ARTICLE pubs.acs.org/Biomac
Efficient, Non-Toxic Hybrid PPV-PAMAM Dendrimer as a Gene Carrier for Neuronal Cells Ana C. Rodrigo,† Ivan Rivilla,† Francisco C. Perez-Martínez,‡ Silvia Monteagudo,‡ Vanessa Oca~na,§ Javier Guerra,†,‡ Joaquín C. García-Martínez,† Sonia Merino,† Prado Sanchez-Verdu,† Valentín Ce~na,*,§,|| and Julian Rodríguez-Lopez*,† †
Facultad de Química, Universidad de Castilla-La Mancha, Avda. Camilo Jose Cela 10, 13071-Ciudad Real, Spain NanoDrugs, S. L., P° de la Innovacion 1, 02071-Albacete, Spain § Unidad Asociada Neurodeath, Facultad de Medicina, CSIC-UCLM, Universidad de Castilla-La Mancha, Avda. Almansa 14, 02006Albacete, Spain CIBERNED, Instituto de salud Carlos III, C/Sinesio Delgado 6, 28071-Madrid, Spain
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bS Supporting Information ABSTRACT: A novel hybrid dendrimer (TRANSGEDEN) that combines a conjugated rigid polyphenylenevinylene (PPV) core with flexible polyamidoamine (PAMAM) branches at the surface was synthesized and characterized. The potential of this material as a nonviral gene delivery system was also examined, and it was observed that dendriplexes formed by TRANSGEDEN and small interfering ribonucleic acids (siRNAs) can be incorporated into >90% of neuronal cells without any toxicity up to a dendrimer concentration of 3 μM. TRANSGEDEN was used to deliver a specific siRNA to rat cerebellar granular neurons (CGNs) to knock down the cofilin-1 protein. Cofilin-1 removal partially protects CGNs from N-methyl D-aspartate (NMDA)-mediated neuronal death.
’ INTRODUCTION Dendrimers are highly branched, monodisperse macromolecules of nanosize dimensions and their shape, size, flexibility, and molecular weight can be controlled during their preparation.1,2 Most of the properties of dendrimers are due to their terminal groups in the periphery, and these can be modified at will. Because of their well-defined structure and the ease with which the surface functionality can be controlled, dendrimers are now the focus of an important interdisciplinary field of research with technical and biochemical applications in catalysis, polymer science, bio-organic chemistry, biotechnology, and medicine.3 One of the most interesting areas of research into dendrimers concerns pharmaceutical and biomedical applications.4-7 Dendrimers are chemical structures that, in some cases, display the ability to cross cell membranes - a possibility that is of great interest for their application in drug and gene delivery.8,9 Because of their commercial availability, PAMAM10,11 and polypropylenimine (PPI)12,13 dendrimers have become the most widely used dendrimer-based vectors for gene transfer, and these can show high transfection efficiency.14,15 The primary amines located on the surface of these dendrimers provide a high cationic charge density that favors DNA or RNA binding through the phosphate backbone. In general, higher generation or structurally fractured systems based on PAMAM (commercially available as Superfect) r 2011 American Chemical Society
are the most efficient systems for gene delivery. One drawback of PAMAM dendrimers, however, is their generation-dependent cytotoxicity.16 The cytotoxicity of PAMAM increases proportionally with the generation number. Modifications of the basic PAMAM or PPI dendritic structures have been investigated in an effort to improve transfection efficiency and reduce cytotoxicity. The modifications investigated include alterations on the surface17-19 and the use of different cores.20,21 In general, the density of amino groups on the dendrimer surface, size, and flexibility are very important parameters that affect transfection efficiency. Intrinsically, these parameters depend on the core on which the dendrimer is constructed.22 However, studies concerning the incorporation into dendritic structures of nonglobular, almost planar cores are much less common in the literature.20 Diederich and coworkers reported low-molecular-mass dendrimers that featured extended rigid cores and showed high transfection activity.23 The use of siRNA24,25 is becoming a very popular method to knock down specific proteins acutely and selectively. siRNAs are double-stranded RNA molecules that induce sequence-specific Received: December 10, 2010 Revised: February 17, 2011 Published: March 10, 2011 1205
dx.doi.org/10.1021/bm1014987 | Biomacromolecules 2011, 12, 1205–1213
