Original Article

Gene Therapy (2008) 15, 668–676; doi:10.1038/gt.2008.16; published online 28 February 2008

Bioadhesive hyaluronan–chitosan nanoparticles can transport genes across the ocular mucosa and transfect ocular tissue

M de la Fuente1, B Seijo1 and M J Alonso1

1Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain

Correspondence: MJ Alonso, Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, Campus Sur, Santiago de Compostela 15782, Spain. E-mail: ffmjalon@usc.es

Received 31 August 2007; Revised 2 January 2008; Accepted 3 January 2008; Published online 28 February 2008.

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Abstract

Gene transfer is considered to be a promising alternative for the treatment of several chronic diseases that affect the ocular surface. The goal of the present work was to investigate the efficacy and mechanism of action of a novel DNA nanocarrier made of hyaluronan (HA) and chitosan (CS), specifically designed for topical ophthalmic gene therapy. With this goal in mind, we first evaluated the transfection efficiency of the plasmid DNA-loaded nanoparticles in a human corneal epithelium cell model. Second, we investigated the bioadhesion and internalization of the nanoparticles in the rabbit ocular epithelia by confocal laser scanning microscopy. Third, we determined the in vivo efficacy of these nanocarriers in terms of their ability to transfect ocular tissues. The results showed that HA–CS nanoparticles and, in particular, those made of low molecular weight CS (10–12kDa), led to high levels of expression of secreted alkaline phosphatase in the human corneal epithelium model. In addition, we observed that, following topical administration to rabbits, these nanoparticles entered the corneal and conjunctival epithelial cells and, then, become assimilated by the cells. More importantly, these nanoparticles provided an efficient delivery of the associated plasmid DNA inside the cells, reaching significant transfection levels. Therefore, we conclude that these nanoparticles may represent a new strategy toward the gene therapy of several ocular diseases.

Keywords:

in vivo, gene transfer, ocular, nanoparticles, hyaluronan, chitosan

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Introduction

The introduction of the therapeutic genes in the target tissue is considered as a promising alternative for the treatment of several chronic diseases that affect the ocular surface such as corneal dystrophies, dry eye, immune-mediated diseases, corneal neovascularization and ocular allergy.1, 2 It is believed that the use of nucleic acid-based drugs may lead to treatments that are more efficient and safer than those consisting of the currently available drugs. These predictions are based on the fact that genes are able to express their products for periods of time that greatly exceed the duration of action of currently available drugs. However, despite the recognized value of DNA-based therapeutics, there are several fundamental problems, such as a poor cellular uptake, a rapid in vivo degradation and a limited transport to the target, which remain to be solved before this technology becomes therapeutically viable.3 Among the strategies developed to overcome these challenges,4 viral vectors have received a great deal of the attention.5, 6 and 7 These vectors have been proposed for the treatment of several disorders affecting the ocular surface due to their capacity to infect cells very efficiently, and provide significant levels of protein expression.8, 9 Unfortunately, these vectors may also elicit strong immune responses and provoke harmful reactions.10 The use of synthetic nanocarriers has been recently proposed as a promising alternative for the delivery of genes to the target tissue.11, 12 In the specific area of ocular gene therapy, nanoparticles are particularly attractive due to their known ability to intimately interact with the ocular surface.13 More specifically, our group has shown that polymeric nanoparticles are capable of adhering to the ocular surface and penetrate through the corneal and conjunctival epithelia,14, 15 thereby achieving a targeted drug delivery after topical administration.16 However, despite this well-known ocular behaviour of nanoparticles and their potential for ocular drug delivery, their application for ocular gene therapy remains to be explored.

