[4]Ilidio 1 Gene Therapy

8/9/2019 [4]Ilidio 1 Gene Therapy

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Nanoparticle mediated delivery of pure P53 supercoiled plasmid DNA forgene therapy

Vtor M. Gaspar a, Ildio J. Correia a, ngela Sousa a, Filomena Silva a, Catarina M. Paquete b,Joo A. Queiroz a, Fani Sousa a,a CICS-UBI Centro de Investigao em Cincias da Sade, Universidade da Beira Interior, Covilh, Portugalb ITQB-UNL Instituto de Tecnologia Qumica e Biolgica, Universidade Nova de Lisboa, Oeiras, Portugal

a b s t r a c ta r t i c l e i n f o

Article history:

Received 2 May 2011Accepted 5 August 2011Available online 12 August 2011

Keywords:

Gene therapyNanoparticlesp53 Tumor suppressorSupercoiled plasmid DNA

The translation of non-viral gene replacement therapies for cancer into clinical application is currentlyhindered due to known issues associated with the effectiveness of plasmid DNA (pDNA) expression vectorsand the production of gene delivery vehicles. Herein we report an integrative approach established on thesynthesis of nanoparticulated carriers, in association with the supercoiled (sc) isoform purication of a p53tumor suppressor encoding plasmid, to improve both delivery and transfection. An arginine-basedchromatographic matrix with specic recognition for the different topoisoforms was used to completelyisolate the biologically active sc pDNA. Our ndings showed that the sc topoisoform is recovered under mildconditions with high purityand structural stability.In addition, to further enhance protection and transfectionefciency, the naked sc pDNA was encapsulated within chitosan nanoparticles by ionotropic gelation. Themild conditions for particle synthesis used in the former technique allowed the attainment of a highencapsulation efciency for sc pDNA (N75%). Moreover, in vitro transfection experiments conrmed thereinstatement of the p53 protein expression and most importantly, the sc pDNA transfected cells exhibitedthe highest p53 expression levels when compared to other formulations. Overall, given the fact that sc pDNAtopoisoform indeed enhances transgene expression rates this approach might have a profound impact on thedevelopment of a sustained nucleic acid-based therapy for cancer.

2011 Elsevier B.V. All rights reserved.

1. Introduction

The development of novel cancer gene therapy approaches basedon the re-establishment of tumor suppressor regulated molecularpathways is raising new prospects on the outcome of an effective anti-cancer therapy[1].

The p53 protein is a unique tumor suppressor, procient in theselective induction of growth arrest and apoptosis in response tooncogenicor damage signaling, actingas a prevailing guardian againstmalignant cell transformation[2]. On the other hand, it is estimatedthat the p53 gene is mutated or deleted in approximately 50% of allhuman cancers and its ubiquitous loss of function contributes as oneof the underling events that trigger and sustain tumorigenesis[3]. Itbecomes therefore reasonable that the reinstatement of the wild-typep53 expression and consequent reactivation of its downstreameffector pathways has impact on cancer therapy, as recently reported[4].

Non-viral delivery, arises as the most suitable approach for genetherapydue to markedly safety advantagesover itsviral counterpart[5].However, an effective application of a p53 DNA-based cancer therapyhas been hampered so far by issues associated with the intracellulardelivery, transfection efciency, and the purication of plasmid DNA(pDNA) expression vectors[5,6]. Purication of pharmaceutical-gradepDNA is a challenging process since the downstream processing mustnot be approached in an individual basis, but in an integrativeperspective that accounts for cell impurities and contaminants such asRNA, genomic DNA (gDNA) and endotoxins that derive from theupstream stages, causing deleterious side effects if delivered to the host[7]. Moreover, despite the fact that pDNA is a very stable biomolecule,during itsproduction andrecovery,it canundergo severaltypes of stressthat may disruptits structural stability [8].Thiseventmustbeaccountedfor, since it mainly affects the sc topoisoform, the only one consideredintact and undamaged[7,8]. Nonetheless, it is important to point outthat the sc pDNA topoisoform possesses the highest transfectionefciency bothin vitro[9], andin vivo[8], when compared to the opencircular (oc) or linear variants, rendering itself as a valuable alternativeto improve transgene expression at the pDNA vector level.

Considering the ever-growing need to meet the preemptive re-quirements of purity and structural stability, our group has recentlydeveloped a high throughput arginine afnity chromatography-based

Journal of Controlled Release 156 (2011) 212222

Corresponding author at: CICS-UBI Centro de Investigao em Cincias da Sade,Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh, Portugal.Tel.: +351 275 329 002; fax: +351 275 329 099.

E-mail address:[email protected](F. Sousa).

0168-3659/$ see front matter 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.jconrel.2011.08.007

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Journal of Controlled Release

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http://dx.doi.org/10.1016/j.jconrel.2011.08.007http://dx.doi.org/10.1016/j.jconrel.2011.08.007http://dx.doi.org/10.1016/j.jconrel.2011.08.007mailto:[email protected]://dx.doi.org/10.1016/j.jconrel.2011.08.007http://www.sciencedirect.com/science/journal/01683659http://www.sciencedirect.com/science/journal/01683659http://dx.doi.org/10.1016/j.jconrel.2011.08.007mailto:[email protected]://dx.doi.org/10.1016/j.jconrel.2011.08.007


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approach to recover sc pDNA for gene therapy [10]. Afnity chroma-tography supports based on immobilized amino acids possess uniquefeatures since they take advantage of naturally occurring biologicalinteractions between DNA and specic amino acids, purifying bio-molecules from the standpoint of their biological function or chemicalstructure[10,11]. In addition, the intrinsic separation selectivity thatalso occurs between pDNA and contaminants, render it a noteworthyapproach to purify plasmid biopharmaceuticals that comply with the

strict regulatory directives issued for gene-based therapies [10].However, despite these major improvements regarding vector trans-fection efciency and safety, its delivery towards and into theintracellular compartment still remains a key setback, since nakedpDNA is vulnerable to degradation (e.g. by serum nucleases and shearforces) [8], is rapidly clearedfromsystemiccirculation (sc pDNAplasmahalf-life of 0.15 min) [12], and is rather inefcient in transposingextracellular barriers and consequently in attaining therapeutic expres-sion levels [13,14]. These limitations regarding intracellular access,protectionand bioavailability maybe overcomeif pDNA is packaged andprotected in nanocarriers that can selectively encapsulate and deliver itin the cytoplasm[15].

