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Review

Journal of Pharmacy And Pharmacology

Dendrimers for gene delivery – a potential approach for ocular therapy? Sahil P. Chaplota and Ilva D. Rupenthalb a Drug Delivery Research Unit, School of Pharmacy and bDepartment of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand

Keywords dendrimers; gene therapy; non-viral vectors; ocular diseases Correspondence Ilva D. Rupenthal, Department of Ophthalmology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: [email protected] Received April 21, 2013 Accepted June 15, 2013 doi: 10.1111/jphp.12104

Abstract Objectives A vast number of blinding diseases have genetic aetiologies and may be treated by molecular based therapies such as antisense oligonucleotides or short interfering RNA. However, treatment success of ocular gene therapy is highly dependent on efficient delivery of such molecules. Key findings The majority of clinical studies for ocular gene therapy utilize viral vectors. While these have proven highly efficient, they show limited loading capacity and pose significant safety risks owing to their oncogenic and immunogenic effects. Non-viral gene carriers have emerged as a promising alternative with dendrimers providing great potential for gene therapy because of their size, shape and high density of modifiable surface groups. However, while dendrimers have been used extensively for drug and gene delivery to other organs, only a few studies have been reported on the eye. Summary This review focuses on the development of dendrimers for gene delivery with special emphasis on ocular gene therapy. Different synthesis approaches and types of dendrimers are discussed. Ocular gene therapy targets are highlighted with an overview of current clinical studies. The use of dendrimers in ocular gene delivery in comparison to liposomes and nanoparticles is also discussed. Finally, future prospects of tailored multifunctional dendrimers for ocular gene therapy are highlighted.

Introduction Historically, the chemistry of polymers mainly focused on linear-shaped molecules. However, in 1978, Buhleier et al.[1] firstly described the synthesis of novel molecules having a globular, nano-sized architecture called ‘dendrimer’ (Dendron = tree and meros = branch (from Greek)), which, in the mid-1980s, was followed by the synthesis of polylysine dendrimers by Denkewalter et al.,[2] cascade molecules by Newkome et al.[3] and star polymers by Tomalia and Dewald.[4] In the same way that dendrites have a central nucleus followed by branches, dendrimers have a central core that is surrounded by overlapping and branched repeating units containing the active sites. As a result, dendrimers generally consist of three main parts: (1) an inner core, (2) highly branched repeating units called layer of generations and (3) peripheral multivalent functional groups which play a key role in gene-complexing (Figure 1).[5–8] Larger dendrimers can be produced by repeating the reaction steps, leading to higher generation 542

dendrimers, which in turn leads to doubling of active sites and molecular weight.[9] Because of the complexity and multibranching properties, which allows attachment of various functional groups, dendrimers have gained increasing interest in the area of drug delivery.[9] A number of recent reviews have described the role of dendrimers in drug and gene delivery as well as their potential as biomimetic artificial proteins, nano-drugs, nano-scale scaffolds and imaging agents.[6,10–14] This review outlines recent advances in dendrimer synthesis and gives an overview over the most common types of dendrimers for gene delivery. Special emphasis is thereby placed on ocular gene targets and recent developments in the use of dendrimers for ocular gene therapy.

Dendrimer synthesis Two main approaches have evolved for the synthesis of dendrimers. The first method developed by Tomalia[15] is known as the ‘divergent method’ in which dendrimers grow

© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 542–556

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Dendrimers for ocular gene delivery

predominantly at advanced generations, to obtain the desired product, which can be costly and timeconsuming.[8] Nevertheless, the divergent method of dendrimer synthesis is currently the most favoured commercial strategy used by international producers such as Dendritic Nanotechnologies Inc. (Michigan, USA), DSM (Heerlen, the Netherlands), Perstorp Holding AB (Perstorp, Sweden) and Qiagen (Hilden, Germany).[9,17]

Convergent growth process

Figure 1 Schematic representation of a generation 3 dendrimer containing an inner core (blue), branching and functional groups (red).

in a fashion originating from an inner core. The second approach by Hawker and Fréchet[16] follows the ‘convergent growth process’. Here, distinct dendrons are fused with a multivalent core to produce a new compound. To date, over 100 structurally different dendrimer families have been synthesized on the basis of these two approaches, and a number of variations and additions have been made to result in more efficient and reproducible dendrimer synthesis.

