Dendron conjugation to graphene oxide using click chemistry for efficient gene delivery

Article abstract of DOI:10.1039/c5ra07004j

Owing to its large surface area and rapid cellular uptake, graphene oxide (GO) is emerging as an attractive candidate material for delivery of drugs and genes. The inherent sp² π-π interaction of GO helps to carry drugs and single stranded RNA (ssRNA) but there is no such interaction with double stranded DNA (dsDNA). In this work, a polyamidoamine (PAMAM) dendron was conjugated with nano GO (nGO) through "click" chemistry to improve the DNA complexation capability of GO as well as its transfection efficiency. The DNA complexation capability of GO was significantly enhanced after dendronization of GO yielding spherical nanosized (250-350 nm) particles of the dendronized GO (DGO)/pDNA complex with a positive zeta potential. The transfection efficiency of GO dramatically increased after conjugation of the PAMAM dendron. Transfection efficiency of 51% in HeLa cells with cell viability of 80% was observed. The transfection efficiency was significantly higher than that of polyethyleneimine 25 kDa (27% efficiency) and also surpassed that of lipofectamine 2000 (47% efficiency). The uptake of the DGO/pDNA complex by the caveolae mediated endocytosis pathway may significantly contribute to the high transfection efficiency. Thus, dendronized GO is shown to be an efficient gene carrier with minimal toxicity and is a promising candidate for use as a nonviral carrier for gene therapy.

Full text of DOI:10.1039/c5ra07004j

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Dendron Conjugated Graphene Oxide for Efficient Gene Delivery
a
a
b*
Kishor Sarkar , Giridhar Madras , Kaushik Chatterjee
a
b
Department of Chemical Engineering and Department of Materials Engineering
Indian Institute of Science, Bangalore 560012 India
*
Corresponding author: kchatterjee@materials.iisc.ernet.in; +91ꢀ80ꢀ22933408
1

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Abstract
Owing to its large surface area and rapid cellular uptake, graphene oxide (GO) is
2
emerging as an attractive candidate material for delivery of drugs and genes. The inherent sp
πꢀπ interaction of GO helps to carry drugs and single stranded RNA (ssRNA) but there is no
such interaction with double stranded DNA (dsDNA). In this work, polyamidoamine
(PAMAM) dendron was conjugated with nano GO (nGO) through “click” chemistry to
improve the DNA complexation capability of GO as well as its transfection efficiency. The
DNA complexation capability of GO was significantly enhanced after dendronization of GO
and yielding spherical nanosized (250ꢀ350 nm) particles of dendronized GO (DGO)/pDNA
complex with positive zeta potential. The transfection efficiency of GO dramatically
increased after conjugation of the PAMAM dendron. Transfection efficiency of 51% in HeLa
cells with cell viability of 80% was observed. The transfection efficiency was significantly
higher than that of polyethyleneimine 25 kDa (27% efficiency) and also surpassed that of
Lipofectamine 2000 (47% efficiency). The uptake of DGO/pDNA complex by the caveolae
mediated endocytosis pathway may significantly contribute to the high transfection
efficiency. Thus, dendronized GO is shown to be an efficient gene carrier with minimal
toxicity and is promising candidate for use as a nonviral carrier for gene therapy.
Keywords: Graphene; Polymer composite; Click chemistry; Gene delivery; Gene therapy
2

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1. Introduction
Gene therapy is an emerging therapeutic tool of modern medicine promising to treat a
1
, 2
variety of genetic disorders and other diseases . The lack of an efficient yet safe carrier is a
significant impediment in the field. High toxicity, immunogenicity, tumorigenicity, lack of
targeting efficiency and high cost of production of viral vectors have underscored the need to
3
, 4
develop safe and efficient alternative nonviral vectors. Cationic macromolecules such as
5
6
7
polyethyleneimine (PEI), polyꢀlꢀlysine, polyamidoamine (PAMAM) dendrimers etc. are
some of the widely studied nonviral carriers. Despite the emergence of several carriers, there
is a need for novel efficient and safe nonviral carriers for successful gene therapy.
Recently, graphene oxide (GO) and functionalized GO has gained tremendous
attention in biomedical applications such as biosensors, bioꢀimaging, tissue engineering, drug
and gene delivery due to its superior water dispersibility, high surface to volume ratio, low
8
ꢀ11
production cost and scope for facile functionalization.
GO has been widely used for drug
2
12
delivery due to favourable sp πꢀπ interaction between GO and the hydrophobic drugs. GO
13, 14
has also been used for imaging.
Thus, the use of GO for gene delivery offers an
additional advantage of potential monitoring cellular uptake and synergistically utilize its
other properties for theranostics.
The πꢀπ interaction of GO can carry single stranded RNA (ssRNA). But, there is no
such interaction with double stranded DNA (dsDNA) and hence GO is unable to transport
15
dsDNA. Few attempts on utilizing modified GO for delivery of plasmid DNA (pDNA) have
16
been reported recently but the transfection efficiency was poor. Kim et al. prepared low
molecular weight branched PEIꢀconjugatedꢀGO using 1ꢀethylꢀ3ꢀ[3ꢀ
dimethylamino)propyl]carbodiimide hydrochloride (EDC) and observed transfection
(
17
efficiency comparable to that of PEI (25 kDa). Chen et al.
conjugated high molecular
3

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weight (25 kDa) branched PEI with GO through EDC and the transfection efficiency of GOꢀ
1
8
PEI was also equivalent to that of PEI (25 kDa). Tripathi el al. reported higher transfection
efficiency (~33%) after conjugation of linear PEI (25 kDa) with GO. The toxicity of
graphene is a general concern for its use in biomedical applications. However, the reports are
19, 20
contradictory and cytotoxicity depends on the concentration, size and functionalization.
2
1
GO is reported to be minimally toxic at low concentration. Thus, it appears that GO may be
used in vivo at low concentration with minimal toxicity although further investigations of
long term effects are warranted.
We hypothesize that conjugation PAMAM to GO can significantly improve the DNA
binding capacity and transfection efficiency of GO. Amine terminated PAMAM dendrimer
has been widely used for drug/gene delivery because of its well defined architecture, high
22
surface charge density, low polydispersity and biodegradability . However, severe toxicity
of PAMAM dendrimer limits its widespread application for drug/gene delivery. The toxicity
2
3, 24
and transfection efficiency are dependent on the generation of PAMAM dendrimer
.
