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1

3 New gene delivery system based on oligochitosan and solid lipid

4 nanoparticles: In vitro and in vivo evaluation

5

6

7 Diego Delgado aQ1 , Ana del Pozo-Rodrguez a, M. Angeles Solins a, Artur Bartkowiak b, Alicia R. Gascn a,

8 a Pharmacokinetics, Nanotechnology and Gene Therapy Group, Pharmacy and Pharmaceutical Technology Laboratory, Pharmacy Faculty, University of the Basque Country9 UPV/EHU, 01006 Vitoria-Gasteiz, Spain0 b Center of Bioimmobilisation and Innovative Packaging Materials, West Pomeranian University of Technology, Szczecin, Poland

12

4

a r t i c l e i n f o

5 Article history:6 Received 18 January 20137 Received in revised form 12 June 20138 Accepted 13 August 20139 Available online xxxx

0 Keywords:1 Solid lipid nanoparticles2 Oligochitosan3 Gene therapy4 In vivo transfection5 Non-viral vector6

7

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 deacetyla-

tion degree of 85%. OligoCh were complexed directly with the pCMS-EGFP plasmid to form

OligoChDNA carriers. Taking into account the features and benefits of both Ch and SLNs, we also com-

bined 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 differ-

ence was detected in transfection after in vivo administration. Moreover, the OligoChC improved the

in vivo transfection efficacy of the DNASLN vector. This work shows the potential utility of the com-

bination of SLNs and OligoCh for the development of new non-viral vectors for gene therapy.

2013 Published by Elsevier B.V.

4

5 1. Introduction

6 The development of new drug delivery systems usually involves

7 the combination of components of different nature, with the aimof

8 taking advantage of beneficial properties of each one. In the field of

9 gene therapy, non-viral systems with different composition are un-

0 der study; e.g. hyaluronic acid and chitosan (de la Fuente et al.,

1 2010), peptides and solid lipid nanoparticles (del Pozo-Rodrguez

2 et al., 2009a; Delgado et al., 2011) or lysine-based peptides and

3 PLGA (Nie et al., 2009), among others.

4 SLN-based vectors developed by our group have shown capacity

5 for transfection in vitro in several cell lines, and in vivo as well6 after intravenous and ocular administration (del Pozo-Rodrguez

7 et al., 2010; Delgado et al., 2012a,b). Their ability to condense

8 and protect DNA (del Pozo-Rodrguez et al., 2007), and their entry

9 efficiency into several cell lines Delgado et al., 2012a), along

0 with the possibility to be decorated with other compounds (del

1 Pozo-Rodrguez et al., 2009a; Delgado et al., 2011, 2012b), make

2 this nanoparticular system an interesting alternative to viral

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 biomed-

ical 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 Chare 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 nanocarri-

ers (Thibault et al., 2011). Although highly deacetylated Ch have

0928-0987/$ - see front matter 2013 Published by Elsevier B.V.http://dx.doi.org/10.1016/j.ejps.2013.08.013

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:alicia.rodriguez@ehu.es(A.R. Gascn).

European Journal of Pharmaceutical Sciences xxx (2013) xxxxxx

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e j p s

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shown better transfection in in vitro studies, the effect of Ch

deacetylation degree is still uncQ2 lear (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 efficacy for gene delivery depends on the

capacity of the Ch to complex genetic material and to cross biolog-

ical 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 fluids

due to their intrinsic pH (Germershaus et al., 2008), and this may

reduce the colloidal stability of the Ch nanoparticles. Several strat-

egies 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 electro-

static 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. Tak-

ing into account the features and benefits 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.

