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European Journal of Pharmaceutical Sciences xxx (2013) xxxxxx 1

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European Journal of Pharmaceutical Sciences

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New gene delivery system based on oligochitosan and solid lipid nanoparticles: In vitro and in vivo evaluation
Q1

Diego Delgado a, Ana del Pozo-Rodrguez a, M. Angeles Solins a, Artur Bartkowiak b, Alicia R. Gascn a,
a Pharmacokinetics, Nanotechnology and Gene Therapy Group, Pharmacy and Pharmaceutical Technology Laboratory, Pharmacy Faculty, University of the Basque Country UPV/EHU, 01006 Vitoria-Gasteiz, Spain b Center of Bioimmobilisation and Innovative Packaging Materials, West Pomeranian University of Technology, Szczecin, Poland

a r t i c l e

i n f o

a b s t r a c t
In the present work, we evaluated the potential utility for gene delivery of three oligochitosans (OligoCh) that differs in the Mn (OligoChA: 6.1 kDa, OligoChB: 11.5 kDa, and OligoChC: 13.7 kDa), with deacetylation degree of 85%. OligoCh were complexed directly with the pCMS-EGFP plasmid to form OligoChDNA carriers. Taking into account the features and benets of both Ch and SLNs, we also combined the OligoCh with SLNs. The three OligoCh presented a great ability to condense and protect the DNA. The OligoCh of highest Mn (OligoChC)) complexed with SLNs at a OligoChC:DNA:SLN ratio 2.5:1:5 induced the highest transfection level in HEK-293 cells at day 3; being transfection 2-fold higher at day 7. After the intravenous administration to mice, OligoChCDNA and OligoChCDNASLN vectors were able to induce the expression of EGFP in the spleen, lung and liver, which was maintained for at least 7 days. In spite of the difference in the in vitro transfection levels between both vectors, no difference was detected in transfection after in vivo administration. Moreover, the OligoChC improved the in vivo transfection efcacy of the DNASLN vector. This work shows the potential utility of the combination of SLNs and OligoCh for the development of new non-viral vectors for gene therapy. 2013 Published by Elsevier B.V.
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Article history: Received 18 January 2013 Received in revised form 12 June 2013 Accepted 13 August 2013 Available online xxxx Keywords: Solid lipid nanoparticles Oligochitosan Gene therapy In vivo transfection Non-viral vector

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1. Introduction The development of new drug delivery systems usually involves the combination of components of different nature, with the aim of taking advantage of benecial properties of each one. In the eld of gene therapy, non-viral systems with different composition are under study; e.g. hyaluronic acid and chitosan (de la Fuente et al., 2010), peptides and solid lipid nanoparticles (del Pozo-Rodrguez et al., 2009a; Delgado et al., 2011) or lysine-based peptides and PLGA (Nie et al., 2009), among others. SLN-based vectors developed by our group have shown capacity for transfection in vitro in several cell lines, and in vivo as well after intravenous and ocular administration (del Pozo-Rodrguez et al., 2010; Delgado et al., 2012a,b). Their ability to condense and protect DNA (del Pozo-Rodrguez et al., 2007), and their entry efciency into several cell lines Delgado et al., 2012a), along with the possibility to be decorated with other compounds (del Pozo-Rodrguez et al., 2009a; Delgado et al., 2011, 2012b), make this nanoparticular system an interesting alternative to viral

Abbreviations: Ch, chitosan; EGFP, green uorescent protein; MM, molar mass; OligoCh, oligochitosan; SLN, solid lipid nanoparticle; RT, room temperature. Corresponding author. Tel.: +34 945013094; fax: +34 945013040. E-mail address: alicia.rodriguez@ehu.es (A.R. Gascn). 0928-0987/$ - see front matter 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ejps.2013.08.013

