P) as a tool for tunable transfection efficiency and apoptosis

Journal Pre-proof Cancer gene therapy mediated by RALA/plasmid DNA vectors: nitrogen to phosphate groups ratio (N/P) as a tool for tunable transfection efficiency and apoptosis A.R. Neves, A. Sousa, R. Faria, T. Albuquerque, J.A. Queiroz, D. Costa

PII:

S0927-7765(19)30754-4

DOI:

https://doi.org/10.1016/j.colsurfb.2019.110610

Reference:

COLSUB 110610

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

14 August 2019

Revised Date:

9 October 2019

Accepted Date:

22 October 2019

Please cite this article as: Neves AR, Sousa A, Faria R, Albuquerque T, Queiroz JA, Costa D, Cancer gene therapy mediated by RALA/plasmid DNA vectors: nitrogen to phosphate groups ratio (N/P) as a tool for tunable transfection efficiency and apoptosis, Colloids and Surfaces B: Biointerfaces (2019), doi: https://doi.org/10.1016/j.colsurfb.2019.110610

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Cancer gene therapy mediated by RALA/plasmid DNA vectors: Nitrogen to Phosphate groups ratio (N/P) as a tool for tunable transfection efficiency and apoptosis

A. R. Neves, A. Sousa, R. Faria, T. Albuquerque, J. A. Queiroz, D. Costa CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal 1

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Corresponding author: Diana Rita Barata Costa Universidade da Beira Interior

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6201-001 Covilhã Portugal

Number of Figures: 6

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Number of Tables: 2

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Number of words: 8480

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E-mail address: [email protected]

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Graphical abstract

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Highlights

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RALA/pDNA vectors were developed at different N/P ratios. Confocal microscopy studies confirmed pDNA uptake and nucleus localization. In vitro transfection of HeLa cells leads to gene release and protein expression. N/P ratio strongly tailors gene transfection efficiency and apoptosis. The great asset of this vector relies on N/P ratio as a tailoring parameter.

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    

Abstract

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Cancer gene therapy based on p53 tumor suppressor gene supplementation emerges as one of the most challenging and promising strategies. The development of a suitable gene delivery system is imperative to ensure the feasibility and viability of cancer gene

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therapy in a clinical setting. The conception of delivery systems based on cellpenetrating peptides may deeply contribute for the evolution of therapy efficacy. In this

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context, the present work explores the p53 encoding plasmid DNA (pDNA) condensation ability of RALA peptide to produce a suitable intracellular delivery platform. These carriers, formed at several nitrogen to phosphate groups (N/P) ratio, were characterized in terms of morphology, size, surface charges, loading and complexation capacity and the fine structure has been analyzed by Fourier-transformed infrared (FTIR) spectroscopy. Confocal microscopy studies confirmed intracellular localization of nanoparticles, resulting in enhanced sustained pDNA uptake. Moreover, in vitro transfection of HeLa cells mediated by RALA/pDNA vectors allows for gene 2

release and p53 protein expression. From these progresses, apoptosis in cancer cells has been investigated. It was found that N/P ratio strongly tailors gene transfection efficiency and, thus, it can be fine-tuned for desired degree of both protein expression and apoptosis. The great asset of the proposed system relies precisely on the use of N/P ratio as a tailoring parameter that can not only modulate vector´s properties but also the extent of pDNA delivery, protein expression and, consequently, the efficacy of p53 mediated cancer therapy. Keywords: RALA peptide; p53 gene; RALA/pDNA vectors; N/P ratio; in vitro transfection; cancer gene therapy; p53 protein; apoptosis.

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Introduction

Cancer gene therapy attracts great and widespread interest due to its biotechnological power, versatility and potential therapeutic effect. 1,2 In particular, gene therapy protocols

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focussed on the restoration of p53 protein function in tumour cells are a priority in this

field.3-6 The p53 tumour suppressor gene keeps the genome integrity under oncogenic

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stress being involved in several cellular pathways such as DNA repair, regulation of the cell cycle and induction of apoptosis. At the molecular level, mutation of the p53 gene is found in greater than 50% of human tumours and it is associated with an

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unfavourable prognosis of cure.7-9 For cancer gene therapy to be feasible and viable in a clinical setting, the development of a convenient and efficient gene delivery system is

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mandatory. Although viral vectors offer the highest transfection rates, they present significant drawbacks such as the given antigenicity, potential oncogenic effects, possible virus recombination or the difficulty in large scale production and storage. 10

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Alternatively, synthetic vectors can be produced in a fast, easy and tailored way, present lack of immune response and unlimited genome-carrying capacity, being considered

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incredible platforms for the intracellular delivery of therapeutic cargos.11-14 Among the broad variety of non-viral carrier materials, Cell-penetrating peptides (CPP) conquered a prominent role due to their structural characteristics, sequence and function.14-16 These peptides are generally short, up to 30 amino acids, and can be separated into two main groups: arginine-rich and amphipathic peptides.17 The latter class contains both hydrophilic and hydrophobic domains that are responsible for the interaction with genetic content and promotes cellular internalization. The cationic peptide RALA, modified from the well studied KALA peptide by the substitution of 3

lysine groups by arginine, contains 7 arginine residues and has an alpha helical structure comprising hydrophobic and hydrophilic amino acids.18 This structural upgrade confers it reduced toxicity and α-helicity at low pH, as arginine groups are protonated in acidic, neutral and most basic environments. 19,20 The interaction of RALA with nucleic acids has been investigated and suggests a condensation of genetic material with the consequent formation of nano-sized complexes exhibiting suitable properties for in vitro delivery.1,18,21,22 In this work, nanoparticles based on RALA and a p53-encoding plasmid DNA (pDNA) were developed at various N/P ratios and adequately characterized. We found that the properties of RALA/pDNA systems can be tailored

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and optimized by N/P variation. A fluorescence microscopy study illustrates the carriers uptake and internalization into HeLa cells with nucleus co-localization. This

phenomenon leads to efficient gene expression, p53 production and effective apoptosis induction in tumour cells. The extent of these events can be tuned by N/P ratio. In fact,

N/P ratio emerges as a crucial tailoring parameter for RALA/pDNA vectors

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performance in p53 gene delivery and therefore, on cancer therapy efficacy. The

findings of the present study strongly instigate further research, exploring the potential

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of the developed carriers for gene delivery applications.

