International Journal of Pharmaceutics 497 (2016) 268–276

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical potential of tacrolimus-loaded albumin nanoparticles

having targetability to rheumatoid arthritis tissues
Le Quang Thaoa , Hyeong Jun Byeona , Changkyu Leea , Seunghyun Leea , Eun Seong Leeb ,
Han-Gon Choic , Eun-Seok Parka , Yu Seok Youna,*
a
School of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon 16419, Republic of Korea
b
Division of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do 14662, Republic of Korea
c
College of Pharmacy, Hanyang University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 15588, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: Albumin is considered an attractive dug carrier for hydrophobic drugs to target inflamed joints of
Received 5 October 2015 rheumatoid arthritis. This study focused on the pharmaceutical potential of albumin-based nanoparticles
Received in revised form 10 November 2015 (NPs) on delivery of tacrolimus (TAC) to enhance targetability and anti-arthritic efficacy. TAC-loaded
Accepted 3 December 2015
human serum albumin (HSA) nanoparticles (TAC HSA-NPs) were prepared using the nabTM technology.
Available online 4 December 2015
The resulting NPs were 185.8
6.8 nm in diameter and had a zeta potential value of 30.5
1.1 mV, as
determined by dynamic light scattering. Particles were uniformly spherical in shape as determined by
Keywords:
transmission electron microscopy. The encapsulation efficacy of TAC was 79.3
3.7% and the water
Albumin
Nanoparticles
solubility was over 46 times greater than that of free TAC. TAC was gradually released from NPs over 24 h,
Tacrolimus which is sufficient time for targeting and treatment of the NPs in inflamed arthritis via intravenous
Rheumatoid arthritis injection. In vitro study using splenocytes excised from spleens of mice following induction of arthritis
Collagen-induced arthritis using collagen clearly demonstrated the anti-proliferative activity of TAC HSA-NPs on activated T cells
Targeting compared with non-activated T cells. Furthermore, TAC HSA-NPs displayed significantly more anti-
arthritic activity than TAC formulations including intravenously administered TAC solution or oral TAC
suspension, as reflected by the incidence of arthritis and clinical score (1.6 vs. 3.2 and 5.0, respectively).
These improvements were due to the targetability of HSA that facilitated the accumulation of TAC HSA-
NPs at inflamed arthritis sites. TAC HSA-NPs are a promising drug delivery system to enhance water
solubility and increase accumulation in joints for treatment of rheumatoid arthritis.
ã 2015 Elsevier B.V. All rights reserved.

1. Introduction in arthritis. Inflamed tissues require much more albumin as a

Albumin is a versatile attractive biomaterial or carrier for many
therapeutics in treating a variety of diseases including cancer,
arthritis and diabetes (Kratz, 2008; Kratz and Warnecke, 2012). It is
used as a conjugation counterpart for many drugs like doxorubicin,
methotrexate and exendin-4 (Bae et al., 2012; Choi et al., 2014;
Stehle et al., 1997; Kim et al., 2010), and is extensively utilized in
preparing nanoparticles (NPs) including anti-cancer agents be-
cause albumin NPs display highly selective targetability to tumor
tissues due to the enhanced permeability and retention effect and
gp60-mediated transcytosis (Sage et al., 1984; Elsadek and Kratz,
2012). Interestingly, albumin has special ability for arthritis
targeting because it markedly accumulates in inflamed tissues
* Corresponding author. Fax: +82 31 290 7724.
E-mail address: ysyoun@skku.edu (Y.S. Youn).
relevant energy source than normal tissues (Wilkinson et al., 1965).
Therefore, synovial cells are highly up-regulated in the up-take of
albumin, and the permeability of blood-joint barrier in inflamed
joint of patients with rheumatoid arthritis (RA) is remarkably
enhanced (Ballantyne et al., 1971; Levick, 1981).
