International Journal of Pharmaceutics 498 (2016) 119–129

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International Journal of Pharmaceutics

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

Effect of particle size on the biodistribution of nano- and microparticles

following intra-articular injection in mice
Julie Pradala , Pierre Maudensa , Cem Gabayb , Christian Alexander Seemayerc ,
Olivier Jordana , Eric Allémanna,*
a
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland
b
Division of Rheumatology, Department of Internal Medicine, University Hospital and Department of Pathology and Immunology, University of Geneva,
School of Medicine, Switzerland
c
Novartis Pharma AG, Postfach, Basel, Switzerland

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

Article history: Intra-articular (IA) injection of extended drug release forms based on biodegradable microparticles holds
Received 6 September 2015 promise for the treatment of joint diseases. However, the fate of microparticles following intra-articular
Received in revised form 13 November 2015 injection is controversial and has not been thoroughly investigated. The aim of this work was therefore to
Accepted 7 December 2015
evaluate the biodistribution of fluorescent poly(lactic acid) particles of different sizes after IA injection in
Available online 10 December 2015
arthritic or healthy mice.
Regardless of the inflammatory status of the joint, 300 nm-nanoparticles leaked from the joint. Due to
Keywords:
inflammation and related increase of vascular permeability, 3 mm-microparticles that were retained in
Microparticles
Arthritic diseases
the non-inflamed synovial membrane leaked from the inflamed joint. Complete retention of 10 mm-
Hyaluronic acid microparticles was observed independently of the joint inflammatory status. Embedding particles in a
Synovial membrane hyaluronic acid gel prolonged the retention of the formulations only in inflamed joints. Depending on
In vivo biodistribution particle’s size, formulations were preferentially eliminated by blood vessels or lymphatic pathways. Poly
Intra-articular administration (lactic acid) particles of 3 mm were biocompatible and retained in knee joints at least for 6 weeks.
This work highlights the need to deliver hyaluronic acid-embedded particles of at least 3 mm to
guarantee their retention in inflamed joints. These results will contribute to the rational design of long-
lasting formulations to treat acute and chronic joint diseases.
ã 2015 Elsevier B.V. All rights reserved.

1. Introduction
Rheumatic diseases are painful conditions usually caused by
inflammation. The inflammatory component can have variable
degrees, such as in rheumatoid arthritis, or can be low as
encountered in osteoarthritis (OA). Historically, treatments for
chronic arthritic diseases were essentially focused on pain relief by
oral dosage forms. However, the poor bioavailability of active drug
substances in the joints and the major side effects that result from
distribution in non-target organs do not allow to efficiently
alleviate the symptoms of the patients. Viscosupplementation and
Abbreviations: AIA, antigen-induced arthritis; CTL, control; DiD, near-red
DilC18(5) fluorescent dye; HA, hyaluronic acid; PLA, poly(D,L)lactide; H&E,
hematoxylin and eosin; OA, osteoarthritis; PBS, phosphate buffered saline; SDS,
sodium dodecyl sulfate.
* Corresponding author at: University of Geneva, Sciences II, 30 quai E.-Ansermet,
CH-1211 Geneva 4, Switzerland.
E-mail address: eric.allemann@unige.ch (E. Allémann).
delivery of the active drug substance directly to the site of action
via intra-articular administration was demonstrated to be a
promising strategy. In most studies, rapid drug clearance from
the joint requires frequently repeated injections, which are at risk
to induce infection (Albert et al., 2006). Thus, maintaining an
effective local therapeutic concentration of the drug over longer
time periods is needed. This can be achieved using IA delivery of
biodegradable particles. Several studies report particles sizes
ranging from nanometers (Rothenfluh et al., 2008) to hundreds of
micrometers (Liggins et al., 2004) for IA administration (Horisawa
et al., 2002a; Liggins et al., 2004; Monkkonen et al., 1995; Pradal
et al., 2015; Ratcliffe et al., 1986; Rothenfluh et al., 2008; Setton,
2008). The formulation, technical and physiological parameters
have to be taken into account to deliver sufficient therapeutic
doses to the site of action. Concerning formulation parameters,
administration of large particles may help reduce the amount
injected and provide extended release properties. Additionally,
drug encapsulation efficiency usually increases with the size of
particles, which is an asset. Small particles are easier to inject, but

http://dx.doi.org/10.1016/j.ijpharm.2015.12.015
0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
120 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129

