Accepted Manuscript

Self-assembled hyaluronic acid nanoparticles: Implications as a

nanomedicine for treatment of type 2 diabetes

Jun Gi Rho, Hwa Seung Han, Ji Hye Han, Hansang Lee, Van Quy
Nguyen, Wang Hee Lee, Seunglee Kwon, Sungeun Heo, Juhwan
Yoon, Han Ho Shin, Eun-young Lee, Hoin Kang, Siyoung Yang,
Eun Kyung Lee, Jae Hyung Park, Wook Kim

PII: S0168-3659(18)30180-9
DOI: doi:10.1016/j.jconrel.2018.04.006
Reference: COREL 9233
To appear in: Journal of Controlled Release
Received date: 22 February 2018
Revised date: 28 March 2018
Accepted date: 5 April 2018

Please cite this article as: Jun Gi Rho, Hwa Seung Han, Ji Hye Han, Hansang Lee, Van Quy
Nguyen, Wang Hee Lee, Seunglee Kwon, Sungeun Heo, Juhwan Yoon, Han Ho Shin, Eun-
young Lee, Hoin Kang, Siyoung Yang, Eun Kyung Lee, Jae Hyung Park, Wook Kim , Self-
assembled hyaluronic acid nanoparticles: Implications as a nanomedicine for treatment of
type 2 diabetes. The address for the corresponding author was captured as affiliation for
all authors. Please check if appropriate. Corel(2018), doi:10.1016/j.jconrel.2018.04.006

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 ACCEPTED MANUSCRIPT

Self-Assembled Hyaluronic Acid Nanoparticles: Implications as a

Nanomedicine for Treatment of Type 2 Diabetes

Jun Gi Rhoa,1 , Hwa Seung Hanb,1 , Ji Hye Hana, Hansang Leeb, Van Quy Nguyenb, Wang Hee
Leea, Seunglee Kwonb, Sungeun Heoa, Juhwan Yoona, Han Ho Shina, Eun-young Leea, Hoin
Kangc, Siyoung Yangd, Eun Kyung Leec, Jae Hyung Parkb,e,2 , and Wook Kima,2
a
Department of Molecular Science & Technology, Ajou University, Suwon 16499, Republic
of Korea.

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b
School of Chemical Engineering, Sungkyunkwan University, Suwon 16419, Republic of
Korea.
c
Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul

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06591, Republic of Korea.
d
Department of Pharmacology, Ajou University School of Medicine, Suwon 16499, Republic

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of Korea.
e
Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University, Suwon
16419, Republic of Korea.
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1
These authors contributed equally to this work.
2
To whom correspondence may be addreseed. Email: [email protected] or
[email protected].
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Abstract

Self-assembled hyaluronic acid nanoparticles (HA-NPs) have been extensively

investigated for biomedical and pharmaceutical applications owing to their bioompatibility
and receptor-binding properties. Here, we report that an empty HA-NP itself not bearing any
drug has therapeutic effects on adipose tissue inflammation and insulin resistance. HA-NPs
inhibited not only the receptor- mediated internalization of low- molecular-weight (LMW) free
HA but also LMW free HA-induced pro- inflammatory gene expression in mouse primary
bone marrow-derived macrophages (BMDMs) isolated from wild-type mice, but not in CD44-
null (CD44-/-) BMDMs. An in vivo biodistribution study showed the distribution of HA-NPs
and their co- localization with CD44 in adipose tissues including epididymal white adipose

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tissues (eWATs), but these were rarely observed in the eWATs of CD44-/- mice. In addition,
CD44 expression and HA-NP accumulation in the eWATs were increased in mice with diet-
induced obesity (DIO) compared to lean mice. Interestingly, treatment with HA-NPs in DIO

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mice suppressed adipose tissue inflammation as indicated by reduced macrophage content, the
production of proinflammatory cytokines and NLRP3 inflammasome activity in eWATs,

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leading to improved insulin sensitivity and normalized blood glucose levels. Collectively,
these results suggest that an empty HA-NP itself can be a therapeutic agent for the treatment
of type 2 diabetes.
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Keywords: self-assembled nanoparticles, hyaluronic acid, nanomedicine, inflammation,
insulin resistance, type 2 diabetes
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1. Introduction
Hyaluronic acid (HA) has excellent biological characteristics such as biocompatible,
biodegradable, non-toxic, and receptor-binding properties [1-4]. Accordingly, HA and its
derivatives have been investigated extensively for biomedical and pharmaceutical applications,
such as tissue engineering, arthritis treatment, ocular surgery, drug delivery, and molecular
imaging [1-5]. In particular, HA and its derivatives have emerged as the constituent of the
drug carrier for the target-specific and long-acting delivery of various chemicals and
biopharmaceuticals including proteins, peptides, and nucleotides. Also, their functional
groups at the backbone allow facile chemical modification, and this has enabled HA to be
used in various formulations to prepare the nanomedicines in the form of conjugates,
nanotubes, dendrimers, liposomes and self-assembled nanoparticles [5-9].