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 olig-

ochtiosans. Chitosans with varying molar masses were prepared

by controlledradical 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 deg-

radation and purification had various molar masses and similar

polydispersities of MM in range 1.52.2. The detailed procedure of

degradation and purification of Ch is described elsewhere

(Bartkowiak and Hunkeler, 2000). The molar mass of final oligoch-

itosan samples was determined by GPC method using Knauer

SmartLine HPLC system (Knauer, Germany) equipped with refracto- metric detection at a flow-rate of 1 cm3/min. Two SEPARON HEMA

BIO columns 1000 and 40 (TESSEK Ltd., Praha, CzechRepublic) 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 calibra-

1tion and as a relative reference for MM calculation of low molar

1mass OligoCh. Table 1 represents the Gel Permeation Chromatogra-

1phy (GPC) results for the three synthesised OligoCh samples.

12.2. Production of solid lipid nanoparticles (SLNs)

1The SLNs, composed by the solid lipid Precirol ATO 5

1(Gattefoss; Madrid, Spain) and the surfactants 1,2-Dioleoyl-3-

1Trimethylammonium-Propane Chloride Salt (DOTAP) from Avanti

1Polar Lipids (0.4% w/v) and Tween 80 (0.1% w/v), were produced

1by a solvent emulsificationevaporation technique previously de-

1scribed (del Pozo-Rodrguez et al., 2007).

12.3. Preparation of vectors

1OligoChDNA vectors were obtained by mixing the pCMS-EGFP

1plasmid, which encodes the enhanced green fluorescent protein

1(EGFP), with an aqueous solution of OligoCh. Different OligoCh to

1DNA ratios (w/w) were ap Q3plied 1:1, 2.5:1, 5:1, 7.5:1, 10:1, 12.5:1

1and 15:1.

1OligoChDNASLN vectors were prepared by first forming

1OligoChDNA complexes at different ratios, and then, incorporat-

1ing the SLN under agitation for 30 min. The SLN to DNA ratio, ex-

1pressed as the ratio of DOTAP to DNA (w/w), was fixed at 5:1.

1These vectors have OligoChDNA complexes adsorbed on the sur-

1face of nanoparticles.

12.4. Study of DNA binding to OligoCh

1The resulting OligoChDNA complexes were subjected to elec-

1trophoresis on a 1% agarose gel (containing ethidium bromide for

1visualisation) for 30 min at 120 V. The gel electrophoresis materials

1were acquired from Bio-Rad (Madrid, Spain). The bands were

1observed with an Uvitec Uvidoc D-55-LCD-20M Auto

1transilluminator.

12.5. Size and potential measurements

1Sizes of OligoChDNA and OligoChDNASLN vectors were

1determined by photon correlation spectroscopy (PCS). Zeta poten-

1tials of OligoChDNA and OligoChDNASLN were measured by la-

1ser doppler velocimetry (LDV). Both measurements were

1performed on a Malvern Zetasizer 3000 (Malvern Instruments,

1Worcesteshire, UK). All samples were diluted in 0.1 mM NaCl

1solution.

12.6. DNase protection study and SDS-induced release in vitro

1Deoxyribonuclease I (DNase I) from SigmaAldrich (Madrid,

1Spain) was added to OligoChDNASLN complexes to a final con-

1centration of 1U DNase I/2.5 lg DNA, and the mixtures were incu-1bated at 37 C for 30 min. Thereafter, a lauryl sulphate sodium

1(SDS) solution was added to the samples to a final concentration

1of 1% to release DNA from the vectors. Samples were then analysed

1by electrophoresis on agarose gel (described above) and the integ-

1rity of DNA in each sample was compared with untreated DNA as

1control.

12.7. Cell culture and transfection protocol in vitro

1In vitro assays were performed with Human Embrionic Kidney

1(HEK-293) cells, obtained from the American Type Culture Collec-

1tion (ATCC). Cell culture reagents were purchased from LGC Pro-

1mochem (Barcelona, Spain).

1HEK-293 cells were maintained in Eagles Minimal Essential1medium with Earles BSS and 2 mM L-glutamine (EMEM)

Table 1

The GPC results for the three OligoCh samples.

