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PHASCI 2840 29 August 2013

European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx 1

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

Diego Delgado a, Ana del Pozo-Rodríguez a, M. Angeles Solinís a, Artur Bartkowiak b, Alicia R. Gascón 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 OligoCh–DNA carriers. Taking into account the features and benefits 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, OligoChC–DNA and OligoChC–DNA–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 efficacy of the DNA–SLN 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. Ó 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 beneficial properties of each one. In the field 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-Rodríguez 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-Rodríguez et al., 2010; Delgado et al., 2012a,b). Their ability to condense and protect DNA (del Pozo-Rodríguez et al., 2007), and their entry efficiency into several cell lines Delgado et al., 2012a), along with the possibility to be decorated with other compounds (del Pozo-Rodríguez et al., 2009a; Delgado et al., 2011, 2012b), make this nanoparticular system an interesting alternative to viral

Abbreviations: Ch, chitosan; EGFP, green fluorescent protein; MM, molar mass; OligoCh, oligochitosan; SLN, solid lipid nanoparticle; RT, room temperature. ⇑ Corresponding author. Tel.: +34 945013094; fax: +34 945013040. E-mail address: (A.R. Gascón). 0928-0987/$ - see front matter Ó 2013 Published by Elsevier B.V.

vectors. From the point of view of the technological application, SLNs have good stability and are subject to be lyophilized (del Pozo-Rodríguez 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 fields, and specifically 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 efficiently 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 efficient 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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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),

000 b Polydispersity 6. The molar mass of final oligochitosan samples was determined by GPC method using Knauer SmartLine HPLC system (Knauer.000 100. The detailed procedure of degradation and purification of Ch is described elsewhere (Bartkowiak and Hunkeler. Mainz.5 M sodium acetate as an eluent.3.08. Size and potential measurements Sizes of OligoCh–DNA and OligoCh–DNA–SLN vectors were determined by photon correlation spectroscopy (PCS). 2. 7. with deacetylation degree of 85%. The gel electrophoresis materials were acquired from Bio-Rad ( 125. USA) were employed as the stationary phase with aqueous solution of 0. 2012). that provides a strong electrostatic interaction with negatively charged mucosal surfaces. 2. The bands were observed with an Uvitec Uvidoc D-55-LCD-20 M Auto transilluminator. we also combined the OligoCh with SLNs and we evaluated these two kinds of vectors.2.013 . obtained from the American Type Culture Collection (ATCC). was fixed at 5:1.4% w/v) and Tween 80 (0.. 2. 2009c). Finally. composed by the solid lipid PrecirolÒ ATO 5 (Gattefossé.1. China) was used as starting material for preparation of three samples of oligochtiosans. http://dx.5. All samples were diluted in 0. 10:1. expressed as the ratio of DOTAP to DNA (w/w).5:1.. Table 1 represents the Gel Permeation Chromatography (GPC) results for the three synthesised OligoCh samples. / European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx No. The reaction was carried out for 2 h at 80 °C. Cell culture and transfection protocol ‘in vitro’ 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 2. Different OligoCh to DNA ratios (w/w) were applied 1:1. 2. Spain). OligoChB: 11. quaternized chitosan) in the polysaccharide backbone.500 13. 2. 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. J.e. Spain) and the surfactants 1. the efficacy for gene delivery depends on the capacity of the Ch to complex genetic material and to cross biological barriers. a lauryl sulphate sodium (SDS) solution was added to the samples to a final concentration of 1% to release DNA from the vectors. 2000). HEK-293 cells were maintained in Eagle’s Minimal Essential medium with Earle’s BSS and 2 mM L-glutamine (EMEM) Please cite this article in press as: Delgado. Czech Republic) with PTFE guard column (Supelco.700 Mn: Number average molecular weight. et al.6 8. Thereafter.2013. 