vectors. From the point of view of the technological application, SLNs have good stability and are subject to be lyophilized (del Pozo-Rodrguez et al., 2009b), which facilities their industrial production. Chitosans (Ch) are polysaccharides comprising copolymers of glucosamine and N-acetylglucosamine. They are biodegradable, biocompatible, nontoxic, and cheap polycationic polymers with low immunogenicity (Jreyssaty et al., 2012). These properties are considered of great interest for many scientists working in biomedical elds, and specically in drug delivery (Dutta et al., 2004; de la Fuente et al., 2008, 2010; Csaba et al., 2006, 2009a). Moreover, the capacity of Ch to cross cell membranes (Thanou et al., 2001) improves the entry of active substances into several types of cells. Due to the positive charge of Ch, it binds DNA efciently and protects it from nuclease degradation; therefore, this polymer is considered very interesting for gene therapy (Ramesan and Sharma, 2012). It has been shown that low molar mass Ch are more efcient for transfection than high molar masses Ch (Csaba et al., 2009b; Turan and Nagata, 2006; Strand et al., 2010; Duceppe and Tabrizian, 2009). This effect can be attributed to the easier release of pDNA from the nanocarrier upon cell internalization (Strand et al., 2010; Thibault et al., 2010). Moreover, low molar mass Ch plays an important role for the endosomal escape of Ch nanocarriers (Thibault et al., 2011). Although highly deacetylated Ch have

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shown better transfection in in vitro studies, the effect of Ch Q2 deacetylation degree is still unclear (Garcia-Fuentes et al., 2012). After in vivo administration, Ch nanocarriers have demonstrated good transfection capacity (Garcia-Fuentes et al., 2012). Most of the Ch-based nanocarriers for gene therapy are based on direct complexation of Ch and the nucleic acid (Leong et al., 1998). As mentioned above, the efcacy for gene delivery depends on the capacity of the Ch to complex genetic material and to cross biological barriers. Additionally, its pH buffering capacity favours the endosomal escape (Chang et al., 2010). It is known that the positive charge of Ch nanocarriers is reduced in some physiological uids due to their intrinsic pH (Germershaus et al., 2008), and this may reduce the colloidal stability of the Ch nanoparticles. Several strategies have been proposed in order to increase the stability of Ch nanocarriers, such as the conjugation with polyethylene glycol (Csaba ET al., 2009c), or the incorporation of amine groups (i.e. quaternized chitosan) in the polysaccharide backbone, making the positive charge independent of pH (Garcia-Fuentes and Alonso, 2012). Finally, its cationic nature, that provides a strong electrostatic interaction with negatively charged mucosal surfaces, joint with its bioadhesive properties, could prolong the contact time with tissues (Mansouri et al., 2004). In the present work, we evaluated the potential utility for gene delivery of three oligochitosans (OligoCh) that differs in the Mn (OligoChA: 6.1 kDa, OligoChB: 11.5 kDa, and OligoChC: 13.7 kDa), with deacetylation degree of 85%. OligoCh were complexed directly with the pCMS-EGFP plasmid to form OligoChDNA carriers. Taking into account the features and benets of both Ch and SLN, we also combined the OligoCh with SLNs and we evaluated these two kinds of vectors, OligoChDNA and OligoChDNASLN, in terms of in vitro and in vivo transfection after intravenous administration to mice.

tion and as a relative reference for MM calculation of low molar mass OligoCh. Table 1 represents the Gel Permeation Chromatography (GPC) results for the three synthesised OligoCh samples. 2.2. Production of solid lipid nanoparticles (SLNs) The SLNs, composed by the solid lipid Precirol ATO 5 (Gattefoss; Madrid, Spain) and the surfactants 1,2-Dioleoyl-3Trimethylammonium-Propane Chloride Salt (DOTAP) from Avanti Polar Lipids (0.4% w/v) and Tween 80 (0.1% w/v), were produced by a solvent emulsicationevaporation technique previously described (del Pozo-Rodrguez et al., 2007). 2.3. Preparation of vectors OligoChDNA vectors were obtained by mixing the pCMS-EGFP plasmid, which encodes the enhanced green uorescent protein (EGFP), with an aqueous solution of OligoCh. Different OligoCh to DNA ratios (w/w) were applied 1:1, 2.5:1, 5:1, 7.5:1, 10:1, 12.5:1 Q3 and 15:1. OligoChDNASLN vectors were prepared by rst forming OligoChDNA complexes at different ratios, and then, incorporating the SLN under agitation for 30 min. The SLN to DNA ratio, expressed as the ratio of DOTAP to DNA (w/w), was xed at 5:1. These vectors have OligoChDNA complexes adsorbed on the surface of nanoparticles. 2.4. Study of DNA binding to OligoCh The resulting OligoChDNA complexes were subjected to electrophoresis on a 1% agarose gel (containing ethidium bromide for visualisation) for 30 min at 120 V. The gel electrophoresis materials were acquired from Bio-Rad (Madrid, Spain). The bands were observed with an Uvitec Uvidoc D-55-LCD-20 M Auto transilluminator. 2.5. Size and potential measurements Sizes of OligoChDNA and OligoChDNASLN vectors were determined by photon correlation spectroscopy (PCS). Zeta potentials of OligoChDNA and OligoChDNASLN were measured by laser doppler velocimetry (LDV). Both measurements were performed on a Malvern Zetasizer 3000 (Malvern Instruments, Worcesteshire, UK). All samples were diluted in 0.1 mM NaCl solution. 2.6. DNase protection study and SDS-induced release in vitro Deoxyribonuclease I (DNase I) from SigmaAldrich (Madrid, Spain) was added to OligoChDNASLN complexes to a nal concentration of 1U DNase I/2.5 lg DNA, and the mixtures were incubated at 37 C for 30 min. Thereafter, a lauryl sulphate sodium (SDS) solution was added to the samples to a nal concentration of 1% to release DNA from the vectors. Samples were then analysed by electrophoresis on agarose gel (described above) and the integrity of DNA in each sample was compared with untreated DNA as control. 2.7. Cell culture and transfection protocol in vitro

