Nephritis is one of the major complications of systemic lupus erythematosus. While glucocorticoids (GCs) are frequently used as the first-line treatment for lupus nephritis (LN), long-term GC usage is often complicated by severe adverse effects. To address this challenge, we have developed a polyethylene glycol-based macromolecular prodrug (ZSJ-0228) of dexamethasone, which self-assembles into micelles in aqueous media. When compared to the dose equivalent daily dexamethasone 21-phosphate disodium (Dex) treatment, monthly intravenous administration of ZSJ-0228 for two months significantly improved the survival of lupus-prone NZB/W F1 mice and was much more effective in normalizing proteinuria, with clear histological evidence of nephritis resolution. Different from the dose equivalent daily Dex treatment, monthly ZSJ-0228 administration has no impact on the serum anti-double-stranded DNA (anti-dsDNA) antibody level but can significantly reduce renal immune complex deposition. No significant systemic toxicities of GCs ( e. g., total IgG reduction, adrenal gland atrophy, and osteopenia) were found to be associated with ZSJ-0228 treatment. In vivo imaging and flow cytometry studies revealed that the fluorescent-labeled ZSJ-0228 primarily distributed to the inflamed kidney after systemic administration, with renal myeloid cells and proximal tubular epithelial cells mainly responsible for its kidney retention. Collectively, these data suggest that the ZSJ-0228's potent local anti-inflammatory/immunosuppressive effects and improved safety may be attributed to its nephrotropicity and cellular sequestration at the inflamed kidney tissues. Pending further optimization, it may be developed into an effective and safe therapy for improved clinical management of LN.
n class="Disease">Nephritispan> is one of the major complications of pan> class="Disease">systemic lupus erythematosus. While glucocorticoids (GCs) are frequently used as the first-line treatment for lupus nephritis (Ln class="Chemical">N), long-term GC usage is often complicated by severe adverse effects. To address this challenge, we have developed a polyethylene glycol-based macromolecular prodrug (ZSJ-0228) of dexamethasone, which self-assembles into micelles in aqueous media. When compared to the dose equivalent daily dexamethasone 21-phosphate disodium (Dex) treatment, monthly intravenous administration of ZSJ-0228 for two months significantly improved the survival of lupus-prone NZB/W F1mice and was much more effective in normalizing proteinuria, with clear histological evidence of nephritis resolution. Different from the dose equivalent daily Dex treatment, monthly ZSJ-0228 administration has no impact on the serum anti-double-stranded DNA (anti-dsDNA) antibody level but can significantly reduce renal immune complex deposition. No significant systemic toxicities of GCs ( e. g., total IgG reduction, adrenal gland atrophy, and osteopenia) were found to be associated with ZSJ-0228 treatment. In vivo imaging and flow cytometry studies revealed that the fluorescent-labeled ZSJ-0228 primarily distributed to the inflamed kidney after systemic administration, with renal myeloid cells and proximal tubular epithelial cells mainly responsible for its kidney retention. Collectively, these data suggest that the ZSJ-0228's potent local anti-inflammatory/immunosuppressive effects and improved safety may be attributed to its nephrotropicity and cellular sequestration at the inflamed kidney tissues. Pending further optimization, it may be developed into an effective and safe therapy for improved clinical management of LN.
n class="Disease">Systemic lupus erythematosuspan>
(n class="Disease">SLE), or lupus, is a chronic complex n class="Disease">autoimmune disease for which
there is no cure. It is characterized by B and T cell hyperactivation,
overproduction of autoantibodies, and the deposition of immune complexes
in various tissues and organs. The symptoms of lupus among patients
are highly heterogeneous, which may include skin rash, arthritis,
pericarditis, neuropsychiatric disorders, and nephritis. The Lupus
Foundation of America estimates that 1.5 million Americans and at
least five million people worldwide have a form of lupus.[1] Lupus affects mostly women of childbearing age
and has a significantly higher prevalence among African Americans.[2] According to a report by the U.S. Centers for
Disease Control and Prevention in 2002, the death rates attributed
to lupus have increased by approximately 70% over a 20-year period
among African American women aged 45–64 years.[3]
n class="Disease">Nephritispan> is one of the most damaginpan>g complications
of lupus. It
is the leadinpan>g cause of morbidity anpan>d mortality among lupus pan> class="Species">patients.
Around 35% adult lupus patients in the U.S. have clinical evidence
of nephritis at the time of diagnosis. An additional 15–25%
patients will develop nephritis within 10 years of their initial diagnosis.[4] Lupus nephritis (LN) is initiated by abnormal
immune complex deposition on the basement membrane of renal glomeruli
and the subsequent activation of the immune effector cells (e.g., macrophages and neutrophils), leading
to damage of the renal tissues.[5] If not
effectively managed, Ln class="Chemical">N can progress rapidly to impair renal function
and eventually result in kidney failure.[6]
Among the limited treatmepan class="Chemical">nt options,[7] glucocorticoid (GC) is one of the most potent anclass="Chemical">pan>d widely used classes
of medication for lupus. Acpan> class="Gene">cording to the American College of Rheumatology’s
recent guidelines,[4,8] a pulse GC treatment followed
by low/high-dose daily GC plus an immunosuppressive is recommended
as a standard treatment regimen for clinical management of LN.[9] Due to their potent anti-inflammatory efficacy
and the lack of alternatives,[10,11] GCs continue to be
the mainstay for clinical management of n class="Disease">lupus symptoms.[4,8] Some lupus pathologies, such as arthritis and skin rash, can be
effectively managed with short-term GC treatment. Serious lupus complications,
including progressive nephritis, however, necessitate long-term GC
therapy, which is often associated with severe adverse events involving
the musculoskeletal, endocrinal, hematopoietic, and cardiovascular
systems.[12]
The diverse biological
effects of GCs are thought to be mediated via transrepressiopan class="Chemical">n,
which elicits GC’s anti-inflammatory
effects, and transactivation, which is responsible for the GC-associated
side effects.[13] Selective glucon class="Gene">corticoid
receptor modulators (SEGRMs) that canpan> preferentially activate the
tranpan>srepression relative to the tranpan>sactivation pathway have been
developed.[14,15] These compounclass="Chemical">pan>ds, however, do
not exhibit strict pathway selectivity anpan>d still produce GC-related
side effects.[13,16]
Recognizipan class="Chemical">ng the therapeutic
potential of GCs in the clinical management
of LN, their accompanpan>yinpan>g severe pan> class="Disease">toxicities, and the limited progress
made in developing SEGRMs, we proposed to address this challenge through
the development of a GC prodrug nanomedicine. Conceptually, this approach
is based upon an inflammation-targeting mechanism, which we have discovered
and termed “ELVIS”.[17] It
involves the Extravasation of the nanomedicine through Leaky Vasculature
at sites of inflammation and its subsequent Inflammatory cell-mediated
Sequestration, which would alter the pharmacokinetics/biodistribution
profile of the parent drug, enabling its inflammatory tissues/organs
specificity. When tested in a spontaneous LNmouse model (female NZB/W
F1 mice), the GC prodrug (ZSJ-0228) nanomedicine we developed demonstrated
superior therapeutic efficacy to dose equivalent dexamethasone 21-phosphate
disodium (Dex) in ameliorating nephritis and improving kidney function,
with no apparent GC toxicities.
Results
The main
objective of this project is to develop a GC prodrug pan class="Chemical">nanomedicine
with organ/tissue specificity to LN. We hypothesize that such anpan> approach
would potentiate the GC’s efficacy anpan>d reduce pan> class="Disease">systemic toxicities.
As shown in Scheme , the prodrug (ZSJ-0228) was designed by conjugating two dexamethasone
molecules to the chain terminus of a short methoxy polyethylene glycol
(mPEG, 1.9 kDa) via a hydrazone/glycine/glutamate
linker system. The apparent amphiphilicity of the prodrug allows its
spontaneous self-assembly into micelles in aqueous media, rendering
the hydrophobic dexamethasonewater-soluble. Additionally, due to
the ELVIS mechanism, the systemically administered ZSJ-0228 would
extravasate/filter at the LN pathology, be sequestered by inflammatory
cells and activated kidney cells, and subsequently release dexamethasone
within the endosomal/lysosomal compartments (via the
acid-cleavable hydrazone bond) to exert its localized anti-inflammatory
and immune-modulating effects without triggering systemic toxicities.
Scheme 1
Design of polyethylene glycol (PEG)-based amphiphilic dexamethasone
prodrug ZSJ-0228, which can self-assemble into micelles in aqueous
media. The oval shape highlights the dexamethasone structure.
Synthesis of Amphiphilic Macromolecular Dexamethasone
Prodrug
(ZSJ-0228)
n class="Chemical">ZSJ-0228pan> was successfully n class="Gene">synthesized acn class="Gene">cording
to the route illustrated in Scheme . The identity of the polymeric prodrug and the absence
of free Dex were confirmed using NMR, MS, and LC-MS/MS. The multistep
synthesis is straightforward with high yield at each step. tert-Butyldimethylsilyl (TBS) was introduced in the first
step to protect the 21-hydroxyl group and to improve solubility. The
bulky presence of TBS sterically hinders access to the C-20 carbonyl
group, resulting in the formation of a hydrazone bond predominantly
at the C-3, not the C-20 carbonyl group. The presence of a local conjugation
system also favors the C-3 hydrazone formation. Because of the use
of hydrazone as the polymeric prodrug’s activation trigger,
multiple configurational isomers of compound 6 were formed,
which could not be separated chromatographically. Mass spectroscopy
(positive ion ESI) of the isomer mixture showed the molecular ion
M + H+ at 1266.4 as a single peak (calculated molecular
weight is 1265.7). After conjugation of compound 6 to
mPEG-COOH and the subsequent removal of the TBS protection group,
the prodrug ZSJ-0228 was prepared. The theoretical dexamethasone content
in ZSJ-0228 was calculated as 26.7 wt %. After complete hydrolysis,
HPLC analysis found 26.4 wt % of the prodrug we synthesized to be
dexamethasone, suggesting ∼99% purity.
As expected,
the conjugatiopan class="Chemical">n of hydrophobic dexamethasone dimer to the hydrophilic
pan> class="Chemical">mPEG led to the amphiphilic ZSJ-0228 prodrug’s self-assembly
into micelles upon direct dissolution in aqueous media. Using the
pyrene-based fluorescence probe method, the critical micelle concentration
(CMC) value of ZSJ-0228 was determined as 2.5 × 10–4 M. It is a relatively high CMC value, which will lead to the micelle’s
disintegration upon i.v. administration due to dilution in the blood.
Dynamic light scattering (DLS) measurements (Figure A, Zetasizer Nano ZS90) revealed that the
ZSJ-0228 can form micelles with an average micelle diameter of 33
nm and a net charge close to neutral. The DLS profile of the micelles
was bimodal, suggesting the formation of heterogeneous particle populations.
As shown in the transmission electron microscope (TEM) images, the
micelles deposited on the substrate showed heterogeneity, with the
majority of micelles showing an average diameter of ∼30 nm
(Figure C) and the
minority having a larger average diameter of ∼100 nm (Figure D). Nanoparticle
tracking analysis (NTA, Nanosight NS300) was employed as an additional
method to measure the micelle size, distribution, and relative concentration.
The NTA result (Figure B) seems to be in agreement with the DLS and TEM findings (as shown
in Figure A, C, and
D), showing that the diameter of micelles was mostly <41 nm (90%)
with a small population around 100 nm (10%, Figure B, inset).
Figure 1
Characterization of ZSJ-0228 micelles.
(A) DLS profile of ZSJ-0228
micelles. The measurement was performed in triplicate. (B) NTA measurement
of ZSJ-0228 micelles. The measurements were repeated five times. (C)
Representative transmission electron microscope (TEM) image of ZSJ-0228
micelles. The average diameter of the micelles is estimated to be
∼30 nm. (D) Representative TEM image of larger ZSJ-0228 micelle
population with an estimated average diameter of ∼100 nm. Scale
bar = 100 nm.
Characterization of papan class="Chemical">n class="Chemical">ZSJ-0228 class="Chemical">pan> class="Species">micelles.
(A) DLS profile of ZSJ-0228micelles. The measurement was performed in triplicate. (B) NTA measurement
of ZSJ-0228micelles. The measurements were repeated five times. (C)
Representative transmission electron microscope (TEM) image of ZSJ-0228micelles. The average diameter of the micelles is estimated to be
∼30 nm. (D) Representative TEM image of larger ZSJ-0228micelle
population with an estimated average diameter of ∼100 nm. Scale
bar = 100 nm.
In vitro release of n class="Chemical">dexamethasone from class="Chemical">pan> class="Chemical">ZSJ-0228
at different pH values. The experiment was done in acetate buffer
(pH 4.5 and 5.0) and phosphate buffer (pH 6.5 and 7.4) at 37 °C.
Pluronic F127 (1 wt % of dexamethasone) was added to create the “sink”
condition. Each experiment was performed in triplicate. Results are
expressed as mean ± SD.
As the n class="Chemical">dexamethasonepan> activation trpan> class="Gene">igger, the hydrazone bond
in
the n class="Chemical">ZSJ-0228 design should only be cleaved under an acidic environment.