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Materials and Methods

Materials. The RALA peptide (N-WEARLARALARALARHLARALARALRACEA-

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C) was synthesized by solid-state synthesis (fluorenylmethyloxycarbonyl, FMOC, Biomatik) and supplied as a lyophilized powder. Peptide stock solutions were prepared in ultrapure water and aliquots were stored at -20 ºC, according to manufacturer’s

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instructions. 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and fluorescein isothiocyanate (FITC) were obtained from Sigma-Aldrich. DAPI was from

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Invitrogen (Carlsbad, CA). The 6.07 kbp plasmid pcDNA3-FLAG-p53 (Addgene plasmid 10838, Cambridge, MA, USA) used in the experiments was produced and purified by a procedure developed by our research group and described in the literature.11 All solutions were freshly prepared by using ultra-pure grade water, purified with a Milli-Q system from Millipore (Billerica, MA, USA). Normal Human Dermal Fibroblasts (NHDF), Ref. C-12302 (cryopreserved cells) and cancer HeLa cells were purchased from PromoCell and Invitrogen, respectively.

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Preparation of RALA/pDNA complexes The nanoparticles were formed from the electrostatic interaction between cationic RALA peptide and negatively charged plasmid DNA. The complexes were conceived at various nitrogen to phosphate (N/P) ratios ranging from 1 to 50. The N/P ratio parameter is the molar ratio of positively charged nitrogen atoms in the peptide to negatively charged phosphates in the pDNA backbone. pDNA is a 6.07 kbp plasmid, with approximately 12,000 negative charges. RALA peptide contains 8 positive charges from arginine residues and 2 negative charges from the two glutamate residues, therefore, exhibiting a global net charge of +6. RALA lyophilized powder was

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suspended in ultrapure water to a concentration of 5.8 mg/ml and aliquots of 0.5 mg/ml were prepared from this stock and stored at -20 ºC. RALA/pDNA nanoparticles were prepared from RALA aliquots of 0.5 mg/ml and a plasmid solution with a concentration of 100 ug/mL. Nanoparticles, at different N/P ratios, were formed by adding variable

quantities of peptide, at vortex for 60 s, to a fixed amount of pDNA (1 µg). For instance,

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to prepare complexes at N/P ratios of 2, 5 or 10, volumes of 5.8 µL, 14.5 µL or 29 µL, respectively, of RALA solution (0.5 mg/ml) were mixed with pDNA and the mixture

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was left for 30 min at room temperature to allow particles formation. The complexes were centrifuged at 10,000 rpm for 20 min and the pellet contained the pDNA based

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vectors. The amount of non-bound pDNA was determined spectrophotometrically measuring the absorbance of the supernatant at 260 nm using a NanoPhotometer™ (Implen, Inc; Westlake Village, CA, USA). The pDNA complexation capacity was

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determined from the equation:

CC (%) = [(Total Amount of pDNA –Non-bound pDNA)/ Total amount of pDNA]

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×100 (1)

Morphology, size and zeta potential measurements Scanning electron microscopy was applied to obtain information regarding the morphology of RALA/pDNA complexes. The various systems were centrifuged (10,000 g, 20 min, 4 ºC) and the pellet was recovered and suspended in an aqueous solution containing 20 µL of tungsten (2%). The solution was placed in roundly shaped coverslip and dried overnight at room temperature. The samples were sputter coated with gold by using an Emitech K550 (London, England) sputter coater. A scanning electron 5

microscope, Hitachi S-2700 (Tokyo, Japan) with accelerating voltage of 20 kV at various magnifications was used to determine the morphology of nanoparticles. The average particle size and the zeta potential of pDNA based vectors have been determined by Dynamic Light Scattering (DLS), at 25 ºC, using a Zetasizer nano ZS. The pellet containing the particles was suspended in 5% glucose with 1 mM NaCl. DLS using a He-Ne laser 633 nm with non-invasive backscatter optics (NIBS) and electrophoretic light scattering using M3-PALS laser technique (Phase analysis Light Scattering) were applied for systems size and charge investigation, respectively. The

Cytotoxicity study

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Malvern zetasizer software v 6.34 was used.

The biocompatibility of the systems was evaluated on fibroblast cells by means of MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium

bromide)

assay.

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colorimetric method quantifies the metabolically active cells. Before cell seeding, the

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96-well plates were ultraviolet irradiated for 30 minutes. Human fibroblast cells were plated at a density of 1 × 104 cells per well and grown at 37 ºC in a 95% air/ 5% CO2

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humidified atmosphere. The vectors were applied to the well plates. After 24 h or 48 h incubation, the redox activity was assessed through the reduction of the MTT. The relative cell viability (%) related to control wells was calculated by [A]test / [A]control

Stability assays

control

is the absorbance

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of control sample.

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× 100, where [A] test is the absorbance of the test sample and [A]

RALA/pDNA complexes were incubated for different time periods (0, 1, 4 or 6 h) with

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25 µL of DMEM medium supplemented with 10% FBS or DNase I solution (4.5 mg mL-1) at 37 ºC. The release and pDNA degradation were monitored by agarose gel

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electrophoresis.

FITC plasmid labelling Plasmid DNA was stained with FITC by assembly 20 μL of pDNA, 63 μL of labelling buffer (0.1 M Sodium Tetraborate, pH 8.5) and 2 μL of FITC (in sterile anhydrous dimethyl sulfoxide, 500 mg/mL). Samples were placed under constant stirring for 4 hours at 4 ºC and protected from light. One volume of 3M NaCl (85 μL) and 2.5 volumes of 100% ethanol (212.5 μL) were added. Samples with the stained plasmid 6

were incubated at -20 ºC overnight. Thereafter, the samples were centrifuged at 4 ºC for 30 min and the pellet was washed with 75% ethanol.

