CHEMICAL AND PHYSICAL CHARACTERISTICS

III. CHEMICAL AND PHYSICAL CHARACTERISTICS

A. General Chemical Structure and Synthesis Dendrimers are composed of concentric, geometrically progressive layers

created through radial amplification from a single, central initiator core molecule containing either three or four reactive sites such as ammonia or ethylene diamine. Like the nucleus of the biotic cell, the core contains the basic information of the dendrimer; it defines the final size, shape, multi- plicity, and functionality of the entire structure. Starting from the center, each layer stores information, which is transferred to the next outer layer or generation via covalent connections, determining that layer’s physical prop- erties (17). This is accomplished by choosing reactants with additional reac- tive sites. Then through a series of protection/deprotection strategies, all the reactive sites on the core molecules are converted without reacting with the reactive sites of the additional reactants. As these steps are repeated, new generations are added to the dendrimer. The radial transfer of information distinguishes the synthetic dendrimer from the biotic cell that transmits information in a linear fashion using DNA and RNA.

The dendrimer amplification process can be described by the simplified synthesis of the basic PAMAM dendrimer (7,17). The first step adds metha- nol to the mixture of ammonia (NH 3 ), the initiator core (I), and methyl

470 Loutsch et al. acrylate (reactant B), causing a molecule of B to substitute for each of the

three hydrogen atoms on I. The second step, ethylene diamine, a primary amine is added to the end of each of the methyl acrylates, completing the first generation of the dendrimer. Each ethylene diamine moiety contains two N-H bonds at the end of each branch of the structure, generating six exposed reactivation sites. The second generation of the dendrimer is cre- ated by adding a methyl acrylate moiety at each hydrogen and then an ethylene diamine molecule to the end of the branch, yielding 12 exposed reactive sites (Fig. 1). Thus, with such successive generation, the number of reactive sites increases depending on the substrate added to B. The ampli- fication process can continue for about 9 or 10 generations, when the den- drimer is no longer able to form perfect branches due to steric hindrance (7,17).

Dendrimer molecules can be assembled via two pathways. The original pathway, the divergent process, involves working outward from the initiator molecule with the addition of each generation (17). This method of assembly can create a number of defects within the dendrimer. The defect can be in an individual dendrimer or between dendrimers within the mixture. The intra-

Fig. 1 Schematic drawing of dendrimer structures. Dendrimers are synthesized by two pathways, either the convergent or the divergent pathway. The convergent path- way consists of building the branches prior to addition to the core molecule (A). The divergent pathway consist of building the branches in concentric ‘‘layers’’ (B). Each layer is a generation (G).

Dendrimers 471 dendrimer faults are usually in the branching and are caused by incomplete

reactions, branch-juncture fragmentation, or abnormal development or sterically induced stoichiometry. The interdendrimer defects usually arise form the incomplete removal of reagents that may function as a core or dendrimer fragmentation, which may act as an initiator species that can act with other dendrimers (Fig. 2). Optimization of the process and synthesis strategies can eliminate these problems (17). In 1989, Hawker and Fre´chet (18) introduced the second pathway, the convergent process, which builds the branch wedge independently and then adds it to the root molecule (Fig. 1). This method allows for using ideal branches because they can be sepa- rated from the defective ones. This method may also allow for the creation of dendrimers that have branches with different functions, such as one branch that targets the dendrimer to a cell and a second branch that con- tains a drug (18). Although this process can generate sufficient dendrimers for the laboratory, it is not conducive to large-scale production (18).

Fig. 2 Defects within dendrimers. Interdendrimer faults can arise form incomplete reactions creating active sites that interact with other dendrimers. The incomplete branching in the top left dendrimer (intradendrimer) has a reactant B, which can react with z from a second dendrimer to interlink the dendrimer structures, creating a defective molecule.

