Nanoparticle mediated delivery of pure P53 supercoiled plasmid DNA for gene therapy

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Journal of Controlled Release 156 (2011) 212–222

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Nanoparticle mediated delivery of pure P53 supercoiled plasmid DNA for gene therapy Vítor M. Gaspar a, Ilídio J. Correia a, Ângela Sousa a, Filomena Silva a, Catarina M. Paquete b, João A. Queiroz a, Fani Sousa a,⁎ a b

CICS-UBI — Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal ITQB-UNL — Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal

a r t i c l e

i n f o

Article history: Received 2 May 2011 Accepted 5 August 2011 Available online 12 August 2011 Keywords: Gene therapy Nanoparticles p53 Tumor suppressor Supercoiled plasmid DNA

a b s t r a c t The translation of non-viral gene replacement therapies for cancer into clinical application is currently hindered due to known issues associated with the effectiveness of plasmid DNA (pDNA) expression vectors and the production of gene delivery vehicles. Herein we report an integrative approach established on the synthesis of nanoparticulated carriers, in association with the supercoiled (sc) isoform purification of a p53 tumor suppressor encoding plasmid, to improve both delivery and transfection. An arginine-based chromatographic matrix with specific recognition for the different topoisoforms was used to completely isolate the biologically active sc pDNA. Our findings showed that the sc topoisoform is recovered under mild conditions with high purity and structural stability. In addition, to further enhance protection and transfection efficiency, the naked sc pDNA was encapsulated within chitosan nanoparticles by ionotropic gelation. The mild conditions for particle synthesis used in the former technique allowed the attainment of a high encapsulation efficiency for sc pDNA (N 75%). Moreover, in vitro transfection experiments confirmed the reinstatement of the p53 protein expression and most importantly, the sc pDNA transfected cells exhibited the highest p53 expression levels when compared to other formulations. Overall, given the fact that sc pDNA topoisoform indeed enhances transgene expression rates this approach might have a profound impact on the development 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 based on the re-establishment of tumor suppressor regulated molecular pathways is raising new prospects on the outcome of an effective anticancer therapy [1]. The p53 protein is a unique tumor suppressor, proficient in the selective induction of growth arrest and apoptosis in response to oncogenic or damage signaling, acting as a prevailing guardian against malignant cell transformation [2]. On the other hand, it is estimated that the p53 gene is mutated or deleted in approximately 50% of all human cancers and its ubiquitous loss of function contributes as one of the underling events that trigger and sustain tumorigenesis [3]. It becomes therefore reasonable that the reinstatement of the wild-type p53 expression and consequent reactivation of its downstream effector pathways has impact on cancer therapy, as recently reported [4].

⁎ Corresponding author at: CICS-UBI — Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, 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

Non-viral delivery, arises as the most suitable approach for gene therapy due to markedly safety advantages over its viral counterpart [5]. However, an effective application of a p53 DNA-based cancer therapy has been hampered so far by issues associated with the intracellular delivery, transfection efficiency, and the purification of plasmid DNA (pDNA) expression vectors [5,6]. Purification of pharmaceutical-grade pDNA is a challenging process since the downstream processing must not be approached in an individual basis, but in an integrative perspective that accounts for cell impurities and contaminants such as RNA, genomic DNA (gDNA) and endotoxins that derive from the upstream stages, causing deleterious side effects if delivered to the host [7]. Moreover, despite the fact that pDNA is a very stable biomolecule, during its production and recovery, it can undergo several types of stress that may disrupt its structural stability [8]. This event must be accounted for, since it mainly affects the sc topoisoform, the only one considered intact and undamaged [7,8]. Nonetheless, it is important to point out that the sc pDNA topoisoform possesses the highest transfection efficiency both in vitro [9], and in vivo [8], when compared to the open circular (oc) or linear variants, rendering itself as a valuable alternative to improve transgene expression at the pDNA vector level. Considering the ever-growing need to meet the preemptive requirements of purity and structural stability, our group has recently developed a high throughput arginine affinity chromatography-based

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from Amersham Biosciences, (Uppsala, Sweden). Chitosan low molecular weight (LMW), pentasodium tripolyphosphate (TPP), Anti-VE Cadherin antibody, fluorescein isothiocyanate isomer I (FITC) and cell culture reagents 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 carcinoma cell line) and HeLa (Human negroid cervix epitheloid carcinoma) cells were purchased from ATCC, Middlesex, UK. Hoechst 33342®, AlexaFluor 546®, AlexaFluor 647®, and transfection reagents including Lipofectamine 2000®, were obtained from Invitrogen (Carlsbad, CA, USA). Analytical grade salts were used throughout this work.

