- Email: [email protected]
Mucosal transfer of wheat germ agglutinin modified lipid–polymer hybrid nanoparticles for oral delivery of oridonin
Mucosal transfer of wheat germ agglutinin modified lipid-polymer hybrid nanoparticles for oral delivery of oridonin Ying Liu, Jinguang Liu, Jun Liang, Meiying Zhang, Zhe Li, Zhi Wang, Beilei Dang, Nianping Feng PII: DOI: Reference:
S1549-9634(17)30079-5 doi: 10.1016/j.nano.2017.05.003 NANO 1577
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
22 October 2016 5 April 2017 4 May 2017
Please cite this article as: Liu Ying, Liu Jinguang, Liang Jun, Zhang Meiying, Li Zhe, Wang Zhi, Dang Beilei, Feng Nianping, Mucosal transfer of wheat germ agglutinin modified lipid-polymer hybrid nanoparticles for oral delivery of oridonin, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.05.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Mucosal transfer of wheat germ agglutinin modified lipid-polymer hybrid
RI P
T
nanoparticles for oral delivery of oridonin
Ying Liu, PhD1, Jinguang Liu1, BSc, Jun Liang, PhD, Meiying Zhang, BSc, Zhe Li, PhD, Zhi
SC
Wang, PhD, Beilei Dang, PhD, Nianping Feng, PhD*
MA NU
School of Pharmacy, Shanghai University of Traditional Chinese Medicine，Shanghai 201203, China *Corresponding author:
Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional
ED
Chinese Medicine, 1200 Cailun Road, Zhangjiang Hi-Tech Park, Pudong New District, Shanghai 201203, P R China. Tel. & Fax: + 86 21 5132 2198; Email: [email protected] These authors contributed equally to this work.
CE
Funding statement:
PT
1
AC
This work was funded by the National Natural Science Foundation of China (No. 81303232, No.81573619).
Conflicts of interest: The authors declare that no competing interests are present. Word count (Abstract): 141 Word count (complete manuscript): 4994 Number of references: 53 Number of figures/tables: 8/0
1
ACCEPTED MANUSCRIPT Abstract Wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles (WGA-LPNs) promote
RI P
T
cellular uptake after oral delivery via receptor-mediated endocytosis and bioadhesion. Understanding the mucosal transport of WGA-LPNs would help to improve bioavailability and
SC
ensure therapeutic efficacy. In this study, WGA-LPNs interacted with mucin, forming larger agglomerates with intact core-shell structure. The interaction of WGA-LPNs with mucin improved
MA NU
enterocyte endocytosis in Caco-2 cells. An in situ intestinal diffusion study in mice confirmed that WGA-LPNs reached enterocytes and underwent endocytosis, despite interference from mucin. Importantly, oral bioavailability of oridonin-loaded WGA-LPNs increased by 1.96-fold compared
ED
with that of LPNs. Furthermore, oral administration of WGA-LPNs inhibited tumor growth in
PT
HepG2 xenograft nude mice. In addition to elucidating interactions between WGA-LPNs and
drugs.
CE
mucin, these results indicated WGA-LPNs might act as promising nanocarriers for oral delivery of
AC
Keywords: Wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles; Mucus diffusion; Bioavailability; Antitumor effect
2
ACCEPTED MANUSCRIPT Low bioavailability of lipophilic drugs presents a challenge for oral administration and has led to the development of numerous nanocarriers in the past two decades.1 Nanocarriers facilitate
RI P
T
solubilization and improve permeability. Efficient diffusion through mucus barriers is an important factor in oral drug delivery. The mucus layer has attracted increasing attention
SC
recently.2,3 Understanding the behavior of nanocarriers in the mucus barrier has significant implications for the development of novel nanocarriers. Mucus provides a selective barrier via size
MA NU
and interaction filtering mechanisms, governed by biological gel properties and features of nanocarriers.4,5 Interactions with hydrophobic regions of mucin glycans occur with many nanocarriers.6-11
ED
Lipid-polymer hybrid nanoparticles (LPNs) have emerged as a sophisticated carrier system to
PT
address the multifaceted challenges in oral drug administration.12 Compared with lipid and polymeric nanocarriers, LPNs show improved drug encapsulation, modulated drug release, and
CE
enhanced cellular uptake.13 Surface modification with biorecognition ligands such as lectin
AC
facilitates cellular uptake and enhances the transport of nanocarriers across the epithelium, improving drug absorption.1 Wheat germ agglutinin (WGA) is a widely studied lectin for oral delivery, targeting N-acetyl-D-glucosamine and sialic acid on the membrane of enterocytes and microfold cells. Decoration of LPNs with WGA yields WGA-LPNs.14 WGA-LPNs promoted in vitro cell uptake via receptor-mediated endocytosis and achieved specific bioadhesion in a study using a ligated intestinal loop model.14 Penetrating the mucus layer is necessary for WGA-LPN activity. In the case of lipid-core polymer-shell systems including PEGylated solid lipid nanoparticles,15 chitosan-coated solid lipid nanoparticles,16 and chitosan-coated liposomes,17 a hydrophilic surface layer assists in overcoming mucus and mucosal barriers. However, for 3
ACCEPTED MANUSCRIPT polymer-core lipid-shell systems such as WGA-LPNs, the hydrophobic surface makes diffusing through the mucus layer more difficult compared to lipid-core polymer-shell systems, as the
RI P
T
hydrophobic groups of the lipids interact with hydrophobic regions in the mucus. In addition to specific binding with receptors located at the cell surface, WGA can reversibly adhere to other
SC
glycosylated components present within the mucus layer.18,19 Mucoadhesion limits the access of WGA-LPN to the cell surface. Although mucoadhesion potentially facilitates bioavailability,
MA NU
dynamic renewal of the mucus layer reduces nanocarrier efficiency. Therefore, reducing the trapping of nanocarriers within mucus and ensuring efficient diffusion is essential for biospecific adhesion of WGA-LPNs, and can be achieved by studying the fate and interactions of WGA-LPNs
ED
in the mucus and optimizing WGA-LPNs accordingly.
PT
Effective diffusion of nanoparticles through mucus layers is accomplished by using various types of surface modification and materials as well as mucin hydration.20-23 Polyethylene glycol
many
nanocarriers
AC
in
CE
modification (PEGylation), reported by Hanes in 2007,20 is widely used with proven effectiveness including
some
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
lipid-based
formulations.
(polyethylene
glycol)-2000]
(DSPE-PEG2000) is an amphiphilic lipid-polymer approved for clinical use by the Food and Drug Administration (FDA). DSPE-PEG2000 is a key formulation component of LPNs and has been used as a functional material to alter the surface characteristics of nanoparticles and improve mucus penetration.24 Therefore, in addition to its role in forming the nanoparticles, we expected that the presence of DSPE-PEG2000 would prevent the nanoparticles from being trapped in the mucus because of reduced interactions between the nanoparticles and mucus. Oridonin is an active ingredient of Rabdosia rubescens (Hemsl.) Hara, which has been used 4
ACCEPTED MANUSCRIPT traditionally for the treatment of hepatic carcinoma.25,26 Molecular mechanisms of antitumor effects of oridonin include induction of apoptosis and G2/M arrest, inhibition of telomerase and
RI P
T
tyrosine kinase activity, and anti-proliferative activity.27-30 However, poor aqueous solubility, low bioavailability, and rapid plasma clearance limit the clinical application of oridonin. Oral delivery
SC
of antitumor agents improves the life quality of patients and meets the needs of follow-up therapy.31 Here, oridonin was loaded in WGA-LPNs to improve oral bioavailability and antitumor
MA NU
efficiency. The pharmacokinetics of oridonin-loaded WGA-LPNs in rats and antitumor effects in HepG2 tumor-bearing nude mice were evaluated. To our knowledge, this is the first study to analyze mucosal diffusion of WGA-LPNs, the influence of mucin-WGA-LPNs interactions on
ED
cellular uptake, in vivo bioavailability, and antitumor efficiency. The results of this study
PT
corroborate the application of polymer-core lipid-shell type LPNs as effective oral drug delivery
AC
CE
systems.
5
ACCEPTED MANUSCRIPT Methods
T
Preparation and characterization of WGA-modified lipid polymeric nanoparticles (WGA-LPNs)
RI P
WGA-LPNs were prepared following our previous work with a slight modification.14 Briefly, an aqueous phase containing DSPE-PEG2000/LIPOID S100 (40/60, or 60/40 weight ratio) was
SC
mixed with the organic phase containing PLGA and oridonin in acetonitrile at a phase volume
MA NU
ratio of 2:1 under gentle stirring for 1 h. After vortexing for 3 min, the mixture was continuously stirred at 100 rpm for 2 h. Then, the LPNs were obtained by removal of acetonitrile using rotary evaporation, filtering through a 0.8-μm nylon filter, and dialysis in pH 7.4 phosphate-buffered
ED
saline (PBS) for 2 h. After incubation of pre-synthesized WGA-DOPE with LPNs for 18 h at room temperature, the WGA-modified LPNs were purified on a Sepharose CL-4B column and eluted
PT
with PBS. To evaluate the effect of PEGylation on the mucus penetration behavior of WGA-LPNs,
termed
CE
two sets of WGA-LPNs with 40 and 60% DSPE-PEG2000 (w/w, DSPE-PEG2000 to total lipids), WGA-LPNs-lower
polyethylene
glycol
modification
(WGA-LPNs-L)
and
AC
WGA-LPNs-higher polyethylene glycol modification (WGA-LPNs-H), respectively, were prepared and used in this study, alongside WGA-free LPNs with 60% DSPE-PEG2000 (LPNs-H). WGA-LPNs with various fluorescent labels (coumarin-6, RhB-MP, Nile red, and Dil), excluding CdSe/ZnS quantum dot-loaded WGA-LPNs, were prepared using the same method, with fluorescent labels in place of oridonin. Particle hydrodynamic diameters and zeta potentials were measured by Zetasizer nano-ZS 90 (Malvern Instruments, Malven, UK). The modification rate of WGA in LPNs was determined as previously reported.32
6
ACCEPTED MANUSCRIPT Interactions of nanoparticles with the mucus layer
T
The hydrodynamic radius of WGA LPNs-H and WGA-free LPNs-H in mucin dispersions was
RI P
measured by nanoparticle tracking analysis (NTA), with nanoparticles labeled with coumarin-6 to avoid interference by intrinsic fluorescence. In particular, 100 μl of coumarin-6-loaded
SC
WGA-LPNs-H and 600 μl of mucus were incubated at 37 °C for designated time. Upon dilution
MA NU
with deionized water (1:10000, v:v), the samples were injected into the chamber of Nanosight NS300 system (Malvern Instruments, Malven, UK), and subjected to data collection and analysis using NTA software (analytical version 2.1). For samples without mucus, WGA-LPNs were diluted with deionized water. Similarly, to observe the changes in the morphology of WGA-LPNs
ED
post incubation with mucus, the samples obtained by the similar treating process were imaged by
PT
field emission transmission electron microscopy (TEM, JEM-2100F, JEOL Ltd., Japan).
CE
Particle motion of the WGA-LPNs in mucus was studied by multiple particle tracking (MPT). Interactions between WGA-LPNs and mucins were further analyzed using fluorescence resonance
AC
energy transfer (FRET) analysis. The interaction of WGA-LPNs with the sialic acid in mucus was revealed by sialic acid content analysis. The methodology of MPT, FRET, and sialic acid content analysis are provided in the Supporting Information.
Cellular uptake studies
Caco-2 cells were seeded in 6-well plates (5 × 105 cells/well). Following culture overnight, the culture medium was replaced with DMEM containing the tested sample (LPNs-H + mucins, LPNs-H + PBS, WGA-LPNs-H + mucins, WGA-LPNs-H + PBS, and WGA-LPNs-L + mucins). The samples were prepared as follows: coumarin-6-loaded nanoparticles were dispersed using
7
ACCEPTED MANUSCRIPT PBS until equal fluorescence intensity was achieved, followed by addition of mucin solution (1%, w/v) or PBS depending on the experimental group. The mixture was gently stirred at 37 °C for 1 h
RI P
T
prior to addition to the cells. At the designated time, the cells were washed with PBS three times, then trypsinized and centrifuged. The cells were collected and resuspended in ice-cold PBS. Flow
SC
cytometry (BD LSRFortessa, BD Biosciences, CA, USA) was used to determine the fluorescence intensity in the fluorescein isothiocyanate (FITC)-A channel with 1 × 105 cell measurements for
MA NU
each sample.
