Colloids and Surfaces B: Biointerfaces 178 (2019) 430–438

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Colloids and Surfaces B: Biointerfaces

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A co-delivery platform based on plasmid DNA peptide-surfactant complexes: T

formation, characterization and release behavior
⁎
Diana Costaa, , Tânia Albuquerquea, João A. Queiroza, Artur J.M. Valenteb
a
CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
b
Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal

A R T I C LE I N FO A B S T R A C T

Keywords: The development of delivery systems based on cell penetrating peptides represents an incredible asset and may
Cell penetrating peptides deeply contribute for the evolution of therapies efficacy. In this context, we explore the plasmid DNA (pDNA)
Cationic surfactants condensation ability of TAT peptide to produce a suitable intracellular delivery platform. The nanoparticles were
pDNA compaction formed at various ratios of nitrogen to phosphate groups (N/P) and the variation of polyplexes properties with
pDNA-surfactant complexes
this parameter was studied. Beyond the large size exhibited by these carriers, their low pDNA immobilization
Controlled/sustained release
Drug/gene co-delivery
profile instigates the need for an additional compacting agent. To maximize the performance of this peptide
delivery system, a series of alkyl trimethyl ammonium bromide surfactants (CnTAB) were employed to further
condense pDNA. In general, not only this strategy promotes the formation of lower sized vectors, but also greatly
enhances particle characteristics such as surface charge and pDNA encapsulation. The magnitude of this effect is
intimately dependent on surfactant chain length. Furthermore, the known cytotoxicity of cationic surfactants has
been dramatically reduced by their incorporation into TAT/pDNA complexes. The release kinetics can be tai-
lored and optimized to promote the controlled/sustained release of pDNA. Following this, the surfactant alkyl
chain length and the N/P ratio are important controlling parameters. In addition, doxorubicin and paclitaxel can
be efficiently loaded and encapsulated into peptide/pDNA/surfactant carriers. The presented platform reveals a
great potential for therapeutic payloads loading and controlled release open advanced and new approaches in
the design/formulation of innovative biomedical systems towards clinical translation.

1. Introduction
The concept of gene therapy offers a promising therapeutic effect in
the treatment of a variety of diseases ranging from hereditary to ac-
quired (infection or cancer) pathologies. Viral vectors are very efficient
in the delivery of therapeutic genes to a desirable target site but their
immunogenicity and the possibility of oncogenesis occurrence limit
their use in gene therapy protocols [1]. On the other hand, non-viral
drug and/or gene delivery systems are widely used in both basic re-
search and translational clinical applications due to their biocompat-
ibility, lack of immune response, high payload content and versatile
preparation method [2,3]. In the last few years, developed innovative
carriers with recognized effect in human disease therapy include
polyplexes, lipoplexes, dendrimers and nanoparticles [4–8]. Along with
the targeting strategy, one of the harder tasks for efficient cell inter-
nalization and gene transfection is the crossing of cell membrane. To
surpass this barrier, peptides derived from natural proteins and pre-
senting ability to penetrate the plasma membrane have been
successfully used [9–12]. These peptides are normally short (less than
30 amino acids), frequently rich in arginine residues and positively
charged. The cationic profile of cell penetrating peptides (CPP) is the
key factor for their interaction with nucleic acids, giving rise to the
formation of micro or nanometer sized complexes. Among the most
common and efficient peptides explored for drug and/or gene release
purposes is the TAT protein of human immunodeficiency virus type 1
(HIV-1). The specific protein domain, TAT49-57 (RKKRRQRRR), also
named protein transduction domain sequence (PTD), has a stronger
ability to translocate the cell membrane and access intracellular en-
vironment [13]. It seems TAT49-57 can cross the membranes through
transient pores favored by the electrostatic attraction with the nega-
tively charged phospholipids [13,14]. Furthermore, it also acts as a
nuclear localization signal [15]. A broad range of interesting and out-
standing studies employing TAT peptide in the formulation of suitable
vectors for the delivery of bioactive payloads can be found in the lit-
erature [11,16–18]. Through the use of TAT, considerable progresses
have been made in the delivery of chemotherapeutic drugs, [13,18]

⁎
Corresponding author at: Universidade da Beira Interior, 6201-001 Covilhã, Portugal.
E-mail address: dcosta@fcsaude.ubi.pt (D. Costa).