approach to recover sc pDNA for gene therapy [10]. Affinity chromatography supports based on immobilized amino acids possess unique features since they take advantage of naturally occurring biological interactions between DNA and specific amino acids, purifying biomolecules from the standpoint of their biological function or chemical structure [10,11]. In addition, the intrinsic separation selectivity that also occurs between pDNA and contaminants, render it a noteworthy approach to purify plasmid biopharmaceuticals that comply with the strict regulatory directives issued for gene-based therapies [10]. However, despite these major improvements regarding vector transfection efficiency and safety, its delivery towards and into the intracellular compartment still remains a key setback, since naked pDNA is vulnerable to degradation (e.g. by serum nucleases and shear forces) [8], is rapidly cleared from systemic circulation (sc pDNA plasma half-life of 0.15 min) [12], and is rather inefficient in transposing extracellular barriers and consequently in attaining therapeutic expression levels [13,14]. These limitations regarding intracellular access, protection and bioavailability may be overcome if pDNA is packaged and protected in nanocarriers that can selectively encapsulate and deliver it in the cytoplasm [15]. Non-viral nanoparticulated carriers usually include cationic liposomes and cationic polymers such as chitosan [16]. Chitosan is a biocompatible and biodegradable polymer [17], with a cationic charged backbone that is responsible for its higher DNA loading capacity [16,18]. In fact, the charge density is its most important physicochemical feature, since it allows the polymer backbone not only to complex with DNA but also to instantly gel upon contact with oppositely charged crosslinkers and surfactants [14,19]. Taking this into account, we recently reported the synthesis of chitosan-pDNA loaded nanoparticles formulated by gelation with a counter polyanionic crosslinker [20]. As schematized in Fig. 1, herein, we report the development of an integrative approach that gathers the recent progresses regarding purification and transfection efficiency of plasmid biopharmaceuticals and the novel generation of gene delivery vehicles, effectively covering the key issues that currently hinder the translation of non-viral gene therapy into clinical applications.

The pcDNA3–FLAG–p53 plasmid was amplified in a bacterial cell culture of Escherichia coli (E. coli) DH5α and recovered as previously described [20]. Chromatography was conducted in a fast protein liquid chromatography system (FPLC), (Amersham Biosciences, Sweden) using a column packed with an arginine–sepharose 4B gel. All experiments were performed under controlled temperature conditions (4 °C) by using a water cooling system. The experiments were performed using two buffer solutions: (i) 10 mM Tris–HCl (pH 8.0) solution; (ii) 300 mM NaCl solution in 10 mM Tris–HCl (pH 8.0). The recovered pDNA isoforms were subsequently precipitated using one volume isopropanol and centrifuged for 30 min, 15,000 g, 4 °C, to remove the salt present in the pDNA samples. Afterwards, the pDNA pellet was then resuspended in 10 mM Tris–HCl (pH 8.0).

2. Materials and methods

2.4. Agarose gel electrophoresis

2.1. Materials

The agarose gel electrophoresis experiments were performed using a 1% agarose gel with ethidium bromide (0.5 μg/mL). Electrophoresis was carried out at 100 V for 45 min in Tris–Acetate–Ethylene Diamine (TAE) buffer. The agarose gels were revealed under UV light. Lane density measurements were performed in the software Bio-Rad Quantity One®, (Hercules, USA).

The 6.07 kbp pcDNA3–FLAG–p53 plasmid was purchased from Addgene (Cambridge, MA, USA). Arginine Sepharose 4B gel was obtained

2.2. Methods A general description of all of the used methodologies is provided as following. Nevertheless, a more detailed description is made available as supplementary material. 2.3. Plasmid biosynthesis and purification by affinity chromatography

2.5. Synthesis of chitosan and formulation of pDNA loaded nanoparticles LMW chitosan with a deacetylation degree (N98%) was purified as formerly reported [20]. Plasmid DNA loaded chitosan nanoparticles were 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 tripolyphosphate (TPP), were prepared. All the solutions were then filtered with a 0.22 μm filter to remove traces of solid particles. In order to promote encapsulation, pDNA (2 mg/mL) was added to the TPP solution prior to particle formation. Afterwards 1 mL of the pDNA–TPP solution was added dropwise to 4 mL of chitosan solution, under magnetic stirring (300 ± 50 rpm), at room temperature, for 30 min. The formulated nanoparticles were then pelleted by centrifugation at 17,000 g for 30 min.

Fig. 1. Schematics of an integrative approach for non-viral cancer gene therapy. (I.) Purification of plasmid biopharmaceuticals and recovery of the sc pDNA topoisoform using arginine affinity chromatography; (II.) Nanoparticle mediated delivery and transfection; (III.) Expression of the p53 tumor suppressor.

2.6. Morphology of the nanoparticles Particle morphology was analyzed by scanning electron microscopy (SEM) using a Hitachi S-2700 (Tokyo, Japan) electron microscope

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working at 20 kV and with different magnifications. Prior to image acquisition one drop of nanoparticles was dispersed through the surface of a cover glass and vacuum dried at 37 °C overnight. Afterwards the samples were mounted in microscope stubs and sputter coated with gold using a sputter coater. 2.7. Nanoparticle size Nanoparticle size was determined by dynamic light scattering (DLS). To determine the hydrodynamic diameter by DLS, chitosan nanoparticles were diluted in 800 μl of milliQ ultrapure water as a dispersant medium, followed by 30 min incubation at room temperature prior to analysis. Size measurements were then performed in a Zetasizer Nano ZS instrument (Malvern Instruments, Worcestershire, UK), in automatic mode and with a scattered light detection angle of 173°. All the measurements were performed in triplicate and measured 40 times. The reported particle size was obtained as an intensity distribution by cumulative analysis performed in the zetasizer software (version 6.20). 2.8. Zeta potential For zeta potential experiments chitosan nanoparticles were prepared as previously described for size measurements. The determination of the zeta potential of the different naked pDNA topoisoforms was also executed, with the pDNA samples dispersed in Tris–HCl 10 mM (pH 8.0). Zeta potential quantification was carried out in a Zetasizer Nano ZS instrument using a zeta dip cell. The experiments were performed in triplicate and an average of 100 measurements was acquired individually for each sample. 2.9. Encapsulation efficiency of pDNA and loading capacity To determine pDNA encapsulation efficiency (EE) nanoparticle samples were isolated by centrifugation, and the supernatant recovered for further analysis. The concentration of unbound pDNA was measured by UV–vis analysis (Shimadzu UV–vis spectrophotometer, Shimadzu Inc, Japan) as reported in the literature [20]. Loading capacity (LC) was determined by weighing blank tubes before the experiment and after nanoparticle recovery. 2.10. Fluorescence labeling of pDNA Plasmid DNA was fluorescently labeled using a FITC–diazonium salt as described elsewhere [21]. 2.11. In vitro transfection and cell culture A549, HeLa cells and rat skin Fibroblasts, isolated as previously described [17], were cultured in 25 cm2 T-flasks in Ham's F12K, Dulbecco's Modified Eagle's Medium (DMEM) and DMEM-F12 respectively, supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics–antimicotics, at 37 °C, in an humidified atmosphere with 5% CO2. One day prior to transfection malignant cells were seeded in 24 well plates (4× 104 cells/well). On the day of transfection, cells at 90– 95% confluence were transfected with either Lipofectamine 2000 or nanoparticles. 2.12. Cellular uptake of nanoparticles To characterize the amount of chitosan/pDNA nanoparticles uptake by the cells flow cytometry experiments were conducted by using fluorescently labeled FITC–pDNA conjugates at different transfection times (2 h and 6 h).The experiments were performed on a BD FACSCalibur flow cytometer and the acquisition of the data was made in the CellQuest TM Pro software where 1 × 10 4 events were counted.