might be rapidly cleared from the joint cavity through fenestra- Staufen, Germany) in a PVAL solution (2% w/v) at an organic/
tions of the synovial membrane. The size of particles was shown to aqueous volume phase ratio of 1:3. Stirring speed and duration of
influence their fate in the joint (Horisawa et al., 2002a; Rothenfluh the emulsification were adjusted to obtain particles of 300 nm,
et al., 2008). To target cartilage or synovium, small nanoparticles 3 mm and 10 mm. The freshly prepared emulsions were then
should be formulated because larger particles would be phagocy- poured in milliQ water and stirred at 500 rpm for 2 h (Eurostar
tized by macrophages (Rothenfluh et al., 2008). Nevertheless, these Digital, IKA Labortechnik, Staufen, Germany).
studies remain unclear about the optimal size of particles to be Particles were centrifuged and washed once with PBS
used for extended residence time in the joints. Specifically, it is still supplemented with 0.1% SDS. The particles were centrifuged a
unclear in how far the joint inflammation itself may influence the second time and washed with milliQ water. Blank particles, i.e.,
biodistribution of nano- and microparticles. without the fluorescent dye, were also prepared using the same
In addition to selecting the appropriate size of particles, method and parameters and were used as control formulations.
embedding them in a hyaluronic acid (HA) gel may prolong their HA-embedded particles were prepared by mixing particles
persistence in the joints and provide viscosupplementation. suspended in PBS with an appropriate amount of concentrated
Injections of HA gels are frequently done in patients suffering HA gel at 3% (w/v) with a magnetic stirrer. The final concentration
from OA to alleviate pain via a cushioning effect. Although positive of HA was 0.6% (w/v) in these formulations.
results have been reported with the use of HA in OA patients
(Bellamy et al., 2006), the long-term clinical efficacy of IA HA
2.3. Characterization of particles
remains controversial (Arrich et al., 2005; Jubb et al., 2003).
The aim of the present study was to determine the optimal size
The size of the microparticles was determined by laser light
of particles that are retained in the mouse joint in the presence or
diffraction (Mastersizer S, Malvern Instruments Ltd., Malvern, UK).
absence of inflammation. Poly(D,L-lactide) (PLA) nano- and micro-
A Nano ZS (Malvern Instruments Ltd., Malvern, UK) was used to
particles containing DiD as a fluorescent dye were formulated and
determine the size of nanoparticles. The Nano ZS was also used to
characterized. The compatibility of the particles with synovial
assess the zeta potential of micro- and nanoparticles. DiD loading
fibroblasts was investigated in vitro. Then, particles were injected
in particles was quantified by spectrofluorometry (Safire, Tecan,
to antigen-induced arthritis (AIA) mice and to naïve mice. Using
Männedorf, Switzerland) after dissolving the particles overnight in
intravital fluorescence and fluorescence quantification in organs,
acetone.
persistence and leakage of particles from mice joints could be
DiD in vitro release kinetics from particles were carried out in
determined. The optimal size of particles was determined, taking
sink conditions by suspending 20 mg of particles in 40 mL of PBS
into account the optional dispersion of particles in HA.
containing 0.5 % SDS (m/v), and also in ethyl oleate to mimic a
physiologic and cell membrane environment, respectively. Liquid
2. Materials and methods
synovial fluid from patients suffering from arthritis was also used
as a release medium. The in vitro release samples were placed on a
2.1. Materials
see-saw rocking plate (60 rpm) at 25  C. Homogenization of the
vials before sampling was realized by upside down shaking.
Poly(D,L)-lactide (PLA, inherent viscosity 0.91 dl g
1; molecular
Aliquots of 50 mL were withdrawn and centrifuged at 26,500  g
weight: 153 kDa; PI: 1.6 determined by GPC) (Resomer1 R 207) was
using an Avanti 30 Centrifuge (Beckman, California, USA) before
provided by Boehringer Ingelheim Pharma GmbH (Ingelheim,
spectrofluorimetric assay of the supernatant. All experiments were
Germany). Hyaluronic acid (HA) (Intrinsic viscosity by Ubbelode
performed in triplicate.
viscometer: 2.76 m3 kg
1, molecular weight: 1.8 MDa) was pur-
chased from HTL (Javène-Fougères, France).
2.4. Viability tests
DiD (1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindodicarbocyanine,
4-chlorobenzenesulfonate salt) was purchased from Life Technol-
Toxicity of fluorescent particles was assessed by viability tests
ogies (Grand Island, New York, USA). Poly(vinyl alcohol) (PVAL)
performed on synovial fibroblasts from OA patients. For these
was a gift from Clariant GmbH (Frankfurt, Germany) (Mowiol1 4-
experiments, cells were deposited in a 96-well plate at a density of
88, hydrolysis degree 88%, molecular weight: 26 kDa). Dichloro-
30,000 cells/well. The following day, all aqueous suspensions of
methane and acetone were of analytical grade. Sodium dodecyl-
dye-loaded or blank particles, as well as particles dispersed in the
sulfate (SDS) and ethyl oleate were provided by Sigma–Aldrich
HA gel, were freshly prepared and adjusted to concentrations of
(Saint Louis, Missouri, United States).
1 mg/mL of polymer material. Then, 200 mL of the formulations
Human synovial fibroblasts were provided by the Division of
were added to the cells for 24 h at 37  C. Each experimental
Rheumatology from The University Hospitals of Geneva and were
condition was replicated 5 times. Then, supernatants were
obtained from a 76-year-old patient undergoing joint replacement
carefully aspirated. Viability was assessed by MTT tests. To perform
for osteoarthritis. Samples were obtained after appropriate
the tests, 50 mL of 0.1% MTT solution were added to the wells and
informed consent, and their use for research was approved by
left in contact for 3 h. Then, 200 mL of dimethyl sulfoxide was
the ethics committee of the University Hospital of Geneva. Cells
added before absorbance was measured at 595 nm.
were cultured in penicillin–streptomycin supplemented with
DMEM according to an established protocol (Pradal et al., 2015).
2.5. Localization of particles in naïve mice and in mice with antigen-
All cell experiments were carried out below passage 10. All cells
induced arthritis
were tested and maintained in mycoplasma-free conditions.
All in vivo experiments were performed in compliance with the
2.2. Formulation of particles Swiss Federal Law on the Protection of the Animals. The protocols
were accepted by the canton of Geneva Authority (Direction
PLA nano- and microparticles were prepared by a solvent- Générale de la Santé, Authorization number GE/4/14).
evaporation method in the dark. PLA R207 (20 mg mL
1) and DiD For the arthritis model, 56 C57Bl/6 male 8–10 week old mice
(2% w/w) were first dissolved in dichloromethane and then (Charles Rivers, France) were divided into 8 groups of 7 mice each
emulsified (homogenizer T25 Ultra-Turrax, IKA Labortechnik, (Table 1). Inflammation was induced as previously described
 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129 121

Table 1
Animal groups for the in vivo studies. Description of injected formulations in inflamed and naïve knee joints with size details. Formulations were suspended in PBS or mBSA in
the case of the naïve model or inflamed mouse models, respectively.