Non-viral nanoparticulated carriers usually include cationic lipo-somes and cationic polymers such as chitosan [16]. Chitosan is abiocompatible and biodegradable polymer [17], with a cationic chargedbackbone thatis responsiblefor its higher DNA loading capacity [16,18].In fact, thechargedensity is itsmostimportantphysicochemical feature,since it allows the polymer backbone not only to complex with DNAbutalso to instantly gel upon contact with oppositely charged crosslinkersand surfactants[14,19]. Taking this into account, we recently reportedthe synthesis of chitosan-pDNA loaded nanoparticles formulated bygelation with a counter polyanionic crosslinker[20].

As schematized inFig. 1, herein, we report the development of anintegrative approach that gathers the recent progresses regardingpurication and transfection efciency of plasmid biopharmaceuticalsand the novel generation of gene delivery vehicles, effectively coveringthe key issues that currently hinder the translation of non-viral genetherapy into clinical applications.

2. Materials and methods

2.1. Materials

The 6.07 kbp pcDNA3FLAGp53 plasmid was purchased fromAddgene (Cambridge, MA,USA).Arginine Sepharose4B gel was obtained

fromAmersham Biosciences, (Uppsala,Sweden).Chitosanlow molecularweight (LMW), pentasodium tripolyphosphate (TPP), Anti-VE Cadherinantibody, uorescein isothiocyanate isomer I (FITC) and cell culturereagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-(4-aminophenyl ethylamine) was purchased from Acros Organics (Geel,Belgium). (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) was obtained from Promega(Madison,WI, USA). A549 (non-small lung carcinomacell line) andHeLa

(Human negroid cervix epitheloidcarcinoma) cellswerepurchasedfromATCC, Middlesex, UK. Hoechst 33342, AlexaFluor 546, AlexaFluor647, and transfection reagents including Lipofectamine 2000, wereobtainedfromInvitrogen(Carlsbad, CA,USA).Analytical grade saltswereused throughout this work.

2.2. Methods

A general description of all of the used methodologies is providedas following. Nevertheless, a more detailed description is madeavailable as supplementary material.

2.3. Plasmid biosynthesis and purication by afnity chromatography

The pcDNA3FLAGp53 plasmid was amplied in a bacterial cellculture ofEscherichia coli(E. coli) DH5and recovered as previouslydescribed[20].

Chromatography was conducted in a fast protein liquid chromatog-raphy system (FPLC), (Amersham Biosciences, Sweden) using a columnpacked with an argininesepharose 4B gel. All experiments wereperformed under controlled temperature conditions (4 C) by using awater cooling system. The experiments were performed using twobuffer solutions: (i) 10 mM TrisHCl (pH 8.0) solution; (ii) 300 mMNaClsolutionin 10 mM TrisHCl(pH 8.0). TherecoveredpDNAisoformswere subsequently precipitated using one volume isopropanol andcentrifuged for 30 min, 15,000 g, 4 C, to remove the salt present in thepDNA samples. Afterwards, the pDNA pellet was then resuspended in10 mM TrisHCl (pH 8.0).

2.4. Agarose gel electrophoresis

Theagarose gelelectrophoresis experimentswere performed using a1% agarose gel with ethidium bromide (0.5g/mL). Electrophoresis wascarried outat 100 V for45 min in TrisAcetateEthylene Diamine (TAE)buffer. The agarose gels were revealed under UV light. Lane densitymeasurements were performed in the software Bio-Rad Quantity One,(Hercules, USA).

2.5. Synthesis of chitosan and formulation of pDNA loaded nanoparticles

LMW chitosan with a deacetylation degree (N98%) was puried asformerly reported[20]. Plasmid DNA loaded chitosan nanoparticleswere synthesized by the ionotropic gelation technique. For this

synthesis a 0.75 mg/ml (pH 4.9) chitosan solution and a 0.5 mg/mL(pH 5.5) solution of the anionic crosslinker, pentasodium tripolypho-sphate (TPP), were prepared. All the solutions were then ltered witha 0.22m lter to remove traces of solid particles. In order to promoteencapsulation, pDNA (2 mg/mL) was added to the TPP solution priorto particle formation. Afterwards 1 mL of the pDNATPP solution wasadded dropwise to 4 mL of chitosan solution, under magnetic stirring(30050 rpm), at room temperature, for 30 min. The formulatednanoparticles were then pelleted by centrifugation at 17,000 g for30 min.

2.6. Morphology of the nanoparticles

Particle morphology was analyzed by scanning electron microscopy

(SEM) using a Hitachi S-2700 (Tokyo, Japan) electron microscope

Fig. 1. Schematics of an integrative approach for non-viral cancer gene therapy. (I.)Purication of plasmid biopharmaceuticals and recovery of the sc pDNA topoisoformusing arginine afnity chromatography; (II.) Nanoparticle mediated delivery and

transfection; (III.) Expression of the p53 tumor suppressor.

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working at 20 kV and with different magnications. Prior to imageacquisitionone drop of nanoparticles wasdispersed throughthe surfaceof a cover glass and vacuum dried at 37 C overnight. Afterwards thesamples were mounted in microscope stubs and sputter coated withgold using a sputter coater.

2.7. Nanoparticle size

Nanoparticlesizewas determinedby dynamiclight scattering(DLS).To determine the hydrodynamic diameter by DLS, chitosan nanoparti-cles were diluted in 800 l of milliQ ultrapure water as a dispersantmedium, followed by 30 min incubation at room temperature prior toanalysis. Size measurements were then performed in a Zetasizer NanoZS instrument(Malvern Instruments,Worcestershire,UK), in automaticmode and with a scattered light detection angle of 173. All themeasurementswere performed in triplicateand measured 40 times. Thereported particle size was obtained as an intensity distribution bycumulative analysis performed in the zetasizer software (version 6.20).