2012). could prolong the contact time with tissues (Mansouri et al.5–2. It is known that the positive charge of Ch nanocarriers is reduced in some physiological fluids due to their intrinsic pH (Germershaus et al.doi. After ‘in vivo’ administration.5% high MM Ch solution. in terms of ‘in vitro’ and ‘in vivo’ transfection after intravenous administration to mice..5 kDa. or the incorporation of amine groups (i. UK). 2. The SLN to DNA ratio. Synthesis of OligoCh Chitosan of Mn of 1. OligoCh were complexed directly with the pCMS-EGFP plasmid to form OligoCh–DNA carriers.1 mM NaCl solution.2. As mentioned above.1 6100 11. Germany) were selected and used for column calibraTable 1 The GPC results for the three OligoCh samples. Mw: Molecular weight.1016/j. 2004). tion and as a relative reference for MM calculation of low molar mass OligoCh. Eur. of Pages 8. ‘In vitro’ assays were performed with Human Embrionic Kidney (HEK-293) cells.1 kDa.200 kDa and degree of deacetylation 85% (Yuhuan Ocean Biochemical CO. In the present work. 2012). (2013). Worcesteshire. 12. 2010)..7 9.. Cell culture reagents were purchased from LGC Promochem (Barcelona. such as the conjugation with polyethylene glycol (Csaba ET al.4. DNase protection study and SDS-induced release ‘in vitro’ Deoxyribonuclease I (DNase I) from Sigma–Aldrich (Madrid.. Both measurements were performed on a Malvern Zetasizer 3000 (Malvern Instruments. Ch nanocarriers have demonstrated good transfection capacity (Garcia-Fuentes et al. Materials and methods 2. its cationic nature.6. and then. Spain) was added to OligoCh–DNA–SLN complexes to a final concentration of 1U DNase I/2. Chitosans with varying molar masses were prepared by controlled radical degradation via continuous addition of various concentration of hydrogen peroxide to 2. Several strategies have been proposed in order to increase the stability of Ch nanocarriers. with an aqueous solution of OligoCh.PHASCI 2840 29 August 2013 2 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 D. Ltd. Sci.1% w/v). OligoCh–DNA and OligoCh–DNA–SLN. Sample OligoChA OligoChB OligoChC a b Mna (g/mol) Mw (g/mol) 40.7 kDa). incorporating the SLN under agitation for 30 min. Two SEPARON HEMA BIO columns 1000 and 40 (TESSEK Ltd..ejps. Model 5G shown better transfection in ‘in vitro’ studies. 1998).5:1 Q3 and 15:1. The water-soluble GPC standards pf dextran (PSS. Preparation of vectors OligoCh–DNA vectors were obtained by mixing the pCMS-EGFP plasmid. and the mixtures were incubated at 37 °C for 30 min. CH-K05011512. joint with its bioadhesive properties. its pH buffering capacity favours the endosomal escape (Chang et al. Study of DNA binding to OligoCh The resulting OligoCh–DNA complexes were subjected to electrophoresis on a 1% agarose gel (containing ethidium bromide for visualisation) for 30 min at 120 V.7. 5:1. Delgado et al. and OligoChC: 13. making the positive charge independent of pH (Garcia-Fuentes and Alonso.2-Dioleoyl-3Trimethylammonium-Propane Chloride Salt (DOTAP) from Avanti Polar Lipids (0.. OligoCh–DNA–SLN vectors were prepared by first forming OligoCh–DNA complexes at different ratios. Spain). 2008). Praha. Additionally. These vectors have OligoCh–DNA complexes adsorbed on the surface of nanoparticles. D. 2.5:1.. after degradation and purification had various molar masses and similar polydispersities of MM in range 1.. Most of the Ch-based nanocarriers for gene therapy are based on direct complexation of Ch and the nucleic acid (Leong et al. 2007). were produced by a solvent emulsification–evaporation technique previously described (del Pozo-Rodríguez et al. Pharm. which encodes the enhanced green fluorescent protein (EGFP). All samples. Germany) equipped with refractometric detection at a flow-rate of 1 cm3/min.5 lg DNA.5 M acetic acid/0. Zeta potentials of OligoCh–DNA and OligoCh–DNA–SLN were measured by laser doppler velocimetry (LDV). Madrid. New gene delivery system based on oligochitosan and solid lipid nanoparticles: ‘In vitro’ and ‘in vivo’ evaluation. Production of solid lipid nanoparticles (SLNs) The SLNs. and this may reduce the colloidal stability of the Ch nanoparticles. Taking into account the features and benefits of both Ch and SLN. we evaluated the potential utility for gene delivery of three oligochitosans (OligoCh) that differs in the Mn (OligoChA: 6. the effect of Ch Q2 deacetylation degree is still unclear (Garcia-Fuentes et al.

