Endocytic Uptake, Transport and Macromolecular Interactions of Anionic PAMAM Dendrimers within Lung Tissue
Endocytic Uptake, Transport and Macromolecular Interactions of Anionic PAMAM Dendrimers within Lung Tissue
Christopher J. Morris 0 1 2
Ghaith Aljayyoussi 0 1 2
Omar Mansour 0 1 2
Peter Griffiths 0 1 2
Mark Gumbleton 0 1 2
Christopher J. Morris 0 1 2
0 Department of Pharmaceutical, Chemical and Environmental Science, University of Greenwich , Medway Campus, Kent ME4 4TB , UK
1 Cardiff School of Pharmacy & Pharmaceutical Sciences , Redwood Building, Cardiff CF10 3NB , UK
2 School of Pharmacy, University of East Anglia , Norwich Research Park NR4 7TJ , UK
Purpose Polyamidoamine (PAMAM) dendrimers are a promising class of nanocarrier with applications in both small and large molecule drug delivery. Here we report a comprehensive evaluation of the uptake and transport pathways that contribute to the lung disposition of dendrimers. Methods Anionic PAMAM dendrimers and control dextran probes were applied to an isolated perfused rat lung (IPRL) model and lung epithelial monolayers. Endocytosis pathways were examined in primary alveolar epithelial cultures by confocal microscopy. Molecular interactions of dendrimers with protein and lipid lung fluid components were studied using small angle neutron scattering (SANS). Results Dendrimers were absorbed across the intact lung via a passive, size-dependent transport pathway at rates slower than dextrans of similar molecular sizes. SANS investigations of concentration-dependent PAMAM transport in the IPRL confirmed no aggregation of PAMAMs with either albumin or dipalmitoylphosphatidylcholine lung lining fluid components. Distinct endocytic compartments were identified within primary alveolar epithelial cells and their functionality in the rapid uptake of fluorescent dendrimers and model macromolecular probes was confirmed by co-localisation studies. Conclusions PAMAM dendrimers display favourable lung biocompatibility but modest lung to blood absorption kinetics. These data support the investigation of dendrimer-based carriers for controlled-release drug delivery to the deep lung.
dendrimer; endocytosis; lung; polymer; scattering; transport; uptake
* Mark Gumbleton
BSA Bovine serum albumin
IPRL Isolated perfused rat lung
MRI Magnetic resonance imaging
Dendrimers represent an important uniform and nanosized
polymer architecture for biomedical and clinical applications
and display a variety of synthetic approaches, chemistries, and
an ability to carry cargo either by encapsulation,
complexation or as pendant groups on the polymer surface. Dendrimers
retain promise as nanosized drug carriers displaying a narrow
polydispersity and an ease of control in the modification of
surface functional groups. The first full family of dendrimer
molecules, spanning a number of growth generations were the
poly(amidoamine) (PAMAM) dendrimers synthesised by the
divergent approach with the iterative addition of methyl
acrylate and ethylenediamine to a polymer core and modified
with hydroxyl (neutral), amine (positive) or carboxyl (negative)
surface functionalities (Scheme 1).
The polyamidoamine (PAMAM) class of dendrimers have
been the most widely studied as carriers for low molecular
Scheme 1 (a) Structure of
exemplar anionic PAMAM
generation 1.5. Iterative branching
of each generation is highlighted by
concentric circles. (b) Molecular
mass (Mw) and the number of
surface functional groups for the
PAMAM generations tested here.
weight drugs, for MRI contrast agents, and as carriers of
biomacromolecules including vaccines, peptides, antibodies
and DNA. A number of groups have used cationic PAMAM
dendrimers to enhance drug delivery across cell membranes,
including avoidance of efflux transporters (1), or for example
enhancing cytoplasmic delivery of nucleic acid therapeutics (2)
or vaccines (3). However, cell membrane disruption and
compromise of tight junction integrity is a significant concern for
the exploitation of cationic dendrimers, although approaches
have been developed to mitigate the biocompatibility issues
associated with the use of these platforms (4).
The dendrimer-enhanced delivery of drugs with low oral
bioavailability has been of particular interest with many studies
(5) reporting dendrimer permeation of cultured intestinal
epithelial cell monolayers, although fewer investigations of
transport in the fully intact ex vivo or in vivo tissue have been
evidenced. Using an everted gut sac model Wiwatanapatapee
et al. (6) reported the effect of dendrimer generation and surface
functionality (anionic and cationic) upon the association of
PAMAM dendrimers with intestinal tissue and upon
dendrimer mucosal to serosal transport. In an in-vivo rat model
Florence and co-workers (7) administered by oral solution
gavage a poly(lysine)15 dendrimer bearing at the surface
covalently linked C12 alkyl chains and studied the subsequent
intestinal and whole body tissue accumulation.
In contrast to the oral route, the pulmonary administration
of dendrimers has received relatively little consideration and
may offer a number of distinct benefits for the localised airway
delivery of drugs that require controlled luminal release, but
also for the transmucosal or systemic delivery of dendrimer
bearing therapeutic cargo. Indeed many reports have
highlighted the favourable lung bioavailability of biologics in
the absence of absorption enhancers (reviewed in (8,9)).
Systematic investigations of lung permeability to nano-sized
polymers such as PAMAM dendrimers are limited, and
particularly those conducted in a model that retains a fully intact
In this study, we utilised an isolated perfused rat lung (IPRL)
model to quantify the rate and extent of absorption from the
deep lung of a series of anionic PAMAM dendrimers spanning
a molecular weight range 3 to 53 kDa. Following airway
administration of the dendrimers in the IPRL model, we
observed size-dependent pulmonary transport kinetics for
three generations (G1.5, G3.5 and G5.5) of anionic PAMAM
dendrimers. In the IPRL, the rate of PAMAM transport from
the lung lumen to the pulmonary circulation tended to be less
than the dextran probes of comparable molecular dimensions.
