Polycation-Based Gene Therapy: Current Knowledge and New ...

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Current Gene Therapy, 2011, 11, 288-306

Polycation-Based Gene Therapy: Current Knowledge and New Perspectives Marcio J. Tiera2, Qin Shi1, Françoise M. Winnik3 and Julio C. Fernandes1,* 1

Orthopedic Research Laboratory, Hôpital du Sacré-Cœur de Montréal, Université de Montréal; 2Departamento de Química e Ciências Ambientais, UNESP-Universidade Estadual Paulista, São José do Rio Preto, Brazil; 3Faculty of Pharmacy and Department of Chemistry, Université de Montréal, Montreal, QC, Canada H4J 1C5 Abstract: At present, gene transfection insufficient efficiency is a major drawback of non-viral gene therapy. The 2 main types of delivery systems deployed in gene therapy are based on viral or non-viral gene carriers. Several non-viral modalities can transfer foreign genetic material into the human body. To do so, polycation-based gene delivery methods must achieve sufficient efficiency in the transportation of therapeutic genes across various extracellular and intracellular barriers. These barriers include interactions with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is a potential obstacle for functional delivery to target cells. Cationic polymers constitute one of the most promising approaches to the use of viral vectors for gene therapy. A better understanding of the mechanisms by which DNA can escape from endosomes and traffic to enter the nucleus has triggered new strategies of synthesis and has revitalized research into new polycation-based systems. The objective of this review is to address the state of the art in gene therapy with synthetic and natural polycations and the latest advances to improve gene transfer efficiency in cells.

Keywords: DNA, gene therapy, nanoparticles, polycations, polymers. 1. INTRODUCTION The development of effective non-viral vectors is a challenge in the field of gene therapy today. The benefits of finding appropriate carriers would be enormous since they present a series of advantages when compared to viral vectors. Viral vectors facilitate a high transfection rate and rapid transcription of foreign material inserted in the viral genome. However, the utility of viruses in gene therapy could be limited by various factors. First and foremost, safety issues have arisen after the death of a patient during a clinical trial that investigated the potential of gene therapy employing viral vectors [1]. Second, gene therapy with viral vectors is limited by the fact that only small DNA sequences can be inserted in the virus genome, and large-scale production may be difficult to achieve. Third, viruses present a variety of potential problems to patients, such as toxicity, immune responses and inflammatory reactions. Lastly, insertional mutagenesis and oncogenic effects can occur in vivo [2]. The limitations of viral vectors, particularly regarding safety concerns, have led to the evaluation and development of alternative vectors based on non-viral systems. Synthetic and natural polycations are good candidates because they have some advantages over viral vectors, e.g., low immunogenicity, relatively large sequences condensed in small particles, good protection of DNA, easy manufacture, and their modification targeting specific cells and/or diseases [3]. However, several problems, such as toxicity, *Address correspondence to this author at the Orthopedic Research Laboratory, Hôpital du Sacré-Coeur de Montréal, 5400 boul. Gouin ouest, Montreal, QC, Canada H4J 1C5; Telephone: 514 338 2222 Extension 2459; Fax: 514 338 3661; E-mail: [email protected] 1566-5232/11 $58.00+.00

lack of biodegradability, low gene transfection yield, poor biocompatibility and, particularly, insufficient transfection efficiency, need to be solved prior to the practical implementation of features to shuttle genes into cells [4,5]. The number of polycations utilized in gene therapy is huge, and many of them have been investigated in fundamental studies to gain a better understanding of DNA-polycation interactions, focusing on the importance of polycation structure, pH and charge ratio (+/-) on particle size and DNA protection. In this review, we summarize the progress made with and perspectives into the most studied natural and synthetic polymers. The present version is an update of research in the last 5 years, from 2006 to 2011, focusing on the most promising systems and the strategies utilized to overcome extracellular and intracellular barriers. 2. POLYPLEXES AND PROPOSED MECHANISM OF THE TRANSFECTION PROCESS The term polyplexes refers to complexes formed when a polycation is mixed with DNA in aqueous solution. Polycation-DNA interaction is driven mainly by electrostatic contact between the polycation and charged phosphate groups, leading to the reversible linear to globule transition of DNA. At a certain critical polycation:DNA ratio, DNA undergoes localized bending or distortion, which facilitates the formation of nanoparticles (NPs) with different shapes, such as rods, toroids [6- 8] and spheroids [9]. The ability of polycations to condense DNA into NPs is often critical for transfection efficiency, since DNA must be protected from DNase degradation. As the DNA wrapped in inter-polyelectrolyte complexes is well-protected, the next step is to reach its target, the cell. In this respect, it is well-accepted that the polye© 2011 Bentham Science Publishers Ltd.

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lectrolyte complex of polycation-DNA exhibiting a net positive charge binds to negatively-charged cell membranes Fig. (1, Step A) [10, 11]. A net positive charge is fundamental in the process, and the level of transfection is shown to increase with the charge ratio (+/-), reaching a maximum and decreasing at higher stoichiometries [12, 13].

plasmid DNA (pDNA) Fig. (1, Step C). This obstacle is also considered crucial, and 2 main mechanisms have been proposed to explain how pDNA enters the nucleus: (i) passive DNA entry into the nucleus during cell division when the nuclear membrane is temporarily disintegrated, or (ii) active DNA transport through nuclear pores. This step has been reviewed by several authors [19-22] and the mechanism is now better understood. In the last 5 years, several authors have shed some light on the subject. Although the transport of macromolecules across the nuclear pore is clearly understood, the suggested mechanisms by which pDNA traverses the nuclear pore complex (NPC), either as a free molecule or during the M phase when the nuclear envelope is disassembled are also well-accepted [23, 24]. However several authors have shown that after intracellular delivery, pDNA binds to cellular proteins and peptides to transverse the nuclear envelope [25, 21]. Recently, Munkoje et al. reported that cytoplasmatic factors may play an important role, helping pDNA nuclear import. These authors demonstrated an increase in gene transfer in non-dividing HeLa cells transiently transfected with pDNA containing binding sequences from 2 DNA shuttle proteins, NM23-H2 and the homeobox transcription factor Chx10 [26]. They argued that their data support the hypothesis that exogenous pDNA binds to cytoplasmic shuttle proteins and is then translocated to the nucleus by minimal import machinery. Glucocorticoid (GC) ligands have been deployed as nuclear localization signals to help the import of polyplexes into the nuclear envelope [2729]. Triamcinolone acetonide (TA) was attached to different molecular weight (MW) PEIs with the aim of investigating the effect of both TA and MW on transfection efficiency. In in vivo and in vitro studies, these authors showed that TA attached to lower MW PEIs elicited high transfection efficiency due to its smaller size and higher translocation ability, which was attributed to the TA ligand [30]. Nuclear factor kappa B (NFB), a transcription factor, has also been exploited to promote pDNA nuclear import [31, 32]. By using plasmids encoding luciferase and bearing the appropriate