https://doi.org/10.1016/j.colsurfb.2019.03.029
Received 12 November 2018; Received in revised form 8 March 2019; Accepted 13 March 2019
Available online 16 March 2019
0927-7765/ © 2019 Elsevier B.V. All rights reserved.
D. Costa, et al.
delivery and expression of genes [11,16] and, consequently, on the
evolution of the design/formulation of peptide based non-viral carriers.
From the acquired knowledge, it has been found that TAT/DNA or RNA
vectors present low transfection efficiency mainly due to both the for-
mation of large complexes and the loss of peptide biological activity
[19–21]. In order to overcome these drawbacks, a variety of strategies
have been explored. Some authors have combined the cationic polymer
polyethylenimine (PEI) with a modified TAT peptide sequence to
achieve significant improvement in gene transfection efficiency [16]
Dong et al. gave a step forward through the development of TAT
modified lipid-PEI hybrid nanoparticles for the simultaneous delivery of
docetaxel and pDNA [22]. Other researchers have condensed TAT/
pDNA complexes with calcium chloride to produce nanoparticles with
high gene delivery performance for lung cancer gene therapy [23].
In line with this, in this work, TAT has been employed to electro-
statically form complexes with a p53 encoding pDNA. Subsequently,
alkyl trimethyl ammonium bromide surfactants (CnTAB) were used to
both further condense pDNA and improve the properties (size, surface
charge, pDNA encapsulation efficiency) of TAT/pDNA vectors. Cationic
surfactants, such as CnTAB, associate strongly with DNA leading to its
compaction and condensation [24]. Such associative behavior is in the
basis of remarkable biotechnological applications ranging from DNA
extraction, purification or gene delivery systems [25,26]. The release
profile of pDNA has been modelling and it was found that both the
surfactant tail length and N/P ratio can tune the in vitro delivery per-
formance. The anticancer drugs doxorubicin (DOX) and paclitaxel
(PTX) were also loaded and efficiently encapsulated into TAT/pDNA/
CnTAB formulations. The developed peptide/surfactant pDNA vector
revealed to be promising as an intracellular co-delivery platform,
greatly contributing for advances in the design/conception of new
systems to operate therapeutically against the most serious and deadly
diseases, such as cancer.
2. Materials and methods
2.1. Materials
The HIV1-TAT49-57 protein domain (RKKRRQRRRR) was obtained
from Biomatik Corporation (Cambridge, Canada). Cationic alkyl-
trimethylammonium bromide surfactants (CnTAB) were obtained from
Serva and used without further purification. 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 group and described in the literature.2 Doxorubicin
hydrochloride (DOX), 579.98 g/mol and paclitaxel (PTX), 853.91 g/
mol, were obtained from Sigma. All solutions were freshly prepared
using Millipore-Q water (Billerica, MA, USA). Normal Human Dermal
Fibroblasts (NHDF), Ref. C-12302 (cryopreserved cells) and cancer
HeLa cells were purchased from PromoCell and Invitrogen, respec-
tively.
2.2. Preparation of complexes
Plasmid DNA stock solution (20 μg/mL) was prepared in sodium
acetate buffer (0.1 mM sodium acetate/0.1 mol/L acetic acid, pH 4.5),
while TAT peptide was dissolved in deionized water (10 mg/mL) im-
mediately before use. Variable concentrations of TAT (100 μL) were
added to a fixed volume of pDNA (300 μL) to formulate TAT/pDNA
complexes at charge ratios (positive charges of peptide to negative
charges of pDNA, N/P) of 1, 2, 4, 8 and 10. The mixture was vortexed
(3000 rpm) for 10 s and incubated for 20 min at 4 °C prior to use.
To prepare TAT/pDNA/CnTAB complexes, CnTAB surfactants (n ꞊ 8,
12, 14 and 16) were dissolved in Millipore Milli-Q de-ionized water.
Aqueous TAT/pDNA mixtures at desirable N/P ratios (200 μL) were
added drop-wise using a 22-gauge needle into gently agitated surfactant
solutions (2 mL). The ratio between pDNA and CnTAB was 2, the
431
Colloids and Surfaces B: Biointerfaces 178 (2019) 430–438
concentrations were determined per charge. The surfactant concentra-
tions were 250 mM, 18 mM, 4.2 mM and 1 mM for C8, C12, C14 and
C16TAB, respectively. The particles were then equilibrated in solution
for 1 h and thereafter were washed in 250 mM sodium phosphate buffer
at pH 7.4. The complexes were centrifuged at 10,000 rpm for 15 min
and the pellet contained the pDNA based nanoparticles. 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 encapsulation efficiency was obtained from the equation:
EE (%) = [(Total Amount of pDNA –Non-bound pDNA)/ Total amount
of pDNA] ×100 (1)
2.3. Loading and encapsulation of drugs
Doxorubicin and paclitaxel have been incorporated into TAT/
pDNA/CnTAB complexes by imbibition. Concentrated solutions of both
DOX and PTX (2 wt.%) were prepared; the dried nanoparticles were
saturated with DOX and PTX by placing the particles in 25 mL of DOX
or PTX solution and left there ca. 48 h. The DOX or PTX loaded vectors
were collected as a pellet after centrifuging (5000 rpm) for 15 min and
the supernatant was recovered. The concentration of DOX and PTX has
been determined by using UV–vis spectroscopy and measuring the ab-
sorbance at 480 nm and at 227 nm, respectively. The drug loading (LE)
and encapsulation efficiencies (EE) were calculated by using the
formalism of Eqs. (2) and (3).
LE = [(total amount of drug – non-bound drug)/ nanoparticle weight]
× 100 (2)
EE(%) = [(total amount of drug – non-bound drug)/ total amount of
drug] × 100 (3)
2.4. Determination of the properties of pDNA complexes
Information concerning the morphology of the various pDNA vec-
tors has been obtained by scanning electron microscopy (SEM). The
different nanoparticles were centrifuged (10,000 rpm, 15 min., 25 °C)
and the pellet was recovered and suspended in an aqueous solution
containing 30 μL of tungsten. The solution was placed in roundly
shaped cover-slip and dried overnight at room temperature. The sam-
ples were sputter coated with gold using an Emitech K550 (London,
England) sputter coater. A Hitachi S-2700 (Tokyo, Japan) scanning
electron microscope (accelerating voltage of 20 kV at various magnifi-
cations) was used. The average particle size and the zeta potential of the
several pDNA formulations has been determined by Dynamic Light
Scattering (DLS) by the use of a Zetasizer nano ZS. The software
Malvern zetasizer v 6.34 was employed for the analysis of the obtained
results.
2.5. Evaluation of the biocompatibility profile
The cytotoxicity of the developed vectors was assessed by means of
MTT assay. This is a colorimetric assay to measure cell viability by
monitoring the metabolically active cells that convert this dye into a
water-insoluble dark blue formazan. The formed crystals can be quan-
tified spectrophotometrically at 570 nm.
Prior cell seeding, the plates were ultraviolet (UV) irradiated for
20 min. The various nanoparticles were applied to a 96-well plate
(Nunc.). Both, fibroblast and HeLa cells were plated at confluency in 96
well plate, with 2 × 104 cells and 1 × 104 per well, respectively, at 37
º
C in 5% CO2 humidified atmosphere, for 24, 48 h, 72 h and 96 h. After
incubation, the redox activity was evaluated by the established proce-
dure [25]. The relative cell viability (%) related to control wells was
calculated by [A]test / [A]control × 100, where [A] test is the absorbance
D. Costa, et al.
of the test sample and [A] control is the absorbance of control sample. All
the experiments were performed in triplicate. The statistical analysis of
experimental data used the Student´s t-test and the results were pre-
sented as mean ± standard deviation. Statistical significance was ac-
cepted at a level of p < 0.05.
2.6. In vitro pDNA and drug release
To determine the release kinetics of pDNA and the two drugs,
150 μL of TAT/pDNA/CnTAB/drug complexes were placed in poly-
styrene tubes and PBS solution (0.5 mol/L, pH 7.4) was added to a final
volume of 2 mL. The tubes were kept in a horizontal shaker at 100 rpm
