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Article
Targeting of cellular organelles by fluorescent plasmid DNA nanoparticles Diana Costa, Carolina Costa, Margarida Vaz Caldeira, Luisa Maria Cortes, João A. Queiroz, and Carla Cruz Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00877 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017
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Biomacromolecules
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Targeting of cellular organelles by fluorescent plasmid DNA nanoparticles
2 3
Diana Costa1, Carolina Costa1, Margarida Caldeira2, Luísa Cortes2, João A. Queiroz1
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and Carla Cruz1
5 6
1
7
D. Henrique, 6200-506 Covilhã, Portugal
8
2
9
Coimbra, Rua Larga, 3004-504 Coimbra, Portugal
CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante
Microscopy Unit-CNC – Center for Neuroscience and Cell Biology, University of
10 11 12
Corresponding author:
13
Diana Rita Barata Costa
14
Universidade da Beira Interior
15
6201-001 Covilhã
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Portugal
17
E-mail address:
[email protected] 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 1 ACS Paragon Plus Environment
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Abstract
2 3
The development of a suitable delivery system and the targeting of intracellular
4
organelles are both essential for the success of drug and gene therapies. The conception
5
of fluorescent ligands, displaying targeting specificity together with low toxicity, is an
6
emerging and reliable tool to develop innovative delivery systems. Biocompatible BSA
7
or pDNA/ligand nanoparticles were synthesized by a co-precipitation method and were
8
shown to display adequate sizes and morphology for delivery purposes, and positive
9
surface charges. Additionally, these fluorescent vectors can target specific intracellular
10
organelles. In vitro transfection mediated by BSA or pDNA based carriers can result in
11
the accumulation of BSA in the cytosol, lysosomes and mitochondria or the expression
12
of the plasmid-encoded protein, respectively. Moreover, the therapeutic effect of
13
pDNA/ligand vectors in cancer gene therapy instigates further research aiming clinical
14
translation.
15 16
Keywords: fluorescent compounds; organelle targeting; nanoparticles; drug/gene
17
delivery; protein expression; cancer therapy.
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Biomacromolecules
1
Introduction
2
During
3
biopharmaceutical research have led to enormous progresses in the development of new
4
carrier systems that can be applied therapeutically in a wide range of clinical
5
applications.1-5 The delivery of drugs, biomolecules, therapeutic and imaging agents,
6
mediated by these carriers, into different tissues and cell organelles acquired
7
considerable interest with recognized clinical benefits.6-8 In line with this, the concept of
8
gene therapy emerges as an exciting approach due to its promising therapeutic effect.9-12
9
For nuclear or mitochondrial gene therapy to be feasible in a clinical setting, the design
10
of an adequate vector is imperative. Despite the efficiency accomplished with viral
11
vectors, the synthetic carriers offer many advantages such as, the easier production, lack
12
of immune response and greater DNA-loading capacity. To be applied in the biomedical
13
field, non-viral systems should be biocompatible, have a suitable size and shape,
14
incorporate large amounts of bioactive agents, ensure protection against enzymatic
15
degradation, have a desirable bio-distribution, and ensure the therapeutic payload to be
16
cell targeted and released in a sustained manner.13,14 Among these requirements,
17
organelle targeting delivery is perhaps the most auspicious of them all. In this sense, the
18
development of ligands and fluorescent probes that can target a specific organelle while
19
maintaining the cell viability, proliferation and membrane permeability,15 emerges as a
20
priority when designing advanced vehicles for targeted drug or gene delivery. The
21
conjugation of these ligands with the biopharmaceutical based carrier has already
22
demonstrated to be a valuable strategy for targeting of a certain organelle improving
23
transfection efficiency and gene expression.16,17 Not only targeting nucleus can bring
24
impressive progresses,18 but also mitochondria and lysosomes attracted the attention of
25
researchers as important intracellular targets.19,20 Therefore, new targeting approaches
26
need to be considered in order to instigate therapeutic development in the protocols
27
centred in these organelles. In this work, fluorescent compounds have been synthesized
28
and the targeting ability of ligand-incorporated protein or pDNA nanoparticles is
29
explored. The cellular toxicity of the compounds is also evaluated on fibroblast and
30
cancer HeLa cells. The fluorescent compounds were shown to cross the plasma
31
membrane and their localization is assessed by fluorescence confocal microscopy. Both
32
confocal imaging analysis and a cell-associated fluorescence study reveal clear evidence
33
of cytosol, lysosomes and mitochondria targeting. In vitro transfection mediated by
34
these fluorescent pDNA nanoparticles can lead to protein expression. The therapeutic
the
last
decade,
the
close
relation
between
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and
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effect on cancer cells, mediated by these carriers, is promising and instigates further
2
research.
3 4 5
Materials and Methods
6
Materials.
7
analytical grade, α cellulose powder (MW: 162.4 g mol−1), 3-(4,5- dimethylthiazol-2-
8
yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), gelatine,
9
naphthalene-1-carbaldehyde; tris(2-aminoethyl)amine were obtained from Sigma (St.
10
Louis, MO, USA). 2-Quinolinecarboxaldehyde, diethylenetriamine, tripropylene-
11
tetramine, p-toluenesulfonyl chloride and sodium borohydride were obtained from
12
Acros Organics. Bovine serum albumin (BSA) (molecular weight of 65000 Da, the
13
radius is around 34.8 Å) and the bicinchoninic acid (BCA) kit were obtained from
14
Sigma-Aldrich.
