Topical Application of Antisense Oligonucleotide-Loaded Chitosan Nanoparticles to Rats

Topical Application of Antisense Oligonucleotide-LoadedChitosan Nanoparticles to Rats

Suna Ozbas-Turan, Julide Akbuga, and Ali Demir Sezer

Skin delivery of antisense oligonucleotides (AsODNs) has exciting potential in the treatment of skin diseases.However, the therapeutic applications of oligonucleotide-based therapies are limited by the instability of thesemolecules toward nucleases, short half-life in vivo, and insufficient cellular uptake. The purpose of this study wasto investigate in vivo antisense effect of AsODN-loaded chitosan nanoparticles after topical application. AsODN-loaded chitosan nanoparticles were topically applied to Sprague Dawley rats (adult and baby). At 1, 3, 6, 9, and 12days posttransfection, animals’ skin samples were taken for measurement of b-galactosidase (b-Gal) expressionand histological control. After topical application of AsODN-loaded chitosan nanoparticles in different doses, b-Gal expression reduced significantly. Highest inhibition was observed after 6 days of transfection of nanoparticles.Free AsODNs exhibited 35% of b-Gal inhibition on the first day. b-Gal expression was inhibited in approximately82–85% with transfection of nanoparticles containing 30 mg AsODNs at 6 days. The antisense effect of AsODN-loaded chitosan nanoparticle in baby skin was evaluated at 6 days: 77–86% of b-Gal suppression was measuredand differences between the doses were not significant. Thus, chitosan nanoparticles are useful carrier for deliveryof AsODNs into skin cells of rats and may be used for topical application on human skin.

Introduction

Antisense oligonucleotides (AsODNs), designed tosuppress a particular gene expression, have considerable

potential as a therapeutic agent. The application of AsODNs asdrug presents serious challenges such as instability, poor cellpenetration, nonspecific binding, pharmacokinetics, and tox-icity. Also, intravenous injection of oligonucleotides presentsparticular challenges (Wraight and White, 2001). Skin deliveryof AsODNs provides an alternative to injection and has excit-ing potential in the treatment of skin diseases including skincarcinomas, melanomas, psoriasis, inflammation, and herpessimplex (Wraight and White, 2001; Tezel et al., 2004). Skin is anattractive target for gene therapy because of its accessibility andimmunological functions for vaccine (Hengge et al., 1996). Inaddition to treating skin diseases, cutaneous AsODN deliverycan be used for systemic effects. Despite all of the advantages,dermal delivery of AsODNs is still severely limited by appro-priate nucleic acid carrier for the skin barriers. Several methodshave been developed to deliver AsODNs to the skin in vitro andin vivo (Regnier and Preat, 1998; Mehta et al., 2000; White et al.,2000, 2002; Dokka et al., 2005).

On the other hand, Dinauer et al. (2004) reported that theentrapment of AsODNs within polymeric matrix of nano-particles could improve AsODN stability, reduce the dose ofAsODNs required for efficacy, and reduce toxicity or non-

specific binding of the molecules. Polylactic acid (PLA), poly-lactic glycolic acid (PLGA) (Lewis et al., 1998; Hussain et al.,2002), protamine–albumin (Mayer et al., 2005), and chitosan(Springate et al., 2005; Enneli et al., 2006) nanoparticles wereused for AsODN delivery.

Chitosan is a biocompatible, nontoxic, and biodegradablepolysaccharide. The amine group of chitosan is protonated atlower pH, producing a polycationic polymer that has beencondensed to deliver genes in vitro and in vivo (Gao et al.,2005). Chitosan and its derivatives have been complexed withAsODNs in vitro and in vivo (Gao et al., 2005; Enneli et al.,2006; Springate et al., 2008). However, limited reports areavailable regarding the application of chitosan for AsODNdelivery and there is not any study about topical applicationof AsODN-loaded chitosan nanoparticles.

