Bacterial magnetosomes as an efficient gene delivery platform for cancer theranostics
Dai et al. Microb Cell Fact
Bacterial magnetosomes as an efficient gene delivery platform for cancer theranostics
Qinglei Dai 3
Ruimin Long 0 1 3
Shibin Wang 0 1 2 3
Ranjith Kumar Kankala 0 1 3
Jiaojiao Wang 4
Wei Jiang 4
Yuangang Liu 0 1 2 3
0 Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University , Xiamen 361021 , People's Republic of China
1 Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University , Xiamen 361021 , People's Republic of China
2 Institute of Pharmaceutical Engineering, Huaqiao University , Xiamen 361021 , People's Republic of China
3 College of Chemical Engineering, Huaqiao University , Xiamen 361021 , People's Republic of China
4 State Key Laboratories for Agrobiotechnology and College of Biological Sciences, China Agricultural University , Beijing 100094 , People's Republic of China
Background: Gene therapy has gained an increasing interest in its anti-tumor efficiency. However, numerous efforts are required to promote them to clinics. In this study, a novel and efficient delivery platform based on bacterial magnetosomes (BMs) were developed, and the efficiency of BMs in delivering small interfering ribonucleic acid (siRNA) as well as antiproliferative effects in vitro were investigated. Results: Initially, we optimized the nitrogen/phosphate ratio and the BMs/siRNA mass ratio as 20 and 1:2, respectively, to prepare the BMs-PEI-siRNA composites. Furthermore, the prepared nanoconjugates were systematically characterized. The dynamic light scattering measurements indicated that the particle size and the zeta potential of BMs-PEI-siRNA are 196.5 nm and 49.5 ± 3.77 mV, respectively, which are optimum for cell internalization. Moreover, the confocal laser scanning microscope observations showed that these composites were at a proximity to the nucleus and led to an effective silencing effect. BMs-PEI-siRNA composites efficiently inhibited the growth of HeLa cells in a dose-as well as time-dependent manner. Eventually, a dual stain assay using acridine orange/ethidium bromide, revealed that these nanocomposites induced late apoptosis in cancer cells. Conclusions: A novel and efficient gene delivery system based on BMs was successfully produced for cancer therapy, and these innovative carriers will potentially find widespread applications in the pharmaceutical field.
Bacterial magnetosomes; siRNA; Polyethylenimine; Gene therapy
Cancer is one of the leading causes of deaths,
accounting for millions of deaths annually. More often,
chemotherapy is the primarily advised therapeutic regimen after
surgery and/or radiation therapy to improve the survival
rate in patients with cancer. However, most of the
chemotherapeutic agents result in several adverse effects due to
their non-specific uptake by healthy cells, poor
bioavailability and multidrug resistance (MDR) attained by
cancer cells, among others [
]. In addition, these undesired
effects result in the lower therapeutic efficacy of
conventional chemotherapeutic agents. To this end, gene
therapy has shown a great potential in the treatment of many
cancers because of the ability of genes in eradicating the
hereditary diseases and replace the defective cell
]. Moreover, the small (or short)-interfering
ribonucleic acid (siRNA), often called as silencing RNA,
is a class of chemically synthesized double-stranded RNA
molecules with 19–23 nucleotides that can trigger the
silencing of homologous gene expression . Inspired by
this fact, the researchers have harnessed the siRNA for
various applications in biomedical field [
there has not been an anticipated success for their
exploration in clinics, due to various reasons such as lack of
stability in organism caused by ribonuclease (RNase)
degradation, poor cellular uptake, and endosomal trapping,
among others . To overcome these issues, a
wide-variety of non-viral vectors have been used to deliver siRNA,
including lipid, cationic polymers, and inorganic
nanoparticles, which are advantageous over virus-based vectors.
Out of various non-viral vectors available, cationic
polymers have gained the significant importance for the
efficient conveyance of genes due to their advantages
such as high stability in physiological fluids, controlled
release of active pharmaceutical agents (APIs) including
genes, large capacity of gene packing and ease of
structural modification to improve the transfection efficiency
and stability of genes. Polyethyleneimine (PEI) is one of
the most promising polymeric substrates explored for
the efficient delivery of DNA [
], siRNA [
]. More often, the genes with desired
