Renal-targeted delivery of triptolide by entrapment in pegylated TRX-20-modified liposomes

Article abstract of DOI:10.2147/IJN.S141095

Previously, 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20)-modified liposomes were reported to specifically target mesangial cells (MCs) in glomeruli. To further gain a better understanding of the characteristics and potential application for glomerular diseases of TRX-20-modified liposomes, we synthesized TRX-20 and prepared TRX-20-modified liposomes (TRX-LPs) with different molar ratios - 6% (6%-TRX-LP), 11% (11%-TRX-LP), and 14% (14%-TRX-LP) - of TRX-20 to total lipid in the present study. All TRX-LPs exhibited concentration-dependent toxicity against the MCs at a lipid concentration ranging from 0.01 to 1.0 mg/mL with IC₅₀ values of 3.45, 1.13, and 0.55 mg/mL, respectively. Comparison of the cell viability of TRX-LPs indicated that high levels of TRX-20 caused severe cell mortality, with 11%-TRX-LP showing the higher cytoplasmic accumulation in the MCs. Triptolide (TP) as a model drug was first loaded into 11%-TRX-LP and the liposomes were further modified with PEG₅₀₀₀ (PEG-TRX-TP-LP) in an attempt to prolong their circulation in blood and enhance TP-mediated immune suppression. Due to specific binding to MCs, PEG-TRX-TP-LP undoubtedly showed better anti-inflammatory action in vitro, evidenced by the inhibition of release of nitric oxide (NO) and tumor necrosis factor-α from lipopolysaccharide-stimulated MCs, compared with free TP at the same dose. In vivo, the PEG-TRX-TP-LP effectively attenuated the symptoms of membranous nephropathic (MN) rats and improved biochemical markers including proteinuria, serum cholesterol, and albumin. Therefore, it can be concluded that the TRX-modified liposome is an effective platform to target the delivery of TP to glomeruli for the treatment of MN.

Full text of DOI:10.2147/IJN.S141095

International Journal of Nanomedicine
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O r I g I N a l r e s e a r c h
renal-targeted delivery of triptolide by
entrapment in pegylatedTRX-20-modified
liposomes
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
8 August 2017
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Zhi-xiang Yuan1,*
lu Jia2,*
Abstract: Previously, 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20)-modified
liposomes were reported to specifically target mesangial cells (MCs) in glomeruli. To further
gain a better understanding of the characteristics and potential application for glomerular diseases
of TRX-20-modified liposomes, we synthesized TRX-20 and prepared TRX-20-modified
liposomes (TRX-LPs) with different molar ratios – 6% (6%-TRX-LP), 11% (11%-TRX-LP),
and 14% (14%-TRX-LP) – of TRX-20 to total lipid in the present study. All TRX-LPs exhibited
concentration-dependent toxicity against the MCs at a lipid concentration ranging from 0.01
to 1.0 mg/mL with IC50 values of 3.45, 1.13, and 0.55 mg/mL, respectively. Comparison of the
cell viability of TRX-LPs indicated that high levels of TRX-20 caused severe cell mortality,
with 11%-TRX-LP showing the higher cytoplasmic accumulation in the MCs. Triptolide (TP)
as a model drug was first loaded into 11%-TRX-LP and the liposomes were further modified
with PEG5000 (PEG-TRX-TP-LP) in an attempt to prolong their circulation in blood and enhance
TP-mediated immune suppression. Due to specific binding to MCs, PEG-TRX-TP-LP undoubt-
edly showed better anti-inflammatory action in vitro, evidenced by the inhibition of release
of nitric oxide (NO) and tumor necrosis factor-α from lipopolysaccharide-stimulated MCs,
compared with free TP at the same dose. In vivo, the PEG-TRX-TP-LP effectively attenuated
the symptoms of membranous nephropathic (MN) rats and improved biochemical markers
including proteinuria, serum cholesterol, and albumin. Therefore, it can be concluded that the
TRX-modified liposome is an effective platform to target the delivery of TP to glomeruli for
the treatment of MN.