Sample Mna (g/mol) Mw

b (g/mol) Polydispersity

OligoChA 6100 40,000 6.6

OligoChB 11,500 100,000 8.7

OligoChC 13,700 125,000 9.1

a

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

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4 supplemented with 10% heat-inactivated horse serum and Normo-

5 cin (InvivoGen; San Diego, California, US). Cells were incubated at

6 37 C with 5% CO2 in air and subcultured every 23 days using

7 trypsin/EDTA. For transfection HEK-293 cells were seeded on 24-

8 well plates at density of 150,000 per well one day before the trans-

9 fection procedure.

0 The plasmid (2.5lg) formulated in the vectors was added in1 75

lL of HBS, and cells were incubated with the vectors for 4 h at

2 37 C. Then the medium containing the complexes in the wells

3 was diluted with 1 mL of complete medium and cells were allowed

4 to grow for 72 h. Moreover, this assay was repeated by growing

5 cells for 1 week.

6 2.8. Flow cytometry mediated analysis of transfection efficacy and cell7 viability

8 At the end of the point times, cells were washed once with PBS

9 and detached with trypsin/EDTA. Cells were resuspended in PBS

0 and introduced to a FACSCalibur flow cytometer (BD Biosciences,

1 San Jose, USA). For each sample 10,000 events were collected.

2 For transfection efficacy the fluorescence of EGFP positive cells

3 was collected at 525 nm (FL1). For cell viability measurements,4 5 lL of BD Via-Probe reagent (BD Biosciences; Belgium) was added5 to each sample, and after 10 min of incubation fluorescence corre-

6 spondent to dead cells was measured at 650 nm (FL3).

7 2.9. Interaction with erythrocytes

8 Hemolysis assay. A hemolysis assay and a hemagglutination as-9 say were conducted following protocols previously described by

0 Kurosaki et al. (2010). In both cases fresh human blood of 0+ type

1 was centrifuged at 4000rpm for 5 min and the plasma and the buf-

2 fy coat were discarded. Erythrocytes were washed three times with

3 PBS by centrifugation at 4000 rpm and were diluted in PBS to a fi-

4 nal concentration of 5% (v/v) for the hemolysis assay, and to a 2%

5 (v/v) concentration for the hemagglutination assay. The naked6 plasmid, OligoChCDNA and OligoChCDNASLN vectors were

7 added to erythrocytes suspension at ratio 1:1 (v/v) and incubated

8 for 60 min (hemolysis assay) or 15 min (hemagglutination assay)

9 at room temperature (RT).

0 In the study of the hemolysis, suspensions were centrifuged at

1 4000 rpm during 5 min and supernatants were taken to quantify

2 the hemolysis by measuring haemoglobin release at 545 nm in a

3 microplate reader. A lysis buffer was employed for the 100% hemo-

4 lysis sample.

5 In the study of the hemagglutination samples were placed on a

6 microscope slide and observed by microscopy.

7 2.10. Intravenous administration of vectors

8 Female Balb/c nude mice weighing 1822 g (5 weeks of age)

9 were purchased from Harlam Interfauna Ibrica S.L. (Barcelona,

0 Spain). Animals were handled in accordance with the Principles

1 of Laboratory Animal Care (http://www.history.nih.gov/laws).

2 Mice were quarantined for at least 1 week prior to the study. They

3 were housed under standard conditions and had ad libitum access

4 to water and standard laboratory rodent diet. a unique dose of

5 60lg of plasmid in 100 lL of the vectors were injected in standard6 way into the tail vein. As controls free DNA and vectors without the

7 plasmid were administered in the same way and volume. The

8 treatment was administered to three mice in each group. After

9 three and 7 days post-injection mice were sacrificed and the liver,

0 lungs and spleen were removed, quick frozen in liquid nitrogen

1 embedded in tissue freezing medium (Jung, Leica) and thin sec-2 tioned on a cryostat (Cryocut 3000, Leica).