Injected formulations Abbreviations of formulations Joint inflammation status Number of mice/group

No formulation, only mBSA CTL mBSA Inflamed 7
Hyaluronic acid gel HA Inflamed 7
300 nm nanoparticles Nano Inflamed 7
HA embedded 300 nm nanoparticles Nano + HA Inflamed 7
3 mm microparticles Micro 3 Inflamed 7
HA embedded 3 mm microparticles Micro 3 + HA Inflamed 7
10 mm microparticles Micro 10 Inflamed 7
HA embedded 10 mm microparticles Micro 10 + HA Inflamed 7
No formulation CTL Naïve 3
300 nm nanoparticles Nano Naïve 4
HA embedded 300 nm nanoparticles Nano + HA Naïve 4
3 mm microparticles Micro 3 Naïve 27
HA embedded 3 mm microparticles Micro 3 + HA Naïve 27
10 mm microparticles Micro 10 Naïve 5
HA embedded 10 mm microparticles Micro 10 + HA Naïve 5

(Brackertz et al., 1977; Pradal et al., 2015). Briefly, to induce the first
immunization, an intradermal injection at 3 weeks before Day
0 was performed at the tail root with 100 mL of 2 mg/mL
methylated bovine serum albumin (mBSA) (Fluka, Switzerland)
emulsified 1:1 with Freund’s complete adjuvant (Sigma–Aldrich,
Switzerland) containing 1 mg/mL heat-inactivated and dried
Mycobacterium tuberculosis. At 2 weeks before Day 0, a second
immunization was performed by intradermal injection of 100 mL of
2 mg/mL mBSA emulsified with Freund’s incomplete adjuvant
(Sigma–Aldrich, Switzerland). On Day 0, inflammation was
induced by an intra-articular injection concomitantly with a
single treatment injection of 10 mL in the left knee. The
formulations of particles or HA-dispersed particles were dispersed
in mBSA (10 mg/kg). In this way, multiple injections were avoided
and the risk of joint infection was reduced. For all experiments, the
left knee was used to test samples. The right knee was injected only
with PBS (no mBSA) and considered as the control non-inflamed
joint (CTL).
To test samples in naïve knees, 49 mice were divided into
9 groups (Table 1). Formulations of particles or HA-dispersed
particles (10 mL) were also injected into the left knee. All
formulations contained particles at a final concentration of
36 mg/mL and in the case of HA-dispersed particles, HA was at a
final concentration of 0.6% (w/v).
2.6. Quantification of inflammation
Joint inflammation was assessed by measuring the accumulation
of 99mTc sodium pertechnetate in the knee on Days 1 and 4 after
induction of inflammation. Briefly, 10 mCi of 99mTc sodium pertech-
netate was subcutaneously injected. Thirty minutes later, external
gamma counting was used to measure accumulation of the isotope in
both knees. The ratio of 99mTc accumulation in the inflamed knee to
the contralateral control knee was calculated. Joints were considered
as inflamed when the ratio was greater than 1.1.
2.7. Fluorescence imaging
Intravital fluorescence was carried out with a Maestro
M1 imaging system (PerkinElmer, Cambridge Research and
Instrumentation Inc., Massachusetts). Images were acquired with
a cooled CCD camera system at exposure times of 5 and 500 ms
from 680 to 950 nm (each 10 nm). Fluorescence emission scans
were performed after 5 min, 1 day, 4 days, 8 days, and then, once a
week for 6 weeks, using the provided excitation and emission filter
set (excitation wavelength between 615 and 665 nm, longpass
emission filter to collect light above 700 nm). In most cases, the
emission wavelength of 720 nm was extracted from the set of
images and further analyzed with Image J software (National
Institutes of Health, Bethesda, Maryland, USA). The mean
fluorescence (FU/pixel2) multiplied by the area of the regions of
interest (pixel2) was expressed as the fluorescence integrated
intensity (FU). Auto-fluorescence images were acquired on mice
from the control group. This background was subtracted from the
corresponding subsequent images.
2.8. Sacrifice and harvest of organs
At 4, 8 or 42 days after the induction of the inflammation, a
number of animals was sacrificed. Practically, deep anesthesia was
induced by isoflurane and the total blood was collected by intra-
cardiac puncture. Then, the mice were sacrificed by spinal cord
dislocation. After animal dissection, organs and tissues of interest
were harvested and rinsed with NaCl 0.9% and blotted dry. Ex vivo
full organ fluorescence was quantified by imaging with the
Maestro with the same settings as previously indicated except
the exposure time, which was 500 ms. Heart, kidney, liver, spleen,
brain and lung were imaged. The organs and other tissues of
interest (lymph nodes) were then frozen at
20  C and kept for
further analysis. Knees were collected for histology.
2.9. Histology
After fixation in 10% formaldehyde and decalcification in 4%
Tris–EDTA solution for 4 weeks, all knee joints were cut in the
sagittal plane. After embedding in paraffin, 4 mm sections were cut
with a microtome. Sections were stained with hematoxylin–eosin
(HE) or toluidine blue and graded by one pathologist (CAS) in a
blinded manner. The severity of the synovial inflammation,
including synovial hyperplasia and the percentage of polymor-
phonuclear cell infiltration, as well as the degree of cartilage
erosion and bone destruction, were evaluated and scored from 0 to
4 (0 = normal, 1 = minimal, 2 = moderate, 3 = severe, 4 = very severe)
(Camps et al., 2005). Particles in the joints were also analyzed by a
reverse fluorescent microscope.
2.10. Biodistribution study
One milliliter of distilled water was added to 100 mL of freshly
harvested blood to induce hemolysis. Hemolyzed blood was freeze
dried and 3 mL of (N,N-dimethylformamide (DMF)) were then
added to extract the fluorescent dye. Mechanical agitation was
performed overnight and was followed by centrifugation
(2800  g, 20 min). Supernatants were collected and centrifuged
122 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129