After internalization, the next crucial step in gene delivery with cationic polymers is the escape of polymer/DNA complexes from the endosome. The inefficient release of DNA/polymer complexes from endocytic vesicles into the cytoplasm is one of the primary causes of poor gene delivery [14]. In this respect, the approach is to enhance endosomal escape with cationic polymers that have a pKa slightly below physiological pH. Endosomal escape is believed to take place through a mechanism known as the “proton sponge”, and the importance of this step has been recognized and reviewed recently [15]. The hypothesis has been proposed to explain the high transfection activity of polyethyleneimine (PEI) [16]. It has been demonstrated that PEI has buffering capacity over a broad pH range; hence, once PEI-based polyplexes are present in the endosome, they can absorb protons that are pumped into the organelle. Swelling of the polymer occurs because of repulsion between protonated amine groups. Moreover, to prevent the build-up of a charge gradient due to the influx of protons, an influx of Cl- ions also takes place. The influx of both protons and Cl- ions increases osmolarity of the endosome and causes water absorption. The combination of polymer swelling and osmotic swelling of the endosome evokes destabilization of the endosome and release of its content into the cytoplasm Fig. (1, Step B) [16, 17]. This hypothesis has been supported recently with the use of N-quaternized (and, therefore, nonproton sponge) versions of PEI and specific cell function inhibitors [18]. These authors reported that both Nquaternization and bafilomycin A1 (a vacuolar proton pump inhibitor) reduced the transfection efficiency of PEI by approximately 2 orders of magnitude. The next step, the nuclear envelope, is the ultimate obstacle to the nuclear entry of

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Fig. (1). Schematic of crucial steps in the gene therapy mechanism: (A) Internalization and endocytosis by the cell membrane and intracellular trafficking. (B) DNA-polycation release from the endosome into the cytoplasm. (C) Internalization of plasmid DNA into the nucleus promoted by nuclear localization signals (NLS). (D) Gene expression: protein can be secreted out of the cell, released into the cytoplasm, or fixed onto the membrane.

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sequence mimicking NFB, Gonçalves et al. determined that the nuclear delivery of 3NF plasmids was more efficient than that of 3NF-free plasmids. Also, their most efficient construct for in vitro transfection had long-lived luciferase expression in vivo [33]. Based on recent studies, it has become clear that carriers presenting a higher ability to enter the nuclear envelope will have a significant impact on transfection efficiency. 3. SYNTHETIC POLYMERS 3.1. Polyethyleneimine PEI has been the most studied polymer in gene therapy because of its relatively high transfection efficiency, and the many strategies developed to improve it have been reviewed recently [34, 11,35]. First reported as a potential gene delivery system by Boussif et al. [16], this polymer is a cationic polyelectrolyte that can be obtained in linear or branched forms. Due to its amino groups, PEI is able to efficiently promote DNA condensation, forming NPs that are easily endocytosed by different cell lines. Its efficiency has resulted in numerous studies and reports which attempted to find ways to improve its properties [36, 37]. Besides high transfection, PEI is reported to efficiently protect DNA from degradation by DNases [16]. However, the main disadvantage of this polymer is its high toxicity, which has been noted by various authors [38, 34, 39]. Its efficiency and cytotoxicity depend on MW, with PEI25-kDa being the most efficient but cytotoxic [40]. The cross-linking of low MW PEIs has been considered as an alternative to circumvent this problem [40]. Cross-linking boosted the gene delivery efficiency of small PEIs by 40- to 550-fold in vitro, and the efficiency of the most potent conjugates even exceeded that of branched PEI 25-kDa by an order of magnitude. It has been observed that at concentrations where 25-kDa PEI resulted in >95% cell death, the conjugates afforded nearly full cell viability. Cross-linked PEIs were 17 to 80 times more effective than unmodified ones in vivo. Furthermore, their efficiencies were up to twice those of 25-kDa PEI. However, Wightman et al. [41] showed that when complexes were generated in salt-free conditions, all forms (linear and branched) of PEI transfected cells inefficiently. These authors concluded that the greater efficiency of linear PEI might be due to inherent kinetic instability under salt conditions. Numerous alternatives have been proposed to decrease PEI cytotoxicity [42]. Fig. (2) summarizes some representative structural strategies adopted to increase both the cytocompatibility and efficiency of PEI. PEI conjugated with poly(ethylene glycol) (PEG) [11] and sugars, such as mannose [43] and galactose [44-45] presented improved properties. In general, PEG attachment masks the surface charge of NPs, decreases toxicity and nonspecific interactions, consequently increasing their half-life in the bloodstream [45-47]. For example, Ogris et al. studied different alternatives to generate surface-shielded formulations, i.e., attaching ligand and PEG molecules to PEI either before or after DNA complex formation [47]. Intravenous (i.v.) injection of transferrin (Tf)-PEG-coated polyplexes resulted in gene transfer to subcutaneous (s.c.) Neuro2a neuroblastoma tumors of syngeneic A/J mice. Also, epidermal growth factor (EGF) was tested to target human hepatocellular carcinoma xenografts in SCID mice. In these models,

