Assembly of plasmid DNA with pyrene-amines cationic amphiphiles into nanoparticles and their visible lysosome localization

Article abstract of DOI:10.1039/c4ra06879c

In this study, we constructed a visible model for drug/gene dual delivery. Firstly, we prepared a series of pyrene fluorophore bearing cationic amphiphiles (Py-amines), which could be further employed as amphiphilic gene carriers and antitumor polyamine drug models. Then, the Py-amines fluorescent amphiphiles were utilized to bind/load plasmid DNA via electrostatic interactions to form nano-sized particles in aqueous solution. The average size, zeta potential and morphology of the self-assembled Py-amines/pDNA complexes were found to be largely dependent on the molecular structures of Py-amines amphiphiles. Moreover, the Py-amines and their pDNA complexes showed an evident cell proliferation inhibition capability in H1299 (human lung cancer) cells. Notably, the lysosomal localization of the Py-amines/pDNA complexes could be directly visualized using fluorescence microscopy in vitro. In summary, this current study could provide new and visible approach to design polyamine-based anti-tumor drug/plasmid DNA dual delivery systems, which could facilitate a greater understanding of the intracellular trafficking/localization of polyamine-based cationic gene/drug payloads.

Full text of DOI:10.1039/c4ra06879c

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PAPER
Assembly of plasmid DNA with pyrene-amines
cationic amphiphiles into nanoparticles and their
visible lysosome localization†
Cite this: RSC Adv., 2015, 5, 12338
a
b
a
a
a
Ruilong Sheng,* Feifei An, Zhao Wang, Mingrui Li and Amin Cao*
In this study, we constructed a visible model for drug/gene dual delivery. Firstly, we prepared a series of
pyrene fluorophore bearing cationic amphiphiles (Py-amines), which could be further employed as
amphiphilic gene carriers and antitumor polyamine drug models. Then, the Py-amines fluorescent
amphiphiles were utilized to bind/load plasmid DNA via electrostatic interactions to form nano-sized
particles in aqueous solution. The average size, zeta potential and morphology of the self-assembled Py-
amines/pDNA complexes were found to be largely dependent on the molecular structures of Py-amines
amphiphiles. Moreover, the Py-amines and their pDNA complexes showed an evident cell proliferation
inhibition capability in H1299 (human lung cancer) cells. Notably, the lysosomal localization of the Py-
amines/pDNA complexes could be directly visualized using fluorescence microscopy in vitro. In
summary, this current study could provide new and visible approach to design polyamine-based anti-
tumor drug/plasmid DNA dual delivery systems, which could facilitate a greater understanding of the
intracellular trafficking/localization of polyamine-based cationic gene/drug payloads.
Received 9th July 2014
Accepted 23rd December 2014
DOI: 10.1039/c4ra06879c
www.rsc.org/advances
6
polyethyleneimine (Cho-OEI) lipids and diammonium con-
Introduction
7
–9
taining Gemini-lipids
possessed remarkably high gene
In recent years, nano-scaled functional delivery systems have transfection efficiencies, even beyond the “gold standard”
10
been developed and employed as potential tools for the efficient transfection agent bPEI-25K. Yang et al. developed 1,12-
transportation of drug/gene substances toward disease treat- diaminododecane-polyamine cationic bolaamphiphiles as gene
1,2
ment. In these delivery systems, various amine-containing carriers and found these simple-structured bolaamphiphiles
cationic building blocks were employed as positively charged possessed highly efficient pDNA transfection capabilities. Yu
11
moieties to bind/load negatively charged gene substances via et al. synthesized some cyclic polyamine-conjugated lipids,
electrostatic forces. Among them, polyamines (such as sper- and revealed that they have high DNA binding affinities, and
2
3
mine and spermidine ) have oen been employed as functional demonstrated proton buffering effects and efficient luciferase
moieties in the construction of gene carriers, due to their rela- gene transfection behaviors. Although several polyamine-based
tively high positive charge density bringing about highly effi- cationic lipids have been developed as efficient gene carriers,
cient DNA binding/loading affinities. Moreover, polyamine- their intracellular trafficking and distribution are still not well
bearing gene carriers could be easily protonated and lead to known due to the lack of optical labeling/imaging moieties. To
4,5
intracellular “lysosomal escape” effects, which is regarded as date, the intracellular localization behaviors of polyamine-
an efficient pathway for enhancing the gene transfection containing cationic gene carriers have only been observed on
capability. To date, the development of new lipid-based poly- some uorescent-labeled polyamine-containing cationic poly-
12,13
amine conjugates with the merit of well-dened and control- mers.