Further, with respect to G3.5 and G5.5 dendrimers we observed
dose-dependent absorption from the airways of the IPRL. Small
angle neutron scattering (SANS) studies indicated that this
dosedependency was not attributable to interaction of G3.5 or G5.5
with either of the two principal components of lung lining fluid,
i.e. albumin and the zwitterionic surfactant,
1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC). Using lung epithelial cell
culture models, we also examined the comparative in-vitro
permeability of the dendrimers as well as their endocytic uptake
and intracellular accumulation within distinct vesicular
compartments of lung epithelia. In summary, these data indicate
that PAMAM dendrimers offer a promising polymeric delivery
platform that could be used to deliver drug cargo either into, or
indeed, across the deep lung respiratory epithelium.
MATERIALS & METHODS
The following were obtained from Sigma-Aldrich (Poole,
UK):anionic PAMAM dendrimer generations (PAMAM) 1.5,
PAMAM 3.5 and PAMAM 5.5 (nominal MWs 3KDa,
13KDa, 53KDa, respectively), N-hydroxysulfosuccinimide
(sulfo-NHS), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC), sodium fluorescein (F-Na) and bovine
serum albumin (>98% purity) and FITC-dextans (FDx, where
x represents the nominal molecular weight in kDa). All tissue
culture plastics were from Corning Costar (Hemel Hempstead,
UK). Anti-caveolin-1 and anti-EEA-1 antibodies were from
BD Biosciences (UK). Oregon Green 488 (OG) cadaverine,
fluorescently labelled probes (10 kDa dextran, bovine serum
albumin and cholera toxin B subunit) were all from
Invitrogen (Paisley, UK). All other chemicals and reagents were
of the highest possible quality from Fisher Scientific
(Loughborough, UK) or Sigma (Poole, UK). Hydrogenous
and deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) were from Avanti Polar Lipids Inc. (USA). Sources of
other exceptional items are mentioned in the text.
Fluorescent Labelling of PAMAM Dendrimers
To allow quantification of pulmonary transport and cell
uptake, dendrimers were labelled with Oregon Green 488
cadaverine (OG) using carbodiimide-mediated coupling
chemistry and purified and characterised as previously described
(10). The extent of dendrimer-fluorophore labelling was
minimised to reduce interference with the surface properties.
Substitution ranged from 1:1.04 to 1:1.31 molar ratio
Primary Alveolar Epithelial Cell Cultures
Isolation and primary culture of type II rat alveolar epithelial
(ATII) cells to an alveolar epithelial type I cell (ATI-like)
phenotype was undertaken according to procedures previously
reported (11). All animal experiments and the protocols
described below adhered to the Animal (Scientific Procedures)
Act 1986 (United Kingdom).
Permeability of Polarised Calu-3 Monolayers
to PAMAM Dendrimers and FITC-Dextrans
Calu-3 monolayers were grown on Transwell® Clear inserts
(0.4 μm pore size) for 10–14 days until the transepithelial
electrical resistance (TEER) values were >500 Ω.cm2 as
measured using ENDohm chambers and an EVOM epithelial
voltohmeter (Word Precision instruments, Sarosota, USA).
Culture media was aspirated, apical and basal chambers were
gently washed with warm PBS and bathing fluid replaced with
DMEM–F12 (without phenol red). After equilibration
(30 min, 37°C) 0.2 mL of dose solution (8 μM or 40 μM
PAMAM-OG in serum-free media) was applied to the donor
chamber. Cumulative PAMAM-OG transport across the
monolayers (37°C, upon an orbital shaker) was measured at
pre-determined intervals by sampling 0.2 mL from the basal
chamber and replacement with an equal volume of warm
media. In addition, transport studies were performed using a
molecular weight range of FITC-dextrans and F-Na (40 μM
donor concentration) as reference solutes. The cumulative
transport of each probe was calculated and an apparent
permeability coefficient (Papp) (cm.s−1) determined according to:
where: δM/δt is the rate of probe accumulation in the basal
chamber (calculated from the initial linear portion of the
transport curve); A denotes the 0.332 cm2 surface area of
the Transwell® inserts and C0 denotes the initial
concentration of probe in the donor chamber.
Imaging of Endocytic Compartments in Primary Rat
Alveolar Epithelial and Calu-3 Cells
Coverslip mounted ATI-like cells were fixed with 3%
paraformaldehyde, quenched with 50 mM NH4Cl, then
permeabilised with 0.2% Triton X-100 in PBS.
Coverslips were incubated with primary antibodies against
Caveolin-1 (Cav-1), early-endosomal antigen-1 (EEA-1)
diluted in blocking buffer (dilutions: Cav-1 1:200; EEA-1 1:200 for
30 min). Lysosomes were labelled by pulse-chase labelling (2 h
pulse-3 h chase) with 0.25 mg/mL
tetramethylrhodaminedextran in serum-free medium.
PAMAM-OG uptake was studied in ATI-like cells seeded
onto glass bottomed 35 mm culture dishes (MatTek
Corporation, Ashland, USA). ATII primary cell were cultured
over 6–8 days to achieve an ATI-like phenotype. To assess
probe uptake, cells were washed with warm PBS (x3), before
addition of PAMAM-OG conjugate (50 nM in DMEM
without phenol red, containing 15 mM HEPES) for
predetermined times. Co-internalisation of PAMAM-OG
conjugates with 100 μg/mL tetramethylrhodamine-BSA (30 min)
or dextran in ATI-like cells was used to co-localise to albumin
positive endosomes and lysosomal compartments,
respectively. Following incubation at 37°C, cells were washed with
prewarmed PBS then immediately imaged by fluorescent
imaging on a Leica DMIRB fluorescent microscope equipped with
a 40x objective and attached to a Digital CCD Retiga 1300
camera, processed using Improvision software. Images were
cropped and annotated for publication using Photoshop CS2
(Adobe Systems Inc.).
Pulmonary Translocation of Solutes in an Ex-Vivo IPRL
An IPRL preparation using a pMDI intra-tracheal solution
instillation technique was used as previously described (12). A
critical appraisal of the IPRL model as well as a schematic
representation has previously been published (13).
