Neuroprotection by chitosan nanoparticles in oxidative stress-mediated injury
Chen et al. BMC Res Notes
Neuroprotection by chitosan nanoparticles in oxidative stress-mediated injury
Bojun Chen 0 2
Jianming Li 0
Richard Ben Borgens 0 1
0 Center for Paralysis Research, Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University , 408 S. University St., West Lafayette, IN 47907 , USA
1 Weldon School of Biomedical Engineering, Purdue University , 206 S Martin Jischke Dr., West Lafayette, IN 47907 , USA
2 University of Southern Indiana , 8600 University Blvd, Evansville, IN 47712 , USA
Objective: Oxidative stress is a critical component of nervous system secondary injury. Oxidative stress produces toxic chemical byproducts including reactive aldehydes that traverse intact membranes and attack neighboring healthy cells. This secondary damage often leads to further patho-biochemical cascades that exacerbate the original insult. In this work, we investigate the therapeutic effects of chitosan nanoparticles on cell cultures exposed to oxidative stress. Results: We found chitosan nanoparticles can rescue BV-2 glial cells from death, but only for cells undergoing necrosis. Necrosis occurred when cultures were challenged with high concentrations of H2O2 (> 110 μM) whereas a slow and progressive loss of cultures was observed in more dilute (50-100 μM) peroxide applications. In the latter case, the primary mode of cell death was apoptosis. These studies revealed that while rescue of H2O2 challenged cultures was achieved for necrotic cell death, no such sparing was observed in apoptotic cells. Based on the current and cumulative data regarding the membrane fusogenic properties of chitosan, we conclude that chitosan neuroprotection arises from its membrane sealing effects. Consistent with this hypothesis is the observation that apoptotic cells did not exhibit early stage membrane damage. These in vitro results elucidate mechanisms by which membrane fusogens may provide therapeutic benefit.
Chitosan; Nanoparticles; Oxidative stress; Neuroprotection
Oxidative stress caused by reactive oxygen species
(ROS) plays a key role in several neurodegenerative
diseases as well as secondary injury in the central
nervous system. ROS are highly toxic and can damage many
biological molecules, including lipids, proteins, and/or
nucleic acids. ROS can react with cell membrane lipids,
leading to the initiation of lipid peroxidation (LPO) and
increased membrane permeability [
]. LPO can in
turn, generate additional toxic species such as aldehydes
(4-hydroxynonenal and acrolein).
The un-regulated generation of H2O2 is a well-known
source of oxidative stress. H2O2 is the intermediate
product in the conversion of O·2− into H2O in the electron
transport chain during mitochondria oxidative
phosphorylation. Disruption of this equilibrium via cell injury can
cause activated oxygen byproducts (O·2− and H2O2) and
overwhelm endogenous antioxidants such as superoxide
dismutase, catalase, glutathione peroxidase, vitamin E
and glutathione .
We previously showed chitosan based nanoparticles
synthesized with and without a drug rescued PC-12 cells
in an acrolein cell death model [
]. The putative mode
of cell preservation by chitosan was restoration of cell
membrane integrity. Recovery of conduction was also
demonstrated with chitosan in guinea pigs subjected to
spinal crush . In this work, we further investigate the
neuroprotective properties of chitosan nanoparticles
on BV-2 rat microglia cells challenged by H2O2. Similar
to prior acrolein studies, this ROS injury model aims to
mimic the biochemical mechanisms associated with CNS
The procedures and analysis of chitosan nanoparticles
have been detailed previously [
]. Briefly, ionic gelation
between chitosan polymer (200 kDa) and dextran
sulfate polymer (DS) or sodium tripolyphosphate (TPP)
polyanion was used. Two types of chitosan nanoparticles
(chitosan-DS nanoparticles (~ 10 kDa) and chitosan-TPP
nanoparticles) were synthesized. For technical reasons
chitosan-DS nanoparticles (Chi-DSNPs) were employed
in this study. Briefly, 0.1% chitosan was dissolved in 1%
acetic acid and mixed for 12–18 h. 0.1% DS was
prepared in DI water and filtered through 0.45 μm syringe
filters. The DS solution was added drop-wise to the
chitosan solution with continuous stirring for 1 h. The
volume ratios for Chi-DSNPs were as follows: 5:3, 5:5, 5:8.5.