again to collect a clear supernatant to enable quantification of the Table 3

amount of DiD by spectrofluorimetric assay (lex: 633 nm, lem: Percentage of DiD released from Nano, Micro 3 and Micro 10 in various release
media after 8 and 42 days (n = 3, SD) (all experiments were performed under sink
644 nm). conditions).
Whole organs and tissues of interest were weighted and
Time point Release media Nano Micro 3 Micro 10
aliquots of them were dissected and weighted. DMF was added to
the organ aliquots in such a way to obtain 5% (m/m) of organs in Day 8 PBS with 0.5% SDS 3.57  0.03 2.32  0.04 0.80  0.06
Ethyl oleate 2.13  0.06 1.18  0.04 b.d.l.a
DMF. Homogenization in DMF was performed until complete
Human Synovial Fluid 1.97  0.09 0.88  0.05 b.d.l.a
disintegration using a Polytron (Ystral Polytron, Scientific. Instru-
ments, Cambridge, UK) for all organs except lymph nodes, which Day 42 Human Synovial Fluid 1.83  0.12 1.05  0.07 0.54  0.10
were homogenized with a sonication probe (20% of amplitude for a
b.d.l.: below detection limit of 2 ng/mL and 2.2 ng/mL in ethyl oleate and human
10 s, Digital Sonifier S450D, Branson, Danbury, United States). After
synovial fluid, respectively.
homogenization, the same mechanical agitation and centrifuga-
tion methods used for blood were applied to the samples. For
groups. As already observed in a previous study (Pradal et al.,
precise quantification of the dye in organs, calibration curves were
2015), large blank particles induced a minor decrease of the cell
performed with organs collected from animals in the control
viability (
13%, p = 0.003). DiD-loaded particles of all sizes did not
group. For that, control organ samples were spiked with known
reduce cell survival, whether or not incorporated in HA gel. The
amounts of DiD.
presence of DiD in particles did not affect cell viability.
Biodistribution data were analyzed using Student’s t-test
Supplementary material related to this article found, in the
(Prism, GraphPad software, La Jolla, CA). A p-value of <0.05 was
online version, at http://dx.doi.org/10.1016/j.ijpharm.2015.12.015.
considered as statistically significant.
3.3. Experimental arthritis model
3. Results
The aim of this study was to explore the fate of various sized
3.1. Particle characterization
particles after IA injection in mice knee joints. The particles were
either embedded in HA gel or were not embedded. They were
Various DiD-loaded PLA particles were formulated to assess the
injected into inflamed and naïve knee joints. To evaluate the
in vivo biodistribution of particles according to their size. For this
inflammatory activity in mouse knees, 99mTc gamma counting was
purpose, particles with a mean size ranging from 300 nm to 10 mm
performed. Inflammation ratios were determined by comparing
were produced by adjusting the stirring speed during the
the inflamed joint to the contralateral naïve knee (Supplementary
emulsification step (Table 2). Solvent evaporation was performed
data, Fig. S2). The mean inflammation ratios at Day 1 were always
at atmospheric pressure. With these optimized process param-
above 1.9 for all animals in all groups treated with particles.
eters, the encapsulation efficiency of DiD was high for all batches,
Considering the high ratios obtained, the inflamed status was
reaching 84% for the Micro 10 (Table 2).
confirmed for all groups in the AIA model. Although not significant,
The release of the lipophilic fluorescent dye from the particles
a slightly lower ratio was obtained for the group injected with HA
was assessed in PBS containing SDS, ethyl oleate and human
without particles (1.56  0.40, p = 0.16).
synovial fluid (Table 3). Dye release was dependent on the size of
Supplementary material related to this article found, in the
particles; smaller particles released their content faster but
online version, at http://dx.doi.org/10.1016/j.ijpharm.2015.12.015.
percentages remained very low. In the lipophilic medium
frequently used to mimic the lipophilic membrane of cells (Bastiat
3.4. Intravital fluorescence
et al., 2013; Simkin, 1995), release of the fluorescent dye was
slower than in PBS. This might be attributed either to the higher
Intravital fluorescence images of the mice were recorded within
viscosity of ethyl oleate or to the slightly solubilizing effect of SDS.
5 min after the intra-articular injection of particles, and then on
Ex vivo human synovial fluid did not contain more than 2% of the
Days 1, 4 and 8 after injection for both models (Fig. 1). Intravital
total amount of DiD encapsulated in nanoparticles after 6 weeks. In
fluorescence was only detected in the region of the left knee. No
all cases, the dye was well retained in the particles for long periods
other region of the body was fluorescent. On Day 0 (within 5 min
of time.
after injection), the fluorescent area was similar for all formula-
tions. Semi-quantitative observations after 24 h highlighted the
3.2. In vitro cell viability
behavior difference of particles in inflamed joints. For inflamed
knees, on Day 1 after the injection, fluorescent areas significantly
Particle compatibility with synovial fibroblasts was tested by a
increased with respect to Day 0 for Nano and Micro 3. By contrast,
MTT viability assay at 1 mg of particles/mL. The results were
the fluorescent areas after injection of Micro 10 did not extend as
compared with untreated OA human synoviocytes (detailed results
much (Fig. 1B, Day 1 vs Day 0, p = 0.0001 for Nano, p = 0.0008 for
are presented in Supplementary data, Fig. S1). Blank PLA particles
Micro 3 and p = 0.0285 for Micro 10). In naïve knees, an increase of
of three different sizes and HA gel 0.6% (w/v) were used as control
the fluorescent area was especially observed for nanoparticles (3-
fold increase) (Fig. 1A, Day 1 vs Day 0, p = 0.0021 for Nano) and to a
Table 2 lesser extent for microparticles (2-fold increase). Although Micro
Process parameters of the emulsification step (speed and duration) and
3 spread into the inflamed joint area, the associated fluorescence
characteristics of formulations (size, zeta potential and DiD encapsulation
efficiency). remained at the same level from D1 to D8 (Fig. 2). This observation
was also valid for Nano with or without HA in both models (Fig. 2).
Characterization Nano Micro 3 Micro 10
Depending on the inflammatory status of the joint, a major
Speed of the emulsification step (rpm) 24000 16000 6200 decrease or a moderate reduction of the mean fluorescence was
Duration of the emulsification step (min) 8 3.5 1
reported over the first 24 h after IA injection in cases of inflamed
Encapsulation efficiency (%) 61.2 75.4 84.3
DiD effective concentration (mg mL
1) 1.2 1.5 1.7 and naïve joints, respectively. In short, the mean fluorescence level
D[4;3] (mm) (Span) 0.37 (0.35) 2.92 (0.29) 9.81 (1.62) was dependent on the mean size of the particles and on the
Zeta potential (mV)
47
47
35 inflammatory status of the joint (Fig. 2). The effect of HA on the
 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129 123