In vitro transfection studies Cancer HeLa cells were grown in 25 cm3 T-flasks with Dulbecco´s Modified Eagle´s Medium with High Glucose (DMEM-HG) (Sigma) supplemented with 10% heat inactivated fetal calf serum, 0.5 g L-1 sodium bicarbonate, 1.10 g HEPES L–1 and 100 μg mL–1 of streptomycin and 100 units mL–1 of penicillin (Sigma), at 37 ºC in a humidified atmosphere, until confluence was attained. Afterward cells were subcultivated each 7 days to maintain their exponential growth. For transfection studies,

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cells were seeded at a density of 1×105 cells/well onto the poly-L-lysine coverslip 12-

well plate and grown in 1.5 mL complete medium. After 24 h and before transfection occurs, the complete medium was replaced by medium supplemented with 10% FBS

and without antibiotic, in order to promote transfection. At confluency (50-60 %), the

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medium was removed and washed with PBS; the cells were transfected with different

particles (100 µL of complexes were added to each well) and incubated for various

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periods of time. Live cell imaging

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HeLa cells were grown in µ-slide 8 well (Ibidi, Martinsried, Germany) until 50-60 % confluence was achieved. Nucleus was stained as described before, FITC-labeled pDNA

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was encapsulated into RALA based formulations and real live transfection was visualized using LSM 710 confocal laser scanning microscope (Carl Zeiss, Germany) under a 63 x magnification and analysed with the Zeiss LSM 710 laser scanning

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confocal microscope (Carl Zeiss SMT, Inc., Oberkochen, Germany). During the

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experiment, HeLa cells were maintained at 37 ºC with 5% CO2.

Reverse transcription polymerase chain reaction (RT-PCR) RT-PCR was used to detect the mRNA expression of the p53 gene. After transfection, the medium was removed and the cells were washed with PBS. Untreated cells were used as control. To extract total RNA, the cells were lysed through the addition of TRIzol (Thermo Scientific, Lisbon, Portugal) (250 µL) and incubated at room temperature for 5 min, followed by the addition of chloroform and vigorous stirring, following manufacturer´s guidelines. The obtained samples were quantified by using a 7

NanoPhotometer™ and additionally they were run on an agarose gel (1%) and analysed by electrophoresis to detect possible contaminations with genomic DNA or RNA degradation. The cDNA synthesis was performed by using the "Xpert cDNA Synthesis Kit" from Grisp (GRiSP, Porto, Portugal), following the manufacturer´s protocol. PCR amplification of p53 cDNA was performed adding in each PCR reaction 3.95 μL of RNase free water, 0.40 μL of primer reverse (5’- CCT CAT TCA GCT CTC GGA AC 3’) and primer forward (5’- CCT CAC CAT CAT CAC ACT GG -3’), 0.5 μL of MgCl2, 6.25 μL of Taq DNA polymerase and 1 μL of cDNA. The samples were homogenized and a mini-spin was performed. Samples were then placed in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc, Hercules, California, USA ), with the

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following conditions: denaturation (95 ºC for 40 s), annealing (60 ºC for 30 s), and extension (72 ºC for 1 min) for 29 cycles. Assays were performed in triplicate.

PCR products were analyzed by electrophoresis on an agarose gel stained with GreenSafe Premium (NZYTech, Lda. Lisbon, Portugal), and were visualized in UVItec

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Gel documentation system under UV light (UVItec Limited, Cambridge, United

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Kingdom).

Western blot

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The p53 and Bax proteins expression in transfected cells with the various vectors was measured by Western blot analysis. The transfected cells were harvested and lysed in lysis buffer (25 mM base Tris, 2.5 mM EDTA, 1% Triton X-100, 2.5 mM

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phenylmethylsulfonyl fluoride (PMSF) and EDTA-free protease inhibitor cocktail (Roche). The homogenate was incubated on ice for 10 min and then centrifuged at 10, 000 g for 1 min at 4 ºC. Supernatant was recovered and protein concentrations

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measured using Pierce 660 nm Protein Assay Reagent (Thermo Scientific USA). Equal amounts of protein (50 µg) from the supernatant fraction were separated by sodium

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dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were denatured (95 ºC for 10 min) and transferred to polyvinylidene difluoride filter (PVDF) membrane (95 V for 15 min; 130 V for 100 min). The membrane was blocked with 5% bovine serum albumin (BSA) at room temperature for 1h and then incubated with antip53 primary antibody (1:200) (Santa Cruz Biotechnology, CA, USA) and anti-BAX primary antibody (1:1000) (Cell Signaling Technology), respectively, overnight at 4 ºC, followed by treatment with p53 anti-mouse secondary antibody (1:5000) (SigmaAldrich) and BAX anti-rabbit secondary antibody (1:5000) (Sigma-Aldrich), 8

respectively, at room temperature for 1 h. The membrane was then incubated in β-actin primary (A3854, Sigma-Aldrich) antibody (1:20 000). ECL substrate (Thermo Scientific, USA) was used to signal detection and images were acquired by using a ChemiDoc™ XRS system (BioRad) and analysed with the Image Lab software (BioRad).

Protein quantification After transfection mediated by the developed RALA systems, cytosol has been isolated

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from the other cellular fractions through the use of the Mitochondria Isolation Kit for Cultured Cells (#89874, Thermo Fisher Scientific Inc., Rockford, USA), following the manufacturer´s instructions in order to separate essentially the mitochondria and the

cytosol. The levels of p53 in the cytosol of HeLa cells were quantified by using the p53

pan ELISA kit (Roche Applied Science), following all the instructions provided by the

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manufacturer. The p53 concentration can be determined by measuring the absorbance at

Caspase- 9 and caspase- 3 activation

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450 nm using Shimadzu UV-Vis 1700 spectrophotometer.

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The activity of caspase-9 and caspase-3 were measured using the Caspase-Glow® 9 Assay (Promega, USA) and the ApoAlertTM Caspase-3 Colorimetric assay kit (Clontech Lab. Inc, A. Takara, USA), respectively, following the provided instructions. Briefly,

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HeLa cells were serum starved and incubated with RALA/pDNA systems for 24 h. Incubation with 1 µM of staurosporine during the same time period has been considered as positive control. The activity of caspase-9 was measured through a homogeneous

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luminescent assay while caspase-3 was assayed by colorimetric detection, at 405 nm, of

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p-nitroaniline (pNA) after cleavage from the peptide substrate DEVD-pNA.