In vitro cellular uptake was qualitatively analyzed by confocal laser scanning microscopy (CLSM). In particular, 400 μl of Nile red-loaded nanoparticles was incubated with 100 μl of FITC-mucin
ED
(2.5%, w/v) at 37 °C by gentle stirring for 1 h, filtered through a 0.8-μm nylon filter, and
PT
ultrafiltered using Vivaspin 6.0 (Sartorius Stedim Biotech SA, Aubagne, France) at 3000 × g for 20 min. The purified nanoparticles were redispersed in PBS (pH 7.4) prior to addition to the cells.
CE
Caco-2 cells were seeded (5 × 104 cells/well) and cultured overnight. The cells were incubated
AC
with DMEM containing the treated nanoparticles or nanoparticles without mucin for 1 h, followed by rinsing with PBS, fixation using 4% paraformaldehyde, and staining of cell nuclei with DAPI. After washing with PBS three times, the cells were observed using a TCS SP8 confocal system (Leica, Mannheim, Germany).
In situ intestinal diffusion study
All animals received humane care and all animal experiments were approved by the Institutional Animal Care and Use Committee, Shanghai University of Traditional Chinese Medicine. Prior to the experiment, a liquid diet was given to the mice for 2 days followed by a 24-h fast with free access to water. The mice were anesthetized by an intraperitoneal injection of chloral hydrate (500 8
ACCEPTED MANUSCRIPT mg/kg), and then placed on a thermostatic surface maintained at 37 °C. An incision (approximately 1 cm) was made in the abdomen. Approximately 2 cm of the jejunum was exposed.
RI P
T
After one end was ligated with a suture, 50 µl of pre-prepared nanoparticle dispersion (composed of an equal volume of coumarin-6-loaded WGA-LPNs (2.87 × 10-2 μmol/ml of coumarin-6) and
SC
Dil-loaded WGA-LPNs (2.70 × 10-2 μmol/ml of Dil)) was sealed within the exposed jejunum by ligation. The jejunum was gently replaced into the abdominal cavity and maintained for 20 min.
MA NU
Subsequently, the sealed segment was washed with saline and cut off along the inside of the suture, and placed on a piece of filter paper to remove the redundant preparations. Then, it was incised along the middle and put onto a piece of filter paper with the mucous layer facing upward. Pieces
ED
(approximately 2 × 5 mm) were prepared and gently placed onto a glass Petri dish and observed
PT
by CLSM. The images were obtained in consecutive parallel XY-sections as focal planes along the Z-axis. The scanning started from the lumen side and moved to the base at 10 μm intervals to
CE
determine the diffusion and penetration depth of the nanoparticles.
AC
In vivo bioavailability study
The WGA-LPNs and LPNs in the in vivo bioavailability study and efficacy evaluation refer to WGA-LPNs-H and LPNs-H, respectively. Fifteen male Sprague-Dawley rats weighting 220 ± 20 g were randomly assigned to three groups. Oridonin suspension, LPNs, and WGA-LPNs were administered via oral gavage at a dose of 10 mg/kg. The oridonin suspension was prepared by dispersing oridonin in PEG400 solution (PEG400 in deionized water, 50:50, v:v). Blood samples (300 μl) of rats were withdrawn at predetermined time points and centrifuged at 3500 × g for 5 min to separate the plasma. Details of the drug content measurement are described in the Supporting Information. Pharmacokinetic parameters were determined using Winnolin 2 software 9
ACCEPTED MANUSCRIPT (Pharsight, USA) using a non-compartmental model.
T
In vivo efficacy evaluation
RI P
The tumor xenograft model was established by inoculating HepG-2 cells suspended in DMEM (5 × 106 cells/mouse) subcutaneously into the right armpit region of male BALB/c nude mice (4-6
SC
weeks old, 18-22 g).33 Once the tumor volume reached approximately 200 mm3, the animals were
MA NU
weighed and randomly divided into five groups (n = 5) receiving saline, 5-fluorouracil solution (22.5 mg/kg), oridonin suspension (7.5 mg/kg), LPNs (7.5 mg/kg), or WGA-LPNs (7.5 mg/kg). The animals were administered the formulations every two days via oral gavage. All treatments
ED
were administered for 24 days. Tumor volume and body weight were recorded every two days during the treatment. Tumor volume was calculated as π/6 × length × (width). The mice were
PT
sacrificed after 24 days, and tumors were isolated and weighed. Histopathological evaluation of
CE
tumor tissues was performed using hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay.
AC
Statistical analysis
Data are presented as the mean ± standard deviation (S.D.). Data were compared using one-way analysis of variance (ANOVA) with Tukey’s post-test. P < 0.05 was considered statistically significant.
10
ACCEPTED MANUSCRIPT Results
T
Nanoparticle characterization
RI P
The characterization of WGA-LPNs-L, WGA-LPNs-H, and LPNs-H is shown in Table S1. The
SC
WGA modification rate was 20.35 ± 7.36% (WGA-LPNs-L) and 35.21 ± 1.62% (WGA-LPNs-H).
Motion of nanoparticles in mucus
MA NU
The motion of CdSe/ZnS quantum dot (QD)-loaded WGA-LPNs-H in mucus or water was recorded and compared with that of LPNs-H using MPT (Figure 1). The ensemble-averaged mean-squared displacement () of WGA-LPNs-H decreased in mucus compared with that
ED
in water (P < 0.05). At a time point of 1 s, the of WGA-LPNs-H was 3.14-fold higher
PT
than the of WGA-LPNs-L, indicating that PEG modification facilitated the mucus penetration of nanoparticles. In addition, compared to WGA-free LPNs-H, WGA-LPNs-H showed
CE
a slight decrease of movement in mucus; however, the difference was not significant (P > 0.05).
AC
The difference could be attributed to the hydrophilic non-WGA surface that protected the particles from interacting with glycosylated glycoproteins.
Interactions of nanoparticles with the mucus layer
The hydrodynamic radius of nanoparticles dispersed in the mucin solution increased over time compared to those dispersed in the mucin-free solution (Figure 2, A, D, F). In addition, the size distribution of particles broadened. In the blank mucin dispersion, 10% of particles had a size of 113 nm. The significant increase in particle size observed in NTA indicates agglomerates composed of nanoparticles and mucin or other adherent components. The particles retained their original core-shell structure and no difference in the thickness of the lipid layer was observed 11
ACCEPTED MANUSCRIPT during the nanoparticle-mucin interaction (Figure 2, C, E, G ). Compared to nanoparticles in the absence of mucin (Figure 2, C), homogeneous components from mucus adhered to the surface of
RI P
T
the particles, with the interactions increasing over time (Figure 2, E, G). In the case of LPNs-H, the lipid shell adhered to the surface of the polymeric core (Figure S1).
SC
Interactions between WGA-LPNs and mucins were analyzed using FRET. FITC and Rhb-MP (synthesized from rhodamine B and α-monopalmitin as previously reported7 ) were used as the
MA NU
FRET donor and acceptor, respectively, with mucins and WGA-LPNs labeled with FITC and Rhb-MP, respectively. As shown in Figure 3, obvious reduction of the donor fluorescence intensity in conjunction with increase of acceptor intensity, indicating energy transfer, suggested an
ED
interaction between WGA-LPNs and mucin. The FRET efficiency was 34.17 ± 1.15% and 17.13 ±
PT
1.96% for WGA-LPNs-H and WGA-LPNs-L, respectively. As sialic acid and sulfate residues from oligosaccharide chains of mucin glycoproteins could form hydrogen bonds with nanocarriers,
CE
we measured the changes in sialic acid content with or without WGA-LPNs. Sialic acid content in
AC
the presence of WGA-LPNs decreased by 1.48-fold compared to that reported for blank mucus, indicating that sialic acid contributes to the interaction of WGA-LPNs with mucus.
In vitro cellular uptake
The FRET results encouraged us to analyze the influence of mucin binding on the cellular uptake of WGA-LPNs. Nanoparticles in the presence or absence of mucin were taken up by Caco-2 cells after 1-h incubation (Figure 4, A). Caco-2 cells treated with WGA-LPNs showed enhanced fluorescence compared with that reported for LPNs (Figure 4, B), indicating that WGA modification improved cellular uptake, in agreement with a previous study.14 Caco-2 cells treated with WGA-LPNs preincubated with mucin showed a marked right shift in the fluorescence curve 12
ACCEPTED MANUSCRIPT compared with that reported for WGA-LPNs without mucin preincubation. The mean fluorescence of cells treated with WGA-LPNs preincubated with mucin increased by 1.4-fold. However, no
RI P
T
significant difference in fluorescence was found between LPNs with or without mucin preincubation, suggesting that the influence of mucin preincubation on nanoparticle cellular
SC
uptake depends on the surface features of the nanoparticles. WGA surface modification enhanced cellular uptake. In addition, WGA-LPNs-H possessing a higher WGA modification rate showed a
MA NU
significant increase in cell uptake compared with that for WGA-LPNs-L, which further suggests that in the presence of mucin, WGA contributes to cellular uptake more than surface hydrophilicity. Cellular uptake was verified by CLSM (Figure 4, C). For WGA-LPNs and LPNs pretreated with
PT
nanoparticles and mucin.
ED
mucin, green fluorescence appeared in the cytoplasm of Caco-2 cells, indicating cellular uptake of
CE
In situ intestinal diffusion study
In situ intestinal diffusion was analyzed to study the distribution of nanoparticles in mucus and
AC
intestinal cells by CLSM (Figure 5, A). Coumarin-6-labeled nanoparticles were used to reveal the penetration and diffusion through the mucus layer and cells, whereas Dil-labeled nanoparticles revealed cellular interactions, as Dil is a lipophilic membrane stain showing a red fluorescent signal when incorporated into the cell membrane. CLSM images in XY-plane micrographs arranged in Z-series tiers were recorded at 10-μm increments from the lumen surface (from top to bottom, Figure 5, B). Fluorescence intensity gradually increased with depth, reached a maximum (as shown in the non-rotated image in Figure 5, B), and then decreased with depth. The average depth of diffusion and penetration of WGA-LPNs-H, WGA-LPNs-L, and LPNs-H was 170.0 ± 7.1 μm (Figure 5, C), 132.0 ± 8.4 μm (Figure S2, A), and 130.0 ± 8.9 μm (Figure S2, B), respectively. 13
ACCEPTED MANUSCRIPT WGA-LPNs-H showed stronger red fluorescence, indicating a greater number of nanoparticles interacting with cells. WGA-modified LPNs reached the surface of cells and achieved specific
RI P
T
bioadhesion despite mucin interference.
In vivo bioavailability
SC
The mean oridonin plasma concentration-time curves of WGA-LPNs after oral administration in
MA NU
rats are shown in Figure 6, whereas the main pharmacokinetic parameters are summarized in Table S2. WGA-LPNs showed a similar maximum plasma concentration (Cmax) (1.1-fold) to LPNs-H. The area under the curve (AUC) of WGA-LPNs was significantly higher than that of LPNs (P <
ED
0.05). In addition, mean residence time (MRT) was 1.5 times longer in WGA-LPNs than in LPNs. The relative bioavailability of WGA-LPNs compared with suspensions and LPNs increased by
CE
oral bioavailability.
PT
9.09-fold and 1.96-fold, respectively, indicating that WGA-LPNs significantly increased oridonin
AC
In vivo efficacy evaluation
As shown in Figure 7, A-D, compared to the control, the positive control group, suspensions, LPNs, and WGA-LPNs showed significant anti-tumor effects (retardation of tumor growth and reduced tumor weight), with WGA-LPNs showing the highest therapeutic efficacy (P < 0.05). Tumor inhibition by WGA-LPNs was significantly higher than that by WGA-free LPNs, indicating that WGA modification enhances antitumor efficacy. In addition, the WGA-LPNs group showed less reduction in body weight compared to the positive control, indicating lower toxicity. H&E staining of the tumor tissue is shown in Figure 7, E. Viable tumor cells and vessels were present in the control group, whereas few necrotic cells were observed in other groups, and a large 14
ACCEPTED MANUSCRIPT necrotic area was identified in the WGA-LPNs group. In addition, based on the TUNEL assay,
RI P
superior effects in the in vivo study compared to the other groups.