During the DS-chitosan formation, the solution clouded
when the volume ratio was above 5:3, indicating presence
of nanoparticles. Following synthesis, the Chi-DSNPs
were purified in 300 kDa dialysis tubing placed in DI
water with stirring. The nanoparticle solutions were kept
in 4 °C before use.
The morphology of ChiNPs were imaged via negative
staining TEM. Briefly, one drop of Chi-NP solution was
placed on a carbon grid and allowed to settle for 2 min.
The grid was swished through a 2% uranyl acetate stain
and the excess liquid removed. Samples were mounted
and imaged using a Phillips CM-100 TEM operated at
100 kV with a 200 μm condenser aperture and 70 μm
Chi‑DSNPS on BV‑2 proliferation and viability
BV-2 mouse microglia obtained via Dr. Jau-Shyong Hong
and Mrs. Belinda C. Wilson of NIH neuropharmacology
group were maintained in DMEM supplemented with
0.044 M sodium bicarbonate, 10% fetal bovine serum
and 100 U/ml penicillin and 100 μg/ml streptomycin. The
cells were cultured in a 5% CO2 and 95% O2 incubator at
37 °C. 0.25 × 105 cells using a 75 cm2 flask. For
proliferation measurements in response to Chi-DSNPs, BV-2 cells
were seeded at a density of 1 × 104 cells/well in a 96-well
plate. After overnight incubation, the cell medium was
replaced with diluted NP solutions at a concentration
of 0, 0.1, 0.2, 0.5 mg/ml, at a volume of 100 μl. For H2O2
challenge, the cell medium was replaced with H2O2 at 0,
50, 100, 200, and 300 μM for 20 h. In these experiments,
cell proliferation was measured by using a WST-assay
(Abcam) per manufacturer’s protocol and wells read with
a plate reader at 450 nm. Four experiments were
conducted in quadruplicate.
To measure BV-2 post-peroxide exposure viability, cells
were seeded at 1 × 105/well in a 12-well plate. After
overnight incubation, the medium was replaced with a
buffered H2O2 solution at 0 (control), 50, 100, and 5500 μM.
Cells were imaged using an environmentally controlled
Olympus IX81 microscope and proliferation tracked
every 30 min for 20 h.
Mode of cell death
BV-2 cells were seeded at 1 × 105/well in 12 well plates.
Cells were exposed to H2O2 at 0 (control), 50, 100, and
5500 μM to induce cell death. The adherent cells were
trypsinized gently and washed with phosphate buffer
solution. Cells were incubated with 5 μl annexin V-FITC
(Abcam) and 5 μl (50 mg/ml) of propidium iodide
(Sigma) for 5 min at 25 °C. Cells were centrifuged gently
and re-suspend in 200 μl binding buffer. 100 μl cell
suspensions were placed on a glass slide, coverslip mounted
and imaged using an Olympus IX81 at incubation times
of 0 and 4 h for H2O2 (5500 μM) and at time 0 and 20 h
for H2O2 (0, 50, 100 μM) with a dual filter set for FITC/
Chi‑DSNPs and neuroprotection
To assess the neuroprotective effects Chi-DSNPs, a
concentration of 5500 μM H2O2 was chosen to induce BV-2
cell death. Here, BV-2 cells were seeded at a density of
0.5 × 105 cells/well in a 24-well plate and cultured
overnight. Afterwards, 10 μl of Chi-DSNPs at 1 mg/ml was
administered at 0 and 15 min after H2O2 addition. Cell
viability was measured by Trypan blue. BV-2 cells were
also pre-incubated with 0.2 mg/ml Chi-DSNPs
(pre-filtered with 1.2 or 5 μm syringe filter) for 4 h and
subsequently exposed to 50 μM H2O2 for an additional 20 h.
No treatment controls were BV-2 cells not exposed
to H2O2, whereas injured controls were cells exposed
to H2O2 but no nanoparticles were applied. Following
20 h culture, the WST-1 assay was used to quantify cell
All data were represented as mean ± standard
deviation. Statistical analysis was conducted using
oneway ANOVA and a Tukey–Kramer post hoc test. A P
value ≤ 0.05 was considered statistically significant.