2.8. Zeta potential

For zetapotential experiments chitosan nanoparticles werepreparedas previously describedfor size measurements. Thedetermination of thezeta potential of the different naked pDNA topoisoforms was alsoexecuted, with thepDNA samplesdispersedin TrisHCl10 mM(pH8.0).Zeta potential quantication was carried out in a Zetasizer Nano ZSinstrument using a zeta dip cell. The experiments were performedin triplicate and an average of 100 measurements was acquired indi-vidually for each sample.

2.9. Encapsulation efciency of pDNA and loading capacity

To determine pDNA encapsulation efciency (EE) nanoparticlesamples wereisolated by centrifugation,and the supernatantrecoveredfor further analysis. The concentration of unbound pDNAwas measuredby UVvis analysis (Shimadzu UVvis spectrophotometer, ShimadzuInc, Japan) as reported in the literature[20]. Loading capacity (LC) was

determined by weighing blank tubes before the experiment and afternanoparticle recovery.

2.10. Fluorescence labeling of pDNA

Plasmid DNA was uorescently labeled using a FITCdiazoniumsalt as described elsewhere[21].

2.11. In vitro transfection and cell culture

A549, HeLa cells and rat skin Fibroblasts, isolated as previouslydescribed [17], were cultured in 25 cm2 T-asks in Ham's F12K,Dulbecco's Modied Eagle's Medium (DMEM) and DMEM-F12 respec-tively, supplemented with 10% fetal bovine serum (FBS) and 1%

antibioticsantimicotics, at 37 C, in an humidied atmosphere with5%CO2. One day prior to transfectionmalignant cells were seededin 24well plates (4 104 cells/well). On the day of transfection, cells at 9095% conuence were transfected with either Lipofectamine 2000 ornanoparticles.

2.12. Cellular uptake of nanoparticles

To characterize the amount of chitosan/pDNA nanoparticlesuptake by the cells ow cytometry experiments were conducted byusing uorescently labeled FITCpDNA conjugates at differenttransfection times (2 h and 6 h).The experiments were performedon a BD FACSCalibur ow cytometer and the acquisition of the datawas made in the CellQuestTM Pro software where 1 104 events were

counted.

Initially, to determine the optimal gating parameters and thepossible auto-uorescence of the malignant cells a screening withnon-labeled cells was performed by accumulating the signalscorresponding to forward and side scatter measurements (FSC andSSC) and dening the region of interest (ROI) according to a giventhreshold level. After the establishment of the ROI the resultinguorescent signals of 2104 events were recorded with the FL-1(530/30 nm) band passlter. Flow cytometry dot plots were analyzed

in FCS Express version 4 Research Edition (De Novo Software

, LA,USA).

3. Immunouorescence and confocal laser scanning microscopy

(CLSM)

Fluorescence experiments were performed with conuent cells.After transfection with either Lipofectamine 2000 or nanoparticles,the cells were xed in 4% paraformaldehyde (PFA) in PBS for 20 min,permeabilized and blocked for 3 h at room temperature. Thecells were then incubated with the primary anti-VE cadherinmonoclonal antibody (dilution 1:250) for 1 h and subsequently rinsed10 times with PBS-Tween 20 (PBS-T) solution. To stain the nucleus ofthe cells a Hoechst 33342 molecular probe was then added andincubated for 15 min followed by 10 washing steps with PBS-T.After nuclear staining, the cells were incubated with an AlexaFluor546 goat anti-(rabbit IgG) conjugate for 1 h at room temperature.The samples were then visualized using a Zeiss AX10 microscope(Carl Zeiss, USA). Axio Vision Real 4.6 software was used for imageanalysis.

To address the intracellular localization and movement of pDNA,CLSM was performed. Conuent cell monolayer's were xed, blockedand stained similarly to immunouorescence however with theexception of the secondary antibody incubation, which was conductedwith a far-red AlexaFluor 647 goat anti-(rabbit IgG) antibody.

Confocal images were obtained with a Zeiss LSM 710 laserscanning confocal microscope (Carl Zeiss SMT Inc., USA) equippedwith a plane-apocromat 63/DIC objective. To obtain enough data

for 3D reconstruction, a series of sequential slices, with different slicesthickness (m), were acquired along the Z-axis using optimizedpinhole parameters in order to comply with the Nyquist-Shannonsampling theorem and minimize image aliasing duringacquisition. Allof the acquired Z-stacks were open as a merged le in the LSM 710software (Carl Zeiss SMT Inc., USA) where subsequent 3D reconstruc-tion was performed. Fast maximum intensity projections (MIP) of thereconstructed images were obtained using Huygens Essential soft-ware (Scientic Volume Imaging, Hilversum, The Netherlands).

3.1. Reinstatement of p53 expression in malignant cells

The re-establishment of the expression of the p53 tumor

suppressor was determined by indirect ow cytometry. Twenty fourhours after transfection, cells were xed with 4% PFA for 15 min atroom temperature, permeabilized with 1% Triton X-100 for 5 min, andtrypsinized using Trypsin/EDTA. The cell suspension was thenpelleted and resuspended in ice cold PBS, 5% FBS, followed byincubation with rabbit anti-p53 antibody (10g/mL) (sc-6243, SantaCruz Biotechnology, CA, USA) for 1 h at room temperature. Afterincubation the cells were spun down and resuspended in PBS 3 timesto remove unbound anti-p53. Afterwards the cells were incubatedwith an anti-rabbit Alexa 647 secondary antibody for 1 h and washedas formerly described. Fluorescence was then detected with the FL-4(661/16) band pass lter on a BD FACSCalibur ow cytometer where1104 events were recorded. Data analysis was performed in FCSExpressversion 4 Research Edition (De Novo Software,LA,USA)and

Treestar FlowJo software version 7.6.1 (Ashland, OR, USA).

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3.2. Quantication of apoptosis

P53 mediated apoptosis in transfected A549 and HeLa malignantcells was assessed by the Annexin VFITC and PI kit (Calbiochem,USA) according to the manufacturer's instructions. Annexin VFITC/PIlabeled cells were excited with a 15 mW laser at 488 nm and theresultinguorescent signals of 2 104 events were recorded with FL-1(530/30 nm) and FL-2 (585/42) band pass lters. Non transfected

cells were used as controls. Flow cytometry dot plots were analyzed inFCS Express version 4 Research Edition (De Novo Software, LA,USA).