T
obvious cellular apoptosis was present in the WGA-LPNs group. Overall, WGA-LPNs showed
Discussion
SC
In a previous study, WGA-LPNs improved enterocyte and goblet cell uptake in an in vitro cell culture model.14 Given the importance of efficient mucus diffusion in effective oral nanocarrier
MA NU
delivery34, this study focused on demonstrating the capacity of WGA-LPNs to overcome the mucus layer and improve bioavailability and therapeutic efficacy. NTA showed interactions between WGA-LPNs and mucus. For lipid nanocarriers, the presence of
ED
hydrophobic groups, which is a characteristic shared with WGA-LPNs, facilitates interactions
PT
with mucin fibers. FRET revealed that mucin was involved in binding of WGA-LPNs to mucus. The higher FRET efficiency in WGA-LPNs-H does not signify a higher amount of mucin adhered
CE
to WGA-LPNs-H, as FRET efficiency reflects donor and acceptor fluorescence at the site of
AC
FRET occurrence. The higher FRET efficiency could have been caused by a higher concentration of acceptor fluorophores located at the shell of WGA-LPNs-H. Mucosal diffusion and interaction between nanocarriers and mucins is influenced by a number of factors.8,10,11,35 As the particle size of WGA-LPNs was comparable to that of LPNs, steric obstruction was not the deciding factor in the present study. The negative charge of WGA-LPNs minimizes possible electrostatic interactions. Lipids in WGA-LPNs could form hydrophobic interactions with hydrophobic lipid-coated domains such as cysteine-rich domains. Hydrophobic interactions are non-specific, occur often in lipid-containing nanocarriers, and play a major role in mucoadhesion. Nanoparticles containing carboxyl, hydroxyl, amino, and sulfate groups form 15
ACCEPTED MANUSCRIPT hydrogen bonds with sialic acid and sulfate residues from mucin oligosaccharide chains.21 Thus, specific interactions between WGA and sialic acid and N-acetyl-D glucosamine are a possibility.
RI P
T
This study focused on the ability of WGA-LPNs to overcome the mucus layer, including their retention of shape integrity, mucus diffusion, and permeation. Despite interacting with mucus,
SC
WGA-LPNs-H remained intact in the mucus after 2 h of exposure. WGA-LPNs-H likely remained intact because the molecular interactions within the lipid shell were stronger than the interactions
MA NU
with the surrounding mucus.7
The influence of PEGylation on the diffusion of WGA-LPNs in the mucus layer was investigated by MPT. WGA-LPNs-H displayed improved particle motion in mucus dispersions. However,
ED
higher PEGylation resulted in more WGA molecules binding to the surface of the nanoparticles,
PT
which decreased particle motion. As the key factors determining nanocarrier motion in mucus are the surface properties of the particles,36 the overall effect should be considered. MPT analyzes
CE
particle motion at a small scale over micrometer distances.37 Considering the high heterogeneity of
AC
mesh porosity in mucus, particle motion was investigated further. In the last decade, several in vitro setups for testing the mucus permeation behavior of nanocarriers have been described,38,39 such as Side-by-Side diffusion setup, Transwell diffusion plates,
and the rotation tubes method. In vitro methods simplify mucus diffusion studies.
However, a number of factors, including source of mucus, freshness, differences in mucus structure, and mucus collection and preparation methods might lead to differences relative to in vivo conditions. Recently, Hanes et al. applied oral gavage and in situ intestinal loop methods combined with ex vivo tracking to study mucus interactions and distribution of nanoparticles.40 Here, the in situ intestinal loop method combined with CLSM vertical scanning was used to 16
ACCEPTED MANUSCRIPT analyze the distribution and diffusion of WGA-LPNs in the mouse intestine. The injection volume was carefully controlled to cover the luminal surface of the intestinal loop and avoid overloading.
RI P
T
Using CLSM vertical scanning, different depths of penetration and diffusion of fluorescently labeled nanoparticle were visualized in real time. Compared to tissue slices, CLSM is more
SC
convenient. The diffusion depth of WGA-LPNs-H was significantly increased compared to that of LPNs-H and WGA-LPNs-L, suggesting that WGA-LPNs-H diffuse through the mucus barrier and
MA NU
are taken up by enterocytes, improving systemic delivery after oral administration. The mucus layer is a dynamically renewing gel layer.34 Upon receiving extracellular stimuli, mucus (primarily MUC2 mucin) is secreted from goblet and Paneth cells via exocytosis.41 Mucin
ED
turnover of goblet cells of small intestine villi occurs in a few hours.42,43 Therefore, we
PT
hypothesize that nanoparticles located in the inner mucus layer near the cell membrane would encounter freshly secreted mucin. We examined the cellular uptake of WGA-LPNs in complex
CE
with mucin using CLSM and showed that WGA-LPNs and LPNs, which interacted with purified
AC
mucin, were taken up by Caco-2 cells. Cells internalize nanoparticles through several routes.44 In a previous study, WGA-LPNs were taken into cells via clathrin-mediated and receptor-mediated endocytosis, and LPNs via clathrin-mediated endocytosis.14 The mucin used in this study is a commercial, purified mucin composed of glycoproteins. Proteins undergo endocytosis via the clathrin-mediated pathway, caveolae-mediated pathway, macropinocytosis, and phagocytosis.45 Once internalized, mucin is localized to the endosome, followed by degradation in lysosomes.46 Membrane and intracellular transport of nanoparticles in complex with mucin are not well understood. Further studies are necessary to ascertain whether mucin perturbs the natural transport pathway of WGA-LPNs. Intracellular transport of mucin is not fully understood and further 17
ACCEPTED MANUSCRIPT research is needed. Mucin is a surfactant and adsorbs at solid-liquid interfaces.47 As binding of mucin with WGA-LPNs was demonstrated in this study, we hypothesize that the
RI P
T
mucin-WGA-LPNs mixture enters the cells via endocytosis. When quantified with flow cytometry, mucin enhanced WGA-LPN uptake and had no influence on WGA-free LPNs. Absence of
SC
WGA-mediated specific interactions in LPNs, coupled with increased particle size, which reduces cellular uptake48 could explain the lack of influence of mucin. In contrast, WGA-LPNs complexed
MA NU
with mucin were recognized at the cell surface, leading to cytoadhesion and enhanced WGA-LPNs uptake.
Recently, oridonin has been encapsulated into galactosylated nanoparticles for intravenous
ED
hepatocellular carcinoma therapy.49-51 Oral administration is of great value for improving the
PT
quality of life and compliance of patients; however, low bioavailability and rapid clearance interfere with successful oral application of oridonin. In this study, oridonin-loaded WGA-LPNs
CE
showed improved bioavailability and antitumor effects, which can be attributed to the following
AC
factors: (1) LPNs are promising carriers encapsulating and improving the solubility of hydrophobic drugs based on their the core-shell structure.12 The particle sizes of nanocarriers, including LPNs and WGA-LPNs, are favorable for enhanced absorption. (2) Following WGA modification, specific bioadhesion facilitates drug absorption by enhancing cellular uptake via multiple routes including receptor-mediated endocytosis. Furthermore, as a lectin, WGA promotes M cell transport, providing opportunities for WGA-LPNs to enter lymphatic tissue.52 (3) DSPE-PEG2000 enhances WGA-LPNs penetration of the mucus layer. (4) PEGylation leads to increased plasma drug concentration and prolonged half-life, which improve the accumulation of drugs in tumors, followed by antitumor effects mediated by the enhanced permeability and 18
ACCEPTED MANUSCRIPT retention (EPR) effect. (5) Use of nanocarriers improves oral absorption and modifies biodistribution,53 which is hypothesized to lead to higher hepatic intake. For efficient oral drug
RI P
T
administration, gastrointestinal toxicity, often indicated by reduction in body weight, is of concern. Animals administered WGA-LPNs showed the least body weight reduction among test groups;
SC
however, the gastrointestinal toxicity should be investigated in more detail.
Taken together, the results of this study demonstrate the potential of WGA-modified LPNs as
MA NU
effective oral nanocarriers combining biospecific adhesion to cellular surfaces and mucus permeation (Figure 8). Based on the in vitro and in situ studies of interactions between nanoparticles and mucus and mucus penetration, the fate of WGA-LPNs in mucus can be
ED
described as follows: upon WGA-LPNs exposure to the mucus layer, WGA-LPNs interact with
PT
mucin. Although the interaction with mucin hinders movement, WGA-LPNs penetrate the mucus layer and are taken up by enterocytes. WGA-LPNs undergo orderly muco- and cytoadhesion. In
CE
addition to the PEGylation evaluated in this study, alternative strategies for improving WGA-LPN
AC
cytoadhesion should be investigated. Oridonin-loaded WGA-LPNs exhibited improved bioavailability with desirable pharmacokinetics and effective antitumor activity in HepG2 tumor-bearing nude mice. These findings suggest that WGA-LPNs are promising oral delivery systems for antitumor therapeutic agents.
19
ACCEPTED MANUSCRIPT
References
T
1. Date AA, Hanes J, Ensign LM. Nanoparticles for oral delivery: Design, evaluation and
RI P
state-of-the-art. J Control Release 2016; 240:504-26.
2. Lundquist P, Artursson P. Oral absorption of peptides and nanoparticles across the human
SC
intestine: Opportunities, limitations and studies in human tissues. Adv Drug Deliv Rev 2016;
MA NU
106(Pt B):256-76.
3. Xu Q, Ensign LM, Boylan NJ, Schön A, Gong X, Yang JC, et al. Impact of surface polyethylene glycol (PEG) density on biodegradable nanoparticle transport in mucus ex vivo
ED
and distribution in vivo. ACS Nano 2015;9(9):9217-27.
4. Pereira de Sousa I, Steiner C, Schmutzler M, Wilcox MD, Veldhuis GJ, Pearson JP, et al.
PT
Mucus permeating carriers: formulation and characterization of highly densely charged
CE
nanoparticles. Eur J Pharm Biopharm 2015;97(Pt A):273-9. 5. Liu M, Zhang J, Zhu X, Shan W, Li L, Zhong J, et al. Efficient mucus permeation and tight
AC
junction opening by dissociable "mucus-inert" agent coated trimethylchitosan nanoparticles for oral insulin delivery. J Control Release 2016;222:67-77. 6. Yang M, Lai SK, Yu T, Wang YY, Happe C, Zhong W, et al. Nanoparticle penetration of human cervicovaginal mucus: the effect of polyvinyl alcohol. J Control Release 2014;192:202-8. 7. Zhang J, Lv Y, Wang B, Zhao S, Tan M, Lv G, et al. Influence of microemulsion-mucin interaction on the fate of microemulsions diffusing through pig gastric mucin solutions. Mol Pharm 2015;12(3):695-705. 8. Groo AC, Saulnier P, Gimel JC, Gravier J, Ailhas C, Benoit JP, et al. Fate of paclitaxel lipid 20
ACCEPTED MANUSCRIPT nanocapsules in intestinal mucus in view of their oral delivery. Int J Nanomedicine 2013;8:4291-302.
RI P
T
9. Yu T, Chan KW, Anonuevo A, Song X, Schuster BS, Chattopadhyay S, et al. Liposome-based mucus-penetrating particles (MPP) for mucosal theranostics: demonstration of diamagnetic
SC
chemical exchange saturation transfer (diaCEST) magnetic resonance imaging (MRI). Nanomedicine 2015;11(2):401-5.
MA NU
10. Karamanidou T, Karidi K, Bourganis V, Kontonikola K, Kammona O, Kiparissides C. Effective incorporation of insulin in mucus permeating self-nanoemulsifying drug delivery systems. Eur J Pharm Biopharm 2015;97(Pt A):223-9.
ED
11. Beloqui A, Solinís MÁ, des Rieux A, Préat V, Rodríguez-Gascón A. Dextran-protamine
PT
coated nanostructured lipid carriers as mucus-penetrating nanoparticles for lipophilicdrugs. Int J Pharm 2014;468(1-2):105-11.
CE
12. Mandal B, Bhattacharjee H, Mittal N, Sah H, Balabathula P, Thoma LA, et al. Core-shell-type hybrid
nanoparticles
as
a
drug
delivery
platform.
Nanomedicine
AC
lipid-polymer
2013;9(4):474-91. 13. Rao S, Prestidge CA. Polymer-lipid hybrid systems: merging the benefits of polymeric and lipid-based
nanocarriers
to
improve
oraldrug
delivery.
Expert
Opin Drug Deliv
2016;13(5):691-707. 14. Liu Y, Zhao YQ, Liu JG, Zhang MY, Yu ML, Feng NP. Wheat germ agglutinin modification of lipid-polymer hybrid nanoparticles: enhanced cellular uptake and bioadhesion. RSC Advances 2016;6:36125-35 15 Yuan H, Chen CY, Chai GH, Du YZ, Hu FQ. Improved transport and absorption through 21
ACCEPTED MANUSCRIPT gastrointestinal tract by PEGylated solid lipid nanoparticles. Mol Pharm 2013;10(5):1865-73. 16. Luo Y, Teng Z, Li Y, Wang Q. Solid lipid nanoparticles for oral drug delivery: chitosan
RI P
T
coating improves stability, controlled delivery, mucoadhesion and cellular uptake. Carbohydr Polym 2015;122:221-9.