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2. Materials and methods 2.1. Synthesis of OligoCh Chitosan of Mn of 1.200 kDa and degree of deacetylation 85% (Yuhuan Ocean Biochemical CO. Ltd. CH-K05011512, China) was used as starting material for preparation of three samples of oligochtiosans. Chitosans with varying molar masses were prepared by controlled radical degradation via continuous addition of various concentration of hydrogen peroxide to 2.5% high MM Ch solution. The reaction was carried out for 2 h at 80 C. All samples, after degradation and purication had various molar masses and similar polydispersities of MM in range 1.52.2. The detailed procedure of degradation and purication of Ch is described elsewhere (Bartkowiak and Hunkeler, 2000). The molar mass of nal oligochitosan samples was determined by GPC method using Knauer SmartLine HPLC system (Knauer, Germany) equipped with refractometric detection at a ow-rate of 1 cm3/min. Two SEPARON HEMA BIO columns 1000 and 40 (TESSEK Ltd., Praha, Czech Republic) with PTFE guard column (Supelco, USA) were employed as the stationary phase with aqueous solution of 0.5 M acetic acid/0.5 M sodium acetate as an eluent. The water-soluble GPC standards pf dextran (PSS, Mainz, Germany) were selected and used for column calibraTable 1 The GPC results for the three OligoCh samples. Sample OligoChA OligoChB OligoChC
a b

Mna

(g/mol)

Mw (g/mol) 40,000 100,000 125,000

Polydispersity 6.6 8.7 9.1

6100 11,500 13,700

Mn: Number average molecular weight. Mw: Molecular weight.

In vitro assays were performed with Human Embrionic Kidney (HEK-293) cells, obtained from the American Type Culture Collection (ATCC). Cell culture reagents were purchased from LGC Promochem (Barcelona, Spain). HEK-293 cells were maintained in Eagles Minimal Essential medium with Earles BSS and 2 mM L-glutamine (EMEM)