472 Loutsch et al.

B. Physical Properties The physical properties of dendrimers depend on the chemical structure/

steric properties of internal and external functional groups. The shape may range from an almost perfect sphere to an ellipsoid to a cylinder-like struc- ture composed of intricately branched ‘‘fans’’ extending out of an elongated base (19). Changes in the core molecule and/or the number of reactive sites present on the core can also influence the shape. Newer dendrimers use phosphorus- or silicone-containing molecules, hydrocarbons, lysine, or thiols as the internal cores (7,17). The dendrimer can also be adjusted based on the selection of reactants. This also can affect the overall size of the dendrimer. Usually size is dependant on the number of generations assembled, but it can also be influenced by the length of the monomers used and the angles between the monomers (7). The average mass of a dendrimer varies from about 500 to 1500 daltons, with the diameter of a spherical dendrimer varying from about 10 A˚ (first generation) to a max- imum of about 130 A˚. Each polymerization step increases the diameter by about 10 A˚ (17).

Dendrimers are relatively stable, covalent macromolecules. They exist independently from each other and from their external environment. As the dendrimer grows, steric congestion eventually prevents further growth. At this point, the dendrimer converts from an open structure to a tight spheroid with open cavities and a dense surface (20). A specific dendrimer’s stability depends on the reactivity of the functional groups located on its surface, which can be modified to achieve the necessary level of stability.

C. Dendrimer Core (the ‘‘Guest-in-Box System’’) Naylor et al. (21) were the first to describe the interior dendrimer space and

to demonstrate the large carrying capacity of PAMAM dendrimers. Generations 4 through 7 were capable of encapsulating three times their weight in aspirin. Tomalia and Esfand (19) showed that the dendrimer interior also contains recognition properties specific to the shape and func- tionality of the guest molecule. The dissolving of water-insoluble molecules, such as ibuprofen, increased in the presence of the dendrimer structure. The ability to encapsulate drugs within the cavities of the dendrimers could result in novel delivery methods.

Jansen et al. (22) reported the synthesis of a ‘‘dendrimer box’’ that is based on a chiral shell of protected amino acids on the dendrimer core. During synthesis of the box, ‘‘guest’’ molecules and small amounts of sol- vent become trapped within the cavities; the molecules dissolve as they become tightly bound to functional groups on the interior ‘‘wall’’ of the

Dendrimers 473 box. Compatible guests such as drugs, DNA, or other small molecules

cannot be extracted, and their spontaneous outward diffusion is extremely slow due to the close packing of the shell. Only medium- to high-generation dendrimers possess dense enough shells to capture and retain guests (22). The release of guest molecules can be controlled by changing the pH of the reaction mix (23).

D. Dendrimer Surface The dendrimer surface ultimately determines the structure’s interactions

with its environment. The surface serves to protect the internal functional groups via steric interactions, which shield these groups from large mole- cules while retaining their accessibility and reactivity to small molecules. These interactions also maintain an outside versus inside position with ionic (polar) chain ends containing a high density of positively charged amino groups and a hydrophobic (nonpolar) interior (19). Dendrimer sur- face properties can be subdivided into endo-receptor properties and exo- receptor properties. Endo-receptor properties are the interior dendritic fea- tures such as size, chemical composition, flexibility, and topology, which are responsible for the so-called convergent recognition of guest molecules by the internal dendrimer surface (17). Exo-receptor properties encompass the exterior features, such as shape, reactivity, stoichiometry, flexibility, and fractal character, which similarly govern the divergent recognition of exter- nal associator molecules by the external dendrimer surface (17).

The dendrimer surface determines its solubility through chemical recognition of different external reagents/solvents and affects the reactivity of the molecule through the number of reactive surface groups (17). The surface can serve as scaffolding for up to thousands of reactive functional groups such as carbohydrate residues and peptidyl epitopes. Functional groups on the surface of dendrimers show higher chemical reactivity than the same groups attached to other polymer molecules (24). This ‘‘charging’’ allows for important covalent coupling reactions with DNA and other mole- cules. Furthermore, biologically active molecules, when complexed with dendrimers, tend to retain their maximum activity even when surface groups on the dendrimer are activated for a reaction.

