Gene Ther Mol Biol Vol 10, 71-94, 2006

Design of functional dendritic polymers for application as drug and gene delivery systems

Review Article

Zili Sideratou, Leto-Aikaterini Tziveleka, Christina Kontoyianni, Dimitris Tsiourvas, Constantinos M. Paleos*

Institute of Physical Chemistry, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece ________________________________________________________________________

*Correspondence: Constantinos M. Paleos, Institute of Physical Chemistry, NCSR "Demokritos"; 15310 Aghia Paraskevi, Attiki, Greece; Tel.: +30 210 6503666; Fax: +30 210 6529792; e-mail: paleos@chem.demokritos.gr

Key words: Dendrimers, Hyperbranched Polymers, Dendritic Polymers, Nanocarriers, Drug Delivery System, Gene Delivery

Abbreviations: Adriamycin, (ADR); arginine-grafted-PAMAM dendrimer, (PAMAM-Arg); Asialo-glycoprotein, (ASGP); betamethasone dipropionate, (BD); betamethasone valerate, (BV); Boron Neutron Capture Therapy, (BNCT); diaminobutane poly(propylene imine) dendrimer, (DAB); diaminobutane poly(propylene imine) fourth generation dendrimer functionalized with 6 guanidinium groups, (DAB-G6 ); diaminobutane poly(propylene imine) fourth generation dendrimer functionalized with 12 guanidinium groups, (DAB-G12); Dynamic Light Scattering, (DLS); epidermal growth factor, (EGF); green fluorescent protein, (GFP); injected dose, (ID); hyperbranched poly(ethylene imine), (PEI); hyperbranched polyglycerol, (PG); Isothermal Titration Calorimety, (ITC); L-lysine grafted-PAMAM dendrimer, (PAMAM-Lys); methotrexate, (MTX); methoxypoly(ethylene glycol)-isocyanate, (PEG-isocyanate); PEGylated diaminobutane poly(propylene imine) dendrimer with 4 PEG chains, (DAB-4PEG); PEGylated diaminobutane poly(propylene imine) dendrimer with 8 PEG chains, (DAB-8PEG); PEGylated polyglycerol, (PG-PEG); PEGylated-Folate polyglycerol, (PG-PEG-Folate); Phosphate Buffer Saline, (PBS); poly(amidoamine) dendrimer, (PAMAM); poly(amidoamine) dendrimer with terminal hydroxyl groups, (PAMAM-OH); poly(ethylene imine)-poly(ethylene glycol)-folate, (PEI-PEG-FOL); poly(ethylene glycol), (PEG); poly(ethylene glycol) monomethyl ether, (M-PEG); poly(propylene imine) dendrimer, (PPI); primaquine phosphate, (PP); pyrene, (PY); quaternized poly(amidoamine) dendrimer with terminal hydroxyl groups, (QPAMAM-OH); tamoxifen, (TAM)

Received: 28 November 2005; Accepted: 10 February 2006; electronically published: March 2006

Summary

The present review deals with the design and preparation of functional and multifunctional dendrimeric and hyperbranched polymers (dendritic polymers), in order to be employed as drug and gene delivery systems. In particular, using as starting materials known and well-characterized basic dendritic polymers, the review discusses the kind of structural modifications that these polymers were subjected for preparing nanocarriers of low toxicity, high encapsulating capacity, specificity to certain biological cells and transport ability through their membranes. Due to the great number of external groups of dendritic polymers either functionalization or multifunctionalization can occur, providing products that fulfill one or more of the requirements that an effective drug carrier should exhibit. A common feature of these dendritic polymers is the exhibition of the so-called polyvalent interactions, while for the multifunctional derivatives a number of targeting ligands determines specificity, other groups secure stability in biological milieu, while others facilitate their transport through cell membranes. In addition, for gene delivery applications these multifunctional systems should be or become cationic in the biological environment for the formation of complexes with the negatively charged genetic material.

I. Introduction

Dendrimers are prepared by tedious synthetic procedures (Bosman et al, 1999; Schlüter and Rabe, 2000; Fréchet and Tomalia, 2001; Newkome et al, 2001) and they are nanometer-sized, highly branched and monodisperse macromolecules with symmetrical architecture. They consist of a central core, branching units and terminal functional groups. The core and the internal units determine the environment of the nanocavities and consequently their solubilizing or encapsulating properties, whereas, the external groups their solubility and chemical behaviour. On the other hand, hyperbranched polymers (Inoue, 2000), including the extensively investigated hyperbranched polyether polyols or polyglycerols (Sunder et al, 1999a, b; 2000a,b; Haag, 2001; Frey and Haag, 2002; Siegers et al, 2004) are conveniently prepared. Hyperbranched polymers are non-symmetrical, highly branched and polydispersed macromolecules, while their main structural feature, also common to dendrimers, is that they exhibit nanocavities. These two types of polymers are called dendritic polymers the nanocavities of which, depending on their polarity, can encapsulate various molecules, including active drug ingredients. The external groups of dendritic polymers can be modified providing a diversity of functional materials (Vögtle et al, 2000) that can be employed for various applications.