Fig. 1. Intravital fluorescence surface areas of knees after IA injection of particles on Day 0 (within 5 min after injection), and Days 1, 4 and 8 (n = 7 except D8: n = 5, SD). A)
naïve model, B) arthritic model. Significant differences were labeled with *(p < 0.05) and **(p < 0.01) and NS indicates a non-significant difference.

Fig. 2. Intravital mean fluorescence of knees after IA injections of particles on Day 0 (5 min after injection), Days 1, 4 and 8 after IA injections. Mean  SD, n = 7, except at Day 8
(n = 5). (A) naïve model, (B) arthritic model.

spreading of particles was only observed as a significant decrease
of the mean fluorescence under inflamed conditions during the
first 24 h (Fig. 2B, Day 1 vs Day 0, p = 3.10
4 for Nano + HA, p = 5.10
4
for Micro 3 + HA). However at Day 1, neither the fluorescent area
nor mean fluorescence were significantly different comparing
particles versus HA-embedded particles, irrespective of the
inflammatory status of the joint.
Based on the initial results (Figs. 1 and 2), with the perspective
of the formulation of a delivery system with extended release
properties over weeks, formulation Micro 3 and Micro 3 + HA were
selected for a longer term study in naïve mice. For that,
biodistribution of particles was assessed for 6 weeks (Fig. 3).
Spreading of the 3 mm particles, as observed by the increase of the
fluorescent area, was also noted within the first day after the IA

Fig. 3. Intravital fluorescence (A) area and (B) integrated intensity from Micro 3 (circle) and Micro 3 embedded in HA (square) in knees of the naïve model over 6 weeks
(mean  SD, n = 7).
124 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129

injection. Then, a slight decrease of the fluorescent areas was the smallest particles injected into naïve joints (Fig. 6 A, Nano vs
observed during the first two weeks for both formulations. Nano + HA, p = 0.01). DiD concentration in the lymph nodes after
Although larger areas were initially obtained with microparticles injection of Micro 3 was lower in naïve joints than in inflamed
embedded in HA, on Day 14, the fluorescent areas converged ones.
toward similar values for both formulations tested. From Day 14 to
Day 28, the areas remained almost constant for both formulations
(Fig. 3A). The integrated intensity of the fluorescence followed a
similar trend. Fluorescence levels then decreased from Day 28 until
the end of the experiment on Day 42 (Fig. 3B). Clearly, the retention
of 3 mm particles injected in knee joints of mice could be observed
for at least 6 weeks.

3.5. Ex vivo fluorescence of full organs

Following sacrifice, organs were harvested and the fluores-

cence intensity of intact organs was quantified (Fig. 4). The liver
accumulated the highest amount of DiD. The fluorescence
intensity of liver after injection of Nano was 3.5 times higher
in mice with inflamed joints compared with mice with no
inflammation. In the inflamed model, Micro 10 were significantly
less present in liver than Nano both at Day 4 and Day 8 (Fig. 4C,
Day 8, p = 0.0046). A similar trend related to the size of the
particles was observed for HA embedded-formulations. The
influence of HA was more pronounced for particles leaking from
inflamed joints (p = 0.019 for Nano vs Nano + HA; p = 0.045 for
Micro 3 vs Micro 3 + HA, p = 0.969 for Micro 10 vs Micro 10 + HA)
than from naïve joints. Minute levels of fluorescence were
detected in the spleen and lungs in both models for all
formulations. Very little levels of fluorescence were detected in
the kidneys, and only in mice with an inflamed joint. Fluorescence
was below detection levels in the brain and heart for both models
(data not shown).
These semi-quantitative data were then confirmed with
spectrofluorimetric quantification of DiD extracted from the same
organs. Additionally, lymph nodes, which were too small to be
imaged as organs, were analyzed in this part of the study. For the
main organs (liver, spleen, kidneys), results obtained by the
fluorescence imaging system were confirmed, except for the Micro
10 in the inflamed model: very low levels were obtained by semi-
quantitative imaging, whereas with DiD quantification, the
percentage of injected DiD dose in organs was similar to the
levels observed in the other formulations. The biodistribution of
particles depended on the mean size of the particles and on the
inflammation status of the animals (Fig. 5). The total amount of DiD
in the main organs from Nano reached 1.9  0.4% and 3.0  1.2% of
the total dose injected in the naïve and inflamed models,
respectively. For Micro 3 injected in mice of both models, the
organs of interest accumulated, in general, less than 2% of the
injected dose. In mice with an inflamed joint, embedding particles
in HA had a clear effect on the reduction of joint leakage. In the
absence of HA, higher levels of fluorescence were detected in the
organs, although in very low amounts compared with the total
dose injected. These leakage differences were also related to the
size of the injected particles, with the most important retention
effect of HA observed for Nano (Fig. 5). Because the leakage of
particles from the joints depended on the inflammatory status of
mice, organs involved in the drainage of knee joints, such as
inguinal and iliac lymph nodes, were explored and their DiD
content quantified.
DiD amount in the lymph nodes, expressed in% of injected dose,
revealed the presence of fluorescent dye in the iliac nodes and, to a
lesser extent, in the left inguinal node in both models (Fig. 6). No
fluorescence was detected in the right inguinal node correspond-
ing to the contralateral negative control joint. Amounts of Fig. 4. Integrated fluorescence intensity of organs (A) from naïve model at D8
formulations dispersed in HA accumulated less in inguinal and (n = 5), (B) from inflamed model at D4 (n = 2) and (C) from inflamed model at D8
iliac nodes than the formulations dispersed in PBS, except for (n = 5, except for both formulations of Nano where n = 4), (mean  SD).
 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129 125