Modifications of the dendrimer surface, specifically the addition of functional groups, occur through the addition of either subnanoscopic or nanoscaled reactants (19). Subnanoscopic reactants can be a variety of small molecules, while nanoscaled reactants are composed of larger molecules such as DNA, antibodies, and proteins that can complex with dendrimers. Scott et al. (25) describe the rapid synthesis of a second-generation dendri-

474 Loutsch et al. mer with primary alcohol groups at the periphery. (The use of modified

dendrimers in bioorganic chemistry is reviewed in Ref. 26.)

IV. TISSUE DISTRIBUTION OF DENDRIMERS For dendrimers to be a viable delivery system for drugs and genes, as well as

imaging contrast agents, it must be possible to administer them systemically or orally with reliability. They also must have low cytotoxicity, low immu- nogenicity, and few adverse side effects.

To determine the potential for dendrimers as drug delivery molecules, Wiwattanapatapee et al. (27) used various [ 125 I]-labeled dendrimers in an ex vivo system, the everted rat intestinal sac model. The distribution of the dendrimers was monitored through the wall of the gut by measuring the radioactivity in the serous fluid and tissues. Anionic PAMAM dendrimers (generation 2.5 and 3.5) had faster, concentration-dependent serosal transfer than cationic PAMAM dendrimers (generation 3 and 4). The anionic PAMAM generation 5.5 and the cationic PAMAM dendrimers had high levels of tissue uptake, thereby slowing the transfer rate (27). These results indicate that these dendrimers have potential as an oral drug delivery system and require an in vivo study.

Sakthivel et al. (28) and Florence et al. (29) gave Sprague-Dawley rats an oral dose of a lipidic peptide dendrimer and monitored tissue distribu- tion. The radiolabeled 2.5 nm dendrimer (generation 4) was given by oral gavage. The first study revealed that the maximum level of the dendrimer was found in the small intestine, with decreasing amounts in the large intes- tines, blood, and other organs. The amount absorbed by the lymphoid tissue of the intestinal tract was greater than the amount in the nonlymphoid tissues, based on organ weight at 3 and 24 hours (28). In the second study, Peyer’s patches were found to absorb more of the dendrimer than normal enterocytes in the small intestine, while the opposite was true in the large intestine, with enterocytes absorbing more (29). The uptake and trans- port of these lipidic dendrimers was lower than expected based on polystyr- ene particle data and may indicate that there is an optimum size for dendrimer uptake in the intestines.

Malik et al. (30) investigated the relationship between dendrimer struc- ture and biocompatibility in vitro. Dendrimers with NH 2 termini had a concentration-dependent hemolysis and red cell morphology after 1-hour incubation with rat red blood cell suspension. The PAMAM dendrimers also demonstrated generation-dependent hemolysis. Those with carboxylate termini had no hemolytic or cytotoxicity in cell culture (30). In vivo analysis of cationic and anionic PAMAM dendrimers in the Wistar rat revealed that

Dendrimers 475 third- and fourth-generation cationic dendrimers are cleared faster than

generations 2.5, 3.5, and 5.5 of the anionic dendrimers. In the case of the anionic dendrimers, the longer clearance times were generation dependent, with the smaller dendrimers remaining in the blood longer (30). The sys- temic clearance correlated with the increased accumulation of the radiola- beled dendrimer in the liver. However, there was no difference between intravenous and intraperitoneal administration. Dendrimers with low toxi- city and carefully tailored surfaces that remain in the circulation with mini- mal hepatic uptake, especially if they need to reach a target organ, are needed if they are to be used for oral drug delivery. The effect of dendrimer degradation products on tissue also needs to be addressed.

