.

O

OH

OH

OH

H 3C

O

O

OH

O

O O

NH2 OH

II. Adriamycin (doxorubicin)

A. D’Emanuele, D. Attwood / Advanced Drug Delivery Reviews 57 (2005) 2147–2162 H2N

N

N

N

N

N

O

uracil (V)), a slower drug release rate (1/6th), and decreased toxicity compared to the nonPEGylated dendrimer.

O

H N

NH2

OH

O

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O

OH

F

HN

III. Methotrexate

O

N H

V. 5-Fluorouracil I -O

O

I

O I

O

Cl

O-

Cl

Cl Cl

IV. Bengal Rose

The influence of the degree of PEG substitution on the encapsulation efficiency and release characteristics of PEGylated G3 PAMAM dendrimers was examined by Pan et al. [34]. Using methotrexate as a model drug, it was found that the degree of substitution had little effect on the encapsulation efficiency suggesting that the drug was localised within the dendrimer rather than the surrounding PEG chains. The degree of substitution was reported to have an effect on release characteristics, but the effect was not dramatic. The encapsulation efficiency of the PEGylated G3 PAMAM dendrimers for methotrexate was similar to that reported by Kojima et al. [28] (approximately 13 methotrexate molecules per G3 dendrimer). The synthesis of PEGylated dendritic systems as nanoparticulate depots for drug delivery was described by Bhadra et al. [30]. G4 PAMAM dendrimers were synthesised and PEGylated using MPEG-5000. A comparison was made of the properties of G4 PAMAM dendrimer and PEGylated dendrimer. The PEGylated systems had a higher drug-loading capacity (12-fold for 5-fluoro-

Yang et al. [31] reported the design of dendritic micelles based on G3 PAMAM dendrimers to which were conjugated PEG chains with molecular weights of 750, 2000 or 5000, with an average of 28, 25 and 23 chains being conjugated per dendrimer, respectively. Although the PEGylated dendrimers all solubilized pyrene, there was no obvious relationship between the amount of pyrene solubilized and PEG chain length, with the PEG 2000 dendrimer solubilizing more than the PEG 750 dendrimer and, surprisingly, more than the PEG 5000 dendrimer (Fig. 2). The authors attributed the latter observation to PEG shell disruption by interpenetration of PEG from adjacent dendrimers, which increases with increase of the length of the PEG chain. One could also argue, however, that in fact repulsion of dendrimers would be expected to take place due to steric hindrance. The synthesis of graft, star-shaped and dendritic polymers based on ethylene glycol was reported by Ooya et al. [29]. So-called polyglycerol dendrimers (Fig. 3) were found to be water-soluble and could

Fig. 2. Effect of PEG arm length on the water solubility of pyrene (x) parent dendrimer; (o) micelle-750; (E) micelle-2000; (x) micelle-5000 (31).

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increase the solubility of paclitaxel (VI) by approximately four orders of magnitude. The release rate of solubilized paclitaxel was related to the shape and generation of the structures.

O O O

NH

O

O

OH

O OH HO O

O O

O O

The larger Bengal Rose molecule remained entrapped and could only be liberated following hydrolysis of the outer shell by 12 mol dm 3 HCl under reflux for 2 h. In order to be feasible for drug delivery applications less harsh conditions would be required for cleavage of the dendrimer surface groups. A similar mechanism of release based on dendrimer surface density was described by Esfand et al. [40]. Using PAMAM based dendrimers, a Dendrilockk structure was described based on a dendrimer with a congested outer shell, the release of guest molecules from which was either immeasurably slow or nonexistent. In contrast, Dendriporesk are lower generation dendritic host structures with less compact surfaces allowing time dependent release of guest molecules. 2.4. Cored dendrimers

