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.

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).

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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

Proceeding with further functionalization and employing a third generation PAMAM dendrimer as a starting compound, multifunctional dendrimers were prepared (Shukla et al, 2003). These derivatives, in addition to the protective PEG-chains they also bear the folate moiety at the end of poly(ethylene glycol) chain which can induce endocytosis into folate receptor-bearing cells (Sudimack and Lee, 2000; Hofland et al, 2002; Antony, 2004; Sabharanjak and Mayor, 2004). The folate receptor is known to be significantly overexpressed over a wide variety of human cancers and, therefore, folate-mediated targeting has been widely applied with liposomes (Lee and Low, 1995; Lee and Huang, 1996; Gabizon et al, 2004), dendrimers (Kono et al, 1999; Konda et al, 2001; Shukla et al, 2003), various polymers and particles (Dauty et al, 2002; Dubé et al, 2002;Aronov et al, 2003; Zuber et al, 2003; Yoo and Park, 2004; Kim et al, 2005b;Licciardi et al, 2005; Wang and Hsiue, 2005) when used as drug delivery systems. In addition, in the previously functionalized dendrimer 12 to 15 decaborate clusters were covalently attached, which can be used for the treatment of cancer in Boron Neutron Capture Therapy(BNCT) requiring the selective delivery of¹⁰B to cancerous cells within a tumor. Varying number of PEG chains of varying length were linked to these boronated dendrimers to reduce hepatic uptake. Among all prepared combinations, boronated dendrimers with 1-1.5 PEG₂₀₀₀units exhibited the lowest hepatic uptake in C57BL/6 mice (7.2-7.7% injected dose (ID)/g liver).

Two folate receptor-targeted boronated third generation poly(amidoamine) dendrimers were prepared, the one shown in Figure 18, one containing ~15 decaborate clusters and ~1 PEG₂₀₀₀ unit with a folic acid moiety attached to the distal end, while the other was containing ~13 decaborate clusters, ~1 PEG₂₀₀₀ unit and ~1 PEG₈₀₀ unit with folic acid attached to the distal end. In vitro studies using folate receptor (+) KB cells demonstrated receptor-dependent uptake of the latter folic acid-functionalized derivative. Biodistribution studies with this derivative in C57BL/6 mice bearing folate receptor (+) murine 24JK-FBP sarcomas resulted in selective tumor uptake (6.0% ID/g tumor), but also high hepatic (38.8% ID/g) and renal (62.8% ID/g) uptake (Table 4), indicating that attachment of a second PEG unit and/or folic acid may adversely affect the pharmacodynamics of this conjugate.

In conclusion, the optimal modification of Boronated dendrimers as well as of dendrimers in general with PEG chains for reducing reticuloendothelial system affinity appears to be a highly complex process that depends on a variety of factors requiring extensive evaluation. The folic acid functionalized PEGylated G3-Boronated Dendrimer showed significantly increased tumor selectivity compared with non-PEGylated Boronated Dendrimeric-antibody and Boronated Dendrimer-EGF (Epidermal growth factor) conjugates previously evaluated for potential application in BNCT (Barth et al, 1994; Yang et al, 1997). However, the hepatic and renal uptake of this conjugate was very high.

III. Drug carriers: from monofunctional to multifunctional hyperbranched polymers

Based on polyglycerol (PG) and also on poly(ethylene imine) (PEI), pH-responsive molecular carriers were prepared (Krämer et al, 2002) through appropriate functionalization. The concept of pH-responsive carriers may have potential application for selective drug delivery in tissues of a lower pH value (for example, infected or tumor tissue). Polyglycerol and poly(ethylene imine), which are commercially available, are randomly branched but have defined dendritic structures with a degree of branching 60 to 75%. Functionalization of these dendritic polymers was achieved through a facile condensation reaction between the 1,2-diol or NH₂moieties at their external surface and various carbonyl compounds as shown in Figure 19. Several dendritic structures originating from these reactions have been prepared, differing in the following: a. the type and molecular weight of the core polymer, b. the structure of the attached peripheral shell and c. the degree of alkylation.