Fig. 5. Percentage of injected dose of DiD assessed by fluorescence spectroscopy in the main organs of (A) naïve and (B) inflamed mice model, 8 days after IA injection.
(mean  SD, n = 5, except for both formulations of Nano in the naïve model where n = 4).

3.6. Histological analysis
The histological analysis of inflamed and naïve joints was
performed to confirm and complete results obtained by gamma
counting and fluorescence methods. Mice were sacrificed on Days
8 (n = 5) and 42 (n = 7). Scores ranging from 0 to 4 were attributed
for levels of inflammation, erosion of cartilage, erosion of bone and
presence of polymorphonuclear cells (PMN) in each treated knee
cross-section (Fig. 7). Absolutely no signs of injury were observed
in contralateral knees (Fig. 7A).
When nano- or microparticles were injected into naïve joints,
all histological scores were below or equal to 1, which corre-
sponded to a minor inflammatory reaction (Table 4). Cartilage was
preserved, as indicated by homogenous toluidine blue staining
(data not shown). A thin synovial membrane, foamy macrophages
in small foci and no inflammatory cell infiltration were observed as
indicated by HE staining (Fig. 7D–F). Although foamy macro-
phages, indicative for ingested particles, were observed in
approximately 60% of the cases irrespective of the particle size,
only Micro 10 particles were identified by histology in the articular
cavity (Table 4, Histology columns) after 8 days (Fig. 7I). However,
observation of histological sections by fluorescence microscopy
revealed the presence of particles in all healthy joints (Table 4,
Fluorescence columns and Fig. 8A–C). Regarding the long-term
study, particles were still observed even after 42 days and
associated histological scores were below 1 (Fig. 8G).
Strong and acute inflammation (synovial hyperplasia and
inflammatory infiltration (granulation tissue)) and focal abscesses
of the control mBSA group (Fig. 7B) were observed and confirmed
by an inflammatory score of 3.5 (Fig. S3), consistently with 99mTc
scintillation counting (Fig. S2). In no case particles increased
inflammation and induced higher erosion in inflamed joints
compared with the mBSA group. By contrast, inflammation on Day
8 was slightly lower in knees injected with Nano or Micro 3,
compared with knees injected with Micro 10, HA or only mBSA
(Fig. S3). Unlike the reduction of inflammation in the presence of
HA revealed by gamma counting, no effect of the gel was observed
by histological scoring on Day 8 (Fig. 7C and Fig. S3). Irrespective of
the particle size, no foamy macrophages were observed by
histology in all inflamed joints (Table 4, Histology columns).
However, fluorescence allowed the identification of particles
(Fig. 8D–F).
Supplementary material related to this article found, in the
online version, at http://dx.doi.org/10.1016/j.ijpharm.2015.12.015.

Fig. 6. Percentage of injected dose of DiD assessed by fluorescence spectroscopy in lymph nodes in naïve model (A) and arthritic model (B) on Day 8 after inflammation
induction (mean  SD, n = 5, except for both formulations of Nano in the naïve model where n = 4).
126 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129

Fig. 7. Histology of mouse knee joints 8 days after intra-articular injection as specified below and stained with H&E: (A) PBS (negative control): normal joint: smooth cartilage,
no inflammatory infiltrate either in the synovial tissue or in the joint cavity, (B) mBSA (positive control) and (C) HA: joint with AIA: strong inflammation of the synovial tissue
and the joint cavity, Injection of (D) Nano, (E) Micro 3, (F) Micro 10 particles (scale bar = 500 mm) in the non-inflammatory setting: smooth cartilage surface, thin synovial
membrane, no inflammatory infiltrate. Enlargement of rectangles drawn on slides B, C and F are, respectively, presented in the following slides: (G) mBSA (scale bar = 200 mm)
and (H) HA (scale bar = 100 mm): thickened synovial membrane, inflammation rich in neutrophils (I) Micro 10 ingested by tissue macrophages organized in a small centralized
area close to the synovial capsule (scale bar = 50 mm). Particles and HA were injected in naïve joints. Mice were sacrificed on Day 8. Magnification: 5 (A-F), 20 (G and H) and
40 (I).