This was confirmed in an in vitro prodrug activation
experiment (Figure ) showing the near zero-order release of the conjugated dexamethasone
with an almost constant rate at ∼1.32%/day and 0.96%/day for
4 weeks in the pH 4.5 and pH 5.0 acetate buffers, respectively. No
dexamethasone release was detected in pH 6.5 and pH 7.4 buffers for
the entire experiment duration.
Figure 2
In vitro release of dexamethasone from ZSJ-0228
at different pH values. The experiment was done in acetate buffer
(pH 4.5 and 5.0) and phosphate buffer (pH 6.5 and 7.4) at 37 °C.
Pluronic F127 (1 wt % of dexamethasone) was added to create the “sink”
condition. Each experiment was performed in triplicate. Results are
expressed as mean ± SD.
ZSJ-0228 Effectively Ameliorated
Proteinuria and Improved the
Survival of NZB/W F1 Mice with Established Nephritis
The
therapeutic effects of n class="Chemical">ZSJ-0228pan> were evaluated inpan> class="Chemical">NZB/W F1 female
n class="Species">mice (∼28 weeks old) with fully developed nephritis (proteinuria
≥100 mg/dL for 2 weeks). The mice were given two monthly intravenous
injections of ZSJ-0228 (28 mg/kg, dexamethasone equivalent). Dose
equivalent daily Dex (1 mg/kg, dexamethasone equivalent, i.v.)[18] and monthly saline administration (i.v.) were
used as controls. At the end of two months, all animals were euthanized.
As shown in Figure A, the mice in the saline group maintained a 100% incident rate of
proteinuria, and 9 out of 12 mice (75%) demonstrated increased proteinuria
level over the course of the experiment, with Albustix readings increasing
from 2 to 4. For the Dex treatment group, the level of proteinuria
increased in 36% of the mice (Albustix reading increased from 2 to
4) and was normalized in 18% of the mice (Albustix reading decreased
from 2 to 1), suggesting that the Dex treatment can partially impede
LN progression. In the ZSJ-0228 treatment group, only one mouse showed
an increased level of proteinuria, with an Albustix reading increased
from 2 to 4. Three mice maintained the same level of proteinuria (Albustix
reading at 2), and the levels of proteinuria for the rest of the mice
(60%) was normalized with an Albustix reading at 0 or 1. This result
is significantly better than the saline control (P < 0.01), suggesting that the prodrug treatment is very effective
in amelioration of LN.
Figure 3
Monthly ZSJ-0228 treatment demonstrates superior therapeutic
efficacy
when compared to dose equivalent daily Dex treatment. (A) Monthly
ZSJ-0228 treatment normalized albuminuria among 60% of NZB/W F1 mice
(with established nephritis), while dose equivalent daily Dex treatment
only normalized 18% at the end of the 2-month treatment. PT = pretreatment.
The percentages shown accounted for those animals with an Albustix
reading of 2 and above at the 8-week time point. Each data point represents
an individual mouse. P = 0.003, Fisher’s exact
test. (B) Kaplan–Meier survival curves for ZSJ-0228, Dex, and
saline groups. Only ZSJ-0228 treatment resulted in 100% survival after
two months of treatment, which is significantly better than saline
controls (*, P < 0.05). No significant difference
between ZSJ-0228 and Dex groups was found (P = 0.17).
Log-rank test with Bonferroni’s pairwise comparison.
Monthly papan class="Chemical">n class="Chemical">ZSJ-0228 treatmenpan>t demonstrates superior therapeutic
efficacy
when compared to dose equivalent daily pan> class="Chemical">Dex treatment. (A) Monthly
ZSJ-0228 treatment normalized albuminuria among 60% of NZB/n class="Gene">W F1 mice
(with established nephritis), while dose equivalent daily Dex treatment
only normalized 18% at the end of the 2-month treatment. PT = pretreatment.
The percentages shown accounted for those animals with an Albustix
reading of 2 and above at the 8-week time point. Each data point represents
an individual mouse. P = 0.003, Fisher’s exact
test. (B) Kaplan–Meier survival curves for ZSJ-0228, Dex, and
saline groups. Only ZSJ-0228 treatment resulted in 100% survival after
two months of treatment, which is significantly better than saline
controls (*, P < 0.05). No significant difference
between ZSJ-0228 and Dex groups was found (P = 0.17).
Log-rank test with Bonferroni’s pairwise comparison.
n class="Chemical">ZSJ-0228pan>-treated n class="Chemical">NZB/n class="Gene">W F1 mice
also had a significantly better
survival rate than the saline control mice (P <
0.05). Before the end of the treatment (mice at 36 weeks of age),
a total of 42% of mice in the saline group died due to severe nephritis
(Figure B). This is
in general agreement with reports in the literature of the median
survival of saline-treated NZB/W F1 female mice (∼35 weeks).[19−22] In comparison, two out of 11 mice treated with Dex died, and all
ZSJ-0228-treated mice survived. The apparent trend of the ZSJ-0228
group toward better survival rates than the Dex group did not reach
statistical significance (P = 0.17). All deceased
animals exhibited the highest level of proteinuria (Albustix reading
of 4) before death.
To further validate the superior therapeutic
efficacy of n class="Chemical">ZSJ-0228pan>,
kidneys isolated at necropsy were sectioned, stainpan>ed with periodic
acid–Schiff, anpan>d evaluated by a pathologist (K.W.F.), who was
blinpan>ded to the groupinpan>g arranpan>gement. The tissue sections were graded
usinpan>g a histopathologic span> class="Gene">coring system with a 4-point scale.[20] Compared to the ZSJ-0228-treated mice, more
than 40% of the saline and Dex-treated mice had a higher percentage
of damaged glomeruli (scoring 3 and 4 points). Evidence of acute glomerular
injury includes endocapillary hypercellularity as well as the presence
of wire-loop lesions, hyaline thrombi (indicating immune complex deposition),
cellular crescents, etc. (Figure ). In contrast, only ∼11% of the mice
in the ZSJ-0228-treated group were graded with severe glomerulonephritis
(with the rest in the mild and moderate categories), which was much
lower than that of the n class="Chemical">saline (∼43%) and Dex (∼44%)
groups. In addition, when individual glomeruli were evaluated histologically,
abnormities were observed in 26% of the glomeruli from the ZSJ-0228-treated
mice, which is close to the frequency (21.6%) found in the NZW mice
(healthy control, which do develop anti-dsDNA antibodies, high serum
levels of retroviral gp70 antigen, and nephritis later in life).[23] Compared to this observation, 40% and 52% of
the glomeruli in the Dex and saline groups were found to be abnormal,
respectively. Though these differences are not statistically significant
(P = 0.19), the apparent trend further supports the
superior efficacy of ZSJ-0228 in treating LN (Figure F).
Figure 4
Histological evaluation of kidneys from different
treatment groups.
The tissues were formalin-fixed, sectioned (3 μm), and stained
with periodic acid–Schiff (PAS) for visual examination and
grading by a pathologist (K.W.F.), who was blinded to the group design.
(A) PAS-stained kidney section from the saline group showing wire-loop
lesions (arrow). (B) PAS-stained kidney section from the Dex-treated
group showing a cellular crescent (arrow). (C) PAS-stained kidney
section from the ZSJ-0228-treated group, showing less severe glomerular
injury with a healthier appearance similar to the NZW control. (D)
PAS-stained kidney section from NZW control group. Scale bar = 50
μm. (E) Fractions of mice in each group with mild, moderate,
and severe renal disease. Results are expressed as percentage of mice
with the indicated disease severity. (F) Average percentage of abnormal
glomeruli found in each group. Results are expressed as mean ±
SD. No statistically significant difference was found among the groups. P = 0.19, one-way analysis of variance (ANOVA) with Tukey’s
pairwise comparison.
Histological evaluation of kidpan class="Chemical">neys from different
treatment groups.
The tissues were formalin-fixed, sectioned (3 μm), anpan>d stainpan>ed
with periodic acid–Schiff (class="Chemical">pan> class="Chemical">PAS) for visual examination and
grading by a pathologist (K.W.F.), who was blinded to the group design.
(A) PAS-stained kidney section from the saline group showing wire-loop
lesions (arrow). (B) PAS-stained kidney section from the Dex-treated
group showing a cellular crescent (arrow). (C) PAS-stained kidney
section from the ZSJ-0228-treated group, showing less severe glomerular
injury with a healthier appearance similar to the NZW control. (D)
PAS-stained kidney section from NZW control group. Scale bar = 50
μm. (E) Fractions of mice in each group with mild, moderate,
and severe renal disease. Results are expressed as percentage of mice
with the indicated disease severity. (F) Average percentage of abnormal
glomeruli found in each group. Results are expressed as mean ±
SD. No statistically significant difference was found among the groups. P = 0.19, one-way analysis of variance (ANOVA) with Tukey’s
pairwise comparison.
ZSJ-0228 Treatment Showed No Apparent GC Toxicities
The
viability of n class="Gene">HK-2pan> cells (an immortalized renal proximal tubule
epithelial cell linpan>e from normal adult pan> class="Species">human kidney) was evaluated
after 72 h of incubation with ZSJ-0228, Dex, and mPEG. As can be seen
in Figure A, ZSJ-0228
and n class="Chemical">Dex showed minimal cytotoxicity to HK-2 cells within the tested
range of dexamethasone equivalent concentrations (0.01–2000
μM). mPEG was also found to be nontoxic.
Figure 5
Safety assessment of
ZSJ-0228 treatment. (A) Impact of mPEG, ZSJ-0228,
and Dex on HK-2 cell viability after 72 h of incubation, as assessed
by the MTT assay. For ZSJ-0228 and Dex, they were tested at dexamethasone
equivalent concentrations (0.01–2000 μM). mPEG was tested
at ZSJ-0228 equivalent concentrations (0.005–1000 μM).
Each ZSJ-0228 has one mPEG and two dexamethasone molecules. (B) Body
weight (% of week 0) of NZB/W F1 mice during the two-month treatments
with ZSJ-0228 and Dex (dexamethasone dose equivalent). Saline was
used as a control. ZSJ-0228 treatment did not affect the mice body
weight. An early trend of decreased body weight was observed in the
Dex group, suggesting a potential adverse effect of the treatment.
(C) ZSJ-0228 treatment is significantly better at preserving bone
mineral density than Dex treatment and the saline controls. (D) ZSJ-0228-treated
mice trended toward a higher bone volume/tissue volume than Dex treatment
and saline controls (P = 0.69). (E) ZSJ-0228-treated
mice have a significantly higher trabecular thickness value than Dex
treatment and saline controls. (F) The average white blood cell (WBC)
count of the ZSJ-0228-treated mice was similar to that of the saline
group, but was significantly higher than the Dex group. (G) Total
serum IgG levels for mice in ZSJ-0228 (n = 10), Dex
(n = 11), and saline (n = 12) groups
were determined by ELISA at pretreatment, 4-week, and 8-week time
points. The ZSJ-0228 treatment did not significantly reduce total
serum IgG levels, whereas Dex treatment did, suggesting potential
immune suppression. (H) Different from Dex treatment, ZSJ-0228 treatment
did not induce adrenal gland atrophy. For bone quality (C, D, and
E), WBC counts (F), and adrenal gland weight (H) evaluations, samples
were available for analysis only from the subset of mice surviving
at the final time point (8-week): 10 mice for the ZSJ-0228 group,
9 mice for the Dex group, 7 mice for the saline group. For total serum
IgG levels (G), the ZSJ-0228 group has 10 mice at 4-week and 8-week
time points; the Dex group has 11 and 9 mice at the 4-week and 8-week
time points, respectively; and the saline group has 11 and 7 mice
at the 4-week and 8-week time points, respectively. Results are expressed
as mean ± SD. Statistical analysis of data in panels C, E, and
H was performed using the Kruskal–Wallis test with Bonferroni’s
pairwise comparison. For panels D and F, the statistical analysis
was performed using one-way ANOVA with Tukey’s pairwise comparison.
For panel G, the statistical analysis was performed using the generalized
estimating equation (GEE) method with Tukey’s pairwise comparison.
*, P < 0.05, **, P < 0.01,
****, P < 0.0001.
Safety assessment of
papan class="Chemical">n class="Chemical">ZSJ-0228 treatmenpan>t. (A) Impact of n class="Chemical">mPEG, n class="Chemical">ZSJ-0228,
and Dex on HK-2 cell viability after 72 h of incubation, as assessed
by the MTT assay. For ZSJ-0228 and Dex, they were tested at dexamethasone
equivalent concentrations (0.01–2000 μM). mPEG was tested
at ZSJ-0228 equivalent concentrations (0.005–1000 μM).
Each ZSJ-0228 has one mPEG and two dexamethasone molecules. (B) Body
weight (% of week 0) of NZB/W F1mice during the two-month treatments
with ZSJ-0228 and Dex (dexamethasone dose equivalent). Saline was
used as a control. ZSJ-0228 treatment did not affect the mice body
weight. An early trend of decreased body weight was observed in the
Dex group, suggesting a potential adverse effect of the treatment.