lee Yong lim3
Ju-chun lin1
gang shu1
ling Zhao1
gang Ye1
Xiao-xia liang1
hongming Ji2
hua-lin Fu1
1Department of Pharmacy,
college of Veterinary Medicine,
sichuan agricultural University,
chengdu, sichuan, 2Department of
Neurosurgery, shanxi Provincial
People’ hospital,Taiyuan, china;
3Pharmacy, centre for Optimization
of Medicines, school of allied health,
The University of Western australia,
crawley, australia
*These authors contributed equally
to this work
Keywords: triptolide, pegylated liposomes, TRX-20, renal targeting, mesangial cells
Introduction
The kidneys are vital organs of humoral regulation, and they maintain the homeostasis
of nutrients and metabolites in the body. Renal dysfunction may lead to chronic
kidney disease, generally acknowledged as a serious problem affecting public health1
as it eventually requires expensive and debilitating renal replacement therapy, such
as dialysis or renal transplantation, to sustain the lives of patients. Drug therapies of
renal diseases often induce complicated extrarenal toxicity. Selective targeting of drugs
to kidneys can realize an increased renal effectiveness combined with a reduction of
generalized side effects.2–4 Targeted therapies can also lower drug doses to further
minimize side effects, an especially attractive option for chronic renal diseases requir-
ing treatment over extended periods.5
correspondence: hongming Ji
Department of Neurosurgery, shanxi
Provincial People’ hospital, No 29,
shuangtasi road, Taiyuan, shanxi 030012,
china
Tel +86 351 496 0343
email hongmingj@sina.com
hua-lin Fu
Department of Pharmacy, college of
Veterinary Medicine, sichuan agricultural
University, No 211, huimin road,
shuangliu District, chengdu, sichuan
611130, china
Renal drug targeting could be directed at the renal tubules or glomeruli depending on
the diseases to be treated. In previous studies, we have developed several drug carriers
Tel +86 133 0816 2816
email fuhl2005@sohu.com
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http://dx.doi.org/10.2147/IJN.S141095

Yuan et al
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with capacity for renal tubular targeting, including low-
To gain a better understanding of the characteristics and
molecular-weight chitosans,6,7 glucosamines,8,9 and peptide potential applications of glomerular diseases of TRX-20-
fragments of human serum albumin.10,11 These carriers have modified liposomes (TRX-LPs), we synthesized TRX-20 and
been shown to achieve effective targeted delivery of pred- prepared TRX-LPs with different molar ratios of TRX-20 to
nisolone or triptolide (TP) to the renal tubules. However, total lipid in this study. Cytotoxicity of TRX-LPs toward the
they may not be effective carriers of drugs required for the MCs was performed for the first time. The binding affinities
treatment of inflammatory and pathogenic glomeruli diseases. to MCs of liposomes modified with different TRX-20 molar
In recent years, a great many studies have demonstrated ratios and loaded with a fluorescent agent were evaluated
a key point for glomerular cell responses in the progression by laser confocal microscopy. Finally, TP as a model drug
of renal disease, which enlightens us that drug delivery to was first loaded into TRX-LP with PEG5000 co-modification
the glomerulus restraining local inflammatory/pathological (PEG-TRX-TP-LP) in an attempt to enhance TP-mediated
reactions could be hoped to produce better therapeutic immune suppression. In vitro anti-inflammatory activity was
results.5,12 Tuffin et al first proposed immunoliposomes as determined by measuring the nitric oxide (NO) and tumor
a glomerular delivery system for the treatment of kidney necrosis factor-α (TNF-α) released from lipopolysaccha-
diseases gradually.13 They developed OX7-coupled immuno- ride (LPS)-stimulated MCs. Additionally, the efficacy of
liposomes by coupling liposomes with Fab’ fragments of OX7 PEG-TRX-TP-LP was evaluated in vivo in a membranous
monoclonal antibody directed against Thy1.1 antigen, which nephropathy (MN) rat model.
is specifically expressed in mesangial cells (MCs). Intrave-
Materials and methods
nous injection of OX-7-IL to rats showed a specific targeting
of all MCs in both kidneys.13 Due to the strong and exclusive Materials and animals
expression of α8 integrin in MCs,14 Scindia et al selected 3,5-dihydroxybenzonitrile, 1-bromopentadecane, choles-
anti-α8 integrin as a ligand to prepare immunoliposomes. terol, collagenase IV, Coumarin-6 (C6), and pluronic 188
Their findings demonstrated the specific delivery of anti-α8 (F68) were from Sigma-Aldrich Co. Ltd. (Gillingham, UK).
integrin immunoliposomes to the mesangium following tail TP was purchased from Xieli Biotechnology Co. Ltd.
vein injection in mice.15 Moreover, Morimoto et al devel- (Sichuan, China). Hydrogenated soybean phosphatidylcho-
oped novel glomeruli-targeting liposome co-modified by line (HSPC) and polyethylene glycol 5000-sn-glycrero-3-
3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20) phosphatydilethanolamine (PEG5000-PE) were obtained from
and PEG5000. PEGylation can prolong blood circulation time Lipoid Co. Ltd. (Ludwigshafen, Germany). 4′,6-diamidino-
of the liposomes and allowed them to accumulate in target- 2-phenylindole dihydrochloride (DAPI) was supplied by
ing tissues where the liposomes repeatedly pass by. The Roche Co. Ltd. (Shanghai, China). Fetal bovine serum and
final liposomes have diameter of around 100 nm,16 positive RPMI 1640 were purchased from Thermo Scientific Co.
zeta potential, and high selection for MCs over vascular Ltd. (IL, USA). All other chemicals and solvents were of
endothelial cells.17
the analytical grade.