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supplemented with 10% heat-inactivated horse serum and Normocin (InvivoGen; San Diego, California, US). Cells were incubated at 37 C with 5% CO2 in air and subcultured every 23 days using trypsin/EDTA. For transfection HEK-293 cells were seeded on 24well plates at density of 150,000 per well one day before the transfection procedure. The plasmid (2.5 lg) formulated in the vectors was added in 75 lL of HBS, and cells were incubated with the vectors for 4 h at 37 C. Then the medium containing the complexes in the wells was diluted with 1 mL of complete medium and cells were allowed to grow for 72 h. Moreover, this assay was repeated by growing cells for 1 week. 2.8. Flow cytometry mediated analysis of transfection efcacy and cell viability At the end of the point times, cells were washed once with PBS and detached with trypsin/EDTA. Cells were resuspended in PBS and introduced to a FACSCalibur ow cytometer (BD Biosciences, San Jose, USA). For each sample 10,000 events were collected. For transfection efcacy the uorescence of EGFP positive cells was collected at 525 nm (FL1). For cell viability measurements, 5 lL of BD Via-Probe reagent (BD Biosciences; Belgium) was added to each sample, and after 10 min of incubation uorescence correspondent to dead cells was measured at 650 nm (FL3). 2.9. Interaction with erythrocytes Hemolysis assay. A hemolysis assay and a hemagglutination assay were conducted following protocols previously described by Kurosaki et al. (2010). In both cases fresh human blood of 0+ type was centrifuged at 4000 rpm for 5 min and the plasma and the buffy coat were discarded. Erythrocytes were washed three times with PBS by centrifugation at 4000 rpm and were diluted in PBS to a nal concentration of 5% (v/v) for the hemolysis assay, and to a 2% (v/v) concentration for the hemagglutination assay. The naked plasmid, OligoChCDNA and OligoChCDNASLN vectors were added to erythrocytes suspension at ratio 1:1 (v/v) and incubated for 60 min (hemolysis assay) or 15 min (hemagglutination assay) at room temperature (RT). In the study of the hemolysis, suspensions were centrifuged at 4000 rpm during 5 min and supernatants were taken to quantify the hemolysis by measuring haemoglobin release at 545 nm in a microplate reader. A lysis buffer was employed for the 100% hemolysis sample. In the study of the hemagglutination samples were placed on a microscope slide and observed by microscopy. 2.10. Intravenous administration of vectors Female Balb/c nude mice weighing 1822 g (5 weeks of age) were purchased from Harlam Interfauna Ibrica S.L. (Barcelona, Spain). Animals were handled in accordance with the Principles of Laboratory Animal Care (http://www.history.nih.gov/laws). Mice were quarantined for at least 1 week prior to the study. They were housed under standard conditions and had ad libitum access to water and standard laboratory rodent diet. a unique dose of 60 lg of plasmid in 100 lL of the vectors were injected in standard way into the tail vein. As controls free DNA and vectors without the plasmid were administered in the same way and volume. The treatment was administered to three mice in each group. After three and 7 days post-injection mice were sacriced and the liver, lungs and spleen were removed, quick frozen in liquid nitrogen embedded in tissue freezing medium (Jung, Leica) and thin sectioned on a cryostat (Cryocut 3000, Leica).

2.11. Immunolabelling of EGFP in tissue sections Cryostat sections (8 lm) were xed with 4% paraformaldehyde during 10 min at RT, and washing in PBS. Then, sections were blocked and permeabilized in PBS 0.1 M, 0.1% Triton X-100 (SigmaAldrich; Madrid, Spain) and 2% normal goat serum (NGS) for 1 h at RT. Then, sections were incubated in primary antibody (polyclonal anti-GFP, IgG fraction) for 2 h at RT. Following adequate washing in PBS, sections were incubated in secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG). Primary antibody and secondary antibody were provided by Invitrogen (Barcelona, Spain), and the NGS from Chemicon International Inc. (Temecula, CA, USA). Finally, sections were washed again in PBS and coverslipped with Dapi-Fluoromount-G (SouthernBiotech; Coultek, Espaa). Images of the immunolabelled sections were captured with an inverted microscopy equipped with an attachment for uorescent observation (model EclipseTE2000-S, Nikon). From each tissue, 12 sections representing the whole organ were analysed. Results were expressed as the percentage of sections in which EGFP was detected. 2.12. Statistical analysis Results are reported as means (S.D. = standard deviation). Statistical analysis was made with SPSS 19.0 for Windows (SPSS, Chicago, USA). Normal distribution of samples was assessed by ShapiroWilks test, and homogeneity of the variance by Levenes test. When variances were homogeneous the statistical analysis between different groups was determined with Bonferroni test, and Tamhanes test was performed when variances were not homogeneous. Results were considered statistically signicant if p < 0.05. 3. Results 3.1. OligoChDNA vectors: size, and zeta potential and binding study OligCh:DNA complexes presented a particle size ranging from 127 nm to 218 nm, and no signicant differences among the three OligoCh and the different proportions were detected. Polydispersity index was always lower than 0.4. Fig. 1 shows the DNA binding capacity of the three OligoCh at different OligoCh to DNA ratios. Lane 1 corresponds to free DNA. On lane 2 (OligoChDNA at ratio 1:1) the intensity of the bands indicates that most of the DNA was free, while on lanes 38 (OligoChDNA at ratios from 2.5:1 to 15:1) bands were absents and the DNA was retained in the point of the gel where complexes were placed. These results show that at least an OligoChDNA ratio of 2.5:1 was needed to bind all DNA. The zeta potential of these formulations was also measured, and the results showed that the smaller the OligoChDNA ratio is, the smaller the zeta potential of the complexes is. When OligoChA was used, the zeta potential was positive for the OligoChDNA ratios from 15:1 to 5:1, but ratios 2.5:1 and 1:1 presented negative values. With OligoChB and OligoChC, positive supercial charge was obtained for the ratios 15:1 to 2.5:1, whereas with the ratio 1:1, the zeta potential was negative. 3.2. OligoChDNASLN vectors: size and zeta potential Table 2 shows the particle size and zeta potential of OligoCh DNASLN vectors. All vectors were similar in size, around 300 nm and there were not signicant differences. The polydispersity index was always lower than 0.4. The higher the OligoCh to DNA ratio was, the higher the supercial charge of the nal vectors was, being differences statistically signicant (p < 0.05).