SC
17. Gradauer K, Barthelmes J, Vonach C, Almer G, Mangge H, Teubl B, et al. Liposomes coated with thiolated chitosan enhance oral peptide delivery to rats. J Control Release
MA NU
2013;172(3):872-8.
18. Bajka BH, Rigby NM, Cross KL, Macierzanka A, Mackie AR. The influence of small intestinal mucus structure on particle transport ex vivo. Colloids Surf B Biointerfaces
ED
2015;135:73-80.
PT
19. Gabor F, Bogner E, Weissenboeck A, Wirth M. The lectin-cell interaction and its implications to intestinal lectin-mediated drug delivery. Adv Drug Deliv Rev 2004;56(4):459-80.
CE
20. Lai SK, O'Hanlon DE, Harrold S, Man ST, Wang YY, Cone R, et al. Rapid transport of large
AC
polymeric nanoparticles in fresh undiluted human mucus. Proc Natl Acad Sci USA 2007;104(5):1482-7. 21. Mansfield ED, Sillence K, Hole P, Williams AC, Khutoryanskiy VV. POZylation: a new approach to enhance nanoparticle diffusion through mucosal barriers. Nanoscale 2015;7(32):13671-9. 22. Liu M, Zhang J, Zhu X, Shan W, Li L, Zhong J, et al. Efficient mucus permeation and tight junction opening by dissociable "mucus-inert" agent coated trimethylchitosan nanoparticles for oral insulin delivery. J Control Release 2016;222:67-77. 23. Popov A, Enlow E, Bourassa J, Chen H. Mucus-penetrating nanoparticles made with 22
ACCEPTED MANUSCRIPT "mucoadhesive" poly(vinyl alcohol). Nanomedicine 2016; 12(7):1863-71. 24. Wang YY, Lai SK, Suk JS, Pace A, Cone R, Hanes J. Addressing the PEG mucoadhesivity
RI P
T
paradox to engineer nanoparticles that "slip" through the human mucus barrier. Angew Chem Int Ed Engl 2008;47(50):9726-9.
SC
25. Zhang DR, Ren TC. Progresses in the pharmaceutical of oridonin. Chin Pharm J 2003; 38:817-20.
MA NU
26. Wang Y, Liu XQ, Liu GP, Guo HJ, Li CY, Zhang YC, et al. Novel galactosylated biodegradable nanoparticles for hepatocyte-delivery of oridonin. Int J Pharm 2016; 502(1-2):47-60.
ED
27. Wang H, Ye Y, Pan SY, Zhu GY, Li YW, Fong DW, et al. Proteomic identification of proteins
PT
involved in the anticancer activities of oridonin in HepG2 cells. Phytomedicine 2011;18(2-3):163-9.
CE
28. Huang J, Wu L, Tashiro S, Onodera S, Ikejima T. Reactive oxygen species mediate
AC
oridonin-induced HepG2 apoptosis through p53, MAPK, and mitochondrial signaling pathways. J Pharmacol Sci 2008;107(4):370-9. 29. Wang H, Ye Y, Chu JH, Zhu GY, Fong WF, Yu ZL. Proteomic and functional analyses reveal the potential involvement of endoplasmic reticulum stress and α-CP1 in the anticancer activities of oridonin in HepG2 cells. Integr Cancer Ther 2011;10(2):160-7. 30. Li CY, Wang EQ, Cheng Y, Bao JK. Oridonin: An active diterpenoid targeting cell cycle arrest, apoptotic and autophagic pathways for cancer therapeutics. Int J Biochem Cell Biol 2011;43(5):701-4. 31. Thanki K, Gangwal RP, Sangamwar AT, Jain S. Oral delivery of anticancer drugs: challenges 23
ACCEPTED MANUSCRIPT and opportunities. J Control Release 2013;170(1):15-40. 32. Liu Y, Wang PF, Sun C, Feng NP, Zhou WX, Yang Y, et al. Wheat germ agglutinin-grafted
RI P
T
lipid nanoparticles: Preparation and in vitro evaluation of the association with Caco-2 monolayers. Int J Pharm 2010, 397:155-63
SC
33. Ding Y, Wang YZ, Opoku-Damoah Y, Wang C, Shen LJ, Yin LF, Zhou JP. Dual-functional bio-derived nanoparticulates for apoptotic antitumor therapy. Biomaterials 2015;72:90-103.
MA NU
34. Ensign LM, Cone R, Hanes J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv Drug Deliv Rev 2012;64(6):557-70. 35. Sheng J, Han L, Qin J, Ru G, Li R, Wu L, et al. N-trimethyl chitosan chloride-coated PLGA
ED
nanoparticles overcoming multiple barriers to oral insulin absorption. ACS Appl Mater
PT
Interfaces 2015;7(28):15430-41.
36. Dünnhaupt S, Kammona O, Waldner C, Kiparissides C, Bernkop-Schnürch A. Nano-carrier
CE
systems: Strategies to overcome the mucus gel barrier. Eur J Pharm Biopharm
AC
2015;96:447-53.
37. Schuster BS, Ensign LM, Allan DB, Suk JS, Hanes J. Particle tracking in drug and gene delivery research: State-of-the-art applications and methods. Adv Drug Deliv Rev 2015;91:70-91. 38. Grießinger J, Dünnhaupt S, Cattoz B, Griffiths P, Oh S, et al. Methods to determine the interactions of micro- and nanoparticles with mucus. Eur J Pharm Biopharm 2015; 96:464-76. 39. Groo AC, Lagarce F. Mucus models to evaluate nanomedicines for diffusion. Drug Discov Today 2014;19(8):1097-108. 24
ACCEPTED MANUSCRIPT 40.Maisel
K, Ensign
L, Reddy
M, Cone
R, Hanes
J.
Effect of surface chemistry on nanoparticle interaction with gastrointestinal mucus and distrib
RI P
T
ution in the gastrointestinal tract following oral and rectal administration in the mouse. J Control Release 2015;197:48-57.
SC
41. McGuckin MA, Lindén SK, Sutton P, Florin TH. Mucin dynamics and enteric pathogens. Nat Rev Microbiol 2011;9(4):265-78. Birchenough
GM, Johansson
ME, Gustafsson
MA NU
42.
JK, Bergström
New developments in goblet cell mucus secretion and function. 2015;8(4):712-9.
JH, Hansson
Mucosal
GC.
Immunol
ED
43. Johansson ME. Fast renewal of the distal colonic mucus layers by the surface goblet cells as
PT
measured by in vivo labeling of mucin glycoproteins. PLoS One 2012;7(7):e41009. 44. Nam HY, Kwon SM, Chung H, Lee SY, Kwon SH, Jeon H, et al. Cellular uptake mechanism
CE
and intracellular fate of hydrophobically modified glycol chitosan nanoparticles. J Control
AC
Release 2009;135(3):259-67. 45. Joshi S, Kumar S, Choudhury A, Ponnusamy MP, Batra SK. Altered Mucins (MUC) trafficking in benign and malignant conditions. Oncotarget 2014;5(17):7272-84. 46. Mayor S, Pagano RE. Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 2007;8(8):603-12. 47. Karin D. Effects of mucin addition on the stability of oil-water emulsions. Colloids Surf B Biointerfaces 1999;15(3):303-12. 48. Iversen TG, Skotland T, Sandvig K. Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 2011;6:176-85. 25
ACCEPTED MANUSCRIPT 49. Wang Y, Liu X, Liu G, Guo H, Li C, Zhang Y,et al. Novel galactosylated biodegradable nanoparticles for hepatocyte-delivery of oridonin. Int J Pharm 2016;502(1-2):47-60.
RI P
T
50. Li C, Zhang D, Guo H, Hao L, Zheng D, Liu G, et al. Preparation and characterization of galactosylated bovine serum albumin nanoparticles for liver-targeted delivery of oridonin. Int
SC
J Pharm 2013;448(1):79-86.
51. Zheng D, Duan C, Zhang D, Jia L, Liu G, Liu Y, et al. Galactosylated chitosan nanoparticles
MA NU
for hepatocyte-targeted delivery of oridonin. Int J Pharm 2012;436(1-2):379-86. 52. Jepson MA, Clark MA, Hirst BH. M cell targeting by lectins: a strategy for mucosal vaccination and drug delivery. Adv Drug Deliv Rev 2004;56(4):511-25.
of
oral
absorption
of
solidified
polymer
micelles.
Nanomedicine
PT
mechanism
ED
53. Abramov E, Cassiola F, Schwob O, Karsh-Bluman A, Shapero M, Ellis J, et al. Cellular
AC
CE
2015;11(8):1993-2002.
26
ACCEPTED MANUSCRIPT
Figure legends
T
Figure 1. Motion of CdSe/ZnS quantum dot (QD)-loaded particles in porcine mucus. (A) Average
RI P
ensemble geometric mean-squared displacement () with respect to time scale. (B) Distribution of individual particle MSD (τ = 1 s) [mean ± S.D. (n = 3), with n ≥ 100 particles
SC
per sample]
MA NU
Figure 2. Particle size analysis. Left row: Nanoparticle tracking analysis (lower left: particle size per relative intensity three-dimensional plot; upper right: particle size per concentration). Right row: TEM images. (A) and (C) WGA-LPNs-H before incubation with mucus. (B) Blank mucus.
ED
(D) and (E) Incubation of WGA-LPNs-H with mucus for 1 h. (F) and (G) Incubation of WGA-LPNs-H with mucus for 2 h.
PT
Figure 3. (A) Changes in fluorescence emission spectra of FITC-labeled mucin (FITC-mucin) as a
CE
function of RhB-MP-labeled WGA-LPNs-H. The ratio represents the volume ratios of FITC-mucin vs. RhB-MP-labeled WGA-LPNs-H; (B) Fluorescence emission intensity ratio of
AC
RhB-MP and FITC with increasing RhB-MP volume and equal amounts of FITC. Figure 4. (A) Cellular uptake determined by flow cytometry analysis of control (untreated cells), WGA-LPNs-L in the presence of mucin (WGA-LPNs-L + mucin), WGA-LPNs-H in the presence of mucin (WGA-LPNs-H + mucin), WGA-LPNs-H in pH 7.4 phosphate-buffered saline (PBS) (WGA-LPNs-H + PBS), LPNs-H in the presence of mucin (LPNs-H + mucin), and LPNs-H in PBS (LPNs-H + PBS) in Caco-2 cells after 1 h of incubation. (B) Fluorescence intensity analysis. Data are shown as the mean ± S.D., n = 3, *P < 0.05, N.S. represents a non-significant difference. (C) Confocal microscopic images of Caco-2 cells treated with LPNs-H and WGA-LPNs-H in the presence of mucin. 27
ACCEPTED MANUSCRIPT Figure 5. (A) Schematic representation of the CLSM vertical scanning approach used to analyze the distribution and diffusion of WGA-LPNs in the small intestine of mice. (B) Representative
RI P
T
confocal micrographs showing in situ diffusion and penetration of WGA-LPNs in mouse jejunum. Coumarin-6-labeled nanoparticles (green signal) and Dil-labeled nanoparticles were mixed in
SC
equal volumes and applied to the intestinal section for 30 min before the images were taken. Sequential stacked XY-plane (rotation angle: x 80°, y 18°, z 270°) in Z-series with 10-μm interval
MA NU
(parallel to the lumen surface). (C) 3D reconstruction images from intestine cross-section (yz plane).
Figure 6. Mean plasma concentration-time profiles of WGA-LPNs-H, LPNs-H, and suspensions in
ED
rats following ig administration at a dose of 10 mg/kg (data are shown as the mean ± S.D., n = 5).
PT
Figure 7. Antitumor efficacy and histological studies in HepG2 tumor xenograft nude mice in vivo. (A) Tumor volumes of HepG2 tumor-bearing mouse groups. (B) Images of xenografts taken by
CE
digital camera after 24-day treatment. (C) Average tumor weight. (D) Body weight curves. (Data
AC
are shown as the mean ± S.D. n = 5, *P < 0.05 and **P < 0.01 for WGA-LPNs versus other groups). (E) Histological analysis of the tumor tissues from each group at the end of the experiment (n = 3).
Figure 8. Schematic illustration of interaction of WGA-LPNs with mucin, mucus penetration, and biospecific adhesion. LPNs and WGA-LPNs are taken up by enterocytes in the presence or absence of mucin. M cell uptake could be an alternative pathway for intestinal absorption of WGA-LPNs.