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luciferase marker gene expression levels in tumor tissues were 10- to 100-fold higher than in other organ tissues. Repeated systemic application of Tf-PEG-PEI/DNA complexes encoding tumor necrosis factor-alpha in tumor-bearing mice induced tumor necrosis and inhibition of tumor growth in 3 murine tumor models of different tissue origins: Neuro2a, M-3 and B16 melanoma [47]. Tang et al. [48] investigated PEG-modified PEI for in vivo gene expression in the central nervous system. Varied numbers of linear PEG (2 kDa) were grafted onto branched PEI (25 kDa) from the average number of PEG per PEI macromolecule at 1-14.5. These authors reported that while higher degrees of PEG grafting did not improve gene expression, a PEI conjugate with 1 segment of PEG was able to mediate transgene expression in the spinal cord up to 11-fold higher than PEI homopolymer after intrathecal administration of its DNA complexes into the lumbar spinal cord subarachnoid space. Improved gene expression with this conjugate was observed in the brain as well after lumbar injection. The study provides in vivo evidence that an appropriate degree of PEG modification is decisive in improving gene transfer mediated by PEGylated polymers. However, many strategies have been considered to improve transfection efficiency. Thomas and Klibanov [49] reported that the chemical modifications of nitrogen atoms of PEIs drastically altered their efficiency as synthetic vectors. N-acylation of PEI with a molecular mass of 25 kDa with alanine nearly doubled its transfection efficiency in the presence of serum and also lowered its toxicity. Furthermore, dodecylation of primary amino groups of 2-kDa PEI yielded a nontoxic polycation whose transfection efficiency in the presence of serum was 400 times higher than that of the parents and which exceeded even that of 25-kDa PEI by 5-fold. Owing to its capacity to interact with DNA, PEI has also been used to modify liposomes to improve transfection [63, 64]. Liposomes modified with cetylated polyethylenimine (PCLs) manifested remarkable transfection efficiency to COS-1 cells in vitro, in comparison to conventional cationic liposome preparations [63]. Effective gene transfer was observed in 8 malignant and 2 normal line cells tested as well as in COS-1 cells. The MW of PEI was shown to affect the transfection efficiency of PCLs-mediated gene transfer, and PEIs with MWs of 600 or 1,800 Da were much more effective than PEI of 25,000 Da. Recently, PEI of 600 Da MW was cross-linked by 2-hydroxypropyl-gamma-cyclodextrin (HP-CD) and then coupled to MC-10 oligopeptide containing a sequence of Met-Ala-Arg-Ala-Lys-Glu. Besides its low cytotoxicity and strong targeting specificity to human EGF receptor (HER2), animal studies revealed that the vector enhanced the anti-tumor effect in tumor-bearing nude mice compared to PEI (25 kDa), indicating that this new PEI derivative could be a potential candidate for cancer gene therapy [65]. The combination of PEI with a variety of other synthethic and natural polymers has been tested, aiming to improve transfection efficiency. In general, attachment of either natural or other biocompatible synthetic polymers provides the new polymer with lower cytotoxicity and good biodegradability. These approaches include the grafting of polymers, such as dextran [66], polycarbonates [67; 68], polyglutamic acid [69], heparin [70], poly(caprolactone) [71] and chitosan [72- 74].



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Fig. (2). Chemical structures of representative PEI derivatives: 1. PEG; 2. maltose; 3. succinic acid; 4. dextran; 5. cyclodextrin; 6. polycarbonates; 7. polyglutamic acids; 8. alkylcarboxylic acids; 9. dithiobis(succinimidyl propionate); 10. cholesterol.

Increased transfection efficiencies have been observed in cell systems by coupling ligands, such as peptides [65, 7578], folic acid [79], galactose [80, 81] and mannose [82]. The nuclear entry of plasmids carried by PEI derivatives can also be improved with nuclear corticoids. Dexamethasone, a steroid that can dilate NPCs and translocate to the nucleus when it is bound to its GC receptor, was attached to PEI to tranfect human embryonic kidney 293 cells and human liver carcinoma HepG2 cells. The results suggested that the conjugation of a GC to PEI could act as a ligand for intracellular GC receptors present in mammalian cells, making the nuclear membrane more vulnerable to NPs by enlarging the nuclear pores [29]. Although several studies disclosed improvements in toxicity and transfection efficiency, the final intracellular fate of PEI, its fragments and more in vivo experiments as well, are necessary to achieve suitable vectors based on PEI. 3.2. Poly[2-(Dimethylamino) Ethyl Methacrylate] (PDMAEMA) Although PEI is the most studied polymer, a huge number of other synthetic polymers have been tested as gene carriers. In general, polymers contain secondary and tertiary amino groups in their structures. This choice, as discussed before, is partially based on previous investigations of PEI, and because it is absolutely necessary to promote electrostatic interaction with the DNA chain. However, buffering capacity is an important property, and polymers containing tertiary amino groups have been postulated to be more ap-

propriate [83]. PDMAEMA and its copolymers have been studied extensively. Comparative experiments on different cationic transfection agents for in vivo gene delivery after i.v. administration have determined that PDMAEMA is more effective than branched PEI and poly(L-lysine) (PLL) [84]. This polymer condenses DNA into small, positively-charged particles that are able to transfect various cell types [85, 86]. Recently, based on in vitro studies, Layman et al. demonstrated that the MW of PDMAEMA has a dramatic influence on transfection efficiency, with gene expression increasing as a function of escalating MW. However, the MW of PDMAEMA did not have any influence on the cellular uptake of polyplexes. These authors also proposed that polyplex sizes had no significant influence on transfection efficiency [87]. However, many barriers must be overcome to make this polymer a good carrier [88, 89]. For example, confocal laser scanning microscopy has revealed that after 24 hours of incubation, a major part of the polyplexes was still present in endosomes [90, 91]. Also, cytotoxicity and nonspecific interactions must be reduced to enhance transfection efficiency. Random copolymers of 2-(dimethylamino) ethyl methacrylate (DMAEMA) with ethoxytriethylene glycol methacrylate or N-vinylpyrrolidone were evaluated as polymeric transfectants in vitro [92]. The advantage of these copolymers was mainly their reduced cytotoxicity when compared with that of the homopolymer. Copolymers with a MW up to 170 kDa had the same transfection capability as a homopolymer of comparable MW. However, higher MW copolymers showed reduced transfection capability com-


pared to the homopolymer, which was ascribed to the diminished capability to condense plasmid size. Diblock copolymers also have been investigated [93]. Two series of DMAEMA-block-2-(methacryloyloxyethyl phosphorylcholine (DMAEMA-MPC) diblock copolymers were synthesized for evaluation, varying independently and systematically in either MPC or DMAEMA block length. DMAEMA-MPC copolymer complexes manifested significantly lower transfection efficiencies than DMAEMA homopolymer. The transfection data in general displayed a similar trend as flow cytometry: the higher the cellular association, the higher the transfection level. Complexes formed with DMAEMA100MPC30 copolymer showed the highest transfection rate, but even this was typically only 10% of that exhibited by DMAEMA homopolymer complexes. The transfection efficiencies of each of the other DNA complexes were not significantly different from free DNA, and neither DMAEMA homopolymer nor DMAEMA100MPC30 copolymer was comparable to Lipofectamine™ [94]. Argawala et al. synthesized pentablock copolymers of poly(diethylamino-ethylmethacrylate) (PDEAEM), poly (ethylene oxide) (PEO) and poly(propylene oxide) (PPO) PDEAEM-b-PEO-b-PPO-b-PEO-b-PDEAEM - and investigated their potential as non-viral vectors for gene therapy [95]. The polymers showed much less cytotoxicity than commercially-available ExGen 500 (linear PEI). It was observed that the cytotoxicity of these copolymers could be tailored by changing the relative lengths of their blocks. In vitro transfection efficiencies of the polymers with green fluorescent protein (pEGFP-N1) and luciferase (pRL-CMV) reporter genes were found to be comparable to that of ExGen 500. Recently, selective chemical modifications of PDMAEMA structure have added benefit, in that they reduce cytotoxicity while improving transfection efficiency. Some of these derivatives are shown in Fig. (3). Among the most promising candidates are branched structures assembled from PDMAEMA linked to more biocompatible polymers and monomers, such as poly (hydroxyethyl methacrylate) [96], N-isopropylacrylamide [97], and other similar polymers based on DMAEMA groups [98, 99]. 3.3. Poly( -Amino Ester)s Poly(-amino ester)s are biodegradable cationic polymers that condense pDNA at physiological pH, and are readily synthesized via the conjugate addition of primary or secondary amines to diacrylates [100]. Systematic investigations have been conducted to accelerate the discovery of new gene carriers. The synthesis and study of more than 500 degradable poly(-amino ester)s led to the identification of 2 polymers with transfection levels 4-8 times higher than those of PEI [101, 102]. This library has fostered correlations between polymer structures and transfection efficiencies [101, 103]. Polymers containing structural elements in which an oxygen atom is 2 carbons removed from an amine are generally unable to form intimate electrostatic complexes with DNA. Characterization of the effective diameters and zeta potentials of polymer/DNA complexes indicates that, in general, small particle sizes and positive surface charges lead to higher levels of cellular uptake in vitro and that polymers

