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Acidic Nanoparticles Are Trafficked to Lysosomes and
Restore an Acidic Lysosomal pH and Degradative
Function to Compromised ARPE-19 Cells
Gabriel C. Baltazar
1
, Sonia Guha
1
, Wennan Lu
1
, Jason Lim
1
, Kathleen Boesze-Battaglia
2
, Alan M. Laties
3
,
Puneet Tyagi
5
, Uday B. Kompella
5
, Claire H. Mitchell
1,4
*
1Department of Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2Department of Biochemistry, University of
Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3Department of Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
of America, 4Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 5Pharmaceutical Sciences and Ophthalmology,
University of Colorado Denver, Aurora, Colorado, United States of America
Abstract
Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to
degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH
is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of
nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked
whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the
cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-
glycolide) (PLGA) 502 H, PLGA 503 H and poly (DL-lactide) (PLA) colocalized to lysosomes of ARPE-19 cells within 60 min.
PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured
1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the
active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like
autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by
the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of
lysosomal pH and improved degradative activity.
Citation: Baltazar GC, Guha S, Lu W, Lim J, Boesze-Battaglia K, et al. (2012) Acidic Nanoparticles Are Trafficked to Lysosomes and Restore an Acidic Lysosomal pH
and Degradative Function to Compromised ARPE-19 Cells. PLoS ONE 7(12): e49635. doi:10.1371/journal.pone.0049635
Editor: Michael E. Boulton, University of Florida, United States of America
Received May 24, 2012; Accepted October 16, 2012; Published December 18, 2012
Copyright: ß2012 Baltazar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by National Institutes of Health grants NIH EY013434 and EY015537 (CHM), EY018705 (KBB), EY017045 (AML), Vision Research
Core Grant EY001583 (CHM and AML), Research to Prevent Blindness (AML), the Paul and Evanina Bell Mackall Foundation Trust (AML), and the Beckman Institute
for Macular Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: chm@exchange.upenn.edu
Introduction
While the propensity of nanoparticles to accumulate in
lysosomes can frustrate many, they are ideally suited to treat
lysosomal defects. In this regard, the lysosomes of retinal pigment
epithelial (RPE) cells represent a prime target. RPE lysosomes
have a high degradative load, processing both the phagocytosed
tips of shed photoreceptor outer segments and considerable
autophagic material [3,4]. The degradative lysosomal enzymes
function optimally at an acidic pH; consequently, elevation of this
pH is predicted to slow enzyme activity and decrease degradation.
Lysosomal pH is elevated in RPE, as well as other cells, by basic
drugs such as chloroquine and tamoxifen [5–7]. In addition, RPE
lysosomes are alkalinized with delay by the bisretinoid N-
retinylidene-N-retinylethanolamine (A2E) [8], and lysosomes of
RPE from ABCA4
2/2
mice having excess A2E are more alkaline
than age matched controls [5]. The elevated pH can impair the
activity of hydrolytic enzymes such as cathepsin D and lysosomal
acid lipase, leading to a decline in lysosomal degradative capacity
[9–11]. Incomplete degradation of phagocytic and autophagic
material leads to the accumulation of autofluorescent lipofuscin
which itself is often associated with retinal degenerations [12–14].
Treatment to restore an acidic lysosomal pH would be of
therapeutic interest. Receptor-mediated pharmacologic interven-
tion has demonstrated promise in lowering lysosomal pH and
restoring the degradative capacity [5,15,16]. However, treatment
that targets lysosomal acidity more directly may be advantageous.
In this regard, polymeric nanoparticles (NPs) may be ideal.
Nanoparticles prepared from biodegradable polymers are consid-
ered an attractive means of drug and gene delivery due to their
non-toxic nature and their ability to be internalized into
mammalian cells [17]. In the eye they have been utilized to
deliver markers to mouse retinal neurons and genes into ARPE-19
cells [18–20]. When injected into the rabbit eye, magnetic
nanoparticles were taken up by RPE cells and caused no
discernable inflammation [21], while delivery of genetic material
to the posterior eye can stop choroidal neovascularization
following laser treatment in rodents [22]. Nanoparticles made
from acidic PLGA nanoparticles are readily taken up into cells via
endo-lysosomal phagocytotic pathways, implying they may be
PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e49635
delivered to both the appropriate cell and organelle [23,24]. We
therefore asked if acidic nanoparticles derived from lactide and
glycolide polymers could acidify the lysosomes of ARPE-19 cells to
prevent the accumulation of autofluorescent material.
Materials and Methods
Ethics Statement
The use of bovine photoreceptor outer segments was approved
by the University of Pennsylvania IACUC.
Materials
Poly (DL-lactide-co-glycolide) (PLGA) ResomerHRG 502 H,
PLGA ResomerHRG 503 H and poly (DL-lactide) (PLA)
ResomerHR 203S were purchased from Boerhinger Ingelheim
Inc., VA. Other material was purchased from Sigma Aldrich (MO)
unless otherwise indicated.
Cell Culture
The human ARPE-19 cell line was obtained from the American
Type Culture Collection (Manassas, VA). [25] Cells were grown to
confluence in 25 cm
2
primary culture flasks in a 1:1 mixture of
Dulbecco’s modified Eagle medium (DMEM) and Ham’s F12
medium with 3 mM L-Glutamine, 100 mg/ml penicillin/strepto-
mycin, 2.5 mg/ml Fungizone, and 10% fetal bovine serum (all
Invitrogen Corp).