It is clear that MCs as an effective target site are adopted
Sprague Dawley (SD) rats (200±20 g, male, 6 weeks old)
in all the studies mentioned above. MCs comprise ~30%–40% were obtained from the Da-Shuo Experimental Animal Ltd.
of the total glomerular cell population, and they play a cen- (Beijing, China). The animals were allowed to acclimatize for
tral role in maintaining the structure and regulating the a few days in environmentally controlled quarters (24°C±1°C,
surface area of the glomerular filtration barrier by virtue 12 h light/dark cycle) and, unless specified otherwise, were
of their contractile capacities.13 MCs are often involved in provided with water and normal diets ad libitum. All animal
multiple biological responses elicited by circulating factors studies were approved by the Animal Ethical Experimenta-
and metabolites, including cytoproliferation, apoptosis, tion Committee of Sichuan Academy of Chinese Medicine
cellular migration, and the elaboration of reactive oxygen Sciences (SYXK[Chuan]2013–100), and were performed
species, cytokines, and chemokines, which undergird many according to the requirements of the People’s Republic of
glomerular diseases. Therefore, the selective delivery of China National Act on the use of experimental animals.
drugs to glomerular MCs could considerably improve the
therapeutic outcome of immunoglobulin A nephropathy and synthesis ofTrX-20
other glomerulopathies than targeting drug delivery to the The synthesis of TRX-20 was carried out according to Harigai
renal tubular cells.
et al’s method with some improvements17 (Figure 1).
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renal-targeted delivery of triptolide
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Figure 1 synthetic route of 3,5-dipentadecyloxybenzamidine hydrochloride.
3,5-dihydroxybenzonitrile (0.405 g), 1-bromopentadecane dissolving C6 (0.5 mg), HSPC, and cholesterol (40 mg,
(1.85 g), and potassium carbonate (0.9 g) were dissolved molar ratio of 4:1) in chloroform in a round-bottom flask
in N,N-dimethylformamide (10 mL) and stirred at 60°C followed by eliminating the organic solvent under reduced
overnight. Then, 50 mL of water was added into the reac- pressure to form the lipid film. After hydration in 5 mL of
tion mixture to precipitate the product followed by thorough phosphate-buffered saline (PBS, pH 7.2) containing 0.2%
washing with water to give 3,5-dipentadecyloxybenzonitrile F68, the dispersion was sonicated using a probe ultrasonicator
with a yield of 61.2%. (Singlet and multiplates were, respec- (JY92-II ultrasonic cell crusher processor, Ningbo, China)
tively, abbreviated as s and m. 1H-NMR (400 MHz, CDCl3): operating at pulse function (200 watts, 20 hertz; pulse on/
ppm δ 6.73 (s, 2H), 6.63 (s, 1H), 3.93 (t, 4H, J=4 Hz), 1.77 off: 20 s/15 s; 10 times), and filtered through a 0.2-μm filter
(t, 4H, J=8 Hz), 1.44 (s, 4H), 1.26 (s, 44H), 0.88 (m, 6H)) to obtain liposomes of around 100 nm.
(Figure S1). TRX-20 was synthesized by dissolving 500 mg
All other liposomes for the study were fabricated using
of 3,5-dipentadecyloxybenzonitrile in 23 mL of methanol similar procedures and by dissolving the requisite starting
and chloroform (3:20), and injecting dry HCl gas into the materials in the chloroform solution prior to lipid film for-
reaction system until saturation. After reaction at ambient mation. TRX-20-modified Coumarin-6-loaded liposomes
temperature overnight, the solvent was evaporated off, and (TRX-C6-LP) were prepared by additionally including
25 mL of saturated ammonia in methanol and chloroform TRX-20 at 2.0, 4.0, and 5.0 mg to give liposomes with
(1:4) were added with stirring for 24 h. TRX-20 was obtained molar ratios of TRX-20 to total lipids of 6%, 11%, and 14%,
as colorless crystals by recrystallization in methanol (yield: respectively. Liposomes with 6%-, 11%-, and 14%-TRX-20
50.4%). (1H-NMR CDCl3, 400 MHz): ppm δ 9.80 (s, 2H), to total lipids are abbreviated as 6%-TRX-LP, 11%-TRX-LP,
7.80 (s, 1H), 6.91 (s, 2H), 6.67 (s, 1H), 4.00 (t, 4H, J=4 Hz), and 14%-TRX-LP, respectively, to ease discussion. Control
1.77 (t, 4H, J=6), 1.44 (s, 4H), 1.26 (s, 44H), 0.88 (m, 6H) TRX-20-modified blank liposomes (TRX-LP) were prepared
(Figure S2).
by omitting the use of C6. TP-loaded TRX-LP (TRX-TP-LP)
was prepared using 4.0 mg of TRX-20 (11 mole%) and
substituting C6 with TP (4.0 mg), while PEG5000-modified
TRX-TP-LP (PEG-TRX-TP-LP) was prepared by also adding
liposome preparation, labeling, and
modification
According to our previous report, all liposomes were PEG5000-PE (1.5 mg, ~0.65 mole% of the total lipids) besides
prepared using a lipid film hydration method.18 Coumarin- 4.0 mg each of TRX-20 and TP into the chloroform solution
6-loaded liposomes (C6-LP) as control were prepared by prior to film formation.