and 37 ºC. Following incubation, and at predetermined time intervals,
the tubes were centrifuged (10 min at 10,000 rpm). The supernatant
was collected and the systems were re-suspended in fresh solution of
PBS medium. The pDNA released into the supernatant was quantified
spectrophotometrically by measuring the absorbance at 260 nm using a
Nanophotometer™(Implen, Germany), while the released DOX and PTX
have been determined by UV/VIS spectroscopy at 480 nm and at
227 nm, respectively.
3. Results and discussion
3.1. The properties of pDNA based vectors
TAT peptide interacts strongly, via electrostatic forces, with pDNA
creating nanometer sized vectors. The TAT/pDNA carriers have been
formulated at various N/P ratios (1, 2, 4, 8 and 10). Scanning electron
microscopy analysis has been performed and provides information
concerning the morphology of TAT/pDNA nanoparticles. As ex-
emplified in the Supplementary Material, Figure S1, for some vectors,
the conceived pDNA formulations present oval or spherical shape. A
deeper investigation on the average size and other properties of TAT/
pDNA vectors has been conducted by dynamic light scattering. The
obtained average size, the polydispersity index and the surface charges
for the various systems are listed in Table 1. The size of TAT/pDNA
complexes ranges from 779 nm to 512 nm, depending on the N/P ratio
considered and, concerning the polydispersity index (PDI), all devel-
oped particles are quite monodisperse with values ranging from ap-
proximately 0.1 to around 0.5. There is a correlation between size and
the N/P ratio employed in the formation of TAT/pDNA particles, with
lower N/P ratios giving rise to higher sized complexes. When N/P ratio
increases, the electrostatic interaction of TAT with pDNA originates a
decrease in the size. Despite N/P ratio can functions as a controlling
parameter of the pDNA vector´s size, based solely on the size analysis,
one can already hypothesized the lower transfection efficiency dis-
played by these formulations. In order to adequately penetrate the cell
membrane and be internalized by endocytosis, complexes should pre-
sent sizes smaller than ˜ 200 nm; systems possessing much higher sizes
should be taken up mostly by phagocytosis mechanism what can
compromises the efficiency of transfection. In addition, the surface
charges of the particles have been determined (Table 1). Negative zeta
potential values have been found for TAT/pDNA carriers at N/P ratios
of 1, 2 and 4, while for the other N/P ratios this parameter increases
and shifts to positive values. Positive surface charges can enhance the
cellular uptake and internalization of TAT/pDNA complexes due to
favorable interactions with the negatively charged cell membrane. The
pDNA encapsulation efficiency (EE) is another relevant subject to
evaluate when designing carriers for gene delivery applications. The
pDNA EE values for the several TAT/pDNA vectors are included in
Table 1. In general, the vehicles produced present lower pDNA EE, what
correlates well with the zeta potential values obtained for each system,
denoting that some pDNA remains free, therefore not encapsulated into
the peptide/pDNA complexes. As the N/P ratio increases, however, one
can observe an improvement in their ability for pDNA encapsulation,
but it is far from desirable. Agarose gel electrophoresis confirms the
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Colloids and Surfaces B: Biointerfaces 178 (2019) 430–438
poor pDNA immobilization achieved at N/P ratios of 1, 2 and 4 (Figure
S2, available in the Supplementary Material). Other authors have also
found unsuitable characteristics on developing TAT/pDNA systems,
mainly the large size of formed complexes which limits the success of
gene transfection. However, contrary to our data, they reported an ef-
ficient DNA encapsulation into the peptide carriers at lower N/P ratios
[20,21,27]. Following this, and beyond the low molecular weight of
TAT which may contribute for a weak pDNA condensation, at the
moment we cannot anticipate a comprehensive explanation to elucidate
the discrepancy between our pDNA EE results and the ones described in
the literature.
Taking into consideration the above properties displayed by TAT/
pDNA particles, such as the large size of the complexes, their negative
superficial charges and the poor capacity for pDNA encapsulation, and
found them inadequate to ensure an effective and efficient cell trans-
fection, CnTAB surfactants have been incorporated into the TAT/pDNA
vectors. Pursuing the goal of improving the characteristics of peptide/
pDNA systems enhancing their performance as gene delivery systems,
C8, C12, C14 or C16TAB have been included in these formulations by the
protocol described in the experimental section. Single-chain surfac-
tants, as the ones considered in this work, self-assemble in spherical
micelles with these globular structures starting to occur at a particular
concentration, the critical micelle concentration (cmc). The cmc
strongly varies with the alkyl chain length of the surfactant; longer tails
present lower cmc values. The interaction of a surfactant with an op-
positely charged polyelectrolyte induces a lowering of the typical cmc,
commonly mentioned the critical association concentration (cac). The
negatively charged pDNA should interact strongly with the oppositely
charged surfactants inducing the formation of particulate systems [28].
As discussed above, it seems TAT does not fully condense pDNA and
some of pDNA chains remain not complexed. With the addition of ca-
tionic surfactants, both electrostatic and hydrophobic interactions took
place and the formation of micelles involving the pDNA chains occurred
with the neutralization of polymer charges [29]. The degree of pDNA
charge neutralization is, however, dependent on surfactant chain length
with longer hydrocarbon tails compacting pDNA more efficiently. Al-
though, the exact mechanism of TAT/pDNA/CnTAB particles is beyond
the scope of the current work, we suppose that this process can be seen
as a surfactant coating of TAT/pDNA particles with CnTAB/pDNA mi-
celles, in a probable hexagonal packing [30,31], formed around the
surface of preformed TAT/pDNA complexes. The possibility of a com-
peting effect between CnTAB and TAT for pDNA binding cannot be
excluded. As presented in Figure S1, available in the Supplementary
Material, spherical nanoparticles can be produced. The properties of
these surfactant polyplexes, such as, size, zeta potential and pDNA EE
are summarized in Table 1. In addition, the cmc values of each sur-
factant are also included in this table. The inclusion of C8TAB in the
TAT/pDNA vectors has no effect in the properties of the systems, as the
size of the complexes remains approximately the same, as well as, the
increment in their zeta potential values is absent or not significant.
Furthermore, the pDNA EE percentages determined for the various
TAT/pDNA/C8TAB systems are very low at lower N/P ratios. This can
be related with the short alkyl chain length of this surfactant, and its
higher cmc, which leads to a weak interaction with the pDNA molecule.
A different situation can be found when considering the C12 or C14TAB
surfactants: not only the formed particles present lower sizes but they
also exhibit positive surface charges for all N/P ratios considered. A
significant improvement in the encapsulation of pDNA can be achieved.
The magnitude of the surfactant effect on the studied properties is
higher as N/P ratio increases and when considering a longer chain
cationic surfactant. In fact, a drastic reduction on the size of the carriers
and a relevant increase in the pDNA EE was found with the in-
corporation of C16TAB into TAT/pDNA complexes. The longest tail
surfactant interacts strongly with pDNA which results in the formation
of particles with very small sizes. This phenomenon also includes the
shifting of ζ to more positive values and the encapsulation of pDNA in
D. Costa, et al. Colloids and Surfaces B: Biointerfaces 178 (2019) 430–438

