Engineered nonviral nanocarriers for intracellular gene delivery applications

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Biomed. Mater. 7 (2012) 054106 (13pp) doi:10.1088/1748-6041/7/5/054106

Engineered nonviral nanocarriers forintracellular gene delivery applicationsIsaac Ojea-Jimenez1,4, Olivia Tort2, Julia Lorenzo2

and Victor F Puntes1,3

1 Institut Catala de Nanotecnologia (ICN), UAB Campus, 08193 Cerdanyola del Valles, Barcelona, Spain2 Institut de Biotecnologia i de Biomedicina and Departament de Bioquımica i Biologia Molecular,Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain3 Institut Catala de Recerca i Estudis Avancats (ICREA), 08010 Barcelona, Spain

E-mail: Isaac.Ojea@icn.cat and Victor.Puntes@icn.cat

Received 10 January 2012Accepted for publication 12 March 2012Published 12 September 2012Online at stacks.iop.org/BMM/7/054106

AbstractThe efficient delivery of nucleic acids into mammalian cells is a central aspect of cell biologyand of medical applications, including cancer therapy and tissue engineering. Non-viralchemical methods have been received with great interest for transfecting cells. However,further development of nanocarriers that are biocompatible, efficient and suitable for clinicalapplications is still required. In this paper, the different material platforms for gene deliveryare comparatively addressed, and the mechanisms of interaction with biological systems arediscussed carefully.

(Some figures may appear in colour only in the online journal)

1. Introduction

Since its development, gene delivery has become a commonlyused technique in a wide range of biological domains such as(i) transforming plasmids into bacteria to obtain high quantitiesof DNA or recombinant proteins, (ii) transducing cells withviruses to obtain stable cell lines for long-term studies or(iii) for genetic therapies in medicine [1]. The major aim ofgene therapy is to deliver genetic material into somatic cellsof a patient, which results into a therapeutic effect by eithercorrecting a genetic defect, by overexpressing proteins that aretherapeutically useful, or by suppressing the expression of agene. On the one hand, the ability to introduce functional genesinto mammalian cells offers many new possibilities for thetreatment of commonly acquired and inherited human diseasessuch as cancers [2], autoimmune disorders, monogenicand cardiovascular diseases and neurodegenerative diseases,among others. For an in-depth overview of disease targetsfor gene therapy, see [3]. On the other hand, for regenerativemedicine and tissue engineering, genetic modification maybe preferable over the exposure of cells to growth factors

4 Author to whom any correspondence should be addressed.

and cytokines, since the half-life and body clearance of thesemolecules may imply the use of either high non-physiologicalconcentrations or repeated administrations to produce thedesired therapeutic effect [4].

Gene therapy approaches currently under developmentcan be classified as in vivo and ex vivo. The in vivo strategyconsists in delivering genes directly to the patient, locallyor systemically, by direct injection or through controlledrelease from a scaffold. However, using this method, it isdifficult to avoid transfection in secondary tissues unless somecell targeting strategy is added to the system. For example,cancer is one of the most popular targets for gene deliverysystems in vivo and addresses much research on siRNAdelivery treatments, which implies silencing of the abnormaloverexpression of defined genes developed in tumors [5, 6].The ex vivo or cell-mediated gene transfer method involvesthe in vitro genetic manipulation of cells followed by thereintroduction of the cells into the injury site. This strategy isespecially suitable for tissue engineering applications, wherestem cells could be grown in cell culture dishes or already in3D environments (such as a matrix or biodegradable scaffoldfor wound repair) and then be implanted in the patient body[4, 7, 8]. After implantation, the transfected cells could recruit

1748-6041/12/054106+13$33.00 1 © 2012 IOP Publishing Ltd Printed in the UK & the USA

Biomed. Mater. 7 (2012) 054106 I Ojea-Jimenez et al

Figure 1. Variables associated with the use of nanocarriers intherapeutic gene delivery applications.

the appropriate host cells to migrate into the scaffold byexpressing the delivered genes that perhaps code for growthfactors, morphogenic proteins, or cell-type-specific adhesionmolecules.