Divergent method This method deals with the formation of the dendrimer from the inner core, which is retorted with a reagent containing a minimum of two protecting branching sites, followed by elimination of the protecting groups. The liberated active sites form the first generation dendrimer. By repeating this process, a dendrimer of the desired size can be obtained. Polyamidoamine (PAMAM) dendrimers were the first synthesized by the divergent method using the Michael addition of amino groups (existing either at the terminal groups of a preformed dendrimer or an initiator inner-core molecule such as ethylenediamine or ammonia) to methyl acrylate, followed by the reaction of ethylenediamine with the intermediate methyl ester (Figure 2).[7] Each repetition of these steps leads to a new generation of dendrimer, which results in the amplification of the molecular size and the active terminal groups by a factor of two. Using this method requires extreme monomer stocking and prolonged chromatographic separations,

The second method for dendrimer synthesis is the convergent growth process, which starts at the periphery of the dendrimer and moves towards the interior by progressively linking surface active units together. Adequate branches are hereby attached to an appropriate inner core to produce a complete dendrimer.[18] Poly(aryl ether) dendrimers are generally synthesized by this technique (Figure 3).[7] The main advantage of this approach is the ability to precisely control the molecular weight and produce dendrimers having functional groups at the accurate and exact position. Moreover, inactive products can easily be removed by purification after each generation of dendrimer. Hence, products are more standardized although the purification of higher generation dendrons may become difficult as a consequence of increasing resemblance between the reactants and the formed products. Therefore, convergent growth approaches are generally restricted to the creation of lower generation dendrimers because of nanoscale steric problems arising when conferring the dendrons to the core.[19]

Recent advances in dendrimer synthesis One of the major challenges for commercial utilization of dendrimers has been the simplification of the synthesis method. Novel synthesis approaches have mainly focused on speeding up the synthesis process by preassembly of oligomeric branches which can then be linked together to reduce the number of synthesis steps involved as well as increase the dendrimer yield. This approach has recently been described as ‘hypercore and branched monomers’ growth.[9] The ‘double exponential’ approach allows the synthesis of monomers for both the convergent and the divergent growth from a single starting material and is very similar to the synthesis used for large linear polymers. The reaction of the two intermediate products results in an orthogonally protected trimer, which can be used for repeating the growth process over and over, resulting in really fast synthesis of larger dendrimers.[9] More recent approaches also include ‘Lego’ and ‘click’ chemistry.[12] In the ‘Lego’ approach, highly active cores and branched monomers are used to prepare phosphorus dendrimers, allowing the multiplication of the number of peripheral

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Sahil P. Chaplot and Ilva D. Rupenthal

MeO2C NH2

H N

CO2Me

H

H N

H2N

CO2Me N

H

N

O

NH2

NH2 N

CO2Me

H N

O

CO2Me N

NH

O

MeO2C

CO2Me

N

O O

HN

CO2Me

NH O

CO2Me

HN H2N

MeO2C

N CO2Me

H2N

NH2 O

NH2

NH O

HN NH2

NH2

N

H N

O

NH O

N

Higher generations of PAMAM dendrimers

NH

N

Repeat Michael addition and amidation steps

O

O NH

O HN

O

N H

NH2

N

NH2 O

Figure 2

NH2

Synthesis of polyamidoamine (PAMAM) dendrimers by the divergent method.[7]

surface active groups from 48 to 250 in just one step. This process requires only a minimal amount of solvent and by-products such as nitrogen and water are environmentally friendly.[20] With ‘click’ chemistry, dendrimers with various surface groups can be produced with high purity and yield due to the near-perfect reliability of the Cu(I)catalysed synthesis of 1,2,3-triazoles from azides and alkynes. All second generation and some third generation dendrimers can be directly isolated as pure solids without chromatographic separation and purification, with sodium chloride being the only major by-product, making this approach not only very efficient, but also environmentally friendly.[21]

Dendrimers for non-viral gene delivery Various viral and non-viral gene delivery systems have been developed for the delivery of genetic materials to 544

N H

different organs, tissues and cells. While viral vectors are the most capable vehicles, their limitations include their high carcinogenicity and immunogenicity in vivo. Nonviral vectors on the other hand generally exhibit lower efficiency but higher safety and tractability.[22,23] A number of recent review articles has discussed the use of various non-viral gene delivery systems including liposomes,[24,25] nanoparticles[26–30] and dendrimers.[6,10–13,23,31–36] Dendrimers have gained great interest as gene delivery vehicles because of their monodisperisity, high density of functional groups, well-defined shape and multivalency.[5,37] They are able to form condensed polycations under various chemical and physiological conditions with the ability to bind to negatively charged nucleic acid molecules.[23,38] Complex formation is thereby not only dependent on the stoichiometry and the concentration of the nucleic acidphosphates and dendrimer-amines, but also on the pH, the salt concentration and the buffer strength.[6] An overall net positive charge of the dendrimer-nucleic acid complex, also