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Fig. 1. Binding and zeta potential of OligoChDNA complexes at different OligoCh to DNA ratios. A = OligoChA (6.1 kDa), B = OligoChB (11.5 kDa) and C = OligoChC (13.7 kDa). Error bars represent SD = standard deviation (n = 3). In electrophoresis gels: lane 1 corresponds to free DNA, and the next lanes correspond to ratios (w/w): 1:1, 2.5:1, 5:1, 7.5:1, 10:1, 12.5:1 and 15:1.

Table 2 Size and zeta potential of OligoChDNASLN vectors. Mean (S.D. = standard deviation) (n = 3). Size (nm) OligoChA:DNA:SLN ratio 1:1:5 339.77 (20.55) 2.5:1:5 355.70 (19.77) 10:1:5 265.43 (35.60) 15:1:5 311.1 (20.17) OligoChB:DNA:SLN ratio 1:1:5 366.10 (31.20) 2.5:1:5 359.10 (15.50) 10:1:5 325.13 (17.38) 15:1:5 316.67 (18.82) OligoChC:DNA:SLN ratio 1:1:5 353.47 (12.31) 2.5:1:5 350.97 (15.50) 10:1:5 307.23 (48.20) 15:1:5 267.13 (14.16) Zeta potential (mV) +45.60 +43.00 +52.61 +62.03 +45.72 +46.80 +52.74 +57.32 +45.03 +47.95 +57.62 +58.02 (1.85) (1.79) (1.81) (3.16) (0.47) (1.25) (1.23) (5.02) (1.82) (0.65) (1.33) (0.43)

1012 (OligoChDNASLN vectors treated only with SDS), the DNA was retained in the loading well, meaning that the DNA in the vector was unable to migrate into the gel. When DNA was formulated in SLN without OligoCh (DNASLN vector) and treated with SDS, the DNA was able to be released and migrate into the gel. 3.4. In vitro transfection and cell viability Transfection capacity and cell viability of vectors were assayed in vitro in HEK-293 culture cells 3 days post-transfection. Fig. 3 features the percentage of transfected cells after the treatment with the vectors OligoChDNA or OligoChDNASLN, at different OligoCh to DNA ratios (1:1, 2.5:1, 10:1 and 15:1). The highest transfection levels were obtained with the OligoChC at ratio 2.5:1; when this vector was complexed with SLNs, the transfection was even higher (p < 0.05). Fig. 4 shows the transfection capacity of the formulations OligoChDNA or OligoChDNASLN at an OligoChC to DNA ratio of 2.5:1 at 3 and 7 days. As it can be seen, the percentage of transfected cells at day 7 was higher than at day 3 (p < 0.01). In all transfection studies cell viability was similar to the observed in the non-treated cells (over 75% of viable cells). 3.5. Interaction with erythrocytes Hemagglutination was evaluated by incubating the vectors with erythrocytes. The photographs in Fig. 5 shows a very light agglutination when erythrocytes were in touch with the OligoChCDNA SLN vector (Fig. 5D). No agglutination was detected when the

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3.3. OligoChDNASLN vectors: DNase protection study and SDSinduced release in vitro For this study, OligoChDNASLN vectors prepared with the three OligoCh at ratio 2.5:1:5 were evaluated by using gel electrophoresis. The absence of bands in lanes 24 in Fig. 2 shows that the DNA was completely bound. The gel also shows the results of the DNase protection study. Lane 5 corresponds to free DNA treated with DNase I; in this lane no band appears because DNA was totally digested by the enzyme. In lanes 68 (OligoChDNASLN complexes treated with DNase I and later with SDS), and in lanes