Among the different polymers of potential interest for the design of ocular gene nanocarriers, the polysaccharides, hyaluronan (HA) and chitosan (CS) are particularly attractive biomaterials. Indeed, HA is a polysaccharide that can naturally be found in the ocular tissues17 and is widely used in ophthalmology due to its biocompatibility, biodegradability and mucoadhesive character.18 In addition, HA exhibits interesting biological properties, that is it promotes adhesion and proliferation in mammalian cells, and is involved in numerous biological processes like cell signalling.19 Furthermore, it is known that many mammalian cells have a variety of HA receptors on their surface, which are probably involved in the HA uptake and in signal transduction. The principal receptor, CD44, has been localized in ocular tissues.20, 21

On the other hand, the cationic polysaccharide CS and nanocarriers based on it offer a particularly optimistic prospect.22 For example, we have previously found that CS-based nanoparticles remain associated to the ocular mucosa for extended periods of time and provide high concentrations of the encapsulated drug on the target site with minimal systemic exposure.15, 23

Overall, this information has led us to the assumption that nanoparticles made of the polysaccharides HA and CS could be of interest for the topical administration of genes onto the eye surface. Consequently, the main goal of the present work was to evaluate the potential of these nanoparticles as gene carriers for the treatment of ocular diseases. With this goal in mind, we first evaluated the nanoparticle transfection efficiency in an organotypic model of the human corneal epithelium (HCE). Second, we investigated the interaction of the nanoparticles with the ocular epithelia and, finally, we determined the capacity of the nanoparticles to transfect the cornea and the conjunctiva after topical administration.

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Results

Physicochemical and plasmid DNA loading properties of hyaluronan–chitosan nanoparticles

The preparation of HA–CS nanoparticles was achieved by the slightly modified ionotropic gelation technique previously developed by our group.24, 25 Table 1 summarizes the characteristics of different nanoparticulate formulations: blank nanoparticles made of CS of different molecular weight alone or in combination with HA, nanoparticles labelled with a fluorescent marker and nanoparticles loaded with different plasmid DNA (pDNA) models.


In general, the size of the nanoparticles was in the range of 100–215nm, this parameter being hardly affected by the pDNA loading. Another general observation is that the positive ζ potential, typically observed for CS nanoparticles, was reduced and even inverted upon incorporation of increasing amounts of HA (ranging from +40 to −30mV). However, the association of pDNA did not impair any significant modification on the surface charge of the nanocarrier. The change in the ζ potential, as the amount of HA in the preparation increases, is easily justified by the fact that HA is negatively charged. Consequently, when HA becomes a predominant polymer in the composition, its disposition at the surface of the nanoparticles leads to a negative charge. Moreover, the fact that the association of different pDNA molecules in a very high amount (7–10% loadings as verified using an agarose gel retardation assay) does not affect the physicochemical properties of the nanoparticles, which indicates that the plasmid is efficiently entrapped within the nanoparticles.

Another interesting observation from the results in Table 1 is that the use of a fluorescence derivative of HA (HA-fl) did not significantly affect the characteristics of the resulting labelled nanoparticles (only a slight decrease was observed in the ζ potential values, which could be explained by the changes induced in the glycosaminoglycan structure due to the introduction of the hydrophobic labelling molecule). Therefore, this labelling approach appears to be acceptable for further studies of the interaction of the nanoparticles with the ocular tissues.

Efficiency of the nanoparticles at transfecting the corneal epithelium model (organotypic human corneal epithelium model)

The use of cell culture models derived from HCE cells model represents nowadays a useful alternative for the evaluation of biomaterial–epithelium interactions, prior to in vivo assessment of ocular bioactive nanocarriers.26, 27 In order to evaluate the potential of HA–CS nanoparticles as gene delivery vehicles to the HCE model, we added nanoparticles loaded with plasmid encoding secreted enhanced alkaline phosphatase (pSEAP) to the culture epithelium, and then determined the amount of alkaline phosphatase secreted by the cells for up to 10 days. As can be noted in Table 2, nanoparticles consisting of low molecular weight CS (chitosan oligomers (CSO)) and HA were led to the highest secreted enhanced alkaline phosphatase (SEAP) expression. More specifically, it was observed that the presence of CSO significantly increased the expression levels and this effect was further improved by the association of HA (Figure 1). Taking into account these results, we chose the formulation composed of HA:CSO (1:2) for the following in vivo experiments. In addition, we selected the composition HA:CS (1:2) in order to further evaluate the effect of CS molecular weight on the in vivo efficacy.