the zeta potential was negative. San Jose.ejps. sections were blocked and permeabilized in PBS 0.5 lg) formulated in the vectors was added in 75 lL of HBS. Results 3. whereas with the ratio 1:1.000 per well one day before the transfection procedure. Statistical analysis Results are reported as means (S. España). Cells were resuspended in PBS and introduced to a FACSCalibur flow cytometer (BD Biosciences. All vectors were similar in size.D. 206 207 208 209 210 211 212 213 214 215 216 272 273 274 275 276 277 278 279 280 281 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 303 304 305 306 307 308 309 Please cite this article in press as: Delgado. OligoCh–DNA vectors: size. (2013). For transfection efficacy the fluorescence of EGFP positive cells was collected at 525 nm (FL1). and washing in PBS.013 .1 M. USA). For each sample 10.5:1 and 1:1 presented negative values. Polydispersity index was always lower than 0. Erythrocytes were washed three times with PBS by centrifugation at 4000 rpm and were diluted in PBS to a final concentration of 5% (v/v) for the hemolysis assay. and after 10 min of incubation fluorescence correspondent to dead cells was measured at 650 nm (FL3). = standard deviation). When variances were homogeneous the statistical analysis between different groups was determined with Bonferroni test. 2. et al.1016/j. 2. and the NGS from Chemicon International Inc. 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. The plasmid (2. 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. sections were incubated in secondary antibody (Alexa FluorÒ 488 goat anti-rabbit IgG).5:1. As controls free DNA and vectors without the plasmid were administered in the same way and volume. Lane 1 corresponds to free DNA. 2.2. while on lanes 3–8 (OligoCh–DNA at ratios from 2. positive superficial charge was obtained for the ratios 15:1 to 2. the zeta potential was positive for the OligoCh–DNA ratios from 15:1 to 5:1. (Barcelona. 12 sections representing the whole organ were analysed. being differences statistically significant (p < 0. a unique dose of 60 lg of plasmid in 100 lL of the vectors were injected in standard way into the tail vein. Immunolabelling of EGFP in tissue sections Cryostat sections (8 lm) were fixed with 4% paraformaldehyde during 10 min at RT. Chicago. this assay was repeated by growing cells for 1 week. These results show that at least an OligoCh–DNA ratio of 2. 5 lL of BD Via-Probe reagent (BD Biosciences.11. cells were washed once with PBS and detached with trypsin/EDTA. US). USA). Coultek. Then. The higher the OligoCh to DNA ratio was. http://dx. The polydispersity index was always lower than 0. Leica) and thin sectioned on a cryostat (Cryocut 3000. and no significant differences among the three OligoCh and the different proportions were detected. the higher the superficial charge of the final vectors was. From each tissue. Following adequate washing in PBS. Pharm. Flow cytometry mediated analysis of transfection efficacy and cell viability At the end of the point times. 1 shows the DNA binding capacity of the three OligoCh at different OligoCh to DNA ratios. Madrid. Cells were incubated at 37 °C with 5% CO2 in air and subcultured every 2–3 days using trypsin/EDTA. Delgado et al. and to a 2% (v/v) concentration for the hemagglutination assay. 3. sections were washed again in PBS and coverslipped with Dapi-Fluoromount-G™ (SouthernBiotech. The zeta potential of these formulations was also measured. In the study of the hemagglutination samples were placed on a microscope slide and observed by microscopy. and homogeneity of the variance by Levene’s test.12. Interaction with erythrocytes Hemolysis assay.000 events were collected. but ratios 2. OligoChC–DNA and OligoChC–DNA–SLN 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). On lane 2 (OligoCh–DNA at ratio 1:1) the intensity of the bands indicates that most of the DNA was free. OligoCh–DNA–SLN vectors: size and zeta potential Table 2 shows the particle size and zeta potential of OligoCh– DNA–SLN vectors. Finally. Moreover. / European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx 194 195 196 197 198 199 200 201 202 203 204 205 No. around 300 nm and there were not significant differences. of Pages 8.PHASCI 2840 29 August 2013 D.L.5:1 to 15:1) bands were absents and the DNA was retained in the point of the gel where complexes were placed.10. and Tamhane’s test was performed when variances were not homogeneous. Model 5G 3 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 supplemented with 10% heat-inactivated horse serum and Normocin (InvivoGen. Normal distribution of samples was assessed by Shapiro–Wilk’s test. (2010). With OligoChB and OligoChC. CA. quick frozen in liquid nitrogen embedded in tissue freezing medium (Jung. Belgium) was added to each sample.nih. Intravenous administration of vectors Female Balb/c nude mice weighing 18–22 g (5 weeks of age) were purchased from Harlam Interfauna Ibérica S. California.4. New gene delivery system based on oligochitosan and solid lipid nanoparticles: ‘In vitro’ and ‘in vivo’ evaluation. 2. and the results showed that the smaller the OligoCh–DNA ratio is. and zeta potential and binding study OligCh:DNA complexes presented a particle size ranging from 127 nm to 218 nm. 2. For transfection HEK-293 cells were seeded on 24well plates at density of 150. Fig. the smaller the zeta potential of the complexes is. Mice were quarantined for at least 1 week prior to the study. sections were incubated in primary antibody (polyclonal anti-GFP. Then.1% TritonÒ X-100 (Sigma– Results were considered statistically significant if p < 0.0 for WindowsÒ (SPSSÒ. When OligoChA was used. Images of the immunolabelled sections were captured with an inverted microscopy equipped with an attachment for fluorescent observation (model EclipseTE2000-S.8. Sci. lungs and spleen were removed.05. and cells were incubated with the vectors for 4 h at 37 °C.2013. J. For cell viability measurements.08. They were housed under standard conditions and had ‘ad libitum’ access to water and standard laboratory rodent diet.. Eur. San Diego. Animals were handled in accordance with the Principles of Laboratory Animal Care (http://www. After three and 7 days post-injection mice were sacrificed and the liver. In the study of the hemolysis. Nikon). The treatment was administered to three mice in each group. Spain).4. 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. (Temecula.5:1 was needed to bind all DNA. Primary antibody and secondary antibody were provided by Invitrogen (Barcelona.05).9. A lysis buffer was employed for the 100% hemolysis sample. Spain). Spain) and 2% normal goat serum (NGS) for 1 h at D. Results were expressed as the percentage of sections in which EGFP was detected. Statistical analysis was made with SPSS 19.history. 0. IgG fraction) for 2 h at RT. USA). 3. Leica). The naked plasmid. A hemolysis assay and a hemagglutination assay were conducted following protocols previously described by Kurosaki et al.

OligoCh–DNA–SLN vectors prepared with the three OligoCh at ratio 2.D.50) 10:1:5 307.5.50) 10:1:5 325.20) 15:1:5 267. J. In all transfection studies cell viability was similar to the observed in the non-treated cells (over 75% of viable cells).85) (1. When DNA was formulated in SLN without OligoCh (DNA–SLN vector) and treated with SDS. Fig. Lane 5 corresponds to free DNA treated with DNase I. Binding and zeta potential of OligoCh–DNA complexes at different OligoCh to DNA ratios.03 +45.00 +52.25) (1.5:1.43) 10–12 (OligoCh–DNA–SLN vectors treated only with SDS).38) 15:1:5 316. 5 shows a very light agglutination when erythrocytes were in touch with the OligoChC–DNA– SLN vector (Fig.13 (17. The photographs in Fig. 3.82) (0.16) (0.43 (35.1 (20.80 +52. OligoCh–DNA–SLN vectors: DNase protection study and SDSinduced release ‘in vitro’ For this study. Eur.10 (31.. ‘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. = standard deviation) (n = 3).02 (1. 1. 12.01).08.5:1. Mean (S.77) 10:1:5 265. B = OligoChB (11. (2013).70 (19.23) (5. New gene delivery system based on oligochitosan and solid lipid nanoparticles: ‘In vitro’ and ‘in vivo’ evaluation. Pharm.05).5:1. 