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Fig. 2. Binding, DNase protection and DNA release from complexes. Lane 1: free DNA; lane 2: OligoChADNASLN; lane 3: OligoChBDNASLN; lane 4: OligoChCDNASLN; lane 5: free DNA treated with DNase I; lane 6: OligoChADNASLN with DNase I; lane 7: OligoChBDNASLN with DNase I; lane 8: OligoChCDNASLN with DNase I; lane 9: DNASLN with SDS; lane 10: OligoChADNASLN with SDS; lane 11: OligoChBDNASLN with SDS; lane 12: OligoChCDNASLN with SDS. The DNA to SLN ratio (w/w) was 1:5 and the OligoCh to DNA ratio (w/w) was 2.5:1.

Fig. 3. In vitro transfection activity and cell viability (small graphics) after treatment with OligoChDNA and OligoChDNASLN vectors assayed in HEK-293 cells. DNA to SLN ratio (w/w) was 1:5 in all cases; the OligoCh to DNA ratio (w/w) varied. (A) OligoCh to DNA ratio 1:1, (B) OligoCh to DNA ratio 2.5:1, (C) OligoCh to DNA ratio 10:1, (D) OligoCh to DNA ratio 15:1. Error bars represent S.D. (n = 3). p < 0.01 respect to the rest of OligoChDNASLN vectors. p < 0.05 respect to the rest of vectors. #p < 0.05 respect to the OligoChCDNA vector.

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erythrocytes were incubated with free DNA or the vector OligoCh DNA. Fig. 5 also features the hemolytic activity of erythrocytes after treatment with vectors prepared with OligoChC. As can be seen, the formulations did not induce hemolysis. 3.6. In vivo protein expression after intravenous administration to mice of OligoChDNA and OligoChDNASLN vectors Free DNA, OligoChCDNA (ratio 2.5:1) and OligoChCDNASLN (ratio 2.5:1:5) vectors were intravenously administered to mice for in vivo assay. In order to ensure that the observed green uorescence was not an artefact of the immunolabelling, we subjected samples of mice treated with formulations without plasmid to the same procedure with the primary and the secondary antibodies, and no green uorescence was detected. The tissue sections of the mice treated with free DNA did not show uorescence due to EGFP; however, hepatic, spleen and lung sections of animals treated with either OligoChDNA or OligoCh DNASLN vectors, showed green uorescence. These sections were obtained from mice sacriced 3 or 7 days after the intravenous

administration. From each tissue, 12 sections representing the whole organ were analysed. At day 3 (Fig. 6A), almost all sections of mice treated with both vectors showed EGFP, and green uorescence remained 7 days after intravenous administration (Fig. 6B). Fig. 7 shows images of the tissues obtained from the animals treated with the OligoChCDNASLN vector. 4. Discussion The need of more efcient non-viral vectors for gene therapy has conducted to the design of a wide variety of materials and systems, including multicomponent systems composed of mixtures of them. In this work, we have studied the potential utility of three OilgoCh, alone or in combination with SLNs, for gene delivery. The three OligoCh presented a great ability to condense the DNA (Fig. 1), and a ratio OligoCh to DNA of 2.5:1 was enough to bind all DNA. Since the Ch is a polycation, the zeta potential of the complexes increased as the ratio OligoCh to DNA increased, which is also dependent on the Mn of the Ch. As the Mn of Ch lowers, more uniform complexes with DNA are formed, so that there are less remaining free cationic charges of primary amino groups

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Fig. 4. In vitro transfection over time (3 and 7 days after addition of vectors) in HEK-293 cells with the vectors OligoChCDNA (2.5:1) and OligoChCDNASLN (2.5:1:5). Error bars represent S.D. (n = 3). p < 0.01 respect to the levels of transfection achieved with the same formulations 3 days after addition of vectors.