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with multiple amines per repeat unit tend to yield complexes with smaller particle sizes and larger zeta potential. Evaluation of the pH environment of delivered DNA suggested that polymers incorporating an imidazole group or 2 amines in close proximity could successfully avoid low pH lysosomes [101]. Among the candidates identified in this screening, poly[(1,6-di(acryloxyethoxy)hexane)-co-(4-amino-butanol)] enhanced pDNA transgene expression by 7-fold and its immunogenicity by 70%. It was found that polymers with moderately hydrophobic backbones and terminal alcohol groups facilitated transfection most effectively in vivo [103]. These authors also tested one of the best performing polymers, in mice, for toxicity and DNA delivery after intratumor and intramuscular (i.m.) injection. The polymer delivered DNA intratumorally 4-fold better than one of the best commercially-available reagents, jetPEI, and 26-fold better than naked DNA [104]. Poly(beta-amino esters) are less cytotoxic when compared with PEI because of their biodegradability, which occurs via hydrolytic reaction of the ester groups. Progress with and the overall analysis of these polymers has been reviewed. For a detailed description of their structures, we refer the reader to Green et al. [105]. However, recent studies show that improved properties can be reached by combining the poly(beta-amino esters) with other polymers. Cherng et al. prepared and characterized poly(b-amino ester) containing branched PEI25k (PEDP-PEI-25kDa) and demonstrated that DNA had a strong electrostatic interaction to self-assemble NPs. The transfection efficiency of PEDP-PEI25kDa into COS-7 cells was higher than that of PEDP without branched PEI25kDa. These authors concluded that the binary mixture of PEDP-PEI-25kDa may be an attractive cationic carrier for gene delivery and an interesting candidate for further study [106]. Bhise et al. synthesized acrylateterminated poly(1,4-butanediol diacrylate-co-5-amino-1pentanol), which was subsequently end-capped with aminecontaining small molecules with primary and tertiary amine groups, aiming to increase intracellular delivery. The synthesized polymers were more effective for gene delivery than FUGENE-HD, one of the leading commercially-available reagents for non-viral gene delivery. These authors claimed that changes in MW within the same polymer structure affected gene delivery. They also showed that for the same base polymer only minor alterations to its terminal groups dramatically affected tranfection efficiency. The most effective polymers were those end-capped with tertiary amine groups, indicating that the presence of tertiary amines could potentially aid in buffering endosomal pH and thereby enhancing transfection efficiency [107]. In another approach, poly(ester amine) (PEA) was conjugated to folic acid, aiming to mediate endocytosis. In vitro and in vivo experiments showed that the ester backbone reduced the cytotoxicity of PEI, which was attached to the PEA backbone to enhace endosomal buffering capacity, leading to the faster endosomal release of polyplexes. These authors demonstrated that FP-PEA/TAM67 complexes could inhibit tumor growth through diverse functions, such as angiogenesis and apoptosis, thus demonstrating target-specific gene delivery [108]. 3.4. Dendrimers Dendrimers are synthetic hyperbranched polymers highly soluble in aqueous solutions with positively-charged termi-



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Fig. (3). Schematic representation of chemical structures: 1. PDMAEMA and its derivatives; 2. grafted isopropyl acrylamide and butyl methacrylate copolymers; 3. vinyl pyrrolidone copolymers; 4. end-capped cholesterol; 5. block copolymers with PEG and butyl methacrylate; 6. block copolymers with PEG; 7. penta block copolymers of hydroethylmethacrylate and PEG; 8. lactosylated poly(ethylene glycol)-bpoly(silamine)-b-poly[2-(N,N-dimethylamino)ethyl methacrylate].


nal groups. Although their structure may vary, polyamidoamine dendrimers are by far the most studied systems. Fig. (4) provides a limited representation of alternatives utilized to generate efficient carriers. They can bind DNA-forming complexes termed dendriplexes [109] and, by analogy, similar complexes formed by liposomes and DNA, called lipoplexes. DNA within dendriplexes is protected from cellular and restriction nucleases [110]. Although dendrimers present a highly-branched structure, it is believed that the formation of DNA/dendrimer complexes is based entirely on charge interaction [110, 111]. Dendrimers from polyamidoamine synthesized from methyl acrylate and ethylenediamine were considered to be well-tolerated by cells and, when complexed to plasmids encoding reporter genes, mediated highly-efficient transfection of a wide variety of cells in culture [120, 121]. Covalent attachment of the amphipathic peptide GALA was also reported to significantly enhance transfection efficiency in both primary cells and cell lines [122]. A comparative study subsequently disclosed that fractured dendrimers and PEI are efficient gene delivery vehicles and can be used for arterial gene therapy via adventitial gene delivery. Fractured dendrimers had the highest in vivo gene transfer efficiency when compared to PEI with a molecular size of 25 kDa [123].