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Following the fabrication of the TP-loaded liposomes, cellular uptake ofTrX-c6-lPs
any unencapsulated TP was removed by gel filtration, and MCs were seeded at a density of 2×104 cells/well with 2 mL
TP loading concentrations in the TRX-TP-LP and PEG- of RPMI 1640 onto six-well chamber slides (Corning Life
TRX-TP-LP were determined by HPLC after demulsification Sciences, NY, USA). After 2–3 days of culture, the cells were
with methanol.
incubated with C6-LP (control) or TRX-C6-LPs at a lipid
The particle sizes of the liposomes were determined concentration of 0.8 mg/mL for 2 h in serum-free RPMI 1640
using a Zetasizer Nano ZS equipment (Malvern Instru- at 37°C. The cells were then washed with PBS to terminate
ments Ltd, Malvern, UK). The morphology of TRX-C6-LP the uptake process. To visualize the cellular uptake, the
(11% mole%) and PEG-TRX-TP-LP was visualized under cells were fixed in 10% neutral buffer formalin for 10 min,
a transmission electron microscope (TEM, H-7500, Hitachi, then counterstained with DAPI for observation under a laser
Japan) following negative staining with 2% sodium phos- confocal microscope (SP5, Leica, Germany).
photungstate solution.
Determination of NO andTNF-α
production in Mcs
Primary culture of Mcs
MCs were isolated from male SD rats weighing 150–200 g To measure the anti-inflammatory activity of TRX-TP-LP
by the sieving method as previously described.19 In brief, and PEG-TRX-TP-LP, their effects on nitric oxide (NO) and
the kidneys were removed from their capsule and the cortex TNF-α production in LPS-stimulated MCs were determined
was separated and cut into fragments (5–10 mm3) followed in vitro.20 MCs were seeded at a density of 1×105 cells/well in
by mechanical sieving to obtain the glomeruli on 100- and 96-well plates and incubated until confluence. The cells were
200-mesh stainless-steel sieves. After enzymatic digestions then exposed to 200 μL LPS (10 μg/mL in DMEM) alone, or
with collagenase IV (0.1% w/v) at 37°C in PBS solution for togetherwithTRX-TP-LPorPEG-TRX-TP-LP(TPequivalent
20–45 min, the MC suspensions were obtained and cultured concentration of 2.0 μg/mL, in DMEM) or TP (2.0 μg/mL in
in RPMI 1640 medium containing 20% heat-inactivated DMEM with 1% DMSO). After incubation for 24 h, 50 μL of
fetal bovine serum, 2 μg/mL insulin, 300 μg/L transferrin, supernatant from each well was aspirated into a new 96-well
100 U/mL penicillin, and 100 U/mL streptomycin at 37°C plate for the determination of NO level using nitric oxide
in a humidified 5% (v/v) CO2 incubator (Thermo Scientific, assay kits (Jiancheng, Nanjing, China). Similarly, 100 μL of
Marietta, OH, USA). Passages of primary cultures were supernatant was collected from each well, and the produc-
performed after 3–4 weeks. To eliminate contamination tion of TNF-α assessed using a commercial rodent TNF-α
by either epithelial or endothelial cells, experiments were enzyme-linked immunosorbent assay kit (Pierce Biotechnol-
performed on cells obtained after passage 5.
ogy, Rockford, IL, USA). Each treatment was repeated for five
separate wells, and the data pooled to obtain the average.
cytotoxicity ofTrX-lPs
MCs (passage 6) were seeded onto 96-well plates (Corning, Treatment of nephropathic rats
NY, USA) at a density of 1×105 cells/well with 200 μL of SD rats were randomly assigned into normal, control, TP,
RPMI 1640. After incubation for 24 h, the culture medium TRX-TP-LP, and PEG-TRX-TP-LP groups (n=5 per group).
was replaced with 200 μL of blank liposomes (control) The MN model was established in all except rats in the
or TRX-LPs at a lipid concentration ranging from 0.01 to normal group according to our previous method.11 In brief,
1.0 mg/mL in RPMI 1640 solutions and the plates were cationic bovine serum albumin (C-BSA, 1 mg emulsified in
returned to the incubator for another 24 h. The test samples 0.5 mL of Freund’s complete adjuvant) was subcutaneously
were discarded and the viability of cells was determined injected into the rats to initiate the immunization. One week
by adding 20 μL of MTT (5 mg/mL in PBS) and 100 μL later, rats were immunized intravenously with 2.5 mg C-BSA
RPMI 1640 into each well. The medium was aspirated after every other day for another 2 weeks to finally establish the
2 h of incubation, and the intracellular formazan dissolved MN rat model. Rats in the normal group followed the same
with 150 μL dimethyl sulfoxide (DMSO) was quantified by injection schedule but were administered with normal saline
measuring the optical density of each well at 490 nm (iMark instead of C-BSA.
microplate-reader, Bio-rad, CA, USA). Percent cell viability
The MN rats were then administered intravenously
relative to control was plotted as a function of total lipid with saline (control group), 100 μg/kg TP (TP group) or
concentration (μg/mL).