Wiener et al. (31) and Konda et al. (32) adapted dendrimers for use as magnetic resonance (MR) imaging agents. Second- and sixth-generation dendrimers were linked to a chelator that enhanced conventional MR ima- ging (31). These dendrimers showed enhanced contrast in the heart, liver, kidneys, and blood vessels of Fisher 344 rats. The higher molecular weight dendrimer may aid in the three-dimensional time-of-flight MR angioplasty due to the prolonged clearance time. A fourth-generation dendrimer was linked to folic acid and a chelating agent for use in targeting tumors with folic acid receptor expression (32). Studies in nude mice with ovarian tumors (positive for folic acid receptor expression) showed enhanced contrast 24 hours following administration of the agent while receptor-negative tumors did not. These new contrast agents will aid in the imaging of tumors and normal tissues.

V. THERAPEUTIC USES OF DENDRIMERS The use of dendrimers in drug and gene therapy is increasing. Table 1

provides a summary of studies that used dendrimers or other small synthetic macromolecules. A discussion of dendrimer-related studies is given below.

A. Dendrimer-Mediated Delivery of Drugs Studies have shown dendritic drug delivery to tissues to be very promising.

Some of the advantages of dendrimers include their capacity to hold a large variety of molecules and to protect these molecules from their surroundings until they are released in a time-dependent, controlled manner. Dendrimers that are synthesized to mimic micelles (hydrophobic inner layer and hydro- philic outer layer) can create a micro-container to transport drugs. The hydroscopic drugs trapped within this space are protected from the envir- onment and have a reduced rate of uptake by the liver and prolonged

476 Table 1 Use of Nanoparticles, Including Dendrimers, Liposomes, and Micelles, for Drug and Gene Delivery In Vivo and In Vitro

Model Ref. PAMAM

Nanoparticle

Drug/Gene/Virus

Application/Target

Epstein-Barr virus plasmid vector

Hepatocellular carcinoma

In vitro, cell culture 49

cells

PAMAM

In vivo, SCID mice 69 PAMAM

Epstein-Barr virus plasmid vector

Intratumoral injection

In vivo, mice 32 PAMAM (G4)

Folate receptor

Ovarian tumors

In vivo, mice 40 PAMAM, Linear

Cisplatin

Cancer chemotherapy

In vitro 35 polyacrylamide polymers,

Influenza viruses

Block sialic acid receptor

and prevent viral

dendron and

attachment

dendrigraft PAMAM (G0-4)

Biotinylated residues

Pretargeting of cancer cells

In vivo, mouse 70

to allow the increase of radioactivity

PAMAM (G2)

71 PAMAM

Folate residues or methyltrexate

Target tumor cells

Inhibit viral challenge in

In vivo, mice 36

vaginal model of HSV

PAMAM, liposomes

Normal cystic fibrosis

Replacement gene therapy

In vitro 61

transmembrane regulator gene

In vitro 72,73 Poly(d,l-lactide)

PAMAM

Cholera toxin

Inhibition of adherence

In vitro and in 74 Loutsch nanoparticles

Platelet-derived growth factor

Prevent restinosis

vivo, rat PAMAM (G5)

inhibitor

In vivo, mice 53 Poly(lactide-co-glycolide)

IL-10

Cardiac graft rejection

In vitro and in vivo 75 et microspheres

Neurotrophic factors and

Blood-brain barrier

antimitotic drugs

al.