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Highly-efficient transfection of a broad range of eukaryotic cells and cell lines was reported with minimal cytotoxicity employing DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers, and the capability of dendrimers to transfect cells appeared to depend on size, shape, and number of primary amino groups on the polymer surface [124]. Also, it was postulated that gene delivery by dendrimers operates via a cholesterol-dependent pathway [125]. Transfection efficiency of the gene delivered by dendrimers was drastically decreased after the extraction of plasma membrane cholesterol. Replenishment of membrane cholesterol restored gene expression. The binding and especially the internalization of dendriplexes were strongly reduced by cholesterol depletion before transfection. Fluorescent dendriplexes co-localized with the ganglioside GM1 present in membrane rafts in both immunoprecipitation assay and confocal microscopy studies. These authors interpreted their data as a strong indication that membrane cholesterol and raft integrity are physiologically relevant for the cellular uptake of dendrimer-DNA complexes. On the other hand, different dendrimers have been investigated to evaluate the effect of structure on complex formation and transfection efficiency. The highest transgene expression and cytotoxicity are also dependent on generation numbers [126, 127], and the presence of addi-

Fig. (4). Schematic representation of dendritic structures of different generations further modified with: 1. amidoamine; 2. phenylalanine; 3. hydrophobic chains; 4. peptides; 5 folate; 6. PEG-folate conjugate; 7. arginine; 8. lactoferrin; 9. PLL; 10. porphyrin; 11. magnetic NPs. The symbol “Gn” indicates the number of generations.


tional compounds, such as DEAE-dextran and surfactants, may increase transfection [124]. The chemical modification of low-generation dendrimers with biocompatible PEG chains was reported to produce a 20-fold increment in transfection efficiency, and the cytotoxicity of PEGylated dendrimers was very low when compared with partiallydegraded dendrimer controls [128]. The post-modification of dendrimer structures was also undertaken to augment transfection efficiency. Poly (amidoamine) dendrimers with phenylalanine or leucine residues at their chain ends provided efficient gene transfection of cells through synergy of the proton sponge effect, which was induced by internal tertiary amines of the dendrimer and hydrophobic interaction by hydrophobic amino acid residues in the dendrimer periphery [129]. Dendrimers having 16, 29, 46, and 64 terminal phenylalanine residues were prepared by reaction of the amine-terminated poly(amidoamine) dendrimer and L-phenylalanine using the condensing reagent 1,3-dicyclo-hexylcarbodiimide. The transfection activity of these phenylalanine-modified dendrimers for CV1 cells, an African green monkey kidney cell line, grew concomitantly with the increasing number of terminal phenylalanine residues. On the other hand, the attachment of L-leucine residues was unable to improve transfection activity of the parent dendrimer, probably because of the relatively lower hydrophobicity of this amino acid. It was also reported that the phenylalanine-modified dendrimer exhibited higher transfection activity and lower cytotoxicity than some widely-used transfection reagents [129]. The modification of polyamidoamine dendrimers on terminal amino groups is still currently under investigation and can be considered a good strategy to improve efficiency and biocompatibility. Many approaches have been attempted [130-132]. Santos et al. [130] tested dendrimers functionalized with peptides to transfect mesenchymal stem cells (MSCs). These authors investigated 2 peptides, a lowaffinity MSC-binding (LAB) peptide and a high-affinity MSC-binding (HAB) peptide, aiming to demonstrate the potential of receptor-mediated gene delivery into MSCs. The gene expression achieved with conjugates attached to the HAB peptide was higher than that obtained with Superfect, a commercially-available gene delivery system made of partially degraded polyamidoamine dendrimers (PAMAM) dendrimers. Receptor-mediated gene delivery into MSCs was confirmed by saturating cell receptors with the peptide prior to transfection, and the new carrier exhibited minimal cytotoxic effects on cultured cells [133]. In a similar approach, a 29-amino-acid peptide derived from rabies virus glycoprotein (RVG29) was exploited as a ligand for efficient braintargeting gene delivery [134]. RVG29 was modified on PAMAM through bifunctional PEG, then complexed with DNA, yielding PAMAM-PEG-RVG29/DNA NPs. NPs were injected in mice and, from in vitro and in vivo studies involving imaging analysis, these authors concluded that PAMAMPEG-RVG29 was efficient for brain-targeting gene delivery. Recently, the different strategies undertaken to improve the efficiency and safety of these systems were reviewed by Ravinã et al., emphasizing the different types of dendritic structures and the modifications performed to impart specific requirements to the carriers [135].


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4. “NATURAL POLYMERS” 4.1. Polylysine and Other Poly (Aminoacids) PLL is considered to be one of the first polycations studied for gene delivery [144]. It is biodegradable, a property that makes it especially suitable for in vivo use; however, the polymer exhibits modest to high toxicity. Its polyplexes are taken up into cells as efficiently as PEI complexes, but transfection efficiencies remain several orders of magnitude lower. A potential reason for this is the lack of amino groups with a pKa between 5 and 7, thus allowing no endosomolysis and low levels of transgene expression [145]. Due to its lower transfection efficiency, much of the work with PLL was initiated by targeting mediated via receptors. In general, DNA/polyamino acid complexes are taken up by cells but require an endosomolytic agent, such as chloroquine, to facilitate transfection. A comparative study reported that DNA/polyornithine (PLO) polyplexes resulted in the highest levels of expression, in comparison to other homopolyamino acids (PLO > PLL = poly-L-lysine > polyarginine) [146]. The abilities of PLL and PLO polymers to interact with pDNA were postulated to be responsible for disparities in cell transfection [147,148]. Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive, even in the presence of chloroquine [146]. Receptor-mediated gene transfer has been indicated as being very promising to overcome this barrier, and was reviewed by Zauner et al. [147]. Transferrin [149], antibodies [150] and folic acid [151] are some ligands attached to PLL to increase transfection efficiency. Improvement of endosomal escape was also shown to be a good way to increase transfection efficacy. PLL containing L-tryptophan [152] and poly(histidine) grafts [153] was able to transfect mammalian cells. The conjugation of histidine to PLL was reported to be a more efficient carrier than a PLLchloroquine mixture [154]. The higher transfection was attributed to greater buffering of the copolymer provided by histidine, which is protonated below pH 6. Toxicity may be decreased by the synthesis of block [155] and graft [156] copolymers of poly(ethylene glycol) co-poly(lysine) (PEG-PLL), which shows spontaneous formation of complexes with DNA. PEG-PLL offers a protective hydrophilic surface to NPs and at an optimal charge ratio, PEG-PLL/DNA complexes presented efficient transfection of 293 cells in vitro [155]. Condensed DNA/PLL complex has been also coated with a lipid bilayer by lipid film hydration. The packaging of DNA/PLL complex with lipids increased transfection activity 10-fold over that of pure DNA/PLL. The coated DNA/PLL, having octaarginine on the envelope as a device for membrane penetration to enhance cellular uptake, demonstrated 1,000-fold higher transfection activity than DNA/PLL [157]. Linking PEG to the polycations PLL and PEI via a pH-sensitive hydrazone bond (PC-HZN-PEG) of acyl hydrazides or 2-pyridyl hydrazines was recently proposed to trigger endosomal de-shielding [158]. The reversibility of the hydrazone bond within conjugates was determined at physiological and endosomal acidic pH. In vitro and in vivo studies disclosed that receptortargeted polyplexes generated with acid-labile bioreversible shielding copolymers have dramatically higher transfection