Preparation and Storage of Nanoparticles
Three formulations of polymeric nanoparticles were developed
by the lab of Dr. Uday Kompella; Nanoparticle 1 (NP1): PLGA
502 H; NP2: PLGA 503 H; and NP3 PLA. See Table 1.
Nanoparticles were labeled with Nile Red for localization
experiments (NP1R, NP2R and NP3R in Table 1). Unlabeled
nanoparticles were used for measurements of lysosomal pH. All
NPs were synthesized using the same process. Briefly, the polymer
solution was prepared by dissolving the polymer in dichlorometh-
ane and transferring this to an aqueous solution of polyvinyl
alcohol and sonicated (Misonix Sonicator 3000, Misonix Inc., NY)
for 1 minute at an energy input of 10 W. The primary emulsion
thus formed was further transferred to a larger volume of aqueous
solution of polyvinyl alcohol and sonicated using a probe sonicator
for 30 seconds at an energy input of 3 W. This step results in
hardening of nanoparticles. The secondary emulsion was kept on
stirring at room temperature for 3 hours to evaporate the organic
solvent present in the nanoparticles. After 3 hours, the nanopar-
ticles were harvested by centrifugation at 27000 g for 30 minutes.
In order to remove the residual amount of polyvinyl alcohol
present, the nanoparticles were washed twice by dispersing into
water each time followed by centrifugation. The final nanopar-
ticles pellet obtained after two washings was suspended in water
and frozen at 280uC. The frozen nanoparticle dispersion was
subjected to freeze drying overnight in a lyophilizer (Labconco
Corporation, MO) to attain lyophilized nanoparticles. Nanopar-
ticles were evaluated for mean particle size and polydispersity
index (variance) using Nicomp 380 ZLSHParticle Sizer (Particle
Sizing Systems, CA). One mg of nanoparticles were uniformly
dispersed in 2 ml water and subjected to analysis. For experiments
with ARPE-19 cells, all particles were desiccated at room
temperature until reconstitution for same-day use.
Visualization of Ingested Nanoparticles
For concentration-dependent uptake studies, ARPE-19 cells
were plated on 12 mm cover glass until reaching 50% confluence.
Solutions of each Nile Red nanoparticle formulation in culture
medium were sonicated and filtered at 0.8 mm (Thermo Fisher
Scientific Inc., Waltham, MA). Initial images in Fig. 1A were
obtained after 24 hr. incubation utilizing a Zeiss confocal
microscope and processing at the University of Pennsylvania
School of Medicine Biomedical Imaging Center. For studies
quantifying the effect of concentration, NPs were present at 0.25,
0.5, 1.0 and 2.0 mg/ml and cells were incubated for 1 hr. For
time-dependent studies, cells were incubated in 1 mg/ml NP
solution for the time indicated. After incubation, cells were washed
3x with isotonic solution (IS; (in mM) NaCl 105, KCl 5, HEPES
Acid 6, Na HEPES 4, NaHCO
3
5, mannitol 60, glucose 5, MgCl
2
0.5, CaCl
2
1.3), then incubated in 5 mM LysoTracker Green
DND-26 (Invitrogen Corp., Carlsbad, CA) for 15 min. Cells were
washed again 3x with IS before visualization of Nile Red
nanoparticles (540 nm ex) or lysosomes with LysoTracker Green
(488 nm ex, Molecular Proves/Invitrogen). For concentration
studies, images were captured with an Eclipse E600 fluorescent
microscope (Nikon Inc.) and Retiga 2000R CCD monochromatic
camera (QImaging, BC, Canada) with processing by ImagePro
(MediaCybernetics Inc., Bethesda, MD). Pearson’s coefficient for
overlap between Nile Red and LysoTracker regions was deter-
mined with one or two cells defined as an area of interest. For
time-dependent assays, cells were processed with a Nikon A1R
confocal microscope system at the University of Pennsylvania Live
Cell Imaging Core with NIS-Elements software (Nikon Inc.) was
used to calculate the Pearson’s correlation coefficient from a cell.
The z-stack with the greatest Pearson’s coefficient in each field was
used for the calculations.
Lysosomal pH Measurements from ARPE-19 Cells
In brief, cells were grown to confluence on black-walled, clear-
bottomed CostarH96-well plates (Corning Inc., Corning, NY) and
grown to 100% confluence. The medium was removed and
replaced with various drugs dissolved in medium: chloroquine
(CHQ, Sigma-Aldrich Co., St. Louis, MO), nanoparticles (NPs) at
various concentrations, or a mixture of CHQ+NPs. The cells were
allowed to incubate for time courses ranging from 1 hour to 12
days. For prolonged experiments, solutions were replaced at day 7
to ensure cell viability.
Lysosomal pH measurements were based on a protocol
described in detail previously [5,15,16]. In brief, ARPE-19 cells
were removed from the incubator and rinsed 3x with isotonic
solution and then incubated with 5 mM LysoSensor Yellow/Blue
DND-160 for 5 min; given the temperamental nature of the dye,
this concentration was the minimum found to provide a consistent
signal-to noise value. The dye exhibits a pH-dependent excitation
at 340 nm and 380 nm and permits the ratiometric assessment of
pH changes in acidic organelles independent of dye concentration.