Initially, to determine the optimal gating parameters and the possible auto-fluorescence of the malignant cells a screening with non-labeled cells was performed by accumulating the signals corresponding to forward and side scatter measurements (FSC and SSC) and defining the region of interest (ROI) according to a given threshold level. After the establishment of the ROI the resulting fluorescent signals of 2 × 10 4 events were recorded with the FL-1 (530/30 nm) band pass filter. Flow cytometry dot plots were analyzed in FCS Express version 4 Research Edition (De Novo Software™, LA, USA).

3. Immunofluorescence and confocal laser scanning microscopy (CLSM) Fluorescence experiments were performed with confluent cells. After transfection with either Lipofectamine 2000 or nanoparticles, the cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min, permeabilized and blocked for 3 h at room temperature. The cells were then incubated with the primary anti-VE cadherin monoclonal antibody (dilution 1:250) for 1 h and subsequently rinsed 10 times with PBS-Tween 20 (PBS-T) solution. To stain the nucleus of the cells a Hoechst 33342® molecular probe was then added and incubated for 15 min followed by 10 washing steps with PBS-T. After nuclear staining, the cells were incubated with an AlexaFluor 546® 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 image analysis. To address the intracellular localization and movement of pDNA, CLSM was performed. Confluent cell monolayer's were fixed, blocked and stained similarly to immunofluorescence however with the exception of the secondary antibody incubation, which was conducted with a far-red AlexaFluor 647® goat anti-(rabbit IgG) antibody. Confocal images were obtained with a Zeiss LSM 710 laser scanning confocal microscope (Carl Zeiss SMT Inc., USA) equipped with a plane-apocromat 63×/DIC objective. To obtain enough data for 3D reconstruction, a series of sequential slices, with different slices thickness (μm), were acquired along the Z-axis using optimized pinhole parameters in order to comply with the Nyquist-Shannon sampling theorem and minimize image aliasing during acquisition. All of the acquired Z-stacks were open as a merged file in the LSM 710 software (Carl Zeiss SMT Inc., USA) where subsequent 3D reconstruction was performed. Fast maximum intensity projections (MIP) of the reconstructed images were obtained using Huygens Essential software (Scientific 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 flow cytometry. Twenty four hours after transfection, cells were fixed with 4% PFA for 15 min at room temperature, permeabilized with 1% Triton X-100 for 5 min, and trypsinized using Trypsin/EDTA. The cell suspension was then pelleted and resuspended in ice cold PBS, 5% FBS, followed by incubation with rabbit anti-p53 antibody (10 μg/mL) (sc-6243, Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. After incubation the cells were spun down and resuspended in PBS 3 times to remove unbound anti-p53. Afterwards the cells were incubated with an anti-rabbit Alexa 647 secondary antibody for 1 h and washed as formerly described. Fluorescence was then detected with the FL-4 (661/16) band pass filter on a BD FACSCalibur flow cytometer where 1 × 10 4 events were recorded. Data analysis was performed in FCS Express version 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. Quantification of apoptosis P53 mediated apoptosis in transfected A549 and HeLa malignant cells was assessed by the Annexin V–FITC and PI kit (Calbiochem, USA) according to the manufacturer's instructions. Annexin V–FITC/PI labeled cells were excited with a 15 mW laser at 488 nm and the resulting fluorescent signals of 2 × 10 4 events were recorded with FL-1 (530/30 nm) and FL-2 (585/42) band pass filters. Non transfected cells were used as controls. Flow cytometry dot plots were analyzed in FCS 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 was performed both in HeLa cells and rat skin Fibroblasts, according to the manufacturer instructions. Twenty four hours prior to the experiment the cells were seeded at a density of 2 × 10 4 cells per well into 96-well flat bottom culture plates with 200 μL of cell culture medium supplemented with 10% FBS, without antibiotics. On the day of the experiment the culture medium was aspirated and replaced by fresh medium. The cells were then incubated with 30 μL of nanoparticle formulations for 24 and 72 h. All the formulations of nanoparticles were resuspended in pre-warmed culture medium containing 10% FBS and then added to each well. A total of five replicates were considered for each formulation. 3.4. Statistical analysis Comparison between multiple plasmid formulations was performed using one-way analysis of variance (ANOVA), with the Student–Newman–Keuls test. Comparisons between plasmid formulations were determined using a two-tailed Student's t-test. A value of P b 0.05 was considered statistically significant. 4. Results

Fig. 2. Selective separation of oc and sc topoisoforms by arginine affinity chromatography. (A) Chromatographic profile. Peak 1: weakly bound species; Peak 2: tightly bound species. (B) Electrophoresis of peaked fractions. Lane MW: molecular weight marker; Lane N: native pDNA sample. Lane 1: Peaked fraction 1; Lane 2: Peaked fraction 2. (C) Density analysis of the corresponding lanes in agarose gel electrophoresis.