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Of the nanomedicines, the amphiphilic HA-based conjugates can be self-assembled
into nano-sized carriers for the delivery of a wide range of hydrophobic drugs as well as
imaging agents. Especially, self-assembled HA nanoparticles (HA-NPs), comprising

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hydrophobic cores surrounded by a hydrophilic HA shell, have been used widely as cancer-
targeting nanocarriers for therapeutic or diagnostic agents, because HA-NPs specifically bind

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to the CD44, the major cell-surface HA receptor overexpressed on cancer cells [6, 10-14].
Moreover, self-assembled HA-NPs have exhibited the potential as the carrier to selectively
deliver the drug to the inflamed joint of rheumatoid arthritis and to the atherosclerotic lesion
after systematic administration, due to the active targeting mechanism via CD44 highly
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expressed on activated macrophages [15, 16]. In addition to drug delivery, HA-NPs also have
utilized as targeted magnetic resonance (MR), positron emission tomography (PET) and
ultrasound imaging agents for biomedical applications [17-19].
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Interestingly, it has been recently demonstrated that CD44 on pro- inflammatory cells
in obese adipose tissue is involved in the development of adipose tissue inflammation and
insulin resistance in rodent models and human patients with type 2 diabetes (T2D) [20-23].
Therefore, it can be hypothesized that the CD44-targeting biomolecules play a role in insulin
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resistance and glycemic control. Here, we report the potential of self-assembled HA-NPs as a
therapeutic agent, not as a drug carrier, for the treatment of T2D. For the in vitro study, the
competitive inhibitory behavior of HA-NPs for free low molecular weight (LMW) HA, which
cause a pro-inflammatory response as an endogenous danger signal, was investigated by
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evaluating the cellular uptake and pro-inflammatory gene expression by free LMW HA-
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treated bone marrow-derived macrophages (BMDMs) in the absence or presence of HA-NPs.

Two kinds of HA-NPs were also systemically administered into diet- induced obese (DIO)
mice to determine their therapeutic effects for T2D. We provide evidence that the HA-NPs
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preferentially accumulate in adipose tissue of DIO mice due to increased CD44 expression,
block LMW HA binding to CD44, and suppress LMW HA- induced pro-inflammatory
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signaling by inducing CD44 clustering, consequently leading to improved insulin sensitivity

and glycemic control (Fig. 1A).

2. Experimental Section
2.1 Materials
Sodium hyaluronate (MW = 235 kDa and 12 kDa) was purchased from Lifecore
Biomedical (Chaska, MN, USA). 5β-Cholanic acid (CA), Propargylamine (98%), sodium
cyanoborohydride (95%), ε-caprolactone, tin(II) 2-ethylhexanoate, sodium azide (NaN3), N-
hydroxysuccinimide (NHS), p-toluenesulfonyl chloride (p-TsCl), 1-ethyl-3(3-
(dimethylamino)propyl)-carbodiimide·hydrochloride (EDC·HCl), ethylenediamine, and
copper(II) sulfate pentahydrate (CuSO 4 •5H2 O) were obtained from Sigma-Aldrich (St. Louis,
MO, USA). Water, used for synthesis and characterization in this experiment, was purified
using an AquaMax-Ultra water purification system (Younglin Co., Anyang, South Korea). All
other chemicals were analytical grade and were used without further purification.
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2.2 Synthesis and characterization of HA-CA NPs.

An amphiphilic HA conjugate, bearing ten CAs per 100 sugar residues of HA, was
synthesized in the presence of EDC and NHS, as described previously [16]. This conjugate
can form self-assembled HA-CA NPs because of the hydrophobic interaction between CAs
[10, 24]. The amphiphilic HA-PCL copolymer, which is capable of being self-assembled into
HA-PCL NPs, was prepared by Huisgen’s 1,3-dipolar cycloaddition, using alkyne-terminated
hyaluronic acid and azide-terminated polycaprolactam in the presence of CuSO 4 •5H2 O and
sodium ascorbate, as previously reported [25]. The chemical structure of the conjugate was
characterized using 1H-NMR (Unity Inova, Varian Technology, CA, USA), which was
operated at 500 MHz. The morphologies of the HA-CA and HA-PCL NPs were observed

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using a HR-TEM (JEOL-2100F, Tokyo, Japan) operated at an accelerating voltage of 200 kV.
For TEM images, samples were dispersed in deionized water (1 mg/ml) and dropped on a
200-mesh copper grid. The nanoparticles were then treated with 1 % uranyl acetate for

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negative staining. The size and zeta potential of the nanoparticles were determined at 25 °C
using a DLS (Nano ZS90, Malvern Instruments, Worcestershire, UK). The particle sizes were

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determined using the nanoparticles in PBS (pH 7.4), whereas zeta potentials were obtained
those in distilled water. 12k-HA-CA and HA-PCL conjugates were synthesized as previously
reported [10, 25]. To analyze the chemical structures using 1H-NMR, 12k-HA-CA and HA-
PCL NPs were dissolved in CD3OD/D2O (1v/1v) and DMSO-d6/D2O (1v/1v), respectively.
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2.3 In vivo biodistribution of HA-CA NPs.
All experiments with live animals were performed in compliance with relevant laws
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and institutional guidelines of Ajou University and Sungkyunkwan University, and

institutional committees approved all experiments (IACUC number: 2014-0007). CD44-/- and
CD44+/+ mice (C57BL/6 background) were obtained from The Jackson Laboratory (Bar
Habor, ME, USA). To observe the in vivo biodistribution of Cy5.5- labeled HA-CA NPs, the
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NPs (1mg/ml) were injected into the tail vein of male C57BL/6 mice (n=3) fed a STD or HFD
for 12 weeks. The organs and tissues were dissected from mice at pre-determined time points,
and the time-dependent biodistribution of the NPs were monitored using the eXplore Optix
system (ART Advanced Research Technologies Inc., Montreal, Canada). All data were
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calculated using the region of interest function of the Analysis Workstation software (ART
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Advanced Research Technologies, Inc., Montreal, Canada), and values are presented as the
means with standard deviations for three animals. For histological analysis, the dissected
eWATs were stained as previously reported [26]. In brief, the tissues were fixed in 4%
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formalin solution, dehydrated with a graded ethanol series, and embedded in paraffin. The
paraffin tissues were sliced into 6 µm in thickness and incubated overnight with anti-CD44
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antibody (BD Bioscience, CA, USA) diluted at 1:200 in a solution containing 3% BSA. After
three washes in PBS, immunofluorescence was detected following incubation with Ale xa
Fluor 488 anti- mouse antibody diluted at 1:200. The fluorescence images were obtained using
an LSM 510 META NLO confocal laser scanning microscope (Carl Zeiss Microimaging
GmbH, Jena, Germany).

