FICHTER ET AL. VOL. 7
’ NO. 1
December 12, 2012
C2012 American Chemical Society
Polymeric Nucleic Acid Vehicles
Exploit Active Interorganelle
Katye M. Fichter,†Nilesh P. Ingle,‡Patrick M. McLendon,§and Theresa M. Reineke‡,*
†Department Chemistry, Missouri State University, Springfield, Missouri, United States,‡Department of Chemistry, University of Minnesota?Twin Cities,
Minneapolis, Minnesota, United States, and§Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio,
ing biological and medical research. Tools
such as small interfering RNA (siRNA), oligo-
deoxynucleotides (ODNs), and plasmid DNA
(pDNA) represent an effective means of
modifying gene expression.1,2To exploit
these tools, drug discovery now includes
novel nanomedicine programs based on
NA therapeutics that hold great promise
for a variety of pathologies.3,4However,
efficient cellular delivery and intracellular
trafficking of NAs are a critical rate-limited
step to their efficacy. While viral-mediated
NA transfer is highly efficient, they are po-
tentially more difficult to modify for various
applications; thus, versatile methods to
deliver a variety of nucleic acids would
greatly benefit the field of nanomedicine.5
biological processes offers unprece-
dented possibilities for revolutioniz-
In response, creative design and develop-
ment of synthetic, nonviral vehicles has
been the subject of extremely vigorous re-
search. Nonviral vehicles6are easier to synthe-
size and can accommodate a variety of nucleic
NA delivery vehicles with different chemical
structures have been developed and exten-
strated that the chemical structure of the
vehicle plays a crucial role in their biological
interactions.16?23In structure-based develop-
ment, the ideal nonviral NA vehicle should
target both a specific tissue type and a specific
intracellular region or organelle while minimiz-
ing interference with native cellular processes.
Therefore, understanding the influence of the
chemical structure of NA vehicles on the intra-
cellular delivery mechanisms is crucial to all
*Address correspondence to
Received for review September 12, 2012
and accepted December 12, 2012.
ABSTRACT Materials that self-assemble with nucleic acids into nanocomplexes
(e.g. polyplexes) are widely used in many fundamental biological and biomedical
experiments. However, understanding the intracellular transport mechanisms of these
vehicles remains a major hurdle in their effective usage. Here, we investigate two
polycation models, Glycofect (which slowly degrades via hydrolysis) and linear poly-
ethyleneimine (PEI) (which does not rapidly hydrolyze), to determine the impact of
polymeric structure on intracellular trafficking. Cells transfected using Glycofect under-
went increasing transgene expression over the course of 40 h and remained benign over
the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h
post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI,
indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides to the Golgi and endoplasmic reticulum, which may be a route towards nuclear
retrograde transport of polyplexes via COP Ivesicles from the Golgi tothe ER. We conclude that slow release and unique trafficking behaviors ofGlycofect
polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery.
KEYWORDS: polyplex.intracellular trafficking.interorganelle.cationic polymer.gene therapy
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FICHTER ET AL. VOL. 7
’ NO. 1
researchers utilizing these materials and refining their
structure toward clinical advancement.
Previous work has shown that cells internalize poly-
plexes (polymer?NA nanocomplexes) through many
active cellular uptake mechanisms (e.g., clathrin- and/
or caveolae-mediated endocytosis and/or macropino-
cytosis).24?28While a multitude of endosomal traffick-
ing pathways exist, the cell often routes exogenous
materials toward the lysosomes, where enzymes de-
grade the therapeutic complexes, rendering them
useless. Therefore, it has been proposed that escape
from endocytic vesicles may be the largest barrier to
intracellular efficacy.29?31To hypothesize upon the
escape of polyplexes from lysosomes, the “proton
sponge mechanism”10has been developed.29,32?34In
brief, polymers with high Hþbuffering capacity (such
during acidification of the vesicle in which they are
located. This theoretically results in vesicular accumu-
osmotic swelling, leading to leaking or lysis of the
However, vehicles employing the proton sponge
mechanism have some notable drawbacks. Most no-
table is the high cellular toxicity associated with these
vehicles, as they are typically membrane-lytic.35Addi-
tionally, NAs are released into the cytoplasm early in
the transfection process, where they face degradation
by nucleases in the cytoplasm.36Furthermore, after
leaving endocytic vesicles, the NAs have little or no
active transport to the target organelle; studies have
shown that DNA intracellularly microinjected into the
cytoplasm undergoes little to no diffusion.37,38Struc-
tural variations in synthetic nonviral vehicles could
exploit alternative intracellular routes involving active
trafficking of the NA nanocomplexes directly to the
targeted organelle, which would greatly enhance their
Several intracellular trafficking studies of polyplexes
have concentrated on two routes that typically
involve trafficking along actin and/or microtubules: (i)
clathrin-mediated endocytosis,25,29,39?43and more re-
cently (ii) caveolae-mediated endocytosis, which may
facilitate higher nuclear delivery efficiency.11,39,44?47
These pathways, however, are known to commonly
sort macromolecules to organelles such as the Golgi
saccharide residues are information-dense biomole-
cules; therefore the galactose residues in Glycofect
could play a role in mediating the active transport of
these polyplexes. Furthermore, because other glyco-
sylated macromolecules, such as hormone receptors,
have been shown to be transported from the ER to
the nucleus, glycosylated NA delivery vehicles, such
as Glycofect, may also be able to exploit the native
intracellular trafficking pathways in the cell. Also,
a recent study suggested that grafting of a nuclear
localization signal, histone H3, on a polyethyleneimine
(PEI)-based delivery vehicle showed a significant pre-
ferential shift toward caveolae-mediated endocytosis
and potential routing through the ER.45These promis-
ing new developments in synthesis and delivery of NA
via nonviral polymeric delivery vehicles ratify the need
for advanced designs. From a therapeutic perspective,
polymer scaffolds are needed that promote efficient
intracellular trafficking and sustained release of NAs.
Here, we explore the potential of information-rich
saccharide moieties on directing polyplex the caveo-
lin-1 (CAV-1), Golgi, ER, and COPI vesicles (vesicles that
intracellular trafficking, and sustained NA delivery.
In the present study, we examine intracellular traf-
ficking of polyplexes through sorting organelles such as
the Golgi and ER in H9c2(2-1) (rat cardiomyoblast) cells;
this work complements our previous work to under-
stand intracellular trafficking pathways of polyplexes.50
infection pathways by viruses; it has been observed that
cholera toxin and some viruses enter cells through
caveolae-mediated endocytosis,48,49which often tar-
get the Golgi.50Current research also shows that
polyplex trafficking though caveolae leads to high
NA efficacy.47,51Once reaching the Golgi, COP I- and
COPII-positive vesicles shuttle cargo between the ER
and Golgi,52?55which is particularly interesting be-
between the inner and outer nuclear membranes.56
While still incompletely understood, studies have un-
covered active trafficking pathways of native glyco-
sylated proteins between the ER and nucleus.57,58
Because some delivery vehicles, such as Glycofect,
contain saccharide moieties, similar intracellular path-
ways could also play a large role in delivery. Under-
standing how the vehicle structure mediates intra-
cellular trafficking mechanisms could facilitate the
de novo design of NA vehicles to exploit these native
pathways, which could greatly increase delivery
Herein, we use two model polycation delivery sys-
tems to investigate the active transport mechanisms
described above. The first is a glycopolymer, (poly-
(galactaramidopentaethylenetetramine), Figure 1A).