LysoSensor was removed from plate wells after ,3 min and cells
washed, followed by addition of either 100 mL control or pH
calibration buffers. Most measurements were made 16–19 min
after dye removal to minimize the slight alkalinizing actions of the
dye. Lysosomal pH was determined from the ratio of light excited
at 340 nm vs. 380 nm (.520 nm em); the measurement of control
and experimental wells simultaneously in 96 well plates minimized
changes attributable to the dye itself. Lysosomal pH values were
calibrated in each plate at the same time as experimental levels
and were determined by exposing cells to 10 mMH+/Na+
ionophore monensin and 20 mMH+/K+ionophore nigericin in
20 MES, 110 KCl and 20 NaCl at pH 4.0–6.0 for 5 min.
Fluorescence was measured with a Fluoroskan 96-well Plate
Reader (Thermo Fisher). In spite of numerous steps taken to
minimize variation (see [15]), some variation in absolute pH level
did occur between plates measured at different days. However,
Acidic Nanoparticles Restore Lysosomal pH
PLOS ONE | www.plosone.org 2 December 2012 | Volume 7 | Issue 12 | e49635
normalization revealed that the relative differences between
experimental and control pH values were constant.
Assessment of Availability of Cathepsin D Active site with
BODIPY FL-pepstatin A Probe
The availability of the cathepsin D active site was measured
with the fluorescent probe BODIPY FL-pepstatin A (Invitrogen).
The probe itself is synthesized by covalently conjugating the
BODIPY (Boron dipyrromethene difluoride) fluorophore to
pepstatin A, a potent and selective inhibitor of cathepsin D [26].
The probe binds directly to the active site of cathepsin D,
providing a measure of access and potential cathepsin D activity.
To localize the stain, ARPE-19 cells were grown on 12 mm cover
glass and incubated in 10 mM BODIPY probe in DMEM/F12 for
30 min at 37uC in the dark, washed, and incubated in 5 mM
LysoTracker Red DND-99 (Invitrogen) for 15 min. Cells were
mounted and examined on the Eclipse microscope (Nikon). To
quantify the availability of the cathepsin D active site, cells were
grown to confluence on black-walled, clear-bottomed 96-well
plates until confluent, and then incubated for 48 hrs. in either
control culture medium, 10 mM CHQ in medium, or 10 mM
CHQ +1 mg/mL NP3 in medium. After incubation, cells were
washed followed by a 30 min incubation in 10 mM BODIPY
probe at 37uC in the dark. Cells were washed again 3x and
fluorescence compared with the Fluoroskan plate reader (Thermo
Fisher), at 488 nm/527 nm (ex/em).
Photoreceptor Outer Segment (POS) Preparation
The isolation of bovine photoreceptor outer segments was based
on published protocols, with approval of the University of
Pennsylvania IACUC [27–29]. Material was handled as previously
published [15,16]. Briefly, fresh bovine retinas were isolated under
sterile conditions and stored at 280uC. Thawed retinas were
agitated in 30% (w/w) buffered sucrose solution (containing 5 mM
HEPES pH 7.4, 65 mM NaCl, 2 mM MgCl
2
) followed by
centrifugation in a Sorval SS-34 rotor (7 min,700 rpm,4uC). The
supernatant was diluted in two volumes of 10 mM HEPES pH 7.4
and further centrifuged (Sorval SS-34 rotor, 20 min, 17500 rpm,
4uC). The resulting pellet was then homogenized and layered on
top of a discontinuous sucrose density gradient solution of 36, 32,
and 26% sucrose (w/w); POS membranes were harvested from the
26%/32% sucrose solution interface. POS prepared this way were
washed in 3 volumes of 0.02 M Tris buffer, pH 7.4 (Sorval SS-34
rotor, 10 min, 13000 rpm, 4uC). The pellet was resuspended in
2.5% (w/w) buffered sucrose solution (Na
2
HPO
4
10 mM,
NaH
2
PO
4
10 mM, NaCl 1 mM) and stored at 280uC for later
use.
Autofluorescence Assay Using Flow Cytometry
ARPE-19 cells were grown to confluence in clear 6-well plates
(BD Biosciences, Franklin Lakes, NJ). On day 0, cells in set one
had culture medium removed and replaced with fresh medium
(control), 10 mM chloroquine in medium, or 10 mM chloroquine
+1 mg/ml NP3 in medium. Cells in set 2 had their medium
removed and incubated with 2 ml POS in culture medium (10
6
outer segments/ml) for 2 hours (pulse); the cells were washed
thoroughly with IS to remove non internalized POS followed by 2
hours chase in medium. Subsequently, medium was removed and
cells incubated for 20 hrs. with one of the following solutions: fresh
medium (control), 10 mM chloroquine in medium, 10 mM
chloroquine +1 mg/ml NP3, or 1 mg/ml NP3. This series of
pulse/chase conditions was repeated for the subsequent 6 days.
After 6 days the cells were washed, detached with 0.25% trypsin in
EDTA (Invitrogen), and analyzed on a flow cytometer (FACS
Calibur; BD Biosciences, Heidelberg, Germany) using the FITC
channel (excitation laser wavelength, 488 nm; detection filter
wavelength, 530 nm). The channel was gated to exclude cell
debris and cell clusters.
Opsin Immunoblotting
ARPE-19 cells were cultured on a 6-well plate until confluent.
Cells were incubated with medium or 1610
6
POS/mL for 2 hrs at
37uC then washed 3x to remove non-internalized POS. Incuba-
tion in culture medium continued for a subsequent 2 hrs., after
which 1 mg/mL NP3 were added and cells incubated as normal.