2.4 Mice.
CD44-/- (C57BL/6J background) mice were obtained from The Jackson Laboratory
(Bar Harbor, ME, USA) and DBA/2 mice were obtained from Orient bio (Seongnam, South
Korea). The mice were housed individually and maintained under a 12-h light-dark cycle and
fed ad libitum (ad- lib) with a STD (NIH-31 rodent diet; 4% of calories from fat). Four-week-
old male C57BL/6J or DBA/2 mice were provided with ad- lib access to water and either STD
or a HFD (D12492, Research Diets, New Brunswick, NJ, USA; 60% of calories from fat) for
12 or 20 weeks, and were randomly allocated to each experimental group. The only criterion
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was that the mean weight was matched among the groups. Mice fed the HFD received
phosphate buffered saline (PBS) as vehicle, HA-CA, or HA-PCL NPs by daily i.v. injection,
according to two different protocols. In the first one, mice fed with a HFD for 12 weeks were
injected with 10 or 20 mg per kg body weight per day HA-CA or PBS for 4 weeks, with age-
matched lean control mice being fed with a STD. In the second protocol, mice fed with the
HFD for 20 weeks received PBS, HA-CA, or HA-PCL NPs daily for 4 weeks by i.v. injection
of 20 mg/kg, with age-matched lean control mice being fed with the STD. Body weight and
food intake were monitored daily. At the end of the treatment period, mice were subjected to
intraperitoneal (i.p.) glucose tolerance and insulin tolerance tests, as described below. Mice
were then euthanized using CO2 gas, and their eWAT, liver, and blood were collected to
determine endocrine and biochemical parameters, macrophage contents, and pro-

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inflammatory gene expression.

2.5 Analysis of metabolic parameters.

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Blood glucose levels were determined using an AGM 4000 glucometer (Allmedicus,
Anyang, South Korea). Plasma insulin was measured using a Mouse Insulin ELISA Kit

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(ALPCO, Salem, NH, USA). HOMA-IR in the mice was calculated using the following
equation: (fasting glucose concentration × fasting insulin concentration)/405. Intrahepatic
triglyceride (TG) content was determined on liver extracts using a serum TG determination kit
(Sigma-Aldrich), according to the manufacturer’s instructions.
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2.6 Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance tests (ITT).
Mice were fasted overnight and then injected with glucose (2 g/kg, i.p.), followed by
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tail blood collection at 0, 15, 30, 60, 90, and 120 minutes. Blood glucose levels were
determined using the AGM-4000 glucometer (Allmedicus). For the insulin tolerance tests,
mice were fasted for 4 h and then i.p. injected with 0.75 U/kg insulin (Sigma-Aldrich); blood
glucose levels were determined at 0, 15, 30, 60, 90, and 120 minutes.
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2.7 Inflammasome characterization.

Caspase-1 activity in the eWAT was determined using a Caspase-1 Colorimetric
Assay Kit (Biovision, Milpitas, CA, USA). IL-1β abundance in plasma and eWAT was
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measured using a Mouse IL-1/IL-1F2 Immunoassay kit (R&D systems). NLRP3, PYCARD,
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caspase-1, IL-1β, and IL-18 mRNA levels in the eWAT were measured by quantitative real-
time PCR analysis, as described below.
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2.8 Histological analysis.

Liver and eWAT samples were fixed in 4% paraformaldehyde solution overnight,
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embedded in paraffin, and sectioned onto glass slides. Tissue slides were stained with
hematoxylin-eosin (H&E) to evaluate the tissue morphology, following standardized
protocols. To evaluate lipid droplets in the liver, frozen liver sections were stained with Oil
Red O and counterstained with hematoxylin. Macrophage infiltration into the eWAT was
evaluated by immunohistochemistry (IHC) using primary antibodies recognizing CD68. The
NLRP3 inflammasome in the eWAT was evaluated by IHC using primary antibodies
recognizing NLRP3 and ASC. Information on the antibodies used for immunostaining is
listed in Table S3. Each bound antibody was revealed using the appropriate Elite ABC
horseradish peroxidase (HRP)-diaminobenzediene (DAB) system (Vector Labs, Burlingame,
CA, USA). Slides were viewed using a Leica DMi8 microscope (Leica, Buffalo Grove, IL,
USA).

2.9 Quantitative real-time PCR analysis.

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Total RNAs were isolated from frozen mouse tissues or cell lines using the TRIzol
reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s
protocol. All extractions were followed by DNase I treatment (Invitrogen). After reverse
transcription (RT) using random hexamers and reverse transcriptase (Toyobo, Osaka, Japan),
the mRNA abundance was assessed using a CFX ConnectTM Real- Time PCR Detection
System (Bio-rad, Hercules, CA, USA) and the SYBR green PCR master mix (Kapa
Biosystems, Wilmington, MA, USA). The 18S or GAPDH gene was used for normalization
to quantify the relative mRNA expression levels. The gene-specific primer sets are listed in
Table S4.