(C) ZSJ-0228 treatment is significantly better at preserving bone
mineral density than Dex treatment and the saline controls. (D) ZSJ-0228-treated
mice trended toward a higher bone volume/tissue volume than Dex treatment
and saline controls (P = 0.69). (E) ZSJ-0228-treated
mice have a significantly higher trabecular thickness value than Dex
treatment and saline controls. (F) The average white blood cell (WBC)
count of the ZSJ-0228-treated mice was similar to that of the saline
group, but was significantly higher than the Dex group. (G) Total
serum IgG levels for mice in ZSJ-0228 (n = 10), Dex
(n = 11), and saline (n = 12) groups
were determined by ELISA at pretreatment, 4-week, and 8-week time
points. The ZSJ-0228 treatment did not significantly reduce total
serum IgG levels, whereas Dex treatment did, suggesting potential
immune suppression. (H) Different from Dex treatment, ZSJ-0228 treatment
did not induce adrenal gland atrophy. For bone quality (C, D, and
E), WBC counts (F), and adrenal gland weight (H) evaluations, samples
were available for analysis only from the subset of mice surviving
at the final time point (8-week): 10 mice for the ZSJ-0228 group,
9 mice for the Dex group, 7 mice for the saline group. For total serum
IgG levels (G), the ZSJ-0228 group has 10 mice at 4-week and 8-week
time points; the Dex group has 11 and 9 mice at the 4-week and 8-week
time points, respectively; and the saline group has 11 and 7 mice
at the 4-week and 8-week time points, respectively. Results are expressed
as mean ± SD. Statistical analysis of data in panels C, E, and
H was performed using the Kruskal–Wallis test with Bonferroni’s
pairwise comparison. For panels D and F, the statistical analysis
was performed using one-way ANOVA with Tukey’s pairwise comparison.
For panel G, the statistical analysis was performed using the generalized
estimating equation (GEE) method with Tukey’s pairwise comparison.
*, P < 0.05, **, P < 0.01,
****, P < 0.0001.
During the two-mopan class="Chemical">nth treatment study, no significant difference
in body weight was observed among ZSJ-0228, pan> class="Chemical">Dex, and saline groups
(Figure B). On average,
all tested groups maintained above 90% of original body weight at
the end of the two-month treatments. Different from the saline and
ZSJ-0228 groups, an early trend of body weight decrease was observed
in the Dex group, suggesting potential adverse effects of the treatment
(Figure B). It is
important to note that several mice from the saline and Dex groups
died earlier (Figure B) due to severe nephritis and significant loss of body weight (>20%).
Their body weight values at the time of death were recorded. If they
would have lived at the end of the two-month treatment period, a significant
difference among the three tested groups may have been observed.
One of the major adverse effects associated with GC use is papan class="Chemical">n class="Disease">osteopenia.
To unclass="Chemical">pan>derstand the impact of pan> class="Chemical">ZSJ-0228 treatment on the skeleton, we
evaluated the femoral bone quality using high-resolution μ-CT
(Skyscan 1172). The bone mineral density and trabecular thickness
in the femoral trabecular bone of ZSJ-0228-treated n class="Species">mice were significantly
higher than those from the saline- and Dex-treated groups (Figure C and E; P < 0.05). A trend toward higher trabecular bone volume/tissue
volume values was also observed (Figure D, P = 0.69).
Chronic
exposure to GC therapy is kpan class="Chemical">nown to be associated with systemic
immunosuppression. To understand if ZSJ-0228 as a GC prodrug would
be similarly immunosuppressive, we evaluated the total serum pan> class="Gene">IgG level
and the peripheral white blood cell (WBC) counts at designated time
points. As shown in Figure F, ZSJ-0228-treated mice exhibited similar WBC counts as the
saline group, but the value is significantly higher than that of the
Dex-treated mice (P < 0.05). As shown in Figure G, total serum IgG
levels were not altered in the ZSJ-0228-treated mice during the treatment.
In contrast, the animals treated with daily Dex administration had
a significant drop of serum IgG value after only one month of treatment
(P < 0.001) and continued this significant decrease
until the end of the experiment (P < 0.0001).
These data collectively suggest that different from the Dex treatment,
ZSJ-0228 did not suppress the immune system of the NZB/W F1mice.
GC exposure, even ipan class="Chemical">n the short term, is known to suppress the hypothalamic–pituitary–adrenal
axis, leading to clinical atrophy of the adrenal glanpan>d.[24] To understanpan>d if pan> class="Chemical">ZSJ-0228 treatment would cause
adrenal gland atrophy in NZB/W F1mice, the adrenal glands from all
treatment groups were isolated and weighed at necropsy. The mean value
of adrenal gland mass in the Dex group was significantly lower than
that from the ZSJ-0228 group (Figure H; P < 0.05). No significant difference
in adrenal gland mass was found between the ZSJ-0228 and saline groups
(Figure H; P = 0.22). These data suggest that the treatment with ZSJ-0228
does not lead to adrenal gland atrophy.
ZSJ-0228 Treatment Ameliorates
Renal Immune Complexes but Does
Not Alter Serum Anti-dsDNA Level
GCs have been showpan class="Chemical">n to exert
some of their therapeutic effects on lupus partially through the down-regulation
of anti-dsDNA anpan>tibody levels.[25] Therefore,
it was importanpan>t to evaluate whether pan> class="Chemical">ZSJ-0228 attained its therapeutic
effect through this mechanism. As shown in Figure , ZSJ-0228 treatment showed no impact on
serum anti-dsDNA IgG during the treatment. However, it was found to
significantly reduce renal immune complex deposition when compared
to the saline control. Daily Dex treatment, on the other hand, was
found to significantly reduce serum anti-dsDNA IgG levels at 4 and
8 weeks post-treatment initiation (P < 0.05),
but had no impact on renal immune complex deposition. The apparent
trend of NZW and ZSJ-0228 groups toward lower renal immune complex
deposition than the Dex group did not reach statistical significance
(P = 0.078 and P = 0.074, respectively).
Collectively, these results suggest that the immunosuppressive effect
of ZSJ-0228 is not systemic but is rather restricted to the inflamed
kidneys.
Figure 6
Effect of different treatments on renal immune complex deposition
and serum anti-dsDNA IgG levels. ZSJ-0228 treatment was found to significantly
reduce renal immune complex deposition. While Dex daily treatment
significantly reduced the serum anti-dsDNA IgG level, it had no impact
on renal immune complex deposition. (A) Representative kidney sections
from each treatment group, immunohistochemically stained for renal
deposition of anti-mouse IgG. Compared to NZW and ZSJ-0228 groups,
more immune complexes were found in the kidney sections from the saline
and Dex groups. Scale bar = 50 μm. (B) Quantification of kidney
immune complex staining. Results are expressed as mean ± SD;
*, P < 0.05, one-way ANOVA test with Tukey’s
pairwise comparison. (C) Serum anti-dsDNA IgG levels at the pretreatment,
4-week, and 8-week time points, as determined by an enzyme-linked
immunosorbent assay. Results are expressed as mean ± SD; *, P < 0.05, **, P < 0.01, GEE method
with Tukey’s pairwise comparison.
Effect of different treatmepan class="Chemical">nts on renal immune complex deposition
and serum anti-dsDNA pan> class="Gene">IgG levels. ZSJ-0228 treatment was found to significantly
reduce renal immune complex deposition. While Dex daily treatment
significantly reduced the serum anti-dsDNA IgG level, it had no impact
on renal immune complex deposition. (A) Representative kidney sections
from each treatment group, immunohistochemically stained for renal
deposition of anti-mouseIgG. Compared to NZW and ZSJ-0228 groups,
more immune complexes were found in the kidney sections from the saline
and Dex groups. Scale bar = 50 μm. (B) Quantification of kidney
immune complex staining. Results are expressed as mean ± SD;
*, P < 0.05, one-way ANOVA test with Tukey’s
pairwise comparison. (C) Serum anti-dsDNA IgG levels at the pretreatment,
4-week, and 8-week time points, as determined by an enzyme-linked
immunosorbent assay. Results are expressed as mean ± SD; *, P < 0.05, **, P < 0.01, GEE method
with Tukey’s pairwise comparison.
ZSJ-0228 Reduced Renal Macrophage Infiltration
To understapan class="Chemical">nd
ZSJ-0228’s workinpan>g mechanclass="Chemical">pan>ism, we examined the inpan>filtration
of macrophages inpan>to the kidney, anpan> inpan>dicator of pan> class="Disease">chronic renal inflammation.
The macrophage marker F4/80 was used in kidney tissue sections from
all the treatment groups (Figure A). Quantification of F4/80 staining suggested that
Dex treatment did not significantly reduce macrophage infiltration
when compared to the saline control. In contrast, ZSJ-0228 treatment
significantly lowered the renal macrophage levels when compared with
Dex treatment and the saline control (Figure B). These results suggested that the n class="Chemical">ZSJ-0228
treatment may partially exert its therapeutic effects through ameliorating
macrophage infiltration to the kidneys.
Figure 7
Impact of different treatments
on renal macrophage infiltration.
(A) Representative kidney sections from each treatment group, immunohistochemically
stained for renal deposition of anti-mouse F4/80. Scale bar = 50 μm.
(B) Quantification of immune complex staining. Results are expressed
as mean ± SD; *, P < 0.05, Kruskal–Wallis
test with Bonferroni’s pairwise comparison.
Impact of different treatmepan class="Chemical">nts
on renal macrophage infiltration.
(A) Representative kidney sections from each treatment group, immunohistochemically
stainpan>ed for renpan>al deposition of anpan>ti-pan> class="Species">mouse F4/80. Scale bar = 50 μm.
(B) Quantification of immune complex staining. Results are expressed
as mean ± SD; *, P < 0.05, Kruskal–Wallis
test with Bonferroni’s pairwise comparison.
ZSJ-0228 Targeted the Nephrotic Kidneys in
NZB/W F1 Mice
To understand the therapeutic efficacy and
reduced GC-associated
toxicities of pan> class="Chemical">ZSJ-0228, the in vivo biodistribution
of ZSJ-0228 was qualitatively analyzed using near-infrared optical
imaging. Both NZB/W F1 and NZW mice (healthy control) received i.v.
injections of IRDye 800 CW-labeled ZSJ-0228 (ZSJ-0228-IRDye). As shown
in Figure A, in NZB/W
F1 mice, ZSJ-0228-IRDye primarily accumulated in the kidneys and could
be detected for at least 4 days. ZSJ-0228-IRDye was also found to
accumulate in NZW mice’s kidneys, but the intensity of the
signal was at a much lower level, especially at 4 days postadministration.
These observations seem to suggest that the severe nephritis of NZB/W
F1 mice may have contributed to the targeting and retention of ZSJ-0228
in the kidneys.
Figure 8
Targeting and retention of ZSJ-0228 in the kidneys of
NZB/W F1
mice. (A) Representative optical images of organs isolated from NZB/W
F1 mice and NZW mice (healthy control). Images were obtained at 1
and 4 days post-i.v. injections of IRDye 800 CW-labeled ZSJ-0228 (ZSJ-0228-IRDye).
Pseudo-color-coded signal intensity reflects the level of ZSJ-0228-IRDye
within the organ examined. All the images were acquired under the
same conditions and share the same pseudocolor scale. Ht, heart; Lv,
liver; Kd, kidney; Sp, spleen; Lu, lung; BL, blood; BM, bone marrow.
(B) Flow cytometry analysis of cells isolated from organs of NZB/W
F1 and NZW mice at 1 and 4 days post-i.v. injections of Alexa Fluor
647-labeled ZSJ-0228 (ZSJ-0228-AF647). Results are expressed as mean
± SD; ****, P < 0.0001, one-way ANOVA with
Tukey’s pairwise comparison.
Targeting apan class="Chemical">nd retention of ZSJ-0228 inpan> the kidneys of
NZB/W F1mice. (A) Representative optical images of organs isolated from NZB/W
F1 mice and NZW mice (healthy control). Images were obtained at 1
and 4 days post-i.v. injections of IRDye 800 CW-labeled ZSJ-0228 (ZSJ-0228-IRDye).
Pseudo-color-coded signal intensity reflects the level of ZSJ-0228-IRDye
within the organ examined. All the images were acquired under the
same conditions and share the same pseudocolor scale. Ht, heart; Lv,
liver; Kd, kidney; Sp, spleen; Lu, lung; BL, blood; BM, bone marrow.
(B) Flow cytometry analysis of cells isolated from organs of NZB/W
F1 and NZW mice at 1 and 4 days post-i.v. injections of Alexa Fluor
647-labeled ZSJ-0228 (ZSJ-0228-AF647). Results are expressed as mean
± SD; ****, P < 0.0001, one-way ANOVA with
Tukey’s pairwise comparison.