VI. Paclitaxel 2.3. Dendritic box Jansen et al. described the synthesis of dendritic boxes based on poly(propyleneimine) dendrimers [36–39]. Guest molecules could be entrapped within the cavities of the dendritic boxes during the synthetic process, with a dense surface shell preventing diffusion from the structures, even after prolonged heating, solvent extraction or sonication. The poly(propyleneimine) dendrimers with primary amine end groups were synthesised by a divergent approach. The rigid shell was obtained through end group modification with a bulky amino acid derivative to yield a dense and rigid chiral shell with solid-phase properties and a flexible core capable of entrapping molecules. The size of 5th generation dendritic boxes was determined to be 5 nm. A number of dye molecules were encapsulated in the dendritic box, for example, up to 4 molecules of Bengal Rose could be encapsulated per dendrimer. The shape selective liberation of guests from dendritic boxes was also described. Hydrolysis of the surface t-BOC groups (with formic acid) of a dendritic box containing 4 molecules of Bengal Rose and 8–10 molecules of 4nitrobenzoic acid resulted in perforation of the dendrimer allowing the release of 4-nitrobenzoic acid.

Zimmerman and coworkers synthesised cored dendrimers that resemble hollow nanospheres and suggested that their potential to encapsulate substances made them candidates for delivery vehicles [41,42]. Encapsulation was achieved by post-synthetic modification of the dendritic architecture. The core unit in a typical dendrimer is essential as it interconnects the dendrons, or branches, of the structure. An alternative approach to maintaining the structural integrity of a dendrimer is to crosslink the peripheral surface groups. In the case of cored dendrimers this was done by a ring-closing metathesis reaction. The interlinking of the dendritic wedges maintained structural integrity on subsequent removal of the core. Removal of the core was via cleavage of ester bonds, with the remaining structure being unaffected as a consequence of robust ether linkages. It is not clear, however, how guest molecules could be loaded into such structures. 2.5. Dendrimer-based block copolymers Dendrimeric di- and tri-block copolymers with linear hydrophilic block(s) and a hydrophobic dendrititic block have been synthesised by a number of groups. The self-association of these amphiphilic molecules in aqueous solution and the ability of the aggregates to solubilize or complex poorly water-

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Fig. 3. Structure of G5 polyglycerol dendrimer [29].

soluble molecules have been examined for several of these systems. The hydrophilic blocks of the majority of the dendrimeric block copolymers reported are linear polyethers, usually PEG. The formation of multimolecular micelles in methanol/water solutions of AB and ABA linear-dendritic block copolymers composed of hydrophobic dendritic aromatic polyethers (generations 2 and 3) and a PEG hydrophilic block has been reported by Gitsov and Fre´chet [43]. A feature of these micelles that distinguishes them from those of classical surfactants is their stability to dilution or prolonged heating, thought to be due in part to the slow establishment of an entanglement– disentanglement equilibrium in the dendritic cores of

the micelles as a consequence of the highly branched structure of the end blocks. Chapman et al. [44] synthesised generation 1–4 diblock dendrimers with a PEG hydrophilic block and a hydrophobic block of poly(l-lysine), aqueous solutions of which formed micelles at well-defined critical concentrations. Di- and tri-block copolymers with PEG as the linear block and poly(benzyl ether) as the hydrophobic dendritic block have been designed and synthesised by Fre´chet et al. [45]. Examination of their solution behaviour by surface tension and light scattering techniques revealed the presence of unimolecular micelles (with the PEG chains forming a hydrophilic corona around the poly(benzyl ether)) below a critical concentration

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and multimolecular micelles at higher concentrations. Chang et al. [46] report the synthesis of an amphiphilic linear-dendritic diblock copolymer based on PEG as the hydrophilic block and polycarbosilane as the hydrophobic block. First and second generation dendrimers of this type formed micelles suitable for the incorporation of hydrophobic molecules. The same group also synthesised triblock copolymers of PEG and polycarbosilane with generations 1 and 2, which formed micelles in aqueous solution. The micelle sizes were large and dependent on generation; mean diameters of 170 and 190 nm were measured for G1 and G2 triblocks, respectively [47]. Micelles with highly branched nanoporous cores were formed in aqueous solutions of amphiphilic linear dendritic ABA block copolymers of PEG and poly(benzyl ether) [48]. Their ability to encapsulate large numbers of hydrophobic solute molecules was demonstrated using a series of polyaromatic compounds. A series of G1-G5 PAMAM-block -PEG-block -PAMAM triblock copolymers were synthesised by Kim et al. and investigated as potential polymeric gene carriers [49]. The copolymers were shown to form highly water-soluble polyplexes with plasmid DNA, which had high transfection efficiencies. Their self-assembly with DNA was a function of the number of surface primary amines, the internal tertiary amines were unable to participate in complexation because of steric hindrance. Citric acid–PEG–citric acid triblock copolymers formed inclusion complexes with a range of guest molecules including 5-aminosalicylic acid (VII), mefenamic acid (VIII) and diclofenac (IX) [32]. The amount of entrapped drug increased with increasing dendrimer generation from G1 to G3 and was greatest for 5-aminosalicylic acid, probably because of its small size and high polarity. The rate of release of complexed drug was pH dependent and increased with dendrimer generation; in all systems release was complete within 6 h. O H2N