Dendrimers PAMAM (G4)

Epidermal growth factor

Target tumor cells for neutron

In vitro and in 52

vivo, rats Cationic dendrimer

capture therapy

Folate-conjugated

Folate receptor–mediated gene

In vitro 50

therapy

PAMAM

Magnetic resonance imaging

Tumor imaging

In vivo 31

contrast agent

PAMAM (G2, G4)

Monoclonal antibody

Neutron capture therapy

In vivo, mice 51

immunoconjugate

PAMAM

Antisense oligonucleotide

Gene expression, rescue

In vitro 46

defective genes

Polyethyleneimine

Gene and oligonucleotide

Target brain tissue

In vitro and in 76

vivo, mice n -(2-Hydroxypropyl)

transfer

In vivo, humans 42 methacrylamide PAMAM

Doxorubicin hydrochloride

Chemotherapeutic agent

Poly(ethylene glycol) grafts

Chemotherapeutic agents

In vitro 41

and anticancer drugs

Dendritic unimolecular

In vitro 33 micelles

Indomethacin

Anti-inflammatory

478 Loutsch et al. systemic half-life (33). The anti-inflammatory drug indomethacin could be

incorporated at a rate of nine to ten molecules per micelle, with sustained in vitro release (33).

1. Antibacterials Dendrimers could assist in counteracting bacterial infections by serving as

transport facilitators for antibiotics and antisense nucleotides or by binding to receptors on the bacterial cell, thereby preventing infection. The anti- sense approach is based on the ability of oligonucleotides to interfere with the biological activity of bacteria by interacting with a necessary protein or hybridizing to the bacterial mRNA. The latter aptameric interaction blocks the synthesis of important bacterial proteins.

Oligonucleotides function at the bacterial ribosome or nucleus and can enter eukaryotic cells via endocytosis. However, the hydrophobic exterior of prokaryotes serves as a barrier to oligonucleotides. Attia et al. (34) reported the use of a small fourth-generation dendrimer combined with ethambutol (a metabolic cell wall inhibitor), which acted as a transport facilitator for oligonucleotides to interact with Mycobacterium tuberculos in vitro. Further investigation is needed to determine the effect of the oligonucleotides on treatment of M. tuberculosis.

2. Antiviral Agents Dendrimers can be used to counter viral activity in several different ways.

They can be modified with external residues that inhibit virus-host cell interactions (35), act as antivirals themselves (36), or serve as delivery vehi- cles.

Influenza viruses infect human cells by binding the viral hemagglutinin to the sialic acid receptor on the host cell and then undergoing receptor- mediated endocytosis of the virus into the cell. The influenza virus can modify the antigenicity of the hemagglutinin and therefore can evade neu- tralizing antibodies as well as antiviral compounds (35). Synthetic dendri- mers containing sialic acid moieties on the surface layer can successfully compete with hemagglutinin for the host cell. This interaction can prevent adhesion to the host cell and subsequent infection. These dendrimers are polyvalent inhibitors, meaning that one dendrimer can interact with many hemagglutinin molecules on one virus at one time. This cooperative inter- action of individual receptors increases the overall binding affinity. Reuter et al. (35) determined that binding of the influenza virus hemagglutinin and neuraminidase glycoproteins with sialic acid residues attached to a dendri- mer resulted in a dose-dependent decrease in influenza infection in vitro.

Dendrimers 479 These dendrimers, which were nontoxic to cells, were the first multivalent

sialic acid inhibitor shown to be effective in vitro. Bourne et al. (36) investigated the potential of five different polylysine PAMAM dendrimers (containing a central benhydrylamine core) as topical microbicides for herpes simplex virus (HSV) types 1 and 2. In vitro studies showed that the dendrimer BRI-2999 could block HSV infection in cyto- pathic effect (CPE) inhibition assays. In vivo studies in the mouse vaginal model of HSV infection determined that the application of a topical solution of BRI-2999 before viral challenge could significantly reduce the occurrence of disease, with no noticeable signs of toxicity to the mice. The dendrimer prevents normal virus-host cell adhesion/adsorption by complexing with the virus. No effect was seen when the dendrimer was applied after the viral challenge. Further evaluation is needed to determine if these compounds can

be used to reduce the spread of sexually transmitted diseases. Additionally, the use of dendrimers has produced promising in vitro inhibitory activity against respiratory syncytial virus. BRI-2999 had potency in the CPE inhi- bition assay but also had some cytotoxic effects on stationary phase cells (37). In HIV studies, polyanionic dendrimers showed inhibition of HIV replication in MT4 cells (38). The interference came during two phases of the viral life cycle: adsorption and reverse transcription. In vitro efficacy was also seen against cytomegalovirus, HSV-1 and HSV-2, thymidine kinase– deficient HSV-1, vesicular stomatitis virus, yellow fever virus, reovirus type

1, and dengue fever virus (38).

