Hindawi Publishing Corporation
Volume 2011, Article ID 918036, 10 pages
Deliveryto the Distal Lung
Jeffrey A. Kazzaz,1Marlene S. Strayer,2Jichuan Wu,3Daniel J. Malone,3Hshi-chi Koo,1
Thomas H.Shaffer,3,4JonathanM. Davis,1,5DavidS.Strayer,2andMarla R.Wolfson3,6
1The Cardiopulmonary Research Institute and Departments of Medicine and Pediatrics, SUNY Stony Brook School of Medicine,
Winthrop University Hospital, Mineola, NY 11507, USA
2Department of Pathology, Thomas Jefferson University Medical Center, Philadelphia, PA 19107, USA
3Departments of Physiology and Pediatrics, Temple University School of Medicine, 3420 North Broad Street, Philadelphia,
PA 19140, USA
4Nemours Research Lung Center, Alfred I. DuPont Hospital for Children, Wilmington, DE 19803, USA
5Department of Pediatrics, The Floating Hospital for Children at Tufts Medical Center, Boston, MA 02111, USA
6Center for Inflammation, Translational, and Clinical Lung Research (CILR) and Temple Lung Center,
Temple University School of Medicine, Philadelphia, PA 19140, USA
Correspondence should be addressed to Marla R. Wolfson, email@example.com
Received 7 March 2011; Accepted 24 May 2011
Academic Editor: Edwin Chilvers
Copyright © 2011 Jeffrey A. Kazzaz et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
We compared lung delivery methods of recombinant adenovirus (rAd): (1) rAd suspended in saline, (2) rAd suspended in saline
followed by a pulse-chase of a perfluorochemical (PFC) liquid mixture, and (3) a PFC-rAd suspension. Cell uptake, distribution,
analyses. Relative to saline, a 4X increase in transduction efficiency was observed in A549 cells exposed to PFC-rAd for 2–4h. rAd
in PFC-rAd suspensions consistently peaked at 24h. These results demonstrate that PFC-rAd suspensions improve distribution
and enhance rAd-mediated gene expression which has important implications in improving lung function by gene therapy.
Gene delivery to distal lung epithelium has proven difficult
because of the extensive system of large and small airways,
mucociliary clearance, and the presence of dense glycocalyx
surfactant proteins act as collectins to remove recombinant
proteins and viruses for gene therapy [1, 2]. The distribution
of the coxsackie-adenovirus receptor (CAR) on basolateral
surfaces of cells has led to the proposed use of reagents that
disrupt tight junctions and improve transduction of airway
epithelial cells [3–5]. As an alternative approach to improve
chase method in the lung to deliver recombinant adenovirus
(rAd) and adeno-associated virus vectors in a small volume
of saline administered followed by a larger volume of
perfluorochemical (PFC) liquid [3, 6–11]. These studies
have demonstrated increased total lung gene expression,
distribution of gene expression, and enhanced delivery to the
distal lung over saline alone. However, uniform distribution
with increased alveolar uptake has not been demonstrated
consistently. In addition, efficacy of these techniques with
respect to differences in overall expression over time has not
PFC liquids are synthetically produced fluorinated hy-
drocarbons that are inert, capable of carrying dissolved
gasses, and dense enough to open areas of collapsed lung.
PFC liquids have long been proposed as a ventilatory treat-
ment as well as a method to homogeneously deliver bioactive
agents to the lung. Studies of liquid ventilation in animal
that PFC liquids have a number of salutary effects in injured
lungs. Following liquid ventilation, animals with experi-
mentally induced acute lung injury demonstrate improved
gas exchange, lung compliance, less hemorrhage, edema,
inflammation, and oxidation compared to similarly injured
animals undergoing conventional gas ventilation [12–20].