Tacrolimus (FK 506; TAC) is a highly lipophilic 23-member
macrolide lactone antibiotic isolated from Streptomyces tsuku-
baensis that is primarily used clinically as an immunosuppressant
(Patel et al., 2012; Spencer et al., 1997). TAC is 10–100 times more
potent than cyclosporine A in inhibiting the production of
lymphokines involved in interleukin (IL)-2 production and so
has become widely used in organ transplantations (Letko et al.,
1999; Tocci et al., 1989). Especially, TAC has demonstrable anti-
arthritic activity in rodents through the suppressing of inflamma-
tion and expression of tumor necrosis factor-alpha (TNF-a) and IL-
1b, which reduces damage to bone and cartilage (Magari et al.,
2003; Sakuma et al., 2000). TAC is therapeutically effective in RA

http://dx.doi.org/10.1016/j.ijpharm.2015.12.004
0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
 L.Q. Thao et al. / International Journal of Pharmaceutics 497 (2016) 268–276
patients following the failure methotrexate treatment (Furst et al.,
2002).
Among many methods to prepare albumin-based NPs including
desolvation, thermal gelation, emulsification, self-assembly or
nano spray drying, the nanoparticle albumin-bound (nabTM)
technology is the most effective and cutting-edge preparation
method (Fanciullino et al., 2013; Min et al., 2015; Elzoghby et al.,
2012). This NP formulation uses only albumin, hydrophobic drugs,
and a small quantity of organic solvents that can be completely
removed by evaporation. The NPs can be easily formed using
several cycles of high-pressure homogenization (Desai et al.,
2006). In particular, the paclitaxel-bound albumin NP formulation
has available commercially as Abraxane1 since 2005, and a variety
of hydrophobic drugs including rapamycin and docetaxel are in
clinical phases of testing in conjunction with the nabTM technology
(Elsadek and Kratz, 2012).
Here, we sought to develop an injection-based TAC-loaded
albumin NP formulation for the treatment of RA. TAC is a good
drug candidate for albumin NPs because it is practically
insoluble and binds well to albumin molecules. Furthermore,
albumin is effectively accumulated in inflamed synovial tissues
in RA; NPs loaded with albumin and TAC should selectively
target arthritis tissues. This study examined the physicochemi-
cal properties, in vitro bioactivity in mouse splenocytes,
targetability and anti-arthritic efficacy of the TAC-loaded human
albumin serum NPs (TAC HSA-NPs) in a collagen-induced mouse
model of arthritis.
2. Materials and methods
2.1. Materials
TAC with a purity of 99.6% was obtained from the Research
Laboratories of ChongKunDang Pharm. Inc. (Yongin, Korea). HSA
(99% pure, 66.5 kDa) was purchased from Sigma–Aldrich (St.
Louis, MO, USA). Cy5.5-NHS ester dye was purchased from GE
Healthcare (Piscataway, NJ, USA). Cell Counting Kit-8 (CCK-8) was
purchased from Dojindo Molecular Technologies Inc. (Gaithers-
burg, MD, USA). Bovine type-II collagen (CII, 2 mg/ml), complete
Freund’s adjuvant (CFA) and incomplete Freund’s adjuvant (IFA)
were purchased from Chondrex, Inc. (Redmond, USA). RPMI
1640 medium, fetal bovine serum (FBS) and penicillin/streptomy-
cin (P/S) were obtained from Corning (Corning, NY, USA). Unless
specified all other reagents and chemicals were purchased from
Sigma–Aldrich.
2.2. Animals
DBA/1 mice (male, 7–8 weeks old, 20–22 g) were purchased
from the Hanlim Experimental Animal Laboratory (Seoul, Korea).
All experiments related to animals were conducted according to
the guidelines of the National Institute of Health (NIH) on the use
of laboratory animals (NIH publication 80–23, revised in 1996).
Animals were acclimated for a week before use and housed with
free access to feed and water. This animal study was approved by
the Ethical Committee on Animal Experimentation of Sungkyunk-
wan University.