The loading capacities (number of encapsulated congo red per polymeric nanocarrier) of dendritic polymers together with their structural features are shown in Table 5. It was found that a minimum core size (ca. 3000 gmol^-1) and a highly branched architecture are required for successful encapsulation of the guest molecules. For efficient encapsulation the degree of alkylation should be about 45-50% and the alkyl chains should have a minimum length (>C10). For example, the conversion of the terminal groups in polyglylcerol, PG (21 000 gmol^-1) with a C16 aldehyde (PGa) containing one alkyl chain per diol unit results in an effective degree of alkyl functionalization of 25% (Table 5) and a poor encapsulation capacity (0.15 congo red molecules). With the same PG core (21 000 gmol^-1), the ketal functionalized carrier, PGb, with two alkyl chains per diol unit and 45% effective alkyl functionalization (Table 5) can encapsulate up to 13 congo red molecules. A higher degree of ketal functionalization (PGc: 55%, Table 5) indicates an optimal shell density of 45-50%. The exact determination of the encapsulation capacities for the amine based poly(ethylene imine) carriers was complicated because of the hydrolytic sensitivity of the imine-bound peripheral shell in the PEI-based systems, for instance in PEIb (Table 5). To avoid hydrolysis the dye was directly encapsulated from the solid/organic solution interface.

The complexation of an antitumor drug, mercaptopurine, several oligonucleotides, as well as bacteriostatic silver compounds (for example, AgI salts and Ag⁰ nanoparticles) (Haag et al, 2002) have been studied for the potential use of these carriers in drug and gene delivery. Successful encapsulation was observed in all cases by the PEI-based carriers while complexation was not observed with the PG-based carriers for the same guest molecules.

The objective to develop a pH-sensitive carrier was tested using several buffer solutions for both the acetal- and imine-bound shells. The encapsulated congo red in the carrier PGb was stable for several months at neutral and basic pH values (pH>7). However, an immediate release of the guest molecules occurred in acidic media (pH<3). The imine-based carriers were even more sensitive to an external decrease of pH. In the case of carrier PEIa (Table 5) the hydrolysis of the shell and the release of the encapsulated guest (namely, congo red) occurs over a period of four days at pH 6. However, it is stable over several weeks at neutral pH. On the other hand, the hydrolysis of the carrier PEIb and the release of the encapsulated guest occurs spontaneously even at pH<7. In the case of PEIb, a slow release can be observed after several hours (pH 8, ca. 3 h, 25 ^oC) or days (pH 12, 2 days, 25 ^oC) even without acidification. For the PEI-based carrier, PEIb, the amount of dye due to imine hydrolysis was followed by IR spectroscopy through the disappearance of the imine signal (Figure 20).

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Figure 18. Boronated third generation PAMAM functionalized with PEG-Folate moiety.

Table 4. Biodistribution of non-pegylated (A), PEGylated (B) and PEGylated with attached folic acid (C) boronated poly(amidoamine) dendrimers.*Reproduced from Skukla et al, 2003 with kind permission from American Chemical Society.

tissue	% ID of A/ g tissue	% ID of B/ g tissue	% ID of C/g tissue
blood	1.1_±1.3	n.m.	n.m.
liver	20.6_±5.0	7.1_±4.0	38.8_±5.9
spleen	14.7_±3.9	20.2_±8.8	25.0_±7.4
kidney	104.2_±20.7	45.0_±13.4	62.8_±14.5
muscle	n.m.	n.m.	n.m.
tumor	n.m.	n.m.	6.0_±1.6

* 24JK-FBP tumor bearing C57BL/6 mice were injected ip with A, B, and C. The % ID/g were determined 6h post injection. Mean ±SDs are based on four animals per group. n.m.: not measurable.

Figure 19. Functionalization of PG and PEI hyperbranched dendritic polymers. Reproduced from Krämer et al, 2002 with kind permission from the authors and Wiley-VCH.

Table 5. Encapsulation capacities of congo red in dendritic nanocarriers based on PG and PEI. Reproduced from Krämer et al, 2002 with kind permission from Wiley-VCH.

Structure	Mn core [gmol^-1]	Shell	Degree of alkylation	Encapsulation capacity
PG	21 000	-	-	-
PGa	21 000		25%	0.15_±0.05
PGb	21 000		45%	13_±4
PGc	21 000		55%	2_±0.5
PEI	25 000	-	-	0.02_±0.005
PEIa	25 000		33%	0.6_±0.1
PEIb	25 000		53%	0.2_±0.05