Within this context, commercially available or custom-made dendrimeric or hyperbranched polymers can be functionalized for being used as effective systems for drug (Liu and Fréchet, 1999; De Jess et al, 2002; Stiriba et al, 2002; Beezer et al, 2003; Gillies and Fréchet, 2005) and gene (Bielinska et al, 1999; Luo et al, 2002; Ohsaki et al, 2002) delivery. Since more than one type of groups can be introduced at the surface of the dendritic polymers, these systems are characterized as multifunctional as shown in Figure 1. Each type of groups plays a specific role in the application of multifunctional dendritic polymers as drug delivery systems. Thus, specificity for certain cells can be accomplished by attaching targeting ligands at the surface of dendritic polymers, while enhanced solubility, decreased toxicity, biocompatibity, stability and protection in the biological milieu can be achieved by the functionalization of the end groups of dendritic polymers, for instance, with poly(ethylene glycol) chains (PEG). The function of PEG-chains is crucial for modifying the behaviour of drug themselves or of their carriers (Noppl-Simson and Needham, 1996; Ishiwata et al, 1997; Liu et al, 1999; Liu et al, 2000; Veronese, 2001; Roberts et al, 2002; Pantos et al, 2004; Vandermeulen and Klok, 2004).

Targeting ligands are complementary to cell receptors (Cooper, 1997; Lodish et al, 2000) and induce the attachment of the nanocarrier to the cell surface. This binding is further enhanced due to the so-called polyvalent interactions (Mammen et al, 1998; Kitov and Bundle, 2003) attributed to the close proximity of the recognizable ligands on the limited surface area of the dendritic molecules. On the other hand, as it has long been established with liposomes (Lasic and Needham, 1995; Crosasso et al, 2000; Needham and Kim, 2000; Silvander et al, 2000), PEG-chains may prolong the circulation of liposomes in biological milieu. Transport through the cell membrane can also be facilitated by the introduction of appropriate moieties at the surface of the dendritic polymers. In addition, modification of the internal groups of dendrimers affects their solubilizing character, making, therefore, possible the encapsulation of a diversity of drugs. In this connection, cationization of dendrimers, and particularly of their external groups, facilitates their application as gene transfer agents (Bielinska et al, 1999; Luo et al, 2002; Ohsaki et al, 2002) due to formation of DNA-Dendritic Polymer complexes.

Monofunctional dendritic drug carriers do not simultaneously show the desired properties that multifunctional derivatives exhibit. Thus, in this review, starting from selected monofunctional systems and specifically from the dendrimeric compounds poly(amidoamine), PAMAM, and diaminobutane poly(propylene imine), DAB, and also from the hyperbranched polymers polyglycerol, PG and poly(ethylene imine), PEI, (Figure 2) a stepwise design of multifunctional systems will be discussed, aiming at obtaining appropriate nanocarriers for drug delivery and gene transfection. This review is by no means exhaustive and only selected examples will be discussed highlighting on work performed recently in our laboratory. The objective of this review is to illustrate the effectiveness of the strategy of molecular engineering, applied on dendritic surfaces, to prepare drug carriers with desired properties.

II. Drug carriers: from monofunctional to multifunctional dendrimers

In an example on molecular engineering of PAMAM surface, poly(ethylene glycol) monomethyl ether (M-PEG), having an average molecular weight of 550 or 2000, was attached at the terminal amino groups of the third and fourth generation polymers as shown in Figure 3. Inside the nanocavities of the so-prepared PEGylated dendrimers, Adriamycin, ADR, or Methotrexate, MTX, anticancer drugs (Figure 4) were encapsulated (Kojima et al, 2000). As the amount of ADR employed for encapsulation inside these PEGylated dendrimers increased, the number of ADR molecules associated with the dendrimer increased and finally reached a plateau. Depending on the generation, the maximum number of ADR molecules encapsulated per dendrimer i.e. by the M-PEG(550)-G3, M-PEG(2000)-G3, M-PEG(550)-G4, and M-PEG(2000)-G4 dendrimeric derivatives are ca. 1.2, 2.3, 1.6, and 6.5, respectively, as shown in Figure 5. Thus, the encapsulation ability varied for these PEG-dendrimers and it was found to depend on the molecular weight of PEG-chains and also on dendrimers’ generation.