4.1. Purification of sc pDNA by arginine affinity chromatography Purification of pDNA by arginine affinity chromatography presents significant advantages over the existing strategies because sc pDNA is recovered with high yield, structural stability and in a single purification step as it was recently reported for a model plasmid (pVAX1–LacZ) [9]. However, given the fact that specific and reversible bioaffinity interactions are involved in the recognition of the different topological isoforms, inherent particularities in arginine–pDNA interactions arise for different pDNA expression vectors. For this reason, the adjustment of the conditions that promote the recovery of the different topoisoforms is essential to achieve high resolution and selectivity profiles that in turn influence the overall recovery yield and purification degree of the plasmid of interest. The obtained chromatographic profile 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 mM NaCl (peak 1) and 300 mM NaCl (peak 2) were detected. In order to establish a correlation between the different plasmid topoisoforms and the eluting peaks in the chromatogram, an agarose gel electrophoresis was performed (Fig. 2B). The results revealed that the elution of the oc topoisoform occurs in the first peak (Fig. 2B, lane 1), at low ionic strength. Whereas, elution of the sc plasmid bound topoisoform only takes place at higher ionic force (Fig. 2B, lane 2), implying that the interaction with the arginine–agarose matrix is stronger. Additionally, the results presented in Fig. 2B also suggest that 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 the result in Fig. 2C depicts, the density peak indeed corresponds only to the sc topoisoform, that is therefore recovered with maximum purity and preserved structural characteristics.

4.2. Synthesis and characterization of pDNA loaded nanoparticles All formulations of pDNA loaded nanocarriers (i.e. with native, oc and sc samples) were synthesized by the ionotropic gelation technique which is based on the electrostatic interactions that occur between the positively charged polymer backbone, pDNA and a counter anionic crosslinker, TPP, that is responsible for the spontaneous gelation of chitosan. Taking into account the possible disturbance of the tertiary superhelical DNA structure and consequent topoisoform conversion (sc to oc or linear conformation), during particle synthesis, a stability study was performed prior to the production of the different particle formulations. Our results demonstrated that the submission of pDNA to the pH range and stirring parameters used does not promote significant topoisoform conversion since the sc pDNA content remained higher than 95% (Supplementary Fig. S1), a value that is approximately the one issued in regulatory directives for the use of plasmid DNA biopharmaceuticals in therapeutic applications (sc pDNA content N 97%) [8]. It is important to point out that the topoisoform conversion in other nanoparticle manufacturing techniques is markedly higher [29].

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Fig. 4. Encapsulation efficiency of the nanoparticles obtained for the different pDNA preparations; native pDNA, oc and sc. Each bar represents the mean ± s.d. (n = 3). *P b 0.05, **P b 0.01. Fig. 3. Characterization of nanoparticle morphology by SEM. (A) Nanoparticles formulated with native pDNA (Scale bar — 500 nm). (B) Nanoparticles formulated with oc pDNA (Scale bar — 500 nm). (C) Nanoparticles formulated with sc pDNA (Scale bar — 1 μm).

The morphology of the different nanocarriers obtained is presented in Fig. 3. Nanoparticulated systems formulated from native and oc pDNA preparations showed randomly shaped particles with spherical, rod and oval-like morphologies as shown in Fig. 3A and B. However, to address the possible influence of the chitosan/ oc pDNA ratio in particle morphology, different formulation ratios were also investigated. Our findings demonstrate that at higher ratios (5:1; 6:1) the synthesized particles possess the same random morphological characteristics, and at lower ratios no particles are formed under the given formulation conditions (Supplementary Fig. S2). Whereas, the nanoparticles obtained from the sc pDNA samples (Fig. 3C) demonstrated well defined spherical shapes. Other relevant characteristics such as particle size, zeta potential, and loading capacity were also determined for the different formulations. As shown in Table 1, all of the nanocarriers demonstrated narrow size distribution with particle sizes suited for delivery in tumoral microenvironments. Moreover, the chitosan nanoparticles formulated with the different topological pDNA isoforms exhibit a strong positive charge on their surface, an important feature that not only influences particle–cell membrane interactions but also particle colloidal stability. Regarding the results of the loading efficiency of the nanocarrier systems it should also be pointed out that all the formulations yielded particles comprised by a significant content of genetic material (44–58%) when compared to the content of chitosan–TPP (42–56%), meaning that the delivery vehicle not only upholds its important chitosan associated features but is also formed by a considerable amount of the therapeutic transgene. The findings related with the process yield are similar for the different formulations and in accordance with those reported in the literature [22].