15
All solutions were freshly prepared using ultra-pure grade water, purified with a Milli-Q
16
system from Millipore (Billerica, MA, USA). Normal Human Dermal Fibroblasts
17
(NHDF), Ref. C-12302 (cryopreserved cells), cancer HeLa and human breast cancer
18
MCF-7 cells were purchased from PromoCell, Invitrogen and ATCC (American Type
19
Culture Collection), respectively.
Acridine, anhydrous calcium chloride, anhydrous sodium carbonate of
20 21
Plasmids
22
The 6.07 kbp plasmid pcDNA3-FLAG-p53 (Addgene plasmid 10838, Cambridge, MA,
23
USA) used in the experiments was produced and purified by a procedure described
24
elsewhere.21 The pCAG-GFP-ND1 (6.5 kbp) was produced by an experimental protocol
25
developed by our group and fully described in a recently published paper.22
26 27
Synthesis of fluorescent compounds
28
The fluorescent compounds, whose structures are represented in Figure 1, were
29
synthesized by the experimental protocols described in the Supporting Information.
30 31
Determination of pDNA fluorescent compounds cytotoxicity by 3-[4,5-dimethyl-
32
thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.
33
Normal human dermal fibroblast (C-12302) and HeLa cells were seeded at a density of
34
1 × 105 and 1 × 106 cells per well, respectively, and grown at 37 ºC in a 95% air/ 5% 4 ACS Paragon Plus Environment
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CO2 humidified atmosphere until 80% confluence. Cells were then incubated with 0.01
2
µM of each fluorescent compound for 24, 48 and 72 h. To test the effect of
3
concentration, cells were treated with five different concentrations (0.01 µM, 0.1 µM, 1
4
µM, 5 µM and 10 µM) of the various compounds for 72 h. After incubation, the redox
5
activity was assessed through the reduction of the MTT, by the established procedure.21
6 7
Fluorescence Microscopy
8
For the fluorescence confocal microscopy assays, human cancer MCF-7 cells were
9
seeded at a density of 10×104 cells/well in a treated µ-slide 8 well (IBIDI, Germany)
10
and grown at 37 °C in a 95% air/5% CO2 humidified atmosphere until 80% confluence.
11
Cells were then incubated with 1 µM or 30 µM each fluorescent compound for 1 h.
12
Cells were then washed two times with phosphate-buffered saline (PBS, 0.137 M NaCl,
13
2.7 mM KCl, 1.4 mM KH2PO4 and 0.01 M Na2PO4, pH 7.4), and mitochondria or
14
lysosomes were labelled with 100 nM MitoTracker Green or Lysotracker Green
15
(Thermo-Fisher Scientific), respectively, for 30 min at 37 °C in the dark. After a gentle
16
wash with PBS, cells were incubated, for 5 min at room temperature in the dark, with 5
17
µM DRAQ5 Fluorescent Probe (Thermo-Fisher Scientific) in PBS to stain the nucleus.
18
Cells were then imaged using a laser scanning confocal inverted microscope (Carl
19
Zeiss, LSM 710 Axio Observer, Germany), equipped with a heating insert stage (Pecon
20
Heating Insert P, Germany), and 5% CO2 supply, and a Plan-Apochromat 40×/1.4
21
objective. Images were acquired using the following laser lines: Diode 405 nm
22
(excitation of nanoparticles), Argon/2 488 nm (excitation of mitoTracker or lysotracker
23
probes) and DPSS 561 nm (excitation of DRAQ5 fluorescent probe).
24 25
Preparation of fluorescent compound/plasmid DNA nanoparticles.
26
A co-precipitation procedure has been used to prepare pDNA based nanoparticles. 5 µg
27
of pCAG-GFP-ND1 or pcDNA3-FLAG-p53, 120 µL of CaCl2 solution (0.03 g/mL) and
28
7.5 µL of fluorescent compound (1 mg/mL) were mixed and then diluted to a total
29
volume of 290 µL. To form the nanoparticles, the solution was gently added dropwise
30
to 255 µL of Na2CO3 solution (0.425 µg/mL). The resultant solution was centrifuged at
31
10 000 rpm for 15 min and the pellet contained the pDNA based nanoparticles. The
32
encapsulation efficiency was determined as described elsewhere21 and using the
33
following equation:
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EE (%) = [(Total Amount of pDNA –Non-bound pDNA)/ Total amount of pDNA] ×100
2
(1)
3 4
Similarly, the fluorescent compounds encapsulation efficiencies were determined by
5
using the equation above. The non-incorporated compound was determined
6
quantitatively by absorption spectral measurements, using 1 cm quartz cuvettes, on a
7
Shimadzu UV-visible 2100 spectrophotometer. The excitation and emission
8
wavelengths for compounds AcridTriamine, Naphtripodal, QuinTriamine and
9
[16]phenN2 are: 318 nm and 322 nm, 276 nm and 334 nm, 320 nm and 323 nm, 230 nm
10
and
270
nm
respectively.
The
compound
concentration
was
determined
11
spectrophotometrically by interpolation in a calibration curve with known ligand
12
concentrations.
13
In order to identify the suitable amount of fluorescent compound for the formulation of
14
pDNA/compound nanoparticles, a screening experiment testing several amounts (from
15
1 µL to 10 µL, with increments of 0.5 unities) of each of the fluorescent compound
16
(1mg/mL) has been performed. The choice of adding 7.5 µL of each compound relates
17
with the onset for higher ligand encapsulation efficiency (EE) obtained; while the main
18
properties of the nanoparticles remain fairly the same for each tested amount of
19
compound.