~1.6 mg total lipids/kg of TRX-TP-LP (TRX-TP-LP group,
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renal-targeted delivery of triptolide
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an equivalent dose of 100 μg/kg TP), or ~1.6 mg total lipids/
kg of PEG-TRX-TP-LP (PEG-TRX-TP-LP group, an equiva-
lent dose of 100 μg/kg TP) every other day for 2 weeks. Urine
samples were collected during the final 24 h of the experiment
from all rats for the measurements of urinary protein (Up)
and urinary creatinine (Ucr) levels (Up and creatinine assay
kits, Jiancheng, Nanjing, China) to determine the level of
proteinuria (ratio of Up/Ucr). Twenty-four hours after the
last injection, the animals were killed and blood samples col-
lected from the abdominal aorta were measured for albumin,
cholesterol, and creatinine (SCr) levels (7020 automated
multiparametric analyzer, Hitachi, Tokyo, Japan).
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Data were analyzed with the SPSS 15.0 statistical package.
Multiple comparisons of mean values were performed by
one-way analysis of variance with the Fisher’s least sig-
nificant difference test applied for post hoc comparisons at
95% CI. P,0.05 was considered statistically significant.
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Figure 2 cytotoxicity of 6%-TrX-lP, 11%-TrX-lP, and 14%-TrX-lP against Mcs
at concentrations equivalent to lipid concentrations between 0.01 and 1.00 mg/ml
for 24 h.
Note: Data represent mean ± stD (n=5).
Abbreviations: Mcs, mesangial cells; lP, lipopolysaccharide; TrX-lP, TrX-20-
modified liposomes; StD, standard deviation.
Results and discussion
toxicity against the MCs at lipid concentration ranging from
0.01 to 1.0 mg/mL, whereas the negative control LPs (without
TRX) showed no obvious cytotoxicity after 24 h incubation
(Figure 2). The IC50 values for 6%-TRX-LP, 11%-TRX-LP,
and 14%-TRX-LP were 3.45, 1.13, and 0.55 mg/mL, respec-
tively, indicating that cytotoxicity increased with increasing
TRX-20 content. Even when incorporated at a low concentra-
tion of 6 mole% into HSPC, the TRX-20 cationic lipid could
provoke cytotoxicity, which was consistent with the results
reported by Koiwai et al.
synthesis and cytotoxicity ofTrX-20
In the study, TRX-20 was synthesized according to the pre-
vious reports, in which it was shown that TRX-20 could be
used to create a liposomal formulation with an MC active
targeting functionality.16,17 The appearance of the signals
from the three aromatic protons of the amidine group at ppm
7.80 and 9.80 in the 1H-NMR spectra indicated the successful
synthesis of TRX-20.
Cationic lipids with good affinity to cells have many
advantages as nonimmunogenic, nononcogenic, and readily
fabricated drug delivery platforms. However, they normally
exhibit significant toxicity intrinsically and because of their
ability to bind a wide variety of biomolecules, which then
limits their biomedical applications.21 A lipid mixture con-
taining 25 mole% TRX-20 that was used for condensing
DNA to enhance transfection was found to be less cytotoxic
against Hep G2 cells than conventional transfection agents.22
However, the effects of TRX-20 on renal cells have yet to
be established. In this study, we examined the cytotoxicity
of TRX-20 against the MCs to ascertain the appropriate
TRX-20 concentration to use that would afford the liposomal
formulation a balance of acceptable toxicity and efficacy in
the treatment of kidney disease.
characteristics of liposomes
The particle size and zeta potential of the different liposomes
are shown in Table 1. C6-LP, which entrapped Coumarin-6
as a trace label, was negatively charged with a zeta poten-
tial of −3.48±0.21 mV and particle size of 90.7±1.1 nm.
Table 1 characterization of all liposomes
Sample
Size/nm ± StD PDI ± StD
Zeta-potential/
mV ± StD
c6-lP
6%-TrX-c6-lP
90.7±1.1
101.8±4.5
0.190±0.005 −3.48±0.21
0.235±0.104 8.61±0.36
11%-TrX-c6-lP 106.4±3.7
14%-TrX-c6-lP 110.5±5.2
0.216±0.127 12.2±0.54
0.243±0.116 15.7±0.60
0.202±0.008 12.6±0.18
0.191±0.007 13.4±0.27
TrX-TP-lP
107.3±3.1
TRX-20 was inserted in the bilayer of the liposomes to
form 6%-TRX-LP, 11%-TRX-LP, and 14%-TRX-LP for the
evaluation of cytotoxicity against MCs isolated from SD rats.