PAMAMOG conjugates were administered to the IPRL in 0.1 mL
20 mM PBS, pH 7.4. Serial aliquots were sampled from the
recirculating perfusate at pre-determined times and
centrifuged (13,000 g, 10 min, 4°C) to remove trace blood cells
and the supernatant stored at −80°C prior to quantification
by microplate fluorescence spectroscopy (λex 485 nm λem
520 nm; FLUOstar, BMG, Aylesbury, UK). A calibration
curve for each PAMAM-OG conjugate was prepared in
control perfusate that had recycled through the lung for 60 min.
Cumulative transfer of dendrimer into the perfusate was
calculated by multiplying the cumulative concentration by the
total perfusate volume (~75 mL). The wet:dry ratio of lung
tissue was calculated by comparison of the tissue weight
immediately after the IPRL experiment (wet weight) and the
tissue weight after lyophilisation for 72 h (dry weight).
Control experiments involved instillation of PBS dose solution
followed by perfusion and ventilation for 60 min.
Lipid Vesicle Preparation
Small unilamellar vesicles comprising h-DPPC or d-DPPC
were prepared using the freeze/thaw extrusion method.
DPPC was dissolved in chloroform (10 mg /mL) in a round
bottom flask then solvent was removed by rotary evaporation
to yield a phospholipid film. Residual solvent was removed
under high vacuum for 1 h. The film was rehydrated in
2 mL deuterated phosphate-buffered saline pH 6.5
Aldrich, Poole, UK) then incubated at 50°C for 1 h. The
hydrated film was then subjected to five freeze/thaw cycles
which involved freezing for 5 min in liquid nitrogen followed
by thawing for 5 min in water at 50°C and then vortex mixing
for 5 min. Finally, the lipid mixture was extruded under high
pressure, firstly through a 200 nm polycarbonate membrane
(10 times) and secondly through a 100 nm polycarbonate
m e m b r a n e ( 2 0 t i m e s ) u s i n g T h e E x t r u d e r ( L i p e x
Biomembranes Inc., Vancouver, Canada) maintained at
SANS Studies on PAMAM Dendrimer in Lung Fluid
SANS experiments were performed on the D11
diffractometer based at the steady-state reactor source at the Institut
Laue-Langevin (ILL) in Grenoble, France. A Q range of
0.01 Å−1 to 0.5 Å−1 was selected by choosing several
instrument settings (sample-to-detector distances, collimation) with
a constant neutron wavelength (λ) of 8 Å, where:
Samples were contained in 1 mm pathlength,
UVspectrophotometer grade, quartz cuvettes (Hellma) and
thermostatically controlled at 25 ± 0.5°C can be achieved.
Experimental measuring times were dependent on the
sampledetector distance, but approximately 40–60 min overall.
All scattering data were normalised for the sample
transmission and the incident wavelength distribution, corrected
for instrumental and sample backgrounds using a quartz cell
filled with D2O, and corrected for the linearity and efficiency
of the detector response using the instrument specific software
package. The data were put onto an absolute scale using a
well-characterized partially deuterated polystyrene blend
Previous SANS studies on PAMAMs have shown that they
form spherical structures (14). Here, the data were fitted to a
model that describes the scatterer as a solid, spherical object. In
this representation, the scattered intensity, I(Q), is expressed as:
Where scale is a volume fraction, V is the volume of the
scatterer, r is the radius of the sphere, Δρ is the difference
between the scattering length density of the scatterer and the
solvent, and Binc is the incoherent background scattering. The
data were mathematically analysed using the open source
software, SasView (15).
A number of mathematical models have been explored in
this work, the one that warrants discussion here being the
unilamellar vesicle one applicable to the scattering from
DPPC based systems. In this formulism, the shape of
the vesicle is modelled as a series of concentric spheres
of varying radii (outermost corona – hydrated
phospholipid headgroups; middle corona – hydrocarbon region,
with thickness typically twice the length of the
hydrocarbon tail; innermost corona - hydrated phospholipid
where ∅ is the volume fraction of shell material, Vshell is the
volume of the shell, Vcore is the volume of the core, Vtot is the
total volume, Rcore is the radius of the core, Rtot is the outer
radius of the shell, ρsolvent is the SLD of the solvent (similar to
the SLD of the core in this model), ρshell is the SLD of the shell
and j1 is the spherical Bessel function. In this study, scattering
from the phospholipids has been modulated by employing
tail-deuterated DPPC, which shows a significantly weakened
scattering as the hydrocarbon corona is now largely invisible
against a D2O background (16).
Statistical / Data Analysis
Non-linear regression analysis of the initial transport kinetics
of PAMAM-OG across the IPL (0–60 min) was performed
using GraphPad Prism (v5.01) according to Eq. 5 with the
recycling perfusate represented as a single accumulating
compartment receiving first-order initial input (Kini).
M ¼ D: F 1−ekini:t
Where M is the cumulative mass of probe transported
across the lung into the perfusate (0–60 min); D is the nominal
dose of PAMAM conjugate, F is the fraction of the dose
deposited in the lung, correcting for losses in the dosing
apparatus, Kini is the rate constant (min−1).
For treatment comparisons results are reported ± S.D.
One-way analysis of variance (ANOVA) was used to compare
cumulative absorption data. Post-hoc analysis was performed
using Newman-Keuls Multiple comparison. Statistical
significance was determined at p < 0.05.
