Nonviral Vectors for the Delivery of Small Interfering RNAs to the CNS

Inmaculada Posadas; Francisco Javier Guerra; Valentín Ceña


Nanomedicine. 2010;5(8):1219-1236. 

In This Article


Dendrimers are branched, multivalent molecules with a well-defined structure that was first reported more than three decades ago and initially termed 'cascade molecules'.[121] Years later, Tomalia and coworkers defined these molecules as dendrimers, with the word formed from the Greek words dendros, meaning tree and meros, meaning part.[122] These macromolecules are globular and nanoscaled with a particular architecture formed by three distinct domains:

  • A central core that is either a single atom or a group of atoms having at least two chemical functionalities that facilitate the linkage of the branches;

  • Branches emanating from the core, composed of repeat units with at least one junction of branching, whose repetition is organized in a geometric progression that results in a series of radially concentric layers termed generations (G);

  • Terminal functional groups, located in the exterior of the macromolecule, which play a key role in their gene-complexing or drug-entrapping ability. The presence of these numerous terminal groups facilitates interactions with solvents, surfaces or other molecules. In general, dendrimers tend to show high solubility, reactivity and binding.

The first report on the use of Starbust® polyamidoamine (PAMAM) dendrimers as transfection vehicles indicated that these molecules could efficiently induce expression of a reported gene in a variety of suspension and adherent cultured mammalian cells, with the G6 (NH3 core) dendrimer having the best efficiency.[123] It was suggested that PAMAM dendrimers had the ability to escape from the endosome due to the ability of the internal dendrimer amine groups to buffer pH changes in the endosome[123] in a mechanism similar to that described for PEIs.

Since then, Starbust PAMAM dendrimers were rapidly studied as carriers for drugs[124–127] and gene delivery into eukaryotic cells.[128,129]

In Rat2 embryonic fibroblast cells, Starbust PAMAM dendrimers (G5-G10) with either an ethylene diamine or ammonium core are capable of efficiently mediating transfection without any cytotoxic effects, with the maximum transfection efficiency achieved with G6 ammonium dendrimer and from there on, decreasing with higher generations.[128] G7 ethylene diamine dendrimers delivered antisense oligonucleotides and plasmid expression vectors coding antisense mRNA efficiently achieving 30–60% inhibition of the expression of the reporter gene depending on DNA concentration, type of dendrimer used and charge ratios of DNA–dendrimer complex.[129]

PAMAM dendrimers showed a generation number-dependent toxicity and were found to be extremely toxic to erythrocytes, causing their rupture and the release of hemoglobin at a concentration as low as 100 µg/ml.[130] Cytotoxicity of dendrimers is directly related to the number of terminal amino groups and the positive charge density. Thus, masking of the amino groups by surface functionalization leads to a decrease in cytotoxicity. Hence, different functionalization strategies have been employed by attachment of targeting groups to the periphery in order to reduce the associated cytotoxicity. Surface-functionalized derivatives have been reported to be nonimmunogenic and noncytotoxic.[27,125,130–132]

In addition to surface modification to reduce cytotoxicity, other structural modifications have been developed to improve delivery of genetic materials. It is well known that there are three main barriers in the intracellular delivery of genetic material, which transfecting vectors have to surmount: cellular membrane, endosomal membrane and nuclear membrane. Introducing arginines onto the surface of the PAMAM G4 dendrimer highly increased transfection efficiency,[133] most likely by increasing endosomal escape. To increase transport to the nucleus of PAMAM dendrimers containing DNA cargo, dexamethasone was conjugated to Starburst PAMAM G4.[134] The glucocorticoid interacted with its receptor, located in cytoplasm in the absence of its ligand, and would translocate into the nucleus, dilating the nuclear pore up to 60 nm and thus facilitating translocation of the polymer/DNA complex into the nucleus.[135] Conjugation of dexamethasone reduced the required amount of the polymeric carrier for optimum transfection, increased transfection efficiency in neuroblastoma Neuro2A cells and showed lower toxicity when compared with 25-kDa branched PEI and PAMAM.[134]

Other dendrimers different from PAMAM have also been used for transfection. Water-soluble carbosilane dendrimers contain peripheral amine and ammonium, and can efficiently interact with DNA, oligonucleotides or plasmids by electrostatic interaction at biocompatible doses.[136] These dendrimers have shown good toxicity profiles in peripheral blood mononuclear cells and erythrocytes over extended periods of time.[136] In addition, carbosilane dendrimers efficiently mediated oligonucleotide, as well as siRNA, internalization in peripheral blood mononuclear cells.[5,137]

Water-soluble polycation dendrimers with a phosphoamidothioate backbone (p-dendrimers) functionalized with N,N-diethyldiamine at the surface, were effective in delivering oligonucleotides into HeLa cells in the presence of serum, but they failed to transfect the HUVEC cell line.[138] In addition, when anionic oligomers were combined with plasmids before dendrimer addition, a marked increase in transfection efficiency could be observed.[139] However, triethanolamine core PAMAM dendrimers are able to effectively deliver Hsp27 siRNA into the prostate cancer cell line PC3 and to produce an efficient silencing of the protein.[4] Polyglycerol-based dendrimers core–shell structures have been used to transfect siRNA to human glioblastoma and murine mammary adenocarcinoma cell lines, suggesting that they could represent a useful tool for siRNA delivery in vivo.[140] In addition, a new polyphenylenevinylene core dendrimer with PAMAM-based branches (IR8) transfects siRNA very efficiently into human prostate cancer LnCAP cells [Monteagudo et al., Unpublished Data].

Transfection of Neurons in vitro

Although dendrimers were first used as transfection vehicles in the early 1990s, no information about their transfection efficiency in primary CNS cultures has been reported until recently.

