Research Paper

Role of serum albumin as a nanoparticulate carrier for

nose-to-brain delivery of R-flurbiprofen: implications for
the treatment of Alzheimer’s disease
Ling Rong Wong and Paul C. Ho
Faculty of Science, Department of Pharmacy, National University of Singapore, Singapore, Singapore

Keywords Abstract
albumin nanoparticles; Alzheimer’s disease;
flurbiprofen; intranasal drug delivery; Objectives R-flurbiprofen (R-FP) was found to offer neuroprotective effects by
mitochondria inhibiting mitochondrial calcium overload induced by b-amyloid peptide toxicity
in Alzheimer’s disease (AD). However, poor brain penetration after oral adminis-
Correspondence
tration posed a challenge to its further development for AD treatment. In this
Paul C. Ho, Faculty of Science, Department
of Pharmacy, National University of
study, we investigated the potential of serum albumin as nanoparticulate carriers
Singapore, Singapore, Singapore. for nose-to-brain delivery of R-FP to improve its brain accumulation.
E-mail: phahocl@nus.edu.sg Methods Mice were subjected to three treatment groups: (1) intranasal R-FP
solution, (2) oral R-FP solution and (3) intranasal R-FP albumin nanoparticles.
Received July 16, 2017 We also investigated whether the in-vivo R-FP level achieved in the brain
Accepted September 21, 2017 afforded by intranasal administration of R-FP nanoparticles had any effect on
mitochondrial respiratory activity in an in-vitro AD model.
doi: 10.1111/jphp.12836
Key findings Our in-vivo experiments demonstrate that the intranasal adminis-
tration of serum albumin-based R-FP nanoparticles achieved higher brain-
to-plasma ratio profile as compared to intranasal and oral administration of a
simple R-FP solution. We observed significantly improved basal and maximal
mitochondrial respiration in cells treated with R-FP albumin nanoparticles at
in-vivo brain concentration.
Conclusions Serum albumin-based nanoparticles administered via the nasal
route may be a viable approach in delivering therapeutic agents to the brain to
alleviate mitochondrial dysfunction in AD.

disappointment also marked the phase 3 trial of a tau

Introduction
Alzheimer’s disease (AD) is the most common cause of
dementia. To date, there is no cure, prevention or disease-
modifying treatment for AD, and nearly one million new
cases are projected each year.[1] AD is characterized by two
key protein abnormalities, namely cerebral plaques laden
with b-amyloid (Ab) peptide and intraneuronal neurofib-
rillary tangles made up of hyperphosphorylated tau pro-
tein.[2] Current AD treatment agents in development are
mainly focused on the inhibition of Ab aggregation and the
acceleration of Ab clearance.[3] However, the evidence sup-
porting Ab lowering approaches are waning. Of note is the
clearance of Ab from the brains of apolipoprotein E
(APOE) e4 carriers in phase 3 clinical trials with bap-
ineuzumab that has failed to provide clinical benefit,[4] sug-
gesting that treatments which alter Ab levels may be
ineffective in mild-to-moderate AD. Similar
protein aggregation inhibitor, TRx 0237 which failed
to slow cognitive or functional decline in mild-to-
moderate patients with AD (ClinicalTrials.gov Identifier:
NCT01689246).
Accumulating evidence suggests that perturbed
neuronal calcium homoeostasis and mitochondrial dys-
function play a significant role in the pathogenesis of
AD.[5–8] The Ab peptide has been shown to inhibit mito-
chondrial respiration.[9] We previously observed that
mitochondrial dysfunction preceded extracellular Ab
accumulation.[10] Furthermore, Ab42 oligomers can
induce a massive entry of calcium ions into neuronal
cells and promote mitochondrial calcium overload
leading to cell death.[11] A series of NSAIDs, including
R-flurbiprofen (R-FP), are able to depolarize the mito-
chondria and inhibit calcium uptake even at low micro-
molar concentrations,[11] likely owing to the ionizable

© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 70 (2018), pp. 59–69 59
R-flurbiprofen-albumin nanoparticles
carboxylic group which resembles mild mitochondrial
uncouplers.[12] Protecting the mitochondria therefore
holds significant promise in AD. A large number of clin-
ical observational studies support the use of NSAIDs for
the prevention of AD.[13] In particular, preliminary evi-
dence for the potential benefit of R-FP in patients with
mild AD was observed in a phase 2 study.[14] However,
subsequent phase 3 trials in patients with mild AD failed
to show efficacy.[15,16] Limited brain penetration after
oral administration was cited as one key reason for the
gap observed between preclinical and clinical studies.[17]
In this aspect, intranasal delivery has been demonstrated
to provide an effective and non-invasive method of
bypassing the blood–brain barrier (BBB) to deliver thera-
peutic agents to the central nervous system (CNS)[18,19]
by leveraging on the existence of a direct transport route
from the nasal cavity to the CNS.[20,21]
High-resolution functional magnetic resonance imag-
ing studies in humans have revealed that AD tends to
initiate in the lateral entorhinal cortex and spreads from
the lateral entorhinal cortex to other areas of the cere-
bral cortex over time.[22] The entorhinal cortex is com-
monly perceived as a gateway to the hippocampus and
transsynaptic progression of neuronal dysfunction
induced by Ab within the entorhinal-hippocampal net-
work may explain the cognitive impairments.[23] The
entorhinal cortex is also an area closely connected to
the olfactory nerves[24] and olfactory dysfunction has
been found to be an early clinical symptom of AD.[25]
It stands to reason that the use of a nasal route of
administration has merit in the case of AD. A number
of intranasally administered proteins and peptides have
been reported to gain access to the brain within a short
period of time.[20] Recently, it was found that 125I-
labelled albumin can reach all regions of the brain
within five minutes following intranasal administra-
tion.[26] In the same study, pretreatment of the mice
with a fluid-phase stimulator selectively increased the
uptake of albumin into the olfactory bulb, suggesting
that the uptake of albumin across the nasal epithelium
may involve fluid-phase transcytosis. Another study
using 111In-labelled ovalbumin as a model protein found
that brain levels of the protein peaked an hour after
intranasal administration, which also indicates rapid
transport from the nose to the brain.[27] The rapid nat-
ure of albumin uptake from the nasal mucosa into the
brain is likely mediated by passage of the protein
through intercellular clefts in the olfactory epithelium to
distribute into the brain directly.[28] Coupled with its
safe and biocompatible nature,[29] these findings suggest
that albumin may function as a good carrier to facilitate
the delivery of therapeutic agents to AD-related brain
regions using the nasal route of administration.
60 © 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 70 (2018), pp. 59–69
Ling Rong Wong and Paul C. Ho
In this study, we investigated the potential of serum
albumin to act as nanoparticulate carriers for R-FP for
nose-to-brain delivery. To the best of our knowledge,
this is the first in-vivo investigation of serum albumin-
based nanoparticulate flurbiprofen applied intranasally.
Our in-vivo experiments demonstrate that higher brain-
to-plasma ratios of R-FP levels were achieved via
intranasal administration of the nanoparticulate drug
formulation compared to intranasal and oral adminis-
tration of a simple drug solution. We concluded that
the intranasal administration of serum albumin-based
R-FP nanoparticles enhanced the accumulation of R-FP
in the brain relative to the blood. Additionally, we
investigated whether the drug level achieved in the
brain afforded by intranasal administration of our
nanoparticulate drug formulation had any effect on
mitochondrial respiratory activity in an AD cell model
using extracellular flux analysis to measure real-time
oxygen consumption rates of the cells. We observed sig-
nificantly improved basal and maximal mitochondrial
respiratory activity in cells treated with R-FP albumin
nanoparticles at in-vivo brain concentration. Drug-
loaded albumin nanoparticles administered via the nasal
route thus represents a useful approach in delivering
therapeutic agents to the brain to alleviate mitochon-
drial dysfunction, which is a common theme in many
neurodegenerative diseases including AD.
Materials and Methods
Chemicals
Albutein 20% was obtained from Grifols (Los Angeles, CA,
USA). R-flurbiprofen was purchased from Glentham Life
Sciences (Wiltshire, UK). Ibuprofen (internal standard, IS)
was purchased from Sigma-Aldrich (St. Louis, MO, USA).
All other reagents were of analytical grade and used as
received.
Animals
Fifty adult male C57BL/6 mice (10–12 weeks of age) were
obtained from InVivos (Singapore). The mice were housed
in groups (maximum of five mice per cage) under standard
conditions of humidity, temperature and 12-h light/dark
cycle with ad libitum access to food and water. All mice
were maintained under constant conditions for 4 days
before experiments. All animal experiments were con-
ducted in accordance with Singapore National Advisory
Committee on Laboratory Animal Research (NACLAR)
guidelines and approved by Institutional Animal Care and
Use Committee (IACUC), National University of
Singapore.
Ling Rong Wong and Paul C. Ho
Preparation of serum albumin-based
nanoparticles
An injectable aqueous 20% (w/v) human serum albumin
solution (Albutein 20%) was diluted to 3% (w/v) with type
1 water, the pH being corrected to between 5.5 and 5.7 with
10% citric acid as described previously.[30] To prepare
drug-loaded albumin nanoparticles, R-FP was separately
dispersed in a minimal amount of chloroform and added
to said albumin solution. The mixture was stirred vigor-
ously for at least an hour and homogenized in an ice-salt
bath to form the nanoparticles upon exposure to high shear
stress.[31] The nanoemulsion obtained was purified by dial-
ysis (20K MWCO) against type 2 water. The purified pro-
duct was rapidly frozen to 80 °C and lyophilized. Drug
loading amounts in the nanoparticles were quantified using
reversed-phase high-performance liquid chromatography
(HPLC). We also evaluated the particle size stability of the