OH OH

VII. 5-Aminosalicylic acid

O OH NH

VIII. Mefenamic Acid Cl NH O-

Cl O

Na+

IX. Diclofenac Several interesting amphiphilic dendrimeric block copolymers have been synthesised in which hydrophilic groups other than the non-ionic PEG have been utilised. Zhu et al. [50] prepared asymmetric lineardendritic block copolymers composed of a polyether dendrimer as the hydrophobic block and an anionic linear chain, poly(N-isopropylacrylamide) as the polar hydrophilic block, which formed spherical aggregates (diameter 40–70 nm) in aqueous solution above a critical concentration of 2  10 6 mol dm 3 by hydrophobic association of the dendritic subunits. At temperatures above 37.5 8C, the hydrophobic interaction increased due to the disruption of hydrogen bonds between the amide groups and the surrounding water, causing the growth of larger entangled tubules, which have potential use as vehicles for drug delivery. Novel AB diblock copolymers in which the hydrophilic A-block is a PAMAM dendrimer with terminal carbohydrate groups and the hydrophobic B-block is a PAMAM dendrimer with phthaloyl terminal groups have been synthesised by Aoi et al. [51]. These socalled dsugar ballT amphiphilic AB-type surface-block dendrimers showed apparent recognition ability towards protein receptors and their use as supramolecular self-assembling globular amphiphiles and intelligent nanocapsules having dual binding surface sectors to proteins and DNA was suggested. This

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same group also synthesised dtadpole-shapedT dendrimers with a highly hydrophilic tail composed of poly(2-methyl-2-oxazoline) and a globular head group of PAMAM with either amine ( G = 4 and 5) or methyl ester ( G = 3.5, 4.5 and 5.5) terminal groups [52]. Surface tension and small-angle neutron scattering measurements demonstrated self-association at a critical concentration into large aggregates. A series of polystyrene (PS)-poly(propyleneimine) dendrimers, PS-dendr-(COOH)n (n = 2, 4, 8, 16, 32) has been synthesised and characterised by van Hest et al. [53]. Aggregation of dendrimers with n of between 8 and 32 was observed above critical concentrations of 10 6 to 10 7 mol dm 3. Single aggregates, identified as curved dworm-likeT micelles, were noted for dendrimers with n = 8; clustering of the aggregates to form large network structures occurred with dendrimers having higher n values.

3. Surface interactions between drugs and dendrimer Although the number of guest molecules incorporated into a dendrimer may be dependent to a limited extent on the architecture of a dendrimer, the loading capacity may be dramatically increased by the formation of a complex with the large number of groups on the dendrimer surface. Thus, the external surfaces of dendrimers have been investigated as potential sites of interaction with drugs. The number of surface groups available for drug interactions doubles with each increasing generation of dendrimer. It should be borne in mind that not all of the surface groups in a dendrimer may be available for interaction, either because of steric hindrance or backfolding of chains into the dendrimer. 3.1. Electrostatic interaction between drug and dendrimer The presence of large numbers of ionizable groups on the surface of dendrimers (a G4 PAMAM dendrimer for example has 64 ionizable NH2 groups) provides an interesting opportunity for electrostatic attachment of numerous ionizable drugs, providing the resultant complex retains sufficient water solubility. In this respect dendrimers compare favourably with other complexing agents where 1 : 1 stoichiom-