3. Antitumor Drug Therapy Duncan and Malik (39) use doxorubicin and cisplatin as model drugs to

study the potential of dendrimer-anticancer therapeutic agents. They found that these compounds retained biological activity in vitro and may be useful in vivo. PAMAM dendrimers were conjugated to cisplatin to create a den- drimer-platinate molecule that was highly water-soluble and released the drug slowly (40). This compound displayed antitumor activity and was less toxic than cisplatin alone. Kojima et al. (41) report that PAMAM dendrimers with poly(ethylene glycol) grafts possessed a space that could carry anticancer drugs with a biocompatible surface, such as adriamycin and methotrexate. Increasing generations and chain length of the poly(ethylene glycol) grafts allowed for increasing amounts of drugs to be encapsulated within the space. The dendrimer carrying methotrexate released the drug slowly in an aqueous solution but rapidly in an ionic solution (41). Vasey et al. (42) used a polymer linked to doxurubicin (adriamycin) in a phase 1 clinical trial. The drug-polymer complex (PK1) was given to patients with refractory solid tumors such as colorectal, breast, ovary, pancreas, and

480 Loutsch et al. others. PK1 was found to decrease the nonspecific organ toxicity and side

effects to the drug while maintaining antitumor activity without polymer toxicity (42). Although this study did not employ a dendrimer, it demon- strates that polymers could play as significant role in future cancer che- motherapy regimens.

B. Dendrimer-Mediated Gene Therapy for Cancer and Genetic Disease

Gene therapy is an exciting new field focusing on the treatment of illnesses via genetic modification. Conventional medicine often manages to increase the patient’s degree of comfort by alleviating the symptoms of the disease while not actually treating the disease. Gene therapy may hold the key to a more permanent solution by seeking to correct the problem at the root of the disease—the defective gene. A reliable gene-delivery vehicle is an indis- pensable component of gene transfer therapy. Current gene-delivery systems use viruses or liposomes as vectors. However, these vectors have many problems such as immunogenicity or carcinogenicity and potential infectiv- ity (43). The characteristics of the dendrimer (nontoxic, nonimmunogenic, and nonviral) make it a very good candidate for gene therapy. (The use of Starburst TM dendrimers in gene transfer is reviewed in Ref. 44.)

The electrostatic interactions between DNA (negative charge) and the dendrimer (positive charge) yield a complex that forms quickly. The com- plex is stable over a wide range of pH (45). The DNA condenses when it contacts the polymer, and the degree of condensation is defined by the generation of the dendrimer, the concentration of the DNA, and the DNA : dendrimer charge ratio (46). As the DNA : dendrimer charge ratio increases, so does the biological function of the complex. Electron micro- scopy shows that DNA complexed with polylysine or whole dendrimer aggregates in solution, whereas DNA complexed with polyethylenimine or fractured dendrimer remains as single discrete units (47). Bielinska et al. (46) and Kukowska-Latallo et al. (45) reported that DNA-dendrimer complexes have a broader concentration range between transfection and cytotoxicity in numerous cell lines.

The efficiency of in vitro transfection increases exponentially with the increase in the generation number of the dendrimer (45). The DNA is pro- tected from nuclease degradation while bound to the dendrimer; however, the DNA is unable to initiate transcription from the promoter. This may be due to the inaccessibility of the promoter to the polymerase enzyme because the secondary and tertiary structure of the DNA is altered when it is com- plexed with the dendrimer. Elongation of the RNA transcript and transla- tion do not appear to be affected in these molecules (48). Additional in vivo

Dendrimers 481 studies are needed to determine cellular localization and transfection effi-

ciency with these dendrimer-DNA complexes.