efficiencies in target cells compared to those generated with stable shielding. However, in the last 5 years, a number of new strategies have been adopted to further improve PLL properties. Among them, changes in PLL structure [159164], ligand-receptor interactions [135,165, 166, ], attaching endosomolytic function groups to increase intracellular delivey [167] as well as new approaches to enhance the nuclear entry of plasmids [168] are the most frequently-employed ways to augment the efficiency of PLL-based carriers. Some of its derivatives and based PLL structures are illustrated in Fig. (5). Cross-linked block copolymers of PEG-PLL (PEG-PLL) were applied with pDNA encoding the soluble form of vascular endothelial growth factor receptor-1 (sFlt-1), a potent antiangiogenic molecule. The polyplexes were tested for their antiangiogenic effect on mice bearing the xenografted BxPC3 cell line derived from human pancreatic adenocarcinoma. The authors noted that the gene expression of sFlt-1 by i.v. injection of polyplex micelles was observed in tumor tissue only, followed by decreased vascular density and significant suppression of tumor growth [169]. Liu et al. tested a specific ligand-receptor-binding strategy with dendrigraft PLL (DGL) to make it cross the blood-brain barrier. DGL grafted with PEG (DGL-PEG) was attached to a 30-aminoacid peptide (leptin30) derived from an endogenic hormone, leptin. DEG-PEG-leptin30 was complexed with pDNA, yielding NPs, and their cellular uptake was tested in brain capillary endothelial cells, which express leptin receptors. From in vitro and in vivo studies, these authors concluded that DGL-PEG-leptin30/DNA NPs demonstrated enough efficiency for brain-targeting gene delivery [135]. 4.2. Chitosan Mumper et al. were the first to report on chitosan as a possible carrier for gene therapy [175]. Since then, it has been established that, besides its immunogenicity, chitosan molecules condense efficiently with DNA, forming tight polyplexes by avoiding its degradation by DNases [176]. Chitosan-DNA interaction is driven mainly by electrostatic interaction between the amino groups of chitosan and the charged phosphate groups of DNA [177]. Transmission electronic microscopy [12] and atomic force microscopy [8] have shown that chitosan condenses plasmid into toroid- and rod-shaped particles. The sizes of the particles were also found to decrease by reducing chitosan's MW and plasmid concentration [178]. Various parameters, such as charge density, Mw, charge ratio (+/-), pH and particle size, have been demonstrated to affect transfection efficiency [179- 181]. Köping-Hoggård et al. [182, 183] prepared monodisperse oligomers of fully deacetylated (6-, 8-,10-, 12- 14- and 24-mer) chitosan with very low polydispersity and ultrapure chitosan (UPC) of 154 kDa. It was established that only UPC and 24-mer chitosan could form stable complexes with DNA, and 24-mer was more efficient in mediating gene expression in vitro and in vivo than was UPC. Sato et al. [184] also found that the molecular mass of chitosan, pH of the medium, and serum concentration are very important for promoting transfection efficiency. Working on chitosan samples whose average MWs were 15, 52, and 100 kDa, they determined that the transfec-

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tion efficiency mediated by 100-kDa chitosan was less than that by 15- and 52-kDa chitosan, but clearly indicating dependence on cell lines. However, Bozkir et al. [185] suggested that formulations with high MW chitosan can be an effective non-viral gene vector method in animal studies. The mechanism of internalization contact and crossing of the cell membrane is only partially understood, and it is wellaccepted that the polyelectrolyte complex chitosan-DNA, exhibiting a net positive charge, binds to negatively-charged cell membranes [10]. Therefore, a net positive charge is fundamental in the process, and the level of transfection is shown to increase with the charge ratio (+/-), reaching a maximum and decreasing at higher stoichio-metries. It has been reported that high transfection levels can be obtained when the charge ratio (+/-) is between 2 and 5 [186]. However, it must be considered that the transfection efficiency of chitosan-DNA NPs is also cell type-dependent and the chitosan:plasmid ratio must be controlled to obtain the appropriate particle size aiming to maximize transfection [187]. Particle size is recognized to be a key parameter since it may affect blood circulation time and cellular uptake [187, 188]. However, the available experimental results are contradictory, with reasonable transfection efficiencies being reported for particles having sizes varying from 100 nm [189] to 2.0 μm [190, 191]. Although chitosan presents limited transfection efficiency because of its low cytotoxicity, it has become one of the most widely investigated polycations for gene therapy [192]. Many strategies have been deployed to improve transfection efficiency, taking into account the biological steps involved in gene delivery Fig. (1). Modifications of chitosan structure to impart properties to NPs, such as to increase endosomal escape [193], attaching ligands to mediate cell internalization or to promote the nuclear entry of DNA, are among the most common ways. Fig. (6) presents representative structures from these chitosan derivatives tested as carriers for gene therapy. Nanospheres modified by introducing PEG5000 chains may be stable during lyophilization and storage for 1 month [194]. These chitosan-DNA nanospheres were effective in tranfecting 293 cells but not HeLa cells, and tranfection efficiency was not affected by PEG derivatization. Poly(vinyl pyrrolidone) (PVP) was also grafted on galactosylated chitosan (GCPVP) and displayed improved physicochemical properties over unmodified chitosan [195]. The binding strength of GCPVP 10k/DNA was superior to that of GCPVP 50k/DNA, which was attributed to its higher flexibility because of its smaller size. However, DNase I protection of GCPVP 10k/DNA complex was inferior to that of GCPVP 50k/DNA. The DNA-binding property was shown to be dependent on the MW of chitosan and the composition of PVP [195]. Targeting mediated by cell surface receptors has also been utilized as an approach to improve the contact and internalization of chitosan-DNA NPs. It is well-known that some kinds of saccharide play important roles in biological recognition on cellular surfaces [196- 199]. Park et al. exploited this specificity to synthesize galactosylated chitosan (GLC), aiming to transfect the HepG2 human hepatoblastoma cell line and HeLa human cervix epithelial carcinoma cells [200- 203]. The results disclosed that transfection effi-



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Fig. (5). Schematic representation of: 1. PLL and other poly (aminoacids) based in the PLL structure; 2. PEGylated block copolymer; 3. reducible linear L-lysine-modified copolymers; 4. PEG-conjugated poly(ketalized serine); 5. poly(aspartamide) derivative; 6. lipid-substituted PLL; 7. PEGylated poly(l-lysine-co-l-phenylalanine) copolymer; 8. tetra(L-lysine)-grafted poly (organophosphazene).