Histopathologic injury and bronchoalveolar lavage (BAL)
fluid content of total white cells, neutrophils, and proinflam-
matory cytokines were reduced in animals or patients with
acute lung injury undergoing liquid ventilation compared to
gas ventilation [19, 21]. These effects have been attributed
to both mechanoprotective and cytoprotective mechanisms
[22, 23]. Several in vitro studies have demonstrated that
intracellularuptake ofPFC altersbehaviorof alveolar epithe-
lial and inflammatory cells, particularly neutrophils and
properties [24–27]. However, physicochemical properties of
PFC liquids such as viscosity, vapor pressure, and lipid sol-
ubility vary depending upon the chemical composition and
arrangement of carbon-fluorine bonds. These characteristics
are important determinants of the degree to which PFC
liquids distribute throughout the lung, evaporate from the
lung, and move across cell membranes [14, 25, 28–31].
With respect to optimal physiological profiles and anti-
inflammatory properties, the most therapeutic PFC liquids
These characteristics allow the PFC liquid to resist redis-
tribution and rapid elimination. These PFC liquids tend to
remain where they are initially administered, and additional
doses are less likely to be required . A recent, novel
approach has been to combine different PFC liquids to
engineer a desired viscosity and vapor pressure. A systematic
of PFC liquids in an in vivo model demonstrated that a 1:3
ratio of PP2 and PP9 had the greatest anti-inflammatory
impact on the lung [13, 15].
In this paper, we used a mixture of PP2:PP9 as a gene
delivery vehicle using rAd both in vitro and in vivo and
compared various methods of delivery: (1) rAd suspended in
saline, (2) rAd suspended in saline followed by a pulse-chase
of PFC liquid, and (3) rAd as a PFC liquid suspension. To
test the hypothesis that rAd delivered as a PFC suspension
would result in higher levels of transgene expression in
lung parenchyma, we examined the expression in A549
cells. In vivo studies in a murine model using rAd-LacZ
construct demonstrated a more uniform distribution than
when delivered in saline or by the pulse-chase method. The
effect of this methodology on temporal expression in vivo
was determined using a rAd-luciferase construct in a murine
model. These data demonstrate that use of PFC suspensions
for the delivery of rAd increases expression in the lung
periphery and gives a more reliable temporal pattern of
2.1. rAd Constructs. Replication-deficient type 5 adenovirus
encoding LacZ (rAd-CMVntLacZ), was obtained from
the Gene Transfer Vector Core at the University of Iowa .
The recombinant adenovirus encoding the firefly luciferase
gene (rAd-CMVLuc) was obtained from the Vector Core
Facility at the University of Pittsburgh.
2.2. PFC-rAd Suspensions. The perfluorochemical used in
this study was 25% perfluorocyclohexane [PP2]/75% perflu-
oromethyldecalin [PP9] obtained from F2 Chemicals, Ltd,
Lancashire, UK. PFC suspensions were prepared using a
homogenous distribution [33, 34]. Briefly, rAd constructs in
(25% PP2/75% PP9; 10mL/kg) were sonicated (Branson
2510, Danbury, CT) for 5–10 minutes.
2.3. rAd Transduction Efficiency In Vitro. Human alveolar
epithelial A549 cells (American Type Culture Collection
CCL-185) were grown in Ham’s F-12-K medium (GIBCO
BRL, Life Technologies, Gaithersburg, MD) supplemented
with 10% heat-inactivated fetal bovine serum, 2mM glu-
tamine, 100U/mL penicillin, and 100μg/mL streptomycin.
Cells (4 × 105) were maintained at 37◦C in 95% room air-
in a humidified chamber, seeded on a 6-well
plate containing coverslips and allowed to adhere overnight.
Subconfluent A549 cells were incubated with rAd at a
multiplicity of infection (MOI) of 100 viral particles/cell
in 0.6mL of complete media or PFC liquid on a rocking
platform for 2–48h at 37◦C. For incubations longer than 8h,
0.6mL of media was added to the PP2:PP9 wells to prevent
the PFC liquid from evaporating and thus protecting the
cells from dehydration. At the end of the transfection period,
PFC liquid was siphoned off, and cells were refreshed with
ing transduction on near confluent cells grown on coverslips
as per manufacturer’s instructions (Sigma Chemicals, St.