2.3. Preparation of TAC HSA-NPs
TAC HSA-NPs were prepared using the nabTM technology (Fu
et al., 2009; Zhao et al., 2015) with slight modifications. Briefly, HSA
(150 mg) was dissolved in 15 ml deionized water (DW). TAC
(7.5 mg) and cholesterol (15 mg) were separately dissolved in
0.3 ml of a 9:1 solution of chloroform and ethanol. Especially,
cholesterol was added to increase the hydrophobic attraction
269
between albumins combined with TAC in the process of
nanoparticle formation (Zhao et al., 2015). These two solutions
were then mixed at low rotating speed to form a crude emulsion
prior to high-pressure homogenization using an EmulsiFlex-
B15 device (Avestin, Ottawa, Ontario, Canada) at 20,000 psi for
nine cycles. The resulting colloid was rotary evaporated to remove
chloroform at 40  C for 15 min under reduced pressure. The
obtained NPs were centrifuged at 6000 rpm for 10 min, the
supernatant was collected and solvent was removed by lyophili-
zation. The product was stored at 70  C until required.
2.4. Characterization of TAC HSA-NPs
The particle size (mean particle diameter and size distribution)
and zeta potential of the NPs were determined by dynamic light
scattering with a Zetasizer Nano ZS90 (Malvern Instruments,
Malvern, UK) using a 633 nm He-Ne laser beam with a fixed
scattering angle of 90 . Measurements were performed in
triplicate with a NP concentration of 1 mg/ml on HSA weight
basis in DW at 25  C. The morphology of the NPs was investigated
by transmission electron microscopy (TEM) using negative
staining with phosphotungstic acid (PTA, 1.0% w/v). Briefly, 20 ml
of NP (1.0 mg/ml) solution was deposited onto the carbon side of
200-mesh, formvar/carbon coated copper grid (TED PELLA,
Redding, CA, USA) and the excess solution was removed using
filter paper after 2 min. The grid was left to dry for 30 min prior to
being floated on the surface of a drop of PTA. The excess was
blotted away after 30 sec using filter paper. Subsequently, the air-
dried grid was examined by TEM using a model H-7600 microscope
(Hitachi, Tokyo, Japan).
2.5. Encapsulation efficiency and the in vitro release profile of TAC
To determine the encapsulation efficiency (EE) and loading
content (LC) of TAC in NPs, 5.0 mg of lyophilized NPs were
dissolved in 2.0 ml of a 9:1 solution of acetonitrile (ACN) and DW,
and thoroughly shaken for 5 min for precipitation protein. The
solution was centrifuged at 13,500 rpm for 10 min. TAC in the
supernatant was collected for measured using high-performance
liquid chromatography (HPLC) with a 1260 infinity system
(Agilent, Sunnyvale, CA, USA) using a LiChrospher 100 RP-
18 reverse phase column (250 mm  4.0 mm, 5 mm particle size;
Merck, Darmstadt, Germany) at 50  C. The mobile phase consisted
of 80% ACN and 20% water, as previously described with slight
modifications (Li et al., 2012). The system was run isocratically at a
flow rate of 1 ml/min and TAC was detected at 215 nm. Each sample
was assayed in triplicate. EE and LC were calculated as:
 
amount of drug entrapped
EE ¼  100:
amount of drug loaded
 
amount of drug entrapped
LC ¼  100:
total amount of NPs
In vitro drug release of TAC from NPs was investigated using the
dialysis diffusion method. Sixty milligrams of NPs were suspended
in 2 ml DW and dialyzed against 200 ml of phosphate buffered
saline (PBS, pH 7.4) containing 0.1% (v/v) Tween 80 at 37  C using a
semipermeable membrane with a 10 kDa cut-off. At predeter-
mined times, 2 ml of released medium was collected and the same
volume replaced with fresh medium. TAC in the released fluid was
measured by HPLC as described above. Each experiment was
performed in triplicate. Cumulative release was expressed as
percentage vs. initial loading amount at each time point.
270 L.Q. Thao et al. / International Journal of Pharmaceutics 497 (2016) 268–276
2.6. Water solubility study
Water solubility of TAC HSA-NPs was evaluated as procedures
described in our previous study with a little modification (Kim
et al., 2011). Briefly, 10 mg of TAC or an equivalent amount of TAC
HSA-NPs was added to 1 ml of PBS. Mixtures were thoroughly
shaken for 5 min, sonicated for 1 min, and centrifuged at 6000 rpm
for 10 min. One volume of supernatant was mixed with nine
volumes of ACN to extract TAC. The concentration of TAC was
determined by HPLC at 215 nm as described above.