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etry is more usual. In addition to the readily accessible surface groups, several classes of dendrimer have ionizable groups within their core which, depending on the nature of the complexing ion, may also be available for complexation. For example, full generation PAMAM dendrimers have primary amine end groups (–NH2) on the surface and tertiary amine groups (N N–) situated at the branching points in the core. Both are titratable having pK a values of 10.7 and 6.5, respectively [54]. Similarly, poly(propylenimine) dendrimers based on a diaminobutane core, (DAB-dendr-(NH2)x ), have 2n + 1 primary amine surface groups (where n is the generation) and 2n+1 2 interior tertiary amines. Studies of the interaction of a series of dendrimers of this type having x = 4, 8, 16, 32 or 64 with several flexible linear polyanions have shown that all the amine groups (of both the dendrimer surface and core) could interact with the anionic groups of the polymer chains, i.e. the dendrimers were fully penetrable by these linear polyanions [55,56]. In contrast the interaction of a linear polycation with carboxylated dendrimers has been found to involve only the surface groups of these dendrimers, possibly due to the presence of tertiary amine groups in the dendrimer core, which would restrict core penetration of the polycation [57]. Factors that affect complexation, such as surface charge density and ionic strength, have been studied by Dubin and coworkers [58,59], for the interaction of small carboxyl-terminated dendrimers with no interior titratable groups, and the strong polycation poly(dimethyldiallyammonium chloride). Complex formation was shown to occur abruptly at a critical surface charge density that was lower for a generation 3 compared to a generation 1 dendrimer, i.e. binding occurred more easily for the larger dendrimer. The drug that has been most widely complexed to full generation PAMAM dendrimers is the nonsteroidal anti-inflammatory drug ibuprofen (X). Electrostatic interaction can occur between the carboxyl groups of this weakly acidic drug and the amine groups of the dendrimers. It has been estimated that approximately 40 ibuprofen molecules interact with G4 PAMAM dendrimer at pH 10.5 causing a considerable enhancement of drug solubility [60]. It was, however, noted that although the

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drug would be fully ionized at this pH (pK a = 5.2), there would be insufficient ionization of the 64 surface amines (pK a = 9.5) to account for the observed solubility enhancement; as the internal tertiary amines would be nonionized at this pH, other causes of solubility enhancement, for example dendrimer aggregation, were suggested. In a similar study involving the interaction of G3 and G4 PAMAM dendrimers with this drug, Kolhe et al. [61] reported the incorporation of 32 and 78 ibuprofen molecules per dendrimer, respectively; NMR and FTIR spectroscopy showed that complexation was due to ionic interaction. No details of the pH of the dendrimer solutions were given and hence the degree of ionization of the surface groups cannot be estimated. The reason for the excess of drug molecules over the total number of surface groups of the G4 dendrimer was attributed to possible encapsulation in the dendrimer core. The number of drug molecules incorporated by the G3 dendrimer was equal to the number of surface amines and it was thought that encapsulation within the smaller core of this dendrimer was unlikely. The dendrimer/drug complexes were stable in water and methanol over a period of 8 h; a slow release of drug from the complex in culture medium was noted. The dynamics of cellular entry of the complexes into A549 human lung epithelial carcinoma cells were explored in a later paper by this group; complexation was shown to facilitate rapid cellular entry of ibuprofen [62].

OH O

X. Ibuprofen Both G3 and G2.5 PAMAM dendrimers were shown to enhance significantly the aqueous solubility of piroxicam (XI) at pH 6 and 8 [63]. In the case of the G3 dendrimer this enhancement was attributed to electrostatic complexation and/or hydrogen bonding. The solubility increase noted in the presence of carboxylated dendrimer (G2.5) was thought to be

simply due to an increase of solution pH by the highly basic dendrimer, since there is no opportunity for electrostatic interaction with these similarly charged compounds.