2.10 Cell culture and treatment.

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BMDMs were obtained from male C57BL/6 CD44+/+ and CD44-/- mice by flushing
the tibia with PBS. The cells (2 x 106 cells/well) were cultured in 6-well plate with a RPMI
1640 medium (Gibco, Grand Island, NY) containing 10 % heat- inactivated fetal bovine serum

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(FBS), 1 % penicillin/streptomycin and 10 μg/ml M-CSF at 37 ˚C in a humidified 5% CO 2
atmosphere. The cells were then incubated for 7 days with medium change every 3 days. For
competitive inhibition studies with free HA (235 kDa) and HA-CA NPs, BMDMs (1 x 105

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cells/well) were seeded in 6-well plates and incubated for 24 h. The cells were pre-treated
with 2 ml of HA-CA NPs or HA-PCL NPs (0.5, 1, or 5 mg/ml) for 60 min. Subsequently,
without the removal of the NPs, Cy5.5-labeled free HA (100 μg/ml) was immediately treated
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in the cells, followed by incubation for an additional 30 min. The cells were then washed
twice with PBS (pH 7.4) and fixed with a 4% paraformaldehyde solution. The intracellular
localization of Cy5.5- labeled free HA was observed using confocal microscopy. To evaluate
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the effects of free HA and HA-CA NPs on pro- inflammatory gene expression, RAW264.7
macrophages (1 x 105 cells/well) and BMDMs (1 x 106 cells/well) were seeded in 6-well
plates for 24 h and washed twice with PBS (p H 7.4). The cells were then treated for 3 h with
free HA (50, 100, 200 and 500 μg/ml) or HA-CA NPs (50, 100, 200 and 500 μg/ml), or with
free HA (100 or 500 μg/ml) in the absence or presence of HA-CA NPs (100 μg/ml) in the
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media (1 ml/well) without FBS. Cells were washed twice in ice-cold sterile PBS before
harvesting. Pro- inflammatory gene expression was determined using quantitative real-time
PCR analysis, as described above.
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2.11 Statistical analysis.

All values are expressed as the mean ± SEM. Group comparisons were assessed with
Student’s t test or analysis of variance (ANOVA). All statistical analyses were performed
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using GraphPad Prism (GraphPad Software). A P value of < 0.05 was considered statistically
significant.
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3. Results and Discussion

3.1 Synthesis and Characterization of Self-Assembled HA-CA NPs.
To prepare a self- assembled HA-NP with hydrophobic CA (HA-CA NP), amphiphilic
conjugates were synthesized by chemical modifications of LMW HA (MW = 235 kDa) with
hydrophobic CA as previously reported (Fig. S1A and 1B) [10]. The amphiphilic nature of
the conjugates allowed them to form self-assembled NPs via hydrophobic interactions among
CAs in an aqueous environment. The chemical structure of HA-CA NPs was confirmed using
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H-NMR (Fig. S2), and the number of CA in a conjugate was estimated to be 61, according to
the integration ratio of characteristic peaks for CA to HA. Transmission electron microscope
(TEM) images showed that HA-CA NPs were spherical in shape (Fig. 1B). Their
hydrodynamic diameter and zeta potential, confirmed by dynamic light scattering (DLS),
were 221.0 ± 3.1 nm and -25.7 ± 1.3mV, respectively (Fig. 1B and Table S1).
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Fig 1. Characteristics of self-assembled HA-CA NPs. (A) Schematic illustration of HA-CA
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NPs for treatment of type 2 diabetes. (B) TEM images and size distribution of HA-CA NPs.
Scale bar, 500 nm (Inset’ 50 nm). (C). Fluorescence images of major organs and adipose
tissues after intravenous administration of Cy5.5-HA-CA NPs at predetermined time intervals.
Li: Liver, Lu: Lung, Sp: Spleen, Ki: Kidney, He: Heart, Pa: Pancreas, eWAT: Epididymal
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adipose tissue, sWAT: Subcutaneous adipose tissue; BAT: Brown adipose tissue. (D) Mean
fluorescence intensities of Cy5.5-HA-CA NPs. Data represent mean ± SD (n = 3 mice per
group). (E) Confocal microscopic images of CD44 and Cy5.5-HA-CA NP in the eWAT of
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HFD-fed DIO mice. Scale bar, 50 μm.

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3.2 In Vivo Biodistribution of HA-CA NPs.

Cy5.5-labeled HA-CA NPs (Cy5.5-HA-CA NPs) were administrated into the tail vein
of lean mice fed a standard diet (STD) and DIO mice fed a high fat diet (HFD), after which
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the in vivo biodistribution was non- invasively monitored for 24 h using a real-time near-
infrared fluorescence (NIRF) imaging technique (Fig. 1C and Fig. S3A). For DIO mice fed a
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HFD, strong NIRF signals were found in the liver and kidney, which might be due to uptake
by the reticuloendothelial system and the renal excretion, respectively [27-29]. Interestingly,
considerable signal was observed for 24 h in all adipose tiss ue, in which epididymal white
adipose tissue (eWAT) exhibited stronger signal, compared to other tissues or organs such as
subcutaneous WAT (sWAT), brown adipose tissue (BAT), lung, spleen, heart, and pancreas
(Fig. 1C and D). This result was further supported by the histological analysis in DIO mice, as
indicated by strong fluorescent signals in the eWAT due to the presence of Cy5.5-HA-CA
NPs (Fig. 1E). The fluorescent signals in the eWAT were increased by HFD feeding and,
conversely, it was largely reduced in CD44- nullt (CD44-/-) mice (Fig. S3A). The
immunohistochemical analysis further confirmed that the fluorescent signals of Cy5.5-HA-
CA NPs and CD44 in the eWAT of HFD- fed wild-type (CD44+/+) DIO mice were much
stronger than those in the eWAT of STD-fed CD44+/+ mice, but both signals were rarely
detected in the eWAT of STD- fed CD44-/- mice (Fig. S3B). Accordingly, the co- localization
of HA-CA NPs with CD44 was obviously detected in HFD- fed CD44+/+ DIO mice and to a
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somewhat lesser degree in STD- fed CD44+/+ mice, but not in STD-fed CD44-/- mice. Given
that CD44 expression was largely induced by the HFD feeding [20, 21, 23], these results
suggest that the interaction between HA-CA NPs and CD44 could be responsible for the
effective targeting of the HA-CA NPs in the eWAT.