2.7. Immunization of collagen-induced arthritis in DBA/1 mice
DBA/1 mice used for collagen induction of arthritis by a slight
modification of previously described procedures (Yoshino et al.,
1999; Byeon et al., 2014). Briefly, equal volumes of high purified
bovine type II collagen (CII, 2 mg/ml) and Complete Freund’s
adjuvant (CFA, 4 mg/ml) were carefully emulsified in an ice water
bath to prevent denaturation of the collagen using an electric
homogenizer at 30,000 rpm for 2–3 min. Male DBA/1 mice were
initially immunized (day 0) by subcutaneous (s.c.) injection at the
base of the tail with 100 ml of emulsion. On day 21, mice received s.
c. booster injection of 100 ml emulsion prepared as described
above, but with incomplete Freund’s adjuvant (IFA) instead of CFA.
2.8. Treatment with TAC HSA-NPs and assessment of arthritis
The anti-arthritis efficacy of TAC HSA-NPs in mice was
investigated as follows. On day 22 after the primary immunization,
mice were randomly divided into four treatment groups of five
mice. The first, third and fourth group of mice received intravenous
injections every 3 days for 3 weeks (22, 25, 28, 31, 34, 37, and
40 days after primary immunization) with 100 ml of sterilized PBS
as the positive control, TAC in an aqueous solution of 6% v/v
cremophor EL and 10% v/v ethanol (20 mg/mouse/day), and TAC
HSA-NPs (20 mg/mouse/day), respectively. The second group was
orally administered everyday with TAC suspension in an aqueous
solution of 0.5% w/v sodium carboxymethyl cellulose (30 mg/
mouse/day). Normal non-treated mice were used as negative
controls. All mice were visually examined three times per week for
the appearance of arthritis in the peripheral joints. The clinical
severity of arthritis was graded as: 0, normal; 1, erythema and
slight swelling confined to the tarsals or ankle joint; 2, erythema
and mild swelling extending from the ankle to the tarsals; 3,
erythema and moderate swelling extending from the ankle to the
metatarsal joints; 4, erythema and severe swelling encompass the
Fig. 1. Schematic diagram preparation of TAC HSA-NPs based on nabTM technology.
ankle and digits. The maximum possible score per mouse was 16
(each limb was scored on a scale of 0–4)
2.9. Histology of joints treated with TAC HSA-NPs
The histology of joints in the arthritic mice was evaluated using
a modification of a previously described procedure (Magari et al.,
2003; Byeon et al., 2014). On day 43, mice were sacrificed and knee
joints were obtained, immediately fixed in 10% neutral buffered
formalin solution and embedded in paraffin. Joint sections were
deparaffinized in xylene, rehydrated through a graded alcohol/
water mixture and stained with hematoxylin and eosin (H&E).
Stained samples were observed by optical microscopy.
2.10. Effect of TAC HSA-NPs on splenocyte proliferation
Anti-proliferative activity of TAC HSA-NPs was investigated
using a slight modification of previously described methods (Jin
et al., 2010; Kwok et al., 2012). Splenocytes were isolated from
spleens of male DBA/1 mice immediately after sacrifice 43 days
after induction of arthritis. Single cell suspension was prepared by
gently teasing the spleens on ice in a petri dish containing
Dulbecco’s PBS (antibiotic supplement) with forceps, filtration
through a 100 mm fine nylon cell strainer (BD Falcon, Franklin
Lakes, NJ, USA) and centrifugation at 1500 rpm at 4  C for 10 min to
pellet the cells. Erythrocytes were lysed by addition of 1 ml (per
spleen) of a red blood cell lysing buffer (Sigma–Aldrich), with
gentle and continuous mixing with a pipet for 2 min on ice. The
buffer was diluted with 20 ml of RPMI 1640 medium containing
10% (v/v) fetal bovine serum and 1% penicillin/streptomycin. After
centrifugation, collected cells were washed two times with
Dulbecco’s PBS and resuspended in RPMI 1640. Cell numbers
and viabilities were first assessed by trypan blue exclusion by using
a hemocytometer, and then seeded in a 96-well plate (80 ml
medium of 4  105 cells/well).