Statistical analysis One-way or two-way analysis of variance (ANOVA), followed by Bonferroni test was used for comparing data of control and multiple experimental groups. A confidence interval of 95% (p <0.05) was considered statistically significant. Data analysis was performed in GraphPad Prism v.7.01 (GraphPad software Inc., CA, USA).

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Results and Discussion

The properties of RALA/p53 complexes. RALA is a 30mer cationic arginine-rich peptide that strongly interacts, mainly by electrostatic forces, with the negatively charged pDNA forming nano-sized complexes. As for other systems reported in the literature, the N/P ratio considered at complex formulation step, was found to deeply influence the pDNA condensation profile. 11,14,15 This behaviour has been investigated by agarose gel electrophoresis. The results, for a broad range of N/P ratios, are

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presented in Figure 1. The interaction of RALA with p53-encoding pDNA leads to the immobilization of pDNA in a molar ratio-dependent manner. For lower N/P ratios, from

0.1 to 1, RALA is unable to condense the plasmid. From N/P ratio of 2, RALA efficiently neutralizes the pDNA charges and pDNA cannot migrate through the agarose gel remaining in the wells. An increment on N/P ratio parameter and, therefore, on

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amine positive charges strengths the interaction between the peptide and pDNA, greatly condensing the latter. This result on pDNA immobilization follows the same trend

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already documented for similar RALA/pDNA based vectors. 1,18,21 Furthermore, the encapsulation of p53-encoding pDNA into the complexes was confirmed by FTIR. In

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Figure S1 are represented the FTIR spectra from the different molecules and the complexes. The spectrum of pDNA (A) presents peaks in the region from, approximately, 1700-1500 cm-1 corresponding to the nitrogen bases, while the narrow

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band at 1053 cm-1 can be attributed to the carbonyl stretching vibration of furanose ring.23 Image S1B shows the RALA characteristic peaks, namely, the band at 3284 cm-1 from the C–H stretching and the bands at 1652 cm-1 and 1547 cm-1 from –COOH and

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amine groups, respectively. The spectrum of RALA/pDNA complexes at N/P ratio of 10 is represented in Image S1C. The nitrogen base region of pDNA overlaps with the

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characteristic amine bands of the peptide, and other regions must be analyzed to infer the interaction between pDNA and RALA. However, in this amine region some band shifts can be observed and are, most probably, a consequence of the complexation process. Moreover, the characteristic peak of RALA at 3284 cm-1 is shifted to lower wavenumber (3195 cm-1) after nanoparticles formation. The absence in Image S1C of the peak from vibration of furanose ring is also indicative of interaction involving the sugar-phosphate backbone of pDNA molecule. The band at 2352 cm-1 present in all

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spectra, certainly, results from incomplete CO2 purging of the spectrometer, as identified and reported by other authors. 24 To investigate the structural conformation of RALA/pDNA complexes, circular dichroism (CD) spectra were recorded for both peptide and RALA/pDNA nanoparticles (Figure S2). Spectrum of RALA peptide free in solution was mainly characterized by minima at 203 nm and 222 nm, and a maximum at 190 nm indicative of a pronounced α-helical structure (maximum peak at around 190 nm and minima at 207 nm and 222 nm).15 CD spectra from the complexes demonstrate that the peptide structure is maintained in the particles, Figure S2. However, the N/P ratio seems to influence the

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helicity of the complexes; while carriers at N/P ratio of 2 and 5 showed clear helical profiles, the spectrum from formulations at N/P ratio of 10 is less defined (Figure S2). This observation may be linked with a different extent of interaction between RALA and pDNA at this ratio.

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Scanning electron microscopy was applied to identify the morphology of RALA/pDNA

complexes. Figure S3 shows images of these carriers prepared at several N/P ratios. All

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the particles exhibit an oval or spherical shape with sizes lower than 500 nm. The mean size and the surface charges of RALA/pDNA formulations were further researched by

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dynamic light scattering. The results are summarized in Table 1. DLS confirms that all carriers exhibit a size below 500 nm. Moreover, the size of the particles strongly decreases as N/P ratio increases (****p < 0.0001). For highest N/P ratios, however, the

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decrease in particle size is less pronounced (*p < 0.05). From N/P ratio of 2, the size of RALA/pDNA vectors become more suitable for cellular internalization, as it has been reported that formulations exhibiting sizes below 200 nm turn easier both the cellular

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uptake and internalization processes, therefore, contributing for efficient gene transfection.25 The analysis of polydispersity index (Table 1) allows to infer that all the

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vectors are monodisperse. Additionally, all carriers present positive surface charges (Table 1). An increase in zeta potential values was found by incrementing the N/P ratio at which RALA/pDNA complexes were formed (***p < 0.001). As for the size parameter, for higher N/P ratios this effect is not significant (n.s). Positively charged nanoparticles interact favourably with the anionic proteoglycans present in the cell surface, being easily attached to the cell what favours the cellular internalization mechanism. Furthermore, it has been stated that delivery systems presenting zeta potential values equal to or higher than +30 mV are considered strongly cationic, fact 11

that enhances their performance to permeate the negatively charged cellular membranes.26 It must be, however, noted that, beyond the promising physicochemical properties exhibited by RALA/pDNA vectors, the success of gene transfection may greatly vary with cell type.27 In addition to the parameters investigated, the monitorization of pDNA complexation capacity is essential to promote an effective therapeutic action. The obtained pDNA CCs, for each particle system, are shown in Table 1. With the exception of the formulations at N/P ratio of 1, pDNA was efficiently complexed. The CC parameter is directly proportional to N/P ratio (***p < 0.001). However, no statistically significant differences were observed between vectors at

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higher N/P ratios (n.s). In particular, the nano-systems prepared at N/P ratios of 20 and 50 showed exactly the same pDNA CC. These results for pDNA complexation are in accordance with the pDNA immobilization profile observed by agarose gel electrophoresis, presented in Figure 1. Furthermore, all formulated complexes remain stable and keep their physicochemical properties over a period of, at least, one month.