3.3 In Vitro Competitive Inhibition Assay of HA-CA NPs.

To examine whether HA-CA NPs competes with LMW free HA (MW = 235 kDa) on
the CD44 binding, we next conducted in vitro competitive inhibition assay using Cy5.5-
labeled free HA and HA-CA NPs in primary BMDMs isolated from CD44+/+ and CD44-/-
mice (Fig. 2A) [25, 30, 31]. Consistent with our previous reports [10-13, 15, 16], strong
fluorescent signals was detected in the cytoplasm when CD44+/+ BMDMs were treated with

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Cy5.5-labeled free HA in the absence of HA-CA NPs. However, the cellular uptake of Cy5.5-
labeled free HA decreased in a dose-dependent manner when the cells were pretreated with an
excess amount of HA-CA NPs. In addition, very weak fluorescence intensities of Cy5.5 were

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detected when CD44-/- BMDMs were treated with free HA and HA-CA NPs. These results
imply that HA-CA NP and free HA compete for binding to CD44. As LMW free HA-

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dependent CD44 signaling induces the expression of pro- inflammatory cytokines and CD44
[21, 32], we compared the effect of LMW free HA on the expression of pro-inflammatory
genes and CD44 in the absence or presence of HA-CA NPs using CD44+/+ and CD44-/-
BMDMs. We found that the expression levels of CD44, TNF-α, NLRP3, and IL-1β were
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increased by LMW free HA, but not by HA-CA NP, in CD44+/+ BMDMs, whereas these
LMW free HA- induced expressions were inhibited by pre-treatment with HA-CA NP (Fig.
2B). Moreover, LMW free HA-induced expression of the pro- inflammatory genes was not
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observed in CD44-/- BMDMs. Taken together, it is likely that HA-CA NP inhibits pro-
inflammatory function of LMW free HA by altering the LMW free HA-binding capacity of
CD44 as a competitive inhibitor.
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Fig. 2. In vitro competitive inhibition assay using LMW free HA in the absence or presence
of HA-CA NPs. (A) Confocal images of CD44+/+ and CD44-/- BMDMs incubated with
Cy5.5-labeled free HA in the absence or presence of HA-CA NPs. BMDMs were treated with
HA-CAs (0.5, 1, or 5 mg/ml) for 60 min, followed by incubation with Cy5.5-labeled free HA
(100 μg/ml) for 30 min. Scale bars, 50 μm. (B) Pro- inflammatory gene expression in
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CD44+/+ and CD44-/- BMDMs treated with free HA and/or HA-CA NP. BMDMs were
treated for 3 h with free HA (500 μg/ml) in the absence or presence of HA-CA NPs (100
μg/ml). Data represent mean ± SEM from at least three indep endent experiments. * P < 0.05,
**
P < 0.01 versus PBS by two-tailed Student’s t test.

3.4 Self-Assembled HA-CA NPs Reduce Body Weight and Improve the Impaired
Glucose Tolerance and Insulin Sensitivity.
It has been recently demonstrated that LMW HA and CD44 are involved in the
pathogenesis of T2D [20-22, 33, 34]. In particular, both LMW HA and CD44 levels in DIO
mice are positively correlated with insulin resistance and glycemic control [20-23, 33, 34].
Thus, these studies have proposed that interrupting LMW HA-CD44 interaction could have

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therapeutic benefits for the treatment of T2D. Indeed, blocking LMW HA-CD44 interaction
in DIO mice by recombinant hyaluronidase PH20, CD44 deficiency or anti-CD44 antibody
treatment reversed HFD- induced insulin resistance and glycemic control [20, 21, 23, 33, 34].

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As HA-CA NPs alter the LMW free HA-binding capacity of CD44 as a competitive inhibitor,
we tested whether HA-CA NPs cause metabolic effects in DIO mice. We first performed daily