Figure 1.
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Secreted levels of enhanced alkaline phosphatase (SEAP) by the corneal epithelial culture model as a function of time, after transfection with hyaluronan (HA):chitosan oligomers (CSO) (1:2; black square), HA:chitosan (CS) (1:2; black circle), HA:CS (2:1; black diamond), CSO (open square) and CS (open circle) nanoparticles or naked plasmid DNA (pDNA) (5μg per insert) (means±s.d., n=5).

Full figure and legend (15K)


Mechanism of interaction of hyaluronan–chitosan nanoparticles with the rabbit ocular mucosa

In order to examine the interaction of the nanoparticles with the corneal and conjunctival epithelia, HA was labelled with fluoresceinamine (HA-fl) and nanoparticles prepared with the HA derivative administered topically to the rabbit eye. The formulations selected were HA-fl:CSO and HA-fl:CS, with a mass ratio of 1:2. A solution of labelled HA-fl was also administered as a control. Post-administration (2h), the animals were killed, and the corneas and conjunctivas excised and freshly observed under the confocal microscope. Figure 2 displays the confocal images of the rabbit conjunctiva. These images (Figures 2b and c) show that the nanoparticles are located inside the cells rather than in the intercellular spaces, suggesting that they penetrate the conjunctival epithelium by a transcellular pathway. Additionally, it can be noted that the appearance of the conjunctiva exposed to the nanoparticles is substantially different from that treated with the HA-fl solution (Figure 2a), in which the intensity of the fluorescent signal was lower.

Figure 2.
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Confocal fluorescence images of the rabbit conjunctiva excised after 2h post-instillation of a solution of fluorescence derivative of hyaluronan (HA-fl) (a), or HA-fl:chitosan oligomers (CSO) (b) and HA-fl:chitosan (CS) (c) nanoparticles (n=4 specimens).

Full figure and legend (101K)

With regard to the corneal disposition, the nanoparticles were able to interact with the cells and penetrate through the corneal epithelium (Figure 3). Moreover, as described for the conjunctival epithelia, it was found that the nanoparticles were distributed inside the cells rather than in the intercellular spaces, thus indicating a transcellular mechanism of transport (Figure 4).

Figure 3.
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Confocal laser scanning micrograph of the corneal epithelium of rabbit after 2h post-instillation of fluorescence derivative of hyaluronan (HA-fl):chitosan (CS) nanoparticles. Image is an x–z cross-section (n=4 specimens).

Full figure and legend (50K)

Figure 4.
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Rabbit corneal epithelium photographed at the confocal microscope at different time points after instillation of a fluorescence derivative of hyaluronan (HA-fl) solution (a) or fluorescent HA-fl:chitosan oligomers (CSO) (b) and HA-fl:chitosan (CS) (c) nanoparticles (n=4 specimens).

Full figure and legend (221K)

In addition, in Figure 4, it can also be noted that at 2h post-administration, the corneal epithelium treated with HA-fl:CSO (1:2) nanoparticles exhibits a stronger fluorescent signal inside the cells than that in the case of HA:CS (1.2) nanoparticles (Figures 4b and c), suggesting a major entry inside the cells.

Given the fact that the nanoparticles are able to enter the cells, and interact intimately with the ocular surface, we decided to assess whether or not the nanoparticles are cleared from the organism. For this purpose, the fluorescent nanoparticles were instilled to the rabbits and their distribution in the rabbit cornea were examined at 4 and 12h post-administration.

It was observed that following instillation of the nanoparticles, the intensity of the fluorescence signals decreased gradually with time (Figure 4). Furthermore, when HA-fl is in a particulate form, its interaction with the ocular surface is stronger and more persistent than in solution (Figure 4a) that is cleared faster from the eye. It has also been observed that the intracellular stability of the nanoparticles was dependent on the nanoparticle composition. Nanoparticles composed by HA-fl:CS (1:2) were visualized longer than those of HA-fl:CSO (1:2) inside the cells (Figures 4b and c).