Higher generation dendrimer (>3.0G) show better transfection efficiency but induce higher
toxicity whereas, low generation dendrimer (<3.0G) showed lower transfection efficiency
2
5, 26
with higher cell viability
. Thus, GO modified with low generation PAMAM can yield a
carrier with good transfection efficiency with low toxicity.
In this work, we report on the synthesis of PAMAM dendrimer conjugated GO
(dendronized GO) by reacting focal point PAMAM dendrimer with GO through “click”
chemistry for DNA delivery. We used low generation PAMAM dendrimers (1.0, 2.0 and
3.0G) to conjugate with GO by “click” chemistry. The use of “click” chemistry offers many
27
advantages for bioconjugation including high specificity with minimal byꢀproducts
.
Importantly, a major limitation of EDC conjugation used previously for PEI modification of
1
2, 15, 28
GO is the lack of specificity
, which may result in a crosslinked mass of many GO
4

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flakes conjugated to a single PEI molecule. Such crosslinking can be minimized by “click”
chemistry utilized herein. The dendronized GO (DGO)/pDNA complex was characterized.
Gene transfection efficiency and cytotoxicity of DGO were assessed in HeLa cells. The rapid
intracellular kinetics and cellular uptake pathways of the DGO/pDNA complex were
characterized to elucidate the mechanisms underlying high transfection efficiency.
2
. Experimental sections
.1. Materials
2
Pristine graphite flake (100 mesh), propargylamine (98%), methyl acrylate (99%),
®
ethylenediamine (ReagentPlus , ≥99%), 3ꢀchloropropylamine hydrochloride (98%), sodium
®
azide (ReagentPlus , ≥99.5%), sodium ascorbate (≥98%), N, Nꢀ dimethylformamide
(anhydrous, 99.8%), ethidium bromide (EtBr), branched PEI (25 kDa), 2,4,6ꢀtrinitrobenzene
sulfonic acid solution (5% w/v) and 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium
bromide) (MTT) were purchased from SigmaꢀAldrich. Chloroacetic acid, methyl alcohol,
copper (II) sulphate and sodium hydroxide were obtained from S. D. Fine Chemicals. 1ꢀ
ethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride (EDC), Nꢀhydroxysuccinimide
(NHS), agarose and sodium salt of 2ꢀ(Nꢀmorpholino) ethanesulfonic acid (MES) were
purchased from SRL Pvt. Ltd. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
serum (FBS), 0.25% trypsin and penicillin–streptomycin were purchased from Life
Technologies. Enhanced green fluorescence protein (EGFP) encoding plasmid DNA
(pEGFPꢀN1, 4.7 kbp) was propagated in Escherichia coli (E. coli) and isolated by pDNA
isolation Kit (Midiprep, Qiagen, USA). All other reagents were used without any purification.
2.2. Synthesis of PAMAM dendrimer
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PAMAM dendrimers were synthesized according to the previous report with slight
29
modification . Briefly, 5.87 g (68 mmol) methyl acrylate (MA) and 20 ml methanol were
taken in 250 ml round bottom three neck flask and put on an ice bath. 1.5 g (27.2 mmol)
propargylamine dissolved in 80 ml methyl alcohol was added drop wise to the above mixture
o
over 1 h under nitrogen atmosphere at 0 C with constant stirring. The reaction mixture was
stirred for another 30 min in ice and then continued at room temperature for 48 h. Then the
o
excess solvent was removed by rotary evaporator under reduced pressure at 40 C to get light
yellow colored ester terminated 0.5G (half generation) PAMAM dendrimer (5.85 g, 94.5%).
To a stirred solution of ethylenediamine (40 g, 0.67 mol) in 80 ml methyl alcohol, 5 g
(22 mmol) 0.5G PAMAM dissolved in 20 ml methyl alcohol was added drop wise over 30
o
o
min under nitrogen atmosphere at 0 C. The reaction was allowed to continue at 0 C for
another 30 min and further for 96 h at room temperature under nitrogen atmosphere. The
o
excess solvent was removed by rotary evaporation at 40 C using toluene/methanol mixture
(9:1 volume ratio) to obtain a light yellow colored amine terminated 1.0G (full generation)
PAMAM dendrimer (6.0 g, 96.3%).The above two consecutive steps were repeated to
synthesis 2.0G and 3.0G PAMAM dendrimers as shown in Figure 1a. The selected NMR
data of 0.5, 1.0, 1.5, 2.0 and 3.0G PAMAM dendrimers are given in supplementary section.
2
.2. Synthesis of nano graphene oxide (nGO)
GO was synthesized from pristine graphite flakes by modified Hummers method .
Briefly, 1.0 g graphite flakes were taken in a 1000 ml beaker. 57.0 ml concentrated H SO
30
2
4
and 6.5 ml H
KMnO was gradually added to the above mixture under ice and the reaction was continued
for another 48 h. 30% H (10 ml) and 140 ml distilled water were added to the reaction
3 4
PO were added to the graphite flakes with constant stirring for 8 h. 5.6 g
4
2
O
2
mixture to terminate the reaction. The reaction mixture was washed with 1.0N HCl solution
6

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and distilled water several times by repeating centrifugation and dispersion methods. Finally,
o
the synthesized GO was dried at 40 C under vacuum for 48 h. The dried GO was dispersed
in distilled water by bath sonication at concentration of 3 mg/ml for 1 h. The GO solution was
o
then ultrasonicated at 400 W for 1 h under ice to obtain nGO and stored at 4 C for future use.
2.3. Synthesis of aminopropyl azide
2
.0 g (15.4 mmol) chloropropylamine hydrochloride was added in a 100 ml round
bottom flask containing 4.0 g (61.6 mmol) sodium azide dissolved in 30 ml distilled water
o
and the reaction mixture was stirred at 75 C for 18 h. The reaction mixture was then
concentrated by rotary evaporator under reduced pressure and KOH pellet was added to the
concentrated mixture followed by extraction using diethyl ether (3×10 ml). The organic layer
was finally dried over anhydrous sodium sulphate followed by rotary evaporation to get
1
colorless volatile aminopropyl azide (1.43 g, 93%). H NMR (CDCl
3
) δ
H
: 3.38 ppm
(
(
2H, t, N −CH
2
−)
,
2.81
ppm
(2H, t, −CH
2
− NH2 )
,
1.74
ppm
3
1
3
2H, m, −CH −CH
2
−CH −) , 1.25 ppm (2H, bs, −CH − N H
2
)
.