All three TRX-LPs exhibited concentration-dependent
Peg-TrX-TP-lP 117.9±1.4
Abbreviations: c6-lP, coumarin-6-loaded liposomes; Peg-TrX-TP-lP, TrX-lP
with Peg5000 co-modification; TP, triptolide; TRX-C6-LP, TRX-20-modified
coumarin-6-loaded liposomes; TrX-TP-lP, triptolide-loaded TrX-lP; stD, standard
deviation; PDI, polydispersity index.
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Incorporation of TRX-20 increased the C6-LP diameter and The glomerular mesangium contains endothelial fenestrations
conferred a positive charge, with larger liposomes having of 130–170 nm, whereas the glomerular basement membrane
higher zeta potential obtained with increasing TRX-20 has much smaller 30–70 nm slit pores. Thus, functionalized
content.
liposomes of 100–120 nm are able to pass into the mesangium
TP as a model drug was also successfully encapsulated but not into the glomerular tubules. The resultant entrapment
into liposomes to obtain TRX-TP-LP and PEG-TRX- of TRX-LP in the mesangial area is further facilitated by its
TP-LP. TP as a bioactive diterpenoid epoxide isolated from ability to bind to the MCs in this region.
Tripterygium wilfordii Hook F has promising potential to
treat a variety of renal diseases23 but its progression into Specific uptake ofTRX-C6-LPs by MCs
clinical use is hampered by low water solubility (log P 1.494) A comparison of the cellular uptake data for C6-LP,
and poor renal discriminative activity that then led to severe 6%-TRX-C6-LP, 11%-TRX-C6-LP, and 14%-TRX-C6-LP
side effects on the circulatory and reproductive systems.24–26 (Figure 4) suggests that TRX-20 modification promoted
To improve its therapeutic potential, much research has the liposomal uptake by MCs, Higher cellular uptake of
been focused on employing appropriate pharmaceutical the liposomes was observed at increasing TRX-20 content,
strategies to attenuate the toxicity and increase the water with 14%-TRX-C6-LP showing the highest accumulation
solubility of TP.11,27–29 In this study, the entrapment of TP and evidence of localization in the cytoplasm. Obviously, it
into 11%-TRX-LP to fabricate TRX-TP-LP did not change is worth noting that the uptake values of the TRX-C6-LPs
the diameter and zeta potential of the liposomes. PEGylation were much higher than those of C6-LP. However, excessive
of TRX-TP-LP led to an increase in diameter but had no TRX-20 modification for liposomes showed severe toxicity
effect on the zeta potential. The obtained liposome solutions to viability of MCs and considerations should be given to
were clear opalescent suspensions without any precipitation balance the toxicity and the uptake efficiency of liposomes.
of unencapsulated TP. The measured TP concentrations in Therefore, TP-loaded liposomes including TRX-TP-LP and
TRX-TP-LP and PEG-TRX-TP-LP were 0.061 mg/mg lipid PEG-TRX-TP-LP for in vivo therapy were modified with
and 0.063 mg/mg lipid after the removal of unencapsulated 11%-TRX-20.
TP by gel filtration, respectively.
Anti-inflammatory activity ofTRX-TP-LP
Under the TEM, 11%-TRX-C6-LP (Figure 3A) and PEG-
TRX-TP-LP (Figure 3B) were observed to contain spherical and Peg-TrX-TP-lP
or ellipsoidal vesicles with good dispersity. TEM micro- TP is a strong anti-inflammatory agent that has been identified
graphs of all TRX-20-modified liposomes, with or without C6 to work in multiple tissues by inhibiting the synthesis and
and TP loading, consistently showed vesicles of narrow size release of inflammatory factors.20 In this study, we measured
distribution within the range of 100–120 nm mean diameter, the levels of extracellular NO and TNF-α as they are relevant
which is beneficial for liposomes traversing through the effector molecules that contribute to glomerular injury in
fenestrated endothelium directly into the mesangial space.5 numerous experimental models of glomerulonephritis.30–32
The renal glomeruli have a unique anatomic structure. ConsistentwithZhou’sreport,thehighlevelsofNOandTNF-α
released by the LPS-stimulated MCs were effectively attenu-
ated by co-incubation with TP for 24 h (Figure 5). TRX-20 has
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%
a high affinity for MCs,16 and this specificity was exploited in
this study to develop renal-targeted therapy of TP. Pegylation
of the resultant TRX-TP-LP was also utilized as a means to
improve therapeutic efficiency in vivo. Entrapment of TP
into the 11%-TRX-LP did not weaken its anti-inflammatory
activity, contrary to the expectation that liposomal encapsula-
tion may hinder TP cellular uptake by MCs.33,34 PEG chains
inserted into the lipid bilayer of TRX-TP-LP also did not
significantly decrease anti-inflammatory action. Indeed,
TRX-TP-LP and PEG-TRX-TP-LP showed better anti-
inflammatory actions than naked TP applied at the equivalent
concentration in the study (P,0.05), which was probably
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Figure 3 Images of 11%-TrX-c6-lP (A) and Peg-TrX-TP-lP (B) by negative
staining with 2% sodium phosphotungstate solution under a transmission electron
microscope.