Efficacy of hyaluronan–chitosan nanoparticles as carriers for ocular in vivo transfection

In order to explore the in vivo potential of the developed nanoparticles in ocular gene therapy, HA:CS (1:2) and HA:CSO (1:2) nanoparticles were loaded with plasmid encoding enhanced green fluorescent protein (pEGFP) and instilled onto the eye surface of rabbits. Figure 5 shows the images of the rabbit corneas following treatment with the naked pDNA and two nanoparticulate formulations. Only detectable fluorescence was noted when naked pDNA was administered at a dose as high as 100μg pEGFP per specimen (Figure 5a). In contrast, the images show gene expression following treatment with the smallest dose tested, 25μg pEGFP per specimen, when the plasmid was associated to HA:CSO nanoparticles (Figure 5b). This level of expression was significantly increased at a dose of 50μg, but apparently maintained when the dose of pEGFP went from 50 to 100μg. In the case of HA:CS nanoparticles, gene expression was only achieved at a dose of 50μg and the intensity of fluorescence was maintained when the dose increased up to 100μg (Figure 5c). With respect to the conjunctiva, the expression of the green protein could not be detected due to the important autofluorescence of this tissue, which overlaps the fluorescence emitted by the expressed protein.

Figure 5.
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Green protein expression in the corneal epithelium of rabbit after topical administration of increasing doses of naked plasmid encoding enhanced green fluorescent protein (pEGFP) (a) and hyaluronan (HA):chitosan oligomers (CSO) (b) or HA:chitosan (CS) (c) pEGFP-loaded nanoparticles. The rabbits were killed and the specimens were excised 2 days post-administration (n=3 specimens).

Full figure and legend (184K)

Taken into account the positive transfection results observed for HA:CSO (1:2) nanoparticles, we decided to evaluate the duration of the gene expression. Interestingly, the positive transfection results were maintained for up to 7 days (Figure 6).

Figure 6.
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Confocal fluorescence images of the corneal epithelium, excised 2, 4 or 7 days after instillation of hyaluronan (HA):chitosan oligomers (CSO) nanoparticles (50μg pDNA per cornea; n=3 specimens).

Full figure and legend (107K)

In order to further assess the in vivo efficacy of the nanoparticles as ocular gene carriers, and to explore their ability to transfect the conjunctiva, a second plasmid model, pDNA encoding β-galactosidase (pβgal), was associated to the nanoparticles and tested for transfection in the rat eye. As noted by the blue tonality of the eye upon X-gal staining, HA:CSO and HA:CS nanoparticles mediated β-gal gene expression on the enucleated eyes at 48h post-topical administration (data not shown).

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Discussion

As indicated in the introduction, the main goal of the present work was to investigate the efficacy and mechanism of action of a novel DNA nanocarrier, specifically designed for ocular gene delivery. This nanocarrier was composed of the natural polysaccharides HA and CS. These biopolymers were selected attending not only to criteria of biocompatibility and biodegradability, but also because of their reported biological functions18, 22 and ability to target CD44 receptor expressed on the ocular tissues.20, 21

The nanoparticles were prepared by a slightly modified ionic gelification technique.24, 25 This technique allows the formation of the nanoparticles in a very mild environment, avoiding the use of high energy, organic solvents or chemical reagents. The formation of these nanoparticles is governed by the controlled gelation of the CS (induced by the crosslinker tripolyphosphate, TPP) and also due to the electrostatic interaction between the CS and the HA.

The obtained nanoparticles exhibit characteristics that are suitable for topical administration to the ocular surface. Namely, (1) they are able to associate great amounts of DNA, thus allowing the amount of polymer used for in vivo transfection purposes to be minimized, (2) they have a very small size, a feature which is known to be critical for the adequate interaction with the ocular mucosa28 and (3) their surface charge can be modulated depending on the HA–CS ratio. This modulation is important since a positive charge is supposed to facilitate the interaction of the nanoparticles with the ocular surface,23, 29 whereas the disposition of HA onto the surface of the nanoparticles may also play a role in their interaction with epithelial cells.20, 21