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As there is no significant advantage in the use of nanoparticles at higher ratios than N/P

of 10, and as N/P ratio of 1 leads to higher sized particles with low pDNA CC, further

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experimental studies were performed considering, solely, carriers formulated at N/P

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ratios of 2, 5 and 10.

Cytotoxicity and stability studies. Cellular toxicity is a relevant subject to investigate when considering a delivery system for gene transfection applications. The

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biocompatibility of the developed carriers was evaluated through MTT assay on fibroblast cells. Fibroblasts have been chosen to evaluate the cytotoxicity of the nanosystems, due to their normal p53 protein levels. Being non-cancerous cells, any

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influence on viability profile can be attributed to the incubation with the formulated RALA/pDNA nanoparticles, and this study allows to unravel unequivocally their

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biocompatibility profile. Contrary, cancer cells present low p53 levels and the incubation with the p53-encoding pDNA based systems is expected to lead to p53 gene supplementation what results in the re-establishment of protein levels, and therefore, into a possible therapeutic effect with decrease of cancer cell viability. This fact has been observed in the current work and it will be discussed later. The results on fibroblast cells, at 24 and 48 h, for several vectors are summarized in Figure 2. As can be observed, none of the nanoparticles are toxic to the cells and, thus, it is not expected that they induce an immune or inflammatory response. However, 12

some differences were observed in the cell viability profile between the systems, related to N/P ratio. Our results seem to show that a more biocompatible formulation can be produced by increasing N/P ratio, as fibroblasts viability slightly increases with this parameter. The higher content of amine groups from the peptide seems to contribute to the exhibited biocompatibility. After 48 h incubation with the nanoparticles, the same trend was found; with however a small decrease in the cellular viability for all formulations (Figure 2). Beyond biocompatibility, it is crucial to evaluate the stability of RALA/pDNA particles into the extracellular compartment and the protection that the system confers to the transgene, as this will significantly affect the success of

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transfection. Hence, in order to get a deep insight into these issues, protection and stability studies have been performed for vectors at N/P ratios of 2, 5 and 10. The vectors were incubated with serum-supplemented DMEM + 10% FBS, for different

time periods (0, 1, 4 and 6 h), and electrophoresis was used to monitor the pDNA

protection. Figure 3 presents the obtained results. The data are consistent with the

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degradation of both the control pDNA and non-encapsulated pDNA (naked pDNA), for

all times of incubation, and with the protection of the genetic material, against serum

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degradation, by the developed carrier. Indeed, nanoparticles are able to ensure protection of the transgene, at least for a 6 h period of serum incubation. Furthermore,

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the protection against nucleases is a relevant aspect to be considered. Following this, the different nanoparticles were incubated with DNAse I for 1h and, in another experiment, with DNAse I + 10% SDS, to promote the de-complexation of pDNA from the vectors.

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The results are shown in Figure S4. The study with nucleases confirms the protection ability of the conceived delivery system, since pDNA seems to remain encapsulated into the carriers and do not suffer any degradation from the action of DNAse. Moreover, the

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assay in the presence of SDS evidences that pDNA is able to keep its integrity and supercoiled isoform after being released from the complexes, Figure S4. The pDNA

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supercoiled isoform is the most bioactive and the one that promotes higher levels of p53 gene expression.28 Therefore, our data suggests that the developed systems are suitable delivery vectors for therapeutic gene release in vivo.

Cellular uptake and intracellular location of complexes. The cellular uptake of various nano-systems into HeLa cells, after 4 h of transfection, was visualized by fluorescence confocal microscopy. The complete study is presented in 13

Figure S5. Nuclei are stained blue and pDNA is green labelled by FITC. The images, corresponding to the transfection mediated by RALA/pDNA carriers, show the presence of stained pDNA into the tumoral cells. Moreover, the extent of internalization seems to be strongly dependent on the considered N/P ratio. For formulations conceived at N/P ratio of 2, the green fluorescent dots from pDNA are very weak, almost undetectable. This seems to indicate a poor ability for cell uptake, internalization, and consequently, gene transfection. Contrary, vectors developed at N/P ratios of 5 and 10 are internalized at a higher extent. As discussed above, the properties displayed by these systems, such as their low size, positively charged surface and higher pDNA encapsulation capacity,

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can strongly promote their cell entry, internalization and pDNA accumulation. The colocalization analysis demonstrates the location of these vehicles, mainly, into the cell

nucleus. However, it should be considered the possibility of some carriers being located into the cytoplasmic compartment and perinuclear space. To further evaluate the

effectiveness of RALA/pDNA particles for pDNA delivery into HeLa cells, live cell

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imaging experiments were performed. Figure S6 illustrates the intracellular distribution of vectors at N/P ratio of 10, after transfection of HeLa cells for a period of 2, 4 and 6 h.

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These nanoparticles are able of cellular internalization and accumulate into the nucleus of cancer cells, already at 2 h. As time proceeds, more pDNA can be internalized and

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accumulates into the nucleus, Image S6 C. Figure 4 shows an orthogonal view of HeLa cells 4 h after transfection has been mediated with RALA/pDNA system and evidences the presence of stained pDNA into the nucleus. Furthermore, from our data, it is not

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evident that a longer time of transfection, 6 h, means that the vehicles are internalized into a higher extent (Supplementary Material, Figure S6). The fluorescence images seem to indicate that the degree of carriers internalization is comparable at 4 and 6 h of

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transfection, Figure S6. The microscopy study reveals, unequivocally, that the nanosystems, at N/P ratios of 5 and 10, are able to overcome both extracellular and

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intracellular barriers transporting the genetic content to the cytoplasm, possibly, followed by the nuclear trafficking to the nuclear periphery. 29 This mechanism is, apparently, controlled by microtubules; the genetic content is then transported into the nucleus by the cell endogenous import machinery, where the gene of interest can be expressed.30 Due to their different properties, the kinetics of gene expression can, however, be different among the developed carriers. Following this achievement, subsequent p53 gene expression is expected when using these vehicles for pDNA

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delivery into cancer cells. The expression of the transgene and the consequent protein production in HeLa cells will be discussed in the next section.