4. Discussion may also be affected by the presence of discontinuities in the

To impede rapid and excessive leakage of the active substance
from the joint cavity, careful design of the particulate drug delivery
systems is essential. Previous studies in various animal models
showed that the fate (i.e., the biodistribution) of particles was size-
dependent (Bonanomi et al., 1987; Bozdag et al., 2001; Horisawa
et al., 2002b; Levick, 1984; Liang et al., 2004, 2005; Liggins et al.,
2004; Nishide et al., 1999; Ratcliffe et al., 1986). The fate of particles
synovial membrane (Knight and Levick, 1984). However, none of
the previous reports have considered the influence of inflamma-
tion on biodistribution of particles.
Therefore, the present study investigated the influence of the
synovial inflammation on particle escape from the joint. Nano
spread out of the joint from both inflamed and naïve joints. Three
micrometer particles spread out of the inflamed joint but not from
the naïve ones (Fig. 1A and B). Micro 10 were well retained in both

Table 4
Histological analysis of mouse knee joints on Days 8 and 42 after intra-articular administration of nano- and microparticles. Inflammation, foamy macrophages and particles
were analyzed by conventional light microscopy and complemented with fluorescence microscopy.

Inflammatory Naïve joint (Day 8) Inflamed joint (Day 8) Naïve joint (Day 42)
status
Method Histology Fluorescence Histology Fluorescence Histology Fluorescence