In order to evaluate the efficacy of the nanoparticles as ocular gene carriers, a cell culture model derived from HCE cells was used prior to the in vivo studies. In general, the closer the culture conditions are to the natural environment of the cells, the more closely the culture epithelium will mimic the in vivo tissue.26, 27 In the present case, the establishment of appropriate growth conditions for the HCE cells led to the formation of a multilayered epithelium with tight junctions between the cells, microvilli, desmosomes and cell layers with apical flat cells.30 The greater efficacy of CSO nanoparticles as compared to CS nanoparticles could be attributed to an early release of the pDNA. Such an expression profile has been described for polyplexes and has also been attributed to the rapid dissociation of the pDNA.31, 32 In addition, the positive effect of HA could be understood by its known capacity of being able to enter the cell nucleus33, 34 and also to its suggested role as a transcription activator.35 Similar results in terms of transfection efficiency have been previously observed for the same nanoparticles in HCE proliferating cells.36

The access of topically applied drugs into the ocular tissues is severely limited by the protective physiological mechanisms that exist in the precorneal area, as well as by the corneal barrier. For an efficient transfer of the therapeutic genes after topical ocular administration, the delivery carrier must first interact with the corneal and conjunctival epithelia, and then overcome the cellular barriers in order to deliver the associated pDNA to the target site. The rational of using bioadhesive nanoparticles in order to overcome these barriers was based upon the assumption that they interact with the ocular surface. Confocal images of both, the cornea and the conjunctiva, show that the instilled nanoparticles (HA-fl:CSO and HA-fl:CS, with a mass ratio of 1:2) were located inside the corneal and conjunctival cells, suggesting that they penetrate the epithelia by a transcellular pathway. This mechanism, which has also been reported for other nanosystems,14, 15 could be reinforced by the known ability of HA to interact with the CD44 receptor expressed on the ocular surface.18, 37 In fact, the fluorescence observed in the cornea exposed to HA-fl solution suggest that the polymer was also internalized into the cells.

Although HA and CS are considered safe and biodegradable polysaccharides, taking into account the capacity of HA:CS nanoparticles to enter the cells, we considered it important to study their intracellular assimilation. This information is also important for the adequate understanding on the mechanisms involved in the transfer of genes. The obtained results suggest that somehow these nanoparticles are assimilated/degraded inside the cells, as the fluorescent signals detected in the rabbit cornea decreased gradually with time. This is logical since HA is naturally present in the ocular tissues, where there is a constant turnover of the glycosaminoglycan. Therefore, we hypothesized that, hyaluronidases and other enzymes, which are responsible for HA degradation,38, 39 could contribute to the rapid clearance of the nanoparticles. With regard to the behaviour of CS, we observed that the intracellular stability and assimilation of the nanoparticles were dependent on the CS molecular weight. In other words, nanoparticles composed by CS could be visualized inside the cells for a longer time than those composed of CSO. This could be justified by the fact that CS takes longer to degrade as compared to CSO. Indeed, it should be considered that the encapsulated pDNA could be released faster from the HA:CSO (1:2) nanoparticles.

A number of chronic disorders which affect the ocular surface, that is corneal dystrophies, immune-mediated diseases, dry eye, and so on, are likely to greatly benefit from gene therapy. Despite the advances made in the gene delivery field, to our knowledge, there are no reports in the literature on the potential of nanoparticles, presented as liquid eye drops, for the transport of genes across the ocular barrier, although other synthetic vectors such as micelles and liposomes have shown a certain potential for this specific application.40, 41 Given the positive results obtained in the present work in terms of the ability of HA–CS nanoparticles to transfect differentiated cells and to enter the ocular epithelia, we further explored in vivo their potential in ocular gene therapy. Nanoparticles loaded with pEGFP or pβgal were administered onto the eye surface, and the cornea and conjunctiva were evaluated for gene expression. A first general observation is that pDNA-associated nanoparticles exhibit a markedly higher level of expression than that of the naked pDNA. A second observation is that, independent to the nanoparticle formulation, the transfected cells were found mainly in the peripheral cornea, probably due to the accumulation of the nanocarriers in these areas. A third observation is the capacity of the nanocarriers to promote transfection and its dependence on the nanocarrier composition. More specifically, higher levels of gene expression were detected for the nanoparticles composed by CSO, and the positive transfection results were maintained for up to 7 days, thus indicating the long-lasting transfection capacity of these nanoparticles. This prolonged response is particularly important for the treatment of some specific corneal disorders which required a prolonged treatment, such as immune-mediated diseases or corneal transplants.1, 2 On the other hand, the greater performance observed for HA:CSO nanoparticles as compared to that of HA:CS nanoparticles is in good agreement with the above-mentioned transfection levels in the in vitro HCE model. This improved efficacy of HA:CSO nanoparticles could be explained by the apparent more important uptake and faster assimilation of HA:CSO nanoparticles in both corneal and conjunctival cells.