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Sahil P. Chaplot and Ilva D. Rupenthal

Dendrimers for ocular gene delivery

O

HO Br

OH

O

CH HO Monomer

K2CO3

O

CBr4/PPh3

K2CO3

+

CBr4/PPh3

O

Br

O

O

Monomer O

O

O

O

OH

O

i. K2CO3 monomer ii. CBr4/PPh3

O

O

O

Br

O

O O

O

Higher generations

O

Repeat coupling to monomer and activation steps

O

O O

O

O

O

O

Br

O O

O O

Figure 3

Poly(aryl-ether) dendrimer synthesis by the convergent growth process.[7]

called dendriplex, is thereby required to allow binding to the negatively charged cell membrane and thus facilitate cellular uptake.[23] However, highly cationic systems may also be cytotoxic,[6] with higher generation dendrimers generally being more toxic. The degree of substitution and amine functionality also play an important role, with primary amines generally being more toxic than secondary or tertiary amines.[39] A balance between cellular uptake and cytotoxicity therefore needs to be established. Moreover, measures for endosomal escape should be in place, as the cargo needs to be released into the cytoplasm to be able to perform its action. Thus, fusogenic peptides such as hemagglutinin 2 may be incorporated into the dendrimer structure, although this is generally not required for polyethyleneimine (PEI) and PAMAM dendrimers acting as a ‘proton sponge’. The proton sponge effect leads to decelerated acidification of the endosome and increased accumulation of osmotically active Cl– ions, eventually resulting in lysis of the endosome wall and release of the cargo. Figure 4 illustrates the schematic uptake of dendriplexes into cells. Dendrimers have been used for various gene delivery applications with an emphasis on cancer therapy. A recent review by Mignani et al.[13] discusses the different routes of

dendrimer administration for drug and gene delivery including intravenous, intratumoral, intraperitoneal, transdermal, oral and ocular. For all of these routes, dendrimer-gene complexes generally have to permeate epithelia to get to the target site. The main factors thereby influencing the permeability across the epithelial barriers include the generation size, surface charge, concentration, incubation time and surface modifications.[13] The following section briefly describes the most common types of dendrimers used for gene delivery and highlights some of the surface modifications employed to reduce toxicity and improve transfection.

PAMAM dendrimers PAMAM are the most widely characterized and commercialized dendrimer species and are generally prepared by the divergent growth process.[40] Their advantages include their water solubility and lack of immunogenicity, while the terminal amine groups can be modified for binding to various hosts or target molecules. PAMAM dendrimers are only hydrolytically degradable under robust conditions as they are comprised of amide backbones. Thus, higher generation dendrimers have shown to be toxic because of

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Dendrimer Cell membrane Nucleic acid Destruction of membrane Nucleic acid release

Dendriplex

Endocytosis Endosome

Figure 4 Schematic transfer of dendrimer-nucleic acid complexes into cells. The dendrimer forms a complex with the nucleic acid in vitro and positively charged dendriplexes bind to the negatively charged cell membrane. They are then internalized by the cell and dendriplex containing endosomes are formed. The proton sponge effect of the dendrimer inside the endosome (pH 5.5) eventually leads to lysis of the endosome wall and release of free nucleic acid into the cytoplasm.

non-biodegradability. PAMAM dendrimers are the most widely researched dendrimers for gene delivery to various organs, and dendrimers with six generations have shown to be the most efficient.[38] A number of surface modifications has been employed to reduce toxicity and enhance tissue targeting. These include carboxylic and hydroxyl surface groups,[41] arginine ester rather than amide bonds,[42] folate for targeting and polyethylene glycol (PEG) for increased circulation.[43] To increase the transfection efficiency of PAMAM dendrimers, b-cyclodextrin addition and electroporation have also recently been utilized. While the addition of b-cyclodextrin produced smaller and more monodisperse particles, electroporation triggered a significant increase in gene expression. Moreover, PAMAM dendrimers functionalized with a-cyclodextrin showed luciferase gene expression about 100 times higher than unfunctionalized dendrimers and noncovalent mixtures of PAMAM and a-cyclodextrin.[44] PEG functionalization of G5-PAMAM dendrimers produced a 20-fold increase in the transfection efficiency using plasmid DNA coding for a reporter protein b-galactosidase with the inclusion of PEG positively influencing the stability of the gene complex.[17] 546