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intravenous (i.v.) injections of HA-CA NPs at 10 or 20 mg/kg/day for 4 weeks in DIO mice
fed a HFD for 12 weeks. At the end of the treatment period, we observed that injection of 20
mg/kg HA-CA NP caused a reduction in body weight, eWAT mass, fasting blood glucose,
and the homeostatic model assessment of insulin resistance (HOMA-IR) index (Fig. S4A-D).
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HA-CA NP at a dose of 20 mg/kg/day also normalized the impaired glucose tolerance and
improved insulin sensitivity (Fig. S4E and F). These effects were not observed at a dose of 10
mg/kg (Fig. S4A-F). To more achieve the full spectrum of T2D symptoms, especially
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inflammation and insulin resistance, and further confirm the metabolic effects of HA-CA NPs,
we performed daily i.v. injections of HA-CA NPs at 20 mg/kg for 4 weeks in DIO mice fed
HFD for 20 weeks and evaluated the metabolic parameters at the end of the treatment period.
Consistent with the results in Fig. S4, HA-CA NPs treatment caused statistically significant
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reductions in body weight and food intake (Fig. 3A-C). HA-CA NPs were also effective in
normalizing fasting blood glucose levels, and attenuating glucose intolerance and insulin
resistance (Fig. 3D-F). These results suggest that HA-CA NPs could be used to interrupt
LMW HA-CD44 interaction, leading to amelioration of insulin sensitivity and glycemic
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control.
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Fig. 3. Effects of self-assembled HA-CA NP treatment on body weight, glucose tolerance,
and insulin resistance in DIO mice. (A–C) Effects of HA-CA NP treatment (20 mg/kg) in DIO
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mice on body weight (A), weight change (B), and food intake (C) compared with PBS-treated
controls. Data represent the mean ± SEM. n = 3 mice per group for the standard chow diet
(STD); n = 5 mice per group for HFD. *P < 0.05, **P < 0.01 versus HFD-PBS group by two-
tailed Student’s t test for bar graphs and two-way ANOVA for comparison of multiple time
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points in line graphs. (D–F) Effects of HA-CA NP treatment (20 mg/kg) in the same DIO
mice on fasting blood glucose (D) along with IPGTT (E) and ITT (F). Data represent the
mean ± SEM. n = 3 mice per group for the STD; n = 4–7 mice per group for HFD. †P < 0.05,
††
P < 0.01 versus STD group; *P < 0.05, **P < 0.01 versus HFD-PBS group by two-tailed
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Student’s t test.

3.5 Self-Assembled HA-CA NPs Treatment Reverses Macrophage Infiltration into

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eWATs of DIO Mice.

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T2D is a chronic inflammatory disease, primarily characterized by insulin resistance in

peripheral tissues. Accumulating evidence indicates a causative link between adipose tissue
inflammation and the development of insulin resistance [35, 36]. Recent studies have
provided strong evidence that CD44 plays a critical role in the development of visceral
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(epididymal) adipose tissue inflammation and insulin resistance in rodents and humans. CD44
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is expressed on inflammatory cells of obese adipose tissue, and CD44 levels are positively
correlated with adipose tissue inflammation, insulin resistance, and glycemic control in both
humans and rodents [20-23]. Accordingly, CD44-/- mice exhibited a significantly improved
adipose tissue inflammation and insulin sensitivity in DIO mice, and anti-CD44 antibody
suppressed adipose tissue inflammation and reduced insulin resistance and hyperglycemia [20,
21, 23]. Given that HA-CA NPs suppress pro- inflammatory function of LMW free HAs by
altering the LMW free HA-binding capacity of CD44 (Fig. 2), we investigated their effects on
eWAT inflammation. In DIO mice, the accumulation of CD68 + macrophages forming crown-
like structures (CLSs) surrounding adipocytes in eWAT was observed frequently, as also
indicated by higher CD68 expression compared with that in STD lean littermates (Fig. 4A).
However, HA-CA NP treatment reduced macrophage infiltration and the e xpressions of CD68.
Consistent with previous reports [20-23], an HFD feeding led to accumulation of CD44 +
inflammatory cells and increased expression of CD44 in the eWAT compared with the STD
lean littermates, suggesting that most infiltrating cells in the CLS are macrophages and are
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positive for CD44 (Fig. 4A). This increase was also reduced by treatment with HA-CA NPs.
We also found that the mRNA expression levels of NF-κB, MCP-1 and TNF-α were
normalized by HA-CA NP treatment (Fig. 4B-D).

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Fig. 4. Effects of self- assembled HA-CA NP treatment on macrophage content in eWAT of

DIO mice. (A) Representative eWAT H&E staining and macrophage content determined by
immunohistochemical staining for CD68 and CD44 in eWATs from lean and HA-CA NP-
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treated DIO mice. Scale bars, 250 μm. Boxed areas were magnified, shown at the bottom, for
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better visualization. CD68 and CD44 mRNA levels measured in eWATs are shown at the
bottom. (B) Expression of NF-κB mRNA in the same eWATs as in (A). (C and D) Pro-
inflammatory gene expression (MCP-1 and TNF-α) in the same eWATs as in (A). Data
represent the mean ± SEM. n = 3 mice for STD; n = 3–7 per group for HFD. †P < 0.05, ††P <
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0.01 versus STD group; * P < 0.05, ** P < 0.01 versus HFD-PBS group by two-tailed Student’s
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t test.

3.6 Self-Assembled HA-CAs Suppress NLRP3 Inflammasome Activation in DIO mice .

The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3)
inflammasome is a large, multimeric, danger-sensing platform formed by the association of
the pyrin domains of NLRP3, the bipartite adaptor ASC (‘apoptosis-associated speck- like
protein containing a caspase-recruitment domain’; also known as PYCARD) and caspase-1
[37-39]. Its activation plays a critical role in the pathogenesis of T2D by controlling the
processing and secretion of IL-1β and IL-18 pro- inflammatory cytokines, which are directly
linked to obesity, insulin resistance, and the initiation and progression of T2D [36, 38, 40-43].
As NLRP3 inflammasome is required for IL-1β production in response to LMW HA-CD44
interaction [44], we investigated the effects of HA-CA NPs on the activation of NLRP3
inflammasome and IL-1β production. Relative to their STD lean littermates, the eWAT from
DIO mice contained higher mRNA and protein levels of NLRP3 and ASC (Fig. 5A and B),
had elevated gene expression of Caspase-1, IL-1β and IL-18 (Fig. 5C), and had similarly
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higher caspase-1 activity in the eWAT, in conjunction with higher IL-1β levels in both the
eWAT (Fig. 5D and E) and blood (Fig. S5). In contrast, the same parameters in HA-CA NP-
treated DIO mice were similar to or only slightly different from their levels in the STD lean
littermates (Fig. 5A-E and Fig. S5). These results indicate that HA-CA NPs have therapeutic
effects on adipose tissue inflammation and insulin resistance by suppressing the activation of
NLRP3 inflammasome.