In-Vitro Transport of PAMAM and Dextran Probes
We firstly examined the transepithelial transport of anionic
PAMAM dendrimers across the Calu-3 bronchial cell line,
and compared the transport to that of a range of
fluoresceinlabelled dextrans (FD) whose molecular size spanned that of the
dendrimers. Scheme 1 includes the generic molecular structure
of low generation number anionic PAMAM dendrimers, as
well as the molecular masses and the number of surface
functional groups of PAMAMs examined herein. Calu-3 cell
monolayers were cultured until they reached a TEER of >500
Ω.cm2, typically around day 10–15 post-seeding. The apparent
permeability coefficients (Papp) determined from the initial
linear transport rates are shown in Table I. The Papp for both
PAMAM3.5 and PAMAM5.5 were dose-independent with,
for example, the Papp for PAMAM3.5 at 40 μM and 8 μM
being respectively, 0.656 vs 0.593 (x 10−6) cm.s−1. Therefore,
over this concentration range the transport of both
PAMAM3.5 and PAMAM5.5 was predominantly one of
passive diffusion. Although the Papp data for the larger molecular
sized PAMAM5.5 was consistently lower than that for the
PAMAM3.5 this was not statistical significant (p > 0.05) despite
the difference in molecular weight and molecular diameters
reported elsewhere (17). The comparative permeability profile
of Calu-3 monolayers to F-Na and a range of FDs, whose
Stokes diameters spanned 1.5 to 12 nm (Table I), was also
studied. The rate of F-Na and FD transport from a 40 μM dose
solution showed a clear inverse relationship to molecular size,
with the smallest probe, F-Na permeating most rapidly, Papp
averaging 0.737 (x 10−6) cm.s−1, whereas FD-70, the largest
solute, permeating some 30 times more slowly with an average
Papp of 0.023 (x 10−6) cm.s−1 (Table I). Figure 1a shows such a
relationship in a plot of Papp against solute molecular diameter
for the FD panel (filled squares). The plot also overlays the
permeability data for PAMAM3.5 and PAMAM5.5 (empty
circles) whose permeabilities are clearly greater than for the
dextrans of a molecular size.
We also undertook comparative transport studies in the
Caco-2 model (Table I). As a reference the Papp to F-Na was
significantly (p < 0.05) lower for the Caco-2 monolayers
compared to the Calu-3 cells not withstanding TEER values
which were comparable ca. 400–500 Ω.cm2. Similarly the
permeability to PAMAM3.5 was significantly (p < 0.05) lower
in the Caco-2 cells, with the transport of PAMAM3.5 some
56% (p < 0.05) of the F-Na permeability in Caco-2, whereas in
Calu-3, the transport of PAMAM3.5 was 89% of that of the
FNa probe (p > 0.05).
Table I Permeability of Calu-3 and
Caco-2 Monolayers. Data Shown
are Expressed as Mean ± SD
(n = 4–6). For the 40 μM PAMAM
Concentration Data the Symbol *1
Indicates Statistical Difference
(p < 0.05) to the Permeation of All
Dextrans (FD10-FD70), While *2
Indicates Statistical Difference
(p < 0.05) to only FD 40 and
FD70. The Symbol *3 Indicates a
Significant Difference (P < 0.05) in
Permeability Between Caco-2 and
Calu-3 Monolayers for the
Probe and cell line
Stokes diameter (nm)
Apparent permeability coefficient
(Papp) (x 10−6 cm.s−1)
0.656 ± 0.102*1
0.593 ± 0.125
0.445 ± 0.154 *2
0.447 ± 0.098
0.737 ± 0.247 *1
0.303 ± 0.087
0.284 ± 0.089
0.047 ± 0.016
0.023 ± 0.011
0.345 ± 0.039 *3
0.194 ± 0.026 *3
We next examined if the anionic PAMAM dendrimers
themselves could alter paracellular permeability by
coapplication to the Calu-3 monolayers of unlabelled
dendrimers (each at 40 μM) together with F-Na as the
paracellular probe. Co-application of PAMAM1.5 caused
no significant change (p > 0.05) in the permeability to F-Na
(Fig. 1b), while co-application of PAMAM3.5 displayed a
trend to reduce F-Na transport although not to any significant
(P > 0.05) difference. In contrast, 40 μM PAMAM5.5 caused
a 35% reduction (p < 0.05) in the Papp of F-Na.
Endocytic Uptake and Trafficking of PAMAMs in Lung
Figure 2a-b demonstrate a degree of separation between the
caveolar membrane system and both the early endosomal
compartment labelled with EEA-1 (Fig. 2a, green) and the
pulse-chase dextran labelled lysosomal compartment
(Fig. 2b, green). As expected, Cav-1 puncta were widely
distributed across the ATI-like cell (18). EEA-1 immunostain
(Fig. 2a) was most often identified in regions of concentrated
puncta in the peri-nuclear region, wherein a small degree of
co-localisation was observed (arrows in zoomed insert,
Fig. 2a). Similarly, in Fig. 2b it is apparent that the majority
of dextran-positive lysosomes (green) did not co-localise with
Cav-1 staining (red). To examine the functionality of these
endocytic compartments we exposed ATI-like cells to two
macromolecular probes – cholera toxin B subunit and
albumin – both of which are used as probes of caveolae-dependent
endocytosis (19,20). ATI-like cell cultures incubated with
CtxB for 10 min (Fig. 2c) displayed a heterogeneous staining
pattern. In some cells, a 10 min internalisation period
produced widespread green staining (left-hand cell, Fig. 2c) and
significant overlap between CtxB puncta and EEA-1 positive
early endosomes (arrowheads, Fig. 2c). In contrast, other cells
contained a more restricted staining intensity (e.g. right hand
cell marked with an asterix in Fig. 2c). This observation is
consistent with the heterogeneous expression of the GM1
sphingolipid receptor reported within other cell types (21).
The speckled CtxB staining pattern persisted after longer
incubation periods (arrows, Fig. 2d), however this was
accompanied by a polarised accumulation of CtxB signal in the
juxtanuclear region (Fig. 2d). Notably, some CtxB vesicles occupied
peripheral locations within the cell (empty arrowheads,
Fig. 2d). Co-localisation of the peri-nuclear CtxB staining to
the trans-Golgi network (TGN) using a polyclonal anti-primate
TGN-46 antibody was impossible due to poor cross-reactivity
to rat antigens. However, CtxB displayed extensive overlap
with the TGN-46 labelled compartment in A549 human
alveolar epithelial cells after 60 min internalisation (Fig. S1).