At this stage, Smad3 AsODN-impregnated chitosan–alginate polyelectrolyte complex was prepared and applied foraccelerated wound healing (Hong et al., 2008).

In vivo characterization of a galactosylated chitosan fordelivering AsODNs into Kupffer cells was reported by Donget al. (2009), who demonstrated inhibitor effect of AsODN=galactosylated chitosan system against the tumor necrosisfactor-a expression.

Dung et al. (2007) suggested that sustained release ofAsODN-loaded chitosan nanoparticles may be suitable for thelocal therapeutic application of periodontal diseases.

Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Marmara University, Istanbul, Turkey.

OLIGONUCLEOTIDESVolume 20, Number 3, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089=oli.2009.0222

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The aim of this study was to investigate skin deliveryability of chitosan-tripolyphosphate (TPP) nanoparticles forAsODNs in adult and baby rats.

Materials and Methods

Materials

Fifteen-nucleotide phosphorothioate oligonucleotides(MWG-Biotech, Ebersberg, Germany) were designed to targetthe sequence of b-galactosidase (b-Gal) gene. All AsODNs areof HPLC grade. The pSV-b-Gal control vector containingSV40 early promoter and enhancer driver transcription of thelac Z gene, which encodes the b-Gal enzyme, was suppliedby Promega (Madison, WI). Chitosan (400 kDa molecularweight) was obtained from Fluka (Darmstadt, Germany). Allthe substances are of molecular grade.

Methods

Preparation of AsODN-loaded chitosan nanoparticles.Nanoparticles were produced based on the ionic gelation ofTPP with chitosan (Calvo et al., 1997). One hundred microli-ters of AsODN was added to 10 ml TPP solution beforedropping to 10 ml chitosan aqueous solution under constantstirring. Nanoparticles were separated by centrifugation at12,000 rpm (Eppendorf 5810 R, Hamburg, Germany) for 10minutes. The supernatant were discarded and particles werewashed with bidistilled water.

Size determination of nanoparticles. Particle size distri-bution was determined by Malvern Nano ZS, Model ZEN3600 (Malvern Instruments, Malvern, United Kingdom). Eachparticle preparation was analyzed in duplicate with 10 read-ings per sample suspended in distilled water and the resultswere expressed as a mean of three batches.

Zeta potential. The zeta potential values of nanoparticleswere determined in bidistilled water after measurement of theelectrophoretic mobility for 20 seconds at 258C using a Mal-vern Nano ZS, Model ZEN 3600 (Malvern Instruments).Measurements were carried out three times.

Animal studies. Experimental design and treatment ofanimals were approved by the Animal Care Committee ofMarmara University. Sprague Dawley rats (200–225 g) at 8weeks (adult; 10 male and 10 female) and 4 weeks old (baby;10 male and 10 female) were used. The hairs on the back ofrats were shaved and then the back area of anesthetized ratswas swabbed with povidone–iodine complex. Before AsODNapplication, DNA-chitosan complex (pSV-b-Gal-Chitosan) wasapplied onto rat skin, and 24 hours after application, AsODN-loaded chitosan nanoparticles (containing different doses ofAsODN; 15, 30, 60, and 90mg) were spread on this hairless areaat the back of the animals (n¼ 40). AsODN-loaded nano-particles were applied with single and double (dosed twotimes) doses on rats skin. Untreated animals were used asnegative control group and free AsODN-applied animals aspositive control group. Animals were sacrificed and skinsamples were taken for measurement of b-Gal expression andhistological control after 1, 3, 6, 9, and 12 days posttransfection.

b-Gal expression was spectrophotometrically measured in thesamples with O-nitrophenol-galactoside (ONPG). Total proteinconcentration was assayed according to Bradford’s method(Bradford, 1976).