To better appreciate the kidney retepan class="Chemical">ntion mechanism of ZSJ-0228
on the cellular level, pan> class="Chemical">Alexa Fluor 647-labeled ZSJ-0228 (ZSJ-0228-AF647)
was i.v. administered to NZB/W F1 and NZW mice. All major organs,
including kidneys, were processed for flow cytometry analysis at 1
and 4 days postinjection. As shown in Figure B, ∼56% of kidney cells from NZB/W
F1 mice were positive for ZSJ-0228-AF647, while only ∼38% of
kidney cells from NZW mice were ZSJ-0228-AF647 positive. The amount
of ZSJ-0228-AF647 positive cells in all the other organs was less
than 10%. For NZW mice, the percentage of ZSJ-0228-AF647-positive
cells at 4 days postinjection decreased significantly (to ∼23%, P < 0.0001) when compared to the value at 1 day postinjection.
No significant ZSJ-0228-AF647 positive cell reduction was observed
in kidneys of NZB/W F1mice from 1 to 4 days postinjection. These
flow cytometry data confirm ZSJ-0228-AF647’s targeting to the
inflamed kidneys of NZB/W F1mice found in the optical imaging study
and attributes the prodrug’s retention in the kidney to cell-mediated
sequestration.
Profiling of Kidney Cells That Internalized
ZSJ-0228
Additional flow cytometry apan class="Chemical">nalysis was performed
to further profile
the ZSJ-0228-AF647 positive cells inpan> the kidnclass="Chemical">pan>eys after systemic administration
of the prodrug. In the pan> class="Disease">nephrotic kidney, ∼70% of CD11b+ (myeloid) cells internalized ZSJ-0228-AF647 at 1 day postinjection;
these cells include CD11c+ (dendritic cell), n class="Gene">NK1.1+ (natural killer cell), Ly6G+ (neutrophil), and
F4/80+ (macrophage) subphenotypes (Figure A).[26] At 4 days
postinjection, ∼50% of these cells still remain positive for
the prodrug. In comparison, ∼30% of the myeloid cells in the
NZW mice group internalized the prodrug at 1 day postinjection, and
only one-third of the cells remained positive for the prodrug at 4
days postinjection. Moreover, ∼60% of the CD326+ (proximal tubular epithelium) and CD146+ (endothelium)
cells in nephrotic kidneys retained ZSJ-0228-AF647 at 4 days postinjection,
which is 50% higher than in the control kidneys. These results suggest
that both inflammatory cells and resident renal cells (including CD326+ and CD146+ cells) in the nephrotic kidneys have
sequestered ZSJ-0228 and retained it as compared to the control. To
account for ZSJ-0228-AF647’s cellular distribution pattern,
∼20–40% of the prodrug was found in the CD11b+ cells of the nephrotic kidney, with less than 10% in the control
kidneys (Figure B).
Around 30–40% of ZSJ-0228-AF647 was internalized by the CD326+ cells of both NZB/W F1 and NZW mice, suggesting that the
prodrugs primarily distribute to the renal myeloid cells and proximal
tubular epithelium. The rest of the ZSJ-0228-AF647 positive cells
remain unidentified due to the lack of specific markers.
Figure 9
Profiling the
cellular internalization and retention of ZSJ-0228
by kidney cells. Flow cytometry was used to analyze cells isolated
from kidneys of NZB/W F1 and NZW mice at 1 and 4 days postinjection
of ZSJ-0228-AF647. (A) Percentage of renal cells that internalized
ZSJ-0228-AF647. (B) Percentage of ZSJ-0228-AF647 internalized by different
cells in the kidney. Results are expressed as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, one-way
ANOVA with Tukey’s pairwise comparison.
Profiling the
cellular ipan class="Chemical">nternalization and retention of ZSJ-0228
by kidney cells. Flow cytometry was used to anpan>alyze cells isolated
from kidneys of pan> class="Gene">NZB/W F1 and NZW mice at 1 and 4 days postinjection
of ZSJ-0228-AF647. (A) Percentage of renal cells that internalized
ZSJ-0228-AF647. (B) Percentage of ZSJ-0228-AF647 internalized by different
cells in the kidney. Results are expressed as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, one-way
ANOVA with Tukey’s pairwise comparison.
To validate renal cells’ sequestratiopan class="Chemical">n of ZSJ-0228-AF647
(red) observed by flow cytometry, pan> class="Disease">nephrotic kidneys were harvested,
sectioned, and immunohistochemically stained at 1 day post-i.v. administration
of the prodrug for confocal microscope analysis. As can be seen in Figure , fluorescent signal
colocalization of CD133 (injured/activated proximal tubular epithelial
cell),[27] CD146 (endothelium), or CD11b
(myeloid cells) with ZSJ-0228-AF647 was observed in both low- and
high-magnification images. This confirms the sequestration of ZSJ-0228-AF647
by these cells in the nephrotic kidney, which further supports the
findings in Figure .
Figure 10
Immunohistochemical analysis of kidney cells’ sequestration
of ZSJ-0228-AF647. NZB/W F1 mice were i.v. administered ZSJ-0228-AF647
(red) and euthanized at 1 day postinjection. Kidneys were sectioned
and stained with anti-mouse CD133, CD146, and CD11b (green). Nuclei
were stained with DAPI (blue). Merged images are shown in the right
panels. The higher magnification (63×) images are shown within
the lower magnification (10×) images at the lower left corner.
Scale bar = 100 μm.
Immunohistochemical apan class="Chemical">nalysis of kidney cells’ sequestration
of ZSJ-0228-AF647. pan> class="Chemical">NZB/W F1mice were i.v. administered ZSJ-0228-AF647
(red) and euthanized at 1 day postinjection. Kidneys were sectioned
and stained with anti-mouseCD133, CD146, and CD11b (green). Nuclei
were stained with DAPI (blue). Merged images are shown in the right
panels. The higher magnification (63×) images are shown within
the lower magnification (10×) images at the lower left corner.
Scale bar = 100 μm.
Internalization Kinetics and Subcellular Location of ZSJ-0228
As an immortalized repan class="Chemical">nal proximal tubule epithelial cell (RPTEC)
line from a normal adult human kidney, pan> class="Gene">HK-2 cells can reproduce experimental
results obtained with freshly isolated RPTECs on the basis of its
histochemical, immunocytochemical, and functional characteristics.[28] As RPTEC represents one of the main cellular
populations identified to sequester ZSJ-0228, an HK-2 cell culture
was used to recapitulate the internalization kinetics of the prodrug
and its subcellular location. Alexa Fluor 488-labeled ZSJ-0228 (ZSJ-0228-AF488)
was used in this particular experiment. As shown in Figure A, HK-2 cells rapidly internalized
ZSJ-0228-AF488 and the fluorescent signal intensity increased over
time. Inflammatory conditions are known to be associated with accelerated
endocytosis.[29,30] The introduction of lipopolysaccharide
(LPS), however, did not accelerate the internalization process as
anticipated, suggesting it may not be sufficient to recapitulate the in vivo inflammatory environment.
Figure 11
In vitro internalization kinetics and subcellular
location of Alexa Fluor 488-labeled ZSJ-0228 in human proximal tubule
epithelial (HK-2) cells. (A) Cellular internalization kinetics of
ZSJ-0228-Alexa 488 by HK-2 cells with or without lipopolysaccharide
(LPS, 10 μg/mL) activation over a 72 h time course. Results
are expressed as mean ± SD. (B) Representative confocal images
exhibiting internalization and subcellular trafficking of ZSJ-0228-AF488
in LPS-activated (10 μg/mL) HK-2 cells. LysoTracker DND-99 signal
(red), ZSJ-0228-AF488 signal (green), DAPI signal (blue). Scale bar
= 20 μm.
In vitro ipan class="Chemical">nternalization kinetics and subcellular
location of Alexa Fluor 488-labeled pan> class="Chemical">ZSJ-0228 in human proximal tubule
epithelial (HK-2) cells. (A) Cellular internalization kinetics of
ZSJ-0228-Alexa 488 by HK-2 cells with or without lipopolysaccharide
(LPS, 10 μg/mL) activation over a 72 h time course. Results
are expressed as mean ± SD. (B) Representative confocal images
exhibiting internalization and subcellular trafficking of ZSJ-0228-AF488
in LPS-activated (10 μg/mL) HK-2 cells. LysoTracker DND-99 signal
(red), ZSJ-0228-AF488 signal (green), DAPI signal (blue). Scale bar
= 20 μm.
To define the subcellular
locatiopan class="Chemical">n of ZSJ-0228 inpan> class="Chemical">pan> class="Gene">HK-2 cells, the
cells were incubated with ZSJ-0228-AF488 and LysoTracker (lysosome
marker, DND-99, red) and then examined under the confocal microscope.
The partial colocalization of internalized ZSJ-0228-AF488 (green)
with DND-99 (red) indicates that ZSJ-0228 was endocytosed and processed
in the acidic lysosomal compartment, which is ideal for the hydrazone-based
acid-cleavable ZSJ-0228 prodrug design (Figure B).
Discussion
Recognizipan class="Chemical">ng
the potent anti-inflammatory and organ-saving efficacy
of GCs in the clinical management of lupus and the notorious toxicities
associated with their chronic use,[31] we
proposed to develop a macromolecular prodrug nanpan>omedicinpan>e of GCs inpan>
this study to inpan>pan> class="Gene">corporate inflammatory tissue-specificity to these
drugs. By restricting GCs’ systemic distribution, we hope to
achieve organ/tissue-specific anti-inflammatory and immunosuppressive
effects, which may potentiate their therapeutic efficacy and mitigate
the adverse effects associated with GCs.
The validity of this
proposed concept capan class="Chemical">n be attributed to the
discovery of an inflammation targetinpan>g mechanpan>ism, which has beenpan> termed
ELVIS.[17] It is fundamentally different
from the well-known EPR (enhanced permeability and retention) effect[32] in that the nanomedicine’s retention
at the inflammation is not mediated by faulty lymphatic drainage,
but through the inflammatory cell (including infiltrates and activated
resident cells)-mediated sequestration.
Acn class="Gene">corpan>ding to the ELVIS
mechanpan>ism, we previously have developed
a macromolecular prodrug of pan> class="Chemical">dexamethasone (P-Dex) using N-(2-hydroxypropyl) methacrylamide (n class="Chemical">HPMA) copolymer as the carrier.
The prodrug was found to provide a potent and long-lasting anti-inflammatory
effect in multiple inflammatory disease models, including the NZB/W
F1 LNmice.[19,33−36] Mechanistically, this pathophysiology-driven
targeting of GC to nephritis is different from the active kidney-targeting
drug delivery,[37,38] in which different targeting
ligands (e.g., peptides, sugar,
and folates) were employed to render the renal specificity.
While n class="Chemical">P-Dexpan>’s therapeutic efficacy in the lupus model was
strong anpan>d sustainpan>ed, it could only circumvent pan> class="Disease">osteopenia, while other
GC toxicities (e.g., immunosuppression
and adrenal gland atrophy) persisted.[19] Optical imaging-based biodistribution study and flow cytometry analyses
of cells from all major organs and tissues suggest that the persisted
side effects may be attributed to the prodrug’s internalization
by circulating WBCs and the high-level deposition to the mononuclear
phagocyte system (MPS, including phagocytic cells of the liver and
spleen). This hypothesis was partially supported by the amelioration
of splenomegaly in the P-Dex-treated animals.[19] We posit that these off-target distributions and gradual activation
of P-Dex may have led to the sustained presence of n class="Chemical">dexamethasone in
the serum. While it may be at a low concentration and not enough to
cause skeletal deterioration, the serum dexamethasone level can be
sufficient to elicit systemic immunosuppression and adrenal gland
atrophy.[39]
With these understapan class="Chemical">ndings,
we proceed to develop the next-generation
macromolecular GC prodrug, which is not only effective in resolving
LN but also able to avoid typical GC adverse effects found with pan> class="Chemical">P-Dex.
The focus of our effort is to further reduce dexamethasone levels
in the serum by limiting the prodrug’s sequestration by WBCs
and its deposition to the MPS.
Our previous findipan class="Chemical">ng suggests
that the use of PEG as a prodrug
carrier may significanclass="Chemical">pan>tly delay cellular internalization when compared
to the use of anpan> pan> class="Chemical">HPMA copolymer carrier of the same size.[40] Therefore, we used PEG as the water-soluble
drug carrier in the ZSJ-0228 design (Scheme ). The selection of low molecular weight
mPEG 1900 was based upon the prior findings that polymeric prodrugs
with the lowest molecular weight demonstrate the highest kidney exposure
and relatively lower liver deposition.[41,42] n class="Chemical">PEGs with
a lower molecular weight of 0.4 or 1 kDa were not selected because
they are mostly in wax or liquid form, which can be difficult to handle
and to purify during the synthesis. The amphiphilic structure of ZSJ-0228
allows its self-assembly into micelles in aqueous media. Using DLS,
TEM and NTA methods, we determined that the ZSJ-0228micelle’s
average hydrodynamic diameter was around 30 nm, which may lead to
a long serum half-life should the micelle remain stable.[43] The CMC value of the micelle, however, was determined
to be relatively high (2.5 × 10–4 M). These
micelles will disintegrate upon i.v. administration and dilution,
significantly reducing its half-life in circulation. We anticipate
that a shorter serum half-life of ZSJ-0228 would limit its distribution
to the liver and spleen when compared to P-Dex,[41,42] but still provide sufficient kidney exposure for therapeutic effects.