This polymer, previously published as G4 and structur-
ally identical to the commercial reagent “Glycofect”,
was characterized to contain nine repeat units and will
hereafter be referred to as its commercial name,
Glycofect, for simplicity. The other polycation used in
this study is linear polyethyleneimine (commercial
name: JetPEI, Figure 1B), which is structurally similar
to Glycofect, minus the saccharide subunits. PEI is a
efficiency, but often exhibits high cytotoxicity.59?61
FICHTER ET AL. VOL. 7
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Glycofect has demonstrated low toxicity, rapid de-
gradation at neutral pH, and high NA transfer
efficiency.11,20,62?67We demonstrate that polyplexes
way involving interorganelle transport via the Golgi
Our results suggest that researchers can exploit effi-
cient native interorganelle trafficking pathways by
optimizing the chemical structure of polymer-based
vehicles to target specific organelles. The analysis of
nonviral NA vehicles.
To investigate temporal changes in cellular delivery,
H9c2(2-1) (rat cardiomyoblast) cells were transfected
with (i) naked plasmid DNA (pDNA) (“no vehicle”), (ii)
polyplexes formulated with linear PEI and pDNA, or
(iii) polyplexes formulated with Glycofect and pDNA.
In each case pDNA encoded the gene for luciferase,
over thecourse of7days. Datafrom luciferase reporter
gene assays are often reported in units of relative light
units (RLU)/mg protein; however, the use of this unit
may result in data interpretation artifacts because
vehicles with high toxicity decrease in the amount
of protein in each sample, falsely reporting higher
“transfection efficiency”.11,62,65,66Data reported in
Figure 2A depict the activity of the luciferase enzyme
in more straightforward terms by normalizing raw RLU
values to the negative control (untransfected cells).
For comparison, data in units of RLU/mg are also
reported in Figure S1.
Figure 2A shows the transfection efficiency of PEI
increases at early time points in the transfection pro-
cess, peaks at 16 h, then decreases. Cells transfected
with Glycofect show lower expression at early time
points, but peaks above PEI at 40 h after transfection.
Glycofect demonstrates almost a 2-fold increase in
gene expression when compared to PEI at longer time
points (4 and 7 days, Figure 2A, inset). Toxicity data
for cells transfected with these vectors are depicted
in Figure 2B and demonstrate the benign nature of
Glycofect over the course of 7 days. PEI demonstrates
excessive cellular toxicity, dropping to about 40%
survival after 16 h and less than 10% survival after
7 days. These results support our hypothesis that
Glycofect promotes slower, more efficacious and be-
nign delivery of pDNA than linear PEI.
The proton sponge hypothesis predicts that the
buffering capacity of the polymer is essential for
endosomal escape and therefore plays a direct role in
of Glycofect and linear PEI were directly compared in
solution withanamineconcentration of0.7744M.This
concentration was selected to model polymer concen-
tration in intracellular endosomes in accordance with
previously published reasoning.20The polymer solu-
tions were titrated with HCl (hydrochloric acid) from
Figure1. Structures of thetwo nonviralNAdeliveryvehiclesexaminedhereinand proposedintracellular pathwaysof study.
(A) Poly(galactaramidopentaethylenetetramine), previously published as “G4” and commercially sold as “Glycofect”, is a
degradable polymer (Mw= 4.6 kDa; degree of polymerization, n = 11).11(B) Linear PEI (commercially sold as “JetPEI”) is a
nondegradable polymer Mw≈ 22 kDa. (C) Proposed intracellular trafficking pathways of polyplexes.
FICHTER ET AL.VOL. 7
’ NO. 1
the titration was calculated. These results demonstrate
that the buffering capacity of Glycofect is much higher
than that of linear PEI in this pH range (Figure 2C). The
residues, in the structure of Glycofect, may allow for
an increased fraction of amine protonation (i.e., the
protonation of one amine electrostatically suppresses
protonation of neighboring amines).20,64
To explore the role of buffering capacity on the
ability of polyplexes to escape acidic vesicles, we mea-
tides within cultured cells via flow cytometry. H9c2(2-1)
cells were transfected with dual-labeled ODNs,
“FITC-ODN-Cy5” [i.e., pH-sensitive FITC (fluorescein
isothiocyanate) and pH-insensitive Cy5 (cyanine 5);
see Materials and Methods]. Previous experiments
have demonstrated that the fluorescence intensity
ratio (FITC/Cy5) is linearly related to the pH environ-
ment of the ODN.29,69A linear calibration plot was
created by measuring the FITC/Cy5 fluorescence in-
tensity ratio of ODNs in transfected cells, suspended
in intracellular clamping buffers (see Materials and
accounts for any effect the complexed polymer may
impart on fluorescence measurements.
We also investigated the effect of vesicular acidifica-
tion on the intracellular pH of ODNs by treating cells
with a vesicular proton pump inhibitor (bafilomycin)
and ion channel inhibitors (ionophores) 8 h after
transfection. This experiment tested for evidence of a
proton sponge mechanism in polymer?ODN delivery
since the use of these drugs inhibits active vesicular
acidification.70,71If the vehicle uses a proton sponge
mechanism, the polymeric vehicles should be able to
absorb excess protons in vesicles as allowed by their
buffering capacity. Because we have measured the
buffering capacity of Glycofect as higher than PEI,
we would expect Glycofect to be able to absorb more
protons than PEI, causing Glycofect?ODNs to be in a
more pH-neutral environment.
Figure 2D depicts the intracellular pH environment
of ODNs calculated from our flow cytometry measure-
ments. It was observed that naked ODNs delivered
without a vehicle (“ODN only”) were in acidic environ-
ments at both 8 and 24 h, presumably due to the
inability to escape actively acidifying vesicles (and the
environment of the naked ODNs increases with the
addition of bafilomycin and ionophores, which was
expected since most ODNs resided in acidic environ-
ments and the presence of these additives inhibits the
Figure 2. Biochemical characterization of PEI and Glycofect (A and B) H9c2(2-1) cells were transfected with luciferase pDNA
and assayedfor luciferaseactivityat theindicatedtime. RawRLUvalueswerenormalizedagainst untransfected cells; erroris
different time points and with or without (() bafilomycin during the transfection process. Intracellular pH calculations were
derived using a standard curve (Figure S2). All transfections were performed with 5 N/P PEI and 20 N/P Glycofect.
FICHTER ET AL. VOL. 7
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proton pumps. The intracellular pH of ODNs delivered
with PEI was neutral after both 8 and 24 h, suggesting
that the majority of PEI?ODN polyplexes exist in pH-
neutral subcellular environments (e.g., the cytoplasm
or neutral organelles). As expected, the pH environ-
ment of PEI?ODN complexes did not change upon
bafilomycin treatment because the pH environment
of these PEI?ODNs was neutral without bafilomycin
treatment. The average intracellular pH of Glycofect?
ODN complexes was 5.7 after 8 h and did not sig-
nificantly change after 24 h (pH = 5.8). Surprisingly, the
significantly change with bafilomycin treatment (from
5.7 to 5.5).