3.3. Cytotoxicity assays

The cellular toxicity of the different formulations of nanoparticles(native, oc and sc) was determined by the MTS assay, which wasperformed both in HeLa cells and rat skin Fibroblasts, according to themanufacturer instructions. Twenty four hours prior to the experimentthe cells were seeded at a density of 2104 cells per well into 96-wellat bottom culture plates with 200 L of cell culture mediumsupplemented with 10% FBS, without antibiotics. On the day of theexperiment the culture medium was aspirated and replaced by freshmedium. The cells were then incubated with 30 L of nanoparticleformulations for 24 and 72 h. All the formulations of nanoparticleswereresuspended in pre-warmed culture medium containing 10% FBS andthen added to each well. A total ofve replicates were considered foreach formulation.

3.4. Statistical analysis

Comparison between multiple plasmid formulations was per-formed using one-way analysis of variance (ANOVA), with theStudentNewmanKeuls test. Comparisons between plasmid formu-lations were determined using a two-tailed Student's t-test. A value ofPb0.05 was considered statistically signicant.

4. Results

4.1. Purication of sc pDNA by arginine afnity chromatography

Purication of pDNA by arginine afnity chromatography presentssignicant advantages over the existing strategies because sc pDNA isrecovered with high yield, structural stability and in a singlepurication step as it was recently reported for a model plasmid(pVAX1LacZ) [9]. However, given the fact that specic and reversiblebioafnity interactions are involved in the recognition of the differenttopological isoforms, inherent particularities in argininepDNA in-teractions arise for different pDNA expression vectors. For this reason,the adjustment of the conditions that promote the recovery of thedifferent topoisoforms is essential to achieve high resolution andselectivity proles that inturninuence the overall recovery yield and

purication degree of the plasmid of interest.The obtained chromatographic prole when a native pDNA sample

(oc+sc) was loaded onto the arginine support is shown in Fig. 2A.Under the reported conditions, two resolved peaks eluting at 112 mMNaCl (peak 1) and 300 mM NaCl (peak 2) were detected. In order toestablish a correlation between the different plasmid topoisoformsand the eluting peaks in the chromatogram, an agarose gelelectrophoresis was performed (Fig. 2B). The results revealed thatthe elution of the oc topoisoform occurs in the rst peak (Fig. 2B, lane1), at low ionic strength. Whereas, elution of the sc plasmid boundtopoisoform only takes place at higher ionic force (Fig. 2B, lane 2),implying that the interaction with the arginineagarose matrix isstronger. Additionally, the results presented in Fig. 2B also suggestthat sc pDNA samples are recovered with 100% homogeneity (i.e.

without any traces of either oc or linear variants). Hence, to further

support this conception, lane density was also determined, and as theresult inFig. 2C depicts, the density peak indeed corresponds only tothe sc topoisoform, that is therefore recovered with maximum purityand preserved structural characteristics.

4.2. Synthesis and characterization of pDNA loaded nanoparticles

All formulations of pDNA loaded nanocarriers (i.e. with native, ocand sc samples) were synthesized by the ionotropic gelationtechnique which is based on the electrostatic interactions that occur

between the positively charged polymer backbone, pDNA and acounter anionic crosslinker, TPP, that is responsible for the sponta-neous gelation of chitosan. Taking into account the possibledisturbance of the tertiary superhelical DNAstructure and consequenttopoisoform conversion (sc to oc or linear conformation), duringparticle synthesis, a stability study was performed prior to theproduction of the different particle formulations. Our results demon-strated that the submission of pDNA to the pH range and stirringparameters used does not promote signicant topoisoform conver-sion since the sc pDNA content remained higher than 95% (Supple-mentary Fig. S1), a value that is approximately the one issued inregulatory directives for the use of plasmid DNA biopharmaceuticalsin therapeutic applications (sc pDNA contentN97%) [8]. It is importantto point out that the topoisoform conversion in other nanoparticle

manufacturing techniques is markedly higher[29].

Fig. 2.Selective separation of oc and sc topoisoforms by arginine afnity chromatog-raphy. (A) Chromatographic prole. Peak 1: weakly bound species; Peak 2: tightlybound species. (B) Electrophoresis of peaked fractions. Lane MW: molecular weightmarker; Lane N:native pDNA sample. Lane 1: Peakedfraction 1; Lane 2:Peakedfraction2. (C) Density analysis of the corresponding lanes in agarose gel electrophoresis.

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The morphology of the different nanocarriers obtained is pre-sented in Fig. 3. Nanoparticulated systems formulated from native andoc pDNA preparations showed randomly shaped particles withspherical, rod and oval-like morphologies as shown inFig. 3A and B.However, to address the possible inuence of the chitosan/ oc pDNAratio in particle morphology, different formulation ratios were alsoinvestigated. Our ndings demonstrate that at higher ratios (5:1; 6:1)the synthesized particles possess the same random morphologicalcharacteristics, and at lower ratios no particles are formed under thegiven formulation conditions (Supplementary Fig. S2). Whereas, thenanoparticles obtained from the sc pDNA samples (Fig. 3C) demon-strated well dened spherical shapes.

Other relevant characteristics such as particle size, zeta potential,and loading capacity were also determined for the different

formulations. As shown inTable 1, all of the nanocarriers demon-strated narrow size distribution with particle sizes suited for deliveryin tumoral microenvironments. Moreover, the chitosan nanoparticlesformulated with the different topological pDNA isoforms exhibit astrong positive charge on their surface, an important feature that notonly inuences particlecell membrane interactions but also particlecolloidal stability.

Regarding the results of the loading efciency of the nanocarriersystems it should also be pointed out that all the formulations yieldedparticles comprised by a signicant contentof geneticmaterial (4458%)when comparedto the contentof chitosanTPP(4256%), meaning thatthe delivery vehicle not only upholds its important chitosan associatedfeatures but is also formed by a considerable amount of the therapeutictransgene. The ndings related with the process yield are similar for the

different formulations and in accordance with those reported in theliterature[22].