drug-loaded albumin nanoparticles for subsequent in-vivo
experiments following reconstitution in normal saline solu-
tion and standing at room temperature over 24 h. Particle
size and polydispersity index (PDI) of the nanoparticles
were determined using the dynamic light scattering method
(Zetasizer Nano-ZS90, Malvern Instruments, UK). The lyo-
philized formulation was reconstituted to 5 mg nanoparti-
cles per ml because this concentration level can generate a
good count rate for dynamic light scattering measurements
and is also similar to the amount used in our in-vivo
experiments.
In-vitro drug release
Lyophilized drug-loaded albumin nanoparticles were
weighed out accurately and reconstituted accordingly to
obtain 2.5 mg nanoparticles per ml. 200 ll nanoparticle
suspension was then placed into a mini dialysis unit con-
structed by sealing the opening of a microfuge tube with
pretreated membrane (12-14K MWCO). The dialysis unit
was inverted into a 150 ml PBS reservoir to simulate the
CSF volume in adults[32] and incubated at 37 °C with gen-
tle magnetic stirring. The amount of drug released into the
reservoir was sampled at predetermined time points and
quantified using HPLC.
In-vivo comparative study of drug
distribution profiles
Mice were split into three treatment groups: (1) R-FP solu-
tion (10 mg/kg, i.n.), (2) R-FP solution (10 mg/kg, p.o.)
and (3) R-FP-loaded albumin nanoparticles (R-FP NP)
(2 mg/kg, i.n.). For administration to the mice, R-FP solu-
tion was prepared by solubilizing the drug in 0.3 M hydrox-
ypropyl-b-cyclodextrin (HPbCD), whereas R-FP NP was
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 70 (2018), pp. 59–69
R-flurbiprofen-albumin nanoparticles
suspended in normal saline solution. For treatment groups
(1) and (3), a dosing volume of 20–30 ll was intranasally
administered to each mouse in standardized steps using
thin gel loading pipette tips.[33] For example, if a dosing
volume of 24 ll was required, we loaded the pipettor with
6 ll, administered to the left nostril first two small droplets
with about half the volume (3 ll) per droplet, followed by
a 15 s hold before repeating another 6 ll for the right nos-
tril in the same manner. After allowing the mouse to rest in
its cage for 2 min, the next 2 9 6 ll administration rou-
tine was repeated for both nostrils, thus giving a total dos-
ing volume of 24 ll for one mouse. For group (2), the dose
was administered via oral gavage. Mice from each treat-
ment group were sacrificed by CO2 euthanasia at predeter-
mined time points (0.5, 1, 1.5, 2 and 4 h) after dosing with
3–4 animals harvested per time point. Blood samples were
collected via cardiac puncture. Whole brains were carefully
removed, washed twice with normal saline solution and
dabbed gently to remove extraneous fluid. Blood samples
were immediately cooled in an ice bath and then cen-
trifuged at 4000g for 10 min to collect the plasma. All brain
and plasma samples were kept at 20 °C immediately after
collection and stored at 80 °C until analysis. On the day
of analysis, all samples were thawed and kept on ice. Brain
tissue samples were homogenized with ultrapure water in a
ratio of 1 part tissue mass to 1 part water. Analytes were
extracted using protein precipitation by adding four vol-
umes of crashing solution (containing 3 lg/ml ibuprofen
as an internal standard in 1 : 1 mixture of methanol and
acetonitrile). All samples were vortex-mixed at high speed
for 5 min and centrifuged at 20,000g for 30 min at 4 °C to
pellet the precipitated protein. Supernatants were then
transferred to glass vials for analysis using a validated
reversed-phase HPLC method. Analyte/IS peak area ratios
of spiked plasma and brain samples were used to construct
linear calibration curves for each sample matrix accord-
ingly, which were then used to quantify R-FP concentra-
tions in experimental samples. Drug concentrations found
in brain and plasma samples were normalized to the lowest
dose, that is, 2 mg/kg with assumption of linear kinetics.
AUC and Cmax values were also dose-normalized to
2 mg/kg.
Cell culture
Chinese hamster ovary (CHO) cells stably transfected with
mouse Ab precursor protein 695 (CHO-APP695) were used
as the AD cell model for our in-vitro experiments. Cells
were incubated at 37 °C in the presence of 5% CO2 for all
cell culture work. Cells were maintained in DMEM culture
medium (Sigma D1152) containing 10% FBS and supple-
mented with 100 units/ml of penicillin and 100 mg/ml of
streptomycin.
61
R-flurbiprofen-albumin nanoparticles
Cytotoxicity of serum albumin-based
nanoparticles in cells
Cells were seeded on 96-well cell culture plates at
1 9 103 cells/well. After overnight incubation, the culture
medium was removed, and the cells were treated with R-FP
solution, R-FP NP and blank NP accordingly. R-FP solu-
tion and R-FP NP were dosed at equivalent incubation con-
centrations of 25, 50, 100 and 200 lM. Blank NP was dosed
at weights equivalent to those of the corresponding R-FP
NP concentrations. 0.2% ethanol and blank culture med-
ium served as vehicle controls for R-FP solution and R-FP
NP, respectively. After 48-h treatment, the culture medium
was removed, and the cells were stained by adding Presto-
Blue reagent (Molecular Probes) to the sample wells and
incubated for 30 min. The fluorescence readings were mea-
sured using a Tecan Infinite M200 microplate reader
(Tecan, Switzerland).
Analysis of mitochondrial function in cells
Mitochondrial function was assessed using the Seahorse
Bioscience XF24 Extracellular Flux Analyzer by measuring
the real-time oxygen consumption rate (OCR) of
CHO-APP695 cells with sequential injection of respiratory
inhibitors, namely oligomycin, carbonyl cyanide-p-trifluor-
omethoxyphenylhydrazone (FCCP) and a mixture of
rotenone + antimycin A. Oligomycin is an ATP synthase
inhibitor, FCCP induces maximal respiration and the rote-
none + antimycin A mixture shuts down mitochondrial
respiration completely. To the best of our knowledge, the
CHO-APP695 cell line has not been used in extracellular
flux analysis before. As such, the optimal cell seeding den-
sity and FCCP concentration required to generate maximal
OCR were first determined through preliminary titration
experiments. CHO-APP695 cells were plated on XF24 cell
culture microplates (Seahorse Bioscience) at a density of
17.5 9 103 cells/well, which was predetermined to be the
optimal cell seeding density. For each microplate, four wells