Table 1
Mean size, polydispersity index (PDI), average zeta potential and pDNA encapsulation efficiency (EE) for the various N/P ratio TAT/pDNA and TAT/pDNA/CnTAB
systems. The size and surface charges of pDNA (20 μg/mL) are also listed. The values were calculated with the data obtained from three independent measurements
(mean ± SD, n = 3).
System Size/nm Zeta Potential/mV PDI pDNA EE (%) CMC/mMa

pDNA 732 ± 17.1 −90 ± 8.2 0.4 ± 0.02

TAT/pDNA N/P 1 759 ± 9.2 −48 ± 2.1 0.2 ± 0.03 18 ± 3.1
TAT/pDNA N/P 2 726 ± 10 −43 ± 6.8 0.2 ± 0.01 20 ± 1.4
TAT/pDNA N/P 4 696 ± 8.3 −38 ± 9.4 0.5 ± 0.02 27 ± 3.7
TAT/pDNA N/P 8 593 ± 11.7 +8 ± 0.6 0.4 ± 0.02 58 ± 1.9
TAT/pDNA N/P 10 512 ± 9.6 +11 ± 1.1 0.1 ± 0.01 60 ± 2.3
C8TAB 140
TAT/pDNA/C8TAB N/P 1 724 ± 5.5 −43 ± 1.8 0.4 ± 0.02 20 ± 3.3
TAT/pDNA/C8TAB N/P 2 718 ± 8.6 −41 ± 2.1 0.3 ± 0.02 21 ± 3.4
TAT/pDNA/C8TAB N/P 4 683 ± 3.2 −33 ± 0.9 0.5 ± 0.01 29 ± 0.5
TAT/pDNA/C8TAB N/P 8 581 ± 5.5 +9 ± 1.7 0.3 ± 0.04 59 ± 4.2
TAT/pDNA/C8TAB N/P 10 504 ± 6.1 +14 ± 8.2 0.4 ± 0.02 60 ± 0.8
C12TAB 15
TAT/pDNA/C12TAB N/P 1 479 ± 4.6 +3 ± 0.8 0.3 ± 0.02 37 ± 3.2
TAT/pDNA/C12TAB N/P 2 500 ± 9.5 +3 ± 1.6 0.4 ± 0.01 39 ± 2.9
TAT/pDNA/C12TAB N/P 4 458 ± 12.2 +9 ± 0.7 0.5 ± 0.03 57 ± 1.2
TAT/pDNA/C12TAB N/P 8 367 ± 6.8 +18 ± 3.1 0.2 ± 0.01 70 ± 0.8
TAT/pDNA/C12TAB N/P 10 264 ± 5.0 +27 ± 4.4 0.6 ± 0.04 76 ± 3.2
C14TAB 3.5
TAT/pDNA/C14TAB N/P 1 457 ± 3.8 +7 ± 1.9 0.4 ± 0.01 52 ± 2.0
TAT/pDNA/C14TAB N/P 2 432 ± 6.6 +9 ± 0.3 0.5 ± 0.03 56 ± 1.6
TAT/pDNA/C14TAB N/P 4 354 ± 2.9 +15 ± 2.0 0.5 ± 0.01 69 ± 1.4
TAT/pDNA/C14TAB N/P 8 282 ± 1.6 +24 ± 1.7 0.3 ± 0.02 85 ± 3.9
TAT/pDNA/C14TAB N/P 10 208 ± 5.5 +34 ± 3.4 0.4 ± 0.03 88 ± 2.7
C16TAB 0.9
TAT/pDNA/C16TAB N/P 1 339 ± 0.8 +16 ± 1.7 0.3 ± 0.02 67 ± 4.2
TAT/pDNA/C16TAB N/P 2 311 ± 3.5 +18 ± 0.5 0.2 ± 0.01 76 ± 1.4
TAT/pDNA/C16TAB N/P 4 257 ± 12.1 +28 ± 1.3 0.4 ± 0.01 87 ± 3.3
TAT/pDNA/C16TAB N/P 8 131 ± 2.8 +41 ± 2.6 0.5 ± 0.03 90 ± 4.5
TAT/pDNA/C16TAB N/P 10 93 ± 2.0 +49 ± 3.3 0.4 ± 0.01 91 ± 1.9

a
From Reference [41].