5:1. when this vector was complexed with SLNs. 2.62 +58.5:1:5 355.4.55) 2. et al.10 (15. No agglutination was detected when the 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 310 311 312 313 314 315 316 317 318 319 3.97 (15.33) (0.ejps.67 (18. the transfection was even higher (p < 0.47) (1. 3. The absence of bands in lanes 2–4 in Fig. meaning that the DNA in the vector was unable to migrate into the gel.7 kDa). and the next lanes correspond to ratios (w/w): 1:1. 2 shows that the DNA was completely bound.5:1:5 were evaluated by using gel electrophoresis.5:1 and 15:1.03 +47. of Pages 8.61 +62. Model 5G Fig.02) (1. 2. 10:1 and 15:1).013 .47 (12. 10:1.2013.79) (1.doi.5:1:5 the percentage of transfected cells at day 7 was higher than at day 3 (p < 0.17) OligoChB:DNA:SLN ratio 1:1:5 366.5:1.5:1:5 359. D. 5D).1016/j.65) (1. the DNA was able to be released and migrate into the gel.81) (3.77 (20. Error bars represent SD = standard deviation (n = 3).32 +45.16) Zeta potential (mV) +45. Size (nm) OligoChA:DNA:SLN ratio 1:1:5 339. Delgado et al. As it can be seen.5 kDa) and C = OligoChC (13. The highest transfection levels were obtained with the OligoChC at ratio 2.74 +57. The gel also shows the results of the DNase protection study. http://dx. Table 2 Size and zeta potential of OligoCh–DNA–SLN vectors.1 kDa).20) 2.72 +46. the DNA was retained in the loading well.60) 15:1:5 311.13 (14.3. 3 features the percentage of transfected cells after the treatment with the vectors OligoCh–DNA or OligoCh–DNA–SLN. 4 shows the transfection capacity of the formulations OligoCh–DNA or OligoCh–DNA–SLN at an OligoChC to DNA ratio of 2. in this lane no band appears because DNA was totally digested by the enzyme. In electrophoresis gels: lane 1 corresponds to free DNA. In lanes 6–8 (OligoCh–DNA–SLN complexes treated with DNase I and later with SDS). / European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx No. A = OligoChA (6. Fig. and in lanes 340 341 342 343 344 Please cite this article in press as: Delgado.60 +43.82) OligoChC:DNA:SLN ratio 1:1:5 353.31) 2. Interaction with erythrocytes Hemagglutination was evaluated by incubating the vectors with erythrocytes.23 (48.5:1 at 3 and 7 days. Sci. 7.PHASCI 2840 29 August 2013 4 D.95 +57. at different OligoCh to DNA ratios (1:1.

The three OligoCh presented a great ability to condense the DNA (Fig. ‘In vitro’ transfection activity and cell viability (small graphics) after treatment with OligoCh–DNA and OligoCh–DNA–SLN vectors assayed in HEK-293 cells. D. J. Error bars represent S.5:1) and OligoChC–DNA–SLN (ratio 2. These sections were obtained from mice sacrificed 3 or 7 days after the intravenous administration. Ãp < 0.5:1 was enough to bind all DNA. In this work. 2. Since the Ch is a polycation. 345 346 347 348 349 erythrocytes were incubated with free DNA or the vector OligoCh– DNA.5:1:5) vectors were intravenously administered to mice for ‘in vivo’ assay. including multicomponent systems composed of mixtures of them. Sci.05 respect to the OligoChC–DNA vector.5:1. lane 3: OligoChB–DNA–SLN.. The tissue sections of the mice treated with free DNA did not show fluorescence due to EGFP. the formulations did not induce hemolysis. (A) OligoCh to DNA ratio 1:1. the OligoCh to DNA ratio (w/w) varied. At day 3 (Fig.6. (C) OligoCh to DNA ratio 10:1. (D) OligoCh to DNA ratio 15:1.05 respect to the rest of vectors. 3. showed green fluorescence. Binding. ÃÃp < 0. so that there are less remaining free cationic charges of primary amino groups 364 365 366 367 368 369 350 351 352 353 354 355 356 357 358 359 360 361 362 363 370 371 372 373 374 375 376 377 378 379 380 381 382 Please cite this article in press as: Delgado. lane 2: OligoChA–DNA–SLN.doi. and no green fluorescence was detected. et al. Model 5G 5 Fig. lane 5: free DNA treated with DNase I. the zeta potential of the complexes increased as the ratio OligoCh to DNA increased. http://dx. As the Mn of Ch lowers.ejps. and green fluorescence remained 7 days after intravenous administration (Fig. 1). 7 shows images of the tissues obtained from the animals treated with the OligoChC–DNA–SLN vector. 