28
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 1
29
AC
Figure 2
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
30
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
Figure 3
31
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
Figure 4
32
AC
Figure 5
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
33
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
Figure 6
34
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
Figure 7
35
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 8
36
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
ED
Graphical abstract
PT
The fate of wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles (WGA-LPNs) in
CE
mucus can be described as follows: upon WGA-LPNs exposure to the mucus layer, WGA-LPNs interact with mucin. Despite the interaction with mucin hindering movement, WGA-LPNs
AC
penetrate the mucus layer and are taken up by enterocytes. Oridonin-loaded WGA-LPNs exhibited improved bioavailability with desirable pharmacokinetics and effective antitumor activity in HepG2 tumor-bearing nude mice.
37
S1549-9634(17)30079-5 doi: 10.1016/j.nano.2017.05.003 NANO 1577
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
22 October 2016 5 April 2017 4 May 2017
Please cite this article as: Liu Ying, Liu Jinguang, Liang Jun, Zhang Meiying, Li Zhe, Wang Zhi, Dang Beilei, Feng Nianping, Mucosal transfer of wheat germ agglutinin modified lipid-polymer hybrid nanoparticles for oral delivery of oridonin, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.05.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Mucosal transfer of wheat germ agglutinin modified lipid-polymer hybrid
RI P
T
nanoparticles for oral delivery of oridonin
Ying Liu, PhD1, Jinguang Liu1, BSc, Jun Liang, PhD, Meiying Zhang, BSc, Zhe Li, PhD, Zhi
SC
Wang, PhD, Beilei Dang, PhD, Nianping Feng, PhD*
MA NU
School of Pharmacy, Shanghai University of Traditional Chinese Medicine，Shanghai 201203, China *Corresponding author:
Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional
ED
Chinese Medicine, 1200 Cailun Road, Zhangjiang Hi-Tech Park, Pudong New District, Shanghai 201203, P R China. Tel. & Fax: + 86 21 5132 2198; Email: [email protected] These authors contributed equally to this work.
CE
Funding statement:
PT
1
AC
This work was funded by the National Natural Science Foundation of China (No. 81303232, No.81573619).
Conflicts of interest: The authors declare that no competing interests are present. Word count (Abstract): 141 Word count (complete manuscript): 4994 Number of references: 53 Number of figures/tables: 8/0
1
ACCEPTED MANUSCRIPT Abstract Wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles (WGA-LPNs) promote
RI P
T
cellular uptake after oral delivery via receptor-mediated endocytosis and bioadhesion. Understanding the mucosal transport of WGA-LPNs would help to improve bioavailability and
SC
ensure therapeutic efficacy. In this study, WGA-LPNs interacted with mucin, forming larger agglomerates with intact core-shell structure. The interaction of WGA-LPNs with mucin improved
MA NU
enterocyte endocytosis in Caco-2 cells. An in situ intestinal diffusion study in mice confirmed that WGA-LPNs reached enterocytes and underwent endocytosis, despite interference from mucin. Importantly, oral bioavailability of oridonin-loaded WGA-LPNs increased by 1.96-fold compared
ED
with that of LPNs. Furthermore, oral administration of WGA-LPNs inhibited tumor growth in
PT
HepG2 xenograft nude mice. In addition to elucidating interactions between WGA-LPNs and
drugs.
CE
mucin, these results indicated WGA-LPNs might act as promising nanocarriers for oral delivery of
AC
Keywords: Wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles; Mucus diffusion; Bioavailability; Antitumor effect
2
ACCEPTED MANUSCRIPT Low bioavailability of lipophilic drugs presents a challenge for oral administration and has led to the development of numerous nanocarriers in the past two decades.1 Nanocarriers facilitate
RI P
T
solubilization and improve permeability. Efficient diffusion through mucus barriers is an important factor in oral drug delivery. The mucus layer has attracted increasing attention
SC
recently.2,3 Understanding the behavior of nanocarriers in the mucus barrier has significant implications for the development of novel nanocarriers. Mucus provides a selective barrier via size
MA NU
and interaction filtering mechanisms, governed by biological gel properties and features of nanocarriers.4,5 Interactions with hydrophobic regions of mucin glycans occur with many nanocarriers.6-11
ED
Lipid-polymer hybrid nanoparticles (LPNs) have emerged as a sophisticated carrier system to
PT
address the multifaceted challenges in oral drug administration.12 Compared with lipid and polymeric nanocarriers, LPNs show improved drug encapsulation, modulated drug release, and
CE
enhanced cellular uptake.13 Surface modification with biorecognition ligands such as lectin
AC
facilitates cellular uptake and enhances the transport of nanocarriers across the epithelium, improving drug absorption.1 Wheat germ agglutinin (WGA) is a widely studied lectin for oral delivery, targeting N-acetyl-D-glucosamine and sialic acid on the membrane of enterocytes and microfold cells. Decoration of LPNs with WGA yields WGA-LPNs.14 WGA-LPNs promoted in vitro cell uptake via receptor-mediated endocytosis and achieved specific bioadhesion in a study using a ligated intestinal loop model.14 Penetrating the mucus layer is necessary for WGA-LPN activity. In the case of lipid-core polymer-shell systems including PEGylated solid lipid nanoparticles,15 chitosan-coated solid lipid nanoparticles,16 and chitosan-coated liposomes,17 a hydrophilic surface layer assists in overcoming mucus and mucosal barriers. However, for 3
ACCEPTED MANUSCRIPT polymer-core lipid-shell systems such as WGA-LPNs, the hydrophobic surface makes diffusing through the mucus layer more difficult compared to lipid-core polymer-shell systems, as the
RI P
T
hydrophobic groups of the lipids interact with hydrophobic regions in the mucus. In addition to specific binding with receptors located at the cell surface, WGA can reversibly adhere to other
SC
glycosylated components present within the mucus layer.18,19 Mucoadhesion limits the access of WGA-LPN to the cell surface. Although mucoadhesion potentially facilitates bioavailability,
MA NU
dynamic renewal of the mucus layer reduces nanocarrier efficiency. Therefore, reducing the trapping of nanocarriers within mucus and ensuring efficient diffusion is essential for biospecific adhesion of WGA-LPNs, and can be achieved by studying the fate and interactions of WGA-LPNs
ED
in the mucus and optimizing WGA-LPNs accordingly.
PT
Effective diffusion of nanoparticles through mucus layers is accomplished by using various types of surface modification and materials as well as mucin hydration.20-23 Polyethylene glycol
many
nanocarriers
AC
in
CE
modification (PEGylation), reported by Hanes in 2007,20 is widely used with proven effectiveness including
some
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
lipid-based
formulations.
(polyethylene
glycol)-2000]
(DSPE-PEG2000) is an amphiphilic lipid-polymer approved for clinical use by the Food and Drug Administration (FDA). DSPE-PEG2000 is a key formulation component of LPNs and has been used as a functional material to alter the surface characteristics of nanoparticles and improve mucus penetration.24 Therefore, in addition to its role in forming the nanoparticles, we expected that the presence of DSPE-PEG2000 would prevent the nanoparticles from being trapped in the mucus because of reduced interactions between the nanoparticles and mucus. Oridonin is an active ingredient of Rabdosia rubescens (Hemsl.) Hara, which has been used 4
ACCEPTED MANUSCRIPT traditionally for the treatment of hepatic carcinoma.25,26 Molecular mechanisms of antitumor effects of oridonin include induction of apoptosis and G2/M arrest, inhibition of telomerase and
RI P
T
tyrosine kinase activity, and anti-proliferative activity.27-30 However, poor aqueous solubility, low bioavailability, and rapid plasma clearance limit the clinical application of oridonin. Oral delivery
SC
of antitumor agents improves the life quality of patients and meets the needs of follow-up therapy.31 Here, oridonin was loaded in WGA-LPNs to improve oral bioavailability and antitumor
MA NU
efficiency. The pharmacokinetics of oridonin-loaded WGA-LPNs in rats and antitumor effects in HepG2 tumor-bearing nude mice were evaluated. To our knowledge, this is the first study to analyze mucosal diffusion of WGA-LPNs, the influence of mucin-WGA-LPNs interactions on
ED
cellular uptake, in vivo bioavailability, and antitumor efficiency. The results of this study
PT
corroborate the application of polymer-core lipid-shell type LPNs as effective oral drug delivery
AC
CE
systems.
5
ACCEPTED MANUSCRIPT Methods
T
Preparation and characterization of WGA-modified lipid polymeric nanoparticles (WGA-LPNs)
RI P
WGA-LPNs were prepared following our previous work with a slight modification.14 Briefly, an aqueous phase containing DSPE-PEG2000/LIPOID S100 (40/60, or 60/40 weight ratio) was
SC
mixed with the organic phase containing PLGA and oridonin in acetonitrile at a phase volume
MA NU
ratio of 2:1 under gentle stirring for 1 h. After vortexing for 3 min, the mixture was continuously stirred at 100 rpm for 2 h. Then, the LPNs were obtained by removal of acetonitrile using rotary evaporation, filtering through a 0.8-μm nylon filter, and dialysis in pH 7.4 phosphate-buffered
ED
saline (PBS) for 2 h. After incubation of pre-synthesized WGA-DOPE with LPNs for 18 h at room temperature, the WGA-modified LPNs were purified on a Sepharose CL-4B column and eluted
PT
with PBS. To evaluate the effect of PEGylation on the mucus penetration behavior of WGA-LPNs,
termed
CE
two sets of WGA-LPNs with 40 and 60% DSPE-PEG2000 (w/w, DSPE-PEG2000 to total lipids), WGA-LPNs-lower
polyethylene
glycol
modification
(WGA-LPNs-L)
and
AC
WGA-LPNs-higher polyethylene glycol modification (WGA-LPNs-H), respectively, were prepared and used in this study, alongside WGA-free LPNs with 60% DSPE-PEG2000 (LPNs-H). WGA-LPNs with various fluorescent labels (coumarin-6, RhB-MP, Nile red, and Dil), excluding CdSe/ZnS quantum dot-loaded WGA-LPNs, were prepared using the same method, with fluorescent labels in place of oridonin. Particle hydrodynamic diameters and zeta potentials were measured by Zetasizer nano-ZS 90 (Malvern Instruments, Malven, UK). The modification rate of WGA in LPNs was determined as previously reported.32
6
ACCEPTED MANUSCRIPT Interactions of nanoparticles with the mucus layer
T
The hydrodynamic radius of WGA LPNs-H and WGA-free LPNs-H in mucin dispersions was
RI P
measured by nanoparticle tracking analysis (NTA), with nanoparticles labeled with coumarin-6 to avoid interference by intrinsic fluorescence. In particular, 100 μl of coumarin-6-loaded
SC
WGA-LPNs-H and 600 μl of mucus were incubated at 37 °C for designated time. Upon dilution
MA NU
with deionized water (1:10000, v:v), the samples were injected into the chamber of Nanosight NS300 system (Malvern Instruments, Malven, UK), and subjected to data collection and analysis using NTA software (analytical version 2.1). For samples without mucus, WGA-LPNs were diluted with deionized water. Similarly, to observe the changes in the morphology of WGA-LPNs
ED
post incubation with mucus, the samples obtained by the similar treating process were imaged by
PT
field emission transmission electron microscopy (TEM, JEM-2100F, JEOL Ltd., Japan).
CE
Particle motion of the WGA-LPNs in mucus was studied by multiple particle tracking (MPT). Interactions between WGA-LPNs and mucins were further analyzed using fluorescence resonance
AC
energy transfer (FRET) analysis. The interaction of WGA-LPNs with the sialic acid in mucus was revealed by sialic acid content analysis. The methodology of MPT, FRET, and sialic acid content analysis are provided in the Supporting Information.
Cellular uptake studies
Caco-2 cells were seeded in 6-well plates (5 × 105 cells/well). Following culture overnight, the culture medium was replaced with DMEM containing the tested sample (LPNs-H + mucins, LPNs-H + PBS, WGA-LPNs-H + mucins, WGA-LPNs-H + PBS, and WGA-LPNs-L + mucins). The samples were prepared as follows: coumarin-6-loaded nanoparticles were dispersed using
7
ACCEPTED MANUSCRIPT PBS until equal fluorescence intensity was achieved, followed by addition of mucin solution (1%, w/v) or PBS depending on the experimental group. The mixture was gently stirred at 37 °C for 1 h
RI P
T
prior to addition to the cells. At the designated time, the cells were washed with PBS three times, then trypsinized and centrifuged. The cells were collected and resuspended in ice-cold PBS. Flow
SC
cytometry (BD LSRFortessa, BD Biosciences, CA, USA) was used to determine the fluorescence intensity in the fluorescein isothiocyanate (FITC)-A channel with 1 × 105 cell measurements for
MA NU
each sample.