higher percentages for all N/P ratios. Therefore, a clear enhancement of Table 2
particles properties is obtained. Moreover, and as stated before for Cytotoxicity profile of the various N/P ratio TAT/pDNA and TAT/pDNA/
TAT/pDNA complexes, the results show that the N/P ratio is a relevant CnTAB systems, evaluated on fibroblast and HeLa cells by means of MTT assay.
parameter to control the characteristics of the developed carriers. Ad- The values were calculated with the data obtained from three independent
ditionally, we found that the surfactant chain length can also deeply measurements (mean ± SD, n = 3).
modulate the properties of the formulations, as increasing the surfac- System Cellular Viability (%)
tant tail length a pronounced effect over the size, surface charges and
N/P NHDF HeLa
pDNA encapsulation performance is observed. The polydispersity index
1 80 ± 0.9 77 ± 3.7
analysis reveals that all the TAT/pDNA surfactant systems can be 2 83 ± 2.6 79 ± 6.0
considered monodisperse (Table 1). TAT/pDNA 4 86 ± 6.3 80 ± 5.5
Additionally, to further characterize the developed formulations, 8 88 ± 2.9 81 ± 2.1
their biocompatibility profile was investigated. The cytotoxicity ex- 10 89 ± 4.3 84 ± 0.9
1 58 ± 0.5 56 ± 3.6
hibited by the different carriers was assessed on fibroblast and HeLa
2 60 ± 3.3 57 ± 6.3
cells by means of the MTT assay. A summary of the study is presented in TAT/pDNA/C8TAB 4 74 ± 1.9 72 ± 2.7
Table 2. As expected, TAT/pDNA carriers, formulated at the studied N/ 8 79 ± 4.0 75 ± 1.9
P ratios, are all biocompatible. After incubation with these systems, 10 81 ± 5.3 78 ± 5.0
1 51 ± 3.1 48 ± 1.9
fibroblast and cancer cells remain viable in a high extent, relative to
2 53 ± 3.6 48 ± 0.9
control cells. A moderate increment in the cellular viability is observed AT/pDNA/C12TAB 4 61 ± 0.7 59 ± 4.4
as N/P ratio increases. Moreover, for all vectors considered, a slightly 8 76 ± 3.1 72 ± 3.7
lower percentage of viable HeLa cells is found. 10 78 ± 5.6 76 ± 1.5
In general, the incorporation of surfactants into TAT/pDNA com- 1 45 ± 4.4 41 ± 3.6
2 48 ± 1.9 42 ± 5.3
plexes results in a decrease of the biocompatibility. This phenomenon
TAT/pDNA/C14TAB 4 59 ± 3.8 56 ± 2.0
becomes dependent on the surfactant tail length, with molecules pos- 8 74 ± 1.6 66 ± 4.3
sessing longer chains leading to a higher cytotoxic effect. In fact, the 10 78 ± 5.1 73 ± 2.7
cytotoxicity seems to increase in the following order: 1 36 ± 5.7 32 ± 4.2
2 38 ± 3.9 33 ± 0.6
C16TAB < C14TAB < C12TAB < C8TAB. To complement this ob-
TAT/pDNA/C16TAB 4 52 ± 1.7 46 ± 2.5
servation, the MTT analysis, at 48 h, on HeLa cells has been performed 8 71 ± 4.7 68 ± 1.9
for TAT/pDNA/CnTAB nanoparticles at N/P ratio of 10 and various 10 75 ± 0.9 71 ± 4.2
CnTAB/pDNA ratios. The results are shown in Fig. 1. Note that, as
mentioned in the experimental section, the particles described above
were formulated at CnTAB/pDNA ratio of 2. For all cationic surfactants, concentration, with cell viability decreasing as the surfactant amount
the results show a relationship between cytotoxicity and surfactant increases. Additionally, this data confirmed the cytotoxic profile

433
D. Costa, et al. Colloids and Surfaces B: Biointerfaces 178 (2019) 430–438

pDNA/CnTAB vectors at N/P ratio of 10 (data not shown). The cyto-

toxicity of cationic surfactants has been largely investigated and is well
documented in the literature [32,33]. Following this, the complexation
of surfactants with agents of recognized biocompatibility has been a
powerful strategy to overcome their cytotoxic effect in the biological
environment [28,33]. In this work, the complexation of surfactants
with TAT/pDNA drastically reduces their cytotoxicity, enabling the use
of the resultant formulations as drug/gene delivery systems. Further-
more, the N/P ratio can also modulate the biocompatibility displayed
by TAT/pDNA/CnTAB particles, as evidenced in Table 2. In the pre-
sence of surfactants, a higher N/P ratio gives rise to an increase in the
biocompatibility of the vectors. This effect is more accentuated as
longer the surfactant alkyl chain. The same trend can be observed for
both fibroblast and HeLa cells.

3.2. In vitro pDNA release profile

The release kinetics of pDNA has been evaluated in vitro by im-

Fig. 1. Cell viability of HeLa cells after 48 h incubation time with TAT/pDNA/
mersion of the carriers into a buffer solution. Fig. 2 presents the cu-
CnTAB formulated at N/P ratio of 10, as a function of CnTAB/pDNA ratio.
mulative release curves of pDNA from the different TAT/pDNA/CnTAB
Percent viability is expressed relative to control cells (control was set to 100%
cell viability). Mean values ± SD are obtained from three experimental de-
systems formulated at various N/P ratios. From the analysis of the data,
terminations; p < 0.05 versus the control (one-way ANOVA with Dunnet`s it can be stated that in general all the surfactant/peptide/pDNA vectors
post-hoc test). are able to release the encapsulated payload after an initial time lag,
which is a common trend for all carriers. It can be seen that the mag-
nitude of time lags is different among the various systems. Longer chain
displayed by the longer chain surfactants. A slight increment in the
surfactant/TAT/pDNA particles present higher time lags. Although, the
cytotoxicity was also observed when a similar study was conducted for
profile for C12, C14 and C16 based carriers is barely the same (with
longer incubation times (72 h and 96 h) of HeLa cells with the TAT/
exception for N/P 1 based systems), the mentioned tendency is still

Fig. 2. Cumulative pDNA release profiles for the TAT/pDNA/CnTAB systems conceived at N/P ratios of 1, 2, 4, 8 and 10.

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tremendously notorious, especially, when comparing TAT/pDNA/