p53 gene and protein expression. After transfection of HeLa cells mediated by the developed delivery systems, p53 gene expression has been evaluated. p53 mRNA expression in transfected cells was determined by RT-PCR assay, using p53 specific primers. Untreated cells were used as control. The results are presented in Figure 5A. Considerable levels of mRNA of p53 were observed for all RALA/pDNA vectors, when compared to the mRNA content

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detected in control cells. Intense bands from p53 transcripts were found for all RALA/pDNA vectors, demonstrating that all systems seem to promote efficient gene

expression. The band intensities are comparable among the samples. The expression levels of p53 mRNA have been confirmed by quantitative real-time PCR (qPCR). Significant levels of mRNA were obtained for RALA/pDNA systems prepared at

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highest N/P ratios (data not shown). In order to determine the p53 expression induced

by the developed carriers in tumoral cells, western blot analysis was performed. As

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evidenced in Figure 5B, p53 protein seems to be produced for all carriers, although in a different extent. Nanoparticles at N/P ratios of 5 and 10 appear to present more intense

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bands, to what may correspond higher levels of protein. Although, control cells present normal mRNA levels, no protein was detected by western blot. This result on control can be justified by the fact that most cervical cancers contain wild type p53. However,

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although p53 mRNA can be identified in HeLa cells, previous works demonstrated that, in most of the cases, p53 protein cannot be detected, probably due to its degradation. Researchers deeply investigated this subject and found out an interesting relationship

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between E6 protein of the oncogenic mucosal-specific HPV types and p53.31,32 It is currently accepted that E6 protein complexes with p53 leading to its rapid proteasome-

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mediated degradation through a process involving a set of enzymes: E1 ubiquitinactivating enzyme, E2 ubiquitin-conjugating enzyme, E3 ubiquitin-protein ligase and the complex E6-AP responsible for the ubiquitination of p53 and its subsequent degradation by the proteasome complex 26S. 32

The results on protein expression were further confirmed by ELISA assay. Table 2 presents the obtained p53 levels in HeLa cells following transfection with naked pDNA and the various RALA/pDNA formulations. p53 protein was not detected when 15

transfection was mediated by naked pDNA, what reinforces the need for pDNA encapsulation into a delivery system in order to ensure the protection and stability of the transgene, thus, guaranteeing the success of transfection process. Different protein content can be produced depending on the N/P ratio considered. Vectors at N/P ratio of 2 present the lowest p53 amount, while as N/P ratio increases, higher p53 levels can be quantified in cancer cells (****p < 0.0001). The effect on p53 expression of increasing the N/P ratio from 5 to 10 is, however, quite small indicating that a plateau on transfection efficiency is perhaps reached at N/P ratio of 5 (not significant). Therefore, our results seem to prove that beyond N/P ratio of 5, there is no significant advantage in

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the increment of peptide amount into the complexes. This observation correlates well with the observed fluorescence images for the cell entry phenomenon, where a dramatic

difference is observed between vectors at N/P ratios of 2 and 5 or 2 and 10 (****p <

0.0001), but not between carriers at N/ ratios of 5 and 10 (Figure S5). Our data demonstrates that the transgene expression promoted by the highest N/P ratios

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RALA/pDNA vectors can lead to considerable p53 protein levels in HeLa cells,

suggesting that these formulations may represent a powerful and valuable tool for p53

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based cancer therapy.

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Vector-mediated apoptosis.

Cancer cell apoptosis induced by the transfection of RALA/pDNA vectors was investigated by TUNEL method. This assay gives information on apoptotic cell death

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by detecting DNA strand breaks.33 A summary of the performed study is presented in Figure S7. Through confocal microscopy observation, HeLa cells treated with DNAse I and staurosporine (positive controls) showed green staining, which indicated DNA

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fragmentation. Evidences of apoptosis induction can also be detected in cancer cells when transfection is mediated by the studied RALA/pDNA formulations. These

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findings agree well with those of DAPI-staining; this dye binds to the minor groove of DNA (AT) sites emitting fluorescence. 34 HeLa cells treated with RALA/pDNA particles exhibited a DAPI-positive phenotype and seem to present chromatin condensation, further demonstrate nuclear fragmentation. Although, through TUNEL, it is hard to predict differences between the systems it seems that increasing N/P ratio a more intense green fluorescence staining can be found in HeLa cells. The obtained images suggest that the carriers developed at N/P ratios of 5 and 10 induce cancer cell apoptosis in a large extent. This fact correlates well with the observed higher cellular 16

internalization for these systems, as well as, the higher levels of produced p53. The TUNEL assay has been supported by a strong evidence of cell induced apoptosis from a set of studies, discussed below. Apoptosis is a complex physiological process responsible for the maintenance of normal cell function, involving cysteinyl aspartate specific proteases.35,36 Initiator caspases (2, 8, 9 and 10) and execution caspases (3, 6 and 7) are the effector molecules of this cell death mechanism by two main pathways. Both extrinsic and intrinsic pathways lead to a cascade of molecular events that culminate into the activation of caspase-3.36 The majority of cells undergo cell death by the intrinsic pathway that uses mitochondria and mitochondrial proteins. The overall

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route is controlled by the action of BCL-2 protein family.36 Several apoptotic stimuli lead to BH3-only proteins, which in turn activate both Bax and Bak that oligomerize

originating the permeabilisation of the outer mitochondrial membrane. This phenomenon is considered the defining event of intrinsic apoptosis. Furthermore, permeabilisation process leads to the release of cytochrome c and other pro-apoptotic

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proteins into the cytosol, culminating with the formation of the apoptosome, the

activation platform for caspase-9.37 In line with this, the expression of Bax protein has

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been also monitored by western blot analysis (Figure 5B). Our results demonstrate the presence of Bax in cancer cells after transfection with RALA/pDNA formulations.