Therefore, to gain a better understanding of the
lable structures as gene delivery carriers has attracted intracellular fate of polyamine-conjugated functional lipid gene
increasing attention, e.g., Bhattacharya et al. disclosed carriers, the development of uorophore-bearing polyamine
that some cholesterol-conjugated low molecular weight cationic lipids for “visible intracellular imaging” is highly
desired.
On the other hand, in addition to the contribution of the
Key Laboratory of Synthesis and Self-assembly Chemistry for Organic Functional polyamines in the construction of cationic gene carriers,
a
Molecules, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences,
recently, it has been revealed that some polyamine containing
amphiphiles or lipids participate in cell metabolism and tumor
3
45 Lingling Road, Shanghai 200032, China
b
National Centre for Nanoscience and Technology, Zhongguancun, Beijing 100190,
China. E-mail: rlsheng@mail.sioc.ac.cn; Tel: +86-21-54925535
1
4,15
proliferation,
role as a “pharmacophore”. For example, some polyamine–
in which the polyamine moiety plays a vital
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4ra06879c
16–18
anthracene conjugates and/or their analogs
were shown to
1
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possess evident anti-tumor activities through the inhibition of Genom Co. Ltd. In this study, all the other reagents and organic
the polyamine transporter (PAT), resulting in tumor cell solvents were of analytical grade and were utilized without
19
apoptosis. Mojzis et al. found that polyamine–naphthalimides further purication. Moreover, the chemical synthesis routes
conjugates have an evident anti-proliferative effect in MCF-7 and structural characterization of the newly synthesized Py-
and A549 cells through a possible non-specic DNA binding amines cationic amphiphiles are described in detail in the ESI
20
mechanism. Wang et al. revealed that some triamine-modied (S1†).
anthraquinones could target lysosome and lead to a possible
mitochondria-mediated cell apoptotic death, and the lysosome
targeting behavior could be indirectly observed by Lysotracker-
red and Hochest 33342 staining. These studies revealed that the
anti-tumor mechanisms of the polyamine-bearing amphiphiles
greatly rely on their molecular structures (such as the aromatic-
ring, charge number, linker, stereo-conguration) and demon-
strated that the polyamine-containing amphiphiles or lipids
could be employed as latent anti-tumor agents for cancer
NMR and MS measurements
1
H NMR spectra were recorded at room temperature on a Varian
VXR 300 FT-NMR Fourier transform NMR spectrometer
1
instrument, operating at 300.0 MHz for the H nuclei, whereas
1
3
C NMR spectra were characterized under room temperature
on a Bruker Avance 300 NMR spectrometer, operating at
1
3
7
5.0 MHz for the C nuclei with tetramethylsilane (TMS) as the
internal chemical shi reference. The mass spectra (ESI-MS)
were measured on a Varian SATURN 2000 spectrometer.
21
treatment. Oupick ´y et al.
recently prepared several
bisethylnorspermine-conjugates as cationic lipid prodrugs,
which could be utilized for gene/drug dual delivery in syner-
gistic breast cancer treatment, indicating that the polyamine-
24
Ethidium-bromide (EB) dye displacement assay
based gene/drug dual delivery system could be further devel- EB (5.0 mg) and pDNA (PCS plasmid DNA, 5.0 mg) were mixed
oped as a potential approach for multi-channel cancer therapy. in 1 mL of 1ꢁ PBS buffer solution (0.01 M, pH ¼ 7.4). Then, the
Moreover, for further investigation on the intracellular fate of mixture solution was further incubated for 2 min at room
polyamine-based gene/drug dual delivery systems, the devel- temperature, and the newly prepared Py-amines solution under
opment of a convenient, easily manipulated, efficient and in situ a calculated N/P charge ratio (N/P ratio of 3–30) was placed into
22
optical labeling/imaging techniques is required.