Peptide dendrimers Peptide dendrimers are branched macromolecules consisting of a peptidyl branching core or covalently attached surface functional units.[45,46] The main advantages of peptide dendrimers include their capability to be endocytosed by cells as well as their biodegradability. Besides drug and gene delivery vehicles, they have been used as protein mimetics, anticancer and antiviral agents, vaccines, contrasting agents for fluorescent imaging, magnetic resonance imaging (MRI) and serodiagnosis.[46–48] Poly-L-lysine (PLL) dendrimers, the most commonly used dendrimers from this category, were first reported by Denkewalter et al. in the early 1980s[2] and are usually synthesized via solid phase peptide synthesis, although chemoselective and orthogonal ligation can also be employed.[46] They have been used extensively for gene delivery, with eight amino acid residues, and thus positive charges, sufficient to form complexes with nucleic acids.[49] They are generally biodegradable and thus show reduced toxicity.[50] However, PLL dendrimers have been shown to get entrapped in the endosome, therefore not delivering their cargo to the site of action. Arginine and histidine modifications[51] due to the proton sponge effect as

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well as addition of fusogenic peptides[52] have shown to increase endosomal escape, while addition of cell penetrating peptides has resulted in increased cellular uptake.[52] Starpharma’s VivaGel (Starpharma Holdings Limited, Melbourne, Australia), a microbicide for prevention of HIV and herpes simplex virus, is based on a polylysine dendrimer and is currently in Phase II clinical trials.[53–55] The use of modified PLL dendrimers for ocular gene delivery will be discussed in detail in a later section.

Glycodendrimers The term ‘glycodendrimer’ is used to describe dendrimers that incorporate carbohydrates into their structures.[45] They can be classified as (1) surface saccharides, (2) core saccharides or (3) saccharide building blocks. Because of their structure and biological relevance, glycodendrimers have been used to study protein–carbohydrate interactions that occur in a number of intercellular recognition events. They have also been utilized for targeting of MRI contrast agents, incorporation into analytical devices, formulation of gels and as gene and drug delivery systems.[9,45,56] Mannosylated dendrimers have shown great success in specific celltargeting because of mannose binding proteins that act as receptors and can mediate uptake and internalization of glycoconjugates. Mannosylated PAMAM-a-cyclodextrin dendrimers have shown gene transfer activity in kidneys 12 h after intravenous injection into mice, which was higher than with the dendrimer or the a-cyclodextrin conjugate alone,[57] while a mannosylated PLL dendrimer enabled mannose receptor-mediated, cell-specific, in-vivo gene transfer of plasmid DNA encoding for the chloramphenicol acetyltransferase gene, with the highest activity found in the liver.[58] A recent review article by Arima et al.[59] gives an overview on sugar-appended PAMAM dendrimer conjugates with cyclodextrins for cell-specific non-viral gene delivery and highlights the potential of achieving celltargeting and active uptake by carbohydrate surface modification of dendrimers.

PEI dendrimers PEI is another efficient and extensively utilized non-viral gene carrier and is known as the gold standard for plasmid DNA (pDNA) delivery. However, PEI has been shown to be less effective for short interfering RNA (siRNA) because of the reduced length of the nucleic acid drug and thus low electrostatic interactions.[60] PEI has been used both in vitro and in vivo in both linear and branched forms. The high efficacy of PEI is attributed to the ability to escape the endosome by the proton sponge effect.[61] Inefficiencies in vivo occur because of the rapid clearance of the positively charged particles in the blood, while the binding strength between PEI and the nucleic acid has great impact on their

Dendrimers for ocular gene delivery

dissociation once inside the cell, with higher molecular weight PEI (>25 kDa) being less effective in dissociating from DNA. Thus, lower weight PEI dendrimers are generally used because of lower cytotoxicity and complexation strength.