The stimulators (each 10 ml) of 500 mg/ml heat-denatured
bovine collagen type II (CII), 80 mg/ml lipopolysaccharide (LPS) or
40 mg/ml concanavalin A (Con A) were added to cells (80 ml
medium/well), which were then treated with aliquots (10 ml) of
Dulbecco’s PBS or TAC and TAC HSA-NPs at predetermined final
concentrations of 0.3 mg/ml (quoted as TAC equivalents). After
2 days incubation at 37  C in a 5% CO2 humidified, cell viability was
determined by adding 10 ml/well of CCK-8 solution and incubating
for 2 h. Cell viabilities (percentages) were calculated by expressing
absorbances at a 450-nm wavelength of treated samples as
percentages of those of untreated controls.
 L.Q. Thao et al. / International Journal of Pharmaceutics 497 (2016) 268–276
2.11. Evaluation of inflamed tissue targeting by TAC HSA-NPs in mice
To evaluate targetability, time-dependent localizations of TAC
HSA-NPs were monitored in arthritic and normal mice. Briefly, the
surfaces of TAC HSA-NPs were covalently modified with Cy5.5 NHS
ester dye in 100 mM phosphate buffer (pH 8.0) for 3 h and
unreacted Cy5.5-NHS was removed from the reaction mixture by
desalting using a Sephadex G-25 column as previously described
(Bae et al., 2012; Kim et al., 2013). A portion (100 ml) of Cy5.5-
labeled NPs was intravenously injected into each arthritic and
normal mouse. Deposition of NPs at 0, 1, 3, 6, 9, 12, and 24 h was
determined using an Optix MX3 in vivo imaging system (Advanced
Research Technologies, Saint-Laurent, Quebec, Canada).
2.12. Statistical analysis
Data are presented as mean
standard deviation. Significances
of differences were determined using the Student’s t-test. P-values
<0.05 were considered statistically significant.
3. Results
3.1. Preparation and characterization of TAC HSA-NPs
TAC HSA-NPs were prepared using the nabTM technology, which
includes the forming of crude emulsion by vortexing two
unmixable liquid phases, using a high pressure homogenizer to
create fine NPs, removal of organic solvent by rotary evaporation
and hardening by lyophilization to improve the physical stability of
NPs (Fig. 1). Consistent with previous observations (Zhao et al.,
2015), the particle diameter and zeta potential of the TAC HSA-NPs
was 185.8
6.8 nm (polydispersity index of 0.18
0.05) and
30.5
1.1 mV, respectively (Fig. 2A and B). TEM revealed the
Fig. 2. Characterization of TAC HSA-NPs according to particle size (A), zeta potential (B) and morphology (C). Scale bars in the TEM images represent 100 nm and 50 nm.
271
uniformly spherical shape. Sizes measured by TEM were similar
with those measured by dynamic light scattering (Fig. 2C). EE and
LC of TAC HSA-NPs was 79.3
3.7% and 1.5
0.1%, respectively.
3.2. In vitro release profile of TAC HSA-NPs
The release profiles of TAC HSA-NPs were evaluated in PBS
containing 0.1% (v/v) Tween 80 at 37  C (Fig. 3A). In particular,
93.8
2.4% of TAC was released from TAC HSA-NPs over the 1-day
test period.
3.3. Water solubility and reconstitutability of TAC HSA-NPs
The water solubility of TAC HSA-NPs was determined by HPLC at
215 nm (Fig. 3C and D). The solubility of TAC in PBS was
3.1
0.2 mg/ml, whereas that of TAC HSA-NPs was
142.4
0.7 mg/ml (quoted as TAC equivalents). The particle size
of TAC HSA-NPs was relatively well maintained after lyophilization
and redispersion. The colloidal solution form of TAC HSA-NPs was
almost same with that before lyophilization (Fig. 3B). NPs were
freeze-dried with no excipient because HSA acted as a cryoprotec-
tant and aided reconstitution.