Even if enzymatic degradation were not a concern, theefficiency of transfecting cells with naked oligonucleotidesin vitro or in vivo would be very low because the molecules’negative charge renders them unlikely candidates for cellularinternalization. In addition, the excessive size of uncondensedgenetic material is an added barrier to cellular uptake.The traditional method to introduce a therapeutic gene intocells involves the use of viral vectors, which posses aninherent ability to inject the nucleic acid into the hostcells. Although viral vectors present high efficiencies ingene transfer and may allow stable gene expression, theirclinical applications are narrow due to their limited packagingcapacity of exogenous DNA, the difficulties of scaling-up procedures and also to potential problems such asoncogenic transformation, pathogenic risk and induction ofimmune responses [9, 10]. These complications gave riseto substantial efforts in the development of alternative non-viral systems, as they offer several advantages such as lowimmunogenicity, low transmission of infectious diseases andlow production costs, among others [11]. Nevertheless, non-viral gene carriers consistently exhibit significantly reducedtransfection efficiency as they are hindered by numerousextracellular and intracellular obstacles [12]. Therefore, theappropriate nanocarrier needs to be carefully developed,optimized and characterized for each use.

While selecting the appropriate genes to deliver is arelatively challenging task, specific to a given application, andis beyond the scope of this discussion, this paper will outlinesome of the more recent advances in gene delivery researchusing non-viral nanocarriers (figure 1). It is intended to providethe reader with a set of design considerations that must beevaluated before selecting the appropriate transfection agentfor a specific application. Specifically, we discuss the use oflipids, polymers and inorganic materials as core platforms with

intrinsic properties and defined structures that can enhance thedelivery of nucleic acids. Subsequently, we discuss the effectof the surface coating on the nanocarrier–cell interactionsand possible modifications with active ligands to improve theoverall delivery efficiency.

2. Material platforms for nucleic acid nanocarriers

Naked oligonucleotides in circulation within the body arerapidly broken down by nucleases and tend to accumulatein the liver and kidneys before clearance from the body[13]. Therefore the design of oligonucleotide nanocarriersimplies selecting a suitable material platform that caneffectively incorporate the nucleic cargo and also integrateadditional functionalities to enhance gene delivery to specifictargets. Due to the diverse nature of disease targets andenvironments for gene therapy, there is no single idealnanocarrier that is universally applicable across the entirespectrum of applications. The effective delivery vehicles needto provide the protection of nucleic acids from degradationby extracellular nucleases, targeting specificity, efficient cellinternalization and the release of the nucleic acid in afunctional form in the cytoplasm or the nucleus. In addition,they also need to have high biocompatibility as well as lowimmunogenicity and cytotoxicity.

2.1. Lipid platforms

An extensive library of lipid-based gene delivery platformshas been constructed from different combinations of lipidicbuilding blocks at different ratios, which gives rise to specificproperties [14] (figure 2). Natural molecules found in the lipidbilayer of the cell such as phospholipids, cholesterols andtheir derivatives have been employed as lipid components.Cationic derivatives of cholesterol, for example, have beensynthesized to improve the stability of some liposomes and alsoto facilitate uptake and endosomal escape [15–17]. In addition,synthetic cationic lipids such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) incorporatedinto unilamellar liposomes [18, 19] as well as a new class oflipid-like molecules termed lipidoids [20] have been developedto improve circulation times as well as to enhance targetingand uptake in transfection studies.

2.1.1. Liposomes. Lipid platforms are suitable to incorporatenucleic acids by encapsulation within hollow interiors ofliposomes. Liposomes can easily be prepared by reverse-phase evaporation in which dried films of amphiphilic lipidcomponents are hydrated to generate multilamellar vesicles,which ultimately undergo sonication to produce unilamellarvesicles in the aqueous phase [21]. The nucleic acids and otherfunctional components can be incorporated during or afterthe liposome formation process. Additionally, liposomes canalso be employed to encapsulate polyplexes of nucleic acids,thus forming so-called lipopolyplexes. For instance, siRNAmolecules complexed with protamines and subsequentlyencapsulated by cationic liposomes have been successfullyemployed for down-regulating specific targets for cancer such

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Figure 2. Chemical structure of common lipids used for gene delivery.

as surviving in model lung cancer cells in vitro [22], as wellas in the antibody-targeted silencing of overexpressed tumorCyclin D1 (CyD1) proteins in mouse gut leukocyte cellsin vivo [23].