Polypropylimine (PPI) dendrimers These dendrimers are based on PPI units with a butylenediamine core and are commercially available as Astramol (DSM, Heerlen, the Netherlands). A study by Schatzlein et al.[62] evaluated different generations of PPI dendrimers as transfection agents and showed that genes were successfully expressed in the liver rather than any other organs. PPI dendrimers have also been used for siRNA delivery. Taratula et al.[63] showed that higher generation PPI dendrimers (G4 and G5) were more effective in forming discrete nanoparticles upon complexation with siRNA than lower generations (G2 and G3). However, the study also revealed that larger size and thus higher positive charge density of G5 dendrimers caused greater toxicity, while G4 dendrimers exhibited maximum effectiveness with regards to siRNA nanoparticle formation, sequence specific gene silencing and siRNA internalization with minimal toxicity. The formulated siRNA–PPI dendrimer complexes exhibited dramatic enhancement in siRNA cellular internalization and a marked knockdown of the target mRNA in human lung cancer cells.[63] PPI dendrimers have also been surface modified to reduce the cytotoxicity and enhance the transfection efficacy. Russ et al.[64] grafted PPI dendrimers via branched oligoethylenimine (OEI) or hexanediol diacrylate, providing bioreversible ester linkages, and found that the transfection levels of OEI-grafted dendrimer-pDNA complexes was higher than those of standard PPI polyplexes. Tack et al.[65] modified PPI dendrimers at the exterior primary amines with acetyl groups or glycol gallate (PEGlike) groups as well as at the interior tertiary amines with methyl iodide or chloride. These dendrimers were stable in aqueous media and enabled delivery of a DNAzyme into ovarian carcinoma cells, while inducing only minimal cellular toxicity. These examples show that surface modifications can reduce the cytotoxicity of even higher generation (ⱖ G4) dendrimers and may thus enable efficient non-viral gene delivery.

Ocular gene therapy Owing to the increasing number of ocular diseases associated with inappropriate protein production, there has been a marked growth in nucleotide-based therapeutics. The eye poses an ideal organ for gene therapy for a number of reasons: (1) direct access of the ocular tissues; (2) small and compact structure only requiring small doses; (3) immuneprivileged status due to the presence of tight junctions

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Sahil P. Chaplot and Ilva D. Rupenthal

Intravitreal Systemic Conjunctiva Suprachoroidal Sclera Choroid Topical

Retina Vitreous

Cornea

Macula

Lens Iris Ciliary body

Optic nerve

Periocular (subconjunctival, subtenon, peribulbar or retrobulbar) Figure 5

Ocular drug delivery routes.

between retinal pigment epithelium (RPE) cells and the blood-retinal barrier preventing leakage of genes or carriers into the circulation; (4) a vast number of target genes have already been identified; (5) adequate animal models are available; and (6) many non-invasive techniques (optical coherence tomography, electroretinography and visual evoked potentials measurement of afferent pupillary light responses) for the assessment of ocular function are available.[66,67] Nevertheless, the eye contains a number of delivery barriers which can be rather impermeable to gene complexes, depending on which delivery route is chosen. Figure 5 gives an overview of the ocular anatomy and the various delivery routes possible for ocular gene therapy. Because large nucleic acid drugs are unable to permeate the cornea and diffuse towards the posterior chamber, topical application of gene therapeutics is only performed if the target is located in the corneal epithelium such as for corneal scaring or persistent epithelial defects (Table 1). However, most ocular diseases require nucleic acid delivery to the neural retina or the RPE and thus other delivery routes have to be employed.[70,71] The most common way of delivering drugs to the posterior segment is by intravitreal injection, and although this delivers the drug closer to its target site, a number of barriers still have to be overcome. The vitreous, for example, can immobilize dendriplexes in the dense proteoglycan filament network by ionic interactions of the cationic dendriplexes with the negatively charged glycosaminoglycans, resulting in particle aggregation.[68] Moreover, the inner and outer limiting membranes at the back of the eye present a tight barrier for gene delivery to the retinal tissues.[69] Pitkanen et al.[72] 548

demonstrated that the diffusion of PEI and PLL polyplexes through the neural retina was restricted by the inner limiting membrane, which contains laminin, collagen and several proteoglycans. Thus, additional surface modifications such a PEGylation may be required to overcome this barrier. While the injection volume for intravitreal delivery is limited to 100 ml to avoid an increase in intraocular pressure, the periocular route allows injection of up to 1 ml into the space surrounding the eye. It is considered the least painful and most efficient route of drug delivery to the posterior segment[73] with the added advantage that molecules of up to 70 000 Da seem to readily penetrate the sclera.[74] However, additional barriers are in place before the gene therapeutic can reach the neural retina. These include the choroid, a network of blood vessels between the sclera and the retina, and the outer blood-retinalbarrier (BRB) comprising the RPE with its tight junctions.[70] The BRB also forms the major barrier for retinal delivery of drugs after systemic administration with only minimal drug concentrations (

Dendrimers for gene delivery--a potential approach for ocular therapy?

A vast number of blinding diseases have genetic aetiologies and may be treated by molecular based therapies such as antisense oligonucleotides or shor...
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