C NMR (CDCl ) δ :
3 C
2
2
2
4
9.23 ppm (N −CH −), 39.37 ppm (−CH − NH ) , 32.45 ppm (−CH −CH −CH −).
3
2
2
2
2
2
2
2
.4. Synthesis of azide functionalized nGO
6.6 ml nGO solution (3 mg/ml) was taken in a 100 ml round bottom flask and the
solution was diluted to 50 ml to make 1 mg/ml final concentration using 5 mM MES buffer
pH= 5.5). Then, 0.27 g EDC and 0.16 g NHS were added to the nGO solution followed by
addition of 0.5 g aminopropyl azide and the reaction was carried out at room temperature for
4 h. The reaction product was purified by dialysis for 3 days using SnakeSkin dialysis
1
(
2
tubing (3.5 kDa MWCO, Thermo Scientific) and finally freeze dried for 3 days to get azide
functionalized GO.
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2.5. Synthesis of PAMAM conjugated GO
1
0 mg nGO azide was dispersed in 12 ml DMF/H
2
O (5:1 v/v) solvent mixture by bath
and 158.4 mg (0.8 mmol) sodium
sonication for 1 h. After that, 98.6 mg (0.4 mmol) CuSO
4
ascorbate was added to the above solution followed by addition of PAMAM dendrimers of
o
different generations (1.0, 2.0 and 3.0G) and the reaction was carried out at 50 C for 24 h
under nitrogen atmosphere. Finally, the reaction mixture was dialyzed against distilled water
for 3 days followed by lyophilisation for another 3 days to get the DGO. The percentage of
primary amine groups of DGO was determined by TNBS assay according to the previous
3
1
report using glycine for standard calibration curve .
2.6. Characterization
The synthesis of dendrimers and DGO was characterized by Fourier transform
infrared (FTIR) spectrophotometer (100 FTIR, Perkin Elmer) using attenuated total reflection
ꢀ1
(
ATR) attachment at a frequency range of 4000ꢀ600 cm . Proton and carbon nuclear
1
13
magnetic resonance ( H and C NMR) spectra were carried out at 400 MHz (Bruker NMR)
NMR spectrometer. All half and full generation PAMAM dendrimers were dissolved in
CDCl
3
and D
2
O, respectively, with tetramethylsilane (TMS) as internal reference. Atomic
®
force micrographs were acquired using NanoWizard 3a NanoScience AFM (JPK Instruments
AG, Germany) in tapping mode by depositing the GO and DGO solution (10 µg/ml) on a
freshly cleaved mica surface and dried in ambient temperature for overnight. Transmission
electron microscopy (TEM) images of GO and DGO deposited on copper grid were captured
using FEI Tecnai T20 UꢀTWIN TEM at 200 kV. Xꢀray Photoelectron Spectroscopy (XPS)
and Raman spectroscopy were carried out in Axis Ultra DLD XPS system (Kratos Analytical
Ltd, UK) and LabRAM HR Evolution (HORIBA Scientific), respectively.
2.7. Preparation and characterization of GO/pDNA complex
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nGO/pDNA and DGO/pDNA complexes were prepared according to a previous study
At first, both GO and DGO solution were prepared in 5 mM MES buffer (pH 6.5) by bath
3
2
.
sonication at a concentration of 1 mg/ml. pDNA stock solution was diluted in 25 mM sodium
sulphate solution at a concentration of 100 µg/ml. Subsequently, equal volume of nGO or
DGO solution and pDNA solution were mixed at different weight ratios (1:1, 5:1, 10:1, 15:1,
2
0:1, 25:1 and 30:1) and vortexed for 20 sec followed by incubation at room temperature for
0 min to prepare GO/pDNA or DGO/pDNA complex (GOplex). The formation of GOplex
3
was confirmed by agarose gel electrophoresis. The GOplexes at different weight ratios
having 0.5 µg DNA in each weight ratio were loaded in 0.8% agarose gel containing
ethidium bromide (10 µg/ml) as DNA visualizer. The gel was run in 1X TAE running buffer
at 100 V for 40 min and subsequently the gel picture was taken by a gel documentation
system (MyECL Image, Thermo Scientific). The GOplex formation was further confirmed by
3
3
ethidium bromide (EtBr) assay according to our previous report . pDNA/EtBr complex was
first prepared at a mole ratio of 10:1 (pDNA:EtBr mole ratio). nGO or DGO solution were
mixed with pDNA/EtBr complex at different weight ratios with respect to pDNA (1:1, 5:1,
10:1, 15:1, 20:1, 25:1 and 30:1) and incubated for 30 min at room temperature. The
fluorescence intensity of the resulting mixture solution at excitation and emission
wavelengths of 510 and 605 nm, respectively was recorded by a multimode microplate reader
(Synergy HT, BioTek).
The hydrodynamic size of nGO/pDNA and DGO/pDNA complexes was measured by
Zetasizer Nano ZS particle size analyzer (Malvern Instruments, UK). Prior to particle size
analysis, GOplexes were prepared at different weight ratios (GO or DGO/pDNA weight
ratioꢀ 1:1, 5:1, 10:1, 15:1, 20:1, 25:1 and 30:1) as discussed above and diluted to 3 ml by
filtered (0.22 µm Axiva syringe filter paper, Axiva Sichem Biotech, India) in double distilled
water. The zeta potential of GO and DGO before and after complexation with pDNA and
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nGOꢀN
3
was measured using the same instrument. The morphology and particle size of
GOplex was further observed by atomic force microscopy (AFM).
2.8. In vitro toxicity assay
Cell viability of nGO and DGO (1.0, 2.0 and 3.0 G) at different concentrations (5, 10,
3
2
0, 30, 40, 50, 100 and 200 µg/ml) on HeLa cell was carried out by MTT assay. 5 × 10 cells
in DMEM medium containing 10% fetal bovine serum (FBS) were seeded in each well of a
o
9
6 well plate and cultured in a humidified chamber supplying 5% CO
2
at 37 C (Thermo
Scientific) for 24 h. GO or DGO solutions at different concentrations were added to the cells
o
in serum free medium and incubated at 37 C in CO incubator. After 4 h, GO or DGO
2
containing medium was replaced by fresh medium containing serum and incubated further for
44 h in the CO
2
incubator. The medium was removed and MTT solution (5 mg/ml) diluted in
100 µl serum free medium (1 mg/ml working concentration) was added to each well. After
incubation for 4 h, the medium containing MTT solution was replaced by 100 µl DMSO to
dissolve the formazan crystals. Finally, the absorbance of the solution was measured at 570
nm using a microplate reader and the cell viability (%) was calculated by the following
equation:
OD570 (Sample)
Cell viability (%) = OD5
×100
70 (Control)
where, OD570 (Control) and OD570 (Sample) are the measurements of the untreated and treated cells,
respectively. All measurements were the mean of three measurements.