2.11. Immunolabelling of EGFP in tissue sections

Cryostat sections (8 lm) were fixed with 4% paraformaldehydeduring 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 ade-

quate washing in PBS, sections were incubated in secondary anti-

body (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 covers-

lipped with Dapi-Fluoromount-G (SouthernBiotech; Coultek,

Espaa). Images of the immunolabelled sections were captured

with an inverted microscopy equipped with an attachment for

fluorescent observation (model EclipseTE2000-S, Nikon). From

each tissue, 12 sections representing the whole organ were ana-

lysed. 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). Sta-

tistical 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 significant 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 significant differences among the three

OligoCh and the different proportions were detected. Polydisper-

sity 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, thesmaller the zeta potential of the complexes is. When OligoChA

was used, the zeta potential was positive for the OligoChDNA ra-

tios from 15:1 to 5:1, but ratios 2.5:1 and 1:1 presented negative

values. With OligoChB and OligoChC, positive superficial 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 2shows the particle size and zeta potential of OligoCh

DNASLN vectors. All vectors were similar in size, around

300 nm and there were not significant differences. The polydisper-

sity index was always lower than 0.4. The higher the OligoCh to

DNA ratio was, the higher the superficial charge of the final vectorswas, being differences statistically significant (p< 0.05).

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3.3. OligoChDNASLN vectors: DNase protection study and SDS-

induced release in vitro

For this study, OligoChDNASLN vectors prepared with the

three OligoCh at ratio 2.5:1:5 were evaluated by using gel electro-

phoresis. 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 to-

tally digested by the enzyme. In lanes 68 (OligoChDNASLN

complexes treated with DNase I and later with SDS), and in lanes

31012 (OligoChDNASLN vectors treated only with SDS), the3DNA was retained in the loading well, meaning that the DNA in

3the vector was unable to migrate into the gel. When DNA was for-

3mulated in SLN without OligoCh (DNASLN vector) and treated

3with SDS, the DNA was able to be released and migrate into the gel.

33.4. In vitro transfection and cell viability

3Transfection capacity and cell viability of vectors were assayed

3in vitro in HEK-293 culture cells 3 days post-transfection. Fig. 3

3features the percentage of transfected cells after the treatment

3with the vectors OligoChDNA or OligoChDNASLN, at different

3OligoCh to DNA ratios (1:1, 2.5:1, 10:1 and 15:1). The highest

3transfection levels were obtained with the OligoChC at ratio

32.5:1; when this vector was complexed with SLNs, the transfection3was even higher (p< 0.05).3Fig. 4shows the transfection capacity of the formulations Olig-

3oChDNA or OligoChDNASLN at an OligoChC to DNA ratio of

32.5:1 at 3 and 7 days. As it can be seen, the percentage of transfec-

3ted cells at day 7 was higher than at day 3 (p< 0.01).3In all transfection studies cell viability was similar to the ob-

3served in the non-treated cells (over 75% of viable cells).

33.5. Interaction with erythrocytes

3Hemagglutination was evaluated by incubating the vectors with

3erythrocytes. The photographs inFig. 5shows a very light aggluti-

3nation when erythrocytes were in touch with the OligoChCDNA3SLN vector (Fig. 5D). No agglutination was detected when the

Fig. 1. Binding andzeta potentialof OligoChDNA complexesat differentOligoCh to DNA ratios. A = OligoChA (6.1 kDa), B = OligoChB (11.5kDa) andC = 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 2Size and zeta potential of OligoChDNASLN vectors. Mean (S.D. = standard deviation)

(n= 3).

Size (nm) Zeta potential (mV)

OligoChA:DNA:SLN ratio

1:1:5 339.77 (20.55) +45.60 (1.85)

2.5:1:5 355.70 (19.77) +43.00 (1.79)

10:1:5 265.43 (35.60) +52.61 (1.81)

15:1:5 311.1 (20.17) +62.03 (3.16)

OligoChB:DNA:SLN ratio

1:1:5 366.10 (31.20) +45.72 (0.47)

2.5:1:5 359.10 (15.50) +46.80 (1.25)

10:1:5 325.13 (17.38) +52.74 (1.23)

15:1:5 316.67 (18.82) +57.32 (5.02)

OligoChC:DNA:SLN ratio

1:1:5 353.47 (12.31) +45.03 (1.82)

2.5:1:5 350.97 (15.50) +47.95 (0.65)

10:1:5 307.23 (48.20) +57.62 (1.33)

15:1:5 267.13 (14.16) +58.02 (0.43)

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5 erythrocytes were incubated with free DNA or the vector OligoCh

6 DNA.

7 Fig. 5also features the hemolytic activity of erythrocytes after

8 treatment with vectors prepared with OligoChC. As can be seen,

9 the formulations did not induce hemolysis.