These measurements of ODN intracellular pH envi-
ronments demonstrate that, while the buffering capa-
city of Glycofect is higher than that of linear PEI
ODNs is lower in all conditions tested, suggesting
that Glycofect may not employ the proton sponge
mechanism as a major mechanism of ODN delivery. At
the same time it should be noted that slower kinetics
of buffering behavior for Glycofect polyplexes and an
effect of polymer hydrolysis (causing the liberation of
galactartic acid and pentaethylenehexamine) could
also be hypothesized to result in delayed Hþbuffering
of endosomes. Because the subcellular pH environment
treatment, we speculate that Glycofect polyplexes may
dwell in a mixture of subcellular compartments, ac-
tively acidifying vesicles, slightly acidic organelles
(i.e., early endosomes, Golgi apparatus, endoplasmic
reticulum), and pH-neutral environments (i.e., cyto-
plasm, nucleus), decreasing the effect of the bafi-
lomycin treatment on overall ODN pH. Conversely,
pH measurements indicate that PEI delivers ODN to
a combination of proton-sponge-mediated release
of ODN to neutral compartments and disruption of
membranes due to direct contact with PEI, causing
the leaking of polyplexes out of vesicles. It has been
previously demonstrated that charge-dense vehicles
such as PEI are very damaging to biological mem-
branes60,72,73and, therefore, may be exploited by this
vector to facilitate rapid release of the ODNs into the
cytoplasm, either directly from the plasma membrane
or from intracellular vesicles. A large body of work by
our lab and others supports these results, demonstrat-
correlate to delivery or transfection efficiency.20,74,75
Because our intracellular pH data from flow cytome-
try is able to provide only an average pH of ODNs in
a large population of cells, microscopy was used to
observe different subcellular populations of ODNs co-
localized with acidic vesicles. Micrographs in Figure 3
in transfected H9c2(2-1) cells, using LysoTracker to
visualize acidic vesicles. Cells were fixed and mounted
in pH-neutral mountant prior to imaging to avoid
quenching of FITC fluorescence in acidic compart-
(Figure 3A?C) generally exhibit punctate staining
without a significant amount of cytoplasmic ODN
staining, which correlates with our previous data
Figure 3. Micrographs of transfected H9c2(2-1) cells with time. Cells were transfected using naked ODN (A?C), PEI?ODN
polyplexes at 5 N/P (D?F), or Glycofect (G4)?ODN polyplexes at 20 N/P (G?I) and fixed at the indicated time. FITC ODNs
the diffuse cytoplasmic location of ODNs. Scale bar = 20 μm.
FICHTER ET AL. VOL. 7
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(Figure 2D) that show these ODNs remain in acidifying
In cells transfected with PEI (Figure 3D?F) we ob-
served that the cytoplasm was brightly and diffusely
stained with ODN 8 h after transfection. This correlates
with our previous data indicating that PEI delivers
the majority of ODN to a neutral pH environment
within 8 h (Figure 2D). Afterward, the cytoplasmic
ODN distribution faded and a significant amount of
ODN was observed in the nucleus after 24 h (Figure 3F
and Figure S3). It should be noted that at both 16 and
24 h some perinuclear co-localization of FITC?ODN
with LysoTracker is observed (Figure 3E, F); however,
the average intracellular pH measurements indicate
that the majority of ODNs reside in pH-neutral envir-
onments (Figure 2D). Additionally, it is not clear from
thesemicroscopy studiesif theODNsco-localized with
LysoTracker in Figure 3E and F are still associated with
PEI encourages quick release of the majority of ODN
into the cytoplasm of the cell within 8 h.
Cells were then transfected with Glycofect under
identical conditions and imaged (Figure 3G?I). A
mixed intracellular population of ODNs was found,
which may explain our average intracellular pH mea-
surements (Figure 2D). For example, one subset of
ODNs appeared punctate (e.g., in membrane-bound
vesicles) and another subset appeared to be diffuse
result in a persistent slightly acidic (pH ∼5.7) average
measurement. Polyplexes formulated with Glycofect
and ODNs appeared to accumulate in the nucleus
of some cells by 16 and 24 h. Importantly, a diffuse,
cytoplasmic fraction of ONDs delivered with Glycofect
persists over the course of 24 h, suggesting the ability
of Glycofect to mediate sustained cytoplasmic ODN
To corroborate these microscopy studies, a flow
cytometry-based Acridine Orange (AO) assay76was
performed to examinethe ability ofpolymeric vehicles
to initiate ODN escape from lysosomes. In this assay,
AO partitions into intact acidic organelles and fluo-
resces brightly intheseacidic environments. Whenthe
membrane integrity of an acidic organelle is compro-
internal acidic environment, and average AO fluores-
cence is quenched (diminished). Our results show that
acidic vesicles remain intact in nontransfected cells
and cells transfected with naked DNA over the course
transfected using PEI exhibit some lysis of acidic
organelles, starting at 8 h post-transfection in up to
6% of the cells, indicating a small percentage of cells
with significant vesicle lysis. This is consistent with
microscopy data in Figure 3D?F, which show diffuse
cytoplasmic presence of ODNs along with some lyso-
somal co-localization. Cells transfected with Glycofect,
While our microscopy data indicates that some Glyco-
fect-delivered ODN is present in lysosomes, these data
correlate with our previous experiments and suggest
that Glycofect may not employ a “proton sponge”
mechanism of endosomal/lysosomal escape and may
use an alternate mechanism.
Actin is implicated in every known form of active
other, less characterized forms of endocytosis.81To
investigate the role of actin on the cellular uptake of
polyplexes, we disrupted actin polymerization with
cytochalasin D (Figure S5A). In addition, we also in-
hibited uptake via caveolae (using Fillipin III) and
uptake via clathrin (using chlorpromazine), to delin-
eate specific information about the primary pathways
of polyplex uptake (Figure S5B, C) in this cell type.82
Glycofect ODN uptake slightly decreased with actin
depolymerization, and PEI?ODN uptake slightly in-
creased with the same, although this difference was
not significant (Figure S5A). Additionally, both clathrin
and caveolae appeared to be major internalization
routes for Glycofect and PEI, but the inhibition of
clathrin- and caveolae-mediated endocytosis was
more pronounced for Glycofect polyplexes, possibly
suggesting the involvement of poorly characterized or
nonendocytic import mechanisms for PEI. These data
are in agreement with previous findings from our
lab and work by other researchers in this field.47,51
In a directly related previous study, we reported
that the structure of the polymer (responsible for the
chemical interactions with the cell surface and net
charge on the surface of the polyplex) significantly
influences interaction with a variety of cell surface
glycosaminoglycans (GAGs) on HeLa cells and also
appears to play a major role in the cellular uptake
efficiency of both Glycofect (previously published as G4)
and JetPEI.83In that study, it was found that sulfated
GAGs are required for cellular internalization of
Glycofect polyplexes but not for JetPEI and that hya-
luronic acid and heparin sulfate were the primary
GAGs that bound in the strongest manner to Glycofect
To investigate polyplex internalization at a subcel-
lular level, we performed immunocytochemistry (ICC)
experiments. In cells transfected with Glycofect?ODN
complexes, minimal co-localization was observed
between FITC?ODN and clathrin, indicating that
ODNs delivered with Glycofect are not associated with
clathrin-coated vesicles at this late (24 h) time point
(Figure 4A, top). This was expected, since the clathrin
coat is removed from internalized vesicles within
minutes of internalization.84Despite previous data
FICHTER ET AL. VOL. 7
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suggesting that uptake of linear PEI (polymer only;
noDNA)is independentofclathrin,85asmalldegree of
co-localization was observed between PEI-delivered
that DNA delivered with PEI can be exocytosed,28a
process that can also involve clathrin; therefore this
observation could also be due to the cellular export of
We also used ICC to investigate co-localization be-
tween caveolae and FITC?ODN in cells 24 h after
transfection (Figure 4B). Significant co-localization was
observed between caveolin-1 and ODN delivered with
both polymers. These results agree with our caveolae
inhibition studies and suggest a role for caveolae in
plasma membrane, Golgi complex, endoplasmic retic-
ulum, and vesicles that bud from these organelles.79
Therefore the co-localization of ODN and caveolin-1
suggests that both Glycofect and PEI polyplexes could
engage in interorganelle trafficking between caveolin-
associated organelles (e.g. Golgi, ER). While many stud-
ies have shown polyplexes are internalized by multiple
avenues of endocytosis,39,47a caveolin-mediated en-
docytic gateway may guide a subset of polyplexes
to caveolin-associated organelles. This observation
may represent a newly discovered active route of
intracellular transport that could enhance transfection
efficiency, due to the spatial proximity of these organ-
elles with the nucleus and the known trafficking routes
between these organelles.45
Cellular internalization of polyplexes by caveolae
and clathrin-mediated uptake can lead to trafficking
to many different organelles within the cell. One
possible destination, particularly associated with
caveolae-mediated trafficking, is the Golgi complex, the
biomolecular cargo. The interior of the Golgi complex
is maintained at a slightly acidic pH (∼6.2?6.6);86,87
therefore polyplex transport to this organelle could
partially contribute to the slightly acidic average
pH environment (∼5.7) measured with Glycofect-
delivered ODN (Figure 2D). Co-localization between
FITC?ODN and the Golgi complex was examined in
H9c2(2-1) cells 24 h post-transfection via ICC with
(Figure 5A and B). A small amount of ODN delivered
with PEI was observed as uniformly small puncta co-
localized with the Golgi complex (Figure 5A and B,
bottom row). ODN delivery with Glycofect (Figure 5A
Figure 4. Co-localizationof ODNsdelivered by PEI (5 N/P)or Glycofect(20 N/P) with clathrin or caveolin-1.(A) H9c2(2-1) cells
were transfected with FITC-ODN; 24 h later, cells were fixed and immunostained for clathrin. The 3D projection depicts a
budding clathrin pit, located at the indicated boxed area in the maximum projection image. The top of the 3D projection
image is near the plasma membrane, and the bottom of the image is the bottom of the coated pit. Magenta pixels indicate
co-localization. (B) Cells were transfected as in A and stained for caveolin-1. Scale bars = 20 μm.