X-Gal study. Briefly, our staining solution consistedof 3.6 ml of 0.1 M Na2HPO4, 40ml of 0.5 M K3[Fe(CN)6],0.5 M K4[Fe(CN)6] 3H2O, and 1% sodium deoxycholate(C24H39NaO4), 8ml of 1 M MgCl2, 40ml of 2% Np-40, and 200mlX-Gal (60 mg=ml in N,N-dimethyl formamide). Care wastaken to add the substances in order, with X-Gal (Sigma,St. Louis, MO; Biotium Inc., Hayward, CA) added as the laststep. Skin samples were dissected, reacted whole in the X-Galsolution at room temperature on a shaker for 24 hours, andhistologically examined with microscope (Olympus BX51,Okaya, Japan) after X-Gal staining (Bancroft and Gamble,2003).

Statistical analysis. Results were expressed as mean�standard deviation. One-way analysis of variance or a t-testwas performed to compare the influence of the variousparameters. A p-value of less than 0.05 was considered asrepresenting a significant difference.

Results

In this article we tried to knock down b-Gal expression inthe skin. Rat skin has low amount of b-Gal expression (Fig. 1);therefore, exogene b-Gal expression was provided by appli-cation of lac 2 gene, which encodes b-Gal (pSV-b-Gal) onto ratskin. pSV-b-Gal plasmid cannot pass alone through the cellmembrane because of its molecular weight, surface charge,and lability in the skin. Plasmid–chitosan complexes havebeen used to improve the cellular uptake of the plasmid andexpression of the cell because of the ability of chitosan to en-hance skin penetration.

Samples were taken at the same area of the skin and thesame day. Suppression of b-Gal expression was calculatedaccording to b-Gal amount on that day. The selected AsODN-loaded chitosan nanoparticles having 221.0� 4.0 nm sizes and20.5� 1.6 mV zeta potential were applied topically to shavedskin of adult and baby rats.

Antisense effect of free AsODNs on adult rats

After free AsODN (100 mg) application on the rat skin, 35%b-Gal inhibition effect was measured on the first day. As seenin Figure 1A, however, this inhibition effect gradually de-creased at 3 and 6 days and disappeared after 6 days. FreeAsODNs remained ineffective after this period ( p> 0.05).

Antisense effect of AsODN-loaded chitosannanoparticles on adult rats

Chitosan nanoparticles containing different doses ofAsODN such as 15, 30, 60, and 90mg were applied to adult ratskin. On the first day, nanoparticles containing low doses ofAsODN (15 and 30mg) did not show antisense effect ( p> 0.05),whereas approximately 29% inhibition effect was obtainedwith high doses of nanoparticles (60 and 90mg) (Fig. 1A). Dif-ferences between the inhibition effect of free, 60mg, and 90mg

148 OZBAS-TURAN ET AL.

nanoparticles are not significant ( p> 0.05), but differences be-tween low and high doses are significant ( p< 0.05).

Antisense effect of nanoparticles significantly increased at 3days. As seen in Figure 1A, 76–80% of inhibition was obtainedwith 60 and 90 mg AsODNs.

Maximum b-Gal inhibition was measured at 6 days aftertransfection but the difference between the doses is not sig-nificant at 6 days (Fig. 1A) ( p> 0.05).

At 9 and 12 days, b-Gal inhibition effect of nanoparticleshas continued and differences are statistically significant( p< 0.05). In general, it can be said that antisense effect wasobtained during 12 days with nanoparticles (Fig. 1A).

When we investigated dose dependency of antisense effectof chitosan nanoparticles, during the 6 days, high antisenseeffect was obtained with 60mg AsODNs ( p< 0.05); however,after this period, differences between the effect of doses arenot significant ( p> 0.05).

Antisense effect of free AsODNs on baby rats

Experiment was repeated with baby rats. On the firstday the highest gene inhibition was obtained after freeAsODN (20mg) application (Fig. 1B). As seen in this figure,antisense effect gradually decreased during 6 days ( p<0.05) and then the effect disappeared at 9 and 12 days( p> 0.05).