A n class="Chemical">hydrazonepan> bond was used as the pan> class="Chemical">ZSJ-0228 prodrug’s activation
trigger. The linker’s in vitro cleavage rate
under acidic pH is relatively slow (Figure ) when compared to other n class="Chemical">hydrazone linker-based
prodrug designs.[44] Such slow activation
kinetics may be explained by the presence of a large conjugation system,
which involves four double bonds including two C=C bonds within
the A ring of dexamethasone, one C=N bond, one C=O bond,
and the lone pair of sp2 electrons of the neighboring nitrogen.
The large delocalization of electrons stabilizes the C-3 hydrazone
and reduces the rate of C=N double-bond cleavage, which is
responsible for the release of dexamethasone. When doxorubicin is
conjugated to HPMA copolymer via a hydrazone bond,[44] such structural stabilization does not exist.
The slow in vitro hydrazone bond cleavage, however,
does not necessarily predict a slow in vivo prodrug
activation. Also contributing to ZSJ-0228’s in vivo activation is the pH value within lysosomal compartments, which
can be significantly reduced under inflammatory conditions[45] and accelerate the hydrazone bond cleavage.
For future prodrug design, other biochemical factors, such as the
elevated presence of reactive oxygen species (ROS), which is often
associated with inflammatory pathologies,[46] may also be considered as a trigger for prodrug activation at the
site of inflammation.[47]
Due to the
use of n class="Chemical">hydrazonepan> as an activation trpan> class="Gene">igger, ZSJ-0228
should have multiple configurational isomers. Since the activation
product from these isomers is the same (i.e., dexamethasone) and their ratio from different batches
remains consistent, the presence of these isomers inn class="Chemical">ZSJ-0228 will
not be a concern during the preclinical Chemistry, Manufacturing,
and Controls development. Though not used in our synthesis, we are
aware of the development of single molecular weight discrete PEG (dPEG),
which has become commercially available. Different from traditional
macromolecular prodrug design, the use of dPEG in the synthesis of
ZSJ-0228 will produce a single molecular weight polymeric prodrug,
which will further reduce potential regulatory hurdles during the
product development.
Previously, daily n class="Chemical">dexamethasonepan> treatment
(1 mg/kg) was found to
attenuate the pan> class="Disease">nephritis and improve survival of NZB/W F1mice.[18] In the present study, it was used as a benchmark
to compare with n class="Chemical">ZSJ-0228 prodrug treatment. Dose equivalent monthly
Dex treatment (28 mg/kg dexamethasone equivalent) was not used as
a control due to adverse effects (e.g., slow movement, eyelids partially closed, changed respiration,
hunched posture, and loss of body weight) observed immediately after
administration. The approved IACUC protocol requests that animals
with such stress behaviors be euthanized immediately to minimize animal
discomfort. As anticipated, when tested in NZB/W F1mice with established
nephritis, ZSJ-0228 monthly treatment effectively attenuated albuminuria
and maintained 100% animal survival for the entire experiment duration.
Dose equivalent daily Dex treatment (1 mg/kg), on the other hand,
only presented with moderate efficacy and 80% survival (Figure ). These observations were
further supported by the glomerular histologic findings in which ZSJ-0228
treatment was found to more effectively prevent glomerular injury
with disease severity rated mostly mild to moderate (Figure ).
n class="Chemical">ZSJ-0228pan> seems to
also possess an improved safety profile. As shown
inpan> Figure A, pan> class="Chemical">ZSJ-0228
showed minimal cytotoxicity in HK-2 cell culture. During the two-month
treatment study, the prodrug treatment did not significantly alter
the mice’s body weight (Figure B). Compared to dose equivalent daily n class="Chemical">Dex treatment,
the monthly ZSJ-0228 treatment did not induce osteopenia (Figure C–E); neither
did it cause immunosuppression, as evidenced by WBC and total serum
IgG levels comparable to the saline group (Figure F,G). Furthermore, mice treated monthly with
ZSJ-0228 were found to have significantly higher adrenal gland mass
than the dose equivalent daily Dex-treated mice, suggesting the absence
of adrenal gland atrophy (Figure H). It is very important to recognize that the WBC
reduction induced by GC treatment is being interpreted as immunosuppression
in NZB/W F1mice.[48] It is well-recognized
that the opposite effect (i.e.,
leukocytosis) is commonly observed in humanpatients when GC is being
used.[49] Therefore, ZSJ-0228’s impact
on WBCs in the NZB/W F1mice should not be directly extrapolated to
humans. Collectively, these data provide clear evidence of ZSJ-0228’s
superior therapeutic efficacy to Dex and significantly improved safety
when compared to Dex and P-Dex[19,34] in the treatment of
NZB/W F1mice.
Inprobipan class="Chemical">ng the working mechanism of ZSJ-0228,
near-inpan>frared imaginpan>g-based inclass="Chemical">pan> vivo biodistribution
data (Figure A) suggest
that the prodrug’s mainpan>
distribution organpan> inpan> pan> class="Chemical">NZB/W F1mice was the inflamed kidney. The distribution
to other organs was limited, which is in stark contrast to the observation
in humans treated with dexamethasone (with fast extrarenal elimination
and high liver deposition)[50] and in mice
with n class="Chemical">P-Dex (with higher liver and spleen deposition).[19,34] Different from P-Dex treatment, ZSJ-0228 treatment has no impact
on splenomegaly (data not shown), which supports the optical imaging
findings. The significantly lower ZSJ-0228 positive cells in other
organs counted by the flow cytometry further validated the nephrotropic
distribution pattern of ZSJ-0228 (Figure B).
Several promising pan class="Chemical">nanoformulations
have been developed for dexamethasone
delivery.[51−53] Comparinpan>g to pan> class="Chemical">ZSJ-0228, they have the advantage of
significantly reduced cost of manufacture and lower barrier for regulatory
approval. Similar to P-Dex, their biodistribution patterns favor the
MPS system. We speculate that the relatively higher liver/spleen distribution
may be attributed to their high molecular weight or large size.[54] ZSJ-0228, on the other hand, is considerably
smaller (∼3 kDa). The water-soluble prodrug consists of an
mPEG 1900 and two dexamethasone molecules (Scheme ). Considering that the hydrodynamic radius
of PEG 3000 is around 15 Å,[55] the
structurally more compact ZSJ-0228 may be even smaller than that.
Our ongoing pharmacokinetic study estimates the t1/2(β) of ZSJ-0228 to be around 12 h, which is significantly
shorter than that of P-Dex.[41,42] We believe the relatively
smaller size and shorter serum half-life may be the main contributing
factor for ZSJ-0228’s nephrotropicity and low MPS distribution.
Two additional factors may also be copan class="Chemical">nsidered in understanding
ZSJ-0228’s high distribution inpan> the kidney: (1) It has been
reported that there is anpan> inclass="Chemical">pan>verse size dependency inpan> the renal clearanpan>ce
of sub-nanpan>ometer gold nanpan>oclusters (Aupan> class="Chemical">NCs), which is distinctly different
from the general understanding that smaller nanoparticles always clear
more rapidly through the kidney than the larger ones.[56] We postulate that the proposed mechanism of physical retention
of AuNCs by the glycocalyx of the glomeruli may have also helped to
slow down the clearance of ZSJ-0228 from the kidney and thus contributed
to its high kidney distribution. (2) According to the ELVIS mechanism,
inflammatory cells’ accelerated sequestration is one of the
major contributors to the high retention of nanomedicine in inflammatory
tissues. As shown in Figure B, the kidney cells of NZB/W F1mice certainly have demonstrated
substantially higher sequestration of ZSJ-0228 than the control NZW
mice.
It is interestipan class="Chemical">ng to note that ZSJ-0228 was also somewhat
retainpan>ed
by kidney/kidnclass="Chemical">pan>ey cells of n class="Chemical">NZW n class="Species">mice (Figures and 9). This is probably
due to the advanced age (>28 weeks) of the animals being used in
this
study. NZB/W F1mice are the offspring of an NZB female (stock # 000684,
Jackson Laboratory) and an NZW male (stock # 001058, Jackson Laboratory).
Both inbred parental strains may occasionally develop autoimmune abnormalities
that are observed in the F1, but not necessarily with the same onset
or severity. While the NZW mice have a normal life span, some do develop
anti-DNA antibodies, high serum levels of retroviral gp70 antigen,
and nephritis at an advanced age.[23]
Further flow cytometry profiling of kidpan class="Chemical">ney cells that internalize
ZSJ-0228 revealed that myeloid cells anpan>d proximal tubular epithelial
cells were the major players inpan> the sequestration anclass="Chemical">pan>d retention of
pan> class="Chemical">ZSJ-0228 in the inflamed kidneys of NZB/W F1n class="Species">mice (Figure ). These findings are not surprising,
as infiltrating myeloid cells in LN are known to be phagocytic.[57] It has been reported that inflammatory insults
could reprogram the myeloid cells’ endocytic machinery from
receptor-mediated endocytosis to macropinocytosis,[58] which would accelerate the rate of cell internalization
in a receptor-independent fashion. Proximal tubular epithelial cells,
on the other hand, recycle the albumin via a receptor-mediated
endocytosis mechanism when exposed to the proteins.[59] One may suggest that the observed endocytosis of ZSJ-0228
(Figures –11) by tubular epithelial cells is a “bystander”
effect associated with the internalization/recycling of albumin by
the cells. Due to the potential binding of ZSJ-0228 to albumin,[60,61] it is also possible that the prodrug may piggyback albumin and be
internalized through the receptor-mediated endocytic process intended
for albumin.
We noticed that while the majority (50%) of repan class="Chemical">nal
CD146+ cells (endothelial cells) inclass="Chemical">pan>ternalized pan> class="Chemical">ZSJ-0228
(Figure B) and IHC
analysis suggests
a high CD146+ cellular uptake of the prodrug (Figure ), the flow cytometry
data show low prodrug distribution (<1%) to this cell population
(Figure B). This discrepancy
may be attributed to the difficulty in isolating endothelial cells
from the kidneys. Typically, a collagenase-based tissue dissociation
reagent, Liberase Blendzyme, can be used to efficiently isolate endothelial
cells from tissues. Its application, however, often results in damage
to other cells.[62−64] Thus, a gentler cell isolation protocol was used
in the present study. We speculate that this limitation of the current
flow cytometry tissue isolation protocol may have inadvertently lowered
the number of endothelial cells isolated from the kidney and underestimated
their contribution to the kidney sequestration of n class="Chemical">ZSJ-0228.
While n class="Chemical">ZSJ-0228pan> demonstrated strong nephrotropicity on the lupus-prone
pan> class="Chemical">NZB/W F1mice, it did not stay in the kidney permanently. The presence
of ZSJ-0228 in the kidney decreased over time (Figures and 9). This is especially
true for the CD11b+ myeloid cells isolated from the kidneys
of NZB/n class="Gene">W F1 mice. The percentage of CD326+ cells (proximal
tubular epithelium) that internalized ZSJ-0228 did not change from
day 1 to day 4 postadministration (Figure ). It has been reported that GC treatment
can induce myeloid cell apoptosis.[65] Renal
epithelial cells, on the other hand, are insensitive to GC treatment.[66] Therefore, it is possible that the ZSJ-0228
sequestered by myeloid cells may have induced their apoptosis, leading
to the prodrug’s gradual clearance from the kidney. Since FcR-bearing
myeloid cells are responsible for triggering LN,[67] ZSJ-0228’s specific sequestration by the myeloid
cells in the kidney may also be a plausible explanation for ZSJ-0228’s
superior efficacy to Dex. Such a notion conforms to the finding shown
in Figure , where
the number of macrophages was significantly reduced after two months
of treatment with ZSJ-0228. Clearly, further investigations are needed
to better understand ZSJ-0228’s working mechanism on the cellular
and molecular levels.
The present study does have its limitatiopan class="Chemical">ns.
ZSJ-0228 was designed
for the improved efficacy anclass="Chemical">pan>d safety of GC treatment for pan> class="Disease">lupus nephritis,
but not for other lupus complications such as n class="Disease">arthritis. Since its
main distribution organ is the kidney, ZSJ-0228 may not be effective
in managing inflammation at other anatomical sites of the body. P-Dex[17] and other GC nanoformulations,[51−53] which have longer serum half-lives, may be better suited for these
conditions. We speculate that the development of a GC prodrug that
would be effective and safe for most of the lupus symptoms may necessitate
a macromolecular prodrug system that is conditionally degradable.[68] Regarding the administration route, ZSJ-0228
was given as a monthly i.v. injection in this study. Though the treatment
schedule is favorable, the i.v. route would require healthcare professionals
for administration in the clinical setting. Previous work[69] indicates that s.c. and i.p. administrated polymeric
prodrugs may enter the circulation through the lymphatic system. Therefore,
we speculate that it is possible that ZSJ-0228 may also be given via these routes and be effective. This approach would enable
self-administration of the medication and further improve the patients’
compliance to the treatment.