ciency into HepG2, which has asialoglycoprotein receptors (ASGP-R), was higher than into HeLa without ASGP-R. Recently, the galactose approach was tested with chitosan derivatives having PEI attached to the main chain, which it is believed to improve endosomal escape. Attaching PEI to the chitosan chain allowed higher transfection efficiency with low cell toxicity, but the system had limited cell specificity [204]. These authors concluded that GLC (Gal-PEG-CHI-gPEI) exhibited lower cytotoxicity compared to PEI25k, and Gal-PEG-CHI-g-PEI/DNA complexes showed good hepatocyte specificity. Furthermore, Gal-PEG-CHI-g-PEI/DNA complexes transfected liver cells more effectively than PEI25k in vivo after i.v. administration [205]. Other studies exploring receptors, such as Tf [206] and folic acid [207-210], have attempted to find alternatives to increase transfection efficiency. KNOB protein was conjugated to NPs with a bis-succinimidyl PEG derivative. KNOB protein conjugation to chitosan-DNA NPs has been reported to increase transfection efficiency in HeLa cells by 130-fold and in HEK293 cells by several fold [187]. Recently, to enhance the transfection efficiency of chitosan, water-soluble chitosan was coupled with urocanic acid (UA) [211]. These

authors reported that the transfection efficiency of chitosan into 293T cells was greatly enhanced after coupling with UA and increased with an elevation of UA content in UAmodified chitosan. Kiang et al. [212] tested poly(propyl acrylic acid) (PPAA) as an approach to enhance the release of endocytosed drugs into the cytoplasmic compartment of cells. The release of pDNA from the endosomal compartment was enhanced by incorporating this polymer in chitosan NPs. In vitro transfection studies confirmed that the incorporation of PPAA into chitosan-DNA NPs enhanced gene expression in both HEK293 and HeLa cells compared to chitosan NPs alone. The dose and time at which PPAA was incorporated during complex formation affected DNA release and transfection efficiency. These authors suggested that PPAA triggered membrane disruption, resulting in DNA release from the endosomal compartment. After endosomal escape, the nuclear entry of pDNA can be overcome by incorporation of nuclear localization signal (NLS) peptides in chitosan NPs. It has been reported that peptide sequences containing charged amino acid residues, such as arginine and lysine, are able to enhance transportation into cells. Arginine-modified


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Fig. (6). Chemical structures of: 1. chitosan and its derivatives; 2. PEG; 3. alkylated; 4. trimethylated; 5. PEI; 6. PEI and PEG grafts; 7. galactosylated; 8. arginine; 9. deoxycholic acid; 10. folic acid; 11. arginine and folate; 12. histidine; 13. grafted PDMAEMA; 14. PEI-PEGgalactose; 15. 6-amino 6-deoxychitosan; 16. O-hydroethyl. The symbols * and ** indicate the positions at which the groups are attached.

trimethylated chitosan labeled with folic acid was used by Morris and Sharma to transfect KB cells in vitro [213]. These authors concluded that due to the large extent of cellular uptake and nuclear trafficking, the derivative exhibited improved transfection efficiency in KB cell lines, even in the presence of 10% serum compared to native polymer without folate. The improvement in transfection efficiency was attributed to targeting ability of the folate group, membrane permeability and the nuclear localization ability of the arginine residue. Opanasopit et al. incorporated the peptide KPKKKRKV during the preparation of chitosan/DNA complexes (CS/DNA) without covalent conjugation to pDNA or chitosan. This peptide is an arginine/lysine-rich NLS from simian virus 40 (SV40) large T antigen (PKKKRKV) recognized by cytoplasmatic transport receptors and mediated nuclear uptake. The results showed that CS/DNA complex containing NLS was able to increase transfection efficiencies in a NLS dose-dependent manner, and transfection efficiency was 74-fold higher than in cells transfected only with CS/DNA complex. These authors suggested that high gene expression with negligible cytotoxicity can be achieved by adding NLS pep-

tide to CS/DNA complexes at an optimal ratio [214]. Sun et al. [215] took a similar approach but attaching a short peptide (SP) to chitosan with the amino acid composition of “LLLRRRDNEY*FY*VRRLL”, which was further combined with GFP/luciferase reporter gene pDNA to form SPCS/DNA complex. The NPs were able to transfect multiple cell lines, and the results revealed that, compared with CS, SP-CS could intensively augment transfection efficiency nearly to the level of Lipofectamine 2000 [215]. However, these authors suggested that the serine and threonine phosphorylation of this short peptide (pSP) could induce intracellular DNA unpacking from the pSP-CS/DNA complex due to electric repulsion. 4.3. Dextran Although transfection mediated by dextran derivatives was reported more than 10 years ago [231-235], systematic investigations were conducted to improve its efficiency as a gene carrier only in the early 2000s. Azzam et al. reported a new class of biodegradable polycations capable of complexing and administering various genes to many cell lines with relatively high yields. More than 300 different polycations


were prepared, starting from various polysaccharides and oligoamines having 2 to 4 amino groups. These polycations were prepared by reductive amination reactions between primary amines and periodate-oxidized polysaccharides. Although most of the conjugates formed stable complexes with various plasmids, as determined by turbidity experiments and ethidium bromide quenching assay, only dextranspermine based polycations were found to be active in transfecting cells in vitro [236]. The same group reported that i.m. injection of dextran-spermine-pSV-LacZ complex in mice induced high local gene expression compared to the low expression of naked DNA. I.v. injection of a dextran-sperminepSV-LacZ complex dispersion resulted in no expression in all examined organs. When the partially PEGylated dextranspermine-pSV-LacZ complex was applied i.v., high gene expression was detected mainly in the liver. Preliminary targeting studies indicated that PEGylated dextran-sperminepSV-LacZ complex bound to the galactose receptors of liver parenchymal cells rather than to the mannose receptors of liver nonparenchymal cells [237]. For these systems, transfection was reported to increase with spermine content. The same system hydrophobized with cholesterol and fatty acids remarkably enhanced gene expression in serum-rich media. Hydrophobized derivatives of other fatty acids and cholesterol disclosed improved transfection yields in comparison to unmodified dextran-spermine, but to a lower extent when compared to oleate modification. The improvement in cell transfection was attributed to oleate residues which probably play a role in increasing stability and uptake of polycationDNA complexes [238]. More recently, confocal microscopy has revealed that fluorescent-labeled polymer concentrates in the Golgi apparatus and around the nucleus during the transfection process, while the cell cytoplasm is free of fluorescent particles, indicating that the polyplexes move in cells toward the nucleus by vesicular transport through the cytoplasm and not by random diffusion. These authors concluded that the plasmids penetrate the cell nucleus without the polymer [239]. Several studies using cationized dextran have emerged in the last decade. Further modified with PEG, dextran-spermine was demonstrated to enhance the suppression of tumor growth by a combination of NK4 plasmid DNAPEG and ultrasound irradiation. The complexes were injected i.v. into mice carrying subcutaneous Lewis lung carcinoma tumor and subsequently submitted to ultrasound irradiation [240]. This same derivative was used to genetically engineer macrophages (M) for biological activation and to evaluate their anti-tumor activity in a tumor-bearing mouse model. NK4-transfected M exhibited stronger inhibition activity in vitro, and when injected i.v. into mice carrying a mass of Meth-A tumor cells, the engineered M accumulated in tumor tissue and presented significant anti-tumor activity [241]. However, dextran has also been shown to be useful when grafted onto other cationic polymers, such as PEI [54, 55] or chitosan, to increase their biocompatibility or colloidal stability [242]. 5. DISCUSSION In general, studies indicate that both synthetic and natural polymers have advantages and disadvantages, which may be circumvented with different strategies. The lower cytotoxicity of some natural carriers, such as chitosan and dextran, is