20 21
Preparation of fluorescent compound/BSA nanoparticles
22
BSA solution containing 5 µg of BSA, 120 µL of CaCl2 solution (0.03 g/mL) and 7.5
23
µL fluorescent compound were mixed and then diluted to a total volume of 290 µL. The
24
preparation of the nanoparticles was then made as described above. The BSA containing
25
nanoparticles were centrifuged for 1h at 18 000 rpm. After centrifugation, the amount of
26
free BSA present in the supernatant was determined by UV/Vis spectroscopy at 460 nm,
27
by interpolation in a calibration curve with known BSA concentrations. Data were given
28
as mean ± standard deviation (SD) based on three independent measurements. The drug
29
loading content (DLC) was calculated as follows.
30 31
DLC (%) = [(total amount of BSA – non-bound BSA)/ nanoparticle weight] × 100 (2)
32 33
The encapsulation efficiency was determined as described above in eq. (1).
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Biomacromolecules
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Morphology, size and zeta (ζ) potential of nanoparticles
2
The morphology of pDNA nanoparticles was investigated by means of Scanning
3
Electron Microscopy (SEM), following the procedure described previously.21 The
4
average particle size of the nanoparticles and the surface charges (zeta potential) were
5
determined by dynamic light scattering using a Zetasizer nano ZS following the method
6
described in the literature.22
7 8
In vitro transfection studies
9
Cancer HeLa cells were grown in Dulbecco´s Modified Eagle´s Medium with High
10
Glucose (DMEM-HG) (Sigma) supplemented with 10% heat inactivated fetal calf
11
serum, 0.5 g/L sodium bicarbonate, 1.10 g/L HEPES and 100 µg/mL of streptomycin
12
and 100 units/mL of penicillin (Sigma), at 37°C in a 95% air/5% CO2 humidified
13
atmosphere. For transfection studies, cells were seeded in 24 well-plates at a density of
14
2 × 105 cells/well in 1 mL of DMEM without antibiotic. When 90 % confluency was
15
attained, the medium was removed and washed twice with PBS. On the day of
16
transfection, confluent HeLa cells were transfected with nanoparticles (100 µL of
17
particles were added to each well). After 6 h incubation period, the cell culture medium
18
was replaced with fresh serum DMEM medium and the cells were allowed to incubate
19
for different times. After transfection, the medium was removed and the cells were
20
washed with PBS.
21 22
Separation of cellular fractions
23
After transfection, and in order to separate the mitochondrial and cytosolic cellular
24
fractions, the Mitochondria Isolation Kit for Cultured Cells (#89874, Thermo Fisher
25
Scientific Inc., Rockford, USA) was employed, as described elsewhere.22
26
Furthermore, in another experiment, after in vitro transfection lysosomes have been
27
isolated by using the Lysosome Isolation kit (LYSOSO 1). This assay provides a
28
protocol for the isolation of an enriched lysosomal fraction by differential centrifugation
29
followed by density gradient centrifugation and/or calcium precipitation. Briefly, HeLa
30
(2 × 106) cells were washed, collected and washed again. Thereafter, cells were break in
31
a Dounce homogenizer and breakage evolution was followed by trypan blue. The cells
32
were centrifuged sequentially at 1000 g and 20 000 g; the pellet from 20 000 g was
33
collected. Calcium chloride (8 mM) was then added and incubated for 15 min followed
34
by centrifugation at 5000 g. The supernatant enriched in lysosomes was recovered. 7 ACS Paragon Plus Environment
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Alternatively, the pellet was placed on an Optiprep step gradient and centrifuged at 150
2
000 g. The fractions were collected and calcium chloride (8 mM) was added and
3
procedures were carried out as described above. The presence and integrity of
4
lysosomes have been confirmed by measuring the acid phosphatase activity with the
5
Acid Phosphatase assay kit (CS0740, Sigma-Aldrich).
6 7 8
Quantification of proteins.
9
BSA amount was quantified using the bicinchoninic acid method, according to the
10
protocol described by the manufacturer (PierceTM BCA Protein assay kit, Thermo
11
Scientific).
12
Green fluorescent protein (GFP) levels were determined by GFP ELISA kit
13
(MitoSciences, ab 117992, Abcam, United Kingdom), as described previously.22
14
The p53 protein levels have been quantified by using the p53 pan ELISA kit (Roche
15
Applied Science), following the procedure described by the manufacturer. The
16
concentration of p53 protein can be determined by spectrophotometrically measuring
17
the absorbance at 450 nm using a Shimadzu UV-Vis 1700 spectrophotometer.)
18
All the experiments were repeated three times in triplicate. Student’s t-test was used to
19
determine significance of the results, and the results were presented as mean ± standard
20
deviation (SD).* p < 0.05, ** p < 0.01.
21 22
Cell growth assay
23
HeLa cells were seeded in 24-well plates at a density of 2 × 105/well. After 24 h, the
24
cells were transfected with pcDNA3-FLAG-p53 alone or the various pcDNA3-FLAG-
25
p53/fluorescent compound nanoparticles. The amount of viable adherent cells were
26
determined by trypan blue exclusion assay performed every other day. Untreated cells
27
were considered as control. Assays were performed in triplicate.