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on the surface of the complexes, leading to overall lower zeta potential. Although the OligoChDNA complexes have positive net charge due to the excess of OligoCh, not all negative charges of the DNA are neutralized. This is because the distance between the excess positive charges of the OligoCh may be higher than the distance between two adjacent phosphate groups of the DNA (negative

charges) (Bonincontro et al., 2008). Therefore, there are free negative charges in the OligoChDNA complex to interact with the positive charges of the SLN. All the OligoChDNASLN complexes presented a similar particle size, irrespective of the OligoCh. However, the greater the OligoCh to DNA ratio in the vectors was, the higher positive supercial charge was, regardless of the OligoCh used (Table 2). Positive charges are known to facilitate the interaction with the negative charged cell surface and, therefore, favour the invagination of the cell plasma membrane and the uptake of the vectors (Elouahabi and Ruysschaert, 2005). We have previously demonstrated the importance of a balance between protection and release of the DNA to achieve transfection with SLNs (del Pozo-Rodrguez et al., 2007). Fig. 2 shows that when formulated with SLNs the OligCh keep the high capacity to bind the DNA since the plasmid was not released even in presence of SDS (Fig. 2, lanes 10, 11 and 12). However, DNA was able to be released from the SLNs in absence of Ch (Fig. 2, lane 9). In order to study the protection capacity of the complexes containing OligoCh, we analysed the integrity of the DNA after the treatment of the vectors with DNase I (Fig. 2). When vectors were rst treated with DNase I and then with SDS (lanes 68), the DNA did not migrate in the gel due to the strong interactions with the OligoCh, although the DNA bands in the loading well indicate that the plasmid was not degraded by the DNase I. In vitro transfection studies in HEK-293 cells showed that the highest transfection level was obtained with the vector OligoChC DNASLNs at a OligoChC to DNA ratio of 2.5:1 (Fig. 3). The same vector prepared without SLNs induced lower transfection; that means that SLNs increased the transfection capacity of OligoChC. This was not observed for the other two OligoCh evaluated, OligoChA and OligoChB. The different transfection level of the three OligoCh when formulated with SLNs cannot be attributed to the vector size or surface charge, since no signicant differences were detected in those features (Table 2). The three OligoCh vary in the

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Fig. 5. Photographs: Agglutination of erythrocytes when blood is untreated (A), treated with DNA (B), with OligoChCDNA vector (C), and with OligoChCDNASLN vector (D). Graphic: Hemolytic activity of erythrocytes after treatment with OligoChCDNA and OligoChCDNASLN vector. Lysis buffer represents 100% hemolysis sample. Error bars represent S.D. (n = 9). p < 0.01 respect to the rest of samples. The SLN to DNA ratio (w/w) was 5:1 and the OligoChC to DNA ratio (w/w) 2.5:1.

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Fig. 6. Percentage of transfected sections of liver, spleen and lung of mice treated with OligoChCDNA and OligoChCDNASLN vectors. The SLN to DNA ratio (w/w) was 5:1 and the OligoChC to DNA ratio 2.5:1. Error bars represent S.D. (n = 3). A = 3 days after administration. B = 7 days after administration.

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Mn, which could lead to a different surface disposition of the OligoCh and the plasmid in the vector. Although the exact disposition of the compounds in a multicomponent non-viral system it is difcult to predict, it could be responsible for differences in condensation, release and protection capacity of the DNA, and therefore, in transfection. Transfection of the vector prepared with the OligoChC at a OligoCh to DNA ratio of 2.5:1 was studied over time, and we observed a 2-fold increase in transfection at day 7, in comparison to transfection at day 3 (Fig. 4). This happened for both the OligoChCDNA vector and the OligoChCDNASLN vector. In a previous work (5), we showed the capacity of transfection of a vector composed by SLN and DNA, and without Ch. The transfection level of that vector in HEK293 cell at the same conditions than in the current study was 40%, higher than transfection achieved with the vector OligoChCDNASLN (16% at day 7). The transfection level obtained with the vector composed by the OligoChC increased from day 3 to day 7; however, the vector prepared without OligoCh produced the same transfection level over time. In our opinion, the high condensation degree of DNA when it is