were left unseeded to serve as background measurements.
After overnight incubation, the culture medium was
removed, and the cells were treated with 1 lM R-FP solu-
tion, 1 lM R-FP NP, 10 lM R-FP NP and 1 lM blank NP
accordingly. Blank NP was dosed at a weight equivalent to
that of 1 lM R-FP NP. 0.2% ethanol and blank culture
medium representing vehicle controls were also included.
Each treatment condition was applied to 3–4 wells per plate
and repeated over several days for a total of 9–12 wells per
treatment group. After 24-h treatment, cells were washed
and incubated for 30 min in unbuffered XF assay medium
(Seahorse Bioscience) containing 2 mM L-glutamine sup-
plemented with 5.5 mM glucose and 1 mM sodium pyru-
vate at 37 °C in a non-CO2 environment. Using the XF Cell
62 © 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 70 (2018), pp. 59–69
Ling Rong Wong and Paul C. Ho
Mito Stress Test Kit (Seahorse Bioscience), OCR of the pre-
treated cells was measured with sequential injection of
1 lM oligomycin, 2 lM FCCP and 0.5 lM mixture of rote-
none + antimycin A (final concentrations are given). Basal
OCR was measured at three consecutive time points for
3 min each separated by a 3 min mix and 2 min wait. Sub-
sequent injection of each inhibitor into was also followed
by three cycles of 3 min mix, 2 min wait and 3 min mea-
surement of OCR.
All OCR measurements were first subjected to back-
ground correction. Next, every three consecutive OCR
readings were averaged to give a single measure of respi-
ration for each state for each well. Outlier wells were
excluded as described previously,[34] especially those wells
with OCR that did not respond to FCCP stimulation.
The mean OCR values were also subjected to Grubb’s test
with significance level set at P < 0.05 and those wells
meeting this criterion were excluded from further analy-
sis. Subsequently, the mean OCR obtained after injection
of the rotenone + antimycin A mixture (which inhibits
all mitochondrial respiration) for each well was sub-
tracted from its corresponding basal, oligomycin and
FCCP rates to report only mitochondrial OCR values.
OCR data were normalized to total protein content which
was determined for each well using the Micro BCA Pro-
tein Assay Kit (Thermo Scientific Pierce) as per the man-
ufacturer’s instructions.
Statistics
Standard unpaired one-way ANOVA was applied for statis-
tical comparison of in-vivo experimental data, and Tukey-
adjusted P-value was used to determine significance. For
analysis of mitochondrial function in cells, each well was
considered as a separate experiment, and statistical signifi-
cance was calculated by standard unpaired one-way
ANOVA with P-values adjusted for multiple comparisons
using the Benjamini–Hochberg method. Significance was
defined as P ≤ 0.05. Analyses were performed using Graph-
Pad Prism 7.
Results
R-FP albumin nanoparticles are stable and
quickly liberate their cargo within nasally
applied time period
The particle size of the drug-loaded albumin nanoparticles
remained relatively stable after reconstituting in normal sal-
ine solution and standing at room temperature over 24 h
(284.4  14.9 nm). PDI was measured to be 0.404 
0.065. The in-vitro drug release profile of R-FP NP is
shown in Figure 1. The albumin nanoparticles released
Ling Rong Wong and Paul C. Ho
Figure 1 In-vitro drug release profile of R-FP NP sampled over 48 h.
Dialysis units were incubated in a PBS reservoir maintained at 37 °C
with gentle magnetic stirring. Data are represented as mean  SD
(N = 3).
more than 60% of their cargo within 2 h, which is similar
to a previous study involving polylactide-based flurbipro-
fen-loaded nanoparticles.[35]
R-FP albumin nanoparticles afford higher
drug distribution to brain after intranasal
administration in vivo
A validated reversed-phase HPLC method was used to
quantify R-FP concentrations in plasma and brain samples
harvested at different time points (0.5, 1, 1.5, 2 and 4 h)
from mice in the three different treatment groups, namely
i.n. R-FP solution, p.o. R-FP solution and i.n. R-FP NP.
The dose-normalized profiles of mean plasma
Figure 2 Dose-normalized R-FP concentrations vs time plots for (a) plasma and (b) brain samples harvested from mice given R-FP solution
(10 mg/kg, i.n.), R-FP solution (10 mg/kg, p.o.) and R-FP NP (2 mg/kg, i.n.). Data are represented as mean  SE with N = 3–4 animals harvested
per time point.
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 70 (2018), pp. 59–69
R-flurbiprofen-albumin nanoparticles
concentrations or mean brain concentrations of R-FP vs
time are shown in Figure 2a and 2b, respectively. Relevant
pharmacokinetic parameters are summarized in Table 1.
Mice administered with i.n. R-FP NP showed a significantly
higher plasma concentration maximum (Cmax,
19.72  2.38 lg/ml) than did mice treated with i.n. R-FP
solution (11.01  0.59 lg/ml, adjusted P = 0.0046) and
p.o. R-FP solution (8.17  0.33 lg/ml, adjusted
P = 0.0014). Significantly higher brain Cmax was also
observed for mice given i.n. R-FP NP (301.2  36.3 ng/ml)
compared to mice given i.n. R-FP solution (121.3 
23.3 ng/ml, adjusted P = 0.0056) and p.o. R-FP solution
(117.0  23.9 ng/ml, adjusted P = 0.0071). The peak time
(Tmax) of plasma and brain R-FP concentrations for mice
from both intranasal treatment groups was 0.5 h after drug
administration. For mice in the oral treatment group,
plasma and brain Tmax were achieved slower at 2 and 1 h,
respectively. The area-under-the-curve (AUC)0–4 h values
of plasma and brain R-FP were 4.50 9 104 and
8.87 9 102 lg
h/l, respectively, for mice administered i.n.
R-FP NP. For mice that received i.n. R-FP solution, the
dose-normalized plasma and brain R-FP AUC0–4 h values
were 2.33 9 104 and 3.31 9 102 lg
h/l, respectively. For
mice that were given p.o. R-FP solution, their plasma and
brain R-FP AUC0–4 h values were 2.74 9 104 and
3.65 9 102 lg
h/l, respectively, after dose normalization.
Additionally, the brain-to-plasma ratios were obtained to
assess brain delivery of R-FP.[36] Mice treated with i.n.
R-FP NP exhibited the highest brain-to-plasma ratios at all
time points tested compared to those treated with i.n. and
p.o. R-FP solution (Figure 3), suggesting the potential of
albumin-based nanoparticulate carriers to increase distri-
bution of R-FP to the brain. This was corroborated by the
63
R-flurbiprofen-albumin nanoparticles Ling Rong Wong and Paul C. Ho