C8TAB and C16TAB vectors. The observed behavior may be related with
the strong interaction of pDNA chains with longer tail surfactants what
makes difficult the release of pDNA from the complexes [28,34]. Fur-
thermore, except for the vehicles based on C8TAB, for each system the
N/P ratio can only slightly modulate the time lag, with the N/P ratio of
1 exhibiting a larger time lag. Different situation can be found for TAT/
pDNA/C8TAB polyplexes where the time lag greatly varies with the N/P
ratio, with vectors conceived at higher N/P ratios showing lower time
lags. This fact suggests a different mechanism for this surfactant-peptide
formulation. Moreover, it was found that the extent of pDNA released
amount greatly varies with the N/P ratio considered. An increment in
the released pDNA amount can be observed by increasing the molar
ratio of nitrogen to DNA phosphate groups. At the highest N/P ratio
studied, N/P of 10, the levels of maximum pDNA released (˜ 90%) are
similar between the different surfactant/peptide/pDNA particles. This
reinforces the fact that the N/P ratio can be a powerful tailoring tool in
pDNA release. In order to have a deeper assessment on the pDNA re-
lease profile, the effect of alkyl chain length of surfactant and N/P ratio
on the time-lag and maximum cumulative pDNA release have been
investigated, assuming a steady-state flux (at least three data points
were used), for the time lag (θ) estimation. The data is presented in
Figure S3, available in the Supplementary Material, and corroborates
well with the main assumptions pointed out above. According to pre-
vious studies available in the literature, the pDNA release kinetics can
be related with the swelling/dissolution profiles exhibited by the dif-
ferent chain surfactant systems, that consequently can predict the re-
lease mechanism [28,34]. In these works, the release curves can be
controlled by swelling or dissolution of the vectors with profound
consequences on the pDNA release profile. In both reports, it was found
that C8TAB based particles exhibited release kinetics by a dissolution
mechanism, what is consistent with the faster pDNA release trend. The
Fig. 3. Dependence of n (Eq. 4) on surfactant alkyl chain and N/P ratio. The initial burst release, observed in the first 24 h, can be attributed to the
horizontal dash lines correspond to n = 0.85 (Case-II transport) and n = 1 (zero release of some pDNA bound weakly on the surface of the carriers. On
order release) (A) and the effect of surfactant alkyl chain length and N/P ratio contrary, in TAT/pDNA/CnTAB systems where longer tail surfactants
on the mean dissolution time (MDT, Eq. 5) (B). were present, the pDNA release showed to be controlled by the swel-
ling/deswelling process. This originates that, after the time lag, the
Table 3 pDNA released amount gradually increases with time until a plateau is
Fitting parameters obtained by fitting Eqs (6) and (7) to the experimental data reached. Moreover, a dependence on the N/P ratio was found and can
(Fig. 3), at 25 °C. deeply modulate the pDNA delivery [28,34].
In order to check these hypotheses, the release mechanism of pDNA
k0 ( ± s) / 10−2 AIC K ( ± s) / 10−1 k1 ( ± s) / h−1 AIC
μ g−1 L h−1 from TAT/pDNA/C8TAB polyplexes has been assessed. Initially, the
modified power law equation (eq. 4) along with the computation of the
C8TAB mean dissolution time (MDT, eq. 5) have been applied for fitting the
N/P1 4.5 ± 0.1 −12.4 −2.4 ± 0.2 1.6 ± 0.3 −7.4
short-range times (Ct/C∞ < 0.6) cumulative release of pDNA, [35,36].
N/P2 5.57 ± 0.05 −20.2 −2.6 ± 0.2 2.2 ± 0.3 −3.7
N/P4 5.74 ± 0.08 −13.9 −2.1 ± 0.2 1.6 ± 0.2 −5.6 Ct
N/P8 5.8 ± 0.1 −17.1 −2.1 ± 0.2 1.6 ± 0.2 −5.6 = k (t − θ)n
C∞ (4)
N/P10 7.2 ± 0.3 −11.8 −1.8 ± 0.2 1.6 ± 0.3 −4.2
C12TAB
N/P1 0.61 ± 0.02 −19.9 −2.8 ± 0.1 0.28 ± 0.02 −9.2 n ⎞ −n−1
MDT = ⎛ k
N/P2 0.62 ± 0.03 −20.1 −2.9 ± 0.1 0.28 ± 0.02 −9.0 ⎝n + 1⎠ (5)
N/P4 0.68 ± 0.01 −24.2 −2.4 ± 0.1 0.22 ± 0.02 −10.8
N/P8 0.85 ± 0.01 −23.8 −2.2 ± 0.1 0.24 ± 0.03 −7.2 where C∞ is the maximum cumulative release, and k and n are fitting
N/P10 0.89 ± 0.02 −20.6 −2.8 ± 0.1 0.22 ± 0.03 −7.3 parameters, giving the latter useful information on the release me-
C14TAB
chanism. The obtained n and MDT values are depicted in Figs. 3A and B,
N/P1 0.65 ± 0.02 −20.9 −2.8 ± 0.2 0.30 ± 0.03 −6.6
N/P2 0.67 ± 0.02 −23.2 −2.6 ± 0.1 0.25 ± 0.02 −9.9 respectively. From the analysis of Fig. 3A it can be seen that n values
N/P4 0.73 ± 0.01 −24.2 −2.2 ± 0.1 0.21 ± 0.02 −11.1 vary between 0.85 and 1; the former characterizes a Case-II transport
N/P8 0.89 ± 0.02 −21.4 −2.0 ± 0.1 0.21 ± 0.02 −8.5 (for spheres – indicative of coupling of diffusional and relaxational
N/P10 0.93 ± 0.02 −19.5 −1.8 ± 0.1 0.20 ± 0.02 −8.7 mechanism [37]) and the latter a zero-order release kinetics. However,
C16TAB
the analysis of MDT shows that for C12 to C16-containing systems,
N/P1 0.77 ± 0.03 −16.5 −2.3 ± 0.1 0.25 ± 0.01 −12.8
N/P2 1.01 ± 0.01 −22.0 −2.1 ± 0.1 0.26 ± 0.03 −7.3 there is a general trend; i.e., MDT decreases by increasing the surfactant
N/P4 1.15 ± 0.02 −16.2 −2.0 ± 0.1 0.29 ± 0.03 −6.5 alkyl chain length and by increasing the N/P ratio. Once the MDT is a
N/P8 1.21 ± 0.03 −14.3 −1.9 ± 0.2 0.28 ± 0.04 −5.5 parameter used to characterize the drug release rate from a dosage form
N/P10 1.26 ± 0.03 −8.7 −1.8 ± 0.1 0.27 ± 0.03 −5.8
and to indicate the drug-release-retarding efficiency of the polymer, it
s is the standard deviation of the fitting parameter; AIC is the Akaike's in-
can be hypothesized that for C8TAB based vectors the formed “skin-
formation criteria. layer” on the complexes is a weak structure allowing for the easy and
fast payload release.