The in vitro toxicity assay of nGO/pDNA and DGO (1.0, 2.0 and 3.0G)/pDNA
complexes at different weight ratios (1:1, 5:1, 10:1, 15:1, 20:1, 25:1 and 30:1) was also
carried out in HeLa cell as mentioned above.
1
0

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2.8. In vitro transfection
The transfection efficiency of nGO/pDNA and DGO/pDNA complexes at different
5
weight ratios was carried out in HeLa cell. Before transfection, 1 × 10 cells in complete
DMEM medium containing 10% FBS were seeded in each well of a 24 well plate. After 24 h,
freshly prepared GOplexes at different weight ratios (5:1, 10:1, 15:1, 20:1, 25:1 and 30:1)
containing 1 µg pDNA in each weight ratio in serum free medium were added to the cells and
o
incubated for 4 h in a CO
2
incubator at 37 C. The transfection medium was replaced by fresh
serumꢀcontaining medium and further incubated for 44 h. Lipofectamine 2000 (LF 2K, Life
Technologies) and PEI (25 kDa) were used as the positive controls. pDNA and PAMAM
(3.0G) were used as negative control. The GFP (green fluorescence protein) expression of the
transfected cells were observed by fluorescence microscope (Olympus IX53, Japan). The
transfection efficiency of nGO and DGO was further quantified by FACS analysis (Bectonꢀ
4
Dickinson) using 1 × 10 cells per analysis.
2.9. Endocytosis pathway
Low temperature or different endocytic inhibitors were used to determine the cellular
uptake pathways of GOplexes. The transfection of DGO (3.0G)/pDNA complex at weight
o
ratio of 30:1 was carried out at 4 C during the 4 h transfection period to inhibit energy
dependent endocytosis. Also, cells were preꢀincubated with genistein (100 mg/mL), amiloride
(10 mg/mL), or chlorpromazine (10 mg/mL) for 1 h prior to transfection to inhibit caveolae,
macroꢀpinocytosis and clathrin mediated endocytosis pathways, respectively. Thereafter,
DGO (3.0G)/DNA complex at weight ratio of 30:1 containing 1 µg DNA was added to the
treated cells and incubated for 4 h. The transfection efficiency was determined by flow
cytometry after 44 h post transfection.
2.10. Intracellular distribution
1
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The intracellular distribution of DGO (3.0G)/pDNA complex at weight ratio of 30:1
®
was observed by labelling pDNA with Cy3 according to manufacturer’s protocol (Label IT
TM
Tracker intracellular nucleic acid localization kit, Mirus Bio, USA). Before transfection,
4
5
×10 cells (HeLa cell) were seeded on four chambered glass slides (Biofil, South Korea).
After 24 h, DGO (3.0G)/pDNA complex at weight ratio of 30:1 containing 1 µg Cy3ꢀlabeled
pDNA for 4 h. LF 2K/pDNA complex was used as positive control. After 4 h, the
transfection medium was removed and the cells were washed with PBS followed by fixing
with 3.7% (w/v) formaldehyde for 30 min. The nucleus was then stained with diamidino
phenylindole (DAPI) for 10 min and the cells were covered with cover slip with mounting
medium containing antifading agent and sealed with colorless nail polish. The intracellular
distribution of GOplex was imaged by confocal laser scanning microscopy (CLSM; Leica
TCS Sp5).
2.11 Statistical analysis
All the data are presented as the average ± the standard deviation. Oneꢀway ANOVA
was used to calculate statistical differences and differences were considered significant for p
values less than 0.05.
3. Results and discussion
3.1. Synthesis of dendronized GO
DGO was synthesized by reaction of alkyne terminated focal point PAMAM
dendrimers (1.0, 2.0 and 3.0G) with azide functionalized nGO through “click” chemistry as
shown in Figure 1. Full generation (amine terminated) focal point PAMAM dendrimers (1.0,
2.0 and 3.0G) were synthesized by repeating the Michael addition reaction followed by
1
2

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amidation reaction using propargyl amine as starting material, as shown in Figure 1a. Figure
S1 shows the FTIR spectra of synthesized PAMAM dendrimers (0.5, 1.0, 1.5 and 2.0G). A
ꢀ
1
strong peak at 1735 cm (C=O stretching of ester) indicates the presence of ester group (ꢀ
COOCH ) in half generation (ester terminated) PAMAM dendrimer (0.5G). A weak
3
ꢀ1
ꢀ1
stretching peak at 2102 cm (inset) and a medium stretching peak at 3279 cm correspond to
ꢀC≡Cꢀ stretching and CꢀH stretching of ꢀC≡CH group, respectively indicating the presence of
alkyne group in 0.5G PAMAM dendrimer. After amidation reaction of 0.5G PAMAM with
ehthylene diamine, the absorption peak for ester group disappeared and two new peaks
ꢀ1
ꢀ1
appeared at 1644 cm and 1554 cm equivalent to the C=O stretching of amide (ꢀCOꢀNHꢀ)
group and NꢀH bending of primary amine (ꢀNH ) group, respectively. The appearance of
2
ꢀ1
other new peaks at 3351 and 3289 cm related to hydrogen bonded NꢀH stretching and free
NꢀH stretching, respectively of primary amine group indicates the synthesis of 1.0G PAMAM
dendrimer (full generation). The reappearance and disappearance of the absorption peak at
ꢀ1
1
731 cm for ester group in the successive steps indicating the synthesis of 1.5, 2.0 (Figure
), 2.5 and 3.0G PAMAM dendrimers. The synthesis of PAMAM dendrimers was further
2
1
13
confirmed by H and C NMR spectra as described Figure S1.
nGO was prepared by oxidation of pristine graphite followed by ultra sonication.