4.3. Nanocarrier pDNA encapsulation ability

Fig. 4presents the EE for the nanoparticulated systems synthesizedwith the different plasmid preparations. Our results showed that theencapsulation of sc pDNA is signicantly higher than that of native or octopologies. Moreover, it is important to point out as well that oc basednanocarrier formulations possess the lowest EE of all tested samples.Interestingly, the results are suggestive of a pattern associatedencapsulation, since the presence of the sc topoisoform (both in nativeand pure sc samples) increases encapsulation. These ndings areimportant for the overall formulation process and may be indicativethat condensed pDNA topology plays somehow an important role inpositive/negative, polymerDNA interactions.

In fact, to further explore this possibility the surface charge of thepDNA biomolecules was determined. The results presented inTable 2indeed illustrate a distinct difference regarding the electrostaticcharacteristics of the various topoisoforms. These striking results willbe further discussed.

4.4. Cellular uptake of nanoparticles

The results presented inFig. 5characterize the cellular internal-ization of the different nanoparticles with either native (oc+sc), ocand sc pDNA topological isoforms. The mean uorescence intensityvalues obtained reveal a slightly increased cellular uptake for sc pDNAnanoparticles in comparison to the other formulations at 2 h (Fig. 5).In addition, this difference in the internalization of the sc pDNAnanoparticles is increased after 6 h of transfection (Fig. 5). Regardingthe cellular uptake of native pDNA and oc nanoparticles, the amountof DNA transported to the intracellular compartment is higher for thenanoparticles synthesized with native pDNA.

4.5. In vitro delivery and intracellular trafcking

Analyzing the in vitro transfection results depicted in Fig. 6 it is clearthat either nanoparticulated carriers or commercial Lipofectamine arecapable of transporting the genetic material to the cell as previouslydemonstrated in cellular uptake experiments. However, a thoroughanalysis of Fig. 6B and F reveals that the dispersion of the green

Fig. 3. Characterization of nanoparticle morphology by SEM. (A) Nanoparticlesformulated with native pDNA (Scale bar 500 nm). (B) Nanoparticles formulatedwith oc pDNA (Scale bar 500 nm). (C) Nanoparticles formulated with sc pDNA (Scalebar 1 m).

Table 1

Characterization of the different formulations of nanocarriers. Data is presented as themeans.d. .(n =3).

Sample Ratio Particle sizeDLS (nm)

Zetapotential(mV)

Particle loadingcapacity (%)

Yield (%)

pDNA(sc+oc)

4:1 157.6197.4 +20.2418.17 51.186.71 26.544.29

Oc 251.3272.0 +34.5514.63 41.440.51 21.420.33Sc 109.8162.5 +22.64.94 42.802.54 20.931.24

Fig. 4.Encapsulation efciency of the nanoparticles obtained for the different pDNApreparations; native pDNA, oc and sc. Each bar represents the means.d. (n =3).*Pb0.05, **Pb0.01.

Table 2

Characterization of the charge distribution of the different plasmid topologicalisoforms.

Sample Solution Zeta potential (mV)

pDNA (oc+ sc) 10 mM TrisHCl (pH=8.0) 16.4Oc 7.31Sc 9.33

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uorescent dots (corresponding to pDNA) is clearly increased whennanocarrier mediated delivery takes place. The intracellular movementand localization of pDNA can be illustrated by the Figures obtained at2 h, 4 h and 6 h of transfection. As demonstrated in the CLSM images scpDNA-based delivery systems are localized within the cytoplasmiccompartment, perinuclear space and nucleus (Fig. 6O,PandQ)at2 h,4and6 h respectively. Whereas, nativepDNA-basednanocarriersareonlylocalized in the perinuclear space, not reaching the nucleus as fast as sc

Fig. 5. Cellular uptake of the nanocarrier systems formulated with different pDNAtopoisoforms. White bars represent cellular internalization after 2 h of transfection.Black bars represent cellular internalization after 6 h of transfection.

Fig. 6. Immunouorescence and CLSM of A549 cells. (A, E) Nuclear staining withHoechst 33342 (blue); (B, F) FITC labeled sc pDNA (green); (C, G) Staining with Anti-VE cadherin-Alexa 546 antibody (red); (D, H) Merged images; (I, J, K) MIP images ofchitosan/native pDNA mediated transfection at different time-frames, 2 h, 4 h and 6 hrespectively; (L, M, N) MIP images of chitosan/oc pDNA mediated transfection atdifferent time-frames, 2 h, 4 h and 6 h respectively; (O, P, Q) MIP images of chitosan/ocpDNA mediated transfection at different time-frames, 2 h, 4 h and 6 h respectively.

White arrows indicate FITC

pDNA.

Fig. 7. Flow cytometry analysis of the reinstatement of p53 expression in A549 cells. (A,D) Half overlay histograms of the re-establishment of p53 with the different pDNAtopological isoforms; (B, E) Mean uorescence intensity (MFI) values of p53 labeledAlexa 647; (C, F) P53 relative expression mediated by the different pDNA topoisoforms

with either nanoparticles and Lipofectamine, respectively.

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pDNA (Fig. 6K). In turn oc-based nanoparticles at 6 h post transfectionare localized in the cytoplasmic compartment.

4.6. Reinstatement of the expression of p53 tumor suppressor

As the results depicted in Fig. 7C and F demonstrate, bothnanoparticle and Lipofectamine 2000 mediated transfection lead tothe restoration of the expression of the p53 tumor suppressor.

Notwithstanding, it is important to denote that different expressionlevels were obtained according to the different plasmid formulations(native, oc or sc). In fact, this is also observed in the histograms andmean uorescence values depicted inFig. 7A, D and B, E respectively,where sc pDNA mediated transgene expression levels are higher thanthose of native and oc formulations, both in nanoparticle andLipofectamine transfected cells. Additionally the same expressionprole associated with the different pDNA topological isoforms is alsoattained in western blot (Supplementary Fig. S3), thus, furtherevidencing the existence of a topoisoform-associated transgeneexpression prole.