1. Cancer Gene Therapy One interesting method for treating certain cancers is to program the tumor

cells to self-destruct. This can be achieved by inserting a viral gene, usually herpes simplex virus thymidine kinase (TK), directly into tumor cells, enabling the cells to produce the enzyme. The tumor is then treated with ganciclovir, the substrate of TK, creating the active form of the drug that causes the cancer cells to ‘‘commit suicide.’’ Harda et al. (49) transfected human hepatocellular carcinoma cells with an Epstein-Barr virus–based plasmid vector containing the TK gene complexed with either PAMAM dendrimers or cationic liposomes. Addition of therapeutic concentrations of ganciclovir resulted in a significant decrease in the number of viable carcinoma cells in the dendrimer-treated cultures. Carcinoma cells trans- fected with the liposome vector alone survived the same dose of ganciclovir. The dendrimer-based vectors showed increased efficiency of gene transfer. These results demonstrate the successful use of dendrimers in the transfer of suicide genes in vitro (49).

Dendrimers are being considered for use in targeting therapy to solid organ tumors. Reddy et al. (50) used a folate-targeted, cholesterol-stabilized liposomal vector linked to a sixth-generation dendrimer to target tumor cells. Certain cancer cells over-express folic acid receptor on their surface, making this a viable mechanism for targeting therapeutic and imaging agents to the tumor. The folic acid–linked dendrimer had a higher transfec- tion efficiency compared to the control dendrimer in cell culture (50).

Another method of cancer chemotherapy is to target boron-10, a stable isotope, to the tumor and then activate it by the use of low-energy irradiation, which excites the boron-10 and causes the death of the cancer cell. Barth et al. (51) used boron-10 coupled to a Starburst dendrimer and conjugated to an antibody against mouse B16 melanoma to treat mice with tumors. They found that the immunoconjugate was localized largely in the liver and spleen rather than in the tumor, even though the antibody is very specific for the tumor in vitro (51). This novel approach to targeting and treating tumor cells could be effective if antibody specific for the tumor in vivo can be developed. Yang et al. (52) exploited the fact that gliomas overproduce the epidermal growth factor receptor on the surface of the cancer cell. They linked epidermal growth factor to a boronated fourth- generation Starburst dendrimer and administered it either intravenously or intratumorally to rats with glial tumors. The intratumor injection resulted in 22% and 16% localization in the tumor at 24 and 48 hours,

482 Loutsch et al. respectively, whereas the intravenous dose showed virtually no localization

to the tumor (52). Additionally, the intravenous dose had a greater localiza- tion to the liver and spleen than the intratumor injection. These results indicate that intracerebellar injection could be the most effective treatment delivery system in epidermal growth factor receptor–positive tumors.

2. Cardiac Gene Therapy Qin et al. (53) used plasmid-mediated gene transfer and subsequent expres-

sion in a mouse cardiac transplant model to improve graft survival. A

MHC-vIL-10 was complexed with G5EDA dendrimer, a 60-fold decrease in DNA resulted in significantly prolonged graft survival to 38.6 days (53). Increasing the amount and time of immunosuppressive cytokine expression in more tissues allowed for further prolongation of graft survival. This novel method of local immunosuppression could be beneficial to patients under- going solid organ transplantation.

A concern for dendrimer-mediated therapy is cytotoxicity at the site of the dendrimer localization. Brazeau et al. (54) report that PAMAM dendri- mers alone had high myotoxicity in an isolated rat muscle cell. The toxicity decreased when the dendrimer was coupled to a DNA plasmid but not to the level of saline or DNA alone (54). While this is an in vitro system, it is imperative that toxicity studies be examined in vivo.