FICHTER ET AL.VOL. 7
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and B, top row) also co-localized with the Golgi com-
were observed to have a more diverse size range than
those of PEI-delivered ODN (Figure 5B, arrows). These
data support the hypothesis that the Golgi complex
may play a role in polyplex sorting and transport
throughout the cell.
The endoplasmic reticulum is another known desti-
nation for caveolae-mediated trafficking. Because the
lumen of the ER is contiguous with the space between
the inner and outer nuclear envelope, trafficking of
nucleic acids to the ER could be an intermediate in
the mechanism of transport to the nucleus. Related
work as indicated cellular trafficking of glycosylated
proteins from the ER to the nucleus,57,58and such a
pathway could play a role in polyplex transport. To
examine the involvement of the ER in nucleic acid
trafficking, ICC was used to observe co-localization
between FITC?ODN and BiP (a luminal ER protein),
24 h after transfection. Significant co-localization was
observed between the ER and ODNs delivered with
the degree of co-localization between the ER and
ODNs delivered with both polymers, the Manders
coefficient88was calculated using the boxed area in
Figure 5C (enlarged in Figure 5D). The Manders coeffi-
cient can be used to analyze the degree of overlap
Co-localization between FITC?ODN and giantin (Golgi complex). (C and D) Co-localization between FITC?ODN and BiP
(ER lumen). (E and F) Co-localization between FITC?ODN and βCOPI (Golgi complex-derived vesicles). Scale bar in A, C, and
E = 20 μm. Scale bar in B, D, and F = 2 μm.
FICHTER ET AL.VOL. 7
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from 0 to 1, where 0 correlates to virtually no overlap
and 1 correlates to virtually complete overlap. The M1
coefficient (greenpixels overlappingred) wasfound to
be 0.889 for Glycofect-transfected cells and 0.457 for
PEI-transfected cells. These values suggest the degree
of co-localization between Glycofect-delivered ODNs and
the ER is twice as much as the degree of co-localization
between PEI-delivered ODNs and the ER, which is addi-
tional evidence of an alternate delivery pathway for Gly-
To investigate active interorganelle trafficking path-
ways of polymer-delivered ODNs, we used ICC to
investigate ODN co-localization with COPI vesicles,
which are responsible for retrograde cargo transport
from the cis end of the Golgi to the rough ER. These
experiments indicate that ODNs delivered with both
polymers co-localize with COPI-coated vesicles 24 h
after transfection (Figure 5E and F).
These data support our hypothesis that polyplexes
undergo active interorganelle transport. Specifically,
these studies suggest potential active interorganelle
routes oftransport tothe ER:(i) via caveolin-associated
vesicles89and/or (ii) via COPI vesicles from the Golgi
was observed between the Golgi and ODNs 24 h after
transfection, it is possible that co-localization between
points. It should also be noted that this organelle is
used for sorting, and so localization at this organelle
will be transient. Trafficking via the ER is particularly
of the ER lumen with the space between the inner and
a route of nuclear transport of nonviral vehicles, which
have previously shown that multiple mechanisms of
cellular uptake are implicated in polymer-mediated
DNA delivery.47These results are corroborated here,
pathways, which may provoke the involvement of the
Golgi complex and ER. More broadly, our results sug-
gest that polymer structure plays a critical role in the
intracellular routes through which exogenous DNA is
trafficked, with potentially profound effects on their
efficacy and cytotoxicity.
Collectively, the average pH environment polymer-
elles implicated in ODN trafficking (Figures 3?5)
“proton sponge mechanism”. We built on these data
by investigating the subcellular pH environments of
ODNs delivered with Glycofect and linear PEI, via live-
cell time-lapse microscopy to visualize spatial and
temporal changes in subcellular pH environments of
ODNs. Similar to the average intracellular pH data
presented in Figure 2D, the pH environment of ODNs
can be assessed at a subcellular level via fluorescence
which H9c2(2-1) cells were transfected with double-
in green) quenches in low pH environments, pixels
representing ODNsin acidic environments(pH <∼5.0)90
have lower, or absent, green intensity. Conversely, in
neutral pH environments, FITC fluoresces brightly and
pixels in the green channel have high pixel intensity.
Because Cy5 fluorescence (pseudocolored in blue) is not
affected by pH, the ratio (FITC/Cy5) of pixel intensity in
the images can be used to infer the relative pH environ-
ment of the ODNs.
Figure 6A depicts a live H9c2(2-1) cell 4 h after
transfection with FITC-ODN-Cy5 using Glycofect (see
also Figure S6A). In the left-most image of Figure 6A,
the boxed area is shown at different time points in
smaller insets to the right. Consistent with the pH
assays (Figure 2D), most FITC-ODN-Cy5 appears blue,
with a pH around 5. However, the time-lapse movie of
this cell (Movie S1) reveals that there are dynamic
ments. Sporadic motion of the puncta is consistent
images on the right of Figure 6A show large clumps of
ODN, which may be polyplexes in the process of being
endocytosed. Thirty hours after transfection with Gly-
cofect (Figure 6B), a subpopulation of slightly acidic
(blue-green) ODNs emerges, indicating that they have
been trafficked to more neutral pH environments (see
also Movie S2). A striking feature of this image is the
environments), one of which appears to possibly leak
small amounts of fluorescent DNA out of a compart-
ment with time (Movie S2).
Figure 6C shows an H9c2(2-1) cell transfected using
PEI?ODN polyplexes 4 h post-transfection. In general,
when compared to Glycofect-delivered ODNs at 4 h
(Figure 6A), a greater fraction of PEI-delivered ODNs
appear to be in pH-neutral environments. However,
similar to Figure 6A, ODNs in neutral pH environments
are located near the plasma membrane. These pH-
neutral ODNs may represent polyplexes on the cell
surface, actively undergoing uptake, or in early stages
of endocytosis. The time-course movies of PEI-delivered
ODN trafficking after 4 h (Movie S3) corroborate this
hypothesis: vesicles containing this ODN appear
to traffic toward the center of the cell and decrease
in green intensity (undergo acidification).
classified into three subpopulations: (1) in pH-neutral
environments within the nucleus, (2) in mildly acidic
environments, accumulated in the perinuclear region,
and (3) in pH-neutral environments near the cell pe-
riphery (Figure 6D). This third subpopulation of ODNs
does not appear to be moving into the cell and may
FICHTER ET AL.VOL. 7
’ NO. 1
represent ODN leaking out of the cell (Movie S4,
arrow). Many studies have demonstrated that PEI in-
duces plasma membrane damage via charge inter-
actions73,91and that PEI is capable of damaging intra-
membrane damage (in addition to the proton sponge
mechanism).This potential for membrane damage
could result in damage to the inner face of the PM
causing DNA to leak from the cell (see DIC overlay
image Figure S6D). We speculate that this may be
observed in Movie S4 (and Figure 6D, right) where the
arrowhead depicts the location of what could be leak-
ing of ODN from the cell.