Table 1 Pharmacokinetic parameters in plasma and brain of mice in the three treatment groups: (A) R-FP solution (10 mg/kg, i.n.), (B) R-FP solu-
tion (10 mg/kg, p.o.) and (C) R-FP NP (2 mg/kg, i.n.)

Plasma Brain
Treatment
a a AUCbrain
group Cmax (lg/ml) Tmax (h) AUC0 ? 4 h (lg
h/l) Cmax (ng/ml)a Tmax (h) AUC0 ? 4 h (lg
h/l)a AUCplasma

A 11.01  0.59 0.5 2.33 9 104 121.3  23.3 0.5 3.31 9 102 1.42%
B 8.17  0.33 2 2.74 9 104 117.0  23.9 1 3.65 9 102 1.33%
C 19.72  2.38 0.5 4.50 9 104 301.2  36.3 0.5 8.87 9 102 1.97%
a
Normalized by the dose.

Figure 4 Cell viability expressed as a percentage of vehicle-treated

Figure 3 Brain-to-plasma ratio vs time plots for mice given R-FP controls after pretreatment with R-FP solution, R-FP NP and blank NP
solution (10 mg/kg, i.n.), R-FP solution (10 mg/kg, p.o.) and R-FP NP at equivalent incubation concentrations or equivalent weights. Data
(2 mg/kg, i.n.). Data are represented as mean  SE with N = 3–4 are represented as mean  SE (N = 18).
animals harvested per time point.