Antisense effect of AsODN-loaded chitosannanoparticle on baby rats

On the first day, approximately 41% of gene inhibition wasmeasured with 15 mg dose of AsODN-containing nano-particles ( p< 0.05); however, 21–23% of inhibition was ob-tained with high doses of nanoparticles (Fig. 1B).

At 3 days, as observed in Figure 1B, nanoparticlescontaining 15 and 30mg AsODNs indicated 76–79% of

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Control (-) pSV-β-Gal AsODN(100 µg)

Nanopart.(15 µg AsODN)








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FIG. 1. Comparison of antisense effect of free AsODNs and AsODN-chitosan nanoparticle (15, 30, 60, and 90 mg) applicationon adult (A) and baby (B) rats. AsODN, antisense oligonucleotide.

TOPICAL APPLICATION OF ANTISENSE OLIGONUCLEOTIDE 149

b-Gal inhibition, whereas 88% of suppression was measuredafter application of 60 mg AsODN-containing nanoparti-cles. Differences between them are statistically significant( p< 0.05).

At 6 days after application, 77–86% of b-Gal suppressionwas measured and differences between the doses are notsignificant ( p> 0.05). Gene inhibition effect of nanoparticleshas continued during 12 days (Fig. 1B).

Comparison of single- and double-dose applicationof free and loaded AsODNs

Free AsODNs were applied to adult rat skin in 100 mg and2�50 mg doses as seen in Figure 2A; significant differenceswere observed between them ( p< 0.001).

Similarly, as seen in Figure 2A, AsODN-loaded chitosannanoparticles were applied in 2�7.5 mg and 2�15mg doses;

however, statistically significant differences were not ob-served between single- and multiple-dose AsODN nano-particle applications on adult rats ( p> 0.05).

In baby rat specimens, higher gene inhibition was obtainedwith double-dose applications when compared with single-dose AsODN nanoparticle applications (Fig. 2B). Particularly,on the first day, 48% of gene suppression was measured after2�15 mg of dose application; this suppression effect is signif-icantly very high when compared with single-dose (30mg)application ( p< 0.05).

Histological study

The histological photographs of skin samples afterstaining with X-Gal are shown in Figure 3. Figure 3B showsdense blue staining of cells because of b-Gal expression, butafter 3 and 9 days the blue color partly disappeared or very

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FIG. 2. Comparison of antisense effect of AsODN-chitosan nanoparticles after single- (15 and 30mg) and double-dose(2�7.5 mg and 2�15 mg) applications on adult (A) and baby (B) rats.


pale blue color cells were present (Fig. 3C, D). These cellsshowed that after chitosan-AsODN application, b-Gal ex-pression reduced significantly.

Discussion

In this study, we tried to study the carriability of AsODN-loaded chitosan-TPP nanoparticles after skin applications andwe have shown that antisense phosphorothioate oligonucle-otides can be used to inhibit gene expression in skin whenapplied topically. In our earlier study, skin gene deliveryability of chitosan-TPP nanoparticles was investigated and wefound that chitosan-TPP nanoparticles can be used for skingene therapy (Akbuga and Ozbas-Turan, 2006).

A number of studies have demonstrated AsODN penetra-tion and=or efficacy using different methods to overcome theskin barrier. These AsODN delivery methods included in-tradermal injection (Wraight et al., 2000), electroporation, andiontophoresis (Regnier and Preat, 1998; White et al., 2002) andit was shown that modified AsODNs in solution could reachthe target cells after topical administration to psoriatic but notnormal cell.

However, none of these methods are well suited to use inthe delivery of nucleic acid because of consideration of cost ordiscomfort (White et al., 2002), and hence, suitable skinAsODN delivery systems are needed.

We have shown skin antisense effect of AsODN-loadedchitosan nanoparticles in both adult and hairless baby rats.We have also investigated the effect of AsODN dose and doseregimens such as single- or double-dose application for b-Galinhibition.