The post-Golgi trafficking of CtxB was examined following an
extended 2 h/3 h pulse-chase co-internalisation with dextran
(Fig. 2e). There was limited evidence of CtxB trafficking to
dextran-positive lysosomes in the peri-nuclear region
(arrowheads, Fig. 2e). Instead, we observed a reticular CtxB
staining pattern, which indicates the retrograde trafficking of
CtxB to the endoplasmic reticulum via the TGN (22).
After 10 min albumin (BSA) internalisation into ATI-like
cells extensive localisation of fluorescent albumin puncta
(green, Fig. 2f) to an EEA-1 positive compartment was
observed. In contrast to the CtxB staining pattern, albumin
staining was less extensive across the cell volume and more
punctate in nature.
A 30 min internalisation of OG-labelled PAMAMs into
ATI-like cells resulted in a decreasing intensity of scattered
cytoplasmic puncta as PAMAM molecular size increased
(Fig. 2g-i). This was not a result of OG-labelling efficiency
between the different PAMAM species as the same labelling
stoichiometry was applied and confirmed in each case. Uptake
of the PAMAM dendrimers into cytoplasmic vesicles was
confirmed by co-localisation with endocytic marker
probes, fluorescent 10 kDa dextran (red, Fig. 2j) and
bovine serum albumin (red, Figs. 2k,l). These probes
were specifically chosen for their comparable molecular
Fig. 1 In-vitro transport of dendrimers and dextran probes in lung epithelial
cells (1a) Permeability of in-vitro Calu-3 cell monolayers showing a plot of Papp
(x 10−6 cm/s) as a function of Stokes diameter, and demonstrating molecular
size-dependent transport of dextrans (FD), with an overlay of F-Na
permeability. The permeability of the dendrimers, PAMAM3.5 and PAMAM5.5,
being considerably greater than expected based on molecular size alone;
(1b) Relative permeability of the paracellular probe F-Na and the impact of
co-incubation with unlabelled PAMAM1.5, PAMAM3.5 and PAMAM5.5. All
data n = 4–6 ± standard deviation. * denotes p < 0.05 compared to control
by one-way ANOVA and Newman-Keuls multiple comparison test.
dimensions to PAMAMs 1.5, 3.5 and 5.5 and well understood
endocytic pathways. A 2 h/3 h pulse-chase co-internalisation
of PAMAM 1.5 and 10 kDa TMR-dextran (Fig. 2j) showed a
fraction of internalised dendrimer co-localised with the
dextran within peri-nuclear lysosomal compartments. A
proportion of the PAMAM 1.5 puncta remained green,
not co-localised and indicating accumulation in upstream
endocytic compartments. Co-internalisation of PAMAM
3.5 and PAMAM 5.5 with BSA (Fig. 2k and l,
respectively) resulted in both noticeable accumulation
of PAMAM puncta in the perinuclear region as well
as co-localisation with BSA positive puncta scattered
through the cell.
Transmucosal Transport of PAMAMs in the Intact Lung
The cumulative absorptive kinetics of PAMAM 1.5,
PAMAM 3.5 and PAMAM 5.5 were determined
following pMDI-mediated solution instillation into the distal
airways of the IPRL model. Figure 3a shows, as an
example, the IPRL absorptive transport profiles for
PAMAM 3.5 following either low dose (20 nmol; empty
symbols) or high dose (130 nmol; filled symbols)
administration; the Figure expresses the absorption data as a
% of lung deposited dose, a parameterisation which
clearly emphasises the different kinetic outcomes.
Specifically, both the transport rate constant (Kini) and
the 60 min extent (%) of PAMAM3.5 dose absorbed
were for the low dose more than double that recorded
for the high dose (p < 0.05, Fig. 3a and Table II). Such
dose-dependent kinetics (low dose showing a faster and
greater extent of absorption compared to high dose) was
also evid ent for th e PAMAM5.5 admin is tration
(Table II). At near equivalent mole doses we observed
what appeared to be a distinct molecular size division in
the absorption of the dendrimer species with the extent
and rate constant of absorption for PAMAM1.5 (3.3 nm
diameter) markedly greater (P < 0.05) than both the
PAMAM3.5 (5.2 nm) and PAMAM5.5 (7.9 nm) species.
Furthermore we found the kinetics of absorption for the
latter two dendrimers species to essentially be
indistinguishable (P > 0.05) following either the low dose
comparison or the high dose comparison (Table II).
To provide a comparative permeability profile within
the IPRL model we also instilled a range of
fluorescently labelled dextrans (FD) whose molecular weights
(3.8 kDa to 75.1 kDa) and diameters (2.8 nm to
12 nm) spanned that of the PAMAM dendrimers. For
the dextran FD20 (20.2 kDa, 6.6 nm) we tested if
dosedependent absorption kinetics were evident across 10 to
100 nmole dose instillations. In contrast to PAMAM3.5
and 5.5 we found no evidence of a dose-dependent
absorption (P > 0.05; Table II). We also found the
same lack of dose-dependency for the small paracellular
probe, sodium fluorescein (F-Na) (Table II). Table II
also shows how the IPRL absorptive parameters for the FDs
varied as a function of size; incremental decreases in the rate
and extent of transport of the FDs within the IPRL evident
with increasing molecular weight and size. For example,
FD40 was transported x5-fold more extensively and with a
rate constant some x8-fold greater than that of FD70
(Table II). Our data are exemplified for the FDs (and F-Na)
in Fig. 3b, which also has an overlay of the equivalent data for
the PAMAM dendrimers. It is clear that PAMAM dendrimers
were somewhat more restricted in their transport than an
equivalent sized FD, which may reflect contrasting molecular
shape between the dendrimers and the dextran polymers.