Figure S2a shows the Xꢀray diffraction pattern of pristine graphite and nGO. Pristine graphite
o
shows characteristic strong diffraction peak at 2θ= 26.5 corresponding to 002 plane having
dꢀspacing of 0.335 nm. This peak disappeared after oxidation of pristine graphite and a new
o
diffraction peak appeared at 2θ= 10.8 with higher dꢀspacing of 0.818 nm confirming the
34
formation of GO . The particle size distribution of nGO is shown in Figure S2b and the
average particle of nGO was 180±55 nm. The size and morphology of nGO was observed by
AFM as shown in Figure S2c and d, respectively. The AFM image shows that the flake
1
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diameter of nGO was about 160ꢀ230 nm and the flake thickness about 0.6ꢀ0.8 nm (Figure
35
S2e) consistent with previous reports .
ꢀ1
GO showed characteristic absorption peaks at 3150ꢀ3500 cm (broad peak of
ꢀ1
ꢀ1
hydrogen bonded OꢀH stretching), 1726 cm (C=O stretching of –COOH), 1621 cm (C=C
ꢀ1
ꢀ1
stretching of aromatic ring), 1043 cm (CꢀO stretching of –CꢀOꢀCꢀ) and 1378 cm (in plane
3
6
ꢀ1
OꢀH bending) as shown in Figure 2a . A strong absorption peak at 2092 cm and a
ꢀ1
3 2
absorption peak at 3300 cm are corresponding to azide (ꢀN ) and primary amine (ꢀNH )
groups indicating the synthesis of aminopropyl azide by reaction of chloropropyl amine and
sodium azide. After reaction of GO with aminopropyl azide, two new absorption peaks
ꢀ
1
ꢀ1
appeared at 2104 cm and 1630 cm analogous to the azide group (ꢀN
of amide group (ꢀCONHꢀ), respectively and the disappearance of C=O stretching peak for
carboxylic acid of GO suggesting the synthesis of azide functionalized GO (GOꢀN ) as
shown in Figure 1b. PAMAM dendrimer of 3.0G showed strong absorption peaks at 1647
3
) and C=O stretching
3
ꢀ1
ꢀ1
cm and 1552 cm for C=O stretching of amide (ꢀCONHꢀ) and NꢀH bending of primary
amine (–NH
2
) groups, respectively, along with NꢀH stretching of primary amine group at
ꢀ1
ꢀ1
3
287 cm . The strong absorption peak at 2104 cm for azide group of azide functionalized
graphene oxide (GOꢀN
3
) disappeared after reaction with focal point PAMAM dendrimer
ꢀ1
through “click” chemistry. The intensity of CꢀH stretching (–CH
2
ꢀ) peak at 2920 cm and
ꢀ1
2
850 cm also increased significantly compared to that of GOꢀN . Along with this, three new
3
ꢀ1
ꢀ1
ꢀ1
peaks appeared at 1742 cm , 1635 cm and 1547 cm related to triazole ring, C=O
stretching of amide group and NꢀH bending of primary amine group, respectively confirming
the synthesis of DGO. In contrast to other conjugation techniques the chemical specificity
imparted by “click” chemistry utilized herein can overcome potential challenges such as
crosslinking of many nGO flakes by a single polycationic chain and electrostatic interactions
between nGO and the polycation.
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Figure 2b shows the UVꢀvis spectra of nGO and DGO (3.0G). GO shows
characteristic strong absorption peak at 229 nm and a broad shoulder at 301 nm due to the πꢀ
3
7
π* transition of aromatic C=C and carboxylic acid group’s C=O, respectively . But the
absorption peak of GO shifted to 272 nm after reaction with PAMAM dendrimer
corresponding to the triazole ring formed during the reaction of GOꢀN and focal point
3
PAMAM dendrimer through “click” chemistry.
1
H NMR spectra further supports the synthesis of dendronized GO as shown in Figure
S3. PAMAM dendrimer (3.0G) showed the characteristic peaks at 2.25ꢀ2.51 ppm
(
−CH
ppm (CH ≡ C − CH
dendrimer with GOꢀN
2
−CONH−), 2.52ꢀ2.58 ppm (−N −CH
−) and 3.36 ppm (−CH
through “click” chemistry, new peaks appeared at 2.70 ppm, 2.86
2
−CH −), 3.20 ppm (−CH − NH2 ) , 3.29
2
2
2
2
−CH − NH ). After reaction of PAMAM
2
2
3
ppm, 3.2 ppm and 3.45ꢀ3.53 ppm (inset) related to the respective protons of PAMAM
dendrimers.
The synthesis of GO followed by DGO was further confirmed by Raman and Xꢀray
photoelectron spectroscopies, as shown in Figures 3a and b, respectively. Figure 3a shows the
Raman spectra of pristine graphite, GO and DGO (3.0G). Pristine graphite shows the typical
ꢀ1
2
Raman band at 1583 cm due to the E phonon of sp hybridized carbon atom of graphite,
2
g
ꢀ1
called G band and a small deflection at 1355 cm corresponding to D band, which is
associated with structural defects. The intensity of G band increased significantly and shifted
ꢀ1
38
to higher frequency (1606 cm ) due to the presence of isolated double in the oxidized GO .
ꢀ1
The D band became more prominent and shifted to lower value (1548 cm ) suggesting
2
generation of small sized inꢀplane sp grains during oxidation of graphite to form GO. The
D G D G
intensity ratio (I /I ) of the D band (I ) and the G band (I ) determines the amount of defect
3
9
and disorder in graphite structure . The I
D
/I value of pristine graphite was only 0.48 that
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40
D G
indicates highly ordered and mean defect free structure . The I /I value of GO increased
significantly from 0.48 to 1.08 during the oxidation of graphite suggesting the formation of
large defects in GO structure. The intensity ratio further increased from 1.08 to 1.11 in DGO
ꢀ1
and the G band shifted to lower frequency (1598 cm ) compared to GO. This indicates
further structural defects after reaction of PAMAM dendrimer with GO.
Figure 3b shows the XPS wide scan spectra of GO and DGO (3.0G). GO shows two
intense peaks at binding energies of 285 and 532 eV for C1s and O1s, respectively indicating
the presence of carbon and oxygen as major elements in GO. A new peak appeared at binding
energy of 401 eV for N1s (coreꢀlevel spectra in inset) after reaction of PAMAM dendrimer
suggesting that PAMAM dendrimer was successfully conjugated with GO through “click”
chemistry.