AUCbrain-to-AUCplasma ratio which is another indication of
the extent of brain uptake.[37] Among the three treatment
groups, mice that received i.n. R-FP NP showed the highest
AUCbrain-to-AUCplasma ratio (1.97%) compared to mice
that were given i.n. R-FP solution (1.42%) and p.o. R-FP
solution (1.33%).
R-FP albumin nanoparticles show no
cytotoxicity at low micromolar
concentrations
The cytotoxicity of R-FP solution and R-FP NP was
investigated in CHO-APP695 cells. To confirm that albu-
min itself does not affect cell viability, CHO-APP695 cells
were also treated with blank albumin nanoparticles, that
is, blank NP at weights equivalent to those of the
corresponding drug-loaded albumin nanoparticles. Cells
treated with R-FP solution and R-FP NP showed
comparable viability up to 200 lM although a slight
decline was observed after 100 lM (Figure 4). These
results confirm the ability of albumin nanoparticles to
deliver R-FP without causing cytotoxicity at low micro-
molar concentrations.
R-FP albumin nanoparticles increase basal
and maximal mitochondrial respiration at
in-vivo brain concentration
We also investigated whether the in-vivo R-FP concentra-
tion (1.23 lM) achieved in the brain afforded by intranasal
administration of our nanoparticulate drug formulation
had any effect on mitochondrial respiratory activity in an
AD cell model using extracellular flux analysis. The vehicle
0.2% ethanol used for R-FP solution was confirmed not to
significantly affect mitochondrial respiration, thus treat-
ment groups were all compared to the blank culture med-
ium control for purposes of uniformity. After discounting
non-mitochondrial respiration, CHO-APP695 cells pre-
treated with 1 lM R-FP solution, 1 lM R-FP NP, 10 lM
R-FP NP and 1 lM blank NP were all found to exhibit
significantly higher basal respiration compared to the blank
control (Figure 5). Pretreatment with 1 lM R-FP solution
showed a significantly higher basal OCR of 16.35 
1.76 pmol/min/lg protein as compared to the control basal
OCR of 9.70  0.95 pmol/min/lg protein (adjusted
P = 0.0146). Significantly higher basal OCRs were also
observed for cells pretreated with 1 lM R-FP NP
(15.90  2.17 pmol/min/lg protein, adjusted P = 0.0146)

64 © 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 70 (2018), pp. 59–69
Ling Rong Wong and Paul C. Ho R-flurbiprofen-albumin nanoparticles

Figure 5 Oxygen consumption rates for CHO-APP695 cells treated with blank culture medium (control), 1 lM R-FP solution, 1 lM R-FP NP, 10 lM
R-FP NP and blank albumin NP before any pharmacological perturbation (basal respiration), followed by sequential injection of oligomycin, FCCP
and rotenone + antimycin A mixture. The oxygen consumption rate (OCR) from each well following rotenone + antimycin A injection has been
subtracted before data analysis to consider only mitochondrial respiration. OCR data are normalized to total protein content and presented as
mean  SE (N = 3–4). Outliers have been omitted. *indicates P < 0.05; **indicates P < 0.01 when compared against the blank control. CHO,
Chinese hamster ovary.

and 10 lM R-FP NP (14.39  1.48 pmol/min/lg protein, Table 2 shows ATP-linked OCR estimates as well as
adjusted P = 0.0344). Even pretreatment with blank albu- coupling efficiency and spare respiratory capacity ratios for
min NP was found to significantly increase basal OCR cells in the various treatment groups. ATP-linked OCR was
(15.89  0.81 pmol/min/lg protein, adjusted P = 0.0146). estimated from the difference between basal OCR and the
Across all the four treatment groups, there was also a trend OCR after oligomycin injection as oligomycin is an ATP
for higher proton leak compared to the blank control (as synthase inhibitor. All four treatment groups exhibited
revealed by the OCR after oligomycin injection), although higher ATP-linked OCRs as compared to the control group
this did not reach statistical significance. Upon stimulation but did not achieve statistical significance. Coupling effi-
by FCCP, the maximal mitochondrial respiration was ciency, which is defined as the proportion of mitochondrial
found to be significantly increased for cells pretreated with respiratory rate used to drive ATP synthesis, was approxi-
1 lM R-FP NP (22.33  3.74 pmol/min/lg protein, mated by the proportion of OCR sensitive to oligomycin
adjusted P = 0.0486) relative to the control group relative to basal OCR. Cells pretreated with 1 and 10 lM
(13.51  1.50 pmol/min/lg protein). Pretreatment with R-FP NP demonstrated higher coupling efficiency values of
an equivalent amount of blank albumin NP also signifi- 21.06  5.94 and 27.84  7.17%, respectively, when
cantly increased the maximal mitochondrial respiration
(21.51  2.09 pmol/min/lg protein, adjusted P = 0.0486).
Table 2 ATP-linked oxygen consumption rates, coupling efficiency
Similarly, cells pretreated with 1 lM R-FP solution and and spare respiratory capacity ratios of CHO-APP695 cells pretreated
10 lM R-FP NP demonstrated higher maximal OCRs of with blank culture medium (control), 1 lM R-FP solution, 1 lM R-FP
18.81  2.04 and 20.66  1.61 pmol/min/lg protein, NP, 10 lM R-FP NP and blank albumin NP
respectively, when compared to the control group, but
ATP-linked OCR Coupling Spare
these did not achieve statistical significance. Non-mito- Treatment (pmol/min/lg efficiency respiratory
chondrial respiration following rotenone + antimycin A group protein) (%) capacity (%)
addition for cells pretreated with R-FP solution, R-FP NP
Control 1.10  0.39 10.25  3.15 126.80  17.77
and blank NP was all lower than the control group,
1 lM R-FP 2.46  0.50 16.20  5.28 115.89  23.22
although only the blank NP treatment group showed signif- solution
icantly lower non-mitochondrial OCR (5.04  0.65 pmol/ 1 lM R-FP NP 3.22  0.87 21.06  5.94 139.96  8.84
min/lg protein, adjusted P = 0.0050) compared to the 10 lM R-FP NP 3.76  0.83 27.84  7.17 146.14  13.51
control non-mitochondrial OCR of 8.02  0.20 pmol/ Blank NP 3.77  1.23 25.05  9.01 138.39  19.97
min/lg protein. CHO, Chinese hamster ovary; OCR, oxygen consumption rate.

© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 70 (2018), pp. 59–69 65
R-flurbiprofen-albumin nanoparticles
compared to pretreatment with 1 lM R-FP solution
(16.20  5.28%). Blank albumin NP treatment resulted in
higher coupling efficiency of 25.05  9.01% as compared
to treatment with 1 lM R-FP NP. Overall, cells pretreated
with 10 lM R-FP NP showed the highest coupling effi-
ciency. The reserve respiratory capacity (estimated by the
ratio of FCCP-stimulated OCR compared to basal OCR) of
the cells pretreated with 1 and 10 lM R-FP NP was found
to be higher (139.96  8.84 and 146.14  13.51%, respec-
tively) than 1 lM R-FP solution (115.89  23.22%). Blank
albumin NP treatment demonstrated similar reserve
respiratory capacity of 138.39  19.97% as 1 lM R-FP NP
treatment but is still lower than cells pretreated with 10 lM
R-FP NP.
Discussion
The main aim of this study was the exploration of safe and
biocompatible serum albumin as a potential nanoparticu-
late carrier for nose-to-brain delivery of R-FP which failed
to delay AD progression in clinical trials despite showing
promise in preclinical studies in part due to its insufficient
brain penetration after oral administration. We believe that
serum albumin may function as a good carrier to facilitate
the delivery of therapeutic agents to AD-related brain
regions using the nasal route of administration. AD trans-
genic mice were not used for our in-vivo study because the
BBB integrity is compromised in these animals.[38] A previ-
ous clinical study found that there exists no generalized
abnormality of the BBB in patients with AD.[39] As such,
healthy C57BL/6 mice with intact BBB serve as an appro-
priate model to investigate R-FP brain penetration for the
purpose of AD treatment.
Numerous techniques to prepare albumin-based
nanoparticles have been reported.[40] In this study, the
approach adopted was based on the technique first demon-
strated by Desai et al.[31] for the production of Abraxaneâ,
a human serum albumin-based paclitaxel nanoparticulate
formulation approved by FDA for the treatment of meta-
static breast cancer. It was proposed that exposure to the
high shear stress generated during high-pressure homoge-
nization causes intense local heating regions in the liquid
which can oxidize free thiol groups as well as disrupt
intramolecular disulfide bonds present in the protein mole-
cules, thereby creating new crosslinking intermolecular
disulfide bonds to produce nanoparticles. The high shear
action also serves to disperse the drug molecules (which are
suspended in a dispersing agent such as chloroform) into
the albumin nanoparticles.
From our in-vivo study, we observed significantly higher
dose-normalized brain Cmax in mice administered i.n. R-FP
NP compared to mice that were given i.n. and p.o. R-FP
solution. Albumin has been reported to be rapidly uptaken
66 © 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 70 (2018), pp. 59–69
Ling Rong Wong and Paul C. Ho
from the nasal mucosa into the brain within five minutes
after intranasal administration via transcytosis across the
nasal epithelium, followed by paracellular transport to
reach further regions within the brain.[26–28] Our R-FP NP
formulation was shown to release more than 60% of its
cargo within two hours in vitro, which is likely attributed to
the non-covalent interactions between the drug molecule
and the albumin carrier. Therefore, the albumin nanoparti-
cles could reach the brain fast enough with its cargo rela-
tively intact and liberate the drug in the brain. Free
flurbiprofen does not exhibit saturable BBB uptake even
when tested at 1 mM,[41] thus supporting the assumption of
linear kinetics in our calculations. Mice that received i.n.
R-FP NP exhibited the highest brain-to-plasma ratio
maximum of 2.3%, whereas mice given i.n. and p.o. R-FP
solution showed brain-to-plasma ratio maxima of 1.8%
and 1.3%, respectively. In a previous study, a comparable
2.2% brain-to-plasma ratio was achieved only when the
mice were given orally 12.5 times the R-FP dose we used
for our i.n. R-FP NP.[42] Another study reported that the
maximum brain penetration of R-FP was approximately
1% in healthy elderly individuals after oral dosing.[43] This
result clearly demonstrates the advantage of serum albumin
as a nanoparticulate carrier for nose-to-brain delivery of a
therapeutic agent such as R-FP which is handicapped by
limited brain penetration after oral administration. Increas-
ing brain penetration of R-FP will result in more drug com-
pound reaching the brain from the same dose given to the
patient, thus therapeutic levels required for the drug to
exert its treatment effects in brain tissue will be more easily
reached. The use of whole brain to determine drug concen-
trations in this study serves as a proof of concept to
demonstrate the feasibility of R-FP NP given intranasally to
enhance drug accumulation in the brain. Further studies
are needed to complete the pharmacokinetic examination
of drug levels in different regions of the brain to establish
the existence of direct nose-to-brain transport of intrana-
sally administered serum albumin-based nanoparticles.[44]
We also investigated whether the in-vivo R-FP concen-
tration achieved in the brain afforded by i.n. R-FP NP had
any effect on mitochondrial bioenergetics in an AD cell
model using extracellular flux analysis. Compromised
mitochondrial bioenergetics have been shown to precede
the development of disease pathology in AD transgenic
mice.[45] Reduced basal and maximal mitochondrial respi-
ration, as well as diminished reserve respiratory capacity
were observed in fibroblasts derived from patients with
AD.[46] The CHO-APP695 amyloidogenic cell model was
employed for our in-vitro experiments. CHO cells have
been used to screen potential therapeutic agents for treating
dementia,[47] and the CHO-APP695 model was shown to be
a suitable in-vitro model for early-stage AD research in
which mitochondrial dysfunction was observed to precede
Ling Rong Wong and Paul C. Ho R-flurbiprofen-albumin nanoparticles