PAMAM has a basic interior and, therefore, it is possible to encapsulate MTX, which is acidic, since it bears two carboxyl groups. The number of MTX molecules associated with one dendrimer molecule, as a function of the MTX/dendrimer ratio during loading is shown in Figure 6. As it was observed in the

encapsulation of ADR, the number of MTX molecules associated with the modified dendrimer increased with increasing amount of MTX employed during loading, and finally reached a constant value. The maximum numbers of MTX molecules associated with the M-PEG(550)-G3, M-PEG(2000)-G3, M-PEG(550)-G4, and M-PEG(2000)-G4 dendrimers are approximately 10, 13, 20, and 26 mol/mol of dendrimer, respectively. Apparently, the number of the encapsulated drugs by the PEGylated dendrimers increased when MTX was used instead of ADR. Since these drugs have similar molecular weights, this result suggests that the electrostatic interaction from the acid-base interaction between the dendrimer and MTX molecules results in an enhanced encapsulation of MTX by these dendrimers. As it was the case with ADR encapsulation, the number of MTX encapsulated by the dendrimer was affected both by generation of the PAMAM and by the chain length of the M-PEG.

Release experiments performed in PBS buffer (Phosphate Buffer Saline) showed that ADR was readily released from the modified dendrimers. Apparently, hydrophobic interaction between ADR and the dendrimer is not strong enough to retain the drug in the interior of the PAMAM dendrimeric moiety. The release of MTX from the M-PEG-functionalized dendrimers was also investigated by the same method. The time dependency of MTX concentration in the outer phase during the dialysis is shown in Figure 7. Apparently, the MTX concentration in the outer phase increased at a slower rate when MTX was encapsulated in the M-PEG-attached dendrimer than in the case of free MTX. This indicates that MTX was gradually released from the modified dendrimer. As mentioned above, MTX was electrostatically bound to the dendrimeric interior and, therefore, dissociation of MTX from the dendrimer was suppressed to some extent. However, when the dialysis was performed in the presence of 150 mM NaCl, no difference in the release rate was observed between MTX encapsulated in the M-PEG-attached dendrimer and free MTX. In this case, MTX can dissociate readily from the dendrimer because the electrostatic interaction is weakened by the shielding effect of Na⁺ and Cl^- (Kojima et al, 2000).

Effective solubilization of hydrophobic drugs was, however, achieved with another PEGylated dendrimeric system (Sideratou et al, 2001), which is analogous to the one previously discussed. PEGylation of dendrimers was performed under facile experimental conditions by the interaction of methoxypoly(ethylene glycol)-isocyanate (PEG-isocyanate) with the external primary amino groups of DAB dendrimers of fifth generation, as shown in Figure 8. Two different PEGylated dendrimeric derivatives were prepared i.e. the DAB-4PEG (weakly PEGylated) and DAB‑8PEG (densely PEGylated). In this manner, the role of PEG-coating on encapsulation and release properties was possible to be assessed.

Comparison of solubilizing ability of the parent and PEGylated DAB dendrimers is shown in Table 1. For this purpose, betamethasone valerate, BV, and betamethasone dipropionate, BD, were used as active drug ingredients (Figure 9). These anti-inflammatory corticosteroids are practically water insoluble and it is, therefore, necessary to encapsulate these compounds in a water-soluble carrier for facilitating their use as drugs. The concentration of encapsulated betamethasone derivatives was significantly increased in PEGylated dendrimers. Thus, for DAB-8PEG the loading was 13 and 7 wt.% for BV and BD, while for DAB-4PEG was 6 and 4 wt.%, respectively. The observed solubility increase was attributed to an additional solubilization of the compounds in PEG-chains by which the dendrimers are coated. This is also verified by the fact that upon protonation they remain solubilized in PEG-chains environment. As expected, by increasing dendrimer concentration, solubilization of drugs analogously increases to a certain limit.