0 3.6. In vivo protein expression after intravenous administration to1 mice of OligoChDNA and OligoChDNASLN vectors

2 Free DNA, OligoChCDNA (ratio 2.5:1) and OligoChCDNASLN

3 (ratio 2.5:1:5) vectors were intravenously administered to mice for

4 in vivo assay. In order to ensure that the observed green fluores-

5 cence was not an artefact of the immunolabelling, we subjected

6 samples of mice treated with formulations without plasmid to

7 the same procedure with the primary and the secondary antibod-

8 ies, and no green fluorescence was detected.

9 The tissue sections of the mice treated with free DNA did not

0 show fluorescence due to EGFP; however, hepatic, spleen and lung

1 sections of animals treated with either OligoChDNA or OligoCh

2 DNASLN vectors, showed green fluorescence. These sections were3 obtained from mice sacrificed 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 fluores-

cence remained 7 days after intravenous administration (Fig. 6B).

Fig. 7shows images of the tissues obtained from the animals trea-

ted with the OligoChCDNASLN vector.

4. Discussion

The need of more efficient non-viral vectors for gene therapy

has conducted to the design of a wide variety of materials and sys-

tems, 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 Mnof the Ch. As the Mn of Ch low-

ers, more uniform complexes with DNA are formed, so that thereare less remaining free cationic charges of primary amino groups

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 withDNase 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< 0.01respect to the rest of OligoChDNASLNvectors. p < 0.05respect tothe rest of vectors.#p< 0.05 respect

to the OligoChCDNA vector.

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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

3charges) (Bonincontro et al., 2008). Therefore, there are free nega-

3tive charges in the OligoChDNA complex to interact with the po-

3sitive charges of the SLN.

3All the OligoChDNASLN complexes presented a similar parti-

3cle size, irrespective of the OligoCh. However, the greater the Olig-

3oCh to DNA ratio in the vectors was, the higher positive superficial

3charge was, regardless of the OligoCh used (Table 2). Positive

3charges are known to facilitate the interaction with the negative

3charged cell surface and, therefore, favour the invagination of the

3cell plasma membrane and the uptake of the vectors (Elouahabi

4and Ruysschaert, 2005).

4We have previously demonstrated the importance of a balance

4between protection and release of the DNA to achieve transfection

4with SLNs (del Pozo-Rodrguez et al., 2007). Fig. 2 shows that when

4formulated with SLNs the OligCh keep the high capacity to bind the

4DNA since the plasmid was not released even in presence of SDS

4(Fig. 2, lanes 10, 11 and 12). However, DNA was able to be released

4fromthe SLNs in absence of Ch(Fig. 2, lane 9). In order to study the

4protection capacity of the complexes containing OligoCh, we ana-

4lysed the integrity of the DNA after the treatment of the vectors

4with DNase I (Fig. 2). When vectors were first treated with DNase

4I and then with SDS (lanes 68), the DNA did not migrate in the gel

4due to the strong interactions with the OligoCh, although the DNA

4bands in the loading well indicate that the plasmid was not de-

4graded by the DNase I.

4In vitro transfection studies in HEK-293 cells showed that the

4highest transfection level was obtained with the vector OligoChC

4DNASLNs at a OligoChC to DNA ratio of 2.5:1 (Fig. 3). The same

4vector prepared without SLNs induced lower transfection; that

4means that SLNs increased the transfection capacity of OligoChC.