extracellular Ab accumulation.[10] We observed significant
improvements in basal mitochondrial respiration in cells
pretreated with 1 lM R-FP solution, 1 lM R-FP NP, 10 lM
R-FP NP and 1 lM blank NP. Notably, the increase in basal
mitochondrial respiratory activity for cells pretreated with
1 lM R-FP NP was larger than that pretreated with 10 lM
R-FP NP. This is consistent with an early study which
showed flurbiprofen to exert stimulatory effect on basal
mitochondrial respiration at around 1 lM but became inhi-
bitory at higher concentrations in isolated rat mitochon-
dria.[48] However, only cells pretreated with 1 lM R-FP NP
and 1 lM blank NP demonstrated significant improve-
ments in maximal mitochondrial respiration, but not those
cells pretreated with 1 lM R-FP solution. The addition of
albumin has been reported to improve the mitochondrial
respiration of isolated rodent brain mitochondria and this
improvement was associated with its antioxidant proper-
ties.[49] As revealed by the OCRs following rotenone + anti-
mycin A injection in Figure 5, we observed significantly
lowered non-mitochondrial OCR after cells were pretreated
with blank albumin NP, which is in agreement with lower
extramitochondrial reactive oxygen species.[50] Coupling
efficiency, which is defined as the proportion of mitochon-
drial rate used to drive ATP synthesis, is higher for cells
pretreated with 1 and 10 lM R-FP NP than 1 lM R-FP
solution, suggesting that there may be additive or synergis-
tic effects on mitochondrial ATP-generating capacity when
R-FP was dosed as serum albumin-based nanoparticles
compared to a simple drug solution. Moreover, the reserve
respiratory capacity of the cells pretreated with 10 lM R-FP
NP turned out to be the greatest, indicating that R-FP albu-
min nanoparticles could confer the cells greater ability to
respond to energetic demands which is thought to be
important for neuronal cells with disrupted mitochondrial
function in AD.[51] Treatment using blank albumin NP
itself was observed to exhibit higher coupling efficiency and
similar reserve respiratory capacity when compared to
1 lM R-FP NP, but increasing the dosing to 10 lM R-FP
NP increased the coupling efficiency and reserve respiratory
capacity. Albumin is well-known for its versatility to bind
many ligands, including reactive redox species and Ab
peptide.[52,53] The binding of drug molecules to albumin
could have shielded these active sites on the biomolecule
and limited the effects of albumin in 1 lM R-FP NP,
although improved coupling efficiency and reserve respira-
tory capacity could still be achieved by increasing the dos-
ing to 10 lM R-FP NP. In summary, the blank albumin NP,
and the R-FP NP at low (1 lM) and high dose (10 lM) of
R-FP have variable effects on the mitochondrial function.
The blank NP has the highest ATP-linked OCR, although
the 10 lM R-FP NP has comparable value (Table 2). The
10 lM R-FP NP has the highest values in both coupling effi-
ciency and reserve respiratory capacity. The whole picture
is quite complicated, and the real functional effects of the
respective blank NP and the R-FP NP at low and high doses
of R-FP can only be confirmed in in-vivo models.
Conclusions
Serum albumin-based R-FP nanoparticles administered via
the nasal route enhanced the relative accumulation of the
drug in the brain. In addition, CHO-APP695 cells were
observed to exhibit greater ability to respond to energetic
demands when R-FP was dosed as albumin-based nanopar-
ticles at in-vivo brain concentration. This combination of
nose-to-brain delivery potential and restoration of mito-
chondrial bioenergetics qualities makes serum albumin-
based nanoparticles a promising approach in delivering
therapeutic agents to the brain to address the mitochon-
drial dysfunction theme underlying the pathophysiology of
AD.
Declarations
Acknowledgements
Ling Rong Wong was the recipient of the Tan Ean Kiam
Graduate Fellowship in Science. This work was supported
by research grants from the Academic Research Funding,
National University of Singapore (R148-000-180-112). We
also thank Mai Gamal Ahmed Ahmed Elhennawy who pro-
vided expertise in the animal experiments.

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