To analyze temporal changes in the pH environ-
ments of ODNs from our ratiometric fluorescence
microscopy experiments, the FITC/Cy5 pixel intensity
ratio was plotted with time. As previously mentioned,
this ratio can be inferred as the relative subcellular pH
environment of ODNs. Figure 6E shows these plots
over the course of about 45 min, starting 4 h after
transfection. As observed, the pH environment of
Glycofect-delivered ODNs was lower than that of PEI-
delivered ODNs, which is consistent with average
intercellular pH measurements in Figure 2D. After
about 4.5 h post-transfection (35 mins after data
collection started), ODNs delivered with PEI exhibit a
spiking pattern (Figure 6E), which may be indicative
of short bursts of ODNs rapidly encountering a neutral
pH environment at this early point in the transfec-
tion process, which is consistent with the diffuse
cytoplasmic distribution observed of PEI-delivered
ODNs at 8 h post-transfection in Figure 3D. As the
FITC-ODN-Cy5 diffuses into the cytosol and out of the
focal plane depicted in the images, the plot returns to
Figure 6F shows plots of the ODN pH environ-
ment over the course of 45 min, starting at approxi-
mately 30 h post transfection. PEI-delivered ODN de-
monstrates a sharp decline in average relative pH.
Figure 6. Confocal images of live H9c2(2-1) cells transfected with double-labeled FITC (green)-Cy5 (blue) ODNs and
polyplexes formulated with either the Glycofect (20 N/P) or PEI (5 N/P). (A) FITC/Cy5-ODNs delivered with Glycofect after
approximately 4 h. (B) FITC/Cy5-ODNs delivered with Glycofect after approximately 30 h. (C) FITC/Cy5-ODNs delivered with
and F) Changes in mean pH environment of ODNs in a single cell with time. The average pixel intensity of FITC and Cy5 was
measured at each time point, and the ratio was plotted against time. E) 4 h post-transfection. (F) 30 h post-transfection.
FICHTER ET AL. VOL. 7
’ NO. 1
This evidence agrees with our hypothesis that the
peripheral population of neutral ODN may be leaving
the cell (potentially through damage to the plasma
membrane or exocytosis) and/or being further acid-
ified en route elsewhere in the cell, decreasing the
overall ODN pH environment. In contrast, after 30 h of
However, the average pH environment of Glycofect-
delivered ODNs fluctuated, exhibiting a similar spiking
pattern seen in theplotofPEI-deliveredODNsafter 4.5h
of transfection (Figure 6F, open circles). These results
also may be interpreted as sustained release of ODN
from acidic compartments at later time points, which
corroborates our hypothesis of prolonged and sus-
tained cytosolic delivery of ODN. The temporal ratio-
metric fluorescence movies of Glycofect-delivered ODN
(Movies S1 and S2) further support our suggestion that
active vesicular transport mechanism within the cell at
later time points. Together, these time-lapse ratiometric
fluorescence measurements support our previous data
demonstrating slower, prolonged, and sustained active
intracellular trafficking of ODNs delivered with Glycofect
Nucleic acid delivery has exhibited tremendous
potential both for therapeutic use, as well as a tool to
understand fundamentals of normal biological pro-
cesses and disease. While it is well known that the
vehicle structure plays a large role in the delivery
mechanisms, kinetics, efficacy, and toxicity, relatively
little is known about how this impacts the intra-
cellular pathways employed by polymer-based vehi-
cles to carry nucleic acids to their desired intracellular
location. Knowledge of these pathways will help re-
searchers to develop vehicles that can efficiently carry
their nucleic acid cargo through specific intracellular
routes while remaining benign to the cell. To this end,
we have examined the intracellular routes taken by
two model polymeric vehicle types, linear PEI and
In cells transfected with PEI, we have shown that
transgene expression occurs quickly, yet decreases as
cellular survival drops drastically, starting at 16 h after
transfection. In contrast, while Glycofect mediates
slower transfectionefficiency, itexhibits sustained cyto-
plasmic release and extended delivery to intracellular
organelles and remains benign to cells in culture over
the course of at least 7 days. Additionally, we have
shown that PEI-delivered ODN is more quickly trans-
to Glycofect (Figure 2C). Furthermore, our subcellular
fluorescence microscopy studies demonstrate delayed,
but more sustained release of Glycofect-delivered ODN
to the cytosol, both with pH-based measurements
(Figures 2D and 6) and subcellular co-localization with
acidic organelles (Figure 3). These results contribute to
and correlate with a body of previously published work
that scrutinize the traditionally held “proton sponge
mechanism” of endosomal release.10In addition, at 4,
8, and 24 h post-transfection, average intracellular
ODN pH is more acidic with Glycofect (Figures 2D and
6A, E), when compared to data at the same time points
with linear PEI (Figures 2D and 6C, E). This acidic
environment would normally suggest that the poly-
plexes may be located inside either late endosomes or
lysosomes; however, upon further exploration, we
found this was not the case. Further studies (Figure S4,
Supporting Information) indicate that PEI has a slightly
higher (p < 0.05) tendency to rupture acidic vesicles
as compared to Glycofect as measured by an AO assay
in whole cells. These data suggest that PEI polyplexes
may be sequestered in and rupture these acidic
vesicles,10,68,69more so than Glycofect. Additionally,
other studies by our group and others have shown
that the tendency of PEI-based polyplexes to cause
membrane rupture also causes significant cellular
toxicity.35,60,93Together these results suggest that vesi-
cle release of polyplexes into the cytoplasm occurs by
different mechanisms depending on vehicle structure.
polyplex population that may be shuttled to other
polyplexes also appeared to remain in vesicles over
longer time periods (Figures 3 and 6), potentially due
to the structural design of this polymer, which could
possibly be responsible for two primary functions: (1)
NA encapsulation and the higher buffering capacity
release of ODN in the perinuclear region of a cell due to
corroborate this possible sustained release in the peri-
nuclear region of a cell. Thus, Glycofect polyplexes may
havea higher portion of intact nucleic acid in the active
transport pathway in the cell; our experiments demon-
strate that between 40 h up to 4 d Glycofect-mediated
sion (Figure 2A).
Despite the fact that Glycofect has a higher buffering
capacity than PEI, the overall subcellular pH environ-
ment of Glycofect-delivered ODN is more acidic than
PEI. These results, as well as results obtained by our
lab and others, have shown that higher buffering ca-
pacity does not necessarily directly correlate to higher
gene expression.94?96This observation lead us to hy-
acidic organelles,29,43,45while cells may favor trafficking
Glycofect polyplexes through an alternative pathway(s)
distinct from the “proton sponge” mechanism.
FICHTER ET AL.VOL. 7
’ NO. 1
In recent studies by our group, we have demon-
strated that PEI induces plasma membrane permeabi-
lization within the first 30 min of transfection and
nuclear membranepermeabilization by 4h post-trans-
fection, which results in higher cellular toxicity. Many
other groups have also shown evidence that PEI can
cause damage to cellular membranes.60,97After 4 h,
PEI present in the cell could also permeabilize other
intracellular organelles, such as mitochondria, causing
be partly attributed to physical membrane disruption.98
Multiple membrane damage events, both at the plasma
membrane and inside the cell, have been shown to
trigger apoptosis and could be a primary means of
material toxicity and cell death.35,46,98,99In contrast,
Glycofect polyplexes are relatively nontoxic and show
sustained gene expression levels up to 48 h (Figure 2A).