In vitro cellular uptake was qualitatively analyzed by confocal laser scanning microscopy (CLSM). In particular, 400 μl of Nile red-loaded nanoparticles was incubated with 100 μl of FITC-mucin
ED
(2.5%, w/v) at 37 °C by gentle stirring for 1 h, filtered through a 0.8-μm nylon filter, and
PT
ultrafiltered using Vivaspin 6.0 (Sartorius Stedim Biotech SA, Aubagne, France) at 3000 × g for 20 min. The purified nanoparticles were redispersed in PBS (pH 7.4) prior to addition to the cells.
CE
Caco-2 cells were seeded (5 × 104 cells/well) and cultured overnight. The cells were incubated
AC
with DMEM containing the treated nanoparticles or nanoparticles without mucin for 1 h, followed by rinsing with PBS, fixation using 4% paraformaldehyde, and staining of cell nuclei with DAPI. After washing with PBS three times, the cells were observed using a TCS SP8 confocal system (Leica, Mannheim, Germany).
In situ intestinal diffusion study
All animals received humane care and all animal experiments were approved by the Institutional Animal Care and Use Committee, Shanghai University of Traditional Chinese Medicine. Prior to the experiment, a liquid diet was given to the mice for 2 days followed by a 24-h fast with free access to water. The mice were anesthetized by an intraperitoneal injection of chloral hydrate (500 8
ACCEPTED MANUSCRIPT mg/kg), and then placed on a thermostatic surface maintained at 37 °C. An incision (approximately 1 cm) was made in the abdomen. Approximately 2 cm of the jejunum was exposed.
RI P
T
After one end was ligated with a suture, 50 µl of pre-prepared nanoparticle dispersion (composed of an equal volume of coumarin-6-loaded WGA-LPNs (2.87 × 10-2 μmol/ml of coumarin-6) and
SC
Dil-loaded WGA-LPNs (2.70 × 10-2 μmol/ml of Dil)) was sealed within the exposed jejunum by ligation. The jejunum was gently replaced into the abdominal cavity and maintained for 20 min.
MA NU
Subsequently, the sealed segment was washed with saline and cut off along the inside of the suture, and placed on a piece of filter paper to remove the redundant preparations. Then, it was incised along the middle and put onto a piece of filter paper with the mucous layer facing upward. Pieces
ED
(approximately 2 × 5 mm) were prepared and gently placed onto a glass Petri dish and observed
PT
by CLSM. The images were obtained in consecutive parallel XY-sections as focal planes along the Z-axis. The scanning started from the lumen side and moved to the base at 10 μm intervals to
CE
determine the diffusion and penetration depth of the nanoparticles.
AC
In vivo bioavailability study
The WGA-LPNs and LPNs in the in vivo bioavailability study and efficacy evaluation refer to WGA-LPNs-H and LPNs-H, respectively. Fifteen male Sprague-Dawley rats weighting 220 ± 20 g were randomly assigned to three groups. Oridonin suspension, LPNs, and WGA-LPNs were administered via oral gavage at a dose of 10 mg/kg. The oridonin suspension was prepared by dispersing oridonin in PEG400 solution (PEG400 in deionized water, 50:50, v:v). Blood samples (300 μl) of rats were withdrawn at predetermined time points and centrifuged at 3500 × g for 5 min to separate the plasma. Details of the drug content measurement are described in the Supporting Information. Pharmacokinetic parameters were determined using Winnolin 2 software 9
ACCEPTED MANUSCRIPT (Pharsight, USA) using a non-compartmental model.
T
In vivo efficacy evaluation
RI P
The tumor xenograft model was established by inoculating HepG-2 cells suspended in DMEM (5 × 106 cells/mouse) subcutaneously into the right armpit region of male BALB/c nude mice (4-6
SC
weeks old, 18-22 g).33 Once the tumor volume reached approximately 200 mm3, the animals were
MA NU
weighed and randomly divided into five groups (n = 5) receiving saline, 5-fluorouracil solution (22.5 mg/kg), oridonin suspension (7.5 mg/kg), LPNs (7.5 mg/kg), or WGA-LPNs (7.5 mg/kg). The animals were administered the formulations every two days via oral gavage. All treatments
ED
were administered for 24 days. Tumor volume and body weight were recorded every two days during the treatment. Tumor volume was calculated as π/6 × length × (width). The mice were
PT
sacrificed after 24 days, and tumors were isolated and weighed. Histopathological evaluation of
CE
tumor tissues was performed using hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay.
AC
Statistical analysis
Data are presented as the mean ± standard deviation (S.D.). Data were compared using one-way analysis of variance (ANOVA) with Tukey’s post-test. P < 0.05 was considered statistically significant.
10
ACCEPTED MANUSCRIPT Results
T
Nanoparticle characterization
RI P
The characterization of WGA-LPNs-L, WGA-LPNs-H, and LPNs-H is shown in Table S1. The
SC
WGA modification rate was 20.35 ± 7.36% (WGA-LPNs-L) and 35.21 ± 1.62% (WGA-LPNs-H).
Motion of nanoparticles in mucus
MA NU
The motion of CdSe/ZnS quantum dot (QD)-loaded WGA-LPNs-H in mucus or water was recorded and compared with that of LPNs-H using MPT (Figure 1). The ensemble-averaged mean-squared displacement (
ED
in water (P < 0.05). At a time point of 1 s, the
PT
than the
CE
a slight decrease of movement in mucus; however, the difference was not significant (P > 0.05).
AC
The difference could be attributed to the hydrophilic non-WGA surface that protected the particles from interacting with glycosylated glycoproteins.
Interactions of nanoparticles with the mucus layer
The hydrodynamic radius of nanoparticles dispersed in the mucin solution increased over time compared to those dispersed in the mucin-free solution (Figure 2, A, D, F). In addition, the size distribution of particles broadened. In the blank mucin dispersion, 10% of particles had a size of 113 nm. The significant increase in particle size observed in NTA indicates agglomerates composed of nanoparticles and mucin or other adherent components. The particles retained their original core-shell structure and no difference in the thickness of the lipid layer was observed 11
ACCEPTED MANUSCRIPT during the nanoparticle-mucin interaction (Figure 2, C, E, G ). Compared to nanoparticles in the absence of mucin (Figure 2, C), homogeneous components from mucus adhered to the surface of
RI P
T
the particles, with the interactions increasing over time (Figure 2, E, G). In the case of LPNs-H, the lipid shell adhered to the surface of the polymeric core (Figure S1).
SC
Interactions between WGA-LPNs and mucins were analyzed using FRET. FITC and Rhb-MP (synthesized from rhodamine B and α-monopalmitin as previously reported7 ) were used as the
MA NU
FRET donor and acceptor, respectively, with mucins and WGA-LPNs labeled with FITC and Rhb-MP, respectively. As shown in Figure 3, obvious reduction of the donor fluorescence intensity in conjunction with increase of acceptor intensity, indicating energy transfer, suggested an
ED
interaction between WGA-LPNs and mucin. The FRET efficiency was 34.17 ± 1.15% and 17.13 ±
PT
1.96% for WGA-LPNs-H and WGA-LPNs-L, respectively. As sialic acid and sulfate residues from oligosaccharide chains of mucin glycoproteins could form hydrogen bonds with nanocarriers,
CE
we measured the changes in sialic acid content with or without WGA-LPNs. Sialic acid content in
AC
the presence of WGA-LPNs decreased by 1.48-fold compared to that reported for blank mucus, indicating that sialic acid contributes to the interaction of WGA-LPNs with mucus.
In vitro cellular uptake
The FRET results encouraged us to analyze the influence of mucin binding on the cellular uptake of WGA-LPNs. Nanoparticles in the presence or absence of mucin were taken up by Caco-2 cells after 1-h incubation (Figure 4, A). Caco-2 cells treated with WGA-LPNs showed enhanced fluorescence compared with that reported for LPNs (Figure 4, B), indicating that WGA modification improved cellular uptake, in agreement with a previous study.14 Caco-2 cells treated with WGA-LPNs preincubated with mucin showed a marked right shift in the fluorescence curve 12
ACCEPTED MANUSCRIPT compared with that reported for WGA-LPNs without mucin preincubation. The mean fluorescence of cells treated with WGA-LPNs preincubated with mucin increased by 1.4-fold. However, no
RI P
T
significant difference in fluorescence was found between LPNs with or without mucin preincubation, suggesting that the influence of mucin preincubation on nanoparticle cellular
SC
uptake depends on the surface features of the nanoparticles. WGA surface modification enhanced cellular uptake. In addition, WGA-LPNs-H possessing a higher WGA modification rate showed a
MA NU
significant increase in cell uptake compared with that for WGA-LPNs-L, which further suggests that in the presence of mucin, WGA contributes to cellular uptake more than surface hydrophilicity. Cellular uptake was verified by CLSM (Figure 4, C). For WGA-LPNs and LPNs pretreated with
PT
nanoparticles and mucin.
ED
mucin, green fluorescence appeared in the cytoplasm of Caco-2 cells, indicating cellular uptake of
CE
In situ intestinal diffusion study
In situ intestinal diffusion was analyzed to study the distribution of nanoparticles in mucus and
AC
intestinal cells by CLSM (Figure 5, A). Coumarin-6-labeled nanoparticles were used to reveal the penetration and diffusion through the mucus layer and cells, whereas Dil-labeled nanoparticles revealed cellular interactions, as Dil is a lipophilic membrane stain showing a red fluorescent signal when incorporated into the cell membrane. CLSM images in XY-plane micrographs arranged in Z-series tiers were recorded at 10-μm increments from the lumen surface (from top to bottom, Figure 5, B). Fluorescence intensity gradually increased with depth, reached a maximum (as shown in the non-rotated image in Figure 5, B), and then decreased with depth. The average depth of diffusion and penetration of WGA-LPNs-H, WGA-LPNs-L, and LPNs-H was 170.0 ± 7.1 μm (Figure 5, C), 132.0 ± 8.4 μm (Figure S2, A), and 130.0 ± 8.9 μm (Figure S2, B), respectively. 13
ACCEPTED MANUSCRIPT WGA-LPNs-H showed stronger red fluorescence, indicating a greater number of nanoparticles interacting with cells. WGA-modified LPNs reached the surface of cells and achieved specific
RI P
T
bioadhesion despite mucin interference.
In vivo bioavailability
SC
The mean oridonin plasma concentration-time curves of WGA-LPNs after oral administration in
MA NU
rats are shown in Figure 6, whereas the main pharmacokinetic parameters are summarized in Table S2. WGA-LPNs showed a similar maximum plasma concentration (Cmax) (1.1-fold) to LPNs-H. The area under the curve (AUC) of WGA-LPNs was significantly higher than that of LPNs (P <
ED
0.05). In addition, mean residence time (MRT) was 1.5 times longer in WGA-LPNs than in LPNs. The relative bioavailability of WGA-LPNs compared with suspensions and LPNs increased by
CE
oral bioavailability.
PT
9.09-fold and 1.96-fold, respectively, indicating that WGA-LPNs significantly increased oridonin
AC
In vivo efficacy evaluation
As shown in Figure 7, A-D, compared to the control, the positive control group, suspensions, LPNs, and WGA-LPNs showed significant anti-tumor effects (retardation of tumor growth and reduced tumor weight), with WGA-LPNs showing the highest therapeutic efficacy (P < 0.05). Tumor inhibition by WGA-LPNs was significantly higher than that by WGA-free LPNs, indicating that WGA modification enhances antitumor efficacy. In addition, the WGA-LPNs group showed less reduction in body weight compared to the positive control, indicating lower toxicity. H&E staining of the tumor tissue is shown in Figure 7, E. Viable tumor cells and vessels were present in the control group, whereas few necrotic cells were observed in other groups, and a large 14
ACCEPTED MANUSCRIPT necrotic area was identified in the WGA-LPNs group. In addition, based on the TUNEL assay,
RI P
superior effects in the in vivo study compared to the other groups.