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D. Costa, et al. Colloids and Surfaces B: Biointerfaces 178 (2019) 430–438

Table 4
Loading content (LC), encapsulation efficiency (EE), mean size, polydispersity index (PDI) and average zeta potential) for the various N/P ratio TAT/pDNA/CnTAB/
DOX and TAT/pDNA/CnTAB/PTX systems. The values were calculated with the data obtained from three independent measurements (mean ± SD, n = 3).
System LC (%) EE(%) Size/nm Zeta Potential/mV PDI

TAT/pDNA/C8TAB/DOX N/P 1 38 ± 2.9 41 ± 0.6 730 ± 5.6 −29 ± 1.9 0.4 ± 0.02
TAT/pDNA/C8TAB/DOX N/P 2 39 ± 1.6 43 ± 1.3 721 ± 8.1 −22 ± 0.7 0.2 ± 0.01
TAT/pDNA/C8TAB/DOX N/P 4 43 ± 0.9 49 ± 2.6 689 ± 4.2 −19 ± 1.1 0.3 ± 0.02
TAT/pDNA/C8TAB/DOX N/P 8 49 ± 2.2 63 ± 1.9 592 ± 6.6 +13 ± 2.3 0.5 ± 0.03
TAT/pDNA/C8TAB/DOX N/P 10 52 ± 1.8 68 ± 3.4 513 ± 5.9 +21 ± 1.5 0.3 ± 0.02
TAT/pDNA/C12TAB/DOX N/P 1 43 ± 2.8 66 ± 1.6 529 ± 6.6 +8 ± 0.4 0.3 ± 0.02
TAT/pDNA/C12TAB/DOX N/P 2 44 ± 2.6 67 ± 3.2 521 ± 7.1 +9 ± 0.4 0.1 ± 0.02
TAT/pDNA/C12TAB/DOX N/P 4 46 ± 0.8 74 ± 4.4 469 ± 5.0 +12 ± 1.2 0.3 ± 0.01
TAT/pDNA/C12TAB/DOX N/P 8 53 ± 3.7 79 ± 1.5 381 ± 6.3 +28 ± 3.8 0.5 ± 0.02
TAT/pDNA/C12TAB/DOX N/P 10 56 ± 1.9 80 ± 5.2 293 ± 3.2 +34 ± 0.7 0.4 ± 0.02
TAT/pDNA/C14TAB/DOX N/P 1 49 ± 3.1 68 ± 2.9 466 ± 4.9 +15 ± 2.0 0.4 ± 0.02
TAT/pDNA/C14TAB/DOX N/P 2 49 ± 4.4 70 ± 4.7 458 ± 3.9 +16 ± 1.6 0.3 ± 0.02
TAT/pDNA/C14TAB/DOX N/P 4 52 ± 2.8 74 ± 0.8 393 ± 4.5 +33 ± 3.9 0.5 ± 0.02
TAT/pDNA/C14TAB/DOX N/P 8 57 ± 1.2 82 ± 4.1 308 ± 2.2 +40 ± 3.5 0.5 ± 0.03
TAT/pDNA/C14TAB/DOX N/P 10 61 ± 6.3 83 ± 3.5 245 ± 5.4 +43 ± 2.7 0.5 ± 0.02
TAT/pDNA/C16TAB/DOX N/P 1 52 ± 2.2 68 ± 1.8 345 ± 3.7 +22 ± 1.1 0.5 ± 0.02
TAT/pDNA/C16TAB/DOX N/P 2 54 ± 3.6 72 ± 4.0 334 ± 5.6 +26 ± 0.5 0.4 ± 0.01
TAT/pDNA/C16TAB/DOX N/P 4 58 ± 4.9 77 ± 6.2 278 ± 5.1 +34 ± 1.3 0.3 ± 0.02
TAT/pDNA/C16TAB/DOX N/P 8 71 ± 3.8 85 ± 1.5 182 ± 4.9 +46 ± 3.4 0.3 ± 0.02
TAT/pDNA/C16TAB/DOX N/P 10 76 ± 2.1 85 ± 3.3 105 ± 4.5 +52 ± 4.7 0.5 ± 0.03
TAT/pDNA/C8TAB/PTX N/P 1 30 ± 3.6 43 ± 1.7 733 ± 6.4 −40 ± 1.4 0.2 ± 0.01
TAT/pDNA/C8TAB/PTX N/P 2 31 ± 4.2 45 ± 2.2 726 ± 5.8 −39 ± 0.4 0.3 ± 0.02
TAT/pDNA/C8TAB/PTX N/P 4 33 ± 2.7 48 ± 4.6 691 ± 8.9 −35 ± 2.6 0.4 ± 0.01
TAT/pDNA/C8TAB/PTX N/P 8 41 ± 1.9 59 ± 2.4 603 ± 6.2 +8 ± 1.8 0.3 ± 0.02
TAT/pDNA/C8TAB/PTX N/P 10 48 ± 3.5 64 ± 0.9 558 ± 5.1 +12 ± 0.5 0.3 ± 0.02
TAT/pDNA/C12TAB/PTX N/P 1 35 ± 3.1 65 ± 2.9 522 ± 4.5 +3 ± 1.2 0.4 ± 0.02
TAT/pDNA/C12TAB/PTX N/P 2 37 ± 4.4 65 ± 3.7 519 ± 8.1 +4 ± 0.7 0.2 ± 0.02
TAT/pDNA/C12TAB/PTX N/P 4 40 ± 4.8 73 ± 1.9 474 ± 5.6 +9 ± 1.4 0.5 ± 0.02
TAT/pDNA/C12TAB/PTX N/P 8 49 ± 2.5 76 ± 5.1 380 ± 5.9 +17 ± 0.8 0.3 ± 0.01
TAT/pDNA/C12TAB/PTX N/P 10 53 ± 2.2 79 ± 0.4 294 ± 7.0 +25 ± 1.7 0.5 ± 0.02
TAT/pDNA/C14TAB/PTX N/P 1 40 ± 3.2 66 ± 1.9 472 ± 5.1 +8 ± 1.2 0.1 ± 0.02
TAT/pDNA/C14TAB/PTX N/P 2 40 ± 2.9 68 ± 3.8 439 ± 9.5 +9 ± 0.2 0.5 ± 0.03
TAT/pDNA/C14TAB/PTX N/P 4 43 ± 3.3 72 ± 1.4 388 ± 7.2 +14 ± 1.1 0.3 ± 0.02
TAT/pDNA/C14TAB/PTX N/P 8 51 ± 1.2 80 ± 5.5 294 ± 8.0 +23 ± 0.5 0.4 ± 0.02
TAT/pDNA/C14TAB/PTX N/P 10 59 ± 4.5 82 ± 4.0 217 ± 2.8 +32 ± 2.3 0.2 ± 0.02
TAT/pDNA/C16TAB/PTX N/P 1 48 ± 2.8 68 ± 4.5 344 ± 5.2 +15 ± 0.7 0.5 ± 0.04
TAT/pDNA/C16TAB/PTX N/P 2 49 ± 4.1 71 ± 4.3 329 ± 8.2 +16 ± 1.0 0.2 ± 0.01
TAT/pDNA/C16TAB/PTX N/P 4 58 ± 5.2 73 ± 1.8 278 ± 6.6 +28 ± 1.9 0.4 ± 0.02
TAT/pDNA/C16TAB/PTX N/P 8 68 ± 4.9 79 ± 4.7 179 ± 3.5 +42 ± 2.2 0.5 ± 0.03
TAT/pDNA/C16TAB/PTX N/P 10 74 ± 2.2 81 ± 2.8 116 ± 4.1 +47 ± 2.7 0.3 ± 0.01