The bare Chi-NPs appeared as dark clustered spheres
during TEM processing (Additional file 1: Figure S1).
Larger globular CNPs were about 100 nm in diameter,
while the majority of the clusters were 50 nm or smaller.
Storage condition tests showed that this globular shape
was maintained even after 2 weeks of air drying (data
not shown). Dose–response characteristics of BV-2 cells
exposed to Chi-DSNPs was assessed with WST-1. Results
showed cell proliferation within 20 h (normalized to
0 mg/ml) was not affected significantly by Chi-DSNPS up
to 0.5 mg/ml (P > 0.05, Additional file 1: Figure S1).
Time and dose dependent inhibition of cell proliferation
induced by H2O2
H2O2 reduced BV-2 cell proliferation within the initial
20 h when assessed with WST-1 (Fig. 1). At H2O2
concentration > 200 μM, no difference was detected with WST
assay as death saturated the populations. Cells at H2O2
challenge time points of 0, 2.5 h, and 18 h were selected
to evaluate changes to cell morphology. In the uninjured
(0 μM H2O2) control groups, (Fig. 1A–C), cells appeared
adherent with elongated processes and normal
proliferation. In 50 μM H2O2 group (D–F), cell morphology was
distorted after only 4 h. In (F), multiple dead cells formed
clumps and altogether appeared as non-viable cultures.
The surface of these cells was very irregular with
globular inclusions. In the 5500 μM hydrogen peroxide group
(G–I), cells started retracting their processes after only
1 h incubation. Cell blebs were formed and the swelling
of the cytoplasm was observed (data not shown). These
cells later darkened, indicating near or actual cell death.
Multiple small bright spots were detected in single dead
cells, suggesting breaches in the cell membranes (I).
Mode of cell death was determined PI and
annexinFITC stains. Photomicrographs Fig. 2d–f show BV-2 cells
treated with 5500 μM H2O2 possessed bright red (PI)
nuclei and small evidence of FITC on their membranes
vs control. We did not detect any cells stained only with
FITC. Interestingly, the treatment of 50 μM (b) and
100 μM (c) H2O2 resulted in some staining with
annexinV/PI, but also led to cells stained only with FITC. This
suggests that a long exposure to a low H2O2 dose induced
mostly apoptotic cell death, with only minor necrosis.
Chi‑DSNPs on BV‑2 viability after H2O2 exposure
BV-2 cell viability after the administration of Chi-DSNPs
at 0 and 15 min after exposure to 5500 μM H2O2 was
measured by Trypan blue. Results (Fig. 3) showed at
a 2.5 h exposure time, 5500 μM H2O2 induced almost
60% cell death vs untreated control groups (P < 0.001)
whereas 5 μm-filtered Chi-DSNPs preserved cells by 20%
(P < 0.01). No difference in the timing of CHI-DSNPs
(0 or 15 min) application was detected. Additionally,
a 30 min delayed administration of Chi-DSNPs did not
provide further beneficial effect (data not shown). At
low dose, WST-1 was used to determine the protective
effects of Chi-DSNPs on H2O2 challenge (Fig. 3b).
Findings show a 20% decrease in cell proliferation after a 20 h
administration of 50 μM H2O2 (P > 0.05). Pre-treating
the cells with either 1.2 or 5 μm filtered Chi-DSNPs at
0.2 mg/ml for 4 h prior to H2O2 challenge did not
statistically improve cell proliferation.
Chitosan is a commonly used polymer in biomaterials
research due to its good biodegradability,
biocompatibility and accessibility for surface modification [
Previously, we showed chitosan nanoparticles exhibited
neuroprotective effects in an acrolein-challenged PC-12
cell model . In this work, we extend the line of
investigation to BV-2 cells, an immortalized rat microglial line
that emulates the characteristics of primary microglia,
displaying similar inflammatory response and phagocytic
]. H2O2 was used as the challenge molecule
since it is a common oxidative stressor for modeling
many neurodegenerative diseases. For instance, Liu et al.