After treating such every day for 7 days, cells were washed and
processed for immunoblotting as published [30]. In brief, washed
cells were lysed in 300 ml RIPA buffer (150 mM NaCl, 1.0%
Triton X-100, 0.5% Na-Deoxycholate, 0.1% SDS, 50 mM Tris,
pH 8.0 and protease inhibitor cocktail) and centrifuged at 13000 g
for 10 min at 4uC. Protein concentrations were determined using
the BCA kit (Pierce). Protein lysate (10 mg per lane) was loaded in
sample buffer (2% SDS, 10% glycerol, 0.001% bromophenol blue,
and 0.05 M Tris-HCl, pH 6.8), separated on a 4–15% gradient
precast gel and transferred to a PVDF membrane. After blocking
with SuperBlock buffer (Themoscientific, Rockford, IL) for 1 hr.,
the membrane was incubated with opsin antibody at 1:1000 (#:
SC-57432, Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
overnight at 4uC. The corresponding secondary antibody was
developed with enhanced chemiluminescence (ECL, Amersham).
Band intensity was detected and analyzed with ImageQuant LAS
Table 1. Physical properties of the polymers used in the study.
NP No Nanoparticle Polymer
Molecular
Weight (Da)
Acid number
(mg KOH/g)
Mean Particle size
(nm) Polydispersity index
1 Blank Nanoparticles PLGA ResomerHRG 502 H 8000–10000 .6 503.9 0.321
2 PLGA ResomerHRG 503 H 30000–35000 .3 387.4 0.277
3 PLA ResomerH
R 203 S
40000 ,1 428.5 0.240
1R Nanoparticles
loaded with Nile red
PLGA ResomerHRG 502H 8000–10000 .6 383.5 0.237
2R PLGA ResomerHRG 503H 30000–35000 .3 379.1 0.210
3R PLA ResomerH
R 203S
40000 ,1 397.8 0.218
doi:10.1371/journal.pone.0049635.t001
Acidic Nanoparticles Restore Lysosomal pH
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4000 system (GE Healthcare Biosciences, Pittsburgh, PA). The
blots were stripped and reprobed with rabbit monoclonal anti-
GAPDH at 1:1000 (#2118, Cell Signaling Technology) overnight
at 4uC, and incubated with secondary antibody for 1hour at room
temperature and processed as above.
Statistical Analysis
Data are reported as mean 6SEM. Statistical analysis used a 1-
way ANOVA with appropriate post-hoc test. Results with p,0.05
were considered significant.
Results
Characteristics of Nanoparticles
As the aim of this project was to acidify lysosomes using
nanoparticles, nanoparticles were constructed using three different
acidic polymers. As shown in Table 1, nanoparticle type 1 (NP1)
was composed of PLGA ResomerHRG 502H and was the smallest
molecular weight, with an acid number of 6 mg KOH/g. NP2 was
composed of PLGA ResomerHRG 503H, intermediate in size and
had an acid number of 3 mg KOH/g. NP3 was composed of PLA
ResomerHR 203S and was the largest, with an acid number of ,
1 mg KOH/g. The particle size was 387.4–503.9 nm for blank
NPs and 383.5–397.8 nm for Nile red loaded NPs. The
polydispersity index ranged from 0.240–0.321 for blank NPs and
0.210–0.240 for Nile red loaded NPs.
Nanoparticles Delivered to Lysosomes
Initial experiments were performed to determine whether
nanoparticles were delivered to lysosomes. Nanoparticles were
stained with Nile Red to facilitate localization. Figures 1A–D
illustrate a typical experiment, where ARPE-19 cells incubated
with NP2R-Nile Red. When cells were examined after a 24 hr.
incubation in NP2R-Nile red, clustered red fluorescence was
detected. This red fluorescence was largely observed in regions
also stained with Lysotracker green, implying NP2R was localized
to lysosomes (Figs 1A–D).
To determine the optimal conditions for nanoparticle delivery,
the experiment was repeated using all three formulations but cells
were examined for overlap between Lysotracker and nanoparticles
after only one hour. Each nanoparticle formulation was tested at
0.25, 0.5, 1.0 and 2.0 mg/ml. To quantify the proportion of
nanoparticles present in the lysosomes, overlap was calculated
using Pearson’s coefficient. While 0.25 mg/ml gave lower levels,
maximal overlap was observed with nanoparticles incubated at
1.0 mg/ml, with no further increase with increasing concentra-
tion. NP1R, NP2R and NP3R all gave similar results (Fig. 1E). As
such, nanoparticles were given at 1 mg/ml in subsequent
experiments.
The preceding experiment indicated that nanoparticle delivery
to the lysosome was largely complete within one hour. To
determine the rate of internalization more accurately, cells were
incubated with NP3R-Nile Red for 15, 30 and 60 min. Following
this, cells were incubated for an additional 15 min with
LysoTracker Green to visualize lysosomes, and the degree of
colocalization determined. In cells exposed to NP3R-Nile Red for
only 15 min, red fluorescence was largely localized on the cell
periphery (Fig. 2A). Fluorescent red staining had extended in cells
exposed to NP3R-Nile Red for 30 min (Fig. 2B), while red staining
Figure 1. Acid nanoparticles are delivered to lysosomes of
ARPE-19 cells. A-D. Live cell images of colocalized nanoparticles with
ARPE-19 lysosomes after a 24 hour incubation with NP2R A. ARPE-19
cell lysosomes as visualized with the dye LysoTracker Green. Images
taken at 40x magnification withaZeissconfocalmicroscope.