4This was not observed for the other two OligoCh evaluated, Oligo-

4ChA and OligoChB. The different transfection level of the three

4OligoCh when formulated with SLNs cannot be attributed to the

4vector size or surface charge, since no significant differences were

4detected in those features (Table 2). The three OligoCh vary in the

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

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5 Mn, which could lead to a different surface disposition of the Olig-6 oCh and the plasmid in the vector. Although the exact disposition

7 of the compounds in a multicomponent non-viral system it is dif-

8 ficult to predict, it could be responsible for differences in conden-

9 sation, release and protection capacity of the DNA, and therefore,0 in transfection.

1 Transfection of the vector prepared with the OligoChC at a Olig-

2 oCh to DNA ratio of 2.5:1 was studied over time, and we observed a

3 2-fold increase in transfection at day 7, in comparison to transfec-

4 tion at day 3 (Fig. 4). This happened for both the OligoChCDNA

5 vector and the OligoChCDNASLN vector.

6 In a previous work (5), we showed the capacity of transfection

7 of a vector composed by SLN and DNA, and without Ch. The trans-

8 fection level of that vector in HEK293 cell at the same conditions

9 than in the current study was 40%, higher than transfection

0 achieved with the vector OligoChCDNASLN (16% at day 7). The

1 transfection level obtained with the vector composed by the Olig-

2 oChC increased from day 3 to day 7; however, the vector prepared

3 without OligoCh produced the same transfection level over time. In4 our opinion, the high condensation degree of DNA when it is

formulated with the OligoChC would led to form a more stable sys-

tem 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 transfec-

tion levels that increase over time. In someway, the new vector

may sequester the DNA and thus, it is not able to release it com-

pletely even after 7 days. Concerning safety, Ch are FDA GRAS

excipients, and have extensively demonstrated intestinal and top-

ical tolerance (24), although the safe use of Ch as a parenteral

excipient is not yet clear (Baldrick, 2010). In our study, Olig-

oChCDNA 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 inFig. 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 de-

tected 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 previ-

ous work, after intravenous administration of DNASLN vector,

transfection in lung was not detected, and EGFP protein expression

in liver and spleen significantly decreased 7 days after administra-

tion (del Pozo-Rodrguez et al., 2010). Therefore, the OligoChC im-

proved the transfection efficacy 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 in-

crease 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 difficult. There-

fore, OligoChC may increase the access of the vector to tissues. Inaddition, 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 pro-

longed residence time of the intact plasmid in the tissues, provid-

ing a sustained release of the DNA and inducing a long-lasting

transfection.

In spite of the difference in the in vitro transfection levels be-

tween the vector OligoChCDNA and OligoChCDNASLN, no dif-

ference was detected in the transfection after the intravenous

administration to the mice. This lack of in vitro-in vivo corre-

lation justifies the convenience of evaluating this kind of vectors

for gene therapy in animal models during early stages of the devel-

opment process. Additional studies are needed to assess the real

potential of these new vectors. It has to be taken into accountthe possibility to functionalize the vectors for specific clinical

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.

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 figure legend, the reader is referred to the webversion of this article.)

D. Delgado et al. / European Journal of Pharmaceutical Sciences xxx (2013) xxxxxx 7

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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 cova-

lently coupled to lipids in SLNs to target to malignant cells (Mulik

et al., 2010), or albumin conjugated SLNs have been used as a strat-

egy to bypass the brain blood barrier (Agarwal et al., 2011). On the

other hand, lactosylated or galoctosylated chitosan for targeting li-

ver cells is an approach to improving the transfeQ4 ction efficiency

(Alex et al., 2011), and conjugation with folate (Ramesan, 2012),

polyethylenimine (Wong et al., 2006), and polyethylenimine-b-

cyclodextrin (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 intrave-

nous 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 be-

tween both vectors, no difference was detected in transfection

after in vivo administration. Moreover, the OligoChC improved

the in vivo transfection efficacy 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-341-

10).

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