Fig. 2 Distribution and
functionality of endocytic
compartments in ATI-like lung
epithelia. Co-localisation of
Caveolin-1 positive vesicles with
(2a) EEA-1 immunopositive
endosomes and (2b) lysosomes
labelled by a 2 h/3 h pulse-chase
internalization of dextran. CtxB
internalized into ATI-like cells
localizes to (2c) early endosomes
after 10 min, (2d) a peri-nuclear
compartment after 45 min. (2e)
only a minor fraction of CtxB puncta
localized to lysosomes following a
2 h/3 h pulse-chase
cointernalisation of CtxB and 10 kDa
dextran. Empty arrowheads in 2D
highlight non-perinuclear puncta
present within upstream
compartments. (2f) TMR-labelled
BSA accumulated in EEA-1 positive
endosomes after a 10 min
incubation. (2g–I) Primary ATI-like
cells show a decrease in the density
of PAMAM-OG staining density
pattern that accompanies an
increase in dendrimer generation
from 1.5 to 5.5 after 30 min
incubation. (j) a fraction of PAMAM
1.5 co-localised with dextran
labelled lysosomes after a 2 h/3 h
pulse-chase co-internalisation. Both
PAMAM 3.5 (k) and PAMAM 5.5 (l)
co-localise with BSA after 30 mins
co-internalisation. Arrows in
zoomed section highlight
colocalised puncta. Scale bar: 5 μm.
Here as part of the IPRL studies we sought to examine
dendrimer biocompatibility, albeit at a gross-level. We found
no acute evidence of the appearance of oedema, a qualitative
observation supported by monitoring lung wet:dry weight
ratios. We found no difference (P > 0.05) in lung wet:dry weight
ratios between control lungs (IPRL run for 60 min following
instillation of an equivalent dosing volume of PBS) at
4.91 ± 0.30, and OG-labelled PAMAM1.5 (4.67 ± 0.02),
PAMAM3.5 (4.78 ± 1.02) and PAMAM5.5 (4.34 ± 0.25)
dosed lungs. Monitoring paracellular permeability of the
IPRL with F-Na we observed that co-instillation of
PAMAM3.5 and F-Na caused no significant change
(p > 0.05) in the permeability to F-Na (Kini, min−1 control
( n = 1 4 ) 0 . 0 3 9 ± 0 . 0 0 9 v s P A M A M 3 . 5 ( n = 4 )
0.043 ± 0.016). H o w e v e r , F - N a c o - i n s t i l l e d w i t h
PAMAM5.5 led to a decrease (P < 0.05) in the permeability
to F-Na (Kini min−1 control (n = 14) 0.039 ± 0.009 vs PAMAM
5.5 (n = 4) 0.018 ± 0.002).
SANS Investigations of PAMAM-Macromolecule
We next employed small angle neutron scattering (SANS) to
investigate the molecular structure of PAMAMs in binary and
tertiary models of lung lining fluid. To establish baseline data
we modelled the SANS from BSA and PAMAM3.5 and 5.5 in
deuterated water (Fig. 4a). The models determined the
molecular radii (RG) for BSA, PAMAM 3.5 and PAMAM 5.5 of
28, 20 and 30 Å, respectively. These PAMAM dimensions are
consistent with hydrodynamic radii data determined by others
using diffusion NMR (17). The use of deuterated DPPC
(dDPPC) in D2O reduces the normally high scattering intensity
of hydrogen-containing lipids (h-DPPC) in D2O. This
Bcontrast^ approach was used to determine that the DPPC
SUVs displayed radii of 580 Å with a bilayer thickness of 30–
40 Å (Fig. 4b, Table III). When we made binary mixtures of
DPPC with BSA (Fig. 4c) or with PAMAM 3.5 or 5.5 (Fig. 4d)
Fig. 4 SANS as a function of (a) PAMAMs/BSA, (b) h and d-DPPC, (c) mixtures of h-DPPC/BSA and d-DPPC/BSA, (d) mixtures of h-DPPC/PAMAM G3.5 and
h-DPPC/PAMAM G5.5, (e) mixtures of 10 mM d-DPPC/4 mg.ml-1 BSA/1.3 mM PAMAM G3.5 and 10 mM d-DPPC/4 mg.ml-1 BSA/1.3 mM PAMAM G5.5.
Solid lines corresponds to the model fits as discussed in the text. Data has been offset for clarity.
Table III Structural Key Parameters of 10 mM h-DPPC / d-DPPC Obtained
from the SANS Model Fitting as a Function of PAMAMs or BSA Concentration at
25°C in D2O. ND: Not Determinable Due to Low Contrast
h-DPPC +1.3 mM PAMAM G3.5
h-DPPC +1.3 mM PAMAM G5.5
h-DPPC +2 mg.ml−1 BSA
d-DPPC +4 mg.ml−1 BSA
d-DPPC +1.3 mM PAMAM G3.5
and 4 mg.ml−1 BSA
d-DPPC +1.3 mM PAMAM G5.5
and 4 mg.ml−1 BSA
(± 10, Å)
thickness (± 5, Å)
and PAMAM5.5 permeate these monolayers at rates higher
than control solutes (FITC-dextrans) of comparable molecular
dimensions. Unexpectedly, the permeability to F-Na
decreased when co-applied with PAMAM 5.5. That the
monolayer TEER remained stable throughout the studies leads us
to interpret this apparent reduced F-Na permeability to reflect
a degree of sequestration of F-Na by the dendrimer, and
hence a reduced F-Na concentration available for free
transport. Nevertheless, together the data supported the conclusion
that tight junctional modulation is not a key factor in anionic
PAMAM transport across Calu-3.
The interaction between PAMAM dendrimers and
epithelial tight junctional proteins has been investigated by a
number of groups, principally using the Caco-2 cell model. Avaritt
and Swann reported the anionic PAMAM3.5 to lead to a
concentration-dependent increase in immunofluorescence
staining intensity for tight junction-associated proteins,
although this was not associated with an increased protein
expression or altered functional paracellular permeability. In
contrast, the cationic PAMAM G4 resulted in significant
increases in paracellular permeability through a mechanism(s)
mediated via intracellular Ca2+ release. Two previous studies
have reported PAMAM3.5 transport across Caco-2 cells, both
studies applying very high dendrimer concentrations
(1000 μM) and with markedly different permeability
outcomes, i.e. Jevprasanephant et al. (23) reporting a Papp of
0.02 (x10−6) cm.s−1 while Kitchens et al. (24) reported a
300fold higher Papp of 6 (x10−6) cm.s−1. Nevertheless, this
reinforces the disconnection that can be apparent between
different in-vitro cellular barrier modelss, the TEER measurements
and the permeability of paracellular probes and the transport
of experimental agents such the dendrimers; a disconnection
that supports studies in a more physiologic lung model that
reflects the epithelial barriers of the deep lung.