LysoTracker detected at 488/500 nm (excitation/emission). B. ARPE-19
ingestion of Nile Red stained nanoparticle NP2R. Cells were incubated
for 24 hours in full culture medium with 1 mg/ml concentration of NPs.
Nanoparticles were detected at 540/580 (excitation/emission). Before
incubation, the nanoparticle suspension was passed through a 0.8 mM
syringe filter to remove clumped particles. After incubation period, cells
were washed thoroughly with isotonic solution in an attempt to further
remove clumps and any extracellular NPs. C. DIC image of the ARPE-19
cells and nanoparticles. With this image the morphology of the ARPE-19
cells are clearly visible. D. Composite image of the LysoTracker Green
(lysosomes), Nile Red (nanoparticles), and DIC exposures. This image
demonstrates the colocalization of the ingested nanoparticles with the
ARPE-19 lysosomes. E. Concentration dependence of lysosomal delivery.
The degree of colocalization of NP1R, NP2R, and NP3R with lysotracker
green as a function of concentration. Each point is the mean +/2SEM
of a calculated Pearson’s coefficient within one area of interest (AOI) in
one microscope field; n = 3 fields. Inserts indicate overlap of NP1R and
NP3R. In 1E, NP2R were incubated for 1 hr. before colocalization was
determined.
doi:10.1371/journal.pone.0049635.g001
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was observed throughout the cells after a 60 min exposure
(Fig. 2C). Quantification supports these observations; only
1363% of the NPs were colocalized in lysosomes after 15 min
and 2664% with lysosomes after 30 min. However, 7268% of
the NPs were in lysosomes after 60 min exposure (Fig. 2D). Of
note, this level is remarkable close to that seen after 60 min in
Figure 1D, supporting the observation that the majority of the
nanoparticles are in the lysosomes after 60 min exposure.
Nanoparticles Acidify Lysosomes
Lysosomal pH was measured to determine if acidic nanopar-
ticles altered pH levels and to compare the efficacy of the different
particles. Cells were incubated with non-fluorescent NP1, NP2
and NP3 for 1 hr. at 1 mg/ml, washed and loaded with the
ratiometric lysosomal pH indicator Lysosensor Yellow/Blue. The
effect of nanoparticles on baseline pH was measured first. NP1 had
no effect on baseline pH. NP2 and NP3 both acidified the
lysosomes, by 0.27 and 0.32 units respectively (Fig. 3A).
Recently we found that the magnitude of acidification can be
greater in cells whose lysosomes are alkalinized by lysoosmotic
agents [15]. Chloroquine has been well documented to elevate
lysosomal pH through its actions as a tertiary amine [31,32].
While the effect of chloroquine was dose dependent over this range
(Supplemental Figure S1), we have previously found 10 mM
produces relatively constant effects on lysosomal pH without
leading to cell death. As such, the ability of nanoparticles to acidify
lysosomes exposed to 10 mM chloroquine for 1 hr. was deter-
mined. The effect of NP1 was minimal, but NP2 and NP3
acidified cells by 0.62 and 0.64 units respectively (Fig. 3B).
To determine the duration of lysosomal acidification by
nanoparticles, chloroquine was given on its own or in the presence
of either NP2 or NP3 on day 0 and lysosomal pH was measured
on various days afterwards. Overall, the mean lysosomal pH in
control was 5.1160.04 and in chloroquine was 5.7560.006
(n = 32). Control cells showed a small 0.1 unit decrease in
lysosomal pH from day 1 to 12 while cells treated with chloroquine
Figure 2. Rapid delivery of nanoparticles to lysosomes. A. Composite image of ARPE-19 cells with internalized NP3R nanoparticles (red, 1 mg/
ml) and Lysotracker (green) after 15 min incubation. B. NP3R and lysosomes after 30 min incubation. C. NP3R and lysosomes after 1 hr. incubation.
Bar = 10 mM in panels A-C. D. Colocalization of NP3R nanoparticles with ARPE-19 cells as a function of time. Each point is the mean +/2standard
deviation of the Pearson’s coefficient in one microscope field; n = 3. Error bars are present but too small to be detected for the 15 min point.
doi:10.1371/journal.pone.0049635.g002
Figure 3. Nanoparticles lower lysosomal pH. A. While NP1 did not alter baseline levels of lysosomal pH (pHL), NP2 and NP3 acidified the
lysosomes significantly. Lysosomal pH was measured 1 hr. after addition of nanoparticles. Here and throughout the figure, nanoparticles were given
at 1 mg/ml. n = 8. * p,0.05 vs. control, ANOVA on ranks, Dunn’s posthoc test. B. Chloroquine (CHQ; 10 mM) raised the lysosomal pH. NP2 and NP3
significantly lowered lysosomal pH, while NP1 had little effect. Chloroquine and nanoparticles were applied concurrently 1 hr. before pH
measurements. *p,0.001 vs. control, **p,0.001 vs. CHQ, ANOVA with Tukey post hoc test. n = 8. C. NP2 and NP3 induced sustained acidification of
lysosomal pH in cells treated with 10 mM chloroquine. The acidification decreased with time but was still detected. Chloroquine and nanoparticles
were added on day 1 and remained in the bath without a solution change. The effect of the nanoparticles was defined as their relative effectiveness
at bringing lysosomal pH towards baseline; the absolute numbers did vary somewhat but this normalization accounted for such differences. %
Reacidification = 100*((CHQ-(CHQ+NP))/(CHQ-Control)). Data from 31 plates.