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the main advantage when compared with synthetic polymers, such as PEI and PDMAEMA. It must also be considered that biodegradability is very important, and in this respect, natural polymers such as poly(aminoacids), chitosan and dextran are more appropriate for in vivo studies [239]. However, if we accept that a net positive charge is fundamental in the process and the level of transfection is shown to increase with the charge ratio (+/-) both synthetic and natural polymers and polyplexes may damage the cell membrane to some extent. On the other hand, the synthesis of new and degradable polymers may circumvent these limitations. The cytocompatibilities of PEI and PDMAEMA have been improved by attaching more biocompatible polymers, such as chitosan [193], dextran and others [11, 243], to these polymers. PEI polyplexes can damage plasma membranes and alter macromolecular permeability. Aiming to circumvent this problem, small PEI units may also be linked to biodegradable bonds, such as disulfide, esters, acetals and orthoesters, which may help carrier fragmentation in endosomal or cytoplasmic compartments [67]. However, concerns regarding the fate of these small fragments remain, since it is believed that PEI could trigger apoptosis in different ways [42]. In vivo studies also show that cationic polymer/DNA polyplexes have short plasma circulation times with rapid hepatic uptake and accumulation or deposit in organs, such as the liver, lungs, spleen, kidneys and heart [244, 245]. Interaction between cationic polyplexes and negativelycharged lung cell membranes may be responsible for accumulation in this organ [88] and represents an important target for gene therapy: for correction of genetic abnormalities, such as cystic fibrosis, for lung cancer therapy, and for vaccination. Aggregation of cationic particles, such as cationic polymer/DNA complexes, can occur after interaction with blood components. Extensive aggregation could lead to physical deposition in the capillary bed, and to prevent unwanted interactions between particles and the dynamic environment of the blood circulation, hydrophilic surfaces can be attached to the cationic particle surface. Also, in the absence of a hydrophilic surface, opsonization prepares the particles for uptake by fixed macrophages of the mononuclear phagocytic system [246]. Therefore, it is necessary to prevent unwanted interactions between particles and the dynamic environment of the blood circulation by introducing hydrophilic surfaces to cationic particles. This procedure decreases toxicity and has been used by many authors but may result in decreased transfection efficiency [93]. Polymer MW has also been shown to affect cytotoxicity and transfection efficiency in a similar way in both natural and synthetic systems. Chitosan [182] and PEI [61, 247] both present decreased transfecion efficiency with escalating MW, DNA protection being compensated by increasing charge ratio of the polyplex. Therefore, the importance of assuring balance between cytotoxicity and transfection activity, both of which are found to be a function of MW, has been recognized [248]. Taking into account that the cellular uptake of polycation-DNA complexes is the first step, attaching different ligands to target cell-surface receptors is very interesting in both ways, increasing cellular uptake and permitting the targeting of different cell types or diseases. The procedure has proven to work effectively [201 203, 211]. In this respect, it is expected that the post-modification of syn-


thetic and natural polymers, to introduce ligands with different structures, could offer improvements in the transfection process. Currently, however, all efforts have been directed to understanding firstly intracellular NP routing and DNA release [20,22] and, secondly, the nuclear import of transfecting pDNA [19]. The entrapment and degradation of pDNA in endolysosomes constitute major impediments to efficient gene transfer [20]. First-generation synthetic and natural polycations, such as PDMAEMA [90], PLL [152-154] and chitosan [211], are relatively inefficient in promoting endosomal escape, and this has led to the development of strategies that enhance the endosomal de-stabilization ability of polyplexes. Even PEI polyplexes, which demonstrated higher buffering capacity when compared with other polymers, had increased endosomal escape by conjugation with the membrane-lytic cationic peptide mellittin [249]. In this respect, modifications of these polymers, by introducing either groups with a higher buffering capacity or membranedestabilizing peptides [91], are promising ways of achieving higher transfection efficiencies. Recently, the utilization of polyplexes assembled with 2 or more polymers able to provide the system with endosome-escaping functions was also shown to be an applicable strategy [159, 250, 164]. Like endosomal escape, pDNA entry into the nucleus is a barrier that is currently not completely resolved. In fact, some results indicate that the transfer of polycation-DNA polyplexes from the lysosomal compartment to the nucleus is the limiting step in cell transfection [251, 20]. In this respect, it has been recognized for more than 20 years that pDNA itself leads to inefficient transfection when injected in the cytosol [22]. On the other hand, transfection mediated by polyplexes and lipoplexes is comparatively more efficient than that obtained with injected DNA. Therefore, taking in account that pDNA may enter the nucleus itself (without the carrier), the better transfection efficiency observed via endocytosis indicates that DNA delivered in the perinuclear zone could have a higher probability of entering the nucleus [144]. Since transfection is reported to work best in dividing populations of cells, mitosis for nuclear import must be considered [19]. However, NLS have been reported as being important in promoting DNA import in post-mitotic cells [252]. Many recent studies have focused on the mechanism by which plasmids, either alone or complexed with polycations, traffic to enter the nucleus [21]. Although many propositions need further investigation, all this information may help the development of new generations of carriers having the ability to efficiently enter the nucleus.

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endocytosis. Currently, gene therapy is being tested in many different health problems, such as cancer, AIDS, and cardiovascular diseases, with mixed results, mainly owing to inefficiency of the gene transfer vectors chosen. Transfection efficiency may depend on several factors, such as the chemical structure of polycations, the size and composition of complexes, interaction between cells and complexes, and cell type. Second- and third-generation polymeric vectors are ideal candidates to overcome these barriers, while retaining low production costs and industrial mass-production characteristics. ACKNOWLEDGEMENTS This study was supported by research grants from the Canadian Institutes of Health Research (CHIR, CCL-92212, CCL-99636 and CCM 104888). Dr. Fernandes holds a clinician scientist scholarship from Fonds de la recherche en santé du Québec. Dr. Tiera holds a post-PhD scholarship from UNESP-Brazil. REFERENCES [1] [2] [3] [4] [5]

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Received: January 28, 2011

Revised: March 20, 2011

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PMID: 21453278

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