2.1.2. Lipoplexes. Lipoplexes are complexes of nucleic acidsand cationic lipid materials which have been widely useddue to their high transfection efficiency. Their preparationcan be easily afforded due to the attractive electrostaticinteractions between the different components upon mixingthem at stoichiometric ratios in an aqueous solution. The mainformulation parameter to obtain the desired size range is givenby the N/P ratio, which is the number of amine groups ofthe polycation divided by the number of phosphate groupsof the nucleic acid. However, the non-permanent character ofthe electrostatic interactions often leads to instability issues,which requires the complexes to be prepared immediately priorto the transfection studies [24]. Although the stability anddelivery efficiency of these lipoplexes could in principle beenhanced by increasing the cationic lipid content, this couldalso result in increased cytotoxicity [25]. The commerciallyavailable Lipofectin is one of the most employed and earliestlipoplexes developed for gene delivery, which consists of amixture of DOTMA and dioleyl phosphatidylethanolamine(DOPE) [18]. Its transfection efficiency has been successfullydemonstrated with RNA for a broad range of cells ofdifferent species [19] and for siRNA for human cell lines[26]. Kim et al synthesized solid lipid nanoparticles usingtricaprin as the solid lipid matrix, Twin-80 and DOPE as thesurfactants and DC-Chol as the cationic agent [27]. When

complexed with plasmid DNA in vitro, transfection efficiencywas higher than Lipofectamine, and in vivo administrationby tail-vein injections showed prolonged gene expression inthe liver, lung, spleen and kidney. In recent studies, cationicsolid lipid nanoparticles were formulated for co-delivery ofpaclitaxel and siRNA, which showed enhanced antitumoreffect in human epithelial carcinoma cells and significantlyreduced the growth of tumors in mice [28]. A comprehensiveoverview of the rational design of lipoplexes along with theirtransfection pathways and mechanisms has been reviewedelsewhere [24, 29].

2.1.3. Synthetic lipids. The progress and increasedunderstanding of lipidic platforms has permitted thedevelopment of novel synthetic nanoparticles. Consequently,numerous structural features have been altered on the basiccationic lipid structure, leading to compositions such as(N-[1-(2, 3-dioleyloxy)propyl]-N′, N′, N′-trimethylammoniumchloride) (DOTAP), which are now commercially availableand under clinical evaluation [30]. For example, hybridmolecules bearing both nucleic acid units and amphiphilicmoieties, such as N-[5′-2′,3′-dioleoyl)uridine]-N′, N′, N′-trimethylammonium tosylate (DOTAU), have been employedto assemble nucleoside-based lipids for efficient transfectionassays of mammalian cell lines in vitro [31]. In addition,specific customizable nanocarriers have been generated froma vast library of over 1200 diverse lipidoid building blocksand screened for the ability to deliver siRNA to a HeLacell line in vitro and to mouse and rat livers in vivo, thusachieving silencing efficiency comparable to commercially

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available Lipofectamine 2000 [20]. Other lipidoids have beenoptimized for the delivery of siRNA to target anti-angiogenicfactor (SHP-1) in human endothelial cell in vitro [32], as wellas silencing Claudin-3, an overexpressed protein in mouseovarian tumors, in vivo [33]. Recently, a co-delivery systemof anticancer drugs and siRNA for effective common therapyhas been reported [34]. The anticancer drug mitoxantrone wasfunctionalized with palmitoleic acid chains to form cationicnanoparticles for siRNA complexation, which showed the highefficiency of the cellular delivery of siRNA and the reductionof tumor cell viability.

2.2. Polymer platforms

Polymers are attractive materials as gene nanocarriers dueto their high versatility and easy manipulation to forma wide range of structures such as polyplexes, micelles,dendrimers, nanocapsules and nanogels. Although the mostnotable are cationic polymers, other types of polymers such ashydrophobic biodegradable polymers and negatively chargedalginage are also used in gene delivery. Polycations differfrom cationic lipids not only in chemical structure, as theydo not contain a hydrophobic moiety and are completelysoluble in water, but also in the interactions with nucleicacids and behavior inside the cell, as a result of the increasedefficiency in condensing genetic material [35]. Both syntheticand biological polycations can form, with nucleic acids,nanosized polyionic complexes named polyplexes. Syntheticpolymers are particularly attractive due to their simple low-costpreparation process as well as to the wide range of structures(differing in length, geometry, substitution and flexibility) andfunctions available starting from a vast library of monomers(figure 3).