4.7. P53 mediated apoptosis in malignant cells

The results in Fig. 8 demonstrate the quantication of p53mediated apoptosis in HeLa cells. As depicted by the dot plots(Fig. 8B, C and D), it is clear that programmed cell death occurred to amuch higher extent in the sc pDNA transfected cells in comparison tothe other formulations. Regarding the apoptosis assay in A549 cells,much lower apoptosis was observed (4% for sc pDNA) when putside by side with that attained for HeLa cells. However, it is importantto point out that our values were in accordance with those obtainedfor p53 transfection with adenovirus [23], and hence demonstratethat the nanoparticles are also quite effective delivery vehicles. Theseresults are likely a consequence of the fact that A549 cells are resistantto p53 mediated apoptosis as recently reported[24]. It is important tounderline that despite the low apoptosis values in A549 cells the sameapoptosis pattern obtained in HeLa cells for the different formulations

was preserved, with the sc pDNA transfected cells yielding the higherpercentage of apoptotic/late apoptotic cells.

4.8. In vitro characterization of the cytotoxic prole of nanoparticles

The cellular cytotoxicity prole of all the chitosan/pDNA formu-lations was characterized to address whether p53 dependentapoptosis is only correlated with the activity of the tumor suppressorprotein and not to a cytotoxic effect of the synthesized formulations.As presented inFig. 9A and B, at 24 h, cellular viability is clearly notaffected by the existence of the native, oc or sc pDNA nanoparticlessince the majority of cells remained viable (N95%) both in malignantand non-malignant cells. However, in order to provide additionalinsights onto the inuence of the therapeutic approach the cytotoxic

index was also determined after 72 h. This approach brought forthsignicant cell viability differences betweennanocarrier formulations.In fact, analyzing the results for malignant cells, those that weretransfected with the sc pDNA nanoparticles exhibit a much lowerviability in comparison to their topological counterparts. This is aninteresting result since this pattern is not observed in non-malignantcells where cell viability remains unchanged and even increases at72 h, hence, suggesting that the drop off in tumoral cells viability

Fig. 8. P53 tumor suppressor mediated apoptosis in HeLa cells induced by differentpDNA topoisoforms. Representative dot plots of Annexin VFITC (x-axis)/PI (y-axis)double staining of (A) Untransfected cells (control); (B) Cells transfected with nativepDNA. (C) Cells transfected with oc pDNA. (D) Cells transfected with sc pDNA. (E)

Percentage of apoptotic/late apoptotic HeLa cells.



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might be correlated with the expression of the p53 therapeutictransgene.

5. Discussion

Nowadays several approaches are being devised in order totranslate non-viral cancer gene delivery into clinical applications[1].However, known issues associated with insufcient transfectionefciency of both pDNA vectors and delivery vehicles, has restrainedthe outcome of a widespread and effective anti-cancer therapy.

In the present study, we propose a novel approach that successfullycovers the limitations associated with non-viral gene therapy, not onlyimproving transfection and delivery of the genetic material, but alsoenhancing purity and transgene expression efciency of plasmidvectors.

To bring about the potential of this approach, native pDNApreparations were initially processed using a high throughput arginineafnity chromatography support, in order to recover the plasmidtopoisoform with higher biological activity and transfection efciency

(sc pDNA) [9]. In fact, our results demonstratethatthe arginine supportwas indeed ableto selectively recognize the differentpDNA topologicalconformations under the established retention/elution conditions,since sc pDNA remained bound to the support, at low salt concentra-tion, whilst oc pDNA was totally eluted, as depicted in the chromato-graphic prole of Fig. 2A. This unique selectivity feature of thechromatographic support is a consequence of the interaction with thenucleotides bases and pairing preference between particular aminoacids and nucleotide bases, namely arginineguanine[25]. Indeed aspreviously reported by our group, arginineguanine pairing via H-bonding is the prevalent interaction when selective separation of bothtopoisoforms occurs [26]. In addition, we also showed that theseinteractions are stronger for sc topologies due to nucleotide baseexposure that occurs as a consequence of torsional strain deformations

[26]. Our results for the pcDNA3

FLAG

p53 plasmid are then in

accordance with those previously reported for pVAX1LacZ[27], sincethesc pDNA topoisoform is elutedat highersaltconcentrations forbothplasmids, suggesting that the chromatographic support specicallyrecognizes and strongly binds the sc topology regardless of the usedplasmid.More importantly,as our results demonstrate the recoveredscpDNA samples presented 100% homogeneity and up kept all of theirdistinctive structural characteristics (Fig. 2B and C), that in turn largelyinuence the transgene expression efciency and thus the therapeutic

outcome.Although sc pDNA percentage is considered the most importantquality attribute of a plasmid preparation[28], in order to translatenucleic acid-based cancer gene therapy into clinical applications,pharmaceutical-grade pDNA must also comply with current goodmanufacturing practice (cGMP) guidelines regarding protein, gDNA,and endotoxin levels. The latter is extremely important since thepresence of endotoxins may cause deleterious side effects such asfever and complement system cascade which may elicit a severeimmunological response [28]. Regarding these issues, we recentlyshowed that the presence of contaminants in the sc pDNA samplerecovered from the arginineagarose was signicantly reduced notonly regarding the gDNA content (up to 117-fold reduction), but alsoendotoxins (95% removal) and proteins (undetectable levels) [9],therefore complying with cGMP for gene therapy applications.

From this stand point it seems undoubtedly clear that majorimprovements regarding the issues associated with pDNA expressionvectors may be outdone if arginine chromatography is employed inpreparation of pharmaceutical grade sc pDNA.