All together, we provide here the first evidence of the capacity of nanoparticles made of HA and CS to enter the corneal and conjunctival epithelial cells and to deliver pDNA in a very effective manner, reaching important transfection levels. Furthermore, we present the ocular fate and bioassimilation of these nanoparticles following topical administration as eye drops. Therefore, these nanoparticles may represent a promising strategy in ocular gene therapy.

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Materials and methods

Chemicals

Ultrapure CS hydrochloride salt (Protasan UP CL 113, having a molecular weight of around 110kDa) was purchased from Pronova Biomedical AS (Oslo, Norway). Sodium hyaluronate ophthalmic grade (having a molecular weight of around 170kDa) was a gift from Bioibérica (Barcelona, Spain). pEGFP and pβgal driven by a CMV promoter were purchased from Elim Biopharmaceuticals (Hayword, CA, USA). pDNA encoding SEAP based on the gWiz high-expression vector system was purchased from Aldevron (Fargo, ND, USA). Pentasodium TPP, acetaldehyde and 5-bromo-chloro-3-indolyl-β-D-galactopyranoside (X-gal) were all obtained from Sigma-Aldrich (Madrid, Spain). Fluoresceinamine and cyclohexyl isocyanide were purchased by Fluka (Madrid, Spain). One KBp DNA ladder was obtained from Life Technologies (Invitrogen, Barcelona, Spain). The Pico Green reagent was purchased from Molecular Probes (Eugene, OR, USA). All other solvents and chemicals were of the highest grade commercially available.

Animals

Male albino New Zealand rabbits weighing between 2.0 and 2.5kg and male Sprague–Dawley rats weighing between 225 and 250g were used for the in vivo studies. The animals were fed a regular diet with no restrictions on the amount of food or water consumed. Experiments were performed by conforming international guidelines for the use of animals in research.

Depolymerization of chitosan

Chitosan oligomers were obtained from Protasan UP CL 113 by sodium nitrite degradation as previously described by Janes and Alonso42 Briefly, 200μl of NaNO2 (0.1M) was added to 4ml of a CS solution of 10mgml−1. The reaction was left overnight to assure completion of the degradation, and oligomers with 10–12kDa molecular weight were recovered by freeze drying. The molecular size of the CSO was verified by size exclusion chromatography–multiangle laser-light scattering (SEC–MALLS). An Iso Pump G1310A (Hewlett Packard) was connected to a PSS Novema GPC column (10μm, 30Å, 8 × 50mm, NOA0830103E1), and a PSS Novema GPC column (10μm, 3000Å, 8 × 300mm, NOA0830103E3). A PSS SLD7000 MALLS detector (Brookhaven Instruments Corporation), operating at 660nm, and a G1362A refractive index detector (Agilent, Santa Clara, CA, USA) were connected on line. A 0.15M NH4OAc/0.2M AcOH buffer (pH 4.5) was used as eluent. Polymer solutions were filtered through 0.2μm pore size membranes (VWR) before injection. Polymer concentration was in the range of 0.30–0.16mgml−1. Refractive index increment of dn/dC was set at 0.188.

Fluoresceinamine labelling of HA

Sodium hyaluronate was labelled with fluoresceinamine, following a modification of the method described by De Belder and Wik.43 In brief, 20ml dimethyl sulphoxide (DMSO) was added to 40ml of HA in water (1.25mgml−1). Subsequently, 0.5ml of fluoresceinamine (50mgml−1 in DMSO), in the presence of 25μl cyclohexyl isocyanide and 25μl acetaldehyde, was added to the HA solution. Magnetic stirring was maintained during 5h in dark. The HA-fl was precipitated with a saturated solution of NaCl and ice cold ethanol, and finally collected by centrifugation. Precipitate fl-HA was dissolved in ultrapure water and extensively dialysed against distilled water for 24h, prior to lyophilization.