T
obvious cellular apoptosis was present in the WGA-LPNs group. Overall, WGA-LPNs showed
Discussion
SC
In a previous study, WGA-LPNs improved enterocyte and goblet cell uptake in an in vitro cell culture model.14 Given the importance of efficient mucus diffusion in effective oral nanocarrier
MA NU
delivery34, this study focused on demonstrating the capacity of WGA-LPNs to overcome the mucus layer and improve bioavailability and therapeutic efficacy. NTA showed interactions between WGA-LPNs and mucus. For lipid nanocarriers, the presence of
ED
hydrophobic groups, which is a characteristic shared with WGA-LPNs, facilitates interactions
PT
with mucin fibers. FRET revealed that mucin was involved in binding of WGA-LPNs to mucus. The higher FRET efficiency in WGA-LPNs-H does not signify a higher amount of mucin adhered
CE
to WGA-LPNs-H, as FRET efficiency reflects donor and acceptor fluorescence at the site of
AC
FRET occurrence. The higher FRET efficiency could have been caused by a higher concentration of acceptor fluorophores located at the shell of WGA-LPNs-H. Mucosal diffusion and interaction between nanocarriers and mucins is influenced by a number of factors.8,10,11,35 As the particle size of WGA-LPNs was comparable to that of LPNs, steric obstruction was not the deciding factor in the present study. The negative charge of WGA-LPNs minimizes possible electrostatic interactions. Lipids in WGA-LPNs could form hydrophobic interactions with hydrophobic lipid-coated domains such as cysteine-rich domains. Hydrophobic interactions are non-specific, occur often in lipid-containing nanocarriers, and play a major role in mucoadhesion. Nanoparticles containing carboxyl, hydroxyl, amino, and sulfate groups form 15
ACCEPTED MANUSCRIPT hydrogen bonds with sialic acid and sulfate residues from mucin oligosaccharide chains.21 Thus, specific interactions between WGA and sialic acid and N-acetyl-D glucosamine are a possibility.
RI P
T
This study focused on the ability of WGA-LPNs to overcome the mucus layer, including their retention of shape integrity, mucus diffusion, and permeation. Despite interacting with mucus,
SC
WGA-LPNs-H remained intact in the mucus after 2 h of exposure. WGA-LPNs-H likely remained intact because the molecular interactions within the lipid shell were stronger than the interactions
MA NU
with the surrounding mucus.7
The influence of PEGylation on the diffusion of WGA-LPNs in the mucus layer was investigated by MPT. WGA-LPNs-H displayed improved particle motion in mucus dispersions. However,
ED
higher PEGylation resulted in more WGA molecules binding to the surface of the nanoparticles,
PT
which decreased particle motion. As the key factors determining nanocarrier motion in mucus are the surface properties of the particles,36 the overall effect should be considered. MPT analyzes
CE
particle motion at a small scale over micrometer distances.37 Considering the high heterogeneity of
AC
mesh porosity in mucus, particle motion was investigated further. In the last decade, several in vitro setups for testing the mucus permeation behavior of nanocarriers have been described,38,39 such as Side-by-Side diffusion setup, Transwell diffusion plates,
and the rotation tubes method. In vitro methods simplify mucus diffusion studies.
However, a number of factors, including source of mucus, freshness, differences in mucus structure, and mucus collection and preparation methods might lead to differences relative to in vivo conditions. Recently, Hanes et al. applied oral gavage and in situ intestinal loop methods combined with ex vivo tracking to study mucus interactions and distribution of nanoparticles.40 Here, the in situ intestinal loop method combined with CLSM vertical scanning was used to 16
ACCEPTED MANUSCRIPT analyze the distribution and diffusion of WGA-LPNs in the mouse intestine. The injection volume was carefully controlled to cover the luminal surface of the intestinal loop and avoid overloading.
RI P
T
Using CLSM vertical scanning, different depths of penetration and diffusion of fluorescently labeled nanoparticle were visualized in real time. Compared to tissue slices, CLSM is more
SC
convenient. The diffusion depth of WGA-LPNs-H was significantly increased compared to that of LPNs-H and WGA-LPNs-L, suggesting that WGA-LPNs-H diffuse through the mucus barrier and
MA NU
are taken up by enterocytes, improving systemic delivery after oral administration. The mucus layer is a dynamically renewing gel layer.34 Upon receiving extracellular stimuli, mucus (primarily MUC2 mucin) is secreted from goblet and Paneth cells via exocytosis.41 Mucin
ED
turnover of goblet cells of small intestine villi occurs in a few hours.42,43 Therefore, we
PT
hypothesize that nanoparticles located in the inner mucus layer near the cell membrane would encounter freshly secreted mucin. We examined the cellular uptake of WGA-LPNs in complex
CE
with mucin using CLSM and showed that WGA-LPNs and LPNs, which interacted with purified
AC
mucin, were taken up by Caco-2 cells. Cells internalize nanoparticles through several routes.44 In a previous study, WGA-LPNs were taken into cells via clathrin-mediated and receptor-mediated endocytosis, and LPNs via clathrin-mediated endocytosis.14 The mucin used in this study is a commercial, purified mucin composed of glycoproteins. Proteins undergo endocytosis via the clathrin-mediated pathway, caveolae-mediated pathway, macropinocytosis, and phagocytosis.45 Once internalized, mucin is localized to the endosome, followed by degradation in lysosomes.46 Membrane and intracellular transport of nanoparticles in complex with mucin are not well understood. Further studies are necessary to ascertain whether mucin perturbs the natural transport pathway of WGA-LPNs. Intracellular transport of mucin is not fully understood and further 17
ACCEPTED MANUSCRIPT research is needed. Mucin is a surfactant and adsorbs at solid-liquid interfaces.47 As binding of mucin with WGA-LPNs was demonstrated in this study, we hypothesize that the
RI P
T
mucin-WGA-LPNs mixture enters the cells via endocytosis. When quantified with flow cytometry, mucin enhanced WGA-LPN uptake and had no influence on WGA-free LPNs. Absence of
SC
WGA-mediated specific interactions in LPNs, coupled with increased particle size, which reduces cellular uptake48 could explain the lack of influence of mucin. In contrast, WGA-LPNs complexed
MA NU
with mucin were recognized at the cell surface, leading to cytoadhesion and enhanced WGA-LPNs uptake.
Recently, oridonin has been encapsulated into galactosylated nanoparticles for intravenous
ED
hepatocellular carcinoma therapy.49-51 Oral administration is of great value for improving the
PT
quality of life and compliance of patients; however, low bioavailability and rapid clearance interfere with successful oral application of oridonin. In this study, oridonin-loaded WGA-LPNs
CE
showed improved bioavailability and antitumor effects, which can be attributed to the following
AC
factors: (1) LPNs are promising carriers encapsulating and improving the solubility of hydrophobic drugs based on their the core-shell structure.12 The particle sizes of nanocarriers, including LPNs and WGA-LPNs, are favorable for enhanced absorption. (2) Following WGA modification, specific bioadhesion facilitates drug absorption by enhancing cellular uptake via multiple routes including receptor-mediated endocytosis. Furthermore, as a lectin, WGA promotes M cell transport, providing opportunities for WGA-LPNs to enter lymphatic tissue.52 (3) DSPE-PEG2000 enhances WGA-LPNs penetration of the mucus layer. (4) PEGylation leads to increased plasma drug concentration and prolonged half-life, which improve the accumulation of drugs in tumors, followed by antitumor effects mediated by the enhanced permeability and 18
ACCEPTED MANUSCRIPT retention (EPR) effect. (5) Use of nanocarriers improves oral absorption and modifies biodistribution,53 which is hypothesized to lead to higher hepatic intake. For efficient oral drug
RI P
T
administration, gastrointestinal toxicity, often indicated by reduction in body weight, is of concern. Animals administered WGA-LPNs showed the least body weight reduction among test groups;
SC
however, the gastrointestinal toxicity should be investigated in more detail.
Taken together, the results of this study demonstrate the potential of WGA-modified LPNs as
MA NU
effective oral nanocarriers combining biospecific adhesion to cellular surfaces and mucus permeation (Figure 8). Based on the in vitro and in situ studies of interactions between nanoparticles and mucus and mucus penetration, the fate of WGA-LPNs in mucus can be
ED
described as follows: upon WGA-LPNs exposure to the mucus layer, WGA-LPNs interact with
PT
mucin. Although the interaction with mucin hinders movement, WGA-LPNs penetrate the mucus layer and are taken up by enterocytes. WGA-LPNs undergo orderly muco- and cytoadhesion. In
CE
addition to the PEGylation evaluated in this study, alternative strategies for improving WGA-LPN
AC
cytoadhesion should be investigated. Oridonin-loaded WGA-LPNs exhibited improved bioavailability with desirable pharmacokinetics and effective antitumor activity in HepG2 tumor-bearing nude mice. These findings suggest that WGA-LPNs are promising oral delivery systems for antitumor therapeutic agents.
19
ACCEPTED MANUSCRIPT
References
T
1. Date AA, Hanes J, Ensign LM. Nanoparticles for oral delivery: Design, evaluation and
RI P
state-of-the-art. J Control Release 2016; 240:504-26.
2. Lundquist P, Artursson P. Oral absorption of peptides and nanoparticles across the human
SC
intestine: Opportunities, limitations and studies in human tissues. Adv Drug Deliv Rev 2016;
MA NU
106(Pt B):256-76.
3. Xu Q, Ensign LM, Boylan NJ, Schön A, Gong X, Yang JC, et al. Impact of surface polyethylene glycol (PEG) density on biodegradable nanoparticle transport in mucus ex vivo
ED
and distribution in vivo. ACS Nano 2015;9(9):9217-27.
4. Pereira de Sousa I, Steiner C, Schmutzler M, Wilcox MD, Veldhuis GJ, Pearson JP, et al.
PT
Mucus permeating carriers: formulation and characterization of highly densely charged
CE
nanoparticles. Eur J Pharm Biopharm 2015;97(Pt A):273-9. 5. Liu M, Zhang J, Zhu X, Shan W, Li L, Zhong J, et al. Efficient mucus permeation and tight
AC
junction opening by dissociable "mucus-inert" agent coated trimethylchitosan nanoparticles for oral insulin delivery. J Control Release 2016;222:67-77. 6. Yang M, Lai SK, Yu T, Wang YY, Happe C, Zhong W, et al. Nanoparticle penetration of human cervicovaginal mucus: the effect of polyvinyl alcohol. J Control Release 2014;192:202-8. 7. Zhang J, Lv Y, Wang B, Zhao S, Tan M, Lv G, et al. Influence of microemulsion-mucin interaction on the fate of microemulsions diffusing through pig gastric mucin solutions. Mol Pharm 2015;12(3):695-705. 8. Groo AC, Saulnier P, Gimel JC, Gravier J, Ailhas C, Benoit JP, et al. Fate of paclitaxel lipid 20
ACCEPTED MANUSCRIPT nanocapsules in intestinal mucus in view of their oral delivery. Int J Nanomedicine 2013;8:4291-302.
RI P
T
9. Yu T, Chan KW, Anonuevo A, Song X, Schuster BS, Chattopadhyay S, et al. Liposome-based mucus-penetrating particles (MPP) for mucosal theranostics: demonstration of diamagnetic
SC
chemical exchange saturation transfer (diaCEST) magnetic resonance imaging (MRI). Nanomedicine 2015;11(2):401-5.
MA NU
10. Karamanidou T, Karidi K, Bourganis V, Kontonikola K, Kammona O, Kiparissides C. Effective incorporation of insulin in mucus permeating self-nanoemulsifying drug delivery systems. Eur J Pharm Biopharm 2015;97(Pt A):223-9.
ED
11. Beloqui A, Solinís MÁ, des Rieux A, Préat V, Rodríguez-Gascón A. Dextran-protamine
PT
coated nanostructured lipid carriers as mucus-penetrating nanoparticles for lipophilicdrugs. Int J Pharm 2014;468(1-2):105-11.
CE
12. Mandal B, Bhattacharjee H, Mittal N, Sah H, Balabathula P, Thoma LA, et al. Core-shell-type hybrid
nanoparticles
as
a
drug
delivery
platform.
Nanomedicine
AC
lipid-polymer
2013;9(4):474-91. 13. Rao S, Prestidge CA. Polymer-lipid hybrid systems: merging the benefits of polymeric and lipid-based
nanocarriers
to
improve
oraldrug
delivery.
Expert
Opin Drug Deliv
2016;13(5):691-707. 14. Liu Y, Zhao YQ, Liu JG, Zhang MY, Yu ML, Feng NP. Wheat germ agglutinin modification of lipid-polymer hybrid nanoparticles: enhanced cellular uptake and bioadhesion. RSC Advances 2016;6:36125-35 15 Yuan H, Chen CY, Chai GH, Du YZ, Hu FQ. Improved transport and absorption through 21
ACCEPTED MANUSCRIPT gastrointestinal tract by PEGylated solid lipid nanoparticles. Mol Pharm 2013;10(5):1865-73. 16. Luo Y, Teng Z, Li Y, Wang Q. Solid lipid nanoparticles for oral drug delivery: chitosan
RI P
T
coating improves stability, controlled delivery, mucoadhesion and cellular uptake. Carbohydr Polym 2015;122:221-9.
SC
17. Gradauer K, Barthelmes J, Vonach C, Almer G, Mangge H, Teubl B, et al. Liposomes coated with thiolated chitosan enhance oral peptide delivery to rats. J Control Release
MA NU
2013;172(3):872-8.