However, other key issues arise since pDNA is susceptible todegradation in the extracellular environment and rather unable totranspose cellular barriers in its naked form [14]. Therefore, itsencapsulation in nanoparticulated delivery systems that are able toprotect and deliver the transgene of interest into the intracellularcompartment is a valuable approach. Hence, in order to synthesizenanocarrier systems that were capable of covering the previous issuesand at the same time preserve the topological characteristics of pDNAvectors we selected ionotropic gelation technique for nanocarrierproduction because DNA encapsulation and particle formation both

take place under milder conditions than other nanoparticle formula-tion techniques (spray-drying, sonication, complex coacervation)[29]. Most importantly since minor sc pDNA topoisoform conversionoccurs, the bulk of the available DNA that interacts with the cationicpolymer backbone during particle production is the biologically activesc pDNA conformation. Regarding particle formulation our resultsshowed that nanocarriers with native and oc DNA samples formedparticles with random shapes (Fig. 3). On the other hand, particlesformed from sc pDNA interestingly presented dened sphericalmorphologies, most likely due to the compact form of the plasmidexpression vector, suggesting that although pDNA molecular weightremains the same, condensed pDNA topology may inuence thethermodynamics of the prevalent electrostatic interactions, andthereof the formation of spherical morphologies. Our results are

consistent with those obtained by Dunlap et al., 1997, that reportedthe synthesis of round and globular shaped polyplexes using sc pDNApreparations, as opposed to those with rather undened morphology,formed from less compact topologies [30]. These ndings assumefurther signicance, since as shown by Chithrani and Chan, 2007,nanoparticles with spherical shapes are wrapped promptly by the cellmembrane (due to surface-to-volume ratio), and as a result exhibitgreater uptake when compared with those with rod-like morphology[31]. Other relevant characteristic of the delivery system thatdenitely inuences the overall transfection efciency is nanoparticlesize. The results herein obtained reveal that allof the nanoparticulatedcarriers have sizes that promote their accumulation in the microen-vironment that encloses tumor cells, since the blood vessels thatsurround tumors possess fenestrations that range between 100 and

600 nm[32]. Nevertheless, it is also important to point out that as

Fig. 9.MTS cytotoxicity index of the nanoparticles formulated with the different pDNAtopoisoforms, native, oc, sc, blank nanoparticles (without pDNA) in: (A) HeLamalignant cells. (B) Rat skin broblasts. White bars represent cell viability at 24h.Black bars represent cell viability at 72 h. Non-transfected cells were used as negativecontrols for cytotoxicity (K). Ethanol treated cells were used as positive controls forcytotoxicity (K+).



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reported by Perrault et al., 2009, smaller sized particles have theability to rapidly diffuse through the tumor matrix and thereforepossibly reach the target cell, increasing the therapeutic effect [33].However, not only particle size but also the surface charge density of thecarrier systeminuencesgene delivery,due to the fact that nanoparticlesmust also possessthe ability to interact with thekey extracellularbarrierto transfection, the cell membrane, and hence be internalized viadifferent uptake pathways[34]. This interaction is mainly governed by

electrostatic forcesinvolving negatively charged proteoglycansin the cellmembrane and positivelycharged particles. In respect, to particle surfacecharge our results demonstrate that all the nano delivery systemformulations possess a positive zeta potential above 20 mV andconsequently colloidal dispersion stability. Analyzing the surface chargedata presentin Table 1 is quiteinteresting to denote thatthe nanocarriersformulated with the sc pDNA topoisoform hold a lower overall value ofsurfacechargeincomparisontotheotherformulations,a ndingthatwillbe further explored.

Despite the inuence of morphology, size and zeta potential, in theoverall transfection efciency, the ability of the carrier system toencapsulate as much genetic material as possible, is also an essentialparameter. For this reason, we also determined the EE of thenanocarriers, in order to address if the presence of different topologicalisoforms would also inuence the carrier's ability to transport pDNA. Infact, as the results inFig. 4depict, EE of the sc pDNA isoform is indeedconsiderably greater than that of the oc pDNA. On the other hand,although the EE attained for nanocarriers formulated with nativeplasmid preparations is smaller than that of the sc pDNA, whencomparedto oc encapsulation,a signicantdifferenceis observed. Theseresults are likely a consequence of the presence of both oc and sctopoisoforms in native samples. Interestingly, akin to the differencesobserved in morphology, also encapsulation is inuenced, and in thisparticular case, also improved with the presence of a densely packedDNA topology. A possible explanation for this event has beenpreviouslyproposed by Turro et al., 1991, that mentioned that the compactgeometrical distribution inuences the outer DNA electric double-layer(Gouy-Chapman layer), mostly composed by ionic phosphate headgroups [35]. Therefore, the enhancement in sc pDNA encapsulation

levels might be explained by the torsional deformations associated withsc topology thatoverexposes nucleotide bases, inuencing the negativecharge of the electrical double-layer and the extent of the interactionwith the positively charged polymer backbone. In fact, it is recognizedthat sc pDNA possesses higher charge density in comparison to itsconformational variables (oc or linear) [36]. Nevertheless this issueremains rather uncertain and to our knowledge the surface chargedensity of a pure sc pDNA biomolecule has yetto be determined. Takingthis into account and in an attempt to further characterize thesupercoiled molecular assembly characteristics, the zeta potential ofthe different pDNA topoisoforms was determined. Remarkably, ourresultsshowtheexistenceofasigni cantdifference in thezeta potentialbetween the relaxed (oc) and compact (sc) isoforms, with the latterpresenting an extra negative charge. As a result, larger amounts of

counterions are condensed by the superhelical backbone and cantherefore be released upon complexation with polycationic polymers,promoting the establishment of favored interactions between sc pDNAand chitosan[37]. This remarkable phenomenon is descriptive of somebiorecognition of this topoisoform from behalf of the polymer duringparticle synthesis[37], and may explain the lower zeta potential valuefor the sc pDNA nanoparticles. Interestingly, the lowest values of LC, EEas well as highestparticle sizeobtained for oc pDNA formulations mightalso be correlated with the negative charge spatial distribution of therelaxed oc biomolecules, since as mentioned forsc pDNA, the density ofthe double-layer largelyinuences theinteractions established withthepolymer. Indeed, due to the fact that oc pDNA possesses smallercounterion displacement capacity it has less ability to condense thecationic chitosan backbone which results in the synthesis of larger

particles with smaller amounts of pDNA, in comparison to the other

formulations.Takingalso into accountthe occurrenceof favorable cross-interactions between sc pDNA and polycations, it is important to pointout that these interactions are responsible for the maintenance of scisoform structural stability after its encapsulation in the nanocarrierssystems when an excessive amount of positive charges from thepolymer are present, as reported by Bronich et al., 2000,[38]. In ourparticular case since the synthesis of chitosan/pDNA nanoparticles isaccomplished in positive overcharging conditions, thermodynamic

stabilization of the sc topological conformation upon complexation istherefore promoted[38].Regarding the cellular uptake of the different nanoparticle formu-

lations the results obtained are in accordance both with the EE and themorphological characteristics of the different carriers. Particularly, scpDNA nanoparticles exhibited slightly higher cellular internalization(Fig. 5). This fact is likely a consequence of the combination of thephysicochemical characteristics of the sc pDNA synthesized nanopar-ticles, since particle size, zeta potential and morphology markedlyinuence the overall particle uptake[39].