This observation strongly corroborates the hypothesis of
this study: that Glycofect begins to moderate the critical
cellular trafficking of polyplexes through native active
intracellular routes (e.g. via Golgi and ER).
The slower kinetics of delivery promoted by
Glycofect and the generally acidic pH environment
experienced by Glycofect polyplexes inspired us to
investigate the alternative active transport pathways
in the cell. Inhibiting clathrin and caveolae-mediated
PEI?ODN and Glycofect?ODN, indicating these path-
ways are major routes of transit in H9c2(2-1) cells,
and others.45,47,51,83An important finding of this study
obtained with the positive co-localization data of ODN
with the Golgi, ER, and COPI vesicles (Figures 4 and 5)
supports our hypothesis that the innate sorting routes
in cells are used to transport exogenous nucleic acids
delivered with nonviral vehicles. In particular, the
strong co-localization of Glycofect?ODNs with the ER
along with the slower delivery kinetics is intriguing
and could indicate that a higher portion of Glycofect-
delivered ODNs are transported via interorganelle
transit. While the co-localization/trafficking results are
similar for linear PEI and Glycofect, the difference in
kinetics and stronger ER localization with Glycofect
suggest that that Glycofect could rely more heavily on
Live cell confocal microscopy (Figure 6) provides
evidence, albeit indirect, to support the hypothesis
that PEI can induce intracellular membrane damage
as a possible means to enhance efficacy (yet, likely
temporal pH data (Figures 6E and 6F), and together
imply that Glycofect utilizes an active transport me-
chanism at later time points post-transfection.
Taken together, data in this study support our
hypothesis thatactivetransport ofnucleicacidsoccurs
within the cell, utilizing the innate sorting and packa-
ging organelles designed for shuttling proteins, over
the time course of the nucleic acid delivery experi-
that labeled ODNs are co-localized with organelles
such as the Golgi, ER, and trafficking vesicles such as
COPI, clathrin-coated pits, and caveolae 24 h after the
initial cellular exposure to polyplexes. As mentioned,
the cytoplasm is a perilous and crowded environment
acids within this environment and into the nucleus. In
glycopolymer vehicle, Glycofect, undergoes different
kinetics of intracellular trafficking, which possibly pro-
motes delivery to a larger degree through this active
As previously mentioned, saccharide residues are
well known for various signaling/routing events in
biological systems, and the galactose residues in Gly-
cofect could play a role in mediating the active trans-
carbohydrate-binding proteins, are well known to ini-
tiate signaling events regulating proliferation, apopto-
sis, and intracellular export.100,101Some studies have
even revealed nuclear trafficking of galectin-3.102Pre-
vious studies on glycosylated polymers indicate that
many carbohydrate moieties strongly affect intracellu-
lar routing; for example, mannosylated polyplexes ap-
pearedto stay longer in endosomes and accumulate in
lysosomes more than lactosylated polyplexes.42,103,104
Therefore, it is possible that Glycofect polyplexes are
targeted to specialized organelles, such as the ER and
Golgi, by virtue of their galactose moiety. Furthermore,
because other glycosylated macromolecules, such as
hormone receptors, have been shown to be trans-
ported from the ER to the nucleus, glycosylated NA
delivery vehicles may also be able to exploit this path-
way. Therefore, future efforts to tailor carbohydrate-
containing vehicles to further exploit this active route
the efficacy of these nonviral nucleic acid delivery
vehicles. This pathway is further supported by findings
from a recent study with the PEI polymer: grafting with
localization signal resulted in rapid accumulation of
polyplexes in ER as early as 5 min post-transfection
and gradually increased over 30 min, as compared to a
low level of accumulation with native PEI.45Thus, the
trafficking route toward an intracellular target. In addi-
tion, it should be noted that this trafficking behavior of
Golgi and ER mimics viruses, such as Simian virus 40,
polio-virus, and hepatitis C virus.105?110
of the polymer used to design a nonviral nucleic acid
FICHTER ET AL.VOL. 7
’ NO. 1
delivery vehicle plays a vital role in its intracellular
trafficking. Both linear PEI and Glycofect delivery ve-
hicles promote alternative active transport routes en
route to the nucleus, rather than just mediated via the
“proton sponge effect”. The unique saccharide-based
structure of Glycofect allows it to initiate cellular entry
via various routes including caveolae, clathrin, macro-
pinocytosis, and perhaps other less-characterized in-
ternalization routes. After cellular internalization, both
linear PEI and Glycofect polyplexes can hitchhike via
intracellular transport machinery, which leads to sort-
ing to the Golgi and likely retrograde transport to the
endoplasmic reticulum. While linear PEI polyplexes
appear to localize to acidic endocytic vesicles, Glycofect
polyplexes appear to bypass lysosomes (more so than
linear PEI) and appear to be, at least in part, trafficked
so than PEI. We hypothesize that this active transport
nucleus and perhaps is the cause of more sustained
gene expression kinetics of this delivery vehicle. In-
deed, polymeric nucleic acid delivery vehicles appear
to mediate delivery, at least in part, via pathways
similar to viruses. Understanding how the structure of
these vehicles impacts transport pathways will aid in
the de novo design of more effective vehicles for
MATERIALS AND METHODS
Materials. Unless otherwise noted, all chemicals and re-
Glycofect (G4) was synthesized according to previously pub-
lished procedures (Mw= 4.6 kDa; degree of polymerization,
n = 11) and was used at an N/P ratio = 20 due to previous
findings that higher N/P ratios promoted the highest transfec-
formed have been found to be around 110 nm, and the zeta
potential has been found to be around 10 mV. Linear PEI (Mw≈
22 kDa) used in titrations was purchased from Polysciences, Inc.
(Warrington, PA, USA). JetPEI (linear PEI, Mw= 22 kDa) used
in cell culture studies (N/P ratio = 5, recommended from
the manufacturer) was purchased from Polyplus Transfection
(New York, NY, USA). All primary antibodies were purchased
from Abcam (Cambridge, MA, USA): clathrin, rabbit polyclonal
antibody to clathrin heavy chain; caveolin, rabbit polyclonal
antibody to caveolin-1; Golgi, mouse monoclonal antibody
giantin; ER, rabbit polyclonal antibody to GRP78 BiP (ER lumen);
COP I, rabbit polyclonal to beta COP. The secondary antibody
was purchased from Molecular Probes (Eugene, OR, USA).
Cell Culture. The H9c2(2-1) cell line was purchased from
American Type Cell Culture Collection (ATCC, Rockville, MD,
USA) and consists of cardiac-like myoblasts derived from BD1X
rat myocardium. Cells were cultured under conditions recom-
mended by ATCC. All cell culture media, antibiotic/antimycotic,
fetal bovine serum (FBS), phosphate-buffered saline (PBS), and
nuclease-free water were purchased from Gibco (Carlsbad, CA,
USA). Plasmid DNA gWiz-Luc (6.7 kb) and pCMV-βGal (8.5 kb)
were purchased from Aldevron Fargo (Fargo, ND, USA).
Time-Course Transfection Efficiency Assay. H9c2(2-1) cells were
seeded into 24-well plates (Corning, MA, USA) at a density of
hour before transfection, polyplexes were formulated at the
appropriate N/P ratio, where “N” is the number of secondary
amines onthe polymer and “P” is the numberof phosphates on
and Glycofect were 5 and 20, respectively. To make polyplexes
Luc plasmid DNA (6.7 kb) solution at0.02 μg/μL andmixed with
gentle pipetting to make 100 μL/well polyplex solution. At
the time of transfection, media was removed and the cells
were washed with PBS (Gibco, Invitrogen, Carlsbad, CA, USA).