Biomacromolecules degradation of homologous single-stranded RNA.26 Therefore, this method allows the role of a specific protein in different physiological and pathological functions to be studied. siRNA technology has several major advantages over other techniques available to perform lack-of-function studies. It is easier to deliver, requires only small doses of siRNA to produce its silencing effect, and can inactivate a gene in almost any stage in development. Moreover, siRNA is specific, it does not allow for the genesis of compensatory pathways during development, and it is cheaper and less time-consuming than knockout mice generation.27 The efficiency of siRNA and its limited side effects have made this technique an attractive alternative to the use of antisense oligonucleotides and ribozymes for therapies based on the inhibition of target genes.28 However, the success of gene silencing applications based on the use of synthetic siRNA critically depends on efficient intracellular delivery.29 siRNAs should be delivered to the cell to perform their inhibitory function using either nonviral30 or viral31 vectors. Neurons are difficult to transfect even with the use of nonviral vectors as carriers.32 Among the nonviral vectors, dendrimers have shown the highest transfection efficiency in neuronal cells.30,33,34 Dendritic PPVs, also called stilbenoid dendrimers, have become one of the most studied families of conjugated dendrimers because of their optoelectronic properties.35 In our search for new layer-block hybrid dendrimers that show different properties,36 we thought that PAMAM dendrimers bearing this kind of unit at their core would lead to new and interesting compounds. Such materials would combine, within the same structure, flexible PAMAM branches with gene delivery properties at the surface and a conjugated rigid PPV that, for example, might be used as an internal chromophoric probe for fluorescence tagging. In this Article, we report the synthesis and characterization of a new PPV-PAMAM hybrid dendrimer (TRANSGEDEN) that is able to bind and release siRNA and, more importantly, is not toxic to neurons at the concentrations used to deliver siRNA. We also demonstrate that dendriplexes formed by TRANSGEDEN and siRNAs can be incorporated into >90% of the neurons, indicating that TRANSGEDEN might be a promising nonviral gene delivery carrier. We have taken advantage of these dendrimer properties to knock down cofilin-1 protein levels to study its role in NMDA-mediated neuronal death in rat CGNs. This protein was chosen because of its crucial role in neurotransmission processes. Altered glutamatergic neurotransmission has been involved in the pathogenesis of chronic neurodegenerative diseases as well as in acute neuronal injury associated with ischemic stroke.37,38 Remarkably, this study is not possible using knockout mice because cofilin-1 deletion is lethal in the embryonic stage.39
’ EXPERIMENTAL SECTION General. Unless otherwise stated, all reagents were used as received and without further purification. Tetrahydrofuran (THF) and dichloromethane (CH2Cl2) (CHROMASOLV) were dried using a solvent purification system. Methanol was dried over calcium oxide, filtered, distilled, and stored over molecular sieves (4 Å). Ethylenediamine (EDA) was dried over CaH2, filtered, distilled under reduced pressure, and stored over molecular sieves (4 Å). Methyl acrylate was distilled before use. NMR spectra were acquired at 25 °C. Chemical shifts are given in ppm relative to TMS (1H, 0.0 ppm) or CDCl3 (13C, 77.0 ppm). IR spectra were recorded on an FT-IR spectrophotometer equipped
ARTICLE
with an ATR accessory. MALDI-TOF mass spectra were registered in positive reflection mode using 20 ,40 ,60 -trihydroxyacetophenone monohydrate (THAP) as the matrix material. Compound 1 has been previously described by some of us.40 All animals were treated and sacrificed in accordance with guidelines of the European Union (86/ 609/EEC) for the use of laboratory animals. Dendrimer Synthesis. Compound 2. To a solution of trialdehyde 1 (300 mg, 0.64 mmol) in anhydrous CH2Cl2 (50 mL) over molecular sieves (4 Å) was added EDA (8.6 mL, 128 mmol) under argon. The mixture was stirred at room temperature for 0.5 h, and the molecular sieves were filtered off. Anhydrous methanol (10 mL) and sodium borohydride (145 mg, 3.84 mmol) were added. The mixture was stirred for an extra 2 h. The solvent was evaporated under vacuum (temperature