] reported a significant elevation of intracellular level of
H2O2 30 min after a weight drop induced SCI. These high
levels of H2O2 were maintained for over 11 h. H2O2 was
suggested to arise from O·2−. The post-injury activation
and sustained increase of H2O2 indicate that it was not
just an immediate response to SCI, and implicate H2O2 in
secondary injury processes associated with SCI [
To mimic the acute release of H2O2 post SCI injury, we
first constructed the H2O2 toxicity profile with BV-2 cells
at exposure ranges estimated in vivo or in other
experimental preparations [
]. The experimental results
show short term exposure to H2O2 (< 50 μM) suppressed
cell proliferation. Further assessment via
morphological analysis and with annexin-V/PI staining (reviewed
]) highlight differences in mode of cell death. Acute
exposure to high peroxide levels (5500 μM) induced cell
membrane damage and rapid necrosis in less than 2.5 h
whereas low levels of peroxide caused mostly apoptosis,
which was evident in the annexin V staining. This data is
not unexpected, and similar to other instances of
ROSSCI induced cell death, which is usually a combination of
necrosis and apoptosis [
]. Administration of
chitosan nanoparticles showed chitosan nanoparticles
protected microglia cells challenged with 5500 μM hydrogen
peroxide (Fig. 3) for 15 min. However, no improvement
in cell proliferation was observed between control and
the chitosan nanoparticle group was found when BV-2
cells were pre-treated with nanoparticles and
subsequently exposed to 50 μM hydrogen peroxide for 20 h.
The chitosan nanoparticles themselves were well
tolerated by the BV-2 cells based on proliferation assays.
The putative neuroprotective mechanism for
chitosan is sealing of damaged cell membranes in a manner
similar to fusogens such as polyethylene glycol [
Therefore, it is consistent to expect necrotic cells to be
sensitive to chitosan rescue. In contrast, low dosages of
peroxide (50 μM) caused cellular apoptosis—a process
that is biochemically driven, primarily irreversible and
does not involve membrane damage at the onset. This
was confirmed both morphologically and via annexin-V
staining. For apoptotic cells, chitosan NPs had no
therapeutic impact. While some studies also report
anti-oxidative properties of chitosan, especially if pre-incubated
with cells, such investigations are impractical outside of
in vitro cultures and may not reveal useful insights into
disease treatment [
]. Again, our current findings
also suggest pre-treatment with chitosan NPs does not
have a meaningful effect on low-dose H2O2 challenge.
Thus, we conclude that neuroprotection by chitosan
nanoparticles is largely due to a physical sealing of cell
membrane breaches, an observation that has been
corroborated by our prior work in chitosan, poloxamers,
poloxamines, and PEG [
3, 4, 6, 19–24
]. Due to the
versatility of chitosan nanoparticles as potential drug delivery
reservoirs/vehicles, these preliminary results offer
support for further therapeutic investigations.
This work was conducted with cell cultures and it
is unknown if the results are applicable in vivo or if
there may be longer-term benefits from nanoparticle
Additional file 1. Chitosan nanoparticles. Transmission electron
micrographs of chitosan nanoparticles. Most particles were 100 nm or less in
diameter. Corresponding table shows Chi-DSNPs did not significantly
inhibit cell proliferation after 20 h incubation at different concentrations
(0, 0.1, 0.2, 0.5 mg/ml).
ROS: reactive oxygen species; LPO: lipid peroxidation; NPs: nanoparticles; DS:
dextran sulfate; TPP: tripolyphosphate; Chi-DSNPs: chitosan-DS nanoparticles;
PS: phosphatidylserine; PI: propidium iodide.
BC and JL drafted the manuscript and analyzed the data. BC designed the
experiments. BC performed the experiments. RBB is the Principle Investigator
and Director of the CPR and is responsible for all elements of the research. All
authors read and approved the final manuscript.
We are grateful for the donation of the BV-2 cells by Dr. Jau-Shyong (John)
Hong and Mrs. Belinda C. Wilson (NIH, neuropharmacology group). We
appreciate the excellent illustrations and graphics by Michel Schweinsberg, and the
administrative assistance of Jennifer Danaher for manuscript preparation.
The authors declare they have no competing interests. There is no competing
interests of any sort in the reporting of these data relative to any author.
Availability of data and materials
All data generated or analyzed during this study are included in this published
Consent to publish
Ethics approval and consent to participate
This research was supported by the General Funds of the Center for Paralysis
Research (State of Indiana HB 1440), and a generous endowment from Mrs.
Mari Hulman George.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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