3. Genetic Disease Therapy Oligonucleotide therapy has limiting factors that may reduce its usefulness.

The majority of these therapies involves the incorporation of antisense oli- gonucleotide sequences that bind to the mRNA of cells and interfere with protein synthesis. Fomivirsen, and antiviral, is a successful antisense oligo- nucleotide therapy that is clinically available for the treatment of cytome- galovirus retinitis (55–57). The factors that limit the usefulness of oligonucleotide therapy are degradation, inefficient cellular uptake and intracellular transport, and binding to the RNA target. The use of dendri- mers could alleviate these problems by the generation of cell-targeting den- drimers that could deliver the gene to the appropriate cell.

Yoo and Juliano (58) used fifth-generation dendrimers labeled with Oregon green 488 and an oligonucleotide labeled with TAMRA, a red fluorophore to determine localization in transfected HeLa cell by fluores- cence microscopy. The oligonucleotide would correct a splicing error in the reporter gene, luciferase, if the transfection was successful and functionally

Dendrimers 483 active. They found luciferase activity in the cells as well as the dendrimer

and oligonucleotide localized in close proximity to one another. Interestingly, the dendrimers with Oregon green 488 enhanced the delivery of the PAMAM dendrimer-oligonucleotide complex to the cells compared to the unlabeled dendrimer-oligonucleotide complex (58). Helin et al. (59) used FITC-labeled oligodeoxynucleotides (ODN) with and without dendri- mers as carriers to determine cellular uptake and intracellular distribution in various cell lines (Table 1). The FITC-ODN had increased cellular fluores- cence when complexed with dendrimers. Some cells had perinuclear fluor- escence and some cells had intense nuclear staining (59). These studies indicate that oligonucleotide can reach the nucleus, the target for gene therapy. Nilsen et al. (60) suggest that dendrimers can be constructed from nucleic acids for use as a ‘‘nucleic acid membrane.’’ This structure could be useful in gene therapy but to date has not been tested.

Cystic fibrosis is a genetic disease caused by a deletion mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that affects the pulmonary system. Patients, mostly children, suffer from respira- tory congestion and numerous lung infections. Goncz et al. (61) used den- drimers to deliver a fragment of DNA that could correct the mutated sequence in CFTR. The small single-stranded DNA fragment (488 nucleo- tides long) was complexed with a Starburst dendrimer and used to transfect primary normal airway epithelial cells (61). The DNA fragment corrected the defective gene in 1 out of 100 cells, a range that could have therapeutic potential. This method of small fragment homologous replacement therapy could be developed into gene therapy strategies for disease caused by site- specific mutations.

C. Additional Therapeutic Uses of Dendrimers Dendrimers linked with DNA can retain their ability to transfect cells after

drying, unlike viral vectors. Bielinska et al. (62) used this novel property of dendrimer-DNA to coat a solid-phase, bioerodable support or to embed the complex in the support. The DNA was a plasmid that expressed either luciferase or green fluorescent protein when transfected into cells. Expression of luciferase or green fluorescent protein was detected following release of the complex from the support or while still attached (62). A membrane that had a dendrimer-DNA complex on the surface was placed on the skin of a hairless mouse. Chloramphenicol transacetylase activity was found in the tissue under the membrane indicating that the DNA was deliv- ered to the skin from the membrane (62).

Kawase et al. (63) created a high-performance, bio-artificial liver sup- port system using fructose-modified dendrimers immobilized on polystyrene

484 Loutsch et al. dishes. The modified dishes were capable of increasing the number of hepa-

tocytes attached to the surface, which in turn increased urea synthesis. The number of cells remained high as the fructose-modified dendrimers appeared to suppress apoptosis (63). Increasing the generation of the dendrimer led to increased numbers of hepatocytes adherent to the support system. This may

be a novel way to treat fulminant hepatic failure, similar to renal dialysis.