2.2.1. PEI. One of the most notable cationic syntheticpolymers known for its high transfection efficiency underin vitro and in vivo conditions is polyethyleneimine (PEI)[36–38]. PEI can be synthesized both in a linear or abranched form with various molecular weights. In addition,its highly cationic charge density aids at condensing nucleicacid molecules, protecting them from nuclease degradation,while its high pH buffering effect facilitates endosomal escapedue to the proton sponge effect. The mean size of PEI/nucleicacid polyplexes depends on the N/P ratio. Although highmolecular weight polyplexes exhibit superior transfectionefficacy, they tend to induce severe cytotoxicity, therebyhampering their therapeutic use [39, 40]. Such toxicity canbe reduced by employing derivatives of PEI obtained viachemical modifications such as ketalization [41], addition ofethyl acrylate or acetylation of amine groups [42] as well as viathe introduction of negatively charged propionic and succinicacid groups [43]. Similarly, poly(L-lysine) (PLL), which wasone of the earliest cationic polymers investigated for antisenseDNA delivery to modulate specifically viral expression [44],shows potential toxicity which was avoided with the use ofglycosylated derivatives [45, 46].

2.2.2. PLGA. Poly(lactic-co-glycolic acid) (PLGA)-basednanocarriers present a significant number of clinicaladvantages such as long-term controlled release, easyadministration and high biocompatibility, and have beenapproved by the US Food and Drug Administration for humanclinical use [47]. As polyesters in nature, these polymersundergo hydrolysis upon implantation into the body, formingbiologically compatible and metabolizable moieties (lacticacid and glycolic acid) that are removed from the body bythe citric acid cycle [48]. In addition, low concentration ofPEI can be incorporated within the PLGA nanoparticle matrixso as to enhance the encapsulation efficiency of short siRNAmolecules, while also improving the release profile, enhancingserum stability and maintaining the low cytotoxic effects [49].

2.2.3. Dendrimers. The use of dendrimers in genedelivery is restricted mainly to the commercially availablepolyamidoamine (PAMAM) and polypropyleneimine (PPI),which are based on an alkyldiamine core with tertiary aminebranches that give a cationic net surface charge [50]. Thesecationic polymers have generated particular interest in nucleicacid delivery for their customizable architecture and size,as well as their potential for tailoring both the type anddensity of functional groups. In addition, these dendrimers arebiocompatible and have high solubility in water, which makesthem very efficient transfection vectors, more markedly in thepresence of more flexible chains or for low-density dendrimer–nucleic acid complexes [51, 52]. For example, the ability ofDNA–dendrimer complexes to transfer oligonucleotides andplasmid DNA to mediate antisense inhibition was assessedin an in vitro cell culture system, which caused ∼25%–50%reduction of baseline activity [53]. Recent studies have shownthat PLL-based dendrons can also mediate efficient antisenseoligonucleotide and siRNA gene knockdown [54].

2.2.4. Chitosan. In recent years, chitosan-based nanocarriershave become one of the natural polymeric vectors that havegained increasing interest as safe and cost-effective deliverysystems for gene materials. Chitosan, a deacetylated formof chitin, has beneficial qualities such as low toxicity, lowimmunogenicity, excellent biodegradability, biocompatibilityand abundant amine groups in its backbone which allow theformation of complexes with nucleotides [55, 56]. In addition,the size (between 150 and 500 nm) and stability of chitosan–nucleic acid polyplexes can be controlled by adjusting themolecular weight of the polysaccharides [57], the degreeof deacetylation and the N/P ratio [58, 59]. Chitosan/DNApolyplexes can be prepared by a complex coacervation processusing sodium sulfate as desolvating agent and they cantransfect a variety of cell types with varying results [55].Some studies have reported an efficient loading of siRNAon chitosan nanocarriers, which resulted in the knockdown ofgene expression by up to 90% in a model of papillary thyroidcarcinoma cells [60]. The transfection efficiency of chitosancan be improved by employing trimethyl chitosan derivatives,and the resulting higher cytotoxicity can be reduced by graftingof PEG [61].

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Figure 3. Chemical structure of the most common synthetic and biological polymers used for gene delivery.

2.2.5. Alginate nanogels. Alginate, a negatively chargedpolysaccharide extracted from brown algae, is anotherbiological polymer widely employed due to its low toxicityand low immunogenicity. The addition of calcium ions to analginate solution under certain conditions leads to gelationand nanoparticle formation as a result of ionic cross-linkagebetween the polysaccharide chains [62]. The resulting Ca–alginate nanoparticles with a mean diameter of 80 nmwere efficiently used as gene carriers. Further investigationsreported a great ability of alginate nanogels to protect antisenseoligonucleotide from degradation in the presence of serum andto modify the biodistribution toward the lungs, the liver andthe spleen after intravenous administration into mice [63]. Theformulation of alginate nanogels often requires the use of apolycation as a platform for gene delivery, which has given riseto diverse studies employing PEI/alginate [64], PLL–alginate[65] and chitosan–alginate nanocomposites [66].