Louis, Mo). Briefly, cells were rinsed with phosphate-
buffered saline (PBS), and then fixed in 0.1% glutaraldehyde
for 5min at room temperature. After three washes with PBS,
cells were stained with 1mg/mL X-gal (Invitrogen) for 2h
at 37◦C. In transfected cells, β-gal cleaved X-gal to produce
a blue stain. Coverslips were then washed and mounted on
microscope slides for viewing. Ten consecutive fields were
with a Sony 3CCD Progressive camera, a PC computer, and
Adobe Photoshop version 7.0 with the appropriate import
plugin using identical settings. Images were then viewed in
Metamorph (Molecular Dynamics), and X-gal positive cells
were counted manually.
2.4. Instillation of rAd In Vivo. Spontaneously breath-
ing, C57BL/6 and Balb/c mice (6–8wks; 15–20gm) ran-
domly assigned to vehicle and time-sequenced groups and
then anesthetized by intraperitoneal injection (ketamine:
40mg/kg; xylazine: 8mg/kg). Once sedated, local anesthesia
a superficial incision. Instillation was performed during
spontaneous breathing by tracheal puncture using a 0.50mL
syringe with 29G needle, approximately 2 cartilaginous
rings below the cricoid. Following instillation, animals
were rotated consistently to augment distribution until the
mice demonstrated normal motor activity and grooming
(i.e., within 30min). All procedures were approved by the
Institutional Animal Care and Use Committee at Temple
University School of Medicine and were in accordance with
National Institutes of Health guidelines.
2.5. rAd Transduction Efficiency In Vivo. Spatial distribution
ofgeneexpressionwasassessedinC57BL/6mice(n = 9)that
received rAd-CMVLacZ in saline (2.7 × 1010viral particles
in 2mL/kg), rAd-CMVLacZ in saline (2mL/kg) followed by
PFC (10mg/kg) (pulsed chase method), or rAd-CMVLacZ
in saline (2mL/kg) suspended in PFC (10mL/kg) (PFC
suspension). Additional animals (n = 6) instilled with
saline alone (2mL/kg) or PFC alone (10mL/kg) were used
as controls. Temporal distribution of gene expression was
assessed by bioluminescence in Balb/c mice (n = 8) that
received rAd-Luciferase in saline (1010or 5 × 1010viral
particles) suspended in PFC (2mL/kg).
2.6. Spatial Distribution. In the lungs of these animals,
spatial distribution was assessed 48h following intratracheal
administration. The trachea was cannulated, and the lungs
were pressure-clamped at 15cm H2O, vascularly perfused
with cold Millonig’s buffer, and removed en bloc. Lungs were
then fixed in glutaraldehyde, incubated in X-gal reaction
solution (1mg/mL of X-gal) at 37◦C for 4h, and postfixed
in formalin. Lung mounts were then paraffin embedded
for imaging and preparation for histological analysis. The
paraffin-embedded tissue blocks of lungs were viewed on a
Nikon SME10 dissecting microscope; images were captured
with a Nikon E5000 Coolpix camera and imported into
Adobe Photoshop for digital output. Thin sections (5μm)
were mounted on slides, stained with hematoxylin (H) and
eosin (E), and examined by light microscopy. Tissue sections
were viewed on a Nikon Optishot microscope; images were
captured with a Spot Insight camera and imported into
Image ProPlus for digital output.