the as-prepared pDNA/EB mixture, and continuously incubated
Herein, we designed and synthesized a series of pyrene u- at ambient temperature for 2 min. Then, the uorescence
orophore bearing cationic polyamine amphiphiles (Py-amines) spectra were measured on a Hitachi F-7000 uorescence spec-
as visible models for drug/gene dual delivery, in which Py- trometer under excitation at 510 nm and an emission at 590 nm.
amines could bind with pDNA via positive–negative electro- The relative uorescence was measured and calculated
25
static interactions; moreover, the interaction of the Py-amines according to our prior study.
and pDNA were determined by EB displacement and DNA gel-
retardation assays, and the average particle size, surface
potential and shape/morphology of the Py-amines/pDNA
complexes were analyzed by a dynamic laser scattering instru-
ment (DLS) and atomic force microscope (AFM), respectively.
Furthermore, MTT cytotoxicity assay and uorescence micros-
copy imaging were employed to explore the possibility of drug/
gene dual delivery using the Py-amines/pDNA complexes.
Agarose gel retardation assay
The pDNA binding affinity of the Py-amines amphiphiles was
further analyzed by an agarose gel retardation assay. Before
the assay, the Py-amines samples were preliminarily dissolved
in pure water to prepare the solution with the concentration of
26
ꢀ
3
3
.3 ꢁ 10 M, and then the Py-amines/pDNA complexes were
prepared by mixing pDNA (1 mg) with a predetermined amount
of Py-amines under a preset N/P charge ratio in 50 mL 1ꢁ PBS
Experimental section
Materials
ꢂ
(
3
0.01 M, pH ¼ 7.4) buffer solution. Aer incubation at 37 C for
0 min, the complex solution was loaded onto a 1% agarose gel
ꢀ
1
containing 0.5 mg mL ethidium bromide dye (EB). Then, the
gel electrophoresis was conducted in 1ꢁ TAE running buffer
under 100 mV for 40 minutes, and the retardant in pDNA
migration was thus recorded on a UVP benchtop 2UV trans-
illuminator system.
Pyrene (97.5%) and polyethyleneimine (M
were purchased from Sigma-Aldrich and were utilized as-
received. Boc-protected amine-containing building blocks
w
¼ 800 Da, PEI-800)
23
were prepared according to previously reported studies .
Dicyclohexylcarbodiimide (DCC, 99.0%), 4-dimethylaminopyr-
idine (DMAP, 98.0%) and triuoroacetic acid (TFA, 97.0%) were
purchased from Shanghai Sinopharm Chemical Reagent Co.
Ltd (Shanghai, China) and were utilized as-received. PCS
Particle size and surface charge of the Py-amines/pDNA
complexes
ꢀ
1
plasmid DNA (pDNA, 1.0 mg mL ) and the H1299 cell lines The average particle size and surface charge of the Py-amines/
were generously gied by Prof. Yuhong Xu from Shanghai pDNA complexes in aqueous solution were characterized at
Jiaotong University, and Lysotracker-red was gied by Dr Hua room temperature on a Malvern Zetasizer Nano ZS90 dynamic
Zhu from Peking University. 96-well microplates and 50 mL cell light scattering instrument with l ¼ 633 nm at a xed scattering
ꢂ
27
cultivation asks were purchased from Corning Co. Ltd. 0.01 M angle of 90 (UK). The Py-amines samples were preliminarily
phosphate buffer solution (PBS), Dulbecco's modied Eagle's dissolved in pure water to prepare the solution with concen-
ꢀ
3
medium (DMEM) and 10% FBS were supplied by Hangzhou tration of 3.3 ꢁ 10 M, and then each sample was mixed with
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pDNA (5.0 mg) in 1 mL water under a predetermined N/P charge Intracellular localization of the Py-amines/pDNA complexes
ꢂ
ratio and then maintained under incubation at 37 C for 20 min.
H1299 and COS-7 cells were seeded into 24-well microplates
Aer that, the average particle size and surface charge of the as-
prepared Py-amines/pDNA complexes were analyzed on a Mal-
vern Zetasizer Nano ZS90 dynamic light scattering instrument.