Conclusions
A n class="Species">micepan>lle-forming pan> class="Chemical">PEG-based
dexamethasone prodrug (n class="Chemical">ZSJ-0228) was
developed with superior and sustained therapeutic efficacy against
nephritis in lupus-prone female NZB/W F1mice. No apparent glucocorticoid
side effects were observed. While as a prodrug of dexamethasone, ZSJ-0228
does not change the parent drug’s anti-inflammatory and immunosuppressive
mechanism on the molecular level, it indeed alters the pharmacology
physiologically by restricting its distribution to the inflamed kidneys,
providing sustained and localized immunosuppressive and anti-inflammatory
effects. We believe ZSJ-0228 can be further developed into a viable
drug candidate for the better clinical management of lupus nephritis.
We further suggest that this GC prodrug may also be explored for the
clinical management of other renal pathologies, such as kidney transplant,
IgA nephropathy, focal segmental glomerulosclerosis, minimal change
disease, and Goodpasture syndrome, in which GCs are frequently used
as first-line therapy.
Methods/Experimental
Materials
and Reagents
n class="Chemical">Dexamethasonepan> was obtained from
Tianpan>jinpan> Pharmaceuticals Group Co., Ltd. (Tianpan>jinpan>, Chinpan>a). pan> class="Chemical">Dexamethasone
21-phosphate disodium was purchased from Hawkins, Inc. (Minneapolis,
MN, USA). Heterofunctional PEGs were purchased from Rapp Polymere
GmbH (Tuebingen, Germany) and Creative PEGWorks (Chapel Hill, n class="Chemical">NC,
USA). Piperidine was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Sephadex LH-20 resins were purchased from GE HealthCare (Piscataway,
NJ, USA). IRDye 800CW NHS ester was purchased from LI-COR Biosciences
(Lincoln, NE, USA). Alexa Fluor 488 NHS ester was obtained from Life
Technologies (Carlsbad, CA, USA). The MTT cell proliferation assay
was purchased from ATCC (Manassas, VA, USA). Penicillin/streptomycin,
trypsin-EDTA, Dulbecco’s phosphate-buffered saline (PBS), and
RPMI 1640 were from Gibco (Grand Island, NY, USA). Fetal bovine serum
was purchased from BenchMark (Gemini Bio-Products, West Sacramento,
CA, USA). All solvents and other reagents if not specified were purchased
from Fisher Scientific or ACROS and used without further purification.
Instruments
n class="Chemical">1Hpan> and pan> class="Chemical">13C NMR spectra
were ren class="Gene">corded on a 500 MHz NMR spectrometer (Varian, Palo Alto, CA,
USA). The mass spectrum analyses were performed with an LC/MS/MS system
composed of an ACQUITY Ultra Performance LC (UPLC) system (Waters,
Milford, MA, USA) and a Sciex 4000 Q TRAP mass spectrometer with an
ESI source (Applied Biosystems, Toronto, Canada). HPLC analyses were
performed on an Agilent 1100 HPLC system (Agilent Technologies, Inc.,
Santa Clara, CA, USA) with a reverse phase C18 column (Phenomenex,
Bondclone H16-441537-C18, 300 × 3.9 mm, 10 μm). In vivo near-infrared fluorescence-based optical imaging
was accomplished on a LI-COR Pearl Impulse small animal imaging system
(Lincoln, NE, USA). Bone qualities were analyzed using a high-resolution
Skyscan 1172 micro-CT system (Bruker microCT, Kontich, Belgium). The
average hydrodynamic diameter, polydispersity index (PDI), and zeta
potential of micelles were determined by DLS experiments using a Zetasizer
Nano ZS90 (Malvern Instruments, Worcestershire, UK). The particle
size distribution was analyzed using NS300 NanoSight (Malvern Instrument).
The micelle morphology was observed using a Tecnai G2 Spirit transmission
electron microscope (FEI, Hillsboro, OR, USA) at an acceleration voltage
of 100 kV. Digital images were acquired using a KeenView high-resolution
camera and analyzed using Soft Imaging Solutions AnalySIS ITEM digital
software. The quantification of fluorescence signal intensities of
IRDye 800 CW, Alexa Fluor 488, Alexa Fluor 647, and pyrene were measured
using a Spectramax M2 spectrofluorometer (Molecular Devices, Sunnyvale,
CA, USA). The flow cytometry experiments were performed on a BD LSRII
Green flow cytometer (San Jose, CA, USA) and analyzed using the FlowJo
software (Treestar, Inc., San Carlos, CA, USA). Confocal microscope
images were acquired under an LSM 800 Zeiss Airyscan microscope (Carl
Zeiss, Oberkochen, Germany) and analyzed using the ZEN Lite software.
Synthesis of ZSJ-0228 (Scheme )
Synthesis of Compound 1
n class="Chemical">Imidazolepan> (2.72
g, 40 mmol) and pan> class="Chemical">dexamethasone (7.84 g, 20 mmol) were dissolved in
anhydrous N,N-dimethylformamide
(n class="Chemical">DMF, 40 mL). After the solution was cooled to 0 °C, tert-butyldimethylsilyl chloride (TBSCl, 3.3 g, 22 mmol)
was added. The solution was maintained at 0 °C for 3 h and at
room temperature for 2 h, with constant stirring. Ethyl acetate (100
mL) was then added and washed with brine (80 mL × 4). The organic
phase was separated and dried over MgSO4. After removal
of the solvent, the residue was purified with flash chromatography
(ethyl acetate/hexane, 1:2) to produce compound 1 (9.98
g). Yield: 98.5%. 1HNMR (500 MHz, DMSO-d6): δ (ppm) 7.28 (d, J = 10.2 Hz,
1H), 6.22 (d, J = 10.2 Hz, 1H), 6.00 (s, 1H), 5.29
(s, 1H), 4.97 (s, 1H), 4.78 (d, J = 19.1 Hz, 1H),
4.29 (d, J = 19.1 Hz, 1H), 4.14 (d, J = 10.1 Hz, 1H), 2.91 (m, 1H), 2.61 (td, J = 13.6
Hz, 5.8 Hz, 1H), 2.36 (m, 2H), 2.11 (m, 2H), 1.76 (m, 1H), 1.62 (q, J = 11.8 Hz, 1H), 1.48 (s, 3H), 1.41 (d, J = 13.5 Hz, 1H), 1.33 (m, 1H), 1.06 (m, 1H), 0.88 (s, 9H), 0.87 (s,
3H), 0.77 (d, J = 7.2 Hz, 3H), 0.04 (s, 3H), 0.03
(s, 3H). 13CNMR (125 MHz, DMSO-d6): δ (ppm) 209.19, 185.45, 167.20, 152.94, 129.17, 124.29,
101.38 (d, JC–F = 174 Hz), 90.52,
70.84 (d, JC–F = 36.9 Hz), 68.14,
48.13 (d, JC–F = 22.5 Hz), 47.65,
36.03, 35.33, 33.82 (d, JC–F =
19.3 Hz), 32.09, 30.46, 27.42, 25.97, 23.11, 23.08, 18.34, 16.86,
15.38, −4.89, −5.04. MS (ESI): m/z 507.2 (M + H+), calculated 506.3.
Synthesis
of Compound 2
n class="Chemical">NH2NH2 monohydratepan>
(750 mg, 15 mmol) and compound 1 (2.53 g, 5 mmol) from
the first step were dissolved inpan> pan> class="Chemical">methanol
(25 mL). After addition of acetic acid (60 mg, 1 mmol), the solution
was stirred at room temperature for 5 h. Ethyl acetate (100 mL) was
then added, and the solution was washed with brine (80 mL × 4).
The organic phase was separated and dried over n class="Chemical">MgSO4. After
solvent removal with rotary evaporation, the residue was purified
with flash chromatography (ethyl acetate/hexane, 1:1) to produce compound 2 (1.14 g). Considering the recovery of unreacted compound 1 (1.24 g), we calculate the final yield at 85.8%. Because
of the hydrazone bond formation, a mixture of two syn/anti configurational
isomers exists in the product. However, they are indistinguishable
on flash chromatography. The two isomers’ molar ratio was determined
to be 1.63:1 according to 1HNMR. 1HNMR (500
MHz, DMSO-d6): δ (ppm) 6.67 (d, J = 10.4 Hz, 0.38H), 6.39 (s, 0.62H), 6.28 (d, J = 10.4 Hz, 0.38H), 6.23 (s, 2H), 6.03 (d, J = 11.5
Hz, 0.62H), 5.98 (d, J = 11.5 Hz, 0.62H), 5.76 (s,
0.38H), 5.09 (dd, J = 10.5 Hz, 3.0 Hz, 1H), 4.96
(s, 1H), 4.74 (d, J = 19.1 Hz, 1H), 4.26 (d, J = 19.1 Hz, 1H), 4.09 (m, 1H), 2.87 (m, 1H), 2.50 (m, 1H),
2.22 (m, 1H), 2.16 (m, 3H), 1.58 (m, 2H), 1.36 (s, 3H), 1.33 (d, J = 13.5 Hz, 1H), 1.26 (m, 1H), 1.02 (m, 1H), 0.85 (s, 9H),
0.81 (s, 3H), 0.74 (d, J = 7.2 Hz, 3H), 0.02 (s,
3H), 0.01 (s, 3H). 13CNMR (125 MHz, DMSO-d6): δ (ppm) 209.71, 170.98, 151.16, 144.73, 140.74,
140.62, 139.03, 132.91, 127.70, 121.61, 116.17, 110.81, 100.71 (d, J = 171.9 Hz), 100.50
(d, J = 171.9
Hz), 90.92, 90.87, 70.00 (d, JC–F = 37.4 Hz), 69.93 (d, JC–F =
37.3 Hz), 68.43, 60.29, 47.21 (d, JC–F = 22.8 Hz), 47.05 (d, JC–F =
22.8 Hz), 44.02, 36.41, 36.35, 35.62, 34.40 (d, JC–F = 19.5 Hz), 34.36 (d, JC–F = 19.5 Hz), 25.28, 25.24, 24.85, 24.82, 21.17, 18.58,
17.15, 15.63, 14.49, −4.67, −4.80. MS (ESI): m/z 521.5 (M + H+), calculated
520.3. LC/MS: two peaks in the chromatography with the retention times
at 5.74 and 6.32 min were found to have the same molecular weight
at 521.356 (conditions: A: 7.5 mM ammonium acetate; B: 95% MeOH and
5% acetonitrile; A:B = 30:70).
Synthesis of Compound 3
n class="Chemical">4-Dimethylaminopyridinepan>
(201 mg, 1.65 mmol) and compound 2 (2.86 g, 5.5 mmol)
were dissolved inpan> anpan>hydrous pan> class="Chemical">DMF (15 mL). After cooling to 0 °C,
Fmoc-glycine (2.12 g, 7.15 mmol) and dicyclohexylcarbodiimide (DCC,
1.70 g, 8.25 mmol) were added. The resulting solution was stirred
at 0 °C for 3 h. Ethyl acetate (100 mL) was then added, and the
solution was washed with n class="Chemical">brine (80 mL × 4). The organic phase
was separated and dried over MgSO4. After removal of the
solvent, the residue was purified by flash chromatography (ethyl acetate/hexanes,
1:1) to produce crude compound 3 (3.72 g). The yield
is 84.5%. TLC indicates the presence of a small amount of side products,
which were difficult to separate. The crude product was used directly
in the next reaction.
Synthesis of Compound 4
Compound 3 (3.0 g, 3.75 mmol) was dissolved ipan class="Chemical">n dichloromethane
(pan> class="Chemical">DCM,
10 mL) and then cooled to 0 °C. After addition of piperidine
(1 mL), the solution was stirred at 0 °C for 3 h. Ethyl acetate
(100 mL) was then added and washed with brine (80 mL × 3). The
organic phase was separated and dried over MgSO4. After
removal of the solvent, toluene (50 mL) was added and then evaporated
to help remove the residual piperidine. After purification with flash
chromatography (ethyl acetate followed by ethyl acetate/methanol,
2:1), 1.96 g of compound 4 was produced. The yield is
calculated as 90.6%. 1HNMR (500 MHz, DMSO-d6): δ (ppm) 7.01 (d, J = 10.3 Hz,
0.23H), 6.86 (d, J = 10.3 Hz, 0.16H), 6.77 (s, 0.26H),
6.66 (d, J = 10.3 Hz, 0.17H), 6.60 (d, J = 10.3 Hz, 0.23), 6.59 (s, 0.25H), 6.46 (d, J =
10.3 Hz, 0.26H), 6.41 (d, J = 10.3 Hz, 0.26H), 6.28
(d, J = 10.3 Hz, 0.25H), 6.19 (d, J = 10.3 Hz, 0.26H), 6.02 (s, 0.16H), 5.92 (s, 0.20H), 5.21 (br, 1H),
4.98 (br s, 1H) 4.77 (d, J = 19.0 Hz, 1H), 4.28 (d, J = 19.0 Hz, 1H), 4.12 (m, 1.0H), 3.25 (s, 2H), 2.90 (m,
1H), 2.60 (m, 1H), 2.25 (m, 2H), 2.14 (m, 1H), 1.71 (m, 1H), 1.58
(m, 1H), 1.41 (d, J = 8.6 Hz, 3H), 1.38 (d, J = 13.5 Hz, 1H), 1.32 (m, 1H), 1.05 (m, 1H), 0.88 (s, 9H),
0.77 (d, J = 7.1 Hz, 3H), 0.04 (s, 3H), 0.02 (s,
3H). 13CNMR (125 MHz, DMSO-d6): δ (ppm) 209.44, 174.80, 169.60, 157.62, 156.88, 152.11,
151.27, 146.38, 146.05,144.35, 143.81, 142.95, 139.56, 138.87, 126.74,
120.70, 116.73, 116.67, 111.46, 111.32, 100.98 (d, JC–F = 173.4 Hz), 100.91 (d, JC–F = 172.3 Hz), 100.76 (d, JC–F = 173.4 Hz), 100.73 (d, JC–F =
172.6 Hz), 90.72, 90.66, 70.13 (d, JC–F = 37.4 Hz), 70.12 (d, JC–F =
37.3 Hz), 68.28, 47.74, 47.49 (d, JC–F = 21.5 Hz), 47.45 (d, JC–F =
21.5 Hz), 47.23, 44.15, 43.77, 42.72, 36.22, 36.17, 34.10 (d, JC–F = 19.5 Hz), 32.16, 31.19, 31.06,
30.20, 30.05, 27.58, 26.08, 24.55, 24.51, 24.19, 24.16, 24.10, 24.06,
18.45, 16.99, 15.52, 15.50, −4.79, −4.93. MS (ESI): m/z = 578.3 (M + H+), calculated
577.3. LC/MS: two peaks in the chromatogram with the retention times
at 3.91 and 4.47 min were found to have the same molecular weight
at 578.347 (conditions: A: 7.5 mM ammonium acetate, B: 95% MeOH and
5% acetonitrile; A:B = 30:70).