Nanoparticle preparation

Nanoparticles were spontaneously obtained by a slightly modified ionotropic gelification.24, 25 In brief, two aqueous phases containing (1) the CS or CSO and (2) the HA solutions with the crosslinker TPP were mixed under magnetic stirring and maintained in agitation for 10min to allow the complete formation of the system. The CS or CSO solution was prepared at a concentration of 0.625mgml−1, and the volume employed was fixed at 0.750ml. On the other hand, 50μl of a TPP solution (0.5mgml−1) was mixed with the HA solution prior to the formation of the nanoparticles. In order to modulate the weight ratio of the polysaccharides that constituted the nanoparticles (HA:CS, 1:2 or 2:1), 0.375ml of a solution prepared at a concentration of 0.625mgml or 0.750ml at a concentration of 1.25mgml−1 was used for the nanoparticle production.

The nanoparticles were loaded with three pDNA models, the pEGFP, the pSEAP and the pβgal, by incorporation of the required amount of the model plasmid in the HA/TPP phase. The theoretical loadings were fixed at 7 and 10% (w/w).

The nanoparticles composed solely by CS or CSO were also prepared by ionic gelification. In that case, CS or CSO were dissolved in ultrapure water at a concentration of 1mgml−1. A 0.2ml of TPP (0.625mgml−1 or 0.42mgml−1) was added over 1ml of CS or CSO solution, under magnetic stirring. Nanoparticles were loaded by including pSEAP in the TPP solution.

Nanoparticle characterization

The mean particle size and the size distribution of the nanoparticles were determined by photon correlation spectroscopy (PCS). The ζ potential values of the nanoparticles were obtained by laser doppler anemometry (LDA), measuring the mean electrophoretic mobility. The PCS and LDA analyses were performed with a Zetasizer 3000HS (Malvern Instruments, UK).

Eventually, the nanoparticles were concentrated by centrifugation (Beckman Avanti 30, Beckman, Madrid, Spain) on a glycerol bed. In order to resuspend the nanoparticles at the required concentration, the amount of nanoparticles in the sediment was calculated by weight upon their freeze drying.

The association of pDNA to the nanoparticles was studied by a conventional agarose gel electrophoresis assay. Samples of the nanoparticles were placed in 1% agarose gel containing ethidium bromide, and ran for 90min at 60V (Sub-Cell GT 96/192, Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) in Tris-acetate-EDTA buffer (TAE; pH 8).

Transfection studies in a culture model of human corneal epithelium

Human corneal epithelium cells were a gift from Professor Arto Urtti (DDTC, University of Helsinki, Finland). HCE cells were cultured in Dulbecco's modified Eagle's medium:F12 (1:1) without L-glutamine, supplemented with 15% fetal bovine serum, penicillin–streptomycin-L-glutamine (100Uml−1, 100μgml−1 and 0.3mgml−1, respectively) (Gibco, Invitrogen, Spain). EGF (10ngml−1; Invitrogen, Barcelona, Spain), 0.5% DMSO (Riedel de Haën, Sleelze, Germany) and 0.1μgml−1 cholera toxin (Gentaur, Brussels, Belgium) were also added to the culture medium. Cells were maintained at 37°C with a 5% CO2 humidified atmosphere. Pass numbers 38–40 were used for the following experiments.

Fibroblasts (3T3 NIH) were donated by Professor Juan Zaldive (University of Santiago de Compostela, Spain). Cells were cultured in minimal essential medium, supplemented with 10% fetal bovine serum and penicillin–streptomycin (100Uml−1 and 100μgml−1, respectively) (Sigma-Aldrich). Cells were maintained at 37°C with a 5% CO2 humidified atmosphere. Pass numbers 11–13 were used for the following experiments.