For a detailed investigation of the solubilization site and release properties of these PEGylated dendrimers the hydrophobic pyrene was employed. This is a very sensitive probe and it is used as a model compound when drugs cannot offer this type of information. By employing the well-known I₁/I₃ fluorescence intensity ratio, which probes the polarity of the medium (Thomas, 1980), it was found that pyrene is solubilized both in the core and in PEG-chains. In addition, upon protonation of the loaded PEGylated dendrimer, pyrene is not released in the bulk aqueous phase as judged again by the I₁/I₃ratio and fluorescence intensity (F/F⁰) results. This is attributed to the fact that as pyrene is leaving the core it is possible to be solubilized inside PEG-chains, as shown schematically in Figure 10. The results of I₁/I₃ fluorescence intensity ratio indicate that pyrene is neither solubilized in the bulk water phase nor in the interior of the dendrimer. Normally, one would expect release of pyrene in water, since, due to protonation, the environment of the nanocavities becomes polar and, therefore, the hydrophobic pyrene cannot remain solubilized. In addition, protonated tertiary amino groups of the core do not exhibit anymore the property to form charge-transfer complexes with pyrene (Sideratou et al, 2000) and, therefore, encapsulation of the pyrene is no longer favoured. It should, however, be noted that complete release of pyrene can be achieved upon exhaustive dilution of the PEGylated dendrimer. The same behaviour was observed for the hydrophobic drugs BV and BD. In conclusion, the enhanced solubilization of these drugs in PEGylated dendrimers secures their application as promising controlled release drug delivery systems.

In another recent report, extending the previous work, a novel multifunctional dendrimeric carrier was designed (Paleos et al, 2004) based on diaminobutane poly(propylene imine) dendrimer of the fifth generation. The synthetic procedure of this derivative is shown in Figure 11. This carrier is intended to simultaneously address issues such as stability in the biological milieu, targeting and very possibly transport through cell membranes. For this purpose, in addition to surface protective poly(ethylene glycol) chains, guanidinium moieties were introduced as targeting ligands. In addition, the accumulation of guanidinium groups at the surface of the dendrimer may also facilitate its transport ability. The functional groups were covalently attached at the dendrimeric surface and it was possible to secure, in principle, desired drug delivery properties due to: a. Protection of the carrier because of the coverage of the dendrimeric surface with poly(ethylene glycol) chains, b. Recognition ability towards complementary moieties; surface guanidinium groups secure the facile interaction with acidic receptors including the biologically significant carboxylate and phosphate groups. Combined electrostatic forces and hydrogen bonding are exercised making this interaction thermodynamically favorable (Hirst et al, 1992), c. Possibility of encapsulation and release of active drug ingredients from the nanocavities, which can be tuned by environmental changes (Sideratou et al, 2001), d. Complexation with DNA for gene therapy applications, e. The occurrence of polyvalency interactions, associated with enhanced binding, due to the accumulation of recognizable moieties on the limited surface area of the dendrimer as schematically illustrated in Figure 12, f. The expected decrease of toxicity due to the facile modification of the toxic amino groups (Malik et al, 2000).

Table 1. Comparative solubility of pyrene (PY), betamethasone valerate (BV) and betamethasone dipropionate (BD) in parent DAB and PEGylated derivatives. Reproduced from Sideratou et al, 2001 with kind permission from Elsevier.

Compound	[dendrimer]/M	[PY]/M	[BV]/M	[BD]/M
DAB	5 x 10^-5	2.15 x 10^-6	2.95 x 10^-5	1.84 x 10^-5
DAB-8PEG	5 x 10^-5	5.40 x 10^-5	3.85 x 10^-4	2.56 x 10^-4
DAB-4PEG	5 x 10^-5	2.14 x 10^-5	2.05 x 10^-4	1.25 x 10^-4
DAB-8PEG	5 x 10^-4	8.75 x 10^-5	3.65 x 10^-3	1.87 x 10^-3
DAB-4PEG	5 x 10^-4	5.25 x 10^-5	1.70 x 10^-3	1.09 x 10^-3

Figure 9. Chemical structure of betamethasone valerate, BV, and betamethasone dipropionate, BD

Figure 10. Schematic representation of the solubilization of pyrene in PEGylated dendrimers. Reproduced from Sideratou et al, 2001 with kind permission from Elsevier BV.

Figure 11. Reaction scheme for the synthesis of a multifunctional dendrimeric derivative. Reproduced from Paleos et al, 2004 with kind permission from American Chemical Society.

