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

Inmaculada Posadas; Francisco Javier Guerra; Valentín Ceña

Disclosures

Nanomedicine. 2010;5(8):1219-1236. 

In This Article

Carbon Nanotubes

These materials consist of seamless graphene sheets rolled concentrically to form capped cylinders composed mostly of carbon hexagons and high-strained regions at the tips, where carbon pentagons are the predominant shape. The number of graphene layers distinguishes amongst single-, double- or multi-walled carbon nanotubes (CNTs; SWNTs, DWNTs and MWNTs, respectively). While SWNTs are composed of a single monolayer of graphene giving rise to a cylinder with a diameter of approximately 0.8–2.0 nm, MWNTs are composed of a concentric arrangement of several nanotubes on an onion-shape structure with diameters of up to 100 nm. Owing to their inherent properties, these nanomaterials are playing an increasing role in different fields of nanomedicine, such as imaging, drug delivery and gene transfer.[156–159]

However, a lack of solubility arises as the main disadvantage inherent to the pristine carbon nanomaterials for most biological applications. This drawback is currently solved by the incorporation of different pendant units that increase solubility.[160] Two general approaches have been used to increase nanotube solubility:

  • A noncovalent approach, where van der Waals and π–π interactions are claimed to be responsible for the functionalization;[161,162]

  • Covalent functionalization of the CNT surface.

Since excessive covalent functionalization damages the CNT surface, the use of dendrimers is a smart answer to solve this problem (Figure 2). Numerous groups have focused on the decoration of CNTs with dendrimers in the search for new properties, such as an increase in the dispersability and an enhancement of the solubility,[163] by introducing metallic NPs[164] or introducing a high number of protonable groups.[165] As expected, the incorporation of dendrimers enhances the solubility without damaging the carbon skeleton of the CNTs.

Figure 2.

Transmission electron microscopy micrograph of a multiwalled carbon nanotube decorated with gold particles (~2 nm) encapsulated in sixth-generation amino-terminated polyamidoamine dendrimers.

Transfection in vitro

The combination of PAMAM dendron fragments and CNTs has proven to be successful for gene transfer.[165] Noncovalent approaches have also been used. DNA has been electrostatically bound to functionalized SWNTs and MWNTs[156,166] and phospholipid–PEG moieties have been anchored to CNTs to allow linking to thiol-terminated siRNA.[156,166] These materials are more active for the inhibition of the protein governed by the gene lamin A/C compared with Lipofectamin.[167] These nanoplexes were also active in vitro into both human T cells and primary cells.[168]

Very little information is currently available on the use of dendrimers coupled to CNTs to transfect genetic material into cultured neurons. However, a polyvinylpyrrolidone core dendrimer (IR8) coupled to MWNT was able to transfect rat granular cerebellum neurons and decrease the mRNA encoding for p42 microtubule-associated kinase protein by approximately 50% (Figure 3), suggesting that CNT-based NPs might also play a relevant role in transfection in the near future.

Figure 3.

Efficiency of a polyphenylenevinylene-based dendrimer coupled to carbon nanotubes (MAHC1) as delivery vector for small interfering RNA in granular cerebellar neurons. Neurons were incubated with nanoparticle alone, p42 MAPK siRNA alone, nanoparticle coupled to specific p42MAPK siRNA or nanoparticle coupled to scramble siRNA. p42MAPK mRNA levels were determined by real-time reverse transcriptase-PCR. Each bar represents the mean ± standard error of at least three experiments.
*p < 0.001, as compared with control values.
siRNA: Small interfering RNAs.

Transfection in vivo

Polyamine-functionalized MWNTs have shown no toxicity in vivo.[157,169]In vivo experiments have been performed with a polyamine coating on the CNT surface targeting tumor suppression. Experiments with mice using CD80siRNA–SWNTs demonstrated an important inhibition of CD80 expression in myeloid immunosuppressive cells.[170] Tumor growth has been inhibited by the use of these materials joined to telomerase reverse transcriptase siRNA.[171] Similar results have recently been obtained by the use of functionalized PAMAM–dendron MWNTs, achieving a suppression of tumor volume combined with the survival of human lung tumor-bearing animals.[169]

An important fact for a therapeutic NP is that it should remain in the body long enough for it to deliver its therapeutic cargo to the target cells. High degree of functionalization of the nanomaterials leads to complete elimination from the host tissues. MWNTs functionalized with diethylentriaminepentaacetic dianhydride (DTPA–MWNT) enter the systemic blood circulation and within 5 min begin to permeate through the renal glomerular filtration system into the bladder, finally being excreted through the urine 24 h after administration.[172]

One of the major issues to be resolved for the future use of CNTs in gene therapy is the possible toxicological effects of CNTs. Toxicity of CNTs depends on inherent physical and chemical characteristics, such as CNT functionalization, coating, length and agglomeration state. However, CNTs are fiber shaped and so might behave similarly to asbestos and other pathogenic fibers, which have toxicity associated with their needle-like shape. However, they are essentially graphitic and are expected to be biologically biopersistent since they do not undergo chemical dissolution and neither weaken, break or dissolve away, and therefore persist in the lungs. Whole or ground MWNTs were administered intratracheally to rats, and their biological activity and biopersistence was characterized at two time points.[173] The longer, unground nanotubes were found to be more biopersistent than the short nanotubes at day 60. This is in agreement with the greater biopersistence of long fibers observed in studies with asbestos and other mineral fibers.[174]

Epidemiologic studies of air pollution suggest that particulate matter has a strong association with cardiopulmonary diseases[175] and that procedures for the handling of CNTs can result in aerosol release of these materials into the surroundings.[176] CNTs produce a toxic response upon reaching the lungs in sufficient quantity; this reaction is produced in a time- and dose-dependent manner. Several in vivo instillation studies have found that CNTs produce granuloma formation.[177,178] CNTs were highly fibrogenic and inflammogenic, approximately equivalent to chrysotile asbestos.[178] Initial toxicologic studies demonstrated that intratracheal or pharyngeal instillation of SWNT suspension in mice caused a persistent accumulation of CNT aggregates in the lung, followed by the rapid formation of pulmonary granulomatous and fibrotic tissues at the site,[177,179] inflammatory reactions of terminal and respiratory bronchioles, and, in some animals, mild fibrosis in the alveolar septa.[180,181]

The unique physical characteristics and the pulmonary toxicity of CNTs raised concerns that respiratory exposure to these materials may be associated with systemic toxicities. Of special concern is the apparent ability of NPs to redistribute from their site of deposition. Thus, following inhalation exposure, NPs have been reported to travel via the nasal nerves to the brain[182,183] and to gain access to the blood and other organs.[184] In addition, it is possible that individual CNTs can translocate from the lung into the systemic circulation causing direct cardiovascular endothelial dysfunction. It has been reported that NPs treated with albumin and/or surfactant proteins cross the alveolo–capillary barrier in order to gain access to the systemic circulation.[185] The proximity between epithelial type I and endothelial cell caveolar membrane structures might play a role in the particle translocation mechanisms.[186] Since CNTs are not well recognized and cleared by lung macrophages,[187] CNTs, dispersed or disintegrated from the agglomerates, may persist in the alveolar space, which will facilitate their access into the systemic circulation.

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