The surface morphology of GO and DGO was characterized by TEM as shown in
Figure S4. GO shows typical wrinkled morphology and transparent thin flake (Figure S4a).
However, the morphology of GO flake changed after surface modification by dendrimer.
After conjugation of PAMAM dendrimers with GO through “click” chemistry, small black
spots (1.0G PAMAM, Figure S4b) and comparatively large black spots (3.0G PAMAM,
Figure S4c) formed on the GO flake due large size of 3.0G PAMAM dendrimer compared to
that of 1.0G PAMAM dendrimer suggesting the successful conjugation of PAMAM
dendrimer on GO surface.
Table 1 also summarizes the change in particle size and thickness of GO flakes after
dendronization. The diameter and thickness of unmodified GO flakes were 206±46 nm and
0.6±0.2 nm, respectively. Both the diameter and thickness of GO flakes increased
progressively with increasing the generation of PAMAM dendrimer due to increasing the size
of the PAMAM dendrimer from low generation to higher generation accordingly.
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Unmodified GO showed negative zeta potential (ꢀ41.5±3.4 mV) as expected due to presence
of negatively charged carboxylic acid functional groups. The zeta potential was ꢀ0.9±3.8 mV
after modification of nGO with the azide group. This reduction in anionic character of nGOꢀ
N is a significant advantage of the chemical synthesis route proposed in this study. It
3
minimizes electrostatic interactions between unmodified nGO and cationic polymers that may
be significant in other techniques such as EDC conjugation. The interactions between
PAMAM dendrimers and the modified nGO can thus primarily be attributed to covalent
interactions between them. The zeta potential became positive (5.4±2.4 mV) after
conjugation of 1.0G PAMAM dendrimer and the value of zeta potential increased gradually
with increasing the generation of PAMAM dendrimer due to increasing the content of
primary amine groups from 2.5±0.5% (DGO 1.0G) to 6.4±1.1% (DGO 2.0G) to 12.8±1.5%
(DGO 3.0G). The zeta potential values of DGO (2.0G) and DGO (3.0G) were 18.3±5.4 and
38.6±3.6 mV, respectively.
3.2. Preparation and characterization of GO/pDNA complex
nGO/pDNA and DGO/pDNA complexes (GOplexes) at different weight ratios (1:1,
:1, 10:1, 15:1, 20:1, 25:1, and 30:1) were prepared by mixing of equal volume of nGO or
5
DGO solution and pDNA solution through electrostatic interaction between the positively
3
3
charged carrier and negatively charged DNA . The formation of GOplexes was confirmed
by agarose gel electrophoresis assay, as shown in Figures 4aꢀd. Figure 4a shows the agarose
gel image of nGO/pDNA complexes at different weight ratios. It is found that nGO has no
complexation capability with pDNA at all weight ratios as expected due to absence of any
cationic functional groups. Previous studies also showed that GO has no complexation
1
6
capability with pDNA due its negative zeta potential value . The DNA complexation
capability of GO started after conjugation of PAMAM dendrimer with GO due to the positive
zeta potential of DGO as shown in Table 1. DGO (1.0G) started to complex almost all pDNA
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at high weight ratio of 30:1 due to its low zeta potential (5.4±2.4 mV). The DNA
complexation capability of DGO increased with increase in the generation of PAMAM
dendrimer due to enhancement of zeta potential. DGO (2.0G) and DGO (3.0G) complexed all
pDNA at 15:1 and 5:1 weight ratio, respectively, where the electrophoretic mobility of pDNA
was completely retarded after complexation with positively charged DGO. The number of
primary amine groups increased with increase in the generation of PAMAM dendrimer as
consequently increased the zeta potential and as a result facilitated the efficient DNA
complexation at low weight ratio.
The DNA complexation capability of GO and DGO was further characterized by EtBr
assay as shown in Figure 4e. In this assay, EtBr was complexed with pDNA at a molar ratio
of 1:10 and gave maximum fluorescence intensity due to intercalation of EtBr molecule into
4
1
the double helix of DNA . After addition of any polycation externally to the EtBr/pDNA
complex, the fluorescence intensity will come down gradually with increase in concentration
of cationic molecule due to replacement of EtBr molecule by foreign cationic molecule
indicating the formation of cationic molecule/pDNA complex. Figure 4e shows that the
fluorescence intensity of EtBr/pDNA complex did not change because of inefficient DNA
complexation by unmodified GO. The fluorescence intensity decreased gradually with
increase in the amount of DGO (1.0G) and reached minimum fluorescence intensity at 30:1
weight ratio. But, DGO (3.0G) most efficiently replaced the EtBr molecules from
EtBr/pDNA complex at very low weight ratio of 5:1 and consequently the fluorescence
intensity decreased significantly at this weight ratio due to its highest zeta potential among
the DGOs. At higher weight ratio, the change in fluorescence intensity remained almost
unchanged because nearly all EtBr molecules were replaced by DGO (3.0) molecules.
3.3. Particle size and zeta potential of GOplex
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Hydrodynamic diameter and zeta potential of nGO/pDNA and DGO/pDNA
complexes at different nGO or DGO to pDNA weight ratios were determined by DLS as
shown in Figures 5a and b, respectively. From Figure 5a, it is found that the particle size of
nGO/pDNA complexes did not change at all weight ratios and the average particle size of
nGO/pDNA complexes was 250±40 nm which was almost equal to the size of nGO only. In
agarose gel and EtBr assay, it was found that the unmodified GO has no DNA binding
capability at all weight ratios and as a result the nGO/pDNA complexes at all weight ratios
showed the diameter of nGO flakes alone. DGO (1.0G)/pDNA complexes showed larger size
(~950 nm) at low weight ratio (5:1) due to inefficient binding of DGO (1.0G) with pDNA.
The hydrodynamic diameter gradually decreased with increase in the weight ratio. However,
the size was quite large compared to that of DGO (2.0) and DGO (3.0). DGO (3.0G) formed
smaller particles among the all dendronized GO due to its highest cationic charge. DGO
(3.0G) formed smallest particles (~285 nm) with pDNA at the weight ratio 20:1 and then the
particle size increased marginally with further increase in weight ratio putatively due to
4
2
17
repulsion of excessive charge offered by DGO . Chen et al. synthesized PEI conjugated
GO by carbodiimide method and they observed similar trends in particle size of PEIꢀ
GO/pDNA complexes at different weight ratios. The particle size of PEIꢀGO/pDNA
complexes was in the range of 250ꢀ500 nm at the weight ratio of 1.3:1 to 20:1. It is suggested
43
that the particle size up to 500ꢀ700 nm can easily be taken up by cells . Therefore, it is
envisaged that the size of DGO/pDNA complex will be suitable for cellular uptake and
consequently may be lead to high transfection efficiency.