2.3. Inorganic platforms

In spite of the substantial progress in the developmentand applications of lipid and polymeric nanocarriers ingene therapy, in some cases they present low transfectionefficiencies associated with problems of cellular uptake,endosomal release and targeting the nucleus [12]. Recentadvances in the synthesis of inorganic nanoparticles have led toa rapid growth of applications in biomedicine, as they presentmany advantages such as ease of synthesis with controlled size,shape and size distribution [67], ready functionalization andtheir unique optical and magnetic properties, among others.The size and shape of nanoparticles has been found to havea great impact on their cellular uptake, since studies with

nanoparticles of different sizes in mammalian cells have foundthat spherical 50 nm-sized nanoparticles were internalizedmore efficiently over 14, 30, 74 and 100 nm nanoparticles,and also than nanorods of different aspect ratios [68]. Inaddition, the surface coating of these nanoparticles allowstuning of the composition, the charge and hydrophobicity inorder to maximize transfection efficiency while minimizingaggregation and toxicity effects [69] (figure 4).

2.3.1. Metallic nanoparticles. Gold nanoparticles (Au NPs)have been widely utilized as a platform for the delivery ofnucleic acids, because of their easy reductive preparation, highchemical stability, biocompatibility and the well-establishedsurface chemistry, which allows easy functionalization viathiol-terminal groups of organic or bioactive molecules [70].The gold surface additionally provides optical properties witha view toward photothermal ablation and imaging applications.From the conceptual point of view, there are two approaches inorder to incorporate nucleic acids onto Au NPs, which include(i) direct attachment via thiol-Au bonds or (ii) a layer-by-layer assembly. The first possibility involves the modificationof nucleic acid strands with terminal thiols for chemisorptiononto Au NPs surface (figure 5(A)). Following this strategy,siRNA conjugated to Au NPs were found to have a significantactivity for gene silencing in HUH-7 cells in vitro [71]. Therelease of siRNA was facilitated by the reductive intracellularenvironment and exchange reaction with glutathione in theendosomes. In parallel studies, oligonucleotide-modified AuNPs, which were highly stable against enzymatic digestionof nucleases, were also employed for intracellular generegulation [72]. Despite their large negative surface coating,the nanoparticles were readily taken by mouse endothelial

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Figure 4. Schematic representation of the most common inorganic platforms used for gene delivery.

(A) (B)

Figure 5. Direct attachment (A) and layer-by-layer assembly (B) to incorporate nucleic acids onto gold nanoparticles.

cells due to the adsorption of serum proteins on the surfacethrough electrostatic interactions [73]. In the layer-by-layerapproach, the nucleic acids can simply be adsorbed on top ofthe shell of stabilizing molecules around the NPs (figure 5(B)).Rotello and co-workers reported that functionalized Au NPswith cationic quaternary ammonium groups, to which theybound plasmid DNA through electrostatic interactions, couldregulate DNA transcription of T7 RNA polymerase [74]as well as provide an effective means of transfection inmammalian 293T cells (up to ∼eightfold more effective thanPEI) [75]. These composite nanoparticles could protect DNAfrom enzymatic digestion [76] and the effective release ofDNA was probed after treatment with glutathione [77]. Lateron, Klibanov and co-workers functionalized Au NPs withbranched PEI and demonstrated that the transfection efficiencyinto monkey kidney (COS-7) cells was ∼12-fold more potentthan the polymer itself [78]. In another approach, alternating

layers of positively charged PEI and negatively charged siRNAwere deposited onto Au NPs, and the siRNA activity ofthe resulting uniform nanoparticles was tested in CHO-K1cells expressing enhanced green fluorescent protein [79]. Inanother example, Au NPs functionalized with cationic 2-aminoethanethiol formed a complex structure with plasmidDNA containing a luciferase gene, which was transfectedinto target HeLa cells [80]. Finally, the electrostatic bindingof siRNA and Au nanorods (Au NRs) has been recentlyreported by Prasad and co-workers. They utilized Au NRsfor plasmonic enhanced dark-field and confocal microscopyimaging of siRNA, which targeted the DARPP-32 gene inthe dopaminergic signalling pathway of neuronal (DAN) cells[81]. This in vitro model study confirmed that Au nanocarrierscan be used as an innovative vehicle to deliver genes intoneuron cells.