5
(
4 ꢁ 10 cells per well in 1 mL DMEM medium with 10% FBS)
ꢂ
and incubated at 37 C under 5% CO
2
for 24 h. Then, the Py-
amines/pDNA complexes were added into each well at a N/P
ratio of 30 and washed with 1ꢁ PBS three times in order to
eliminate the uorescence background aer 4 h incubation.
Subsequently, the cells were stained with Lysotracker-red
Morphology of the Py-amines/pDNA complexes by atomic
force microscopy
ꢀ
1
(250 ng mL ) for 15 min to localize the position of the lyso-
some and washed with 1ꢁ PBS three times. Finally, the uo-
rescent images were observed and recorded on a Nikon Ti–S
invert uorescence microscope.
The Py-amines/pDNA complex morphology measurements were
conducted at room temperature on a Nanoscope IVa atomic
force microscope (AFM, Veeco Instrument) in tapping mode
with an Olympus AC160TS cantilever (frequency: 300 kHz,
ꢀ1
28
stiffness: 42 N m ). The Py-amines/pDNA complexes solution
ꢀ3
were rst prepared by mixing Py-amines (3.3 ꢁ 10 M) with Results and discussion
pDNA (5.0 mg) under a N/P charge ratio of 20 in 1 mL of pure
Synthesis of Py-amines amphiphiles
water, and then the complexes solution were preliminarily
dropped onto fresh cleaved mica and air-dried at room
temperature prior to the AFM measurements.
Facile, efficient and modular synthetic methods were acquired
for the diversely oriented preparation of bioactive substances
31
and pharmaceutics. In this study, a precursor compound of 1-
amino-pyrene (2) was synthesized by catalyzed reduction
(
NH
2
NH
2
$H
2
O with 10% Pd/C as the catalyst) of 1-nitropyrene
Proton buffering capacity measured by acid–base titration
25
(1) and served as a uorophore/hydrophobe dual functional
The proton buffering capacity for the newly synthesized Py- moiety. Then, the as-prepared (2) was coupled with various Boc-
amines in aqueous solution was measured by acid–base titra- protected amine/polyamine-containing building blocks (3, 4, 5,
tion on a Schott Titroline Easy instrument at room tempera-
29
ture with NaCl (0.1 M, negative control) and PEI-800
polyethyleneimine (800 Da) as a positive control) as refer-
(
ences. The cationic Py-amines and PEI-800 were separately
dissolved in distilled water to prepare the solution with total
amine concentration of 0.01 M, and then 1.0 M NaOH was
added to set the initial pH to 11.0–11.5, and then the solution
was gradually titrated with 0.1 M HCl solution. The proton
buffering curves were measured manually on a Schott Titroline
Easy instrument.
MTT cytotoxicity assay of the Py-amines and Py-amines/pDNA
complexes
30
MTT assays were conducted with H1299 cell lines in order to
evaluate the cytotoxicities of the Py-amines as well as their
pDNA lipoplexes. Firstly, the cells were seeded into 96-well
3
microplates with 5 ꢁ 10 cells per well in 100 mL RPMI-1640
ꢂ
medium (10% FBS added) and cultivated under 37 C and 5%
CO for 24 h. Subsequently, the medium was replaced with fresh
2
RPMI-1640 (10% FBS), and then the Py-amines and Py-amines/
pDNA complex under various concentrations (N/P charge ratios)
were individually added into the wells and further incubated for
ꢀ1
another 24 h. Then, 20 mL of MTT (5.0 mg mL ) was added into
each well and kept under incubation for 4 h. Aer removing the
medium, DMSO (100 mL per well) was added to dissolve the
formed MTT formazan. Finally, with a gentle shaking of the
microplates for 10 min in order to dissolve the formazan, each
sample with six replicates (n ¼ 6) was analyzed on a microplate
reader (BioTek, ELX800, USA) at labs ¼ 490 nm (labs ¼ 630 nm as
reference wavelength).
Fig. 1 Synthesis routes and chemical structures of the Py-amines
cationic amphiphiles.