Table 1 Characterization of the different formulations of nanocarriers. Data is presented as the mean ± s.d. .(n = 3). Sample

Ratio

Particle size DLS (nm)

Zeta potential (mV)

Particle loading capacity (%)

Yield (%)

pDNA (sc + oc) Oc Sc

4:1

157.6–197.4

+ 20.24 ± 18.17

51.18 ± 6.71

26.54 ± 4.29

251.3–272.0 109.8–162.5

+ 34.55 ± 14.63 + 22.6 ± 4.94

41.44 ± 0.51 42.80 ± 2.54

21.42 ± 0.33 20.93 ± 1.24

4.3. Nanocarrier pDNA encapsulation ability Fig. 4 presents the EE for the nanoparticulated systems synthesized with the different plasmid preparations. Our results showed that the encapsulation of sc pDNA is significantly higher than that of native or oc topologies. Moreover, it is important to point out as well that oc based nanocarrier formulations possess the lowest EE of all tested samples. Interestingly, the results are suggestive of a pattern associated encapsulation, since the presence of the sc topoisoform (both in native and pure sc samples) increases encapsulation. These findings are important for the overall formulation process and may be indicative that condensed pDNA topology plays somehow an important role in positive/negative, polymer–DNA interactions. In fact, to further explore this possibility the surface charge of the pDNA biomolecules was determined. The results presented in Table 2 indeed illustrate a distinct difference regarding the electrostatic characteristics of the various topoisoforms. These striking results will be further discussed. 4.4. Cellular uptake of nanoparticles The results presented in Fig. 5 characterize the cellular internalization of the different nanoparticles with either native (oc + sc), oc and sc pDNA topological isoforms. The mean fluorescence intensity values obtained reveal a slightly increased cellular uptake for sc pDNA nanoparticles in comparison to the other formulations at 2 h (Fig. 5). In addition, this difference in the internalization of the sc pDNA nanoparticles is increased after 6 h of transfection (Fig. 5). Regarding the cellular uptake of native pDNA and oc nanoparticles, the amount of DNA transported to the intracellular compartment is higher for the nanoparticles synthesized with native pDNA. 4.5. In vitro delivery and intracellular trafficking Analyzing the in vitro transfection results depicted in Fig. 6 it is clear that either nanoparticulated carriers or commercial Lipofectamine are capable of transporting the genetic material to the cell as previously demonstrated in cellular uptake experiments. However, a thorough analysis of Fig. 6B and F reveals that the dispersion of the green

Table 2 Characterization of the charge distribution of the different plasmid topological isoforms. Sample

Solution

Zeta potential (mV)

pDNA (oc + sc) Oc Sc

10 mM Tris–HCl (pH = 8.0)

− 16.4 − 7.31 − 9.33

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

fluorescent dots (corresponding to pDNA) is clearly increased when nanocarrier mediated delivery takes place. The intracellular movement and localization of pDNA can be illustrated by the Figures obtained at 2 h, 4 h and 6 h of transfection. As demonstrated in the CLSM images sc pDNA-based delivery systems are localized within the cytoplasmic compartment, perinuclear space and nucleus (Fig. 6O, P and Q) at 2 h, 4 and 6 h respectively. Whereas, native pDNA-based nanocarriers are only localized in the perinuclear space, not reaching the nucleus as fast as sc

Fig. 6. Immunofluorescence and CLSM of A549 cells. (A, E) Nuclear staining with Hoechst® 33342 (blue); (B, F) FITC labeled sc pDNA (green); (C, G) Staining with AntiVE cadherin-Alexa 546 antibody (red); (D, H) Merged images; (I, J, K) MIP images of chitosan/native pDNA mediated transfection at different time-frames, 2 h, 4 h and 6 h respectively; (L, M, N) MIP images of chitosan/oc pDNA mediated transfection at different time-frames, 2 h, 4 h and 6 h respectively; (O, P, Q) MIP images of chitosan/oc pDNA 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 pDNA topological isoforms; (B, E) Mean fluorescence intensity (MFI) values of p53 labeled Alexa 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 transfection are 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, both nanoparticle and Lipofectamine 2000 mediated transfection lead to the restoration of the expression of the p53 tumor suppressor. Notwithstanding, it is important to denote that different expression levels were obtained according to the different plasmid formulations (native, oc or sc). In fact, this is also observed in the histograms and mean fluorescence values depicted in Fig. 7A, D and B, E respectively, where sc pDNA mediated transgene expression levels are higher than those of native and oc formulations, both in nanoparticle and Lipofectamine transfected cells. Additionally the same expression profile associated with the different pDNA topological isoforms is also attained in western blot (Supplementary Fig. S3), thus, further evidencing the existence of a topoisoform-associated transgene expression profile. 4.7. P53 mediated apoptosis in malignant cells The results in Fig. 8 demonstrate the quantification of p53 mediated apoptosis in HeLa cells. As depicted by the dot plots (Fig. 8B, C and D), it is clear that programmed cell death occurred to a much higher extent in the sc pDNA transfected cells in comparison to the other formulations. Regarding the apoptosis assay in A549 cells, much lower apoptosis was observed (≈4% for sc pDNA) when put side by side with that attained for HeLa cells. However, it is important to point out that our values were in accordance with those obtained for p53 transfection with adenovirus [23], and hence demonstrate that the nanoparticles are also quite effective delivery vehicles. These results are likely a consequence of the fact that A549 cells are resistant to p53 mediated apoptosis as recently reported [24]. It is important to underline that despite the low apoptosis values in A549 cells the same apoptosis pattern obtained in HeLa cells for the different formulations was preserved, with the sc pDNA transfected cells yielding the higher percentage of apoptotic/late apoptotic cells. 4.8. In vitro characterization of the cytotoxic profile of nanoparticles The cellular cytotoxicity profile of all the chitosan/pDNA formulations was characterized to address whether p53 dependent apoptosis is only correlated with the activity of the tumor suppressor protein and not to a cytotoxic effect of the synthesized formulations. As presented in Fig. 9A and B, at 24 h, cellular viability is clearly not affected by the existence of the native, oc or sc pDNA nanoparticles since the majority of cells remained viable (N95%) both in malignant and non-malignant cells. However, in order to provide additional insights onto the influence of the therapeutic approach the cytotoxic index was also determined after 72 h. This approach brought forth significant cell viability differences between nanocarrier formulations. In fact, analyzing the results for malignant cells, those that were transfected with the sc pDNA nanoparticles exhibit a much lower viability in comparison to their topological counterparts. This is an interesting result since this pattern is not observed in non-malignant cells where cell viability remains unchanged and even increases at 72 h, hence, suggesting that the drop off in tumoral cells viability