To a deeper insight on the release mechanism, the model analysis 3.3. Plasmid DNA-drug based systems
was extended to long-range times (Ct/C∞ < 0.85 [38]). Following this,
two different models have been applied to fit the experimental data: the DOX and PTX, therapeutic drugs commonly applied in cancer
zero-order and the first-order release equations (eqs. 6 and 7, respec- treatments, have been encapsulated into TAT/pDNA/CnTAB vectors by
tively): the procedure described in the experimental section. The drugs loading
content and encapsulation efficiency have been determined and the
Ct = k 0 (t − θ) (6)
results are presented in Table 4. Along with pDNA, both drugs can be
ln Ct = K − k1 (t − θ) (7) loaded and efficiently incorporated into the nanosystems at various N/P
ratios. The variation of DOX EE or PTX EE with the N/P ratio parameter
where K is a constant and k0 and k1 are the zero-order and first-order
follows the same trend observed for the pDNA encapsulation. In com-
rate constants, respectively. The use of these equations has the ad-
parison to pDNA EE, presented above, DOX and PTX EE values are
vantage of no knowledge of initial or borer conditions is needed.
higher for all surfactant TAT/pDNA carriers, except for the TAT/pDNA/
Table 3 shows the fitting parameters for eqs. (6) and (7). Using the
C14TAB at higher N/P ratios and the system where C16TAB is present. In
Akaike's information criteria (AIC) [39] for the assessment of the best
addition, the pDNA EE percentages remain fairly the same or slightly
fitting, it comes out that, in all systems, the release mechanism follows
decrease for all the TAT/pDNA/CnTAB/drug formulations, and there-
a zero order release. It can also be seen that there is a good correlation
fore the encapsulation of a drug seems do not significantly interfere
between k0 and MDT values; i.e., the rate constant for C8-containing
with the efficiency of pDNA encapsulation (data not shown). Moreover,
systems are higher than those obtained for other systems; on the other
the effect of the presence of a drug into TAT/pDNA/CnTAB properties,
hand, for other systems, the rate constant k0 increases by increasing the
namely, the size and zeta potential, has been also examined and is re-
N/P ratio and, in general, also increases by increasing the surfactant
ported in Table 4. An increase in the size of the complexes can be found
alkyl chain length. Zero-order release systems are not quite often, but
when DOX or PTX is encapsulated into the nanoparticles. Concerning
can be developed by using, e.g., membrane diffusion controlled release;
the surface charges, the presence of DOX increases the zeta potential
i.e., assuming a reservoir device, with a saturated drug in the core, a
values due to its positive charge at lower and neutral pH; DOX has a
steady state drug concentration profile occurs through in the rate
pKa around 8.2, its positive charge arises from the –NH3+ groups. On
controlling membrane. Knowing that cationic surfactants can form a
contrary, the incorporation of PTX into the nanocarriers has no sig-
skin-like membrane by interaction with DNA, [28,34,40] it is antici-
nificant effect on their surface charge as this drug is hydrophobic and
pated that such electrostatic complex is controlling the pDNA release.

436
D. Costa, et al.
uncharged.
The in vitro release kinetics of both anticancer drugs for the various
systems has also been evaluated. As pDNA, DOX and PTX can be re-
leased from the various peptide/surfactant vectors (data not shown).
Similarly, the same dependence on both the N/P ratio and the surfac-
tant chain length were observed and, along with the formed “skin-
layer” discussed above, dictate the release trend of drugs. Ongoing
work is focussed on the investigation of the co-delivery system ability
for cellular uptake and internalization processes, as well as, its trans-
fection efficiency and therapeutic effect, namely, on cancer cells. We
strongly hope that the in vitro studies, along with in vivo experiments,
can reveal the suitability of this formulation for drug/gene delivery
protocols aiming cancer clinical translation. Progresses on this topic,
fortunately, will be reported soon.
4. Conclusions
Peptide (TAT)/p53 encoding plasmid DNA systems have been con-
ceived based on the electrostatic interaction between the two mole-
cules. This report evidenced the limitations of these vectors as delivery
systems, mainly, concerning their large size and low pDNA im-
mobilization ability. We found that the subsequent compaction of
pDNA by cationic CnTAB surfactants enhances the properties (size,
surface charge, pDNA or drug encapsulation efficiency) of developed
TAT/pDNA/CnTAB formulations turning them suitable for gene de-
livery protocols. Moreover, these characteristics can be tailored, by
manipulating both the N/P ratio and the surfactant tail length and show
to also deeply influence the pDNA release profile. in vitro release ki-
netics demonstrated that pDNA release follows a zero-order release.
Such kinetics describes the dissolution of active substances in, e.g.,
coated matrices. This is in close agreement with significant time-lag
values showing an interaction between cationic surfactants and DNA.
Such relationship can be used to describe the drug dissolution of several
types of modified release pharmaceutical dosage forms.
Along with pDNA, anticancer drugs such as doxorubicin and pacli-
taxel can both be loaded and incorporated into peptide/pDNA/surfac-
tant systems without significant changes on their properties. The drugs
can be released from the carriers by a release pattern similar to pDNA
release kinetics. This innovative nanoplatform represents a great
achievement in the design/formulation of advanced co-delivery systems
to be further explored for the most challenging therapeutic demands,
such as, cancer therapy.
Declarations of interest
None.
Acknowledgements
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-FEDER-041068 and FEDER funds
through the POCI −COMPETE 2020 - Operational Programme
Competitiveness and Internationalisation in Axis I - Strengthening re-
search, technological development and innovation (Project POCI-01-
0145-FEDER-007491) and FCT - Foundation for Science and
Technology (Project UID/Multi /00709/2013). This work was also
partially supported by “Programa Operacional do Centro, Centro 2020”
through the funding of the ICON Project (Interdisciplinary Challenges
on Neurodegeneration; CENTRO-01-0145-FEDER-000013)”.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.03.029.
437
Colloids and Surfaces B: Biointerfaces 178 (2019) 430–438
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