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) under the catalysis of DCC/DMAP. Then, the Boc protection
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6
groups were removed in excess triuoroacetic acid at room
temperature to obtain a series of pyrene-derived cationic
amphiphiles (Py-amines) bearing different numbers of amino
groups as the nal products, with the isolation yields of 50–
65%. The amphiphiles were denoted as Py-1N (monoamine), Py-
2N (diamines), Py-3N (triamines) and Py-4N (tetramines),
respectively, and their synthetic routes are depicted in Fig. 1.
The synthesized Py-amines cationic amphiphiles and important
intermediates were characterized by NMR, MS and FTIR (see
ESI, S1†). The results indicated that the Py-amines cationic
amphiphiles were successfully prepared via facile, efficient and
modular synthetic approaches.
pDNA binding affinity of the Py-amines (EB and DNA-
retardation assays)
Fig. 3 Agarose-gel pDNA retardation assay for determining the pDNA
In order to examine the gene binding/loading capability of the binding affinity of the synthesized Py-amines cationic amphiphiles
Py-amines cationic amphiphiles, ethidium bromide (EB) under various N/P charge ratios.
32
28
displacement
employed. As shown in Fig. 2, it could be found that the relative
uorescence intensities of EB decreased gradually with the
and DNA gel-retardation assays
were
ca. 22 (Py-1N), 11 (Py-2N), 4 (Py-3N) and 5 (Py-4N), which might

8
inuence the condensation/decondensation of pDNA. More-
increasing amount of Py-amines (N/P from 0 to 30). Among
them, Py-3N (BC50 ¼ 3.3) showed a higher pDNA binding affinity
over, the trends of pDNA binding affinity for the Py-amines
amphiphiles analyzed by DNA gel-retardation assay were in
accordance with the EB displacement results. In other words,
these results indicated that the pDNA binding affinity could be
tuned by selecting cationic moieties/headgroups with proper
charge numbers, which should provide a possible approach for
controlled gene loading/release for drug/gene dual delivery
systems.
than that of Py-1N (BC50 ¼ 7.5) and Py-2N (BC ¼ 5.1), indi-
50
cating that the increasing positive charge number in cationic
amphiphiles could enhance the pDNA binding affinity;
however, lower binding affinity was observed on Py-4N (BC50
¼
4.2), which might due to the longer polyamine chains
23
decreasing the hydrophobicity of the cationic amphiphile.
Furthermore, the pDNA binding capabilities of the Py-amines
amphiphiles were examined using a pDNA retardation assay. As
shown in Fig. 3, it could be seen that Py-1N and Py-2N inhibited Particle size, surface potential and morphology of the Py-
the migration of pDNA at an N/P ratio of 21–24, Py-3N showed amines/pDNA complexes
higher pDNA retardation capability at a N/P ratio of 12–15, and
Py-4N showed lower pDNA retardation efficiency at a N/P ratio of
The size and shape, surface potential, as well as the particle
distribution of nano-scaled particles are already known to be
18–21). These results demonstrated that the number of amino
essential factors for determining the related endocytosis
groups in the Py-amines inuenced the pDNA binding affinity.
In addition, it could be estimated that the numbers of Py-
amines molecules interacting/binding with per DNA unit were
33
pathway, intracellular trafficking and localization behaviors.
Herein, dynamic laser scattering (DLS) method was utilized to
characterize the average particle size and surface potential of
the Py-amines/pDNA aggregates in distilled water with various
N/P charge ratios. As depicted in Fig. 4a, it can be seen that the
Py-amines and pDNA formed nano-scaled aggregates (diameter
ca. 176–296 nm) at a low N/P charge ratio of 5, while when the N/
P charge ratio was increased to 10–15, the particle sizes of the
Py-amines/pDNA aggregates decreased to ca. 135–226 nm,
indicating that the Py-amines amphiphiles possessed pDNA
34
condensation abilities in water. However, with the continuous
increase in the N/P charge ratio from 15 to 30, the average
particle sizes of Py-amines/pDNA aggregates were drastically
raised (about 81–196%). Notably, the pDNA complexes formed
by Py-3N and Py-4N showed much larger particle size than that
of the Py-1N and Py-2N counterparts at a higher N/P ratio (see
Fig. 4a and ESI, S2†), which might be attributed to their higher
hydration capabilities (see ESI, S3†). Moreover, for the Py-
amines/pDNA complexes, the surface zeta potential converted
from negative to positive with increase in the N/P charge ratio
Fig. 2 Ethidium bromide (EB) displacement assay of the plasmid DNA
binding affinity of the synthesized Py-amines amphiphiles in 0.01 M
PBS buffer.