The effect of free AsODNs on skin application is limitedwith time. Free AsODNs showed 35% suppression on the firstday and this was the highest inhibition effect of free AsODNsin skin application of rats. After free nucleic acid application,as expected, the expression was limited because of in vivolability of these molecules.

AsODN-loaded chitosan nanoparticles, however, showedlong and sustained antisense effect in adult and baby rats. Asseen in Figure 1, on the first day, the antisense effect of na-noparticles except high doses (60 and 90 mg) showed lowsuppression, but then gene suppression increased rapidly andreached maximum on 6th day and the effect continued until12th day. Differences between the applied doses disappearedafter 6 days. Comparing adult and baby rats’ results, higher b-Gal inhibition was obtained with the baby specimens, par-ticularly on the first and third days (Figs. 1 and 2). Similarresults were obtained with skin DNA application (Akbugaand Ozbas-Turan, 2006). The results of our histological studyare in accordance with b-Gal data. As seen in Figure 3, b-Galexpression gradually decreased after AsODN-loaded chit-osan nanoparticle application. Moreover, as seen in Figure 3B,skin samples show b-Gal expression not only in the epidermis,but also frequently in the dermis and underlying muscle. Thesame fact is also available for AsODN-chitosan nanoparticleapplied skin samples (Fig. 3C, D). On the other hand, thesedata also reveal important local and systemic effects ofAsODNs. Brand and Iversen (2000) reported that oligonucle-otides pass through the living layers of skin and so systemicdelivery should be considered. In our study, systemic deliveryof AsODNs may be possible with chitosan nanoparticles,because of the antisense effect of formulation after skin ap-

FIG. 3. Histological photographs of the skin samples of adult rats stained with X-gal (A, negative control; B, positive control[only applied pSV-b-Gal]; C, applied nanoparticles containing 90 mg AsODNs [3 days]; D, applied nanoparticles with 90 mgAsODNs [9 days]). b-Gal, b-Galactosidase. Color images available online at www.liebertonline.com=oli.


plication. Presence of oligonucleotides in the deeper part ofskin such as dermis may be related to the effect of chitosan. Asreported earlier, chitosan affects tight junctions and increasesdrug permeation. Our histology results are also similar to thedata of Dokka et al. (2005), who suggested dermal delivery ofAsODN formulated in cream preparation via folliculartransport.

AsODNs provide a gene-targeted approach for the pre-vention and treatment of diseases due to overexpression ofspecific genes. They have hydrophilic property and do noteasily pass biological membranes. Cutaneous delivery ofAsODNs provided an alternative to intravenous injection andhas exciting potential in the treatment of skin diseases.

In this study, in vivo AsODN delivery ability of chitosan-based nanoparticles with topical application was investi-gated. These results indicate that a topical applicant ofAsODN-loaded chitosan-TPP nanoparticles is a suitable formfor AsODN delivery to the skin, yielding higher antisenseeffect for protein inhibition.

In conclusion, chitosan-TPP nanoparticles successfully de-livered phosphorothioate oligonucleotides to the skin cellsafter topical application to the adult and baby rats. With thischitosan-TPP nanoparticle, long and sustained antisense ef-fect may be obtained. Moreover, systemic antisense effect maybe possible after skin application of AsODN-loaded chitosannanoparticles.

Acknowledgments

This study was supported by the Commission of MarmaraUniversity Scientific Research Project (BAPKO, SAG-BGS-120707-01 43). The authors thank Naziye Ozkan and OmerOzdogmus for their help in histological study.

Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Dr. Ali Demir Sezer

Department of Pharmaceutical BiotechnologyFaculty of PharmacyMarmara University

Haydarpasa, UskudarIstanbul 34668

Turkey

E-mail: [email protected]

Received for publication October 28, 2009; accepted afterrevision January 10, 2010.


Topical Application of Antisense Oligonucleotide-Loaded Chitosan Nanoparticles to Rats

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