O

OH

S O

N

N H

N

O

XI. Piroxicam Solubility enhancement of the poorly watersoluble drug indomethacin in the presence of a series of G4 and G4.5 PAMAM dendrimers and also G4 dendrimers with surface hydroxyl groups was examined by Chauhan et al. [64]. In the case of the G4 dendrimer, increased solubility was explained on the basis of electrostatic bonding between the carboxyl group of indomethacin and the amino groups of the dendrimer. As no electrostatic bonding can be expected between the G4.5 and drug, the increased solubility was thought to be a consequence of molecular encapsulation resulting from non-specific, non-covalent interactions. Possible involvement of weak hydrogen bonding was also thought to be operative in causing an increase of solubility in the presence of the hydroxylterminated G4 dendrimer. All three types of dendrimer were shown to be effective in increasing the flux of indomethacin across skin in both in vitro and in vivo experiments. The interaction of benzoic acid at neutral pH (virtually water-insoluble) with neutral PAMAM dendrimers formed by conversion of the terminal groups into hydroxyls was reported by Beezer et al. [65]. Stable water-soluble complexes were formed in which the benzoic acid was thought to be bound to the internal tertiary nitrogens of the dendrimer by simple ion-pairing; there was no evidence from NMR of hydrogen bonding between the carboxyl groups of the benzoic acid and the internal amides.

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3.2. Conjugation of drug to dendrimer

O

The covalent attachment of drugs to the surface groups of dendrimers through hydrolysable or biodegradable linkages offers the opportunity for a greater control over drug release than can be achieved by electrostatic complexation of drugs to the dendrimers. Yang and Lopina have conjugated penicillin V (XII) with both G2.5 and G3 PAMAM dendrimers through a PEG spacer via amide and ester bonds, respectively [66]. The use of an amide linkage provided bond stability, whereas ester linkage of the drug to the dendrimer provided a means of controlling drug release via hydrolysis. The microbial activity of the penicillin released by ester hydrolysis of the PEG-PAMAM (G3) conjugate was approximately the same (within 3%) as that of non-modified penicillin. In a second study by these authors [67], an extended release formulation is described in which the third generation antidepressant venlafaxine (XIII) was covalently linked to a G2.5 anionic PAMAM dendrimer via a hydrolysable ester bond, with the aim of overcoming the problem of poor patient compliance associated with the multiple daily administration of this drug. Almost complete conjugation of the surface sites (32 surface carboxylate groups) was achieved by a direct coupling reaction. An in vitro release study indicated sustained release of drug over a period of 120 h with approximately 50% release within 18 h. Further control of release rates was achieved by incorporating the conjugated dendrimers into semi-interpenetrating networks prepared by cross-linking acrylamide in the presence of PEG. Wiwattanapatapee et al. [68] have designed dendrimer conjugates for colonic delivery of 5aminosalicylic acid, in which the drug was bound to G3 PAMAM dendrimers using either p-aminoH N

O O

O

H

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N OH

XIII. Venlafaxine benzoic acid or p-aminohippuric acid spacers. The amount of drug released over 24 h when incubated was between 45% and 57% for these conjugates compared with 80% in 6 h from the commercial prodrug, sulfasalazine (Fig. 4). We have reported an enhancement of the transport of propranolol across monolayers of the human colon adenocarcinoma cell line (Caco-2) following conjugation to G3 PAMAM dendrimers and to G3 PAMAM dendrimers with attached lauroyl chains (which lower cytotoxicity and enhance permeation) [69,70]. In addition to enhancing its aqueous solubility, conjugation of propranolol (XIV) (a P-glycoprotein substrate) to these den-

S

N O

OH Fig. 4. Release profiles of 5-ASA from PAMAM-PABA-SA conjugate (x), PAMAM-PAH-SA conjugate ( ) and sulfasalazine (E) during incubation with rat cecal content at 37 8C. Data shown are the mean F S.D (n = 5) (68).

.

XII. Penicillin V

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drimer carriers increased its epithelial permeation by circumventing the P-glycoprotein efflux transporter (Fig. 5).