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Results and Discussion
2 3
Cytotoxicity analysis. A brief discussion concerning the synthesis of fluorescent
4
compounds is available in the Supporting Information. One relevant issue to consider a
5
probe suitable to be used in living cells is its cellular toxicity. The cytotoxicity of the
6
compounds was evaluated using fibroblast cells (NHDF) and cancer HeLa cells. The
7
experiment fully described and presented in the Supporting Information (Figure S1)
8
shows that compounds AcridTriamine, Naphtripodal and QuinTriamine are not toxic to
9
the cells, while QuinPentamine is cytotoxic to both fibroblast and HeLa cells for tested
10
concentration (0.01 µM). To evaluate the effect of ligand concentration on cell viability,
11
NHDF cells were incubated, for 72 h, with AcridTriamine, Naphtripodal and
12
QuinTriamine, with concentrations ranging from 0.01 µM to 10 µM and the results are
13
summarized in Figure 2. All compounds induce a decrease in cellular viability by
14
increasing its concentration. Concentrations higher than 1 µM, drastically decrease cell
15
viability and when a concentration of 10 µM is considered, a remarkable cytotoxicity is
16
observed for all compounds studied, being AcridTriamine the most cytotoxic to NHDF
17
cells. A previous study focused on the targeting ability of fluorescent macrocyclic
18
compounds have revealed the low toxicity of [16]phenN2 in NHDF, MCF-7 and in
19
human adult dermal skin fibroblasts from a breast cancer patient (P14). This compound
20
show decreased levels of cytotoxicity at concentrations ranging from 10 nM to 10 µM.15
21
Therefore, this low cytotoxicity observed for the studied fluorescence compounds
22
suggests that they may have a potential utility as targeting probes.
23 24
Sub-cellular localization. An important characteristic of the synthesized compounds is
25
their sub-cellular localization. The intracellular localization of the fluorescent
26
compounds was accessed through colocalization studies using nuclei, mitochondria and
27
lysosomes specific labelling under confocal microscopy. As shown in Figure 3, the
28
QuinTriamine compound colocalizes with the lysosomes after 1 hour of incubation. The
29
AcridTriamine and Naphtripodal compounds are localized in the cytoplasm of the cells,
30
and no colocalization in the mitochondria nor in lysosomes was observed (data not
31
shown). None of the compounds studied localize in the nucleus. Previous study on the
32
evaluation of macrocyclic compounds as potential molecular probes, indicates that
33
[16]phenN2 compound exhibits sub-cellular localization pattern on mitochondria and
34
endoplasmic reticulum.15 9 ACS Paragon Plus Environment
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1
Formulation and characterization of pDNA or BSA loaded nanoparticles. Plasmid
2
DNA or BSA based nanoparticles have been formulated by a co-precipitation method.17
3
CaCO3 is an inorganic and biocompatible component that can be dissolved in acidic
4
environment and release Ca2+ and CO32-, ions naturally found in the body and safe to be
5
incorporated in the blood.23 In this work, pCAG-GFP-ND1, pcDNA3-FLAG-p53 or
6
BSA have been encapsulated into calcium carbonate co-precipitates by a fast and easy
7
protocol. Additionally, the fluorescent compounds have been included in the procedure
8
of nanoparticles formation. As shown by scanning electron microscopy analysis (Figure
9
S2, available in the Supporting Information), the co-precipitation method gives rise to
10
spherical or ellipsoidal nanoparticles with rough surfaces displaying sizes lower than
11
500 nm. Information concerning the mean size and the surface charges of these
12
nanoparticles was obtained by dynamic light scattering and data is summarized in Table
13
1 and Table S1 (available in the Supporting Information). For plasmid DNA/ligand
14
systems (Table 1), diameters of the nanoparticles are higher than 170 nm but lower than
15
340 nm. For both tested plasmids, the inclusion of [16]phenN2 gives rise to
16
nanoparticles with the lowest sizes, while large diameter vectors are synthesized when
17
QuinTriamine is added to co-precipitate in the nanoparticles formation protocol.
18
Comparison between the two plasmids shows the synthesis of particles with lower sizes
19
when using pcDNA3-FLAG-p53. Considering that the size of the plasmids is identical,
20
this fact seems to indicate a stronger electrostatic interaction of Ca2+ with the negatively
21
charged p53 plasmid, despite the slightly higher negative charges displayed by the
22
pCAG-GFP-ND1 plasmid (Table 1). Concerning the surface charges, all the
23
nanoparticulate systems exhibit positive zeta potential values, and higher than +30 mV.
24
According to previous studies, nanoparticles with zeta potentials greater than +30 mV
25
are considered strongly cationic and this property can be related with an enhanced
26
ability to permeate cellular membranes, as the latter are known to be negatively
27
charged.24 Not only the charges of nanoparticles can influence the success of cellular
28
uptake, but also their size and morphology are important parameters. In fact, it is the
29
intricate balance between all the particle properties that can dictate the efficiency of
30
vector internalization and, ultimately, gene transfection efficiency.25 It has been
31
demonstrated that spherical and positively charged nanoparticles with sizes less than
32
300 nm generally favors cellular uptake. It should be mentioned, however, that the
33
optimal physicochemical carrier properties for efficient cellular uptake may be
34
dependent on cell type.26 Furthermore, the quantification of pDNA encapsulation 10 ACS Paragon Plus Environment
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Biomacromolecules
1
efficiency (EE) is an essential parameter in the characterization of therapeutic carriers.