18. Bajka BH, Rigby NM, Cross KL, Macierzanka A, Mackie AR. The influence of small intestinal mucus structure on particle transport ex vivo. Colloids Surf B Biointerfaces
ED
2015;135:73-80.
PT
19. Gabor F, Bogner E, Weissenboeck A, Wirth M. The lectin-cell interaction and its implications to intestinal lectin-mediated drug delivery. Adv Drug Deliv Rev 2004;56(4):459-80.
CE
20. Lai SK, O'Hanlon DE, Harrold S, Man ST, Wang YY, Cone R, et al. Rapid transport of large
AC
polymeric nanoparticles in fresh undiluted human mucus. Proc Natl Acad Sci USA 2007;104(5):1482-7. 21. Mansfield ED, Sillence K, Hole P, Williams AC, Khutoryanskiy VV. POZylation: a new approach to enhance nanoparticle diffusion through mucosal barriers. Nanoscale 2015;7(32):13671-9. 22. Liu M, Zhang J, Zhu X, Shan W, Li L, Zhong J, et al. Efficient mucus permeation and tight junction opening by dissociable "mucus-inert" agent coated trimethylchitosan nanoparticles for oral insulin delivery. J Control Release 2016;222:67-77. 23. Popov A, Enlow E, Bourassa J, Chen H. Mucus-penetrating nanoparticles made with 22
ACCEPTED MANUSCRIPT "mucoadhesive" poly(vinyl alcohol). Nanomedicine 2016; 12(7):1863-71. 24. Wang YY, Lai SK, Suk JS, Pace A, Cone R, Hanes J. Addressing the PEG mucoadhesivity
RI P
T
paradox to engineer nanoparticles that "slip" through the human mucus barrier. Angew Chem Int Ed Engl 2008;47(50):9726-9.
SC
25. Zhang DR, Ren TC. Progresses in the pharmaceutical of oridonin. Chin Pharm J 2003; 38:817-20.
MA NU
26. Wang Y, Liu XQ, Liu GP, Guo HJ, Li CY, Zhang YC, et al. Novel galactosylated biodegradable nanoparticles for hepatocyte-delivery of oridonin. Int J Pharm 2016; 502(1-2):47-60.
ED
27. Wang H, Ye Y, Pan SY, Zhu GY, Li YW, Fong DW, et al. Proteomic identification of proteins
PT
involved in the anticancer activities of oridonin in HepG2 cells. Phytomedicine 2011;18(2-3):163-9.
CE
28. Huang J, Wu L, Tashiro S, Onodera S, Ikejima T. Reactive oxygen species mediate
AC
oridonin-induced HepG2 apoptosis through p53, MAPK, and mitochondrial signaling pathways. J Pharmacol Sci 2008;107(4):370-9. 29. Wang H, Ye Y, Chu JH, Zhu GY, Fong WF, Yu ZL. Proteomic and functional analyses reveal the potential involvement of endoplasmic reticulum stress and α-CP1 in the anticancer activities of oridonin in HepG2 cells. Integr Cancer Ther 2011;10(2):160-7. 30. Li CY, Wang EQ, Cheng Y, Bao JK. Oridonin: An active diterpenoid targeting cell cycle arrest, apoptotic and autophagic pathways for cancer therapeutics. Int J Biochem Cell Biol 2011;43(5):701-4. 31. Thanki K, Gangwal RP, Sangamwar AT, Jain S. Oral delivery of anticancer drugs: challenges 23
ACCEPTED MANUSCRIPT and opportunities. J Control Release 2013;170(1):15-40. 32. Liu Y, Wang PF, Sun C, Feng NP, Zhou WX, Yang Y, et al. Wheat germ agglutinin-grafted
RI P
T
lipid nanoparticles: Preparation and in vitro evaluation of the association with Caco-2 monolayers. Int J Pharm 2010, 397:155-63
SC
33. Ding Y, Wang YZ, Opoku-Damoah Y, Wang C, Shen LJ, Yin LF, Zhou JP. Dual-functional bio-derived nanoparticulates for apoptotic antitumor therapy. Biomaterials 2015;72:90-103.
MA NU
34. Ensign LM, Cone R, Hanes J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv Drug Deliv Rev 2012;64(6):557-70. 35. Sheng J, Han L, Qin J, Ru G, Li R, Wu L, et al. N-trimethyl chitosan chloride-coated PLGA
ED
nanoparticles overcoming multiple barriers to oral insulin absorption. ACS Appl Mater
PT
Interfaces 2015;7(28):15430-41.
36. Dünnhaupt S, Kammona O, Waldner C, Kiparissides C, Bernkop-Schnürch A. Nano-carrier
CE
systems: Strategies to overcome the mucus gel barrier. Eur J Pharm Biopharm
AC
2015;96:447-53.
37. Schuster BS, Ensign LM, Allan DB, Suk JS, Hanes J. Particle tracking in drug and gene delivery research: State-of-the-art applications and methods. Adv Drug Deliv Rev 2015;91:70-91. 38. Grießinger J, Dünnhaupt S, Cattoz B, Griffiths P, Oh S, et al. Methods to determine the interactions of micro- and nanoparticles with mucus. Eur J Pharm Biopharm 2015; 96:464-76. 39. Groo AC, Lagarce F. Mucus models to evaluate nanomedicines for diffusion. Drug Discov Today 2014;19(8):1097-108. 24
ACCEPTED MANUSCRIPT 40.Maisel
K, Ensign
L, Reddy
M, Cone
R, Hanes
J.
Effect of surface chemistry on nanoparticle interaction with gastrointestinal mucus and distrib
RI P
T
ution in the gastrointestinal tract following oral and rectal administration in the mouse. J Control Release 2015;197:48-57.
SC
41. McGuckin MA, Lindén SK, Sutton P, Florin TH. Mucin dynamics and enteric pathogens. Nat Rev Microbiol 2011;9(4):265-78. Birchenough
GM, Johansson
ME, Gustafsson
MA NU
42.
JK, Bergström
New developments in goblet cell mucus secretion and function. 2015;8(4):712-9.
JH, Hansson
Mucosal
GC.
Immunol
ED
43. Johansson ME. Fast renewal of the distal colonic mucus layers by the surface goblet cells as
PT
measured by in vivo labeling of mucin glycoproteins. PLoS One 2012;7(7):e41009. 44. Nam HY, Kwon SM, Chung H, Lee SY, Kwon SH, Jeon H, et al. Cellular uptake mechanism
CE
and intracellular fate of hydrophobically modified glycol chitosan nanoparticles. J Control
AC
Release 2009;135(3):259-67. 45. Joshi S, Kumar S, Choudhury A, Ponnusamy MP, Batra SK. Altered Mucins (MUC) trafficking in benign and malignant conditions. Oncotarget 2014;5(17):7272-84. 46. Mayor S, Pagano RE. Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 2007;8(8):603-12. 47. Karin D. Effects of mucin addition on the stability of oil-water emulsions. Colloids Surf B Biointerfaces 1999;15(3):303-12. 48. Iversen TG, Skotland T, Sandvig K. Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 2011;6:176-85. 25
ACCEPTED MANUSCRIPT 49. Wang Y, Liu X, Liu G, Guo H, Li C, Zhang Y,et al. Novel galactosylated biodegradable nanoparticles for hepatocyte-delivery of oridonin. Int J Pharm 2016;502(1-2):47-60.
RI P
T
50. Li C, Zhang D, Guo H, Hao L, Zheng D, Liu G, et al. Preparation and characterization of galactosylated bovine serum albumin nanoparticles for liver-targeted delivery of oridonin. Int
SC
J Pharm 2013;448(1):79-86.
51. Zheng D, Duan C, Zhang D, Jia L, Liu G, Liu Y, et al. Galactosylated chitosan nanoparticles
MA NU
for hepatocyte-targeted delivery of oridonin. Int J Pharm 2012;436(1-2):379-86. 52. Jepson MA, Clark MA, Hirst BH. M cell targeting by lectins: a strategy for mucosal vaccination and drug delivery. Adv Drug Deliv Rev 2004;56(4):511-25.
of
oral
absorption
of
solidified
polymer
micelles.
Nanomedicine
PT
mechanism
ED
53. Abramov E, Cassiola F, Schwob O, Karsh-Bluman A, Shapero M, Ellis J, et al. Cellular
AC
CE
2015;11(8):1993-2002.
26
ACCEPTED MANUSCRIPT
Figure legends
T
Figure 1. Motion of CdSe/ZnS quantum dot (QD)-loaded particles in porcine mucus. (A) Average
RI P
ensemble geometric mean-squared displacement (
SC
per sample]
MA NU
Figure 2. Particle size analysis. Left row: Nanoparticle tracking analysis (lower left: particle size per relative intensity three-dimensional plot; upper right: particle size per concentration). Right row: TEM images. (A) and (C) WGA-LPNs-H before incubation with mucus. (B) Blank mucus.
ED
(D) and (E) Incubation of WGA-LPNs-H with mucus for 1 h. (F) and (G) Incubation of WGA-LPNs-H with mucus for 2 h.
PT
Figure 3. (A) Changes in fluorescence emission spectra of FITC-labeled mucin (FITC-mucin) as a
CE
function of RhB-MP-labeled WGA-LPNs-H. The ratio represents the volume ratios of FITC-mucin vs. RhB-MP-labeled WGA-LPNs-H; (B) Fluorescence emission intensity ratio of
AC
RhB-MP and FITC with increasing RhB-MP volume and equal amounts of FITC. Figure 4. (A) Cellular uptake determined by flow cytometry analysis of control (untreated cells), WGA-LPNs-L in the presence of mucin (WGA-LPNs-L + mucin), WGA-LPNs-H in the presence of mucin (WGA-LPNs-H + mucin), WGA-LPNs-H in pH 7.4 phosphate-buffered saline (PBS) (WGA-LPNs-H + PBS), LPNs-H in the presence of mucin (LPNs-H + mucin), and LPNs-H in PBS (LPNs-H + PBS) in Caco-2 cells after 1 h of incubation. (B) Fluorescence intensity analysis. Data are shown as the mean ± S.D., n = 3, *P < 0.05, N.S. represents a non-significant difference. (C) Confocal microscopic images of Caco-2 cells treated with LPNs-H and WGA-LPNs-H in the presence of mucin. 27
ACCEPTED MANUSCRIPT Figure 5. (A) Schematic representation of the CLSM vertical scanning approach used to analyze the distribution and diffusion of WGA-LPNs in the small intestine of mice. (B) Representative
RI P
T
confocal micrographs showing in situ diffusion and penetration of WGA-LPNs in mouse jejunum. Coumarin-6-labeled nanoparticles (green signal) and Dil-labeled nanoparticles were mixed in
SC
equal volumes and applied to the intestinal section for 30 min before the images were taken. Sequential stacked XY-plane (rotation angle: x 80°, y 18°, z 270°) in Z-series with 10-μm interval
MA NU
(parallel to the lumen surface). (C) 3D reconstruction images from intestine cross-section (yz plane).
Figure 6. Mean plasma concentration-time profiles of WGA-LPNs-H, LPNs-H, and suspensions in
ED
rats following ig administration at a dose of 10 mg/kg (data are shown as the mean ± S.D., n = 5).
PT
Figure 7. Antitumor efficacy and histological studies in HepG2 tumor xenograft nude mice in vivo. (A) Tumor volumes of HepG2 tumor-bearing mouse groups. (B) Images of xenografts taken by
CE
digital camera after 24-day treatment. (C) Average tumor weight. (D) Body weight curves. (Data
AC
are shown as the mean ± S.D. n = 5, *P < 0.05 and **P < 0.01 for WGA-LPNs versus other groups). (E) Histological analysis of the tumor tissues from each group at the end of the experiment (n = 3).
Figure 8. Schematic illustration of interaction of WGA-LPNs with mucin, mucus penetration, and biospecific adhesion. LPNs and WGA-LPNs are taken up by enterocytes in the presence or absence of mucin. M cell uptake could be an alternative pathway for intestinal absorption of WGA-LPNs.
28
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 1
29
AC
Figure 2
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
30
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
Figure 3
31
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
Figure 4
32
AC
Figure 5
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
33
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
Figure 6
34
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
Figure 7
35
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 8
36
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
ED
Graphical abstract
PT
The fate of wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles (WGA-LPNs) in
CE
mucus can be described as follows: upon WGA-LPNs exposure to the mucus layer, WGA-LPNs interact with mucin. Despite the interaction with mucin hindering movement, WGA-LPNs
AC
penetrate the mucus layer and are taken up by enterocytes. Oridonin-loaded WGA-LPNs exhibited improved bioavailability with desirable pharmacokinetics and effective antitumor activity in HepG2 tumor-bearing nude mice.
37