doi:10.1371/journal.pone.0049635.g003
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had a 0.1 unit rise over this time. However, the variability between
plates dictated that the effect of nanoparticles was best determined
on a plate by plate basis, with normalization used to reduce
variation found from multiple plates over numerous days. The
effectiveness of nanoparticles was defined by the extent they could
lower the pH in chloroquine treated cells with respect to that in
untreated cells. To that end, both NP2 and NP3 proved capable of
prolonged lysosomal acidification, lowering the pH in ARPE-19
lysosomes for over a week (Fig. 3C, see also Supplemental Figure
S2). Thus acidic nanoparticles were capable of prolonged
lysosomal acidification. Of note, cells undergoing various treat-
ments appeared similar under gross examination, suggesting
extended exposure to acidic nanoparticles or this concentration
of chloroquine was not itself detrimental; this may be of interest
with regard to the lethal effects of increased levels of chloroquine
on these cells reported recently [33].
Nanoparticles Improve Lysosome Function
While the absolute magnitude of the acidification induced by
acidic nanoparticles was not huge, lysosomal enzymes are
particularly sensitive to pH at this level [11]. As such, even a
relatively modest acidification can have a substantial effect on the
ability of the lysosomes to degrade material. Cathepsin D is a
major protease in the lysosomes of RPE cells and contributes to
the degradation of phagocytosed photoreceptor outer segments
[34]. The availability of the cathepsin D active sight was
monitored in situ with BODIPY FL-pepstatin A; the active site
for cathepsin D is identified by the binding of fluorescently tagged
substrate pepstatin A and availability is pH sensitive [11]. Co-
incubation of ARPE-19 cells with BODIPY FL-pepstatin A
demonstrated that the majority of the probe colocalized with
LysoTracker Green (Fig. 4A–C). The amount of red probe was
quantified to determine the effect of pH manipulation on the
availability of the cathepsin D active site. As expected, chloroquine
decreased the fluorescence readout, consistent with a decrease in
potential cathepsin D activity with lysosomal alkalinization
(Fig. 4D). Substrate binding to the cathepsin D active site was
restored to baseline levels after treatment with NP3. These
changes in the cathepsin D active site parallel the changes in
lysosomal pH and suggest that the reacidification by nanoparticles
may be sufficient to increase enzyme activity.
Acidic Nanoparticles Increase Clearance of Photoreceptor
Outer Segments
RPE lysosomes are required to degrade engulfed photorecep-
tors through heterophagy and extraneous internal material
through autophagy. Elevation of lysosomal pH is predicted to
be detrimental to both types of degradation, and acidic
nanoparticles have the potential to improve both. To examine
the impact of nanoparticles on heterophagy, ARPE-19 cells were
fed photoreceptor outer segments for 2 hrs., followed by a two
hour wash to allow for internalization. After this interval, half of
the wells were exposed to NP3 for 20 hrs. The cycle was
repeated for 5 more days (6 exposure cycles in total), after which
Figure 4. BODIPY FL-pepstatin A: Probing cathepsin D activity. A. Lysosomes of ARPE-19 cells as stained with LysoTracker Red B. BODIPY FL-
pepstatin A staining. C. Composite image demonstrating colocalization of BODIPY fluorophore with RPE lysosomes D. CHQ administration raises the
lysosomal pH, inactivating cathepsin D and hindering the binding of the BODIPY probe to the enzyme. This is seen quantitatively as a lowering in the
amount of fluorescence (measured in arbitrary light units, ALU). Nanoparticles reverse this process, with NP3 significantly restoring cathepsin D
activity. * p,0.05 vs. control, ** p,0.05 vs. CHQ; one-way ANOVA with Tukey post hoc test. n = 5 wells from 1 plate. Similar results seen in 2 plates.
doi:10.1371/journal.pone.0049635.g004
Acidic Nanoparticles Restore Lysosomal pH
PLOS ONE | www.plosone.org 6 December 2012 | Volume 7 | Issue 12 | e49635
cells were detached and autofluorescence at 488 nm was
determined using FACS analysis. NP3 was used as it demon-
strated the greatest promise for sustained decrease of lysosomal
pH. Treatment with photoreceptor outer segments increased
autofluorescence 4 fold, but subsequent exposure to NP3 reduced
this autofluorescence (Supplemental Figure S3). Interestingly,
exposure to chloroquine alone increased the autofluorescence
four-fold, indicating a decreased processing in material of cellular
origin. However, inclusion of NP3 in the treatment substantially
reduced the autofluorescence. Finally, to test if the effects were
additive, chloroquine or chloroquine+NP3 was added to the cells
2 hrs. after washing off the outer segments for 6 days. Together,
chloroquine and photoreceptors increased autofluorescence ten-
fold, suggesting the effects were, at a minimum, additive (Fig. 5A).
However, NP3 greatly reduced the autofluorescence seen when
both challenges were added. The mean changes in total
autofluorescence at 488 nm induced by combinations of POS,
chloroquine and NP3 are presented in Fig. 5B. In each of the
flow cytometry experiments conducted, NP3 consistently reduced
autofluorescence.