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Fig. 5. Effects of self-assembled HA-CA NP treatment on the assembly of NLRP3-ASC

inflammasome in DIO mice. (A and B) Representative immunohistochemical staining for
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NLRP3 (A) and ASC (B) in eWATs from lean and HA-CA NP-treated DIO mice. Scale bars,
250 μm. NLRP3 and PYCARD mRNA levels measured in eWATs are shown on the right.
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(C–E) Caspase-1, IL-1β, and IL-18 mRNA levels (C) as well as caspase-1 activity (D) and IL-
1β protein (E) in the same eWATs as in (A). n = 3 mice for STD; n = 3–7 mice per group for
HFD. Data represent the mean ± SEM. †P < 0.05, ††P < 0.01 versus STD group; * P < 0.05, ** P
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< 0.01 versus HFD-PBS group by two-tailed Student’s t test.

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3.7 Other Self-Assembled HA-NP, HA-PCL NP, Has Similar Effects with HA-CA NP on
Adipose Tissue Inflammation and Insulin Resistance.
Hydrophobic CA used in HA-CA NP synthesis is a bile acid derivative [45]. Bile acids
and their derivatives play critical roles in the regulation of glucose and energy metabolism,
and inflammation [46]. In addition, pro-inflammatory response can be affected by the MW of
HA in the conjugate [32, 47]. To confirm the effect of the hydrophobic moiety and MW of
HA, we have prepared two more HA-NPs, consisting of HA (MW = 12 kDa) as the
hydrophilic shell and CA or polycaprolactone (PCL) as the hydrophobic core (Fig. S1, S6A,
and S7A). The chemical structures of 12k-HA-CA and HA-PCL NPs were analyzed using 1 H-
NMR (Fig. S2A and B), and TEM images indicated that they are spherical in shape (Fig. S6B
and S7B). Compare to HA-CA NP, 12k-HA-CA and HA-PCL NPs had similar particle sizes
with slightly lower zeta potential values (Tables S1 and S2). In addition, 12k-HA-CA and
HA-PCL NPs showed similar effects with HA-CA NP all in vitro experiments. Briefly, in
vitro competitive inhibition assay showed that both the NPs and LMW free HA compete for
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binding to CD44 (Fig. S6C and S7C). Similar to HA-CA NP, 12k-HA-CA NP suppressed
LMW free HA- induced expressions of CD44, TNF-α, and NLRP3 in RAW264.7
macrophages (Fig. S6D). For in vivo experiment using DIO mice, HA-PCL NP significantly
reduced body weight and food intake, normalized fasting blood glucose, and attenuated
glucose intolerance and insulin resistance (Fig. S8A-F). HA-PCL NPs also reversed
macrophage infiltration into eWATs and suppressed pro-inflammatory gene expression and
NLRP3 inflammasome activation in DIO mice (Fig. S9 and S10). These results imply that,
irrespective of hydrophobic constituents (CA and PCL) and the molecular weights of free HA
(12 kDa and 235 kDa), the therapeutic effects of HA-CA NPs on adipose tissue inflammation,
glycemic control and insulin resistance are attributed to the presence of self- assembled
hydrophilic HA shell with multiple binding sites to CD44.

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In normal tissue, the most widely distributed native form of HA is a high- molecular-
weight (HMW, MW > 1000 kDa) polymer present in all tissues and body fluid [1, 48]. Under
pathological conditions, including inflammation and tissue injury, HMW HA undergoes

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dynamic degradation, resulting in accumulation of LMW species (MW < 500 kDa), which
trigger and promote a pro- inflammatory response as an endogenous danger signal [1, 48-51].

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In contrast, HMW HAs exert protective anti- inflammatory effects through reducing gene
expression and synthesis of pro- inflammatory cytokines [1, 48-51]. These differential
biological activities of HMW and LMW HAs are likely due to the ability of HA to induce
CD44 clustering on cell surface. Recent studies reported that CD44 has two different
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conformational forms and HA size-dependent conformational switching between the low- and
high-affinity binding states induce clustering of CD44 [52-55]. In contrast to a LMW HA, a
HMW HA binds simultaneously to many CD44 receptors through multivalent or repeated
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binding sites, leading to CD44 clustering and subsequent activation of differential

downstream signaling events distinct from a LMW HA, consequently which is likely to be
counter- inflammatory [54-57]. Given that, similar to a HMW HA, a HA-coated NP contains
multiple CD44 binding sites and induces CD44 clustering [58], we propose that a self-
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assembled HA-NP exerts anti- inflammatory effects through the same mechanism with a
HMW HA (Fig. 1A).