Zeta potential of carrier/DNA complex is an important parameter for cellular uptake.
The overall positive zeta potential of the complex will help the attachment of complex with
negatively charged cellular membrane through electrostatic interactions facilitating for
4
4
cellular uptake through endocytosis . As shown in Figure 5b, nGO/pDNA complexes at all
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weight ratios showed negative zeta potential due to incomplete complexation of nGO with
pDNA. DGO (1.0G)/pDNA complexes also showed negative zeta potential at lower weight
ratio but the value of zeta potential changed from higher negative to lower negative value
with increase in weight ratio. The positive zeta potential (5.3±1.4 mV) was obtained at
weight ratio of 30:1 indicating complete DNA complexation, which is also showed in agarose
gel and EtBr assay. DGO (3.0G) was more efficient among the all dendronized GOs and it
showed positive zeta potential (5.6±2.6 mV) at very low weight ratio (5:1). The zeta potential
of DGO (3.0G)/pDNA complexes increased with increase in weight ratio up to the weight
ratio of 20:1 and then reached plateau after the weight ratio of 20:1. The zeta potential of
DGO (3.0G)/pDNA complexes at 20:1 and 30:1 was 25.3±2.3 and 27.3±1.3 mV, respectively
whereas the maximum zeta potential of DGO (2.0G)/pDNA complexes at 30:1 was 20.5±3.4
42
mV. In a previous study a similar trends in zeta potential of dendronized chitosan/pDNA
1
6
complexes was observed. In another study, Kim et al. also obtained negative zeta potential
of GO/pDNA complexes at all N/P ratios (nitrogen to phosphate ratios) from 2:1 to 20:1
ratios with constant positive zeta potentials with branched PEIꢀGO/pDNA complexes after
the N/P ratio of 5:1.
The morphology and particle size of DGO (3.0G) before and after complexation with
pDNA was characterized by AFM, as shown in Figure 6. Figures 6a, c and e show the AFM
images of DGO (3.0G) before complexation with pDNA. The particle diameter and thickness
of DGO (3.0G) before complexation were 150±45 nm and 4.0±1.0 nm, respectively. Both the
particle diameter and thickness of DGO (3.0G) increased after complexation with pDNA at
weight ratio of 20:1 and the sizes were 170±35 and 6±1 nm, respectively (Figure 6b, d and f).
Along with these, the morphology of DGO (3.0G0/pDNA complex was spherical in shape,
which was different from DGO before complexation with pDNA (Figure 6d).
3.4. In vitro cytotoxicity assay
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The in vitro toxicity of nGO and DGO at different concentrations (5, 10, 20, 30, 40,
50, 100 and 200 µg/ml) and weight ratios (1:1, 5:1, 10:1, 15:1, 20:1, 25:1 and 30:1) before
and after complexation with pDNA, respectively was assessed by MTT assay against HeLa
cells as shown in Figure 7. Figure 7a shows the percent cell viability of nGO and DGOs at
different concentrations. nGO was less toxic compared to that of dendronized GOs at all
concentrations. nGO showed >80% cell viability at high concentration (200 µg/ml) which
45
corroborates findings of previous studies . The toxicity of nGO may be attributed to damage
46
to cell membrane by sharp edges of graphene sheets . The toxicity of nGO increased after
dendronization and the cell viability of DGO decreased with increase in the generation of
PAMAM dendrimer. DGO (1.0G) showed lowest toxicity among the all dendronized GOs.
However, the cell viability was above 60% in the presence of DGO (3.0G) at high
concentration (200 µg/ml). The toxicity of dendronized GOs increased due to the positive
charge of DGO. It is reported that the toxicity of polycation is related to its surface positive
charge which interacts with the negatively charged plasma membrane and consequently
47
impairs the function of cellular membrane through aggregation on the cell membrane . The
toxicity of DGO (3.0G) decreased after complexation with pDNA (Figure 7b) due to
reduction in zeta potential of DGO (3.0G) from 38.6±2.6 mV to 27.3±1.3 mV after
complexation with pDNA at the weight ratio of 30:1 through charge neutralization by
negatively charged pDNA. However, the toxicity increased for all DGO/pDNA complexes
with increase in the weight ratio although the cell viability of DGO (3.0G)/pDNA complexes
was above 80% even at high weight ratio of 30:1.
3.5. In vitro transfection efficiency
The transfection efficiency of nGO/pDNA and DGO/pDNA complexes at different
weight ratios from 5:1 to 30:1 using EGFP pDNA was observed in HeLa cells. PEI (25
kDa)/pDNA complex at N/P ratio of 10 and LF 2K were used as positive control. As shown
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in Figure 8, nGO/pDNA and PAMAM (3.0G)/pDNA complexes at weight ratio of 30:1 did
not show any transfection efficiency as expected. It is shown above that nGO has poor
complexation ability with pDNA. The transfection efficiency of GO improved after
dendronization of GO although the transfection efficiency of DGO increased with increase in
the generation of dendrimer. DGO (3.0G0/pDNA complex at weight ratio of 30:1 showed
largest number of GFP positive cells and the number of GFP positive cells was even higher
that of LF 2K.
The transfection efficiency of GOplexes was further quantified by FACS analysis, as
shown in Figure 9a. It was observed that nGO/pDNA complexes at all weight ratios did not
show any transfection as noted in fluorescence micrographs. DGO (1.0G)/pDNA complex at
weight ratio of 30:1 showed only 10% transfection efficiency whereas DGO (2.0G)/pDNA
and DGO (3.0G)/pDNA complex showed 35% and 51% transfection efficiency, respectively
at the same weight ratio. The transfection efficiency of DGO (3.0G)/pDNA complex at the
weight ratio 30:1 was significantly higher that of PEI (25 kDa)/pDNA complex (28%). It was
higher than that of LF 2K/pDNA complex (47%) although the differences were not
statistically significant. Thus, the transfection efficiency of GO significantly increased after
conjugation of PAMAM dendrimer to GO and it scaled with increase in both weight ratio as
18
well as generation of the dendrimer. Previously, Tripathi et al.
prepared linear PEIꢀ
conjugatedꢀGO (LPꢀGO) for gene delivery and observed approximately 33% transfection
48
efficiency in HeLa cells at N/P ratio of 97:1. In another study, Liu et al.
synthesized
PAMAM dendrimer (4.0G) conjugated GO through oleic acid using carbodiimide and they
also obtained 18.3% transfection efficiency in HeLa cells. In comparison to these reported
studies, the synthesized DGO (3.0G) in this work showed markedly higher transfection
efficiency and therefore, it is a significant improvement over currently available GOꢀbased
gene carrier.