Note: Original magnification for both images was ×100 k.
Abbreviations: c6, coumarin-6; lP, lipopolysaccharide; Peg-TrX-TP-lP, TrX-lP
with Peg5000 co-modification; TRX-C6-LP, TRX-20-modified Coumarin-6-loaded
liposomes; TRX-LP, TRX-20-modified liposomes.
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renal-targeted delivery of triptolide
Figure 4 confocal micrographs showing the uptake by Mcs of c6-lP (A), 6%-TrX-c6-lP (B), 11%-TrX-c6-lP (C), and 14%-TrX-c6-lP (D) after 24 h incubation
(c6: green).
Notes: Mcs were incubated with c6-lP (control) or TrX-c6-lPs at a lipid concentration of 0.8 mg/ml for 2 h in serum-free rPMI 1640 at 37°c and then washed with
PBS to terminate the uptake process. After fixation in 10% neutral buffer formalin, the cells were counterstained with DAPI (blue) for observation under a laser confocal
microscope. Magnification ×400.
Abbreviations: c6, coumarin-6; c6-lP, coumarin-6-loaded liposomes; DaPI, dihydrochloride; Mcs, mesangial cells; PBs, phosphate-buffered saline; TrX-c6-lP,
TRX-20-modified Coumarin-6-loaded liposomes.
attributed to the accentuated uptake of TRX-20-modified and 51%, respectively) that were induced by LPS treatment
liposomes by the MCs. As shown in Figure 5, TRX-TP-LP of the MCs. The results encouraged us to compare the
and PEG-TRX-TP-LP significantly inhibited the release of therapeutic efficiency of TRX-TP-LP and PEG-TRX-TP-LP
NO (by 71% and 66%, respectively) and TNF-α (by 52% in vivo because the previous studies from Morimoto et al and
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Figure 5 Anti-inflammatory activity of free TP, TRX-TP-LP, and PEG-TRX-TP-LP as measured by an inhibition of NO and TNF-α production by lPs-stimulated Mcs.
Notes: lPs-stimulated Mcs without any treatment served as lPs group while normal Mcs as blank. Data represent mean ± stD (n=5) *P,0.05 versus lPs.
Abbreviations: lPs, lipopolysaccharide; Mcs, mesangial cells; NO, nitric oxide; Peg-TrX-TP-lP, TrX-lP with Peg5000 co-modification; TNF-α, tumor necrosis factor-α;
TP, triptolide; TRX-TP-LP, triptolide-loaded TRX-LP; TRX-LP, TRX-20-modified liposomes; StD, standard deviation.
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Yuan et al
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Scindia et al reported that only PEGylated liposomes could is desirable for nephropathy as it limits the immunological
display better specific accumulation in animal models.15,16 events only to the kidneys, thereby reducing systemic side
It is also worth noting that the anti-inflammatory activity of effects.3 Morimoto et al were the first to apply TRX-20-
TP was elicited by applying TRX-TP-LP and PEG-TRX- modified liposomes to renal-targeted therapy, loading the
TP-LP at equivalent total lipid concentration of lower than liposomes with prednisolone for the treatment of anti-Thy-1
0.05 mg/mL, which was 22.6 times lower than the IC50 nephritic rats.16 MCs are the primary targets of immune-
of 11%-TRX-LP, and at which the viability of MCs was mediated glomerular diseases and they may also respond
96.51% (Figure 2).
to other glomerular injuries that involve the podocytes,
endothelial cells, or the glomerular basement membrane.35
Therefore, MC is an ideal targeting site for the treatment of
renal-targeted therapy
Modified liposome is an enabling technology for the creation glomerular nephropathy.
of tissue-/cell-specific therapeutics. The kidney, specifically
In this study, preliminary pharmacodynamics evaluation
the glomerulus, is one of the accessible sites for the targeted was performed to ascertain whether TRX-TP-LP and PEG-
delivery of drug-loaded liposomes. Renal-targeting therapy TRX-TP-LP enhanced the renal-targeted pharmacological
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Figure 6 Biochemical markers of normal sD rats (normal) and sD rats induced with membranous nephropathic (MN) by c-Bsa injection over 4 weeks. MN rats received
intravenous injections of saline (control), TP solution, or TrX-TP-lP or Peg-TrX-TP-lP dispersions.
Notes: (A) serum creatinine; (B) proteinuria expressed as the ratio of urinary protein (Up) and urinary creatinine (Ucr) levels; (C) serum albumin; (D) serum cholesterol.
Data represent mean ± stD (n=5). *P,0.05 versus TP group; **P,0.05 versus TrX-TP-lP group.