Cells were transfected with 100 μL/well of polyplex solution in
200 μL/well of Opti-MEM (1 μg of plasmid DNA per well) and
returned to the incubator at 37 ?C and 5% CO2. All transfections
were performed in triplicate and included negative controls
of both untransfected cells and cells transfected with naked
plasmid DNA. After 4 h, the Opti-MEM (reduced serum media)
solution was aspirated and exchanged with Dulbecco's mini-
mum essential medium (D-MEM) supplemented with 10% FBS,
amphotericin. (Gibco, Invitrogen). At the appropriate time point,
the cells were removed from the incubator, washed with PBS,
and lysed with100 μL/well of 1? lysis buffer (Promega,WI, USA).
The amount of protein in each sample well was analyzed using a
Bio-Rad DC protein assay kit (Hercules, CA, USA) and determined
using a standard curve created with bovine serum albumin
gene activity using a luciferase assay kit (Promega). Lumines-
cence was measured in duplicate over 10 s with a Tecan GENios
for each sample. Cell viability is reported as the average amount
cells. The transfection efficiency of each vector is reported as
relative lightunits(RLU)/mg ofprotein for each triplicate normal-
ized against a control of plasmid DNA only.
Oligonucleotide Synthesis and Annealing. Two strands of 20 bp
DNA oligonucleotide were synthesized via standard phos-
phoramidite chemistry. The sense strand sequence was 50
CCTTGAAGGGATTTCCCTCC 30, and the antisense strand se-
phosphorothiolated completely and fluorescently labeled with
FITC (sense strand) and/or Cy5 (antisense strand) at the 50end.
To anneal, the two oligos were dissolved in nuclease-free water
and diluted to a concentration of 1 μg/μL in annealing buffer
[0.05 M NaCl, 10 mM EDTA (ethylenediaminetetraacetic acid),
10 mM Tris pH 7.5]. Thesolution was then immersed in an 80 ?C
room temperature over the course of several hours.
Buffering Capacity. The protocol for titration of polymer solu-
tions was slightly modified from a previously published
method.20Briefly, the polymers were dissolved in PBS (pH 7.4)
and the concentration of secondary amines was held constant
at 0.7744 M for both polymer solutions. This concentration was
chosen in an attempt to model the physiological concentration
of polymer in endosomes during intracellular trafficking
1 plasmid DNA internalized by an endosome approximately
200 nmin diameter). Polymer solutions were titrated with 0.1 M
standard HCl, and pH was monitored using an Accumet Basic
AB15 pH meter (Pittsburgh, PA, USA). The titration data were
analyzed via nonlinear regression using GraphPad Prism 4.0
(San Diego, CA, USA).
Intracellular pH Assay. H9c2(2-1) cells were seeded in six-well
plates at a density of 200,000 cells/well in D-MEM containing
10% FBS, 100 units/mg penicillin, 100 μg/mL streptomycin, and
0.25 μg/mL amphotericin and cultured for 24 h before transfec-
tion. A few hours before transfection, polyplexes were formu-
lated at N/P = 5 for PEI and N/P = 20 for Glycofect by adding
polymer solution to annealed FITC-Cy5-labeled oligonucleo-
tides (0.02 μg/μL); polyplexes were allowed to form for at least
FICHTER ET AL.VOL. 7
’ NO. 1
washed with PBS, and 1 mL of Opti-MEM was added to each
well. Then 500 μL of polyplex solution (5 μg of DNA) was
immediately added to each well, and the plates were swirled
to evenly distribute the DNA and replaced in the incubator.
After 4 h, the Opti-MEM solution was aspirated, the cells were
washed extensively with PBS three times, and 2 mL of D-MEM
to allow DNA to traffic for the necessary amount of time
(measurements taken at 8 and 24 h post-transfection). D-MEM
was removed, and cells were again washed with PBS. The cells
were trypsinized at 37 ?C for 10?15 min and pelleted in an
amber 1.5 mL Eppendorf tube. The cells were washed again in
PBS and repelleted. Supernatant was removed, and cells were
resuspended in either PBS or one of four pH clamping buffers
of NaOH or HCl. The buffers were sterilized by filtration through
a 0.2 μm syringe filter. In indicated experiments, the buffers
were supplemented with ionophores (0.010 mM valinomycin
and 0.010 mM nigericin; Fluka, Sigma-Aldrich) and 200 nM
bafilomycin. The cells were then transferred to Falcon tubes
and kept on ice until analyzed via flow cytometry on a FACS-
were measured in each sample. A negative control of untrans-
fected cells was used to gate the level of positive fluorescence
such that 1% or less of the untransfected cells was positive for
either FITC or Cy5 fluorescence.
Confocal Time-Course Microscopy. H9c2(2-1) cells were seeded
into six-well plates containing a 25 mm No. 1 coverslip at a
density of 15,000 cells/well in supplemented DMEM (10% FBS,
amphotericin). Cells were cultured for 2 days to allow them to
attach to coverslips. One hour before transfection, polyplexes
were formulated by adding a solution of polymer to a solution
of FITC-labeled annealed oligonucleotide at an N/P ratio of 5 for
was removed from wells and cells were washed with PBS. Cells
were transfected with 200 μL of polyplex solution (2 μg of
oligonucleotide) in 2 mL of Opti-MEM and returned to the
incubator. After 4 h, 2 mL of supplemented D-MEM was added
LysoTracker Red (Molecular Probes) in supplemented D-MEM
was added to each well. Cells were returned to the incubator for
30 min. Afterward, media was removed from cells, washed again
with PBS, and fixed with 1% paraformaldehyde solution over-
night. Before imaging, nuclei of cells were counterstained with
DRAQ5 (Biostatus Limited, Leicestershire, UK) and mounted in
ProLong Gold Antifade Reagent mounting media (Molecular
Probes). Cells were imaged on a Zeiss LSM510 confocal system
(Zeiss, Thornwood, NY, USA) fitted onto an Axioplan 2 upright
microscope (Zeiss). Images were collected using the appropriate
BP 505?550 nm; LysoTracker Red: excitation 543 nm, emission BP
560?615 nm; DRAQ5: excitation 633 nm, emission LP 650). Cells
imaged were chosen based on being representative of the entire
population observed. The confocal pinhole was opened to 1.5 μm
for all channels in order to capture the cytoplasmic distribution of
a fourth channel to depict the morphology of transfected cells.
Images were enhanced for brightness and contrast using ImageJ
analysis software (National Institutes of Health, Bethesda, MD).111
Endocytic Inhibition Experiments. H9c2(2-1) cells were seeded
at 200,000 cells/well in supplemented D-MEM (10% FBS, 100
units/mg penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL
were formed with Cy5-labeled ODN as described above. Media
Opti-MEM containing 2 μg/mL cytochalasin D, chlorpromazine
at 10 μg/mL, and filipin III at 1 μg/mL (2 mL) was added to each
well. Plates were returned to the incubator, and cells were
preincubated with the drugs for the amount of time indicated
(Figure S5). Polyplex solution (300 μL) was added directly to
each well and swirled to mix. Negative controls were treated
with 300 μL of Opti-MEM (“cells only”) or uncomplexed plasmid
to each well, and cells were incubated an additional 30 min.