For evaluating the loading capacity and release properties of the above multifunctional dendrimer, pyrene (PY) and betamethasone valerate (BV), were used as model compounds. The dendrimeric derivative encapsulated significantly higher concentrations of the above compounds compared to the parent dendrimer, as determined by UV spectroscopy and shown in Table 2. This is particularly significant for betamethasone valerate, of which seven molecules are solubilized per dendrimeric molecule. As previously mentioned (Sideratou et al, 2001), this was attributed to the presence PEG-chains. Additionally, in the case of betamethasone valerate the loading capacity is 11 wt% for the multifunctional dendrimer, i.e. almost double compared to the loading capacity of the simply PEGylated dendrimer (6 wt%) and more than five times compared to the loading capacity of the parent dendrimeric solution (1.7 wt %) (Sideratou et al, 2001). This is quite beneficial for its use as drug delivery system and it can only be attributed to the other two functional groups introduced at the surface of the multifunctional derivative. As it will be discussed below, they may act synergistically enhancing solubilization of betamethasone valerate.

Pyrene, as in the previous work (Sideratou et al, 2001), was in first place employed as model hydrophobic compound for probing the solubilization properties of the multifunctional dendrimer. For this reason fluorescence intensity (F/F⁰) changes and I₁/I₃ratio were monitored, which are sensitive parameters and their values depend on the medium of solubilization of the probe. These parameters were monitored by a titration-like addition of the dendrimer to an aqueous pyrene solution. A significant quenching of fluorescence intensity (F/F⁰) was observed and the I₁/I₃ ratio decreased (Figure 13). Fluorescence quenching was attributed (Sideratou et al, 2001) to the formation of a charge-transfer complex between pyrene and tertiary amino groups, as evidenced by the appearance of a weak exciplex fluorescence centered at approximately 485 nm (Lakowicz, 1983). As the concentration of the dendrimer increases, the I₁/I₃ ratio decreases to a value of about 0.90, which is close to the one observed in the hydrophobic environment usually encountered in the conventional micelles. Thus, pyrene is mainly incorporated inside the nanocavities of the dendrimer, in order to avoid contact with the hydrophilic external groups.

The release of the active ingredient from the dendrimer when it reaches the target site enhances its bioavailability and efficacy. In addition, drug release from endosomal compartment appears a limiting factor for several targeted drug delivery formulations (Boomer et al, 2003). These requirements impose the need for developing drug delivery systems in which the release of drug can be triggered by appropriate stimulus. For this purpose pH-triggered, enzymatic, thermal and photochemically induced processes have been reported (Boomer et al, 2003). For instance low pH within endosomal and ischemic tissue environments renders acid triggerable delivery systems attractive for controlled release.

The multifunctional poly(propylene imine) dendrimers prepared, due to the presence of tertiary amino groups in their core fulfill at least one of these requirements, i.e. being pH responsive (Sideratou et al, 2000; Sideratou et al, 2001; Paleos et al, 2004). As found in the previous experiment pyrene is solubilized in the interior of dendrimer and also within PEG chains, while upon protonation of tertiary amines of the nanocavities pyrene is repositioned in the PEG coat.

For achieving the release of the encapsulated pyrene from the PEG protective coat another method has, therefore, to be explored. We were prompted to use aqueous sodium chloride solution for triggering pyrene release since, as it has been established in independent studies (Wang et al, 2000; Bogan and Agnes, 2002), ions of alkali metals cationize poly(ethylene glycol) moieties through complexation. The designed multifunctional dendrimer, due to the attachment of PEG chains at its surface, is susceptible to analogous interactions and, therefore, it could be possible for metal cations to replace solubilized pyrene releasing it to the bulk aqueous phase. Indeed, by titrating dendrimeric solutions with sodium chloride solution, pyrene was released and dispersed in the bulk solution in the form of crystallites. The isolated crystallites were indentified by ¹H NMR and proved to be pure pyrene.

The two-step triggered release from the multifunctional dendrimer was also investigated using the lipophilic drug betamethasone valerate. Release of the drug with hydrochloric acid has not been observed since betamethasone valerate remained solubilized within the dendrimeric environment and preferably within the poly(ethylene glycol) chains. Betamethasone valerate encapsulated in the multifunctional dendrimer was completely released upon addition of sodium chloride as shown in Figure 14. However, within the concentration

Table 2. Comparative solubility of pyrene (PY) and betamethasone valerate (BV) in the parent fifth generation DAB and multifunctional dendrimer. Reproduced from Paleos et al, 2004 with kind permission from American Chemical Society.

Compound

[dendrimer] /M

[PY] /M

PY/Dendrimer molar ratio

[BV] /M

BV/Dendrimer molar ratio

DAB

1.0 x 10^-3

2.1_±0.2 x 10^-5

0.021_±0.002

2.5_±0.4 x 10^-4

0.25_±0.04

Multifunctional Dendrimer

2.5 x 10^-4

1.9_±0.08 x 10^-5

0.076_±0.002

1.80_±0.4 x 10^-3