12 sections representing the whole organ were analysed.1016/j. lane 7: OligoChB–DNA–SLN with DNase I. #p < 0. for gene delivery. 6A). we have studied the potential utility of three OilgoCh. lane 10: OligoChA–DNA–SLN with SDS.013 . Fig. (n = 3). From each tissue.2013. alone or in combination with SLNs. of Pages 8. which is also dependent on the Mn of the Ch. DNA to SLN ratio (w/w) was 1:5 in all cases. New gene delivery system based on oligochitosan and solid lipid nanoparticles: ‘In vitro’ and ‘in vivo’ evaluation. hepatic. lane 6: OligoChA–DNA–SLN with DNase I. lane 11: OligoChB–DNA–SLN with SDS. Fig. we subjected samples of mice treated with formulations without plasmid to the same procedure with the primary and the secondary antibodies. 6B).org/10. lane 9: DNA–SLN with SDS. Delgado et al. 5 also features the hemolytic activity of erythrocytes after treatment with vectors prepared with OligoChC.5:1. (2013). OligoChC–DNA (ratio 2. The DNA to SLN ratio (w/w) was 1:5 and the OligoCh to DNA ratio (w/w) was 2. In order to ensure that the observed green fluorescence was not an artefact of the immunolabelling. Pharm. and a ratio OligoCh to DNA of 2. DNase protection and DNA release from complexes. Fig. 3.D. spleen and lung sections of animals treated with either OligoCh–DNA or OligoCh– DNA–SLN vectors. more uniform complexes with DNA are formed. ‘In vivo’ protein expression after intravenous administration to mice of OligoCh–DNA and OligoCh–DNA–SLN vectors Free DNA. however. lane 4: OligoChC–DNA–SLN. Discussion The need of more efficient non-viral vectors for gene therapy has conducted to the design of a wide variety of materials and systems. (B) OligoCh to DNA ratio 2.08. almost all sections of mice treated with both vectors showed EGFP. As can be seen. lane 8: OligoChC–DNA–SLN with DNase I. lane 12: OligoChC–DNA–SLN with SDS.PHASCI 2840 29 August 2013 D. Lane 1: free DNA. Eur. 4.01 respect to the rest of OligoCh–DNA–SLN vectors. / European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx No.

there are free negative charges in the OligoCh–DNA complex to interact with the positive charges of the SLN. 383 384 385 386 387 388 389 on the surface of the complexes. The same vector prepared without SLNs induced lower transfection. treated with DNA (B).org/10. All the OligoCh–DNA–SLN complexes presented a similar particle size. Photographs: Agglutination of erythrocytes when blood is untreated (A). Lysis buffer represents 100% hemolysis sample. ‘In vitro’ transfection over time (3 and 7 days after addition of vectors) in HEK-293 cells with the vectors OligoChC–DNA (2. DNA was able to be released from the SLNs in absence of Ch (Fig. that means that SLNs increased the transfection capacity of OligoChC. the DNA did not migrate in the gel due to the strong interactions with the OligoCh. favour the invagination of the cell plasma membrane and the uptake of the vectors (Elouahabi and Ruysschaert. Although the OligoCh–DNA complexes have positive net charge due to the excess of OligoCh. Delgado et al.5:1:5).2013. / European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx No. Eur. 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.. 2005). (2013). the higher positive superficial charge was. However.08. with OligoChC–DNA vector (C). http://dx. This was not observed for the other two OligoCh evaluated. 2. 5. regardless of the OligoCh used (Table 2). Therefore. we analysed the integrity of the DNA after the treatment of the vectors with DNase I (Fig. J. In order to study the protection capacity of the complexes containing OligoCh.doi. irrespective of the OligoCh. 11 and 12).ejps.5:1 (Fig. The three OligoCh vary in the 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 Fig. 2. ÃÃp < 0. Positive charges are known to facilitate the interaction with the negative charged cell surface and. ÃÃp < 0. although the DNA bands in the loading well indicate that the plasmid was not degraded by the DNase I. since no significant differences were detected in those features (Table 2). 