2
The pDNA/fluorescent compound nanoparticles resulted in high pDNA encapsulation
3
efficiencies, especially for p53 based vectors (Table 1). Nanoparticles where
4
[16]phenN2 is present encapsulate more efficiently both plasmids, with slightly larger
5
EE found for p53 systems, while, AcridTriamine incorporated particles leads to the
6
lowest pND1 and p53 EE values. The association of this result with the smallest size
7
presented by pDNA/[16]phenN2 nanoparticles turns these vectors the most promising,
8
of all the studied systems, for the cellular internalization/uptake process and for gene
9
delivery applications. The fluorescent ligand encapsulation efficiencies were also
10
determined. The EE (%) data, obtained from spectrophotometric analysis, are for
11
AcridTriamine: 68 ± 2.3 and 72 ± 3, for Naphtripodal: 62 ± 2.8 and 67 ± 4.1, for
12
QuinTriamine: 70 ± 4.6 and 73 ± 2.5, for [16]phenN2: 74 ± 4.4 and 76 ± 3.9, relative to
13
pCAG-GFP-ND1/compound
14
respectively. Therefore, we found that, besides pDNA, also fluorescent compounds can
15
be easily and efficiently encapsulated into calcium carbonate precipitates forming
16
pDNA/fluorescent compound based particles. Moreover, the cytotoxicity profile of
17
these nanosystems was also investigated in different cell lines by means of MTT
18
colorimetric assay. The results, after 72 h of cells incubation with the different
19
nanoparticles (50 µL), are presented in Table 1. All systems demonstrated to be
20
biocompatible for NHDF, HeLa and MCF-7 cells, since no significant loss of cell
21
viability have been observed.
22
BSA protein has also been encapsulated into nanoparticles. The main physicochemical
23
properties of BSA loaded particles were investigated and are listed in Table 1 of
24
Supporting Information (Table S1). The mean size of BSA vectors depends on the
25
fluorescent compound incorporated in the preparation procedure, however, and for all
26
ligands considered, BSA nanoparticles show larger sizes when compared to the
27
corresponding pDNA vectors. Similarly to what has been observed for the size of
28
pDNA based nanoparticles, small particles are formed in the presence of [16]phenN2.
29
Furthermore, BSA particles exhibit positive surface charges, with small variations
30
between the different systems. Higher encapsulation efficiency values have been
31
obtained for all studied BSA carriers. The results demonstrate that BSA can be more
32
efficiently encapsulated into calcium carbonate nanoparticles than pCAG-GFP-ND1 or
33
pcDNA3-FLAG-p53 plasmid. Moreover, fluorescent compounds can also be
and
pcDNA3-FLAG-p53/compound
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incorporated into BSA nanoparticles. The EE (%) data, are for AcridTriamine: 62 ± 5.1,
2
for Naphtripodal: 60 ± 5.3, for QuinTriamine: 66 ± 3.9 and for [16]phenN2: 68 ± 4.4
3
An MTT analysis revealed that, similarly to pDNA based nanoparticles, BSA carriers
4
are biocompatible for NHDF, HeLa and MCF-7 cells (data not shown).
5
Targeting of cellular organelles. The phenomena of pDNA/ligand nanoparticles
6
cellular uptake and internalization have been examined by the quantification of
7
compound fluorescence intensity in the various cellular fractions, such as, cytosol,
8
mitochondria and lysosomes of HeLa cells. Figure 4 presents the cell-associated
9
fluorescence intensity of each fluorescent compound and after 48 h of transfection
10
mediated by pCAG-GFP-ND1 or pcDNA3-FLAG-p53/compound based nanoparticles.
11
The results obtained are in clear agreement with the fluorescence microscopy study
12
presented above. The compounds AcridTriamine and Naphtripodal do not localize in the
13
mitochondria neither in the lysosomes, instead, they can both be detected in the cytosol
14
of HeLa cells. The AcridTriamine or Naphtripodal fluorescent levels in mitochondria
15
and lysosomes are insignificant compared with their higher fluorescence intensity
16
detected in the cytosolic cellular fraction. Moreover, the extent of cytoplasm
17
accumulation of fluorescent compound is increased when the transfection process is
18
mediated by pCAG-GFP-ND1 or pcDNA3-FLAG-p53/AcridTriamine or Naphtripodal
19
based particles (Figure 4A and Figure 4B). Comparing the two plasmid systems,
20
transfection mediated by p53/AcridTriamine or Naphtripodal nanoparticles is more
21
efficient, as it promotes higher fluorescence intensity levels. Conversely, as expected by
22
the previous fluorescence microscopy study, QuinTriamine appears to localize in the
23
lysosomes of HeLa cells, as evidenced in Figure 4C. Residual fluorescence levels of this
24
compound are present in both cytosol and mitochondria. Moreover, the fluorescence
25
intensity levels, detected in the lysosomes, are increased when transfection is mediated
26
by pCAG-GFP-ND1 or pcDNA3-FLAG-p53/QuinTriamine carriers. The compound
27
[16]phenN2 reveals mitochondrial affinity since higher levels of this ligand were
28
detected in this organelle, Figure 4D. This result is in agreement with a previous work
29
on the cellular targeting evaluation of macrocyclic probes.15 As previously observed, the
30
levels of each fluorescent compound in the different cellular organelles are increased
31
when transfection is mediated by the plasmid/ligand based nanoparticles. Furthermore,
32
when p53 plasmid is used, higher fluorescence intensity can be monitored in the various
33
cellular fractions of HeLa cells. As these p53 based nanoparticles already demonstrated 12 ACS Paragon Plus Environment
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Biomacromolecules
1
to possess a set of properties concerning size, morphology, surface charge, loading and
2
encapsulation capacity suitable for gene transfection purposes, in addition to their
3
targeting ability, these p53 carriers seem to be very promising to be applied in cancer
4
therapeutics.
5
Quantification of proteins. The main aim of the prepared formulations is the delivery
6
of a bioactive drug or gene to a desired cellular organelle for a therapeutic action. The
7
presence of BSA and the expression of proteins through the cellular transfection
8
mediated by the described fluorescent compound nanoparticles have been investigated.