IV. Gene carriers: from monofunctional dendrimers to multifunctional dendritic polymers

Numerous gene delivery systems based on viral (Verma and Somia, 1997; Lotze and Kost, 2002) and non-viral (Li and Huang, 2000; Brown et al, 2001; Nishikawa and Huang, 2001) vectors have been developed and tested so far. Recently, several recurring issues about safety of viral vectors have led to a careful reconsideration of their application in human clinical trials and prompted the use of synthetic systems. Moreover, viral vectors experience significant limitation in large-scale production and the available size of DNA they can carry. For addressing these problems, non-viral gene delivery systems such as cationic polymers or cationic lipids, liposomes or cationic dendrimers have attracted great attention for achieving a breakthrough in the development of an effective gene carrier. Specifically, synthetic non-viral carriers of genetic material present insignificant risks of genetic recombinations in the genome. Transfection with synthetic vectors, through appropriate tailoring, may exhibit low cell toxicity, high reproducibility and ease of application. However, the currently known synthetic vectors present disadvantages, which are due to their generally low effectiveness compared to viral vectors and to their inability for targeted gene expression. For an effective gene expression, genes must be transferred in the interior of the nucleus of the cell and this procedure has to circumvent a series of endo- and exo-cell obstacles. Among these obstacles are included cell targeting, effective transport of the carriers together with attached genetic material through cell membranes and the need of carriers release from the endosome following endocytosis. For the synthetic carriers that have been described in the literature, some or all of these difficulties have been addressed, without, however, completely achieving this objective yet.

The strategy employed for the delivery of the conventional drugs through the preparation of functional dendritic polymers can also be applied for the delivery of genetic material. Specifically, the method involves molecular engineering of dendritic surface and/or the core aiming at obtaining polymers, which should be positively charged, biologically stable, non-toxic, exhibit targeting ability, and have the ability to be effectively transported through cell membranes. In addition, the dendrimer-DNA complex should have the possibility of being released from the endosome following endocytosis.

Dendrimers and hyperbranched polymers are stable nanoparticles in contrast to liposomes, which, as a rule, are unstable. Additionally, the dependence of dendrimers’ size on their generation can affect transfection efficiency. Several studies (Boas and Heegaard, 2004) have reported the use of unmodified amino-terminated PAMAM or DAB dendrimers as non-viral gene transfer agents, enhancing the transfection of DNA into the cell nucleus. The exact structure of these host–guest binding motifs has not been determined in detail, but it is presumably based on acid‑base interactions between the anionic phosphate moieties on the DNA backbone and the primary and tertiary amines of the dendrimers, which are positively charged under physiological conditions. It has also been found (Tang et al, 1996) that partially degraded (or fragmented) dendrimers, are more appropriate for gene delivery than the intact dendrimers and a fragmentation (or activation step) consisting of hydrolytic cleavage of the amide bonds is needed to enhance the transfection. These dendrimers are characterized as activated and are shown in Figure 24. It has been concluded from several investigations that the spherical shape of dendrimers is not advantageous in gene delivery. This agrees with earlier work, where ‘fragmented’ PAMAM dendrimers show superior transfection efficacy in comparison with the spherical ‘intact’ dendrimers (Boas and Heegaard, 2004).

In comparison to the intact dendrimers, the partially degraded dendrimers have a more flexible structure (fewer amide bonds) and form a more compact complex with DNA, which is preferable for gene delivery by the endocytotic pathway (Dennig and Duncan, 2002). In addition, it is generally found that the maximum transfection efficiency (Figure 25) is obtained with an excess of primary amines to DNA phosphates, yielding a positive net charge of the complexes The more flexible higher generation DAB dendrimers (containing no amides) are found to be too cytotoxic for use as non-viral gene vectors, however, the lower generation dendrimers are well-suited for gene delivery (Zinselmeyer et al, 2002).

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Constantinos M. Paleos

Drug	Dendrimer	Drug transfer (%) in aggregates		Drug transfer (%)after redispersion
Drug	Dendrimer	Water	Phosphate Buffer	Water	Phosphate Buffer
	DAB-G0	24.4_±2.4	19.8_±1.2	15.8_±0.9	12.1_±1.1
BD	DAB-G6	62.5_±1.9	48.5_±1.6	28.1_±1.7	24.5_±1.3
	DAB-G12	84.5_±2.1	68.4_±1.5	45.1_±1.8	40.0_±1.4
	DAB-G0	32.9_±2.0	27.1_±1.0	15.9_±1.2	14.1_±0.9
BV	DAB-G6	59.0_±1.5	39.5_±2.1	29.0_±1.0	26.1_±1.5
	DAB-G12	78.1_±2.3	57.5_±2.0	42.0_±1.5	38.2_±1.2

Hyperbranched Polymer	C_Polymer [M]	C_Tamoxifen [M]
PG	1.0 x 10^-3	9.6 x 10^-6
PG-PEG	1.0 x 10^-3	1.23 x 10^-4
PG-PEG-Folate	1.0 x 10^-3	2.48 x 10^-3

Review Article

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

I. Introduction

Shell

Figure 21. Functionalized PG-PEG and PG-PEG-Folate hyperbranched dendritic polymers.

IV. Gene carriers: from monofunctional dendrimers to multifunctional dendritic polymers

V. Concluding remarks

References