2.7. Temporal Gene Expression. Temporal gene expression of
the rAd-CMV vector was assessed on days 1, 2, 4, 7 and,
10 following administration of the rAd encoding the firefly
luciferase gene (rAd-CMVLuc). This bioluminescent assay
system is used to indirectly measure a gene of interest where
the luciferase gene is placed downstream of the relevant
promoter. The substrate, luciferin, reacts with oxygen in the
presence of the enzyme luciferase, resulting in the forma-
tion of light. In vivo luciferase expression was visualized
in anesthetized Balb/c mice (n
Animals were administered 100μL (150mg/kg) of luciferin
by intraperitoneal (IP) injection, placed under gas anesthesia
(isoflurane), and imaged 15–20min after administration
with the IVIS 50 imaging system (Xenogen Corp., Alameda,
Callif) and the Living Image software package (Caliper Life
Sciences, Hopkinton, Mass). An initial dose-response curve
= 3-4 animals/group).
determined that signal peaked in the lung at 15min and
began to subside at 25min (data not shown). Image settings
of a 3min exposure at high sensitivity (8 × 8 binning)
were used except when these settings resulted in a saturated
image. The images were quantified using identical region
sizes. Background values were determined from sham-
2.8. Statistical Analyses. For viral transduced cells, a Stu-
dent’s t-test was performed comparing groups within each
time period. Differential β-gal and luciferase expression
in the lungs was compared using one-way ANOVA with
a Fisher- or Bonferroni-adjusted posthoc comparison of
means between groups. All analyses were performed using
the SAS software package version 8.1 (SAS Institute, Cary
NC, 2001). Results are reported as mean ± standard
3.1. PP2:PP9 Improves rAd Transduction Efficiency In Vitro.
Transduction of lung epithelial cells by rAd in saline
compared with rAd suspended in PFC was first examined
using A549 cells, a human lung carcinoma type II cell line.
rAd-CMVLacZ was added to A549 cells for 2, 4 or 8h, and
β-gal activity was assayed 48h after the addition of rAd for
all time points. The timeframe of A549 cell transduction was
different for rAd delivery in PFC liquid compared to rAd
in saline (Figure 1). Transduction in PFC liquid occurred
sooner than in saline; at2 and 4h therewerefour-to-five fold
more β gal (+) cells with PFC liquid (P < 0.05) compared to
saline controls. By 8h, >95% of cells in both cultures were
β-gal (+) (data not shown).
3.2. PP2:PP9:rAd Suspensions Improve Distribution to Murine
Distal Lung. Transduction in vivo was tested using three
different rAd-CMVLacZ administration methods: (a) in
saline, (b) pulse-chase—in saline followed by PFC liquid,
or (c) in PFC liquid suspension. Forty-eight hours after
instillation lungs were removed en bloc and stained for β-gal
activity (Figure 2: left panel). X-gal stained lungs were then
embedded in paraffin and examined by light microscopy
to compare the cell-type distribution of rAd-CMVLacZ-
mediated β-gal expression resulting from the different meth-
ods of delivery (Figure 2: right panel). Representative lungs
(Figure 2) showed that rAd-CMVLacZ delivered in saline
alone (Figure 2(a)) resulted in β-gal activity in central and
proximal large airways only. No staining was observed in
the lungs of saline-alone or PFC-alone control mice (data
not shown). Low magnification photos of lungs instilled
by pulse-chase (Figure 2(b): middle panel) and PFC liquid
suspension (Figure 2(c): middle panel) illustrated expression
in large airways (see arrows). When delivered by pulse-
chase method (Figure 2(b)), more cells expressed β-gal,
and the level of expression was increased in the distal
airways; however, there was little evidence of expression
in the peripheral lung, especially the alveolar epithelium.
β-gal positive cells/field
Figure 1: PFC liquid increases transduction efficiency in cell
culture. (a) Representative fields of A549 cells transduced with rAd-
CMVLacZ (MOI = 100 viral particles/cell) in either saline (Control,
left) or PFC liquid (right) for 2h (top) or 4h (bottom) followed
by X-gal staining. (b) Consecutive low-power fields were acquired
under identical settings, and β-gal positive cells were enumerated
manually. Valuesrepresentmean ±SDof10fields.∗P < 0.05versus
In contrast, when delivered as a PFC liquid suspension
(Figure 2(c)), X-gal staining was clearly more intense, more
homogenous and reached the extreme peripheral lung thus
reflecting the most uniform, highest, and widest distribution
of β-gal expression. Supplementary images of lungs of mice
transfected by the three techniques are shown in Figure 2(d).