3.4. Anti-arthritic efficacy of TAC HSA-NPs in the mouse model of
arthritis
Collagen-induced arthritis developed rapidly in mice immu-
nized with CII. Clinical signs of the disease first appeared in the
hind paws at approximately 23 days post-collagen induction, and
all treated mice were influenced on day 28. At 25 days after the first
immunization, the clinical incidence in PBS-treated mice was 80%.
The incidence in mice treated with oral TAC and intravenous TAC
solution was decreased to 60% and 40%, respectively. Mice that
272 L.Q. Thao et al. / International Journal of Pharmaceutics 497 (2016) 268–276

Fig. 3. (A) In vitro release profiles of tacrolimus from TAC HSA-NPs (n = 4). (B) Redispersability of TAC HSA-NPs in PBS; (C) HPLC determined solubilities of TAC and TAC HSA-
NPs in PBS (n = 3); (D) Images showed the insolubility of TAC and solubility of TAC HSA-NPs in PBS (10 mg/ml). Data represent mean
SD.

received intravenous TAC HSA-NPs showed no significant clinical
signs of arthritis at 25 days following induction (Fig. 4A). Hind-paw
erythema and swelling increased in frequency and severity in a
time-dependent manner, and a mean maximum clinical score was
reached 8.2 at 43 days following collagen induction in PBS-treated
mice. However, the clinical scores of mice treated with TAC HSA-
NPs were significantly reduced than those of mice treated with TAC
suspension or TAC solution (1.6 vs. 5 and 3.2, respectively) at
43 days (Fig. 4B). The degree of hind-paw erythema and swelling in
TAC HSA-NPs-treated mice appeared to be negligible and similar to
that of normal mice without CIA (Fig. 5). Furthermore, the weight
of the animals was not affected by immunization and drug
treatment (data not shown).
3.5. Histological evaluation of arthritic mice treated with TAC HSA-NPs
To investigate the effects of TAC HSA-NPs on pathological
changes in the inflamed joints, H&E staining was performed on
joint tissue sections obtained from hind paws of the treated or
untreated CIA mice. Images of H&E-stained knee joints from
untreated mice showed severe signs of arthritis including
penetration of inflammatory cells into joint spaces, extensive
synovitis, pannus formation, cartilage and bone erosion. Mice
treated with TAC suspension or TAC solution displayed similar
signs, but they were less pronounced. In contrast, images from
mice treated with TAC HSA-NPs revealed negligible histology

Fig. 4. In vivo anti-arthritic efficacy studies of oral TAC suspension, intravenous TAC solution and intravenous TAC HSA-NPs in DBA/1 male mice until 43 days post-CIA (5 mice
in each group). (A) Incidence (%) and (B) clinical score. Data represent mean
SD.
 L.Q. Thao et al. / International Journal of Pharmaceutics 497 (2016) 268–276 273

Fig. 5. (A) Scale of clinical scores for evaluation. (B) Images of hind paws of normal-mice and collagen-induced arthritis mice treated with PBS, oral TAC suspension,
intravenous TAC solution and intravenous TAC HSA-NPs at 21, 28, 35 and 41 days post-induction.

changes, except slight synovitis due to infiltration of immune cells
(Fig. 6).
3.6. Anti-proliferative activity of TAC HSA-NPs in mouse splenocytes
To assess the effect of TAC HSA-NPs on proliferation as reflected
in cell number, splenocytes isolated from spleens of mice were
stimulated with various autoantigens. The proliferative responses
of splenocytes cultured with CII (50 mg/ml), LPS (8 mg/ml), or Con A
(4 mg/ml) in the presence of TAC HSA-NPs at 0.3 mg/ml was
reduced by 23.4%, 39.9% and 56.7%, respectively, versus PBS-
treated controls, comparable to those of TAC solution at the same
concentration (28.1%, 43.6%, and 58.8%, respectively) (Fig. 7).