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2.3.2. Carbon nanotubes. The use of carbon-basednanostructures, such as carbon nanotubes (CNTs), inbiomedicine is increasingly attracting attention. One keyadvantage of carbon nanotubes is their ability to translocatethrough plasma membranes by receptor-mediated endocytosis,allowing their use for the delivery of therapeutically activemolecules [82–84]. Moreover, exploitation of their uniqueelectrical, optical, thermal and spectroscopic properties in abiological context is hoped to yield great advances in thedetection, monitoring and therapy of disease. The toxicityinvolving the use of CNTs still remains a controversial issuesince, on the one hand, preliminary toxicology studies on micerevealed no signs of toxicity after four months [85], while onthe other hand, CNTs introduced into the abdominal cavity ofmice showed asbestos-like pathogenicity [86].

The most common strategy to render CNTs viable as aplatform for gene delivery is to use a vector system ableto associate with nucleic acids by self-assembly and assistits intracellular translocation. The surface functionalizationplays an important role since only positively charged CNTs,covalently functionalized with amine-terminal groups, are ableto complex and deliver nucleic acids [87]. CNT-mediatedgene delivery and expression leading to the production ofmarker proteins has been investigated by Kostarelos andco-workers [88, 89], such as expression of β-galactosidasein Chinese hamster ovary (CHO) cells, which was found∼ 5–10-fold higher than the naked pDNA alone. In parallelstudies, Liu and co-workers also reported a non-covalentassociation of pDNA with PEI-functionalized CNTs and foundthat the levels of expression of luciferase in different celllines were much higher for the complexes incorporatingCNTs than pDNA or PEI-pDNA alone [90]. Other verydifferent approaches make use of magnetic CNTs containingNi particles enclosed in their tips [91] or microwave irradiationto open up temporary nanochannels across the cell envelope[92] to deliver pDNA into either mammalian cells or E. coli,respectively. Finally, CNTs have also been conjugated withsiRNA and some promising initial results have been reportedin siRNA-mediated gene silencing. Dai and co-workers havelinked siRNA through disulfide bonds to PEG-functionalizedphospholipids coating the CNT surface [93]. These siRNA-CNT conjugates were internalized by human T-cells andprimary cells, and the siRNA was released intracellularlyupon cleavage of disulfide bonds due to the reducingenvironment of the endosome, which resulted in a 60%–70%knockdown in phenotypic expression [94]. Further examplesemployed cationic amine-functionalized CNTs to complex anddeliver siRNA, which allowed to suppress the expression oftarget proteins in primary cardiomyocytes [95], or suppresstumor growth and prolong survival of tumor-bearing animals[96, 97].

2.3.3. Semiconductor quantum dots. Quantum dots (QDs)are semiconductor nanoparticles that exhibit size-dependentphotoluminiscent properties due to the effect of quantumconfinement, which provides many advantages over traditionalorganic dyes such as narrow emission spectra, largermolar extinction coefficients and enhanced stability with

minimal photobleaching [98, 99]. These QDs can be usedas the core platform onto which nucleic acids and otherfunctional components can be conjugated, therefore givingrise to multifunctional nanocarriers combining therapeutic anddiagnostic imaging functions in a single vehicle toward thetreatment of focal human diseases such as cancer. For example,siRNA and F3 tumor-targeting peptides were conjugated ontoPEGylated QDs using labile and non-labile cross-linkers, andthese nanocarriers were tested toward targeted internalizationand gene knockdown in tumor HeLa cells [100, 101].In addition, QDs covered with proton-absorbing polymericcoatings [102] as well as amphipols [103] have been used forsiRNA delivery in order to facilitate cellular internalizationand endosomal escape.