in tissue sections under higher magnification (Figure 2:
right panel). Airway epithelial cells were identified by
their location lining the airway. While cell-specific staining
was not performed, transgene expression was evident in
what appears to be alveolar epithelial cells by shape and
location within alveoli; type I cells with an elongated
appearance and type II cells with a cuboidal appearance
are located in corners. The distribution of β-gal expression
appeared different depending upon the method of delivery,
particularly in distal portions of the lung. rAd-CMVLacZ
delivery by pulse-chase appeared to result in expression
primarily in airway and less so in alveoli with most alve-
olar expression occurring in type I cells (Figure 2(b)). In
contrast, administration in PFC liquid suspension appeared
to result in greater expression in alveolar epithelial cells
3.3. PP2:PP9 Improve Distribution and Early Expression of
rAd. The light microscopy data demonstrate better delivery
to the peripheral lung parenchyma with virus suspended
in PFC. To determine if this translates into more transgene
expression in the lung, we used luciferase as a reporter for
two reasons: (1) transgene expression could be measured
noninvasively and repeatedly over time and (2) the half-life
of luciferase is short (∼3h) so new transgene expression,
rather than protein accumulation, is measured. In these
studies, rAd-luciferase in saline or as a PFC suspension
was instilled intracheally, and a longitudinal study was
performed using a low-light imaging system. Balb/c mice
were used for these experiments because their white coat
color minimizes absorbsion of light emitted from the vector.
Two viral titers were tested (1 × 1010viral particles and 5 ×
1010viral particles); images were acquired at various times
after administration, and luciferase activity was quantified.
Representative animals from each group are shown in
Figure 3. Animals receiving virus suspended in PFC had
better bilateral distribution at both low and to a greater
degree, higher viral titers (Figure 3(a)). At later times, the
appearance of luciferase activity in the diaphragm area
of the mice from the saline-delivery group suggests that
the virus was cleared by mucociliary transport and then
ingested (see arrows in Figure 3(b)). While 50% of the saline
group had signal in the abdominal area, no expression was
detected in the abdominal area of animals from the PFC
group suggesting less clearance and better retention in the
lung. Quantitation of expression was then used to compare
transgene expression in the thoracic region of the mice.
Figure 4 shows the temporal pattern of individual animals,
and Table 1 provides the expression when analyzed as a
group.No consistentpatternwasevidentin thesalinegroups
(Figures 4(a) and 4(c)) with thoracic expression peaking
at 4d, 2d, and 1d in Mouse 4, Mouse 5, and Mouse
6, respectively (Figure 4(c)). In the high viral titer group,
animals in the PFC liquid group had a consistent pattern
of expression peaking on day 1 then diminishing daily
(Figure 4(d)). The PFC liquid animals in the low viral titer
group (Figure 4(b)) exhibited the same pattern of expression
Figure 2: PP2/PP9 suspensions improve transgene expression in the distal lung. Left Panel: whole mounts of lungs from mice transduced
with rAd-CMVLacZ (2.7 × 1010viral particles) administered intratracheally in (a) saline alone, (b) saline followed by a chase of PP2:PP9
(10mL/kg), or (c) rAd/PP2:PP9 suspension. (a) Proximal airway expression is seen predominantly when rAd is delivered in saline alone.