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Additionally, the mitochondrial membrane potential (ΔΨM) of HeLa cells has been determined. The measurement of ΔΨM, assessed by the fluorescence of the cationic dye 5,5,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1)38

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(Figure S8), indicates a reduction on this parameter when the transfection of HeLa cells is mediated by the developed nanoparticles (****p < 0.0001). The effect is more pronounced as N/P increases. Moreover, assaying for caspase-3 and caspase-9 activity

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further allows apoptosis detection giving information on the induced apoptosis pathway. Figure 6 shows that the transfection mediated by the nano-systems leads to the

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activation of both caspase-3 and caspase-9 activity and, thus, to apoptosis in HeLa cells, most probably through mitochondrial route. A remarkable difference in the fluorescence (Figure 6A) and luminescence intensity (Figure 6B) was found between carriers conceived at N/P ratio of 2 and the ones at N/P ratios of 5 and 10 (**p < 0.01). The lowest N/P ratio vectors lead to lower caspase-3 and caspase-9 activity to what may also correspond a lower extent of apoptosis in cancer cells. The difference between the highest N/P ratios is very small (not significant), with transfection using nanoparticles at N/P ratio of 10 promoting slightly higher caspases activity. 17

Altogether, our data deeply support the evidence of intrinsic apoptosis pathway as the main route for the RALA/pDNA vectors induced apoptosis. Additionally, an MTT assay on HeLa cells further supports the evidence of apoptosis. Table S1 presents a decrease in cellular viability after the incubation of HeLa cells (24 h and 48 h) with the conceived formulations (*p < 0.05). The extent of this phenomenon is dependent on the N/P ratio considered; a more significant inhibition of cell growth is achieved with systems prepared at higher N/P ratios and after two days incubation (*p < 0.05). This data indicates that the observed cell death can be a signal of apoptosis induced by p53 gene expression mediated by the nano-systems and not a cytotoxic

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effect promoted by the formulation itself. As demonstrated earlier on this report in a study on fibroblast cells, RALA/pDNA vectors are biocompatible. Therefore, at this stage we can then correlate the obtained results on Hela cells with efficient transfection,

effective gene expression and p53 protein supplementation in cancer cells with consequent apoptosis (Table S1). Other authors found the same phenomenon on HeLa

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cells when studying p53-encoding pDNA based nano-platforms.28,39 Altogether, these results on apoptosis agree well with the tendency, already observed, between the

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different N/P ratio vehicles, that is consistent with enhanced physicochemical properties, high ability for cell uptake/internalization, higher produced protein levels

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and large extent of apoptosis for the RALA/pDNA vectors developed at higher N/P ratios. Therefore, and strongly supported by our data, N/P ratio can be considered a powerful controlling parameter when exploring the suitability of RALA/pDNA carriers

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Conclusions

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to supplement p53 levels in cancer therapy.

RALA peptide/pDNA complexes have been developed at several N/P ratios and

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revealed to possess a set of properties that strongly vary with this parameter. This fact clearly opens the possibility of engineering tunable RALA/pDNA systems to optimize their characteristics, increasing their performance as delivery vehicles. In vitro studies on HeLa cells showed that RALA/pDNA nanoparticles are able of cellular uptake/internalization and localize in the cell nucleus. This ability resulted in effective p53 gene delivery and protein expression, in an extent dependent on the N/P ratio considered. The investigation of apoptosis demonstrated that the supplementation of p53 protein, mediated by the developed vector, induces cancer cell death. Furthermore, 18

this process can be modulated by adjusting N/P ratio parameter. Our collective approach brings significant improvements on the application of cell-penetrating peptides in the development of tailored and high-performance p53 gene based platforms for translational cancer therapy.

Declaration of Interests Authors have no competing or conflict interests to declare.

Acknowledgements

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D. Costa acknowledges the FCT program contract IF/01459/2015 supported by Fundo Social Europeu e Programa Operacional Potencial Humano. This work was supported

by FCT - Foundation for Science and Technology (project FCOMP-01-0124-FEDER041068 and FEDER funds through the POCI - COMPETE 2020 - Operational

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Programme Competitiveness and Internationalisation in Axis I - Strengthening research,

technological development and innovation (Project POCI-01-0145-FEDER-007491) and FCT - Foundation for Science and Technology (Project UID/Multi /00709/2013).

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This work was also partially supported by “Programa Operacional do Centro, Centro 2020” through the funding of the ICON Project (Interdisciplinary Challenges on CENTRO-01-0145-FEDER-000013)”

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Neurodegeneration;

and

PPBI-Portuguese

Platform of BioImaging through the Project POCI-01-0145-FEDER-022122. The authors would like to thank to C. Ferreira e A. Borges for their support in confocal

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[26] Clogston, J. D.; Patri, A. K. Methods in Molecular Biology 2011, vol. 697, Springer Science Business Media, LLC. Scott E. McNeil (Ed.). Characterization of nanoparticles intended for drug delivery. [27] Liu, X.; Wu, F.; Tian, Y.; Wu, M.; Zhou, Q.; Jiang, S.; Niu, Z. Size dependent cellular uptake of rod-like bionanoparticles with different aspect ratios. Sci. Rep. 2016, 6:24567. [28] Valente, J. F. A.; Sousa, A.; Gaspar, V. M.; Queiroz, J. A.; Sousa, F. The biological performance of purified supercoiled p53 plasmid DNA in different cancer cell lines. Process. Biochem. 2018, 75, 240-249. [29] Lam, A.; Dean, D. Progress and prospects: nuclear import of nonviral vectors. Gene Ther. 2010, 17, 439-447. 21

[30] Teo, P. Y.; Cheng, W.; Hedrick, J. L.; Yang, Y. Y. Co-delivery of drugs and plasmid DNA for cancer therapy. Adv. Drug Deliver Rev. 2016, 98, 41-63. [31] Scheffner, M.; Huibregtse, J.; Vierstra, R.; Howley, P. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 1993, 75, 495-505. [32] Thomas, M.; Pim, D.; Banks, L. The role of the E6-p53 interaction in the molecular pathogenesis of HPV. Oncogene 1999, 18, 7690-7700. [33] Majtnerová, P.; Roušar, T. An overview of apoptosis assays detecting DNA fragmentation. Mol. Biol. Rep. 2018, 45, 1469-1478.

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[34] Estandarte, A. K.; Botchway, S.; Lynch, C.; Yusuf, M.; Robinson, I. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes. Sci. Rep. 2016, 6:31417. [35] Baig, S.; Seevasant, I.; Mohamad, J.; Mukheem, A.; Huri, H. Z.; Kamarul, T. Potential of apoptotic pathway-targeted cancer therapeutic research: where do we stand? Cell Death Dis. 2016, 7, e2058.