2007). (n = 9). leading to overall lower zeta potential.013 .. Fig.01 respect to the rest of samples.D. 2). et al. therefore. Error bars represent S. Please cite this article in press as: Delgado. and with OligoChC–DNA–SLN vector (D).5:1. Model 5G Fig. the greater the OligoCh to DNA ratio in the vectors was. However. Graphic: Hemolytic activity of erythrocytes after treatment with OligoChC–DNA and OligoChC–DNA–SLN vector.D. Error bars represent S. New gene delivery system based on oligochitosan and solid lipid nanoparticles: ‘In vitro’ and ‘in vivo’ evaluation.PHASCI 2840 29 August 2013 6 D.5:1) and OligoChC–DNA–SLN (2.1016/j. When vectors were first treated with DNase I and then with SDS (lanes 6–8). Pharm. D. Sci. ‘‘In vitro’’ transfection studies in HEK-293 cells showed that the highest transfection level was obtained with the vector OligoChC– DNA–SLNs at a OligoChC to DNA ratio of 2. 4.01 respect to the levels of transfection achieved with the same formulations 3 days after addition of vectors. 2008). lane 9). 3). (n = 3). The different transfection level of the three OligoCh when formulated with SLNs cannot be attributed to the vector size or surface charge. We have previously demonstrated the importance of a balance between protection and release of the DNA to achieve transfection with SLNs (del Pozo-Rodríguez et al. of Pages 8. OligoChA and OligoChB. 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. not all negative charges of the DNA are neutralized.. The SLN to DNA ratio (w/w) was 5:1 and the OligoChC to DNA ratio (w/w) 2. lanes 10.

2010).. in transfection. After the intravenous administration to mice of the OligoChC– DNA and OligoChC–DNA–SLN vectors. Ch are FDA GRAS excipients. after intravenous administration of DNA–SLN vector. http://dx. and the opening of tight junctions of cells (Holme et al. 2000). 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. transfection in lung was not detected. higher than transfection achieved with the vector OligoChC–DNA–SLN (16% at day 7).ejps. Sci. although the safe use of Ch as a parenteral excipient is not yet clear (Baldrick.) Please cite this article in press as: Delgado. This lack of ‘‘in vitro’’-‘‘in vivo’’ correlation justifies the convenience of evaluating this kind of vectors for gene therapy in animal models during early stages of the development process.2013. Error bars represent S.08. This happened for both the OligoChC–DNA vector and the OligoChC–DNA–SLN vector. which could lead to a different surface disposition of the OligoCh and the plasmid in the vector. In a previous work (5). deacetylation degree over 50% has been related to the increase in the cell permeability. In someway. 3). J. The transfection level of that vector in HEK293 cell at the same conditions than in the current study was 40%. where the uptake of the vectors is more difficult. 5). Images were captured at 60x. (n = 3). In addition. However.D. such as the lung. this light agglutination is not expected to be clinically relevant. in comparison to transfection at day 3 (Fig.5:1.1016/j. spleen and lungs at day 3.5:1 was studied over time. 6. The transfection level obtained with the vector composed by the OligoChC increased from day 3 to day 7. It has to be taken into account the possibility to functionalize the vectors for specific clinical Fig. Cell nucleuses were labelled with DAPI (blue). Model 5G 7 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 Fig. Concerning safety. after the immunolabelling of EGFP (green). the reader is referred to the web version of this article. / European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx 2012). 2010). Moreover. Eur. release and protection capacity of the DNA. the OligoChC improved the transfection efficacy of the DNA–SLN vector. the new vector may sequester the DNA and thus. OligoChC–DNA and OligoChC–DNA–SLN vectors did not decrease cell viability (Fig.. however. 