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Fig. 4 The particle sizes (a) and surface potentials (b) of the Py-amines/pDNA complexes under various N/P charge ratios in pure water,
determined using a dynamic light scattering instrument (DLS, Malvern Nano ZS290, UK) with a laser wavelength of l ¼ 633 nm and a scattering
ꢂ
angle of 90 .
from 0 to 5 (see Fig. 4b), and then gradually increased with electrostatic interactions and form nano-scaled complexes in
increase in the N/P ratio from 10 to 30. In addition, it could be water. Moreover, the particle size observed by AFM was found to
found that the surface potentials were in a trend of Py-1N/pDNA be smaller than that measured by DLS (around 130–490 nm).
(from +20.4 to +27.0 mV) > Py-2N/pDNA z Py-3N/pDNA (from This could be explained as the Py-amines/pDNA aggregates
+7.9 to +20.4 mV) > Py-4N (from +5.5 to +11.8 mV). The results measured by DLS were in their hydrated form, which made
might be attributed to the comparatively high amino group them observable as bigger-sized nanoparticles; moreover, the
numbers on Py-3N and Py-4N, which increased the hydrophi- smaller-sized nanoparticles observed by AFM were due to the
licity of the amphiphiles and further resulted in decrease in the shrinkage of the Py-amines/pDNA complexes during the drying
35
36
surface potential and stability of the pDNA payloads.
Furthermore, we studied the morphology of the Py-amines/
process in the AFM samples preparation.
pDNA aggregates (N/P ¼ 20) by means of atomic force micros- Proton buffering capacity of the Py-amines
copy (AFM). As shown in Fig. 5, it could be noticed that the Py-
It had been revealed that the proton buffering capacity of
amines/pDNA complexes were spherical-shaped nanoparticles
with an average particle size of 90–195 nm, indicating that
the Py-amines amphiphiles could bind with pDNA via
multiamine-containing cationic gene carriers is regarded as an
important factor because this inuences the lysosome
localization/release behaviors of their gene payloads through a
37
“
proton sponge” effect. Herein, the proton buffering capabil-
ities of the Py-amines amphiphiles were examined by acid–base
titration with NaCl (0.1 M, negative control) and PEI-800
(positive control) as the references, and the titration curves
were obtained by the plot of pH values against the added HCl
38
volume. As shown in Fig. 6, it could be seen that the negative
control NaCl solution did not show any buffering effect.
Notably, Py-2N, Py-3N and Py-4N showed higher buffering
capacities than that of Py-1N, which might be attributed to their
Fig. 5 AFM image of the morphology of Py-amines/pDNA complexes Fig. 6 Acid–base titration curves of the Py-amines amphiphiles for
determined under an N/P ratio of 20 (inset: size distribution of Py- determining the proton buffering capacities, with NaCl (0.1 M) as the
amines/pDNA measured by DLS at the same N/P charge ratio).
negative control and PEI-800 as the positive control.
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ꢂ
ꢂ
attached primary and secondary (1 , 2 ) amine groups, which cell proliferation and division. The results indicated that the
lead to higher protonation abilities in solution. Moreover, the cytotoxicity depended largely on the molecular structures of the
positive control PEI-800 showed the highest buffering effect Py-amines and that the Py-amines/pDNA complexes could be
among the cationic amphiphiles, due to its three different types further optimized by changing the number of attached amino
ꢂ
ꢂ
ꢂ
of amino groups (1 , 2 and 3 ). It could be expected that the groups. Moreover, the Py-amines/pDNA complexes could be
distinct proton buffering capacities of the Py-amines amphi- expected to serve as antitumor drug/pDNA dual delivery systems
philes would lead to different ‘Lysosome escape’ effects, in practical applications.
which might further cause different inter-lysosomal pDNA
dissociation/releasing behaviors.