Fig. 8. P53 tumor suppressor mediated apoptosis in HeLa cells induced by different pDNA topoisoforms. Representative dot plots of Annexin V–FITC (x-axis)/PI (y-axis) double staining of (A) Untransfected cells (control); (B) Cells transfected with native pDNA. (C) Cells transfected with oc pDNA. (D) Cells transfected with sc pDNA. (E) Percentage of apoptotic/late apoptotic HeLa cells.

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

might be correlated with the expression of the p53 therapeutic transgene. 5. Discussion Nowadays several approaches are being devised in order to translate non-viral cancer gene delivery into clinical applications [1]. However, known issues associated with insufficient transfection efficiency of both pDNA vectors and delivery vehicles, has restrained the outcome of a widespread and effective anti-cancer therapy. In the present study, we propose a novel approach that successfully covers the limitations associated with non-viral gene therapy, not only improving transfection and delivery of the genetic material, but also enhancing purity and transgene expression efficiency of plasmid vectors. To bring about the potential of this approach, native pDNA preparations were initially processed using a high throughput arginine affinity chromatography support, in order to recover the plasmid topoisoform with higher biological activity and transfection efficiency (sc pDNA) [9]. In fact, our results demonstrate that the arginine support was indeed able to selectively recognize the different pDNA topological conformations under the established retention/elution conditions, since sc pDNA remained bound to the support, at low salt concentration, whilst oc pDNA was totally eluted, as depicted in the chromatographic profile of Fig. 2A. This unique selectivity feature of the chromatographic support is a consequence of the interaction with the nucleotides bases and pairing preference between particular amino acids and nucleotide bases, namely arginine–guanine [25]. Indeed as previously reported by our group, arginine–guanine pairing via Hbonding is the prevalent interaction when selective separation of both topoisoforms occurs [26]. In addition, we also showed that these interactions are stronger for sc topologies due to nucleotide base exposure that occurs as a consequence of torsional strain deformations [26]. Our results for the pcDNA3–FLAG–p53 plasmid are then in

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accordance with those previously reported for pVAX1–LacZ [27], since the sc pDNA topoisoform is eluted at higher salt concentrations for both plasmids, suggesting that the chromatographic support specifically recognizes and strongly binds the sc topology regardless of the used plasmid. More importantly, as our results demonstrate the recovered sc pDNA samples presented 100% homogeneity and up kept all of their distinctive structural characteristics (Fig. 2B and C), that in turn largely influence the transgene expression efficiency and thus the therapeutic outcome. Although sc pDNA percentage is considered the most important quality attribute of a plasmid preparation [28], in order to translate nucleic acid-based cancer gene therapy into clinical applications, pharmaceutical-grade pDNA must also comply with current good manufacturing practice (cGMP) guidelines regarding protein, gDNA, and endotoxin levels. The latter is extremely important since the presence of endotoxins may cause deleterious side effects such as fever and complement system cascade which may elicit a severe immunological response [28]. Regarding these issues, we recently showed that the presence of contaminants in the sc pDNA sample recovered from the arginine–agarose was significantly reduced not only regarding the gDNA content (up to 117-fold reduction), but also endotoxins (95% removal) and proteins (undetectable levels) [9], therefore complying with cGMP for gene therapy applications. From this stand point it seems undoubtedly clear that major improvements regarding the issues associated with pDNA expression vectors may be outdone if arginine chromatography is employed in preparation of pharmaceutical grade sc pDNA. However, other key issues arise since pDNA is susceptible to degradation in the extracellular environment and rather unable to transpose cellular barriers in its naked form [14]. Therefore, its encapsulation in nanoparticulated delivery systems that are able to protect and deliver the transgene of interest into the intracellular compartment is a valuable approach. Hence, in order to synthesize nanocarrier systems that were capable of covering the previous issues and at the same time preserve the topological characteristics of pDNA vectors we selected ionotropic gelation technique for nanocarrier production because DNA encapsulation and particle formation both take place under milder conditions than other nanoparticle formulation techniques (spray-drying, sonication, complex coacervation) [29]. Most importantly since minor sc pDNA topoisoform conversion occurs, the bulk of the available DNA that interacts with the cationic polymer backbone during particle production is the biologically active sc pDNA conformation. Regarding particle formulation our results showed that nanocarriers with native and oc DNA samples formed particles with random shapes (Fig. 3). On the other hand, particles formed from sc pDNA interestingly presented defined spherical morphologies, most likely due to the compact form of the plasmid expression vector, suggesting that although pDNA molecular weight remains the same, condensed pDNA topology may influence the thermodynamics of the prevalent electrostatic interactions, and thereof the formation of spherical morphologies. Our results are consistent with those obtained by Dunlap et al., 1997, that reported the synthesis of round and globular shaped polyplexes using sc pDNA preparations, as opposed to those with rather undefined morphology, formed from less compact topologies [30]. These findings assume further significance, since as shown by Chithrani and Chan, 2007, nanoparticles with spherical shapes are wrapped promptly by the cell membrane (due to surface-to-volume ratio), and as a result exhibit greater uptake when compared with those with rod-like morphology [31]. Other relevant characteristic of the delivery system that definitely influences the overall transfection efficiency is nanoparticle size. The results herein obtained reveal that all of the nanoparticulated carriers have sizes that promote their accumulation in the microenvironment that encloses tumor cells, since the blood vessels that surround tumors possess fenestrations that range between 100 and 600 nm [32]. Nevertheless, it is also important to point out that as