6 and 7). providing a sustained release of the DNA and inducing a long-lasting transfection. Therefore. spleen and lung of mice treated with OligoChC–DNA and OligoChC–DNA–SLN vectors.PHASCI 2840 29 August 2013 D. and therefore. Therefore.. Percentage of transfected sections of liver.doi. of Pages 8. Additional studies are needed to assess the real potential of these new vectors. (For interpretation of the references to colour in this figure legend. spleen (B) and lung (C) of mice treated with OligoChC–DNA–SLN vectors. The high DNA condensation provided by the OligoChC would maintain the DNA protected. In spite of the difference in the ‘‘in vitro’’ transfection levels between the vector OligoChC–DNA and OligoChC–DNA–SLN. D. and without Ch. in fact. New gene delivery system based on oligochitosan and solid lipid nanoparticles: ‘In vitro’ and ‘in vivo’ evaluation. which was maintained for at least 7 days (Figs. may justify prolonged residence time of the intact plasmid in the tissues. no difference was detected in the transfection after the intravenous administration to the mice. Images of transfected sections of liver (A). 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. In our study. B = 7 days after administration.013 . 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. 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. Although the exact disposition of the compounds in a multicomponent non-viral system it is difficult to predict. and when vectors were put in contact ‘in vitro’ with erythrocytes only a light agglutination was observed with the OligoChC–DNA–SLN vector (photograph D in Fig. EGFP expression was detected in liver. which may induce a longer stay of the plasmid in the organism. A = 3 days after administration. Transfection of the vector prepared with the OligoChC at a OligoCh to DNA ratio of 2. and have extensively demonstrated intestinal and topical tolerance (24). OligoChC has a deacetylation degree of 85%. and EGFP protein expression in liver and spleen significantly decreased 7 days after administration (del Pozo-Rodríguez et al. and we observed a 2-fold increase in transfection at day 7. Pharm. The SLN to DNA ratio (w/w) was 5:1 and the OligoChC to DNA ratio 2. it is not able to release it completely even after 7 days. it could be responsible for differences in condensation. In a previous work. OligoChC may increase the access of the vector to tissues. we showed the capacity of transfection of a vector composed by SLN and DNA. (2013).. 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 Mn. This is especially important for tissues other than those of the reticuloendothelial system (RES). In our opinion. 4). the vector prepared without OligoCh produced the same transfection level over time. No difference in the transfection capacity of the two formulations studied was detected. 2012a). et al. Delgado et al. 7.

R. Sharma.T. Chondroitin sulfate capsule system for efficient and secure gene delivery..M. J. H..J. Dornish..K. 410. A. Verhoef.. Junginger. M. a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo.. Fernández. et al. M.. S. Solinís. C. Sharma.R. 57. Chang.C..R... 23. Drug Deliv..ejps..A. S.. W. N. Pharm. M. Acknowledgement This work was supported by the Basque Government’s Department of Education. Lavertu. K. Alonso. Gene Ther. On the one hand. Drug Deliv. Winnik. J.. 503–512. Biopharm. Gascón. Riguera. S. 2008. A. 369270. J.L. Ind. H. 496–504. 8328–8341.PHASCI 2840 29 August 2013 8 497 498 499 500 501 502 503 504 505 506 507 508 509 D. Sci...L. Understanding the mechanism of protamine in solid lipid nanoparticle-based lipofection: the importance of the entry pathway... Delgado.C. Kawakami... 2009. Zhang. (Ed. 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Khew. 2011). 4639–4646. 18. Model 5G application. 2009. Y. 975–987. 162–171. S91–S101.J.A. A. Ramesan. Hu.. K. 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 Please cite this article in press as: Delgado.L. Hum. Drug Deliv. Wang.. 2001. R.. Cationized albumin conjugated solid lipid nanoparticles as vectors for brain delivery of an anti-cancer drug.... Pharm. Fernandes.. Juvonen. Duceppe. J.. M. both the solid lipid nanoparticles and the OligoCh offer several possibilities. Delgado.. Rodríguez. Liu. Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes.. Merzouki.. Pujals. V... 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