Abbreviations: c-Bsa, cationic bovine serum albumin; Peg-TrX-TP-lP, TrX-lP with Peg5000 co-modification; TP, triptolide; TRX-TP-LP, triptolide-loaded TRX-LP;
TRX-LP, TRX-20-modified liposomes; StD, standard deviation.
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renal-targeted delivery of triptolide
activity of TP in vivo. For this purpose, a nephropathic rodent MCs because of specific binding of the TRX-LP to MCs.
model was established through repeated C-BSA exposure in Pegylation to give PEG-TRX-TP-LP resulted in further
the SD rats. The nephropathic rodent model follows a similar improvement in the anti-inflammatory action of TP in vitro in
clinical course and histopathology to human MN. MN is an the LPS-stimulated MCs, and in vivo in the MN rodent model.
autoimmune-mediated glomerulonephritis characterized by It can be concluded that TRX- and PEG co-modification of
the presence of diffuse thickening of the glomerular base- HSPC liposomes could be used for the specific delivery of
ment membrane and subepithelial in situ immune-complex TP to renal glomeruli for the treatment of MN.
deposition, and is the most common of nephrotic syndrome
Acknowledgments
This work was supported by the project (2016M602706)
funded by China Postdoctoral Science Foundation and the
National Natural Science Foundation of China (81202489).
in adult humans. Presently available immunosuppressive
therapies are not always effective and often present with
persistent comorbidities.36
The three formulations comprising free TP solution,
TRX-TP-LP, and PEG-TRX-TP-LP dispersions were admin-
istered intravenously on alternate days over 2 weeks at
equivalent TP dose of 100 μg/kg to the rodent MN model.
Compared to rats in the normal group that were not immu-
nized with C-BSA, the MN rats developed normal SCr levels
(Figure 6A) and the characteristic clinical symptoms of overt
proteinuria (Figure 6B), hypoalbuminemia (Figure 6C),
and hypercholesterolemia (Figure 6D), which were con-
sistent with the biochemical data in our previous study.11
Treatment with TP over 2 weeks seemed to alleviate the
biochemical abnormalities to some extent, but its efficiency
did not match that observed in MN rats treated with TRX-
TP-LP or PEG-TRX-TP-LP. Treatment with TRX-TP-LP
or PEG-TRX-TP-LP effectively attenuated the symptoms
and improved biochemical markers including proteinuria,
serum cholesterol, and albumin. Relative to the MN rats in
the TRX-TP-LP group, treatment of the MN rats with PEG-
TRX-TP-LP significantly further reduced the proteinuria
and serum cholesterol level, while concomitantly raising
the serum albumin level (P,0.05). These three biochemical
markers in the PEG-TRX-TP-LP group were brought close to
the baseline levels of the normal rats, suggesting that PEG-
TRX-TP-LP has the potential to reverse the inflammation
induced by C-BSA in the SD rats. Histological sections of
kidney tissues in each group were also observed under micro-
scope (Figure S3). The histopathological findings supported
the biochemical data in advocating the superior efficacy of
PEG-TRX-TP-LP.
Disclosure
The authors report no conflicts of interest in this work.
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renal-targeted delivery of triptolide
Supplementary materials
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Figure S1 Nuclear magnetic resonance spectrum of 3,5-dipentadecyloxybenzonitrile.
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Figure S2 Nuclear magnetic resonance spectrum of 3,5-dipentadecyloxybenzamidine hydrochloride.
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renal-targeted delivery of triptolide
Figure S3 histological sections of kidney tissues (hematoxylin and eosin staining) harvested on week 6 from euthanized normal sprague Dawley rats (A) and rats induced
with MN by c-Bsa injection over 4 weeks, and receiving intravenous injections of control saline solution (B), Peg-TrX-TP-lP dispersions (at equivalent TP dose of
100 μg/kg) (C), 100 μg/kg TP (D), or TrX-TP-lP dispersions (at equivalent TP dose of 100 μg/kg) (E). scale bar =100 μm.
Notes: Kidney section for normal group exhibited a histologically normal glomerular architecture (A). In comparison, the kidney section for the control group showed typical
nephropathic characteristics of a diffuse basement thickening (thin arrow) of the length of glomerular basement membrane (gBM) (B). after the treatment Peg-TrX-TP-lP
(C) or TrX-TP-lP (E), a certain alleviation of pathological changes can be observed, whereas a renal tubular injury (thick arrow) was evident after free TP treatment (D).
The histopathological findings supported the biochemical data in advocating the superior efficacy of PEG-TRX-TP-LP.
Abbreviations: c-Bsa, cationic bovine serum albumin; lP, lipopolysaccharide; MN, membranous nephropathic; Peg-TrX-TP-lP, TrX-lP with Peg5000 co-modification;
TP, triptolide; TRX-TP-LP, triptolide-loaded TRX-LP; TRX-LP, TRX-20-modified liposomes.
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