After removing the media and rinsing with PBS, 500 μL of
Trypsin-EDTA was added to each well and the cells were
incubated until detached from the well (5?10 min). D-MEM
containing 10% FBS (1 mL) was added to inactivate trypsin, and
the contents of each well were transferred to a Falcon Tube
(BD Biosciences). Cells were centrifuged at 4 ?C and 1000 rpm
for 10 min, then resuspended in 2% FBS in PBS. Cellular uptake
was measured on a BD FACSCanto II Flow Cytometer (BD
Biosciences). Propidium iodide (PI) was added to each tube at
a concentration of 5 μg/mL and volume 2.5 μL/tube, and
the tubes were gently vortexed 2?5 min prior to analysis.
Appropriate gating was performed using a negative control
(untreated H9c2(2-1) cells) to ensure that autofluorescent and
PI-positive cells were excluded from subsequent analysis. Com-
piled data are an average of at least two replications (with the
exception of caveolae inhibition, which was a single experiment).
with a 660/20 nm emission bandpass filter; propidium iodide
was excited with a 488 nm solid-state laser, and fluorescence
emission was detected by a 670 nm long-pass filter. A total of
analysis was done using BD FACSDiva software (BD Biosciences).
Immunocytochemistry. H9c2(2-1) cells were seeded into 12-
well plates containing a 15 mm No. 1 coverslip at a density
of 12,000 cells/well and incubated at 37 ?C under a 5% CO2
atmosphere for 24 h prior to transfection. Polyplexes were
formed by adding 50 μL of 0.02 μg/μL FITC-labeled annealed
ODN to 50 μL of polymer solution diluted to the appropriate
concentration to reach N/P = 5 (JetPEI) or N/P = 20 (Glycofect).
After incubation for 1 h at room temperature, cells were
aspirated of media, washed with 0.5 mL of PBS, and transfected
with 400 mL of Opti-MEM/polyplex solution (corresponding to
1 μg of plasmid DNA/well). Cells were allowed to incubate at
37 ?C under a 5% CO2atmosphere for 4 h, and then 930 μL of
fully supplemented D-MEM was added to each well. After an
additional 20 h incubation, cells were aspirated of media and
washed three times with 1 mL of PBS. One milliliter of 1% PFA
and cells were washed three times with 1 mL of PBS. Cells were
then incubated at room temperature with 0.25% Triton X-100
(Integra Chemical Co., Kent, WA, USA) for 10 min and then
washed with 1 mL of PBS for 5 min. Cells were blocked in a
solution of 1% BSA in PBS at room temperature for 40 min.
Afterward, BSA solution was removed from each well and
500 μL of the specified primary antibody dissolved in 1% BSA
in PBS solution wasadded to each well andallowed toincubate
at room temperature for 1 h. (Concentrations used: clathrin,
1 μg/mL; caveolin, 1/500 dilution of provided manufacturer
solution; Golgi, 2 μg/mL; ER, 1 μg/mL; COPI, 1/2000 dilution of
the provided manufacturer solution.) Afterward, the primary
antibody solution was removed from each well and cells were
washed three times with 1 mL of PBS for 5 min. Coverslips were
then carefully placed cell-side down into 100 μL of 5 μg/mL
secondary antibody solution in a humidified chamber and
allowed to incubate for an additional hour. Afterward, cells
were again washed three times with 1 mL of PBS for five times
and placed cell-side down into 50 μL of DRAQ5 (Biostatus
Limited) nuclear counterstain solution (10 μM) and allowed
to incubate at room temperature for 5?10 min. Coverslips
were carefully removed from DRAQ5 solution and washed with
1 mL of PBS before being mounted in ProLong Gold Antifade
Reagent (Molecular Probes).
Cells were imaged on a LSM510 confocal system (Zeiss)
fitted onto an Axioplan 2 upright microscope (Zeiss). Images
were collected using the appropriate laser excitation and filter
sets (FITC: excitation 488 nm and emission BP 505?550 nm;
Alexa Fluor555 dye: excitation 543 nm and emission BP 560?
615; DRAQ5: excitation 633 nm, emission LP 650). The cells
FICHTER ET AL.VOL. 7
’ NO. 1
of the entire population observed. The confocal pinhole was
Images were initially processed using the despeckle algorithm
(through ImageJ),111and then appropriate background subtrac-
tion was applied to each image. Images were then minimally
processed for brightness and contrast enhancement using
ImageJ. Co-localization highlighting was also processed using
the Colocalization Highlighter plugin for ImageJ. Manders'
coefficient88(M1, for green (ODN) pixels overlapping red (ER)
shown in eq 1. Manders' coefficient ranges from 0 to 1, where
1 denotes complete co-localization and 0 represents none:
where Gi,co‑locis the intensity of green pixels overlapping red
pixels and Giis the total intensity of green pixels
Confocal Live Cell Imaging. H9c2(2-1) cells were seeded into
six-well plates containing a 25 mm No. 1 coverslip (Mattek
to culture for 24 h before transfection. A few hours before
transfection, polyplexes were formulated by adding a solution
of polymer to a solution of double-labeled (FITC-Cy5) annealed
oligonucleotide at an N/P ratio of 5 for JetPEI and 20 for
Glycofect. At the time of transfection, media was removed from
wells and cells were washed with PBS. Cells were transfected
with 200 μL of polyplex solution (2 μg of oligonucleotide)
in 2 mL of Opti-MEM and returned to the incubator. For cells
allowed to transfect for 30 h, after the initial 4 h of transfection,
were imaged on an LM510 confocal system (Zeiss, Thornwood,
NY) fitted onto an Zeiss Axiovert 100 M inverted microscope
appropriate laser excitation and filter sets (FITC: excitation
488 nm and emission BP 505?550; Cy5: excitation 633 nm
representative of the entire population observed. Images were
enhanced for brightness and contrast using ImageJ analysis
Live Cell Time-Lapse Image Processing. Images were initially
processed using the despeckle algorithm (through ImageJ),
and then appropriate background subtraction was applied to
used to increase the clarity of the presented images; however,
Ratio Calculations of Live Cell Time-Lapse Images. A cell to be
analyzed was cropped out of the original image, and FITC and
Cy5 brightness and contrast were adjusted simultaneously in a
merged image. The pixels representing intracellular plasmid
DNA to be analyzed were subjected to threshold, and back-
ground (black) pixels were converted to “not a number” to
remove them from the calculation. The mean pixel intensity of
FITC and Cy5 in each cell at each time point in the time series
was then measured separately. Dividing the mean FITC pixel
intensity by the mean Cy5 pixel intensity and plotting this ratio
pH graphs depicted in Figure 6E, F.
Acridine Orange Assay. H9c2(2-1) cells were seeded in six-well
cell culture plates at a density of 100,000 cells/well. A lower
number of cells was used to reduce cell-to-cell confluency at
extended time points in the experiments. The polyplexes were
formulated by adding 125 μL of polymer to 125 μL of NFkB
oligonucleotide in DNase RNase-free H2O and incubated for 1 h
at room temperature. Prior to transfection, the polyplexes were
diluted in 500 μL of Opti-MEM. The transfected cells were
incubated at 37 ?C for 15 min. After 4 h, 1 mL of DMEM was
the cells were treated with 5 μg/μL of AO solution in D-MEM at
37 ?C for 15 min and then prepared for flow cytometry experi-
FACS CantoII flow cytometer (BD Biosciences) for 20,000 cells.
The 488 nm argon laser was used to excite red lysosomal
fluorescence and detected at 670 nm. The FACSDiva software
(BD Biosciences) was used to acquire data. The loss in detected
fluorescence was attributed to rupture of lysosomes, causing
a reduction in intensity. These cells were referred to as “pale
Conflict of Interest: The authors declare the following com-
peting financial interest(s): T.M.R. is a consultant to and has
stock options in Techulon, Inc.
Supporting Information Available: Figures S1?S6 and
Movies S1 to S4. This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. The authors wish to thank NIH 1-R21-
EB3938-01 and the Director's New Innovator Award program
(DP2-OD006669-01) for funding of this project.
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