Fig. 6. Histological observations of hind paws collected from normal mice and CIA-mice that treated with PBS, oral TAC suspension, intravenous TAC solution, intravenous TAC
HSA-NPs at 43 days post-induction by H&E staining (magnification  250). I.V. TAC nanoparticle treated mouse (1. Wide joint space, 2. Smooth cartilage surface); I.V. TAC
solution treated mouse (1. Narrow joint space, 2. Cartilage and bone erosion, 3. Inflammatory cell infiltration); oral TAC suspension treated mouse (1. Ultra-narrow joint space,
2. Cartilage and bone erosion, 3. Inflammatory cell infiltration, 4. Synovial hyperplasia); PBS control (severely deformed joint).
274 L.Q. Thao et al. / International Journal of Pharmaceutics 497 (2016) 268–276
Fig. 7. Effect of TAC and TAC HSA-NPs on splenocyte proliferation in mice at 43 days
post-induction. (*P < 0.04 vs. control (PBS); n = 6).
3.7. In vivo inflamed tissues targeting in arthritic mice using Cy5.5-
labelled TAC HSA-NPs
TAC HSA-NPs were mainly distributed in the livers of normal
mice from 3 h post-injection (Fig. 8). In contrast, TAC HSA-NPs
primarily located in inflamed joints of collagen-induced mice,
while an obvious decrease in the liver was evident. Localization of
TAC HSA-NPs in inflamed hind paws was maintained continuously
with only a slight decrease in Cy5.5 NIR signal until 48 h post-
injection.
4. Discussion
RA is a chronic autoimmune-related disease that causes severe
inflammation in the tissue around the joints of human, resulting in
joint deformity, destruction and loss of function. Animal models of
Fig. 8. Location of TAC HSA-NPs in normal mice and in RA tissue sites of collagen-induced arthritis mice one day after administration.
arthritis are used to study the pathogenesis of disease and to test
the anti-arthritic potential of new drugs for clinical application.
Among the most widely used models of RA is the collagen-induced
arthritis mouse. The model is highly reproducible and shows
similarities to human RA in both immunological and pathological
features (Kannan et al., 2005; Bendele, 2001). In this study, we
selected male DBA/1 mice as the animal model of collagen-induced
arthritis to investigate the anti-arthritic activity of TAC HSA-NPs.
As shown in Figs. 4 and 5, signs of RA including erythema and
swelling were clearly evident in the hind paws from 4 weeks post-
induction in 80–100% of the mice.
Oral bioavailability of TAC is poor and highly variable due to the
low solubility, high but site-dependent permeability, extensive
first-pass metabolism in the liver and phosphorylated glycopro-
tein-mediated drug efflux (Patel et al., 2012). A marketed
intravenous formulation of TAC, Prograf1, formulated in alcohol
and polyoxyl 60 hydrogenated castor oil (HCO-60) is used for the
treatment of RA in humans. However, a major disadvantage of this
formulation is potential toxicity cause by HCO-60 as leukocytosis,
hyperpyrexia (Patel et al., 2012; Ali et al., 2010). On the contrary,
our formulation based on the nabTM technology assisted by high-
pressure homogenization associated with physical linkage of
hydrophobic drugs made NPs suitable for injection without the
presence of surfactant. In this formulation, albumin plays a role as
stabilizer when in physical conjunction with a poorly water-
soluble drug as TAC, facilitating drug dispersal in the aqueous
phase (Fig. 3B). Moreover, the negative charge of albumin on the
surface of nanoparticles contributed to stabilization of the NPs due
to electrostatic force repulsion against aggregation. Water
solubility study indicated that the solubility of TAC HSA-NPs
was 46-fold higher than that of TAC (142.4 vs. 3.1 mg/ml; Fig. 3C).
On the other hand, the binding of TAC to albumin tends to delay
TAC release from NPs until 24 h, which was considered sufficient
for intravenous injection (Fig. 3A).