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[36] Pfeffer, C. M.; Singh, A. T. K. Apoptosis: a target for anticancer therapy. Int. J. Mol. Sci. 2018, 19, 448-458.

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[37] Würstle, M. L.; Laussmann, M. A.; Rehm, M. The central role of initiator caspase9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome. Exp. Cell. Res. 2012, 318, 1213-1220.

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[38] Sivandzade, F.; Bhalerao, A.; Cucullo, L. Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe. Bio. Protoc. 2019, 9 (1): doi:10.21769/BioProtoc.3128.

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[39] Gaspar, V. M.; Correia, I. J.; Sousa, A.; Silva, F.; Paquete, C. M.; Queiroz, J. A.; Sousa, F. Nanoparticle mediated delivery of pure p53 supercoiled plasmid DNA for gene therapy. J. Control Release 2011, 156, 212-222.

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Figure 1. RALA/pDNA complexation behaviour at various N/P ratios investigated by agarose gel electrophoresis. The samples were loaded at the application site at the upper end of the image and the lower end is the cathodic end.

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Figure 2. Cellular viability of fibroblast cells after, 24 h or 48 h, incubation with RALA/pDNA nanoparticles at various N/P ratios. Percent viability is expressed relative to control cells. Non-transfected cells were used as negative control (K-) and ethanol treated cells were used as positive control (K+). Data is presented as mean ± SD, n = 3, and analysed by one-way ANOVA with the Bonferroni test. A statistically significant difference was noticed between the positive and negative control (*p < 0.05). For all RALA/pDNA systems, comparison with negative control is not significant (n.s). Figure 3. Eletrophoretic analysis of the vectors protection of pDNA after its incubation with serum supplemented DMEM + 10% FBS. The samples were loaded at the 22

application site at the upper end of the image and the lower end is the cathodic end. Image A - Lane 1: pDNA control; lane 2: non-encapsulated pDNA, 0 h; lane 3: nonencapsulated pDNA, 1 h; lane 4: non-encapsulated pDNA, 4 h; lane 5: RALA/pDNA N/P 2, 0 h; lane 6: RALA/pDNA N/P 5, 0 h; lane 7: RALA/pDNA N/P 10, 0 h; lane 8: RALA/pDNA N/P 2, 1 h; lane 9: RALA/pDNA N/P 5, 1 h; lane 10: RALA/pDNA N/P 10, 1 h; lane 11: RALA/pDNA N/P 2, 4 h; lane 12: RALA/pDNA N/P 5, 4 h; lane 13: RALA/pDNA N/P 10, 4 h. Image B – Lane 1: pDNA control; lane 2: non-encapsulated pDNA, 6 h; lane 3: RALA/pDNA N/P 2, 6 h; lane 4: RALA/pDNA N/P 5, 6 h; lane 5: RALA/pDNA N/P 10, 6 h. Figure 4. Orthogonal view of HeLa cells after 4 h of transfection mediated by RALA/pDNA vectors at N/P ratio of 10. Scale bar = 20 µM.

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Figure 5. PCR analysis of p53 mRNA in HeLa cells after 24 h of transfection mediated by RALA/pDNA nanoparticles prepared at N/P ratios of 2, 5 and 10 (A) and evaluation of p53 protein and Bax expression by western blot analysis after 48 h transfection with RALA/pDNA nanoparticles prepared at N/P ratios of 2, 5 and 10 (B). Untreated cells were shown as control. MW- DNA ladder molecular weight marker.

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Figure 6. Caspase-3 (A) and caspase-9 (B) activity in HeLa cells after 48 h of transfection mediated by RALA/pDNA vectors at N/P ratios of 2, 5 and 10. Negative control (K-) corresponds to untreated HeLa cells while incubation with 1 µM of staurosporine for 48 h was used as positive control (K+). Data are presented as mean ± SD, n = 3, and analysed by one-way ANOVA followed by Bonferroni test. The difference between positive e negative controls is statistically significant (****p < 0.0001). The vectors induced caspase-3 and caspase-9 activity relatively to negative control (****p < 0.0001).

23

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Table 1. Mean size, polydispersity index (P.I.), average zeta potential and pDNA complexation capacity (CC) for RALA/pDNA complexes formulated at several N/P ratios. The values were calculated with the data obtained from three independent measurements (mean ± SD, n = 3). Data were analysed by one-way ANOVA followed by Bonferroni test, p < 0.05 was considered statistically significant.

System

Size (nm)

P.I.

26

Zeta Potential (mV)

CC (%)

406 ±9.2

0.2 ±0.01

+2 ±0.8

47 ±3.9

RALA/pDNA N/P 2

331 ±4.7

0.3 ±0.01

+19 ±2.7

79 ±5.0

RALA/pDNA N/P 5

212 ±6.3

0.1 ±0.01

+31 ±1.8

91 ±4.8

RALA/pDNA N/P 10

139 ±4.9

0.3 ±0.01

+37 ±0.7

94 ±4.5

RALA/pDNA N/P 15

117 ±3.7

0.2 ±0.01

+38 ±2.8

96 ±3.6

RALA/pDNA N/P 20

102 ±3.9

0.2 ±0.02

+40 ±3.2

98 ±5.1

RALA/pDNA N/P 50

98 ±4.2

0.3 ±0.01

+40 ±1.9

98 ±2.7

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RALA/pDNA N/P 1

0

Naked pDNA

0

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Control cells

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p53 Content (ng/mL)

120 ±6.6

RALA/pDNA N/P 5

465 ±4.2

RALA/pDNA N/P 10

482 ±5.8

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RALA/pDNA N/P 2

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Table 2. Quantification of p53 protein levels (ng/mL) in the cytosol of HeLa cells after 48 h of transfection mediated by RALA/pDNA complexes at N/P ratios of 2, 5 and 10. The values were calculated with the data obtained from three independent measurements (mean ± SD, n = 3) and analysed by one-way ANOVA with the Bonferroni test. For all RALA/pDNA systems, ****p < 0.0001 relatively to control cells.

27