(b) Increased expression in proximal and distal lung is seen when delivery of the virus in saline is followed by a PP2:PP9 chase; (c) Greatest
staining compared to the other methodologies in the alveolar spaces and the distal lung with delivery of rAd in PP2:PP9 suspensions. Middle
Panel: lungs were removed, embedded in paraffin, and sectioned to the same approximate depth. Inset in middle panel C demonstrates a
distinctly alveolar pattern in the PFC liquid/rAd lungs and a lack of staining in the corresponding region of the virus in saline is followed
by a PP2:PP9 chase (inset, middle panel (b)). Right Panel: sections were mounted on slides and counterstained with eosin.. When rAd is
delivered in saline (right panel (a)), the signal was predominantly detected in the central airways with very little β-gal expression seen in
the lung parenchyma. With virus delivered in saline followed by a PP2:PP9 chase (right panel (b)), there is an increase in the intensity of
staining in the corresponding regions of the airways with more evidence of peripheralization With virus delivered as PFC suspension (right
panel (c)), β-gal expression is seen in more global regions of the parenchyma. Based on the location and morphology, these cells appear to
be type II cells. Panel D displays additional examples of whole mounts of lungs from mice transduced with rAd-CMVLacZ (2.7 × 1010viral
particles) administered in saline alone, by saline followed by a chase of PP2:PP9 (10mL/kg), or as a rAd/PP2:PP9 suspension.
Table 1: Temporal pattern of thoracic luciferase expression.
VehicleViral load (viral particles)
Days after instillation
Animals were instilled with either 5 × 1010viral particles (n = 3/group) or 1 × 1010viral particles (n = 4/group) rAd-Luc intratracheally using either saline
or as a PP2:PP9 PFC suspension. Mice were anesthetized, and images were acquired at the time indicated using a cooled CCD camera and imaging system
(IVIS50, Xenogen Corp). Luminescence was quantified using Living Image software with a constant region of interest. Values represent the mean ± standard
as in the high viral titer group with one notable exception
(Figure 4(b), Mouse 3). When viewed and analyzed as a
group, the most dramatic differences were observed when
comparing the PFC liquid and saline groups at the high viral
titer (Table 1). Due to the variability in the temporal pattern
of the saline group with the low viral titer (Figure 4(a)), no
statistical differences were found between the PFC liquid and
saline groups at any of the time points (Table 1).
This study demonstrates that a specific combination of
PFC liquids (PP2:PP9), that we have previously shown
to significantly improve gas exchange, lung function, and
attenuate inflammation in animal models of lung injury
[13, 15, 35], also significantly improves the efficiency of rAd
gene delivery to the peripheral lung. The generation of PFC
6 Pulmonary Medicine
∗ ∗∗∗ ∗∗
Color scale: min = 1 ×106, max = 2 ×107
Day 2Day 4Day 7
Figure 3: Comparison of spatial distribution of rAd-saline and -PP2/PP9 suspensions. (a) Expression at 1d after instillation. Overlay of
photographs and luminescent images of three representative animals per group. Mice were instilled with 5 × 1010viral particles of rAd.luc
intraperitoneal delivery of luciferin (100μL; 150mg/kg). Luminescent images were acquired under identical settings (3min acquisition).
Images from day 1 are scaled linearly with the range of signal indicated. Asterisks denote animals whose images are shown at later time
points in panel (b). (b) Delivery with PP2/PP9 results in better retention in the lung. Overlay of photographs and luminescent images of
representative animals from the high-dose groups is denoted in panel (a). The mice were imaged on successive days after instillation as
indicated. Images are scaled logarithmically (note scale on the left).