It is well documented that TAC suppresses collagen-induced
arthritis effectively in dose- and time-dependent manners when
administered subcutaneously or intraperitoneally. Also in our
study, oral TAC suspensions and intravenous TAC solutions were
selected to comparatively demonstrate the anti-arthritic
 L.Q. Thao et al. / International Journal of Pharmaceutics 497 (2016) 268–276
Fig. 9. Schematic diagram delivery and targeting of TAC HSA-NPs to RA tissue sites.
domination of the albumin NP formulation. Because of low oral
bioavailability of TAC (23%), oral TAC suspension at a dose of
1.5 mg/kg (daily treatment) was considered to be equivalent of
intravenous TAC solution (1.0 mg/kg), but the dosing interval was
3 days, similar to TAC HSA-NPs (1.0 mg/kg). Focusing on the anti-
arthritic activity of our NPs, as shown in Fig. 4A, TAC HSA-NPs
significantly decreased the clinical scores in the arthritic mice (>2
times compared with TAC solution, >3.1 times compared with TAC
suspension and 5.1 compared with PBS treatment, respectively) as
reflected by the level of erythema and swelling in inflamed hind
paws (Fig. 5). Destruction of articular cartilage is associated with
loss of joint specific function, which leads to decreased quality of
life in arthritis patients. In this regard, TAC HSA-NPs were clearly
more effective than control saline and TAC suspension or TAC
solution in terms of suppression of the progression of arthritis,
which included penetration of immune cells, inflamed synovial
membrane, pannus formation and destruction of bone and
cartilage (Fig. 6).
T-cells play a crucial role in the pathogenesis of human RA as
well as collagen-induced arthritis. A previous study suggested that
inflammatory cytokines in arthritis are produced through contin-
uous activation of T cells and interaction between the activated T
cells and other immune cells (monocytes, macrophages) (Panayi
et al., 1992). TAC selectively inhibits the transcription of IL-2, a
cytokine necessary for activation of T cells, which hinders the
progression of T cells from the G1 to the S phase of the cell cycle. T-
cell anti-proliferation activity of TAC HSA-NPs on splenocytes
isolated from the arthritic mice was observed clearly by co-culture
with stimulators like CII (50 mg/ml), LPS (8 mg/ml), or Con A (4 mg/
ml). TAC HSA-NPs markedly reduced proliferation of activated T
cells without affecting normal cell proliferation (Fig. 7). It seems
plausible that the binding and incorporation of TAC to albumin in
NPs formulation may result in delayed absorption of TAC in NPs
that leads to a slightly decreased anti-proliferation of TAC HSA-NPs
compared to TAC at the same concentration. In fact, collagen is not
a strong T-cell antigen and T-cell population specific to collagen is
low. Thus, we used a high concentration of highly purified CII to
achieve T-cell responses to collagen and to restrict impaction of
275
pepsin, a strong T-cell antigen that commonly remaining in
collagen preparations.
An effective strategy to enhance bioavailability and to decrease
side effects of TAC in treatment of RA is based on targeted drug
delivery to sites of inflamed joint. A preclinical study revealed that
radio-labeled and fluorescence-labeled albumin uptake by in-
flamed hind paws increased markedly in contrast with non-
inflamed paw (Wunder et al., 2003). These observations can be
explained by significantly increasing the permeability of the blood-
joint barrier in the arthritic mice compared with normal mice in
the same manner as human RA. The present results confirm the
finding of albumin-based arthritis targeting ability. Cy5.5-labeled
TAC HSA-NPs markedly accumulated at inflamed hind paws of
mice with collagen-induced arthritis until 24 h, whereas it did not
significantly accumulate at the paw site in normal mice, but
instead extensively distributed into the liver and other organ
tissues (Fig. 8).
5. Conclusions
Albumin-based TAC NPs were obviously efficacious in sup-
pressing the progression of collagen-induced arthritis compared
with oral TAC suspension or intravenous TAC solution formula-
tions, as reflected in the reduced clinical score and incidence. In
addition, TAC HSA-NPs showed markedly improved water
solubility of TAC and displayed delayed release profile over a
24 h-period that made them suitable for injection. TAC HSA-NPs
displayed remarkable targetability to inflamed joints in the
arthritic mice versus normal mice, which might play a pivotal
role in treating collagen-induced arthritis (Fig. 9). Albumin-based
TAC NPs have promising pharmaceutical potential as a new
prototype of targeted delivery system in the treatment of RA.
Acknowledgment
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea
276 L.Q. Thao et al. / International Journal of Pharmaceutics 497 (2016) 268–276
funded by the Ministry of Science, ICT, and future Planning
(#2014002133).
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