A number of published reports have concluded that
endocytic uptake and / or transcytosis across epithelial
barriers could play a significant role in the net transmucosal
dendrimer transport (6,25). We next gathered spatiotemporal
information on the expression and functionality of
macromolecular uptake pathways to examine their role in dendrimer
uptake by lung alveolar cells. A combination of fixed cell
immunolabelling and live cell imaging of primary rat
ATIlike cells confirmed limited co-localisation of intracellular
populations of Cav-1 positive, EEA-1 positive and dextran-loaded
lysosomes. Spatial segregation is a key feature of polarised
epithelial cells such as lung alveolar epithelia because it affords
proper control of many key processes including, but not
limited to, cell adhesion, migration and nutrient uptake (26).
Each of these distinct endocytic compartments was shown to
fulfil a functional role in the uptake of probes with molecular
dimensions comparable to the size range of PAMAM
dendrimers (3.3–7.9 nm) that were studied here i.e. 10 kDa
dextran (4.6 nm), CtxB (6.5 nm (27)) and albumin (6.8 nm
(28)). Co-localisation of internalised dendrimer with
immunolabelled compartments was not performed due to
fixation-associated artefacts that can misrepresent polymer
CtxB binds to GM1 ganglioside within ordered
microdomains termed lipid rafts and the toxin B subunit specifically
serves to deliver the catalytic A subunit of the holotoxin to the
endoplasmic reticulum and subsequently to the cytosol (29).
Demonstrable uptake and trafficking of CtxB within primary
rat alveolar epithelia indicates Cav-1 functionality in the
pulmonary epithelial barrier. To complement this, BSA was used
as a probe for non-specific fluid-phase endocytosis as it results
in considerably less background fluorescence on the glass
coverslips after short incubation periods compared to, for
example dextrans. This can be particularly challenging when
imaging ATI-like cells due to the highly attenuated cell periphery
(30). The internalisation of albumin has been linked to the
gp60 (albondin) receptor-mediated uptake of proteins in lung
alveolar epithelial cells (31) although this has not been widely
Lung ATI cells represent 95% of the lung absorptive
surface area and express caveolin-1 (Cav-1) as a phenotypic
biochemical marker. Cav-1 (32,33) and macromolecular
trafficking via caveolae has been targeted for enhanced delivery of
nanoparticles and macromolecules (34). A detailed
investigation of the trafficking mechanisms of PAMAMs in lung
epithelia was beyond the scope of this study. Nonetheless, these
data provide a novel insight into the endocytic capacity of ATI
cells and lead us to conclude that the endocytic machinery of
alveolar epithelia, including Cav-1 positive vesicles (35,36),
fulfil a role in the uptake and intracellular trafficking of
macromolecules such as PAMAM dendrimers at the air-blood
barrier, as described previously (37) for other cellular barriers.
The contribution of endocytic/transcytotic mechanisms to the
bulk transport of polymers across epithelial barriers is
extremely challenging to deconvolute. The use of chemical
and/or genetic knockdown techniques inevitably leads to the
modulation of the paracellular transport route, which is most
likely the predominant transport pathway for unmodified
polymeric carriers displaying sub-10 nm dimensions.
W e n e x t e m p l o y e d t h e I P R L m o d e l t o s t u d y
transpulmonary PAMAM transport. This model gives insight
into the complex parallel dispositional barriers to absorption
from the airways within an intact lung architecture without
the confounding whole body distribution and elimination
processes such as mucociliary clearance (MCC). The absorption
kinetics of PAMAMs and control FD probes displayed a
reciprocal dependency between transport and solute size,
consistent with a restricted diffusional process. More importantly,
anionic PAMAM dendrimers displayed modest lung transport
kinetics cf. similarly sized FD solutes. We have previously
demonstrated (38,39), using a range of small molecule probes,
that the IPRL model discriminates between rapidly absorbed
lipophilic compounds such as digoxin (absorption t1/2, 6 min)
from polar, paracellular probes such as mannitol (absorption
t1/2, 63 min). The range of absorption half-lives recorded for
PAMAMs here spanned 100–600 min, illustrating the
prolonged transpulmonary absorption cf. smaller solutes. A
rate of polymer clearance from the lung airways that is
inversely proportional to molecular weight is, of course, not
unique, with comparatively recent studies reporting similar
profiles with polylysine dendrimers (40), polyethylene glycol
polymers (0.5 to 20 kDa) (41) and hyaluronic acid polymers (7
to 741 kDa) (42). In the case of dendrimer administration to
the lung Ryan et al. (40) recently reported a relative lung
bioavailability of 20–30% for pegylated polylysine dendrimers in
the range of 11-22 kDa (3.0–5.7 nm) after in vivo intratracheal
instillation. da Rocha and colleagues (43,44) administered to
mice G3 PAMAM and its pegylated derivatives by pharyngeal
aspiration and reported absorption kinetics that were
significantly faster than those reported here i.e. absorption half-lives
in the range of 120 min. Direct comparison of our data with
others is precluded by contrasting approaches such as the use
of different PAMAM types, deposition in the central lung
in vivo cf. peripheral lung ex vivo, as well as dispositional
disparities, including the presence of mucociliary clearance
in vivo, which permits a contribution to bioavailability by
gastrointestinal absorption. It should be highlighted that the
tracheobronchial circulation is not perfused in the IPRL model
and the forced solution instillation delivers >90% of the
instilled dose into the peripheral lung regions, with the
remaining fraction largely confined to the dosing apparatus
(10). In combination, these two key features of our IPRL
model allow us to interpret the appearance of solutes in the
recirculating pulmonary perfusate as a direct consequence of
absorption across the deep lung epithelium, with negligible
impact of MCC. In contrast to other earlier works in other
mucosal models (45) our data indicate that deep lung delivery
of anionic PAMAMs is unlikely to enhance drug absorption
kinetics. Hubbard et al. (46) concluded that PAMAMs exhibit
only modest transepithelial transport across intact human
intestinal epithelial sheets and that, similar to our results,
transport rate is overestimated by in vitro (Caco-2) monolayers. We
would therefore advise that caution should be exercised when
interpreting only in vitro permeability profiles of dendrimeric
carriers for lung delivery. Our observation does not exclude
the opportunity to exploit PAMAMs for controlled release of
low molecular weight drugs into or across the lung epithelia as
previously demonstrated with PEG polymers (47).