Thoracic luminescence (photons ×106)
Mouse 5 saline
Mouse 6 saline
Mouse 7 saline
Mouse 8 saline
Time following instillation (day)
Thoracic luminescence (photons ×106)
Mouse 1 PP2/PP9
Mouse 2 PP2/PP9
Mouse 3 PP2/PP9
Mouse 4 PP2/PP9
Time following instillation (day)
Thoracic luminescence (photons ×106)
Mouse 4 saline
Mouse 5 saline
Mouse 6 saline
Time following instillation (day)
Thoracic luminescence (photons ×106)
Mouse 1 PP2/PP9
Mouse 2 PP2/PP9
Mouse 3 PP2/PP9
Time following instillation (day)
Figure 4: Comparison of luciferase activity in the lungs of mice. Mice were instilled with rAd-luc, and thoracic luciferase activity was
quantified noninvasively over the course of 7d. Two viral titers were administered 1 × 1010viral particles (Low) or 5 × 1010viral particles
(High) in either saline or PFC liquid suspension. (a) 1 × 1010viral particles delivered in saline; (b) 1 × 1010viral particles delivered in PFC
liquid; (c) 5 × 1010viral particles delivered in saline; (d) 5 × 1010viral particles delivered in PFC liquid. Each line represents the luciferase
activity of a single animal. The images were acquired 10min after intraperitoneal delivery of luciferin (150mg/kg). Luminescent images
were acquired under identical settings (3min acquisition), and activity was assessed using an identical area (region of interest) and analyzed
using the Living Image software package. The PFC liquid groups demonstrate a more consistent temporal pattern of expression compared
liquid/rAd suspensions specifically improved expression in
the alveolar epithelial cells.
Recombinant adenovirus can induce high levels of tran-
sient transgene expression. Several important improvements
have been made in this vector resulting in enhanced gene
delivery to the lung. One of earliest improvements was to
change from serotype 7 to serotype 5. Since serotype 5
binds the CAR receptor found in airways more efficiently,
the viral titer required for gene expression is attenuated,
and inflammation significantly reduced. Next, reagents such
as EGTA that disrupt tight junctions have been shown to
improve expression in the airways by exposing CAR on
the basolateral surfaces of airway epithelium. Various PFC
liquids have been utilized to improve expression in the
distal airways of both healthy and diseased lungs [3, 6–
10]. Our findings reflect at least two mechanisms thought
to be involved with improved expression. First, it has
been previously shown that PFC liquid facilitates transient
disruption of tight junctions, increasing exposure to CARs,
that are distributed throughout the A549 cell membrane
. Second, based on the physicochemical property of this
specific PFC liquid combination (with unique kinematic
viscosity, vapor pressure, and lipophilicity profiles) [14,
36], the PFC suspension fosters increased and uniformed
distribution of the virus relative to saline, such that more
cells are exposed to virus. As such, clinical use of PFC
liquids for the delivery of rAd could reduce the amount
of virus necessary for the treatment of lung disease. This
possibility is an important consideration given the inherent
inflammatory response associated with adenovirus. The
unique physicochemical properties of the PFC suspension
may support further improvement over viscoelastic gels
which have been recently shown to improve rAd and AAV
expression only in the central airways without increases
in alveolar expression . Our data document significant
versus saline at early time points both in vitro and in vivo. In
vitro we demonstrate a higher earlier transduction efficiency
and in vivo a more consistent pattern of temporal expression.
We have demonstrated the effect of different PFC liquids
on membrane fluidity and packaging using liposomes as
model systems . When the amount of luciferase activity
was assessed in the present study, significant differences in
when PFC liquid suspensions were compared to other
methods. We speculate that temporal differences between
methods of administration (favoring PFC suspension) may
actually be greater than the present analysis; however, we
believe that more detailed analyses may be confounded by
the high level of focal expression in upper airways with saline
instillation as compared to the more peripheral distribution
with PFC suspension. As such, this report demonstrates
that the major advantages of PFC liquid-suspension delivery
of rAd are increased early expression (Figure 4, Table 1),
better distal lung expression, and better retention in the lung
While this study was not designed to comprehensively
assess ectopic expression, our method of introduction by
minitracheal puncture is likely to oppose this possibility
since this method bypasses the nasal epithelium as well
as minimizes the potential for the virus to move up the
mucocililiary tract to transduce the gut. In addition, the
alveolar surfaces appeared intact thereby minimizing access
for ectopic expression through translocation across the
alveolar-capillary membrane. In addition, there was very
Previous studies using PFC liquid vehicles for rAd-gene
delivery utilized a pulse-chase method of administration,
that is, delivery of rAd in saline followed immediately by
instillation of PFC liquid [7, 9, 11, 38]. We reasoned that
the distribution of the vector compared to the pulse-chase
method. We have demonstrated previously that formation of
nanocrystal suspensions with PFC liquids improves delivery
of biological agents (recombinant proteins and antibiotics)
to the lung [33, 35, 39]. However this technology is
of interest. Since rAd is relatively labile and loses viability
when lyophilized, we limited our methods to creating liquid
suspensions. There have been reports of formulations that
stabilize rAd for freeze-dry methods, but our attempts to
create nanocrystals using this particular method proved
unsuccessful (data not shown). More stable gene delivery
vectors or compounds (plasmid DNA, rSV40, siRNA) would
be viable candidates for PFC liquid nanocrystal suspension
formation. The efficacy of siRNA strategies, delivered as
either oligonucleotides or in other gene vector systems,
provides the possibility of suppressing gene expression.