formulated with the OligoChC would led to form a more stable system that maintain the DNA protected and active for a longer period of time. The increase of transfection over time seems to indicate a sustained release of the DNA that initially induces lower transfection levels that increase over time. In someway, the new vector may sequester the DNA and thus, it is not able to release it completely even after 7 days. Concerning safety, Ch are FDA GRAS excipients, and have extensively demonstrated intestinal and topical tolerance (24), although the safe use of Ch as a parenteral excipient is not yet clear (Baldrick, 2010). In our study, OligoChCDNA and OligoChCDNASLN vectors did not decrease cell viability (Fig. 3), and when vectors were put in contact in vitro with erythrocytes only a light agglutination was observed with the OligoChCDNASLN vector (photograph D in Fig. 5). However, this light agglutination is not expected to be clinically relevant; in fact, the intravenous administration to mice of vectors based on SLNs with similar agglutination effect in vitro did not result in apparent signs of toxicity (Delgado et al., 2012a). After the intravenous administration to mice of the OligoChC DNA and OligoChCDNASLN vectors, EGFP expression was detected in liver, spleen and lungs at day 3, which was maintained for at least 7 days (Figs. 6 and 7). No difference in the transfection capacity of the two formulations studied was detected. In a previous work, after intravenous administration of DNASLN vector, transfection in lung was not detected, and EGFP protein expression in liver and spleen signicantly decreased 7 days after administration (del Pozo-Rodrguez et al., 2010). Therefore, the OligoChC improved the transfection efcacy of the DNASLN vector. The high DNA condensation provided by the OligoChC would maintain the DNA protected, which may induce a longer stay of the plasmid in the organism. Moreover, OligoChC has a deacetylation degree of 85%; deacetylation degree over 50% has been related to the increase in the cell permeability, and the opening of tight junctions of cells (Holme et al., 2000). This is especially important for tissues other than those of the reticuloendothelial system (RES), such as the lung, where the uptake of the vectors is more difcult. Therefore, OligoChC may increase the access of the vector to tissues. In addition, the bioadhesive properties of Ch and the proved capacity of Ch-based nanosytems to load and provide a sustained release of several actives (Garcia-Fuentes and Alonso, 2012), may justify prolonged residence time of the intact plasmid in the tissues, providing a sustained release of the DNA and inducing a long-lasting transfection. In spite of the difference in the in vitro transfection levels between the vector OligoChCDNA and OligoChCDNASLN, no difference was detected in the transfection after the intravenous administration to the mice. This lack of in vitro-in vivo correlation justies the convenience of evaluating this kind of vectors for gene therapy in animal models during early stages of the development process. Additional studies are needed to assess the real potential of these new vectors. It has to be taken into account the possibility to functionalize the vectors for specic clinical

Fig. 7. Images of transfected sections of liver (A), spleen (B) and lung (C) of mice treated with OligoChCDNASLN vectors, after the immunolabelling of EGFP (green). Images were captured at 60x. Cell nucleuses were labelled with DAPI (blue). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Delgado, D., et al. New gene delivery system based on oligochitosan and solid lipid nanoparticles: In vitro and in vivo evaluation. Eur. J. Pharm. Sci. (2013), http://dx.doi.org/10.1016/j.ejps.2013.08.013

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application. In this sense, both the solid lipid nanoparticles and the OligoCh offer several possibilities. On the one hand, the inclusion of galactosylated lipids in the solid lipid nanoparticles has been used to target to the liver (Wang et al., 2010), transferrin may be covalently coupled to lipids in SLNs to target to malignant cells (Mulik et al., 2010), or albumin conjugated SLNs have been used as a strategy to bypass the brain blood barrier (Agarwal et al., 2011). On the other hand, lactosylated or galoctosylated chitosan for targeting liQ4 ver cells is an approach to improving the transfection efciency (Alex et al., 2011), and conjugation with folate (Ramesan, 2012), polyethylenimine (Wong et al., 2006), and polyethylenimine-bcyclodextrin (Ping et al., 2011) are other strategies to increase the transfection capacity of chitosan. 5. Conclusion The highest in vitro transfection level was obtained with the OligoChC, the OligoCh with higher Mn. The SLNs increased the in vitro transfection capacity of this OligoChC. After the intravenous administration to mice, OligoChCDNA and OligoChCDNA SLN vectors were able to induce the expression of EGFP in the spleen, lung and liver, which was maintained for at least 7 days. In spite of the difference in the in vitro transfection levels between both vectors, no difference was detected in transfection after in vivo administration. Moreover, the OligoChC improved the in vivo transfection efcacy of the DNASLN vector. This work shows the potential utility of the combination of SLNs and OligoCh for the development of new non-viral vectors for gene therapy. Acknowledgement This work was supported by the Basque Governments Department of Education, Universities and Investigation (IT-34110). References
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