4. Conclusions
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In summary, we report a novel therapeutic role of empty HA-NPs without any drug in
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adipose tissue inflammation, insulin resistance and glycemic control. The in vitro studies
demonstrated that HA-NPs effectively suppressed the LMW free HA- induced expressions of
pro-inflammatory mediators by acting as a competitive inhibitor of HA-CD44 interactions in
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macrophages. These results suggest that HA-NPs can exert anti- inflammatory effects by
interrupting the LMW HA interaction with CD44. Indeed, treatment with HA-NPs in DIO
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mice inhibited macrophage infiltration into adipose tissue and production of pro-inflammatory
cytokines, leading to an attenuated adipose tissue inflammation, and normalized fasting blood
glucose levels and insulin sensitivity. Therefore, our results identify novel function of a self-
assembled HA-NP, which could be a therapeutic agent for the treatment of T2D by breaking
the links between adipose tissue inflammation and insulin resistance. Ultimately, we can
expect that self-assembled HA-NP loaded with specific drugs might further improve the
therapeutic outcome, exert simultaneous multiple effects or reduce side effects.

Acknowledgements
This work was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future
(2016R1E1A1A01941213, 2015R1A2A2A05001390, and 20100027955).
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Figure caption

Fig 1. Characteristics of self-assembled HA-CA NPs. (A) Schematic illustration of HA-CA

NPs for treatment of type 2 diabetes. (B) TEM images and size distribution of HA-CA NPs.
Scale bar, 500 nm (Inset’ 50 nm). (C). Fluorescence images of major organs and adipose
tissues after intravenous administration of Cy5.5-HA-CA NPs at predetermined time intervals.
Li: Liver, Lu: Lung, Sp: Spleen, Ki: Kidney, He: Heart, Pa: Pancreas, eWAT: Epididymal
adipose tissue, sWAT: Subcutaneous adipose tissue; BAT: Brown adipose tissue. (D) Mean
fluorescence intensities of Cy5.5-HA-CA NPs. Data represent mean ± SD (n = 3 mice per
group). (E) Confocal microscopic images of CD44 and Cy5.5-HA-CA NP in the eWAT of
HFD-fed DIO mice. Scale bar, 50 μm.

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Fig. 2. In vitro competitive inhibition assay using LMW free HA in the absence or presence
of HA-CA NPs. (A) Confocal images of CD44+/+ and CD44-/- BMDMs incubated with

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Cy5.5-labeled free HA in the absence or presence of HA-CA NPs. BMDMs were treated with
HA-CAs (0.5, 1, or 5 mg/ml) for 60 min, followed by incubation with Cy5.5-labeled free HA
(100 μg/ml) for 30 min. Scale bars, 50 μm. (B) Pro-inflammatory gene expression in

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CD44+/+ and CD44-/- BMDMs treated with free HA and/or HA-CA NP. BMDMs were
treated for 3 h with free HA (500 μg/ml) in the absence or presence of HA-CA NPs (100
μg/ml). Data represent mean ± SEM from at least three independent experiments. * P < 0.05,
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**
P < 0.01 versus PBS by two-tailed Student’s t test.

Fig. 3. Effects of self-assembled HA-CA NP treatment on body weight, glucose tolerance,

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and insulin resistance in DIO mice. (A–C) Effects of HA-CA NP treatment (20 mg/kg) in DIO
mice on body weight (A), weight change (B), and food intake (C) compared with PBS-treated
controls. Data represent the mean ± SEM. n = 3 mice per group for the standard chow diet
(STD); n = 5 mice per group for HFD. *P < 0.05, **P < 0.01 versus HFD-PBS group by two-
tailed Student’s t test for bar graphs and two-way ANOVA for comparison of multiple time
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points in line graphs. (D–F) Effects of HA-CA NP treatment (20 mg/kg) in the same DIO
mice on fasting blood glucose (D) along with IPGTT (E) and ITT (F). Data represent the
mean ± SEM. n = 3 mice per group for the STD; n = 4–7 mice per group for HFD. †P < 0.05,
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††
P < 0.01 versus STD group; *P < 0.05, **P < 0.01 versus HFD-PBS group by two-tailed
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Student’s t test.

Fig. 4. Effects of self- assembled HA-CA NP treatment on macrophage content in eWAT of

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DIO mice. (A) Representative eWAT H&E staining and macrophage content determined by
immunohistochemical staining for CD68 and CD44 in eWATs from lean and HA-CA NP-
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treated DIO mice. Scale bars, 250 μm. Boxed areas were magnified, shown at the bottom, for
better visualization. CD68 and CD44 mRNA levels measured in eWATs are shown at the
bottom. (B) Expression of NF-κB mRNA in the same eWATs as in (A). (C and D) Pro-
inflammatory gene expression (MCP-1 and TNF-α) in the same eWATs as in (A). Data
represent the mean ± SEM. n = 3 mice for STD; n = 3–7 per group for HFD. †P < 0.05, ††P <
0.01 versus STD group; * P < 0.05, ** P < 0.01 versus HFD-PBS group by two-tailed Student’s
t test.

Fig. 5. Effects of self-assembled HA-CA NP treatment on the assembly of NLRP3-ASC

inflammasome in DIO mice. (A and B) Representative immunohistochemical staining for
NLRP3 (A) and ASC (B) in eWATs from lean and HA-CA NP-treated DIO mice. Scale bars,
250 μm. NLRP3 and PYCARD mRNA levels measured in eWATs are shown on the right.
(C–E) Caspase-1, IL-1β, and IL-18 mRNA levels (C) as well as caspase-1 activity (D) and IL-
1β protein (E) in the same eWATs as in (A). n = 3 mice for STD; n = 3–7 mice per group for
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HFD. Data represent the mean ± SEM. †P < 0.05, ††P < 0.01 versus STD group; * P < 0.05, ** P
< 0.01 versus HFD-PBS group by two-tailed Student’s t test.

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 ACCEPTED MANUSCRIPT

Graphical abstract

PT
RI
SC
NU
MA
ED
T
C EP
AC