2.3.4. Magnetic nanoparticles. Magnetic nanoparticleshave also been employed as platforms for gene deliverydue to their appealing intrinsic magnetic properties thatallow both imaging and tracking. Although the synthesisof magnetic nanoparticles with controlled size and shapeis already well established, their stability in physiologicalsolutions and the degree to which their surfaces may bechemically functionalized still remains a big challenge, whichhampers the extent of their biomedical applicability [104].Nevertheless, iron oxide magnetic nanoparticles have beenfunctionalized with siRNA, a dye molecule and a dextrancoating, which allowed monitoring of cell internalizationduring in vitro studies by magnetic resonance imagingand near-infrared fluorescence optical imaging, resulting insuppression of the targeted gene [105]. Similarly, oleic acid-coated magnetic nanoparticles could be stabilized with acationic lipid shell with siRNA molecules, and the resultingformulation could be magnetically guided in vivo to deliverand silence genes in cells and tumors in mice [106]. Furtherstudies by Bhatia and co-workers made use of dendrimer-conjugated magnetofluorescent nanoworms as a modularplatform for siRNA delivery in vivo [107]. SiRNA moleculestargeting against the epidermal growth factor receptor (EGFR)reduced protein levels of EGFR in human glioblastomacells by 70%–80%. Finally, the multifunctional characterof magnetic nanoparticle platforms conjugated with siRNA,cancer-cell-specific targeting moieties and fluorescent dyeswas demonstrated for simultaneous delivery and multimodalimaging of the location and trafficking of siRNA in vivo [108].

2.3.5. Silica nanoparticles. Mesoporous silica nanoparticles(MSNPs) have been investigated as potential carriers of nucleicacids largely due to their inherent biocompatibility and lowtoxicity, easy surface modification through silanol groups onthe surface and the large pore volume which facilitates nucleicacid loading [109]. Diverse polycationic polymers have beenemployed for pore capping of silica nanoparticles [110];among them PAMAM-dendrimer capped MSNPs could bindto plasmid DNA and showed enhanced transfection efficiencyin neural glia and HeLa cells [111]. The same deliveryvehicle was used for the simultaneous delivery of doxorubicinand siRNAs for chemotherapy in multidrug-resistant humanovarian cancer cells [112]. Alternatively, PEI-functionalized

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have also been used for the delivery of siRNA resulting in upto 60% knockdown of green fluorescent protein (GFP) whileexhibiting low cytotoxicity [113, 114].

2.3.6. Calcium phosphate nanoparticles. Particles formed bycalcium phosphate present excellent biocompatibility, sincethis inorganic biological material is the main componentin bones and teeth. While the size-controlled synthesisof calcium phosphate nanoparticles still remains elusive,commonly employed methods make use of the high affinityof this material for binding to nucleic acids [115]. Theoligonucleotide is first dissolved in a phosphate buffer andthen a calcium chloride solution is added, resulting in a calciumphosphate precipitate containing the DNA on its surface [116].Neuronal cells are typically transfected by calcium phosphatemethods optimized to decrease toxicity, but only 1%–5%of cells are efficiently transfected. Improvements in thistechnique lead to up to 60% transfection efficiencies in low-density primary neuronal cultures [117]. These improvementsbasically consisted in the formation of smaller precipitateparticles easier to endocytose. For example, the self-assemblyof a PEG-based block copolymer with calcium phosphateentrapping nucleic acids yielded nanocarriers with diametersin the range of hundreds of nanometers [118, 119]. The calciumphosphate core dissociates in the intracellular environment dueto the lowered Ca+2-concentration, thus allowing the release ofthe cargo [120]. Similarly, lipid-coated calcium phosphateformulations for siRNA delivery have also been developedfor controlled disassembly in the acidic environment of theendosomes [121].

3. Binding and uptake of the nanocarriers by thecells

The lipid bilayer membrane is a common obstacle forthe delivery of nucleic acids into the cell. Unlike small-molecule drugs, macromolecules including DNA, RNA andtheir nanocarriers are prohibited from passive diffusion into thecell [122]. While neutral functional groups on nanoparticlesare excellent in preventing nanocarrier–biological interactions,most charged moieties are responsible for active interactionwith cells. It is generally accepted that interaction of lipoplexesand polyplexes with cells in culture, in the absence of serum,is non-specific and involves mainly electrostatic interactionsbetween the positively charged complexes and the negativelycharged cell surface [36, 123]. Endocytosis or endocytosis-like mechanisms have been proposed as the main pathways ofinternalization of lipoplexes and polyplexes rather than fusionof membranes [124], which was evidenced by microscopyanalysis and endocytosis-interfering drugs. In parallel studies,it has been observed that cancer cell membranes absorbmuch less neutral and negatively charged Au NPs, withconsequently lower levels of internalization, than positivelycharged particles [125]. The internalization of negativelycharged nanoparticles is believed to occur through non-specificbinding and clustering of the particles on the cell membrane,which induces endocytosis. By contrast, positively chargednanoparticles are rapidly internalized via clathrin-mediated

pathway [126]. The presence of slight changes in surfacefunctionalities such as hydrophobic structures can lead tovarying amounts of cellular internalization [127].