The ability of PLA NP3 to enhance outer segment clearance
was tested directly by quantifying the amount of opsin present in
the cells with the immunoblotting technique. Confluent ARPE-19
cells were exposed to photoreceptor outer segments with or
without PLA NP3 nanoparticles using the pulse chase protocol
described above. No opsin was detected in cells not exposed to
outer segments, although the band intensity increased in cells
challenged with outer segments, consistent with the increased
autofluorescence above (Fig. 5C). However, treatment with NP3
decreased the band intensity substantially. Quantification indicat-
ed that NP3 treatment reduced mean opsin levels in cells by over
90% (Fig. 5D). This supports the concept that acidifying lysosomes
Figure 5. Nanoparticles reduce autofluorescence and opsin levels associated with ingestion of photoreceptor outer segments. A.
Sample readout of the FACS analysis demonstrating treatment with NP3 greatly reduced the mean autofluorescence at 488 nm in RPE cells treated
with chloroquine (CHQ) and phagocytosed photoreceptor outer segments (POS). B. Nanoparticles reduced the autofluorescence in ARPE-19 cells
given POS, CHQ, and or POS+CHQ. Bars represent the mean 6SEM of autofluorescence detected at 488 nm. * p,0.05 vs. control; #p,0.05 vs.
POS+CHQ. ANOVA. C. Immunoblot for opsin detected in ARPE-19 cells in the absence of photoreceptor outer segments (Control), after exposure to
outer segments over 7 days (POS), and with a delayed addition of PLA NP3 after each outer segment feeding (POS+NP3). The blot was at the
predicted size of ,40 kDa. GAPDH binding of the blot is demonstrated below. D. Quantitation of opsin levels in immunoblots. Levels were first
controlled for GAPDH staining, and then normalized to the mean POS value in each blot to control for variation. * p,0.001, n = 4.
doi:10.1371/journal.pone.0049635.g005
Acidic Nanoparticles Restore Lysosomal pH
PLOS ONE | www.plosone.org 7 December 2012 | Volume 7 | Issue 12 | e49635
with acidic nanoparticles can enhance the degradation and
clearance of photoreceptor outer segments by RPE cells.
Discussion
Nanoparticles have great promise for drug delivery, but their
delivery to the correct target is critical. This study turns their
propensity for lysosomal accumulation into an advantage, and
demonstrates that acidic nanoparticles can lower the pH of
compromised lysosomes to improve degradative function. Nano-
particles localized to lysosomes over the course of one hour, with a
saturating concentration of 1 mg/ml (Figs 1–2). Acidic particles
seemed to function in lysosomes; pH was significantly reduced one
hour after treatment, with acidification remaining after 12 days
(Fig 3). Acidic nanoparticles restored the availability of the
cathepsin D active site (Fig. 4), and greatly increased the clearance
of autofluorescent material and opsin (Fig. 5). Together, these
observations lead us to propose a model whereby the ingested
acidic nanoparticles can reduce the accumulation of partially
degraded autofluorescence material in RPE cells (Fig. 6). Given
the critical role that lysosomal enzymes play in general cellular
maintenance, the potential for acidic nanoparticles to improve
degradative function has implications for a broad range of cell
types.
Rapid and Sustained Delivery of Nanoparticle to
Lysosome
The results above imply that acidic nanoparticles are delivered
rapidly to lysosomes and remain active for at least a week.
Measurement of lysosomal pH taken one hour after adding acidic
nanoparticles to the bath demonstrated a clear acidification.
Functional evidence is consistent with microscopic data confirming
the majority of nanoparticles localized to lysosomes 60 min after
addition. Although the pathways used to these deliver nanopar-
ticles are unknown, it is of interest that the colocalization rate was
not affected by the acid number of the material used to construct
the nanoparticle nor the molecular weight. It was recently
reported that most nanoparticles given to macrophages ended
up in the lysosome regardless of the endocytotic pathways used
[35], consistent with our findings. It is also intriguing that the
particle with the lowest acid number was most effective at lowering
lysosomal pH.
While the lysotracker dye labels acidic organelles, and
nanoparticles are acidic, several observations testify to the
independence of the staining. First, if Lysotracker just went to
the nanoparticles, lower concentrations of nanoparticles would
have more than enough Lysotracker and display increased
colocalization; Figure 1E shows the opposite occurs. Likewise, if
Lysotracker was just labeling nanoparticles, then rates of
colocalization would be instantaneous, and not delayed, as
illustrated in Figure 2D. A recent study using MDCK and
Caco-2 cells exposed to coated microbeads suggested that
internalization required 23–32 min, phagosomal acidification took
3–4 min and fusion of the phagosome to endosome/lysosomes was
complete within 74–120 min [36]. The similar time course for
colocalization of Lysotracker and nanoparticles strongly suggests
delivery of the nanoparticles into the lysosomes. Third, the degree
of costaining was not affected by the different acid number of NP1-
Figure 6. Model of enhanced photoreceptor degradation. A. RPE cells with compromised lysosomes cannot sufficiently degrade
photoreceptor outer segments. The undigested material accumulates inside the cell as autofluorescent lipofuscin. B. After treatment with acidic
nanoparticles, RPE lysosomes are more capable of breaking down the POS. The end result is a substantial decrease in undigested debris and
lipofuscin.
doi:10.1371/journal.pone.0049635.g006
Acidic Nanoparticles Restore Lysosomal pH
PLOS ONE | www.plosone.org 8 December 2012 | Volume 7 | Issue 12 | e49635
3, suggesting the fluorescence was not a direct reflection of acidity
but a more complex reaction. Finally, there was a clear increase in
degradative activity of lysosomal enzymes in the presence of the
nanoparticles. Together, these observations imply that nanopar-
ticles are present in the lysosomes.