Lysosomal colocalization of the Py-amines/pDNA observed by

uorescent microscopy
Cell proliferation inhibition of the Py-amines and Py-amines/
pDNA by MTT assay
The “Lysosomal escape” effect of gene carriers is regarded as a
key factor for gene transfection, and the “endocytosis–
The cancer cell proliferation inhibition of Py-amines and Py- endosomal/lysosomal
escape-nuclear
gene
expression”
39
amines/pDNA complexes was examined by MTT assay in an pathway is widely regarded as the dominant intracellular
H1299 (human lung cancer) cell line. As shown in Fig. 7, the cell trafficking route of cationic gene carriers because this facilitates
viabilities of Py-1N (8.8%) and Py-2N (7.3%) were lower than the release of gene payloads trapped in lysosomes/endosomes
+
that of Py-3N (18.6%) at the dose of N/P ¼ 5 (pDNA 1 mg per well), via endosome swelling and breaking by buffering H and the
ꢀ
40
whereas a much higher cell viability was observed with Py-4N at subsequent accumulation of Cl . In order to investigate the
around 58%. Aer loading with pDNA (1 mg per well) at N/P ¼ 5, intracellular localization of Py-amines/pDNA complexes, the
it could be found that the cell viability greatly increased (Py-1N lysosome organelles of H1299 cells were stained with
3
8.5%, Py-2N 30.4%, Py-3N 36.8% and Py-4N 78.8%), possibly Lysotracker-red, a uorescent agent for lysosomal-specic
due to the binding of positively charged Py-amines with nega- labeling. As shown in Fig. 8, aer 4 h incubation of the Py-
tively charged pDNA diminishing their cell membrane perme- amines/pDNA complexes, an evident blue uorescence emis-
ation effect. Moreover, with the incubation of a higher dosage of sion located in the cytoplasm could be seen, and only weak blue
Py-1N, Py-2N, Py-3N and their pDNA complexes (N/P > 10), very uorescence could be found around the plasma membrane.
low H1299 viabilities (6.5–8.8%) could be observed, which The blue uorescence was attributed to the emission of pyrene
41–43
indicated their H1299 tumor cell inhibition properties could uorophore.
Our previous study on pyrene-based cationic
not be interrupted by the loading of pDNA. However, the H1299 lipids also demonstrated that the strong blue-uorescence
cells incubated with Py-4N (23.2–46.5%) and Py-4N/pDNA (26.2– emission of pyrene uorophore could be observed even under
6
7.5%) showed higher cell viabilities, indicating their lower high ionic strength conditions (in PBS buffer or cell culture
25
tumor suppression efficiency. As far as the antitumor mecha- medium).
The intracellular blue uorescence emission
nism was concerned, prior studies revealed that some exoge-
nous polyamines compounds could block the overexpressed
19
polyamine (such as spermine and spermidine) transporter in
the tumor cells. Thus, we supposed that the synthesized Py-
amines might go through a similar pathway to block the poly-
amine transporter and then result in the inhibition of cancer
Fig. 7 The cytotoxicity of H1299 cells incubated with Py-amines/ Fig. 8 Fluorescence images of Py-amines/pDNA (blue fluorescence)
pDNA (+pDNA) complexes at various charge ratios of N/P (pDNA 1 mg complexes in H1299 cell lines under an N/P ratio of 30, Lysotracker-
per well), and with the Py-amines (ꢀpDNA) at the same dosage as the red was used as the lysosomal specific labeling agent (red
control.
fluorescence).
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indicated that
a large amount of the Py-amines/pDNA
Acknowledgements
complexes could be efficiently taken up into the cytoplasm.
Notably, a signicant colocalization of red (Lysotracker-red) and The authors are indebted to the nancial supports partially
blue (Py-amines/pDNA complexes) uorescence were observed from the national science foundation of China (21174160,
in the H1299 (Fig. 8, merged images) and COS-7 cells (ESI, S4†), 21002116 and 21372251).
suggesting that the Py-amines/pDNA complexes tended to
localize inside the lysosome/endosome, which might due to the
Notes and references
44
“proton sponge” effect of their attached amino groups.
Moreover, it could be seen that the uorescence of Py-3N/pDNA
and Py-4N/pDNA partially overlapped with Lysotracker-red,
indicating that they might possess a stronger “lysosomal
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