A model of the HCE was developed by establishing the growing conditions suitable for the in vitro differentiation of the HCE cells.30 Hence, the HCE cells were seeded at a density of 400000 cells per insert, on transwell tissue culture inserts (24mm diameter, polyester filters, with a pore size of 0.4μm) obtained from Costar (Corning Life Sciences, Schiphol-Ruk, The Netherlands). The inserts were previously coated with 0.275ml of collagen (1.33mgml−1, type I rat tail, BD Biosciences, Madrid, Spain) containing fibroblasts (94000 cells per filter). Cells were allowed to grow throughout 1 week with culture medium in both the apical (1.5ml) and the basolateral (2.5ml) sides. On the eighth day, the airlift conditions were achieved by removing medium from the apical side of the inserts, and culture medium on the basolateral side was replaced with 1.5ml of fresh medium. The medium was replaced every 2 days. The differentiation stage during cultivation was followed by measuring the transepithelial resistance (TER). After 2–3 weeks, the differentiated cells with TER values >400cm−2 were used in the transfection studies.

Transfections were performed with nanoparticles loaded with 7% (w/w) gWizpSEAP or naked gWizpSEAP (5μg per insert), placed on the apical compartment and incubated for 5h. The yield of gene expression was evaluated non-invasively by monitoring concentrations of SEAP in the basolateral compartment at different time points. Samples of the culture medium were taken during 10 days and analysed for SEAP quantification with chemiluminescences assay using the Great EscAPe SEAP Reporter System Kit protocol (Clontech, Mountain View, CA, USA) and a luminometer (Ultra Evolution, Tecan Iberica, Barcelona, Spain).

Interaction with the ocular epithelia

Fluorescence derivative of HA and the nanoparticles were prepared following the protocol described above. Nanoparticles were concentrated by centrifugation at 16000 relative centrifugal force for 30min, and then resuspended in ultrapure water. The final concentration of the nanoparticle suspension was adjusted at 3mgml−1. Four instillations of 25μl of the nanoparticles suspension were administered, in 10min intervals, to the cul-de-sac of normal conscious rabbits. An aqueous solution of HA-fl was used as control. During the administration of the nanoparticles, animals were maintained in an upright position using restraining boxes. After 2, 4 or 12h post-administration, rabbits were killed with an intraperitoneal overdose of sodium pentobarbital, and the cornea and conjunctiva dissected. The freshly excised specimens were directly mounted on a glass slide and examined by confocal laser scanning microscopy (CLSM) (Leica TCS SP2, Leica Microsystems) without additional tissue processing.

In vivo transfection

Nanoparticles loaded with pEGFP (10% loading) were concentrated by centrifugation, and resuspended in a small volume of ultrapure water in order to obtain 0.6μg pDNA per μl of the nanoparticle suspension (concentration of nanoparticles, 3mgml−1). Normal conscious rabbits were placed in a restraint box and 15μl of the nanoparticles suspension was topically administrated to the cul-de-sac with a frequency of 10min between each instillation. In order to evaluate the yield of transfection efficiency of the nanoparticles, the assayed doses of pDNA per eye were 25, 50 and 100μg. The expression of the green protein was observed after 2, 4 or 7 days. Animals were killed with an intraperitoneal overdose of sodium pentobarbital. The corneas and conjunctivas were excised and analysed for green protein expression by observance of CLSM.

Additionally, the in vivo efficacy of the nanoparticles was evaluated in rats. pβgal-loaded nanoparticles (10% loading) were concentrated and 2.5μg pDNA per eye were administered to the eye surface by single instillation (5μl). Post-administration (48h) the eyes were enucleated and the expression of β-gal was studied by X-gal histochemistry, according to the following protocol: The eyes were exhaustively washed with phosphate-buffered saline (PBS) and fixed in paraformaldehyde 4% in 0.1M PBS during 90min at 4°C. The specimens were rinsed with PBS and incubated in a solution containing 2mgml−1 X-gal, 5mM K4Fe(CN)6, 5mM K3Fe(CN)6, 2mM MgCl2, 0.01% sodium deoxycholate and 0.02% Tween 20, during 20h at 37°C. The eyes were rinsed several times in PBS and eventually examined in a stereomicroscope connected to a digital camera (Olympus, Japan).

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References

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Acknowledgements

This work has been supported by the Spanish Ministry of Science and Technology (MAT 2004-04792-C02-02; NAN 2004-09230-C04-04). The first author acknowledges a grant from the Spanish Government (FPU-MEC). We thank Elisa Toropainen and Margit Hornof for their help in the development of the HCE model, and Rafael Romero for his help with the manipulation of animals.