Although endocytosis itself may initiate a series of changes to
lysosomes, it is unlikely that nanoparticle internalization can itself
lower lysosomal pH as all three nanoparticle formulations were
delivered to the lysosome but only two of them lowered lysosomal
pH. It is unclear why the PLA-based NP3 was more effective at
restoring the cathepsin D active site than NP2 given their similar
effects on pH, although the enhanced performance of NP3 over
the long term suggests it would be a better drug overall.
Functional Restoration by Acidic Nanoparticles
While the absolute decrease on pH values induced by acidic
nanoparticles was relatively small, this shift is expected to have an
impact on degradation because of the sharp relationship between
pH and enzyme activity over this range. For example, the activity
of cathepsin D increases 3 fold when pH falls from 5.0 to 4.5 [11]
and acid lipase activity more than doubles when pH falls from 5.2
to 4.5 [10]. The magnitude of the acidification induced by
nanoparticles was larger in compromised lysosomes with elevated
pH than at baseline levels. This is desirable with regards to
treatment, as it will enable the particles to preferentially target the
more disturbed organelles in a mixed population.
The effectiveness of the acid nanoparticles was confirmed by the
increase in binding of Bodipy-pepstatin A to the cathepsin D active
site, by the decrease in opsin, and by the decrease in
autofluorescence. The fluorescent Bodipy-pepstatin-A binds to
the active site of cathepsin D, so the enhanced signal observed
after treatment with acidic nanoparticles could reflect an increase
in the pH-sensitive maturation of cathepsin D [37], a direct effect
of pH on active site availability [11], or both. In rat RPE cells,
opsin was shown to disappear from RPE phagosomes that
costained for cathepsin D, but blockage of the vHATPase with
bafilomycin prevented this opsin degradation [38]. The ability of
NP3 to decrease immunoblotting for opsin is consistent with
improved degradation by cathepsin D once acidity is restored.
The reduced lipofuscin-like autofluorescence observed after
nanoparticle treatment further supports the functional effective-
ness of the acid nanoparticles. The increased autofluorescence
detected in cells treated just with chloroquine is consistent with an
incomplete degradation of autophagic material following elevated
lysosomal pH, although this needs confirmation. The increased
autofluorescence in cells treated with photoreceptor outer
segments is consistent with a retinoid component and is supported
by changes in cellular opsin levels. The ability of acid nanopar-
ticles to greatly reduce both forms of autofluorescence may have
important implications for the treatment of macular degeneration,
as the accumulation of autofluorescent lipofuscin has been
associated by some with disease progression [12–14]. The ability
of NP3 treatment to significantly reduce the levels of cellular opsin
detected in immunoblots provides direct evidence that the
nanoparticles enhance degradation and clearance of photorecep-
tor outer segments. In this regard, the sustained acidification
observed following treatment in Figure 3 may be of benefit.
Nanoparticles composed of PLA remained in RPE cells without
being degraded for up to 4 months following a single injection
[39], consistent with the slower degradation of these particles in
lower pH [40]. The degradation of PLA into non-toxic lactic acid
suggests their appropriateness as a chronic treatment, although it
remains to be determined if acid nanoparticles can lead to a
sustained improvement of opsin degradation in vivo.
Supporting Information
Figure S1 Increasing concentrations of chloroquine
lead to proportional increases in lysosomal pH in
ARPE-19 cells. Addition of 1, 10 or 20 mM chloroquine let to
increasing magnitudes of lysosomal alkalinization when examined
15 (upper panel) or 60 (lower panel) min after addition.
(EPS)
Figure S2 Long term acidification of ARPE-19 cells by
nanoparticles. These figures were meant to show the long term
effects of the nanoparticles ranging from 1–12 days. Of note is the
observance that NP1(A) after 1 day never acidified the lysosomes,
explaining why the % acidification is always negative. NP2 (B) and
NP3 (C) were much more promising, with maximum acidification
in the range of 50%. NP2 seemed to peak earlier and acidification
dropped rather predictably over the 12 days, while NP3 seemed to
slowly peak at day 7 days and then drop.
(TIF)
Figure S3 FACS histograms of nanoparticles reducing
autofluorescence in ARPE-19 cells given outer segments
OR CHQ. ARPE-19 cells were fed bovine POS for 2 hours,
washed, and two hours were allowed for outer segment delivery to
the lysosomes. At this point, nanoparticles were added to the cells.
Adding the particles after the two hour interval ensured effects
were restricted to outer segment digestion and did not alter
binding or phagocytosis. This two stage treatment was repeated
every day for multiple days. Cells were then dissociated and the
autofluorescence at 488/520 (ex/em) was determined using flow
cytometry. Nanoparticle 3 lowered the lipofuscin-like autofluores-
cence that the cells acquired from digesting POS. NP3 lowered the
fluorescence to almost baseline levels.
(TIF)
Acknowledgments
The authors would like to thank Bruce Shenker and the University of
Pennsylvania SDM Flow Cytometry Facility for advice on the FACS
analysis.
Author Contributions
Conceived and designed the experiments: GCB KBB AML UBK CHM.
Performed the experiments: GCB SG PT WL JCL. Analyzed the data:
GCB SG WL UBK CHM. Wrote the paper: CHM.
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