Arginine-grafted PAMAM G4 dendrimers achieve transfection levels of 35–40% in primary cortical neurons. These transfection levels are significantly higher than those previously reported for Lipofectamine, 25-kDa branched PEI or native PAMAM.[141] More recently, it has been described that biodegradable polycationic PAMAM esters, in which arginine is ester bound to PAMAM-OH functionalized dendrimers, successfully allows siRNA delivery to a primary culture of mixed cortical cells containing neurons and glia, achieving a reduction of approximately 80%, 12 h post-transfection, without any toxic effects enacted onto the neurons.[142]

The second generation of ammonium-terminated carbosilane dendrimers containing 16 positive charges (NN16) was able to efficiently transfect siRNA into rat cortical neurons. At 18 h post-transfection, approximately 85% of neurons incorporated the fluorescein-labeled siRNA/2G-NN16 complex. Analysis of target protein (hypoxia-inducible factor [HIF]) showed a reduction of approximately 80% of the total protein content, without toxic effects on neurons.[143] Moreover, a polyphenylenevinylene core dendrimer (IR8) can deliver siRNA to rat cortical neurons, markedly decreasing both mRNA and protein levels of the target protein (HIF) (Figure 1).

Figure 1.

Efficiency of IR8 dendrimer as a delivery vector for small interfering RNAs in rat cortical neurons. (A) HIF-1α protein levels (top panel) in neurons treated with either V or 200 µM CoCl2 for 4 h, alone or after the neurons were preincubated with IR8 dendrimer alone, HIF-1α siRNA alone, HIF-1α siRNA/IR8 dendrimer complex or scrambled siRNA/IR8 dendrimer complex. β-actin (bottom panel) was used as the protein loading control. (B) Densitometric analysis of HIF-1α protein levels expressed as a ratio of HIF-1α OD and β-actin OD. Each bar represents the mean ± standard error of at least three experiments.
*p < 0.001, as compared with CoCl2-treated cells.
HIF: Hypoxia-inducible factor; OD: Optical density; siRNA: Small interfering RNAs; V: Vehicle.

Transfection of Neurons in vivo

As mentioned previously, the delivery of therapeutic nucleic acids to the CNS is one of the major challenges for gene therapy of neurological diseases. Reports of polycationic dendrimers as efficient gene-delivery vehicles in vitro with low toxicity[123] certainly suggested that dendrimers could be suitable vehicles for gene-transfer in vivo. Initial in vivo experiments were carried out for topical delivery to hairless mouse skin.[144]

A great number of modifications of native PAMAM have been attempted in order to increase transfection efficiency in vivo. Fractured dendrimers, obtained by thermal degradation of high generation PAMAM and commercialized under the name Superfect®, have been reported to deliver the gene in vivo to established mice tumors.[145] However, PEGylation of G5 PAMAM resulted in a marked increase in transfection efficiency compared with Superfect.[146]

However, a more efficient way of increasing transfection efficiency consists of coupling either proteins or peptides to dendrimers. Transferrin-conjugated PEG–PAMAM and lactoferrin-conjugated PEG–PAMAM, when administered intravenously in the tail vein of mice, both efficiently deliver genetic material into brain tissue. Target protein expression (GFP) was detected in the cortex, hippocampus, caudate putamen and sustantia nigra, with lactoferrin-conjugated PEG–PAMAM more efficient at dendriplex translocation into the brain across the BBB. However, maximal gene expression was detected in the kidneys from both vehicles studied.[147] Covalent linkage with rabies virus glycoprotein peptide (RVG29) to enable viral entry into neurons[148] was injected intravenously, allowing accumulation of the target gene in the brain in a similar extension to that observed in the spleen and heart. In the brain, gene expression was much higher in the hippocampus and substantia nigra.[149] The mechanism involved in particle internalization appeared to be clathrin- and caveola-mediated energy-dependent endocytosis.

Angiopep-2 is one of the peptides derived from the Kunitz domain that possesses a high brain penetration capability.[150] Intravenous administration of surface-modified PEG–PAMAM with angiopep-2 in the tail vein of mice resulted in a high accumulation of the target gene in the brain and the spleen.[151] Here, internalization of the dendriplex into brain capillary endothelial cells occurred mainly through clathrin- and caveolae-mediated endocytosis, although also partly via macropinocytosis.[151]

The modifications described above were performed with the aim of improving transfection efficiency of PAMAM dendrimers in vivo for the delivery of genes or antisense oligonucleotides. However, the delivery of siRNA using dendrimers has so far been challenging and poor efficiencies have been reported.[152,153]

Nevertheless, dendrimers synthethized by polyvalent conjugation of lower generation dendrimers onto an elongated magnetic NP (dendriworms) have been proposed as efficient carriers for siRNA delivery in the brain.[154] It has been proposed that dendriworms would induce a high proton sponge effect and enable efficient endosomal escape of siRNA.[155] Dendriworms efficiently knockdown the target protein in GBM-6 glioma cells in culture and, interestingly, intracranial infusion for a period of 3 and 7 days using an osmotic pump demonstrated that NPs were able to penetrate through the brain parenchyma.[154] Unfortunately, intravenous administration of dendriworms in mice preferentially accumulated in the lungs and reticuloendothelial filtration organs, with low amounts reaching the brain, suggesting that further modifications should be performed to facilitate siRNA delivery into the brain. This could be achieved using intravenous administration in order to overcome the BBB, and thus avoiding aggressive and highly invasive methods, such as a craniotomy or intracerebral administration.

Dendrimers appear to be very promising tools with a future potential for specific delivery of genetic material to the nervous system both in vitro and in vivo, although more work is needed to design structures that could support genetic material delivery to the CNS from a therapeutic point of view.[155]


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