O OH

N H

dendrimer-platinate which released platinum slowly in vitro [75]. The dendrimer-Pt conjugate was between 3- and 15-fold less toxic than cisplatin and its selective accumulation in solid tumour tissue by the enhanced permeability and retention (EPR) effect was appreciably higher. NH3 Cl Pt NH3 Cl

XIV. Propranolol Several workers have developed dendrimer conjugates with potential application as vehicles for the delivery of anticancer agents [71]. Zhuo et al. have synthesised a series of dendritic polymers having cyclic cores of 1,4,7,10-tetraazacyclododecane with full and half generations ranging between 0.5 and 5.5, and reported their conjugation with 5-fluorouracil [72]. Hydrolysis of the conjugates resulted in a slow in vitro release of drug over a period of several days, so reducing the toxic side effects of this potent antitumour drug. The design, synthesis and evaluation of dendritic polyester systems based on the monomer unit 2,2-bis(hydroxymethyl)propanoic acid have been described [73,74]. The potential of these polymers to act as scaffolds in the design of drug carriers was demonstrated by covalently binding (using an acidlabile hydrazone linkage) the anticancer drug doxorubicin to one of the polymers. The anticancer agent cisplatin (XV) has been conjugated to a G3.5 PAMAM dendrimer giving a highly water-soluble

XV. Cisplatin A problem that may arise as a consequence of coupling large numbers of drugs to the dendrimer surface is the insolubility of the resultant conjugate; this problem can often be resolved through the concomitant attachment of PEG chains. It was found, for example, that insoluble complexes were formed when five or more ibuprofen molecules were covalently attached to the surface of G4 PAMAM dendrimers, but as many as 32 ibuprofen molecules could be conjugated to the PEGylated dendrimers [76,77]. Similarly, Liu et al. synthesised several poly(aryl ether) dendrimers and demonstrated their potential as drug carriers by the covalent attachment of a number of model compounds (e.g. cholesterol and two amino acid derivatives) [78]. These dendritic carriers were made soluble by the surface attachment of short PEG chains.

4. Conclusions

Fig. 5. AB (5) and BA (n) permeability across Caco-2 cell monolayers at 37 8C of free propranolol (P), propranolol-G3 dendrimer conjugates (G3P2, G3P4 and G3P6) and propranolollauroyl (L)-G3 dendrimer conjugates (G3L2P2, G3L6P2 and G3L2P6), (mean F SD, n = 4) [69].

Dendrimers offer several advantages over conventional polymers with respect to drug interactions, including the fact that they are well-defined molecules, allow the development of well-defined drug/ polymer systems, and have potential for high drug payloads. There has been considerable interest in the use of dendrimers for drug delivery applications in the past 5 years. Although this interest is likely to continue, one topic that requires further addressing is the full biological evaluation of these novel polymeric materials. Whilst the biological properties of unmodified dendrimers have been evaluated, it is

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clear that modifying, or surface engineering, dendrimers is likely to significantly modify the biological profile of dendrimer-based delivery systems [79– 83]. The majority of drug/dendrimer studies to date have been based on PAMAM and polyethyleneimine (PEI) dendrimers. Whilst the synthesis of novel dendritic architectures is a major challenge, new biodegradable and biocompatible materials that are water-soluble are emerging. For example, the synthesis of biodegradable chitosan-dendrimer hybrids [84] and of biodegradable branched poly(l-glutamic acid) based on PAMAM or PEI cores [85] have been reported. Other potential dendrimers for biological application include those based on peptides [86] and melamine [87]. Another area that has attracted recent interest is the development of triggered release systems based on a dendrimer carrier. pH dependent release of pyrene from the interior of poly(propyleneimine) dendrimers has been demonstrated [88]: incorporation of pyrene was favoured at high pH, but at low pH protonation of internal dendrimer amines occurred thus creating a polar environment, resulting in release (expulsion) of pyrene. Hydrophilic quaternary groups on the surface of the poly(propyleneimine) dendrimers effect release over a narrower pH range [89] and PEG chains improved the biological properties [90]. The attachment of isobutyramide groups to the chain ends of PAMAM and poly(propylenimine) dendrimers increased the temperature sensitivity of their water solubility [91]. Archut et al. have reported the development of dendrimers containing light switchable units [92]. It is possible that responsive dendritic structures may be designed in the future that are triggered by specific chemicals.

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