No method of gene delivery is ideal, and there are sig-
nif-i-cant problems associated with the use of rAd (e.g.,
inflammation; cell specificity limited to cells expressing
the appropriate receptor for viral entry), especially in the
clinical setting. Several strategies have been implemented
to try to minimize these problems, including generating
“gutted virus”  and the use of chimeric fiber [41, 42].
In spite of the potential difficulties of rAd, these vectors
still hold promise for delivering transient overexpression of
genes. As demonstrated in the present study, PFC liquid
promotes uniform delivery to the lung and may minimize
the inflammatory response to rAd gene products . This
would be particularly important in nonhomogeneous and
inflammatory types of lung injury processes where PFC
liquids could facilitate distribution of genes or other bio-
logic agents while facilitating gas exchange and improving
pulmonary function for management/treatment (i.e., cystic
fibrosis, COPD, or ARDS). In contrast, in disease states that
affect the central or proximal airways, delivery by saline or
the pulse-chase method might be advantageous.
The PFC liquid formulation used in the present studies
was selected based on the greatest improvement in gas
exchange and lung mechanics and attenuation of lung
inflammation in models of acute lung injury [13, 15, 35].
Within this context, the use of PFC suspensions to enhance
gene expression challenges the notion that minor inflamma-
tion is required for gene uptake. The major improvement in
gene expression in vivo appears to be in the delivery of the
virus to alveolar epithelial cells of the distal lung. Why this
increase is observed in these cell types remains unclear. The
most likely explanation is that the specific physicochemical
properties (e.g., kinematic viscosity, vapor pressure, and
lipophilic nature) of PFC liquid enhance delivery to and
assist cells in the transduction of the alveoli (i.e., similar
to how surfactant is recycled, alterations in membrane
dynamics). We suggest that delivery into this compartment
prevents clearance by the mucociliary tract.
In summary, PP2:PP9 PFC rAd suspensions increased
expression in distal portions of the lung. The suspensions
may facilitate receptor-mediated and independent entry of
the virus which significantly increases transduction effi-
ciency. This has important therapeutic implications for
diseases where expression in the central airways is not
sufficient and expression in the alveoli is required. The use of
PFC liquid holds promise as a vehicle to deliver gene delivery
vectors and other biologic agents in order to more directly
and specifically prevent and treat high-risk lung disease
processes in critically ill patients. Others have implicated
a “surfactant” mediated uptake of PFC into alveolar type
II cells, ostensibly associated with an endocytotic process
[31, 36, 43]. If these particles are then secreted out into
the lung surface, it would foster recycling of the virus to
transduce additional or adjacent alveolar epithelial cells.
If this theory holds true, the use of PFC suspensions for
gene vector delivery prevents a novel and forward-thinking
contribution to the field of gene therapy. Further study
of PFC-assisted gene vector delivery methods, including
nebulization of PFC liquid suspensions, is warranted to
potentially overcome many of obstacles currently associated
with gene delivery to the lung epithelium.
tance with the statistical analyses. This work was supported
in part by Grants from the National Institutes of Health
(HLBI 64158 and 1 P20 RR020173), the PA Department of
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