The ability and the specific roles of different types ofendocytosis in the uptake depend, in part, on the cell type andare important parameters that should be taken into account.For example, in primary cell cultures, such as epithelial airwaycells, the binding and uptake are important rate-limiting stepsfor the transfection efficiency [128, 129], while in establishedcell lines such as fibroblast-like COS or HeLa cells, the uptakeis not a rate-limiting step [130, 131]. The presence of serumhas a dramatic inhibitory effect on the transfection efficiency[132], since polycationic nanocarriers become negativelycharged after interaction with plasma proteins, thus decreasingthe interactions with the cell membrane and consequently theuptake [133].

4. Targeting specificity

The delivery of oligonucleotide nanocarriers to specific tissuesin vivo can be accomplished via two approaches, passive andactive targeting. Passive targeting of tumor tissues can beachieved by the enhanced permeability and retention effect(EPR) [134], in which a combination of leaky vessels and poorlymphatic drainage, both characteristic of the rapid onset ofangiogenesis in solid tumors, results in improved extravasationand enhanced retention in tumor tissues. Nanoparticles witha diameter of less than 500 nm can achieve passive tumortargeting via the hypothetical EPR effect [135]. However,passive targeting is a limited strategy, since not all tumorsexhibit the EPR effect and even amongst those that do, thedegree of permeability may differ significantly within a singletumor.

Alternatively, active targeting of specific tissue and cellscan be achieved by modification of the delivery platform withtarget-recognition molecules that facilitate localization of thenanocarrier via specific ligand–receptor interactions at thecell surface. A great number of ligands have been developedand can be incorporated into nanocarriers for improvedtargeting specificity or enhanced cellular uptake. Among them,antibodies and aptamers are typically used for targeting cell-specific receptors as they present high binding affinities andtarget specificities, while cell-penetrating peptides (CPPs)provide enhanced cellular internalization as they facilitatethe translocation across the membrane. For example, CPPssuch as HIV-TAT protein or VP22 from Herpes simplex virustype I (obtained from the protein transduction domains ofvirus) [136, 137], nuclear-localization-signal (NLS) peptides[138], model proteins such as transferrin [139] as well asother peptide motifs such as Arg–Gly–Asp (RGD) [140], PLL[141] and arginine-rich peptides [142] have been shown aseffective mediators of nanocarrier internalization into variouscell types. Most of such biological motifs have positivelycharged residues assisted by hydrophobic moieties for efficientcellular internalization, which correlates very well with thebehavior of positively charged functionalized nanocarriers[143]. Recently, Wood et al have shown tumor-targeted genedelivery exceeding that of PEI by conjugating a PAMAM

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dendrimer with a short peptide ligand that targets the tumorantigen glucose regulated protein-78 [144].

5. Conclusions and perspectives

The development of gene delivery systems using non-viralnanocarriers presents multiple challenges such as overcomingphysiological barriers, conjugating targeting ligands orinteracting with the molecular targets. The mechanisms oftargeting, binding, interaction or release depend on severalfactors, including the vector used, the type of cells and thetransfection conditions employed (e.g. in vitro or in vivo).As a consequence, a wide range of material platforms iscurrently being used as transfection vectors and a hugeamount of fundamental research has been performed in thisdirection. Almost at the same time, many research groups arepushing forward with experiments that have direct clinicalimplications either on the molecular level or in treatmentsin the entire organism. Approaches to eventual therapiesrange from the permanent repair of chromosomal mutationto silencing the expression of a given gene using antisenseoligodeoxynucleotides or siRNA. Unfortunate events inclinical gene delivery trials have raised serious concernsregarding the safety of using gene delivery as medicaltreatment, which has somehow hampered a smooth and rapidprogress in this field.

A rational design of highly efficient nanocarriers requiresa deep understanding of the interactions between the vector andthe nucleic acid, and the cellular pathways and the mechanismsinvolved in the entry into the cell. In this paper, we providean overview of the different material platforms used for genedelivery together with the most recent developments in thisfield, which is under constant progress and expansion. Whilesignificant improvements have been made, there is still muchwork that has to be performed before any nanocarrier can beused as a viable option for the treatment of certain humandiseases.

Acknowledgment

We thank Dr Elena Bellido from Centro de Investigacion enNanociencia y Nanotecnologıa (CIN2, ICN-CSIC) for helpwith figures.

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