Synthesis of Compound 5
Compound 4 (444 mg, 0.768 mmol) was
dissolved ipan class="Chemical">n anhydrous DMF (3 mL),
followed by the addition of pan> class="Chemical">Fmoc-glutamic acid (135 mg, 0.366 mmol),
hydroxybenzotriazole (HOBt, 148 mg, 1.098 mmol), and DCC (226 mg,
1.098 mmol). The resulting solution was stirred at room temperature
for 4 h. Ethyl acetate (100 mL) was then added to the solution and
washed with brine (80 mL × 3). The organic phase was separated
and dried over MgSO4. After the solvent removal, the residue
was purified by flash chromatography (ethyl acetate/methanol, 10:1)
to afford crude compound 5 (471 mg). The yield is 86.5%.
TLC indicates the presence of a small amount of side products, which
were difficult to separate. The crude product was used directly in
the next reaction.
Synthesis of Compound 6
Compound 5 (450 mg, 0.3 mmol) was dissolved ipan class="Chemical">n DCM (4.5
mL) anpan>d cooled
to 0 °C. After additionclass="Chemical">pan> of n class="Chemical">piperidine (1.5 mL), the resulting
solution was stirred at 0 °C for 3 h. n class="Chemical">Ethyl acetate (100 mL)
was then added and washed with brine (80 mL × 3). The organic
phase was separated and dried over MgSO4. After solvent
removal, the residue was purified by flash chromatography (ethyl acetate
followed by ethyl acetate/methanol, 3:1) to produce compound 6 (330 mg). The yield is 86.4%. 1HNMR (500 MHz,
DMSO-d6): δ (ppm) 10.83 (m, 1.12H),
10.64 (m, 0.80H), 8.38 (m, 0.30H), 8.28 (m, 0.62H), 8.18 28 (m, 0.42H),
7.99 (m, 0.60H), 7.01 (m, 0.29H), 6.93 (m, 0.16H), 6.77 (s, 0.91H),
6.68 (s, 0.73H), 6.62 (m, 0.34H), 6.45 (m, 1.52H), 6.25 (m, 1.53H),
6.01 (s, 0.15H), 5.96 (0.31H), 5.17 (s, 1.0H), 5.15 (s, 1.0H), 4.95
(s, 2H), 4.77 (d, J = 19.1 Hz, 2.0H), 4.28 (d, J = 19.1 Hz, 2.0H), 4.19 (m, 0.53H), 3.95–4.15 (m,
4.91H), 3.70–3.90 (m, 1.68H), 3.26 (m, 1.54H), 2.90 (m, 2.15H),
2.55–2.75 (m, 3.93H), 2.15–2.35 (m, 5.23H), 2.05–2.15
(m, 5.04H), 1.87 (m, 1.10H), 1.72 (m, 3.07H), 1.59 (m, 2.23H), 1.41
(m, s, 6.87H), 1.31 (m, 3.38H), 1.05 (m, 1.99H), 0.87 (s, 18H), 0.85
(s, 6H), 0.77 (d, J = 6.9 Hz), 0.03 (s, 6H), 0.02
(s, 6H). MS (ESI): m/z 1266.4 (M
+ H+), calculated 1265.7. LC/MS: three peaks in the chromatogram
with the retention times at 5.07, 5.80, and 6.54 min were found to
have the same molecular weight of 1266.782 (conditions: A: 7.5 mM
ammonium acetate, B: 95% MeOH and 5% acetonitrile; A:B = 18:82).
Synthesis of ZSJ-0228
n class="Chemical">DCCpan> (107 mg, 0.52 mmol), n class="Chemical">HOBt
(70.2 mg, 0.52 mmol), and n class="Chemical">mPEG-COOH (100 mg, 0.052 mmol) were dissolved
in anhydrous DMF (3 mL) at room temperature and stirred for 1 h. After
addition of compound 6 (428 mg, 0.338 mmol), the solution
was stirred at room temperature for 24 h. An LH-20 column was used
to obtain the polymer fraction. After removal of the solvent, the
polymeric residue was dissolved in tetra-n-butylammonium
fluoride (1 M, 2 mL) and stirred for 2 h. The resulting solution was
again applied to an LH-20 column to separate the polymeric fraction.
After dialysis against DI water overnight (MWCO = 2 kDa), the solution
was lyophilized to produce ZSJ-0228 (118.7 mg). The yield is 77.8%. 1HNMR (500 MHz, DMSO-d6): δ
(ppm) 10.95 (s, 0.28H), 10.91 (s, 0.27H), 10.86 (s, 0.80H), 10.82
(s, 0.37H), 10.58 (m, 0.67H), 8.39 (m, 0.35H), 8.21 (s, 0.59H), 8.14
(m, 0.35H), 7.99 (m, 0.61H), 7.78 m, 0.30H), 7.71 (m, 0.69H), 7.54
(m, 0.1H), 7.02 (d, J = 10.3 Hz, 0.53H), 6.90 (m,
0.26H), 6.78 (s, 0.69H), 6.60–6.71 (m, 1.25H), 6.42–6.47
(m, 1.14H), 6.27 (m, 0.41H), 6.21 (m, 0.73H), 6.00 (m, 0.26H), 5.96
(s, 0.53H), 5.14 (m, 1.98H), 4.93 (s, 1.85H), 4.66(m, 1.86H), 4.48
(dd, J = 19.2 Hz, 5.8 Hz, 1.80H), 4.40 (m, 0.67H),
4.34 (m, 0.40H), 4.00–4.25 (m, 6.66H), 3.92 (s, 2.55H), 3.86
(m, 0.52H), 3.83 (m, 0.36H), 3.51 (m, 183H), 3.25 (s, 3.24H), 2.93
(m, 1.92H), 2.63 (m, 1.43H), 2.36 (m, 0.66H), 2.20–2.35 (m,
5.71H), 2.05–2.15 (m, 4.46H), 1.97 (m, 1.45H), 1.84 (m, 1.16H),
1.71 (m, 2.15H), 1.60 (m, 2.48H), 1.42 (s, 7.95H), 1.31 (m, 2.55H),
1.23 (s, 1.07H), 1.05 (m, 2.12H), 0.84 (s, 6.00H), 0.77 (d, J = 7.1 Hz, 5.74H). MS (ESI): Two clusters of peaks were
observed. One cluster is at around 1550, which are diion peaks. For
example: n = 44, calculated: (M + 2Na+)/2 = 1546.4, found 1546.7; n = 40, calculated: (M + 2Na+)/2 = 1458.38, found 1458.3. The other cluster is at around 1100,
which are triion peaks, n = 46 calculated (M + 3Na+)/3 = 1068.0, found
1068.5. MS (MALDI-TOF): One symmetric cluster of peaks (at about 3000)
was observed, which represent the molecular weights of ZSJ-0228 plus
sodium ion. For example: M + Na+ = 2937.83, found 2938.28; M + Na+ = 3025.84, found 3026.342; M + Na+ = 3201.96, found 3202.425.
Synthesis
of Fluorescent-Labeled ZSJ-0228
The n class="Gene">synpan>thetic
routes for IRDye n class="Chemical">800CW-, n class="Chemical">Alexa Fluor 488-, and Alexa Fluor 647-labeled
ZSJ-0228 are similar to the unlabeled ZSJ-0228 (Scheme ), except the mPEG-COOH was replaced with
Fmoc-NH-PEG-COOH. After Fmoc deprotection, the resulting NH2-containing polymeric prodrug (100 mg) was dissolved in anhydrous
DMF (1 mL) together with the NHS esters of IRDye 800CW (1 mg), Alexa
Fluor 488 (1 mg), or Alexa Fluor 647 (1 mg). The solution was stirred
at room temperature for 15 h. After LH-20 column purification, the
fluorescent-labeled prodrugs were obtained. Using a Spectramax M2
spectrofluorometer, the IRDye 800CW, Alexa Fluor 488, and Alexa Fluor
647 contents were determined as 126.94 ± 0.75, 145.88 ±
3.1, and 85.13 ± 1.49 μmol/g, respectively.
Micelle Characterization
The n class="Chemical">pyrenepan>-based fluorescence
probe method was used to determinpan>e the CMC of pan> class="Chemical">ZSJ-0228. For sample
preparation, a stock solution of pyrene (0.02 mg/mL, in acetone) was
added to a 96-well plate. After adding the aqueous solution of n class="Chemical">ZSJ-0228
at different concentrations to the wells, the plate was left at room
temperature for 2 h to allow acetone evaporation. The final pyrene
concentration was maintained at 0.6 μM, which is slightly below
its water solubility at room temperature. After transferring to a
quartz 96-well plate, the fluorescence intensity was measured using
a fluorescence microplate spectrofluorometer. The excitation wavelength
was set at 334 nm, and the emission wavelength at 373 nm (I1) and
384 nm (I3). The ratio of fluorescence intensity I1/I3 was plotted
against prodrug concentration to obtain the CMC value.[70]
The hydrodynamic diameter (Dh), ζ-potepan class="Chemical">ntial, and PDIs of micelles were determinpan>ed
with a Malvern Zetasizer class="Chemical">pan> class="Chemical">Nano-ZS at 25 °C at a 90° angle
in triplicate. ZSJ-0228 was dissolved in ddH2O at designed
concentrations, filtered through a 20 μm syringe filter, and
vacuumed in the desiccator for 10 min to eliminate air bubbles before
measurement. Data were analyzed by the Zetasizer software version
7.12.
The concepan class="Chemical">ntration and the size distribution of micelles
were determinpan>ed
usinpan>g anpan> pan> class="Chemical">NS300 NanoSight with a low-volume flow cell plate and a 405
nm laser. The micelle solutions were pumped into the chamber using
the installed syringe pump. The measurements were conducted at room
temperature, replicated five times, and further analyzed using the
NTA analytical software, version 3.2. Each video sequence was captured
over 60 s.
To understapan class="Chemical">nd the micelle morphology, TEM was used
to visualize
pan> class="Species">micelles at 80 kV. For sample preparation, one drop of the ZSJ-0228micelle solution (2 mg/mL) was deposited on the Formvar/silicone monoxide-coated
200 mesh copper grid surface and then dried overnight at room temperature.
The analysis was performed at 25 °C.
To quantify the papan class="Chemical">n class="Chemical">dexamethasone
conclass="Chemical">pan>tent inpan> pan> class="Chemical">ZSJ-0228, the prodrug
was dissolved in HCl (0.5 mL, 0.1 N) and stirred overnight at room
temperature. The sample (50 μL) was withdrawn and neutralized
by addition of n class="Chemical">NaOH (50 μL, 0.1 N), then diluted in acetonitrile
(0.9 mL). The sample (in triplet) was analyzed using an Agilent 1100
HPLC system equipped with a reverse phase C18 column (Phenomenex,
Bondclone H16-441537-C18, 300 × 3.9 mm, 10 μm).
Mobile phase: acetonitrile/water, 30:70; detection wavelength, 240
nm; flow rate, 1 mL/min; injection volume, 10 μL. The dexamethasone
content in ZSJ-0228 was then calculated based on the HPLC analysis
result.
To understapan class="Chemical">nd the impact of pH values on the release
of dexamethasone
from pan> class="Chemical">ZSJ-0228, the prodrug (3 mg/mL) was dissolved in acetate buffers
(pH = 4.5, 5.0, 6.5, and 7.4). Pluronic F127 (1 w/v % of buffer) was
added as a surfactant to create the “sink” condition.[71] The micelle solutions were incubated at 37 °C
with gentle agitation (60 r/min). At selected time points, the sampled
releasing solution (0.1 mL) was neutralized with NaOH (0.1 N) and
analyzed with HPLC in triplicate. The experiment was repeated three
times in each pH buffer. The accumulative release of dexamethasone
from ZSJ-0228micelles was calculated according to the following equation,
where 2 mL refers to the total volume of release medium, 0.1 mL is
the sampling volume, and C refers to
the concentration of dexamethasone at sampling time point n.