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reported by Perrault et al., 2009, smaller sized particles have the ability to rapidly diffuse through the tumor matrix and therefore possibly reach the target cell, increasing the therapeutic effect [33]. However, not only particle size but also the surface charge density of the carrier system influences gene delivery, due to the fact that nanoparticles must also possess the ability to interact with the key extracellular barrier to transfection, the cell membrane, and hence be internalized via different uptake pathways [34]. This interaction is mainly governed by electrostatic forces involving negatively charged proteoglycans in the cell membrane and positively charged particles. In respect, to particle surface charge our results demonstrate that all the nano delivery system formulations possess a positive zeta potential above 20 mV and consequently colloidal dispersion stability. Analyzing the surface charge data present in Table 1 is quite interesting to denote that the nanocarriers formulated with the sc pDNA topoisoform hold a lower overall value of surface charge in comparison to the other formulations, a finding that will be further explored. Despite the influence of morphology, size and zeta potential, in the overall transfection efficiency, the ability of the carrier system to encapsulate as much genetic material as possible, is also an essential parameter. For this reason, we also determined the EE of the nanocarriers, in order to address if the presence of different topological isoforms would also influence the carrier's ability to transport pDNA. In fact, as the results in Fig. 4 depict, EE of the sc pDNA isoform is indeed considerably greater than that of the oc pDNA. On the other hand, although the EE attained for nanocarriers formulated with native plasmid preparations is smaller than that of the sc pDNA, when compared to oc encapsulation, a significant difference is observed. These results are likely a consequence of the presence of both oc and sc topoisoforms in native samples. Interestingly, akin to the differences observed in morphology, also encapsulation is influenced, and in this particular case, also improved with the presence of a densely packed DNA topology. A possible explanation for this event has been previously proposed by Turro et al., 1991, that mentioned that the compact geometrical distribution influences the outer DNA electric double-layer (Gouy-Chapman layer), mostly composed by ionic phosphate head groups [35]. Therefore, the enhancement in sc pDNA encapsulation levels might be explained by the torsional deformations associated with sc topology that overexposes nucleotide bases, influencing the negative charge of the electrical double-layer and the extent of the interaction with the positively charged polymer backbone. In fact, it is recognized that sc pDNA possesses higher charge density in comparison to its conformational variables (oc or linear) [36]. Nevertheless this issue remains rather uncertain and to our knowledge the surface charge density of a pure sc pDNA biomolecule has yet to be determined. Taking this into account and in an attempt to further characterize the supercoiled molecular assembly characteristics, the zeta potential of the different pDNA topoisoforms was determined. Remarkably, our results show the existence of a significant difference in the zeta potential between the relaxed (oc) and compact (sc) isoforms, with the latter presenting an extra negative charge. As a result, larger amounts of counterions are condensed by the superhelical backbone and can therefore be released upon complexation with polycationic polymers, promoting the establishment of favored interactions between sc pDNA and chitosan [37]. This remarkable phenomenon is descriptive of some biorecognition of this topoisoform from behalf of the polymer during particle synthesis [37], and may explain the lower zeta potential value for the sc pDNA nanoparticles. Interestingly, the lowest values of LC, EE as well as highest particle size obtained for oc pDNA formulations might also be correlated with the negative charge spatial distribution of the relaxed oc biomolecules, since as mentioned for sc pDNA, the density of the double-layer largely influences the interactions established with the polymer. Indeed, due to the fact that oc pDNA possesses smaller counterion displacement capacity it has less ability to condense the cationic chitosan backbone which results in the synthesis of larger particles with smaller amounts of pDNA, in comparison to the other