The biocompatibility of inhalation delivery platforms is a
critical consideration in the future development of
nanomedicines. The decreased permeability observed in both
Calu-3 and IPRL studies indicates that PAMAM 5.5 reduces
the concentration of diffusible F-Na at the epithelial surface.
Bonizzoni et al. (48) recently showed that cationic PAMAM
dendrimers reversibly interact with, and induce aggregation
of, the structurally related fluorescein dye,
5,6-carboxyfluorescein leading to dye sequestration. Anionic dendrimers would
not be expected to drive such an exact binding event. More
likely could be the interaction between fluorescein and the
secondary and tertiary amine groups within the dendrimer core.
The capacity for lung-instilled solutes to cause epithelial
barrier disruption was reported previously. Intra-tracheal
instillation of cationic PAMAM G3 has been associated with
acute lung injury (49). Similarly, we recently showed that
instillation of select cationic, amphipathic peptides into the
IPRL causes dose-dependent increases in the permeability to
mannitol (12). Our data complement a number of in vitro
cytotoxicity studies (50,51) which conclude that anionic
PAMAMs offer greatly reduced potential to cause cellular
damage compared to cationic dendrimers.
We reported above that at higher doses both the
PAMAM3.5 and 5.5 species showed a reduced transport rate
constant (Kini) and extent (%) of absorption within the IPRL.
Intriguingly, transport of the similarly sized FD20 polymer
was identical across a 10-fold dose range, indicating that
dose-dependent phenomenon for PAMAM3.5 and 5.5 is not
a generalised issue within the IPRL model but rather a feature
of the PAMAM polymer species. A technical point is that size
exclusion chromatography performed as previously described
(10) upon all labelled PAMAM and FD dosing solutions and
upon the respective 60 min IPRL perfusate samples (data not
shown), confirmed the integrity of all fluorescent conjugates
and as such the validity of the kinetic transport interpretation.
We wondered if dose-dependent PAMAM transport may be
due to PAMAM aggregation within the lung lining fluids.
Using SANS analysis of PAMAMs in a relatively simple model
of the lung lining fluid we observed that PAMAM generations
3.5 and 5.5 do not aggregate or adsorb to DPPC vesicles. The
20 Å increase in the bilayer thickness of d-DPPC vesicles in the
presence of 4 mg/ml BSA or PAMAM G5.5 + 4 mg/ml BSA
is not interpretable as a binding or membrane insertion. Using
the simplest conceptual model of albumin or PAMAM
binding with DPPC vesicles, we predict that the average
DPPC vesicle radii would increase by at least one BSA/
PAMAM molecular diameter (40–60 Å). The pulmonary
(alveolar) epithelial surface is covered with surfactant
composed predominantly of lipid (90%) and with the remainder
protein. Dipalmitoylphosphatidylcholine (DPPC) is the major
lipid component, comprising 30–60% of the total lipid
surfactant content (52). Interactions between PAMAM dendrimers
and model lipid membranes have been previously investigated
using a range of biophysical methods to examine membrane
interaction and cytotoxicity (53–55). The majority of
published studies on dendrimer-membrane interactions have
focussed upon cationic PAMAM dendrimers. However, of
particular relevance to our data Lombardo et al. (56) recently
observed DPPC alkyl chain disruption by PAMAM 2.5, which
was mirrored by increases in vesicle zeta potential and
modulated SAXS spectra. The technique of co-dissolution of
PAMAMs with DPPC prior to liposome extrusion, as used
by Lombardo et al., is likely to increase the probability of
intermolecular interactions between the components and the
entrapment of dendrimer within the interfacial lipid region.
This is highly unlikely in our system when PAMAM were
mixed with colloidally stable DPPC vesicles. Here we have
excluded PAMAM aggregation with DPPC or albumin fluid
components. The possibility remains that PAMAM
aggregation or sequestration with other lung luminal components, e.g.
complex formation with innate cell membranes (57), innate
defence proteins, other serum proteins or surfactant proteins,
could explain the dose-dependent transport kinetics.
Transport of PAMAM 3.5 and 5.5 across in-vitro Calu-3
monolayers was faster than predicted by solutes of
comparable molecular dimensions but slower than that reported by
others in different epithelial monolayer systems e.g. intestinal
models. The expression and functionality of endocytic uptake
pathways, including caveolae, in primary alveolar epithelial
cells were confirmed for the uptake of PAMAM dendrimers
as well as model macromolecular probes. In an ex-vivo IPRL
model dendrimers did not compromise epithelial barrier
integrity, although the airway-to-blood transport kinetics were
dose-dependent and restricted compared to solutes of
comparable size. Neutron scattering experiments confirmed that
PAMAMs do not aggregate in a model lung lining fluid
supporting the conclusion that PAMAM self-association is
unlikely to be a significant factor in the pulmonary disposition of
dendrimer carriers. In summary, anionic PAMAM
dendrimers have a favourable lung biocompatibility profile,
display rapid uptake into respiratory epithelia and prolonged
lung transport kinetics. These findings strongly support the
future development of inhaled PAMAM-based drug delivery
systems for modified local drug delivery to lung epithelia as
well as transpulmonary drug delivery.
ACKNOWLEDGMENTS AND DISCLOSURES
CJM was supported, in part, by EPSRC Platform Grant EP/
C013220/1. This work benefitted from SasView software,
originally developed by the DANSE project under NSF
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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