Observation Inflammation Foamy Particles Particles Inflammation Foamy Particles Particles Inflammation Foamy Particles Particles
score macro- score macro- score macro-
phages phages phages
PBS 0.0 0/1 0/1 0/1     0.0 0/2 0/2 0/2
mBSA     3.5  0.6 0/5 0/5 0/5    
HA     3.5  0.6 0/5 0/5 0/5    
Nano 0.6  0.3 2/4 0/4 4/4 2.6  0.5 0/5 0/5 2/5    
Nano + HA 0.8  0.3 4/4 0/4 4/4 2.8  0.5 0/5 0/5 3/5    
Micro 3 0.8  0.7 4/5 0/5 5/5 2.5  0.5 0/5 0/5 5/5 0.5  0.1 7/7 7/7 7/7
Micro 3 + HA 0.3  0.2 3/5 0/5 5/5 2.5  0.5 0/5 0/5 4/5 0.4  0.2 6/7 6/7 7/7
Micro 10 0.7  0.3 4/5 2/5 5/5 3.6  0.8 0/5 0/5 4/5    
Micro 10 + HA 1.0  0.0 5/5 4/5 5/5 3.6  0.4 0/5 0/5 5/5    
 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129
Fig. 8. Histology of mouse knee joints under fluorescence microscopy: 8 days after intra-articular injection : (row 1) in healthy knee joints at Day 8, (row 2) in arthritic knee
joints at Day 8, (row 3) in healthy knee joints at Day 42, (column 1) Nano, (column 2) Micro 3, (column 3) Micro 10. Magnification  40.
inflamed and naïve joints (Fig. 7F and I, naïve joint and Table 4).
This was also confirmed by very low DiD levels in the liver and
demonstrates retention of particles in the joint cavity as a function
of their size. In short, Nano escaped from inflamed and naïve joints,
whereas Micro 3 escaped only from inflamed joints. The capillary
permeability is thought to be the reason for this joint efflux
(Kushner and Somerville, 1971). Synovial fluid and serum tend to
equilibrate (Simkin, 1995) through synovium intercellular gaps
(Knight and Levick, 1984) under naïve conditions. This physiologi-
cal turnover drives the leakage of the Nano and the limiting
permeability (or “mesh”) of the healthy synovium prevents the
escape of Micro 3. Actually, retention of Micro 3 into the healthy
joint cavity for at least 6 weeks was confirmed by both intravital
imaging and by fluorescence microscopy (Fig. 8G).
Joint inflammation is characterized by enhancement of the
capillary permeability. In our study, this was demonstrated by the
significant accumulation of 99mTc in inflamed joints (Fig. S2).
Inflammation also leads to an increase of the synovial membrane
volume and a rise of the metabolic activity (Loeuille et al., 2009).
However, the high amount of inflammatory cells limited the
detection of particle carrying cells by classical histology. Nano- and
microparticles could still be detected by fluorescence microscopy
in all specimens. These physiopathological modifications were
distinctly illustrated by the spreading of Nano and Micro 3 out of
the synovial cavity (Fig. 1A: Nano  HA and Fig. 1B: Nano  HA and
Micro 3  HA). We deduced that the synovial fluid/serum
equilibrium was displaced toward the serum in cases of
inflammation as also suggested earlier by Simkin (1995). In
127
addition to considering their size for the development of particles
providing extended drug release properties, their biocompatibility
is of importance. In our study, the scores remaining low even after
6 weeks confirms the biocompatibility of PLA particles with the IA
joint tissues (Table 4, Fig. S3 and Fig. 8G).
The size of particles is important not only with regard to
retention or leakage from the joint, but also with regard to
interaction with cells present in the joint. According to Edwards
and Dong (Dong et al., 2013; Edwards et al., 2007), the size of
particles needs to be less than 10 mm in order to be phagocytized
by macrophages, which delays their clearance from the joint cavity
and increases the drug concentration directly in the inflammatory
cells to be treated (Butoescu et al., 2009). After sacrifice of the
treated mice, histological observations showed that the presence
of Nano  HA, Micro 3  HA and Micro 10  HA in the joint,
regardless of arthritic status, did not increase inflammation scores,
which indicates their innocuity in this particular site (Fig. S3). As
previously observed by the authors (Pradal et al., 2015) and also
reported by Horisawa et al. (2002a), administration of particles
significantly larger than 10 mm (e.g., 20–50 mm) led to the
production of giant cells and foreign body reactions (Anderson
and Shive, 2012). However, in the present study, the presence of
giant cells around 10 mm particles was not observed.
In addition to an appropriate selection of the particles’ sizes,
embedding them into hydrogel could be beneficial in prolonging
the drug residence time in the joints and to reduce sedimentation
and possible aggregation of particles in the joint cavity. Although
the long term favorable effect of HA itself for OA patients remains
128 J. Pradal et al. / International Journal of Pharmaceutics 498 (2016) 119–129
controversial (Altman et al., 2000; Brandt et al., 2000; Felson and
Anderson, 2002; Pendleton et al., 2000), the commercial for-
mulations are well tolerated by patients. In our study it did not
alter the viability of synovial fibroblasts (Fig. S1). Moreover, a non-
significant trend toward decreased 99Tc sodium pertechnetate
level was observed, only for HA without particles (Fig. S2). This
might suggest a decreased inflammation, consistent with some
literature reports (Fujii et al., 2001; Mccourt et al., 1994).
Additionally, HA down-regulates TNF-alpha and proinflammatory
cytokines (Takahashi et al. 1999; Wang et al., 2006). Intravital
imaging indicated that HA gels improved the retention of small
particles that tend to leak in the inflamed knee model (Fig. 4A and
C). Transient improvement of HA was observed on the retention of
the particles over 1 week (Fig. 3). The semi-quantitative imaging of
the full organs following sacrifice and DiD quantification both
confirmed a favorable effect of HA against the leakage of particles
(Nano and Micro 3) in the inflamed model on Days 4 and 8 (Figs. 4 B
and C and 5 B and Fig. S4). All HA embedded formulations injected
in an arthritic joint were found in lower concentrations in
abdominal organs compared with formulations of the same sizes
suspended in saline (Fig. 5). This improved retention may be linked
to specific interaction of HA with CD44 expressed by chondrocytes
and, to a lesser extent, by synoviocytes (Elron-Gross et al., 2009;
Laroui et al., 2007). This might also be related to HA-induced
viscosification leading to reduced turnover.
Supplementary material related to this article found, in the
online version, at http://dx.doi.org/10.1016/j.ijpharm.2015.12.015.
The biodistribution of particles in various organs after IA
administration was assessed and is discussed with regard to
particles’ size. Material elimination from the joints can go through
the microvasculature and/or through the lymphatic pathway.
Independently of the elimination route, particulate material tends
to accumulate in the liver, which was effectively observed (Fig. 4).
After injecting particles into inflamed knees, buildup of particles in
the liver remained very low but was slightly higher for the smallest
particles. This confirmed that leakage from the joint is dependent
on the particles' sizes (Figs. 4 B and C and 5). Presence of DiD in
distant organs was assessed. Concentrations in brain and lungs
were below the quantification limit (data not shown). Actually,
since none of the investigated particles had a smaller size than
300 nm, nothing was expected to cross the blood–brain barrier but
deserved to be confirmed with regard to safety (Kreuter, 2001;
Kreuter, 2014; Olivier, 2005).
The microvascular pathway represents the main elimination
pathway for very small particles from the naïve joint. In our study,
on Day 8, all nanoparticles were cleared from the blood (Fig. 5).
This clearance is attributed to efficient opsonization and uptake by
the mononuclear phagocyte system (Harashima et al., 1994; Ishida
et al., 2002; Moghimi et al., 2001). Because of this rapid clearance
from the blood, the microvascular pathway may be discussed only
on indirect facts such as the amount of DiD detected in the liver and
in the lymph nodes (Figs. 4 and 5). Although no particles were
observed in muscles or knee-surrounding tissues at sacrifice, the
very small amount of Nano detected in the lymph nodes (Fig. 6)
indicates that the main elimination route for nanoparticles is
vascular. For Micro 3 under inflamed conditions, the permeability
of the capillary bed occurred together with a relatively high
accumulation in the lymph nodes (Fig. 6). This suggests that Micro
3 are eliminated by both microvascular and lymphatic elimination
pathways.
The lymphatic pathway removes particles and debris from the
joint space independently of their size (Wallis et al., 1987). Harell
et al. highlighted that approximately half of the hind leg lymphatic
drainage enters the inguinal lymphatic node and the remaining
goes through the iliac lymphatic nodes near the aortic bifurcation.
In our study, particles and HA-dispersed particles were
preferentially drained through the iliac route (Fig. 6). Afterwards,
both ways drain in the rostral direction into the subclavian vein via
the thoracic duct (Harrell et al., 2008). Although Micro 3 were read
(Fig. 4A and B) or quantified (Fig. 5A and B) in similar amounts in
the liver, they seemed to escape differently from the joint
according to the inflammatory status. In the case of inflamed
conditions, the elimination of Micro 3 by the lymphatic pathway
was increased. As discussed above, 10 mm particles were massively
retained in the joint but still some escaped and were found in the
lymph nodes (Fig. 6). This is not surprising considering lymphatic
drainage, which is physiologically able to remove cartilage debris
from the joint (Horwitz, 1948; Saito et al., 2002).
5. Conclusion
Intra-articular administration of drug-releasing particles has
encountered growing interest, prompting the need to better
understand particle retention in the joints and biodistribution in
remote organs.
In this report, size-dependent transport of the particles was
observed and is consistent with an inflammation-dependent
capillary permeability. Nanoparticles may diffuse rapidly out of
the joint in both inflamed and naïve models, whereas large 10 mm-
particles are essentially retained. The mid-size 3 mm-particles are
well retained for 6 weeks in the naïve model. Embedding particles in
hyaluronic acid favored the retention of particles. Depending on the
size, the presence of HA and the inflammatory status of the joint,
biodistribution through blood circulation or lymphatic pathways
was modulated. Long-term administration of DiD-loaded PLA
particles demonstrated biocompatibility and dye entrapment over
6 weeks that opens possibilities for the development of drug delivery
systems for chronic arthritic diseases.
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