formulations. Taking also into account the occurrence of favorable crossinteractions between sc pDNA and polycations, it is important to point out that these interactions are responsible for the maintenance of sc isoform structural stability after its encapsulation in the nanocarriers systems when an excessive amount of positive charges from the polymer are present, as reported by Bronich et al., 2000, [38]. In our particular case since the synthesis of chitosan/pDNA nanoparticles is accomplished in positive overcharging conditions, thermodynamic stabilization of the sc topological conformation upon complexation is therefore promoted [38]. Regarding the cellular uptake of the different nanoparticle formulations the results obtained are in accordance both with the EE and the morphological characteristics of the different carriers. Particularly, sc pDNA nanoparticles exhibited slightly higher cellular internalization (Fig. 5). This fact is likely a consequence of the combination of the physicochemical characteristics of the sc pDNA synthesized nanoparticles, since particle size, zeta potential and morphology markedly influence the overall particle uptake [39]. The encapsulation capacity of the nanoparticulated systems is also emphasized in Fig. 6 since the amount of fluorescently labeled pDNA that reaches the intracellular compartment is superior when chitosan based nanoparticulated systems are employed as delivery vehicles, in detriment of cationic lipid-based nanocarriers. In fact as previously reported, chitosan is able to condense higher amounts of pDNA than cationic lipids [14]. All the results shown in Fig. 6 not only further evidence that the nanocarriers formulated with the different topological isoforms efficiently transpose the extracellular and intracellular barriers but also that the transport of the genetic material does not affect the carrier transfection capacity. Actually, our group recently reported that chitosan-based delivery systems also protect pDNA from the deleterious action of DNA degrading enzymes present in serum, this is an important feature since as mentioned earlier, the material must upkeep its structural stability [20]. Upon intracellular localization the genetic material is expected to follow the routes depicted in the schematics in Fig. 1. In fact, as the MIP reconstruction images at different time-frames depict, 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 cytoplasm and then the intracellular trafficking to the nuclear periphery is initiated (Fig. 6J and P), a transport that is thought to be essentially controlled by microtubules [40]. Finally the genetic material is then shuttled into the nucleus by the cell endogenous import machinery [41], (Fig. 6Q) where it will ultimately be expressed. However, it is important to denote that the intracellular transit of the oc pDNA /chitosan particles is relatively slower than that of the native or sc, a fact clearly noticeable at 6 h where these particles are still localized in the cytoplasm, whilst the sc pDNA nanoparticles are extensively localized in the nucleus and the native pDNA nanoparticles in the perinuclear space. These findings may partially explain the increased biological activity of the sc pDNA formulation due to the fact that the kinetics of gene expression may be different among the studied formulations. Altogether, as the results of Fig. 7 demonstrate, p53 gene expression is indeed re-established in malignant cells. As illustrated by Fig. 7C and F, higher p53 protein levels are attained with sc pDNA mediated transfection either in nanoparticulated or liposome transfected cells, in comparison to other topologies. It should also be emphasized that despite transfection levels of nanoparticles and Lipofectamine 2000 are comparable, the slightly higher levels of protein expression obtained for liposome transfected cells might be correlated with the differences in pDNA vector unpacking properties of the nanocarriers at these transfection times [32]. Taking this into account, it is also essential to point out that Lipofectamine mediates transient transfection, whilst, the transgene expression promoted by chitosan nanoparticles can achieve quantifiable protein levels in an extended time-frame as recently reported [19], thus, rendering it a more powerful and valuable approach for a future p53 cancer-based therapy. In accordance with our p53 sc

pDNA dependent transgene expression, Chandok et al., 2010 also reported that gene expression in mammalian cells is enhanced by the superhelicity of DNA, demonstrating that this topological conformation is also responsible for the localized unwinding of the DNA molecule and effective recruitment of the cell replication machinery [42]. Apart from this, it is relevant to point out that this was a key experimental finding since it emphasizes the impact of the plasmid topological conformation not only in the synthesis of the nanoparticulated carriers but also in the expression of the therapeutic transgene. Indeed, the analysis performed for the p53 mediated apoptosis has revealed that the sc pDNA transfected cells were those where higher apoptosis was attained, when compared to the other formulations (Fig. 8). The latter results are further supported by the cytotoxic profile of the different isoforms, since malignant cell viability decreased over time with sc pDNA-mediated transgene expression (Fig. 9). Albeit, to exclude the possibility that the cellular viability and consequently apoptosis of malignant cells would be correlated with cytotoxicity of sc pDNA topoisoform non-malignant cells were also assayed for viability and as our results demonstrate cell viability has even increased over time, suggesting that p53-sc pDNA mediated transgene expression indeed instigates a therapeutic response. In summary, we have demonstrated that pharmaceutical-grade sc pDNA is successfully recovered in a one step process, with high yield, and topological stability using arginine affinity chromatography, a cost effective and simple approach for purifying pDNA that can subsequently be encapsulated in biocompatible nanocarriers. These carriers have shown the ability to encapsulate pDNA and mediate its delivery to the location where it will ultimately exert its therapeutic effect. To our knowledge this was the first time that a pure sc pDNA sample was encapsulated onto chitosan-based nanoparticulated systems, hence this strategy may therefore also provide the foundations for novel research developments in non-viral gene therapy. In conclusion, our collective approach provides valuable improvements regarding the delivery of genetic material as well as vector associated transfection and will in a near future be used for a translational p53-based anti-cancer therapy. Acknowledgments The authors would like to thank to Ana Martinho for her help with fluorescence and western blot experiments and Dr. Thomas Roberts for providing the pcDNA3–FLAG–p53 construct trough Addgene, ref: 10838. The authors would also like to thank Dr. Olga Lourenço for her advice in flow cytometry experiments. This work was supported by the 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. References [1] J. Roth, J. Nemunaitis, L. Ji, R. Ramesh, Tumor suppressor gene therapy, GeneBased Therapies for Cancer, Springer, London, 2010, p. 275. [2] M. Junttila, G. Evan, p53—a Jack of all trades but master of none, Nat. Rev. Cancer 9 (2009) 821–829. [3] G. Evan, K. Vousden, Proliferation, cell cycle and apoptosis in cancer, Nature 411 (2001) 342–348. [4] A.S. Azmi, M. Millard, D. Pathania, F. Grande, S. Xu, N. Neamati, H. Shen, C.G. Maki, P. Secchiero, R. Bosco, Hot topic pharmaceutical reactivation of p53 pathways in cancer, Curr. Pharm. Des. 17 (2011). [5] D. Glover, H. Lipps, D. Jans, Towards safe, non-viral therapeutic gene expression in humans, Nat. Rev. Genet. 6 (2005) 299–310. [6] P.H. Oliveira, K.J. Prather, D.M.F. Prazeres, G.A. Monteiro, Structural instability of plasmid biopharmaceuticals: challenges and implications, Trends Biotechnol. 27 (2009) 503–511. [7] F. Sousa, D. Prazeres, J. Queiroz, Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA, Trends Biotechnol. 26 (2008) 518–525.

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