Evaluation of ZSJ-0228’s Therapeutic
Efficacy on NZB/W
F1 Mice
Beginpan class="Chemical">ning at 20 weeks of age, NZB/pan> class="Gene">W F1 female mice
(Jackson Laboratories, Bar Harbor, ME, USA) were randomized into three
groups (saline control, Dex, and ZSJ-0228). The urine protein level
was monitored weekly using Albustix reagent strips (Siemens Healthineers,
Erlangen, Germany). Only mice with established nephritis, as evidenced
by sustained albuminuria (≥100 mg/dL) over 2 weeks, were enrolled
in the study. ZSJ-0228 treatment (106 mg/kg, containing 28 mg/kg of
dexamethasone, n = 10) and saline (n = 12) were administered as a monthly i.v. injection. A total of
two injections of ZSJ-0228 were given during the two-month treatment
period. The Dex treatment (dexamethasone 21-phosphate disodium, 1.32
mg/kg, containing 1.00 mg/kg of dexamethasone, n =
11) was given as a daily i.v. injection. All treatments continued
for 8 weeks. The body weight and proteinuria level of the animals
were monitored weekly. Every 4 weeks, peripheral blood was sampled
from the saphenous vein for serum analyses. Mice that developed severe
proteinuria (≥2000 mg/dL) or showed signs of distress (e.g., reduced mobility, weight loss >20%,
edema, unkempt appearance) were sacrificed immediately. At two months
post-treatment initiation, all surviving mice were euthanized by CO2 asphyxiation, with major tissues and organs isolated, weighed,
and processed at necropsy. The animal procedures were approved by
the Institutional Animal Care and Use Committee (IACUC) of the University
of Nebraska Medical Center (UNMC).
Histological Evaluation
n class="Chemical">Paraffinpan> sections of the pan> class="Species">mouse
kidneys were deparaffinized, rehydrated, washed with n class="Chemical">water, and stained
with periodic acid–Schiff for evidence of glomerulonephritis
by light microscopy. Glomerulonephritis was assessed by a pathologist
(K.W.F.) using a semiquantitative scale as described previously.[20] Briefly, a score of 0 represents healthy condition;
a score of 1 represents mild focal disease; a score of 2 represents
moderate focal disease; scores of 3 and 4 represent diffuse disease
(namely, severe glomerulonephritis). Scores of 0, 1, 2, 3, and 4 indicate
that 0, 1–19, 20–50, 51–75, and >75% of the
glomeruli
were affected. Fifty glomeruli per mouse were evaluated.
Immune
complexes apan class="Chemical">nd macrophage infiltration were observed via immunohistochemistry. Rabbit anpan>ti-pan> class="Species">mouse IgG (Abcam) and rabbit anti-mouseF4/80IgG (Abcam) were used as the primary antibodies. After deparaffinization
and rehydration, a rabbit-specific HRP/DAB (ABC) detection IHC kit
(Abcam) was applied. Briefly, the slides were first incubated in pH
6.0 citrate buffer (0.1 M), washed, and then incubated in H2O2. The slides were blocked and incubated with the primary
antibodies. Antibody binding was visualized using the DAB chromogen.
The staining intensity (represented as arbitrary gray units) of 50
glomeruli per mouse was quantified using Zeiss AxioVision software
(version 4.6.3.0). Another set of slides stained for immune complexes
and macrophages were counterstained with hematoxylin; these slides
were used for illustration purposes only.
For confocal micpapan class="Chemical">n class="Chemical">roscopy,
kidnclass="Chemical">pan>eys were dissected, fixed, dehydrated,
and frozen inpan> pan> class="Chemical">2-methylbutane in a dry ice bath. Sections (20 μm)
were cut on a Bright’s cryostat (Bright Instrument Co., Huntingdon,
UK), thaw-mounted onto slides, and stored at −80 °C until
stained. The sections were stained with anti-mouseCD11b (Santa Cruz),
n class="Gene">CD 133 (Abcam), and CD146 (Abcam) and then incubated with Alexa Fluor
488-labeled secondary antibody (Thermo Fisher Scientific, Bremen).
Cells were visualized via immunofluorescence under
a Zeiss LSM 800 confocal microscope. The images were processed using
the Carl Zeiss Zen 2.3 software.
Analysis of Bone Quality
Femoral bone quality was apan class="Chemical">nalyzed
using a Skyscan 1172 micro-CT system. The scanning parameters were
set as follows: voltage 48 kV, current 187 μA, exposure time
620 ms, resolution 6.07 μm, and aluminum filter 0.5 mm. Three-dimensional
reconstructions were achieved usinpan>g the pan> class="Chemical">NRecon and DataViewer software
(Bruker micro-CT). A consistent polygonal region of interest of trabecular
bone at the distal femur, from 20 slices (0.25 mm) to 100 slices proximal
(1.25 mm) to the growth plate, was selected for bone quality analysis.
The mean bone mineral density, bone volume/tissue volume, trabecular
number, and thickness were quantified using the Bruker CTAn software.
Analysis of Serum Immunoglobulin and Autoantibody Levels
Serum levels of immunoglobulipan class="Chemical">n were assessed via an enzyme-linked immunosorbent assay (ELISA). Total serum IgG levels
were measured usinpan>g a commercial ELISA kit (Innovation Research, pan> class="Chemical">Novi,
MI, USA). Serum anti-dsDNA IgG levels were determined by ELISA (Alpha
Diagnostic, San Antonio, TX, USA) as described previously.[34]
Near-Infrared Optical Imaging
After
the n class="Disease">proteinuriapan>
was established, n class="Chemical">NZB/n class="Gene">W F1 mice and NZW mice were given ZSJ-0228-IRDye
(IRDye 800 CW dose of 148 nmol/kg, dexamethasone equivalent dose of
28 mg/kg) via tail vein injection. At selected time
points (1 and 4 days postinjection), the mice were euthanized and
perfused with saline. All major organs (i.e., heart, lungs, liver, spleen, kidneys, and adrenal gland)
were isolated and imaged using a LI-COR Pearl Impulse imager. The
two imaging time points (1 and 4 days postinjection) were selected
to avoid the early dynamic distribution phase when the prodrug concentrations
in different organs and tissues change dramatically within a short
period of time. All images were collected under the channel of 800
nm with the same resolution (170 μm) settings.
Flow Cytometry
Analysis
After the n class="Disease">proteinuriapan> was established,
n class="Chemical">NZB/n class="Gene">W F1 mice (n = 3 for each time point) and NZW
mice (n = 5 for each time point) were given ZSJ-0228-AF647
(Alexa Fluor 647 dose of 300 nmol/kg, dexamethasone equivalent dose
of 28 mg/kg) via tail vein injection. The animals
were euthanized and perfused at 1 and 4 days postadministration. Blood,
bone marrow, heart, lungs, kidneys, liver, and spleen were harvested
and processed to obtain single-cell suspensions. These cells were
marked by the following antibodies: BV711-labeled anti-mouseCD11b,
BV786-labeled anti-mouseCD3e, BUV395-labeled anti-mouseNK1.1, PerCP-Cy5.5
labeled-CD146, PE-Cy7-labeled CD19, BV510-labeled anti-mouseCD11c,
APC-eFluor 780-labeled anti-mouseLy-6G (BD Biosciences), APC-labeled
anti-mouseF4/80 and PE-labeled anti-mouseCD326 (eBioscience Inc.).
The cells were then analyzed using a BD LSR II Green flow cytometer
with FlowJo software.
Cell Culture Study
The cell viability
was quantified
usipan class="Chemical">ng the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(pan> class="Chemical">MTT) cell proliferation assay from ATCC. Briefly, human proximal
tubule epithelial (HK-2) cells[28] were seeded
in 96-well plates (1 × 104 cells/well) overnight and
treated with different concentrations of ZSJ-0228 (0.01–2000
μM dexamethasone equivalent), Dex (0.01–2000 μM
dexamethasone equivalent), and mPEG (0.005–1000 μM ZSJ-0228
equivalent). PBS was used as a control. The cells were incubated for
24, 48, and 72 h. At each time point, 10 μL of MTT was added
to each well and incubated at 37 °C for 4 h. MTT detergent reagent
(100 μL/well) was added to dissolve the insoluble formazan,
then incubated for 2 h in the dark at room temperature. A microplate
reader (Fisherbrand accuSkan FC filter-based microplate photometer)
was then used to measure the UV absorbance at 570 and 660 nm (reference
wavelength). The cell viability results are presented as the percentage
of PBS control (100%).
To investigate the ipan class="Chemical">nternalization of
Alexa Fluor 488-labeled pan> class="Chemical">ZSJ-0228 (ZSJ-0228-AF488), HK-2 cells were
seeded on a 24-well plastic plate and incubated overnight with LPS
(10 μg/mL) to mimic the lupus nephritis condition. ZSJ-0228-AF488
(final concentration of 200 μg/mL) was added to LPS-stimulated
HK-2 cells. The HK-2 cells without LPS treatment were used as controls.
At 1, 4, 24, 48, and 72 h time intervals, the cells were rinsed, fixed,
and analyzed using a flow cytometer.
For the subcellular localization
studies, papan class="Chemical">n class="Gene">HK-2 cells were cultured
overnclass="Chemical">pan>ight with n class="Disease">LPS and then incubated with n class="Chemical">ZSJ-0228-AF488 (200 μg/mL)
for 24 h on glass coverslips in the 24-well plates. Cells were rinsed
and incubated with LysoTracker DND-99 (Invitrogen, 75 nM) for 3 h.
Then the cells were rinsed, stained with DAPI, fixed, mounted, and
observed under a confocal microscope.
Statistical Analysis
IBM SPSS 17.0 (IBM n class="Gene">Corpan>p., Armonk,
pan> class="Chemical">NY, USA) or SAS 9.4 (SAS Institute, Cary, NC, USA) were used for statistical
analyses in this study. Continuous outcomes were compared among ≥3
groups using the analysis of variance (An class="Chemical">NOVA) or Kruskal–Wallis
tests. Tukey’s post hoct test or the Mann–Whitney U test with the Bonferroni method
for multiple comparisons was used for the pairwise comparisons. The
outcome of survival rate was compared among three groups using the
log-rank test. Multiple comparisons were corrected with Bonferroni’s
method. The binary outcome of having proteinuria reading values of
2 and above at the 8-week time point was compared among the three
groups using Fisher’s exact test. Bonferroni’s pairwise
comparison was used to control for multiple testing. The generalized
estimating equation (GEE) method was used to model the repeated measurements
of a continuous outcome to account for the correlation within the
subject. The GEE method is robust to the misspecification of the distribution
of the outcome. Tukey’s pairwise comparison between groups
was performed to control for multiple testing in the GEE model.
Authors: Jai Radhakrishnan; Dimitrios-Anestis Moutzouris; Ellen M Ginzler; Neil Solomons; Ilias I Siempos; Gerald B Appel Journal: Kidney Int Date: 2009-11-04 Impact factor: 10.612
Authors: Fang Yuan; Dana E Tabor; Richard K Nelson; Hongjiang Yuan; Yijia Zhang; Jenny Nuxoll; Kimberly K Bynoté; Subodh M Lele; Dong Wang; Karen A Gould Journal: PLoS One Date: 2013-11-28 Impact factor: 3.240
Authors: Lingdong Quan; Yijia Zhang; Bart J Crielaard; Anand Dusad; Subodh M Lele; Cristianne J F Rijcken; Josbert M Metselaar; Hana Kostková; Tomáš Etrych; Karel Ulbrich; Fabian Kiessling; Ted R Mikuls; Wim E Hennink; Gert Storm; Twan Lammers; Dong Wang Journal: ACS Nano Date: 2013-12-27 Impact factor: 15.881
Authors: Zhenshan Jia; Gang Zhao; Xin Wei; Dexuan Kong; Yuanyuan Sun; You Zhou; Subodh M Lele; Edward V Fehringer; Kevin L Garvin; Steven R Goldring; Dong Wang Journal: J Control Release Date: 2020-05-20 Impact factor: 9.776
Authors: Sarika Chaudhari; Grace S Pham; Calvin D Brooks; Viet Q Dinh; Cassandra M Young-Stubbs; Caroline G Shimoura; Keisa W Mathis Journal: Front Physiol Date: 2022-06-13 Impact factor: 4.755
Authors: Zhifeng Zhao; Zhenshan Jia; Kirk W Foster; Xin Wei; Fangfang Qiao; Haochen Jiang; Yan Jin; Guojuan Li; Ningrong Chen; Gang Zhao; Geoffrey M Thiele; Jennifer L Medlin; James R O'Dell; Dong Wang Journal: Nanomedicine Date: 2020-09-24 Impact factor: 5.307
Scheme 1
Scheme 2
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11