9
The results are summarized in Table 2. The GFP, p53 and BSA in the different cellular
10
fractions of HeLa cells, have been quantified by the experimental protocols described
11
above. In the absence of incorporated fluorescent compounds into pDNA nanoparticles,
12
both pCAG-GFP-ND1 and pcDNA3-FLAG-p53 based vectors are able of GPF and p53
13
protein production, respectively. However, the obtained protein levels when transfection
14
of HeLa cells is mediated by free ligand nanoparticles is significantly lower (Table 2).
15
Incubation with AcridTriamine, Naphtripodal, QuinTriamine and [16]phenN2/BSA
16
based nanoparticles in HeLa cells results in the accumulation of BSA in the cytosol,
17
lysosomes and mitochondria of cancer cells, respectively. This confirms the specific
18
targeting ability displayed by the BSA/fluorescent ligand carriers and demonstrates the
19
potential utility of these particles for the targeted delivery of drugs to individualized
20
cellular organelles. Therapies centred in the release of pharmaceutics to lysosomes or
21
mitochondria can greatly benefit from this strategy. Targeted delivery to lysosomes is a
22
particularly interesting therapeutic option for many lysosomal storage diseases or for the
23
direct enzyme replacement in this organelle.27,28 Concerning cancer therapy, the
24
inconvenient of multidrug resistance can also be outcome by the agents targeting
25
lysosomes strategy.20,29 In the same way, the targeting of macromolecules and
26
anticancer drugs to mitochondria may provide an effective tool for the treatment of
27
mitochondrial diseases and cancer.30,31 Transfection mediated by pCAG-GFP-
28
ND1/AcridTriamine and pCAG-GFP-ND1/Naphtripodal based nanoparticles results in
29
the expression of GFP in the cytosol. Similarly, HeLa cells transfection with pcDNA3-
30
FLAG-p53/AcridTriamine and pcDNA3-FLAG-p53/Naphtripodal nano-carriers leads to
31
the production of p53 protein. The formulated CaCO3/pDNA/fluorescent ligand vectors
32
are able to cross the cell membrane by an endocytosis process.32 Due to the acidic
33
environment of endosomes, nanoparticles can be dissolved being able to release the 13 ACS Paragon Plus Environment
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1
bioactive genetic content into the cytoplasm. To be incorporated into the nucleus, where
2
pDNA can be expressed, this payload must cross the nuclear barrier, which is facilitated
3
during mitosis, when the nuclear pores become wide open. Thus, the high rate division
4
of cancer cells may contribute to a higher efficiency of pDNA entrance into the nucleus
5
of these cells. Additionally, the strong electrostatic interaction between the Ca2+ ions
6
and pDNA protect this latter from nuclease degradation, enhancing the nuclear uptake
7
of pDNA. Therefore, efficient transfection and gene expression can occur with the
8
ultimate production of proteins in the cytoplasm. In line with this, transfection mediated
9
by
pCAG-GFP-ND1/QuinTriamine
and
pcDNA3-FLAG-p53/QuinTriamine
10
nanoparticles also results in the expression of GFP and p53 protein, as shown in Table
11
2. The low pH (4.5 – 5.5) inside lysosomes promotes the degradation of CaCO3
12
matrices facilitating the release of the genetic material that, thereafter, can be
13
internalized into the nucleus and able of gene expression by the mechanism presented
14
above. Although both proteins can be produced when the two pDNA/QuinTriamine
15
nano-systems are considered, their levels are lower when compared to the quantified
16
GFP and p53 protein amounts when transfection is mediated by pDNA/AcridTriamine
17
and pDNA/Naphtripodal nanoparticles. Therefore, these results suggest that cellular
18
transfection is less efficient when QuinTriamine is present in the nanoparticles.
19
Besides the nucleus, mitochondria also possess a genome: the mitochondrial
20
DNA, mtDNA.33 Mitochondria present a different codon system from the nucleus and
21
some codons correspond to different amino acids in mitochondria than in universal
22
nuclear code.33 Therefore, and as expected, no green fluorescent protein neither p53
23
protein
24
ND1/[16]phenN2 and pcDNA3-FLAG-p53/[16]phenN2 based carriers. The same kind of
25
observation was found before.22 Evolution in this topic can be achieved by the
26
conception of artificial mitochondrial genomes and the production of recoded proteins.34
27
The phosphoprotein p53 is known to be involved in malignancy, apoptosis and
28
other abnormal cell proliferation processes. It regulates the cell cycle preventing tumour
29
development. The therapeutic effect induced by the treatment of cancer cells with
30
pcDNA3-FLAG-p53/fluorescent ligand nanoparticles has been evaluated through an in
31
vitro growth assay. Figure 5 presents the rate of HeLa cell growth for a period of 8 days
32
after transfection with the pcDNA3-FLAG-p53 or the various pcDNA3-FLAG-
33
p53/ligand vectors. From this study, it was found that the growth rate of HeLa cells
34
transfected with p53, p53/AcridTriamine, p53/Naphtripodal and p53/QuinTriamine
can
be
expressed
when
transfection
is
mediated
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systems was significantly inhibited, in comparison with control cells. However, the
2
effect of free ligand p53 loaded vehicles is lower when compared to the inhibitory effect
3
promoted by ligand p53 based carriers. Among the fluorescent pDNA vectors, it was
4
observed that p53/AcridTriamine and p53/Naphtripodal nanoparticles are more efficient
5
in promoting the suppression of cell growth and proliferation, which is in agreement
6
with the previous described results. Following this, the lower levels of p53 protein
7
produced when transfection is mediated by p53/QuinTriamine also corroborate well
8
with lower cancer therapeutic role demonstrated by this vector. Therefore, the
9
optimization of the design of gene delivery systems conjugated with AcridTriamine and
10
Naphtripodal compounds seems to be quite promising for cancer gene therapy
11
applications. In contrast, p53/[16]phenN2 loaded nanoparticles show to be ineffective in
12
preventing cell growth. As discussed, these nanoparticles are targeted to the site of
13
mitochondria where, due to a different genetic code, p53 protein cannot be expressed. It
14
can thus be stated that [16]phenN2 conjugated vectors are not appropriate for nuclear
15
gene therapy. Instead, they can found useful biomedical applications in the emerging
16
area of mitochondrial gene therapy where the treatment of mtDNA mitochondrial
17
disorders, can greatly evolve through the development of a mitochondria targeting gene
18
carrier.