The encapsulation capacity of the nanoparticulated systems is alsoemphasized inFig. 6since the amount ofuorescently labeled pDNAthat reaches the intracellular compartment is superior when chitosanbased nanoparticulated systems are employed as delivery vehicles, indetriment of cationic lipid-based nanocarriers. In fact as previouslyreported, chitosan is able to condense higher amounts of pDNA thancationic lipids [14]. All the results shown in Fig. 6 not only furtherevidence that thenanocarriers formulatedwith thedifferent topologicalisoforms efciently transpose the extracellular and intracellularbarriersbut also that the transport of the genetic material does not affect thecarrier transfection capacity. Actually, our group recently reported thatchitosan-baseddelivery systems alsoprotect pDNA fromthe deleteriousaction of DNA degrading enzymes present in serum, this is an importantfeature since as mentioned earlier, the material must upkeep itsstructural stability [20]. Upon intracellular localization the geneticmaterial is expected to follow the routes depicted in the schematics inFig. 1. In fact, as the MIP reconstruction images at different time-framesdepict, initially the pDNA is internalized in cell vesicles (Fig. 6I, O)Afterwards since vesicle disruption is promoted by the nanocarrier

systems (proton sponge effect) the pDNA is localized in the cytoplasmandthenthe intracellulartrafcking to thenuclear periphery is initiated(Fig. 6J and P), a transport that is thought to be essentially controlled bymicrotubules[40]. Finally the genetic material is then shuttled into thenucleus by the cell endogenous import machinery [41], (Fig. 6Q) whereit will ultimately be expressed. However, it is important to denote thatthe intracellular transit of the oc pDNA /chitosan particles is relativelyslowerthanthatof the native or sc,a fact clearly noticeable at 6 h wherethese particles are still localized in the cytoplasm, whilst the sc pDNAnanoparticles are extensively localized in the nucleus and the nativepDNA nanoparticles in the perinuclear space. These ndings maypartially explain the increased biological activity of the sc pDNAformulation due to the fact that the kinetics of gene expression maybe different among the studied formulations.

Altogether, as the results ofFig.7 demonstrate, p53 gene expressionis indeed re-establishedin malignantcells.As illustratedby Fig.7CandF,higher p53 protein levels are attained with sc pDNA mediatedtransfection either in nanoparticulated or liposome transfected cells,in comparison to other topologies. It should also be emphasized thatdespite transfection levels of nanoparticles and Lipofectamine 2000 arecomparable, the slightly higher levels of protein expression obtainedfor liposome transfected cells might be correlated with the differencesin pDNA vector unpacking properties of the nanocarriers at thesetransfection times[32]. Taking this into account, it is also essential topoint out that Lipofectamine mediatestransient transfection,whilst,thetransgene expression promoted by chitosan nanoparticles can achievequantiable protein levels in an extended time-frame as recentlyreported [19], thus, rendering it a morepowerful andvaluable approach

for a future p53 cancer-based therapy. In accordance with our p53 sc



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pDNA dependent transgene expression, Chandok et al., 2010 alsoreported that gene expression in mammalian cells is enhanced by thesuperhelicity of DNA, demonstrating that this topological conformationis also responsible forthe localized unwinding of the DNAmolecule andeffective recruitment of the cell replication machinery[42]. Apart fromthis, it is relevant to point out that this was a key experimentalndingsince it emphasizes the impact of the plasmid topological conformationnot only in the synthesis of the nanoparticulated carriers but also in the

expression of the therapeutic transgene. Indeed, the analysis performedfor the p53 mediated apoptosis has revealed that the sc pDNA trans-fected cells were those where higher apoptosis was attained, whencomparedtotheotherformulations(Fig.8). Thelatterresultsare furthersupported by the cytotoxic prole of the different isoforms, sincemalignant cell viability decreased over time with sc pDNA-mediatedtransgene expression (Fig. 9). Albeit, to exclude the possibility that thecellular viabilityand consequently apoptosisof malignantcells wouldbecorrelated with cytotoxicity of sc pDNA topoisoform non-malignantcells were also assayed for viability and as our results demonstrate cellviability has even increased over time, suggesting that p53-sc pDNAmediated transgeneexpression indeedinstigates a therapeuticresponse.

In summary, we have demonstrated that pharmaceutical-grade scpDNA is successfully recovered in a one step process, with high yield,and topological stability using arginine afnity chromatography, a costeffective andsimple approachfor purifying pDNA that can subsequentlybe encapsulated in biocompatible nanocarriers. These carriers haveshown the ability to encapsulate pDNA and mediate its delivery to thelocation where it will ultimately exert its therapeutic effect. To ourknowledge this was the rst time that a pure sc pDNA sample wasencapsulated onto chitosan-basednanoparticulated systems, hence thisstrategy may therefore also provide the foundations for novel researchdevelopments in non-viral gene therapy.

In conclusion, our collective approach provides valuable improve-ments regarding the delivery of genetic material as well as vectorassociated transfectionand will ina near futurebe used fora translationalp53-based anti-cancer therapy.

Acknowledgments

The authors would like to thank to Ana Martinho for her help withuorescence and western blot experiments and Dr. Thomas Robertsfor providing the pcDNA3FLAGp53 construct trough Addgene, ref:10838. The authors would also like to thank Dr. Olga Loureno for heradvice in ow cytometry experiments. This work was supported bythe Portuguese Foundation for Science and Technology (FCT), (PTDC/EME-TME/103375/2008 and PTDC/EBB-BIO/114320/2009).

Appendix A. Supplementary data

Supplementary data to this article can be found online at doi:10.1016/j.jconrel.2011.08.007.

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