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3.6. Mechanism of cellular uptake
The transfection efficiency of nonviral vector not only depends on complexation
capability with pDNA but also depends on the cellular uptake mechanism. It is reported that
nonviral vectors enter into the cell through one or more of the following endocytosis
pathways including clathrin, caveolae and macroꢀpinocytosis mediated endocytosis pathways
4
9, 50
.
To determine the pathway for transfection of DGO, we measured transfection of DGO
(3.0G)/pDNA complex at weight ratio of 30:1 in the presence of the following endocytotic
inhibiting drugs and at low temperature (Figure 9b). Chlorpromazine (CPZ) is known to
5
1
inhibit clathrin mediated endocytosis by preventing the recycling of clathrin proteins
genistein (GEN) inhibits tyrosine kinase involved in caveolae mediated endocytosis
,
,
52
5
3
whereas amiloride (AMIL) inhibits macroꢀpinocytotic uptake . In addition, low temperature
o
54
(
4 C) inhibits energy dependent pathway, that is, overall endocytosis pathway . It was
o
observed that the transfection efficiency significantly decreased at low temperature (4 C)
suggesting that DGO (3.0G)/pDNA complex enters into the cell by energy dependent
pathway, that is, endocytosis pathway was involved during cellular uptake. Furthermore, the
transfection efficiency was inhibited by the presence of chlorpromazine and genistein. But
genistein inhibited 67% transfection efficiency whereas chlorpromazine inhibited only 25%.
Therefore, it appears that the cellular uptake of DGO (3.0G)/pDNA complex occurred
primarily by caveolae mediated endocytosis pathway supported by clathrin mediated pathway
whereas the macroꢀpinocytosis was not involved in the cellular uptake. It is reported that the
particles of size <200 nm are internalized into the cell through the clathrin mediated
endocytosis pathway and the caveolae mediated endocytosis is involved in uptake of particles
5
5
>
200 nm . It has also been reported that the clathrin mediated endocytosis pathway involves
the degradative acidic lysosomes during nuclear transportation and this is the main drawback
associated with this pathway leading to lower transfection efficiency. In contrast, caveolae
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mediated pathway avoids the lysosome compartment in nuclear transportation resulting in
4
4
56
higher transfection efficiency . Recently, Zhi et al.
prepared multifunctional GO
nanocomplex composed of PEI/poly(sodium 4ꢀstyrenesulfonates) (PSS)/GO (PPG) for the
delivery of adriamycin along with siRNA. They also showed that the multifunctionalized GO
nanocomplexes were internalized primarily by caveolae mediated endocytosis pathway
during cellular uptake in MCFꢀ7 cells.
3.7. Intracellular distribution
After cellular uptake, intracellular distribution of carrier/DNA complex is an
important parameter for efficient transgene expression because the carrier/DNA complex has
to overcome several obstacles in the form of transport and degradative mechanisms in the
cell. For intracellular distribution of DGO (3.0G)/pDNA complex at weight ratio of 30:1, the
nucleus of HeLa cell was stained with DAPI and the plasmid DNA was labeled with Cy3 and
finally observed using CLSM after 4 h of transfection as shown in Figure 10. PEI (25 kDa)
and LF 2K were used as positive controls. Figure 10a1 shows that the numbers of red
particles were much higher, that is, more pDNA was carried out by DGO (3.0G) in to the cell
compared to that of either PEI (Figure 10b1) or LF 2K (Figure 10c1) after 4 h of transfection.
It is also observed that more numbers of pDNA localized in the nucleus by DGO (3.0G)
within 4 h of tranfection whereas most of pDNA remained far away from the nucleus
resulting in low transfection efficiency for PEI. On the other hand, LF 2K was able to
transfect fewer pDNA into the nucleus compared to that of DGO (3.0G). It has been reported
that the transfection of both branched and linear PEI/DNA complex may involve either
clathrin or caveolae mediated endocytosis but clathrin mediated endocytosis plays a critical
57
role for transfection of PEI/DNA complex . Uptake of lipoplex (liposome/pDNA complex)
58
is mainly through clathrin mediated pathway . The uptake of DGO (3.0)/pDNA complex
reported in this study is largely through the caveolae mediated endocytosis pathway, which
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helps the DGO/DNA complex to avoid the degradative endosomal compartment facilitating
more effective transport to the nucleus and results in high transfection efficiency compared to
both PEI (25 kDa) and LF 2K.
4. Conclusion
DGO was synthesized by reaction of focal point PAMAM dendrimer of different
generations (1.0, 2.0 and 3.0G) with azide functionalized nGO through “click” chemistry.
The synthesis of DGO was confirmed by FTIR, NMR, XPS, Raman spectroscopy and TEM
analysis. The DNA complexation capability of nGO was significantly improved after
conjugation of PAMAM dendrimer with nGO. DGO formed spherical nano sized complexes
with pDNA through electrostatic interaction having the particle diameter of 250ꢀ350 nm with
positive zeta potential. The transfection efficiency of nGO was significantly increased from
1.2% to 51% in HeLa cells with minimal toxicity and also surpassed the transfection
efficiency of both LF 2K (47% efficiency) and high molecular weight branched PEI (27%
efficiency). Caveolae mediated endocytosis pathway is shown to play an important role
underlying the high transfection efficiency of DGO. Therefore, it may be concluded that
DGO may be an efficient and safe novel nonviral vector in gene therapy application.
Acknowledgements
This work was funded by the Department of Science and Technology (DST), India.
K.S. was supported by the D.S. Kothari fellowship (BSR/EN/13ꢀ14/0005) from the
University Grants Commission (UGC), India. K.C. acknowledges the Ramanujan fellowship
from DST.
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