19
Conclusions
20
The synthesized fluorescent compounds display low cellular toxicity and can localize in
21
one of the following cellular fractions: cytosol, lysosomes or mitochondria. Fluorescent
22
compounds have been incorporated into BSA or plasmid DNA calcium carbonate
23
nanoparticles. The vectors possess suitable properties (size, morphology, surface charge
24
and encapsulation efficiency) to be applied in both drug and gene delivery applications.
25
Furthermore, the specific sub-cellular localization of fluorescent compounds confers
26
targeting ability to nanoparticles enhancing their performance, namely, as gene carriers.
27
The expression of green fluorescent and p53 proteins, mediated by fluorescent plasmid
28
DNA systems, opens the possibility to further explore them for clinical therapeutic
29
purposes. Following this achievement, an in vitro cell growth assay, demonstrate that
30
gene therapy mediated by the formulated vectors appears to be very promising in the
31
treatment of cancer. The present study is a remarkable contribution for the design of
32
targeted drug and gene delivery systems contributing for progresses in the therapeutic
33
efficacy of synthetic carriers. 15 ACS Paragon Plus Environment
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1
Supporting Information
2
The supporting information is available free of charge via the internet at
3
http://pubs.acs.org. Fluorescent compounds synthesis protocols; cytotoxicity analysis of
4
fluorescent ligands in Figure S1; scanning electron micrographs of pDNA/fluorescent
5
compound nanoparticles in Figure S2; properties of BSA/fluorescent ligand loaded
6
nanoparticles in Table S1.
7 8
Acknowledgements
9
D. Costa and C. Cruz acknowledge the FCT projects “Projeto de Investigação
10
Exploratória” references IF/01459/2015 and IF/00959/2015, respectively.
11
This work was supported by FEDER funds through the POCI - COMPETE 2020 -
12
Operational Programme Competitiveness and Internationalisation in Axis I -
13
Strengthening research, technological development and innovation (Project POCI-01-
14
0145-FEDER-007491) and National Funds by FCT - Foundation for Science and
15
Technology (Project UID/Multi /00709/2013).
16 17 18
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Figure Captions
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 1. Chemical structures of AcridTriamine, Naphtripodal, QuinTriamine, QuinPentamine and [16]phenN2.
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Figure 4. Ligand fluorescence intensity in HeLa cells after 48 h incubation with each ligand or after transfection with pCAG-GFP-ND1/ligand (X) or pcDNA3-FLAGp53/ligand (X) based nanoparticles. The data were obtained by calculating the average of 3 experiments. The respective errors were determined and were below 0.05%. Oneway ANOVA analysis was performed followed by Bonferroni´s multiple comparison test that indicated that differences of control versus pCAG-GFP-ND1/ligand (X) or pcDNA3-FLAG-p53/ligand (X) nanoparticles and differences of ligand versus pCAGGFP-ND1/ligand (X) or pcDNA3-FLAG-p53/ligand (X) nanoparticles were statistically significant (p ˂ 0.05). X is AcridTriamine (A), Naphtripodal (B), QuinTriamine (C) or [16]phenN2 (D).
Figure 2. Cell viability of fibroblast cells after 72 h incubation time with Naphtripodal, QuinTriamine and AcridTriamine, in concentrations ranging from 0.01 µM to 10 µM. Percent viability is expressed relative to control cells (control was set to 100% cell viability). Mean values ± SD are obtained from three experimental determinations; p < 0.05 versus the control (one-way ANOVA with Dunnet`s post-hoc test).
Figure 3. Confocal microscopy co-localization studies of QuinTriamine with lysosomes and mitochondria on MCF7 cell line. MCF7 cells untreated [QT(-)] or treated [QT(+)] with 1µM QuinTriamine for 1h followed by 30 min incubation with 0.1µM Lysotracker, for lysosomes staining (A), or 0.1µM MitoTracker, for mitochondria labeling (B). White arrows indicate colocalization between lysosome labeling and QuinTriamine. Scale bar = 10µM.
Figure 5. Cell growth assay in HeLa cells after transfection with pcDNA3-FLAG-p53, pcDNA3-FLAG-p53/AcridTriamine, pcDNA3-FLAG-p53/Naphtripodal, pcDNA3FLAG-p53/QuinTriamine and pcDNA3-FLAG-p53/[16]phenN2 based nanoparticles compared with untreated cells. The data represent the average of 5 independent experiments; p ˂ 0.05 in comparison with the control.
38 39 40 41 42 43 44 45 21 ACS Paragon Plus Environment
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Figures
2
3 4 5
Figure 1. 100
80
Cell Viability (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Naphtripodal * * *
60
*
40
* 20
0
0.01
0.1
1
5
Concentration (µM)
6
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Page 23 of 28
100
QuinTriamine
Cell Viability (%)
80
* *
60
* 40
* *
20
0 0.01
0.1
1
5
10
Concentration (µM)
1
100
AcridTriamine 80
* Cell Viability (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
60
*
40
* *
20
* 0
0.1
1
5
Concentration (µM)
2 3
0.01
Figure 2.
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Biomacromolecules
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B 2 3 4 5 6 7 8 9
Figure 3.
10 11 12 13
Figure 3
14 A Fluorescence Intensity (a.u.)/µg AcridTriamine
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Cytosol
AcridTriamine pND1/AcridTriamine nanoparticles p53/AcridTriamine nanoparticles
4
3
2
1
Mitochondria
Lysosomes
0
15
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Fluorescence Intensity (a.u.)/µg NaphTripodal
B Naphtripodal pND1/Naphtripodal p53/Naphtripodal
5
Cytosol 4
3
2
1
Mitochondria
Lysosomes
0
1 Fluorescence Intensity (a.u.)/µg QuinTriamine
C 5
4
QuinTriamine pND1/QuinTriamine p53/QuinTriamine
Lysosomes
3
2
1
Cytosol
Mitochondria
0
2 D Fluorescence Intensity (a.u.)/µg [16]phenN2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Mitochondria
[16]phenN2 pND1/[16]phenN2 p53/[16]phenN2
4
3
2
1
Cytosol
Lysosomes
0
3 4
Figure 4.
5
25 ACS Paragon Plus Environment
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Control p53 p53/AcridTriamine p53/Naphtripodal p53/QuinTriamine p53/[16]phenN2
100
80 4 Number of Cells (x 10 )
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60
40
20
0 0
2
4
6
8
Time (days)
1 2
Figure 5.
3 4 5 6 7 8 9 10 11 12 13
Tables Table 1. Mean size, average zeta potential, pDNA encapsulation efficiency and cell viability, at 72 h, in different cell lines (NHDF, HeLa and MCF-7) of fluorescent compound/pCAG-GFP-ND1 (pND1) or fluorescent compound/pcDNA3-FLAG-p53 (p53) based nanoparticles with 5 µg pDNA loading amount. Average zeta potential values of the pCAG-GFP-ND1 and pcDNA3-FLAG-p53 were also presented. The values were calculated with the data obtained from three independent measurements (mean ± SD, n = 3). Size (nm)
Zeta Potential (mV) pDNA EE (%)
System pCAG-GFP-ND1
Cell viability (%) NHDF
HeLa
MCF-7
-102.3 ± 5.4
pND1/AcridTriamine
223 ± 4.5
+41.3 ± 4.2
58.2 ± 3.3
78 ± 5.2
73 ± 2.2
70 ± 3.7
pND1/Naphtripodal
290 ± 7.8
+34.1 ± 2.9
64.1 ± 4.9
82 ± 2.3
76 ± 4.1
70 ± 2.9
pND1/QuinTriamine
326 ± 11
+47.6 ± 8.3
67.6 ± 4.5
85 ± 4.1
85 ± 3.3
72 ± 4.2
pND1/[16]phenN2
201 ± 5.1
+44.9 ± 5.1
73.8 ± 5.0
85 ± 5.3
86 ± 4.9
82 ± 5.1
pcDNA3-FLAG-p53
-98.4 ± 8.6
p53/AcridTriamine
194 ± 6.9
+42.2 ± 7.1
64.2 ± 3.9
74 ± 4.6
72 ± 3.8 70 ± 2.1
p53/Naphtripodal
205 ± 5.5
+30.8 ± 6.6
68.5 ± 6.2
79 ± 3.9
80 ± 1.3 72 ± 4.4
p53/QuinTriamine
277 ± 7.1
+46.1 ± 9.5
79.3 ± 5.7
81 ± 2.9
80 ± 4.0 79 ± 1.9
p53/[16]phenN2
178 ± 4.3
+38.4 ± 8.3
80.1 ± 4.9
84 ± 4.2
81 ± 2.6 78 ± 2.9
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Biomacromolecules
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Table 2. Quantification of BSA, GFP and p53 protein content in HeLa cells after 48 h of incubation with BSA/ligand (X), pCAG-GFP-ND1, pCAG-GFP-ND1/ligand (X), pcDNA3-FLAG-p53 or pcDNA3-FLAG-p53/ligand (X) based nanoparticles, where X = AcridTriamine, Naphtripodal, QuinTriamine or [16]phenN2. The values are calculated with the data obtained from three independent measurements (mean ± SD, n = 3). BSA (ng/mL) Cytosol
Lysosomes
GFP (ng/mL)
p53 (ng/mL)
Cytosol
Cytosol
Mitochondria
System BSA/AcridTriamine
663 ± 9.2
BSA/Naphtripodal
598 ± 10.9
BSA/QuinTriamine
508 ± 9.4
BSA/[16]phenN2
602 ± 7.1
pND1
272 ± 5.5
pND1/AcridTriamine
463 ± 8.5
pND1/Naphtripodal
449 ± 12.6
pND1/QuinTriamine
328 ± 7.4 0
pND1/[16]phenN2 p53
416 ± 9.1
p53/AcridTriamine
584 ± 8.9
p53/Naphtripodal
609 ± 10.2
p53/QuinTriamine
478 ± 9.9
p53/[16]phenN2
0
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Biomacromolecules Page 28 of 28 Lysosomes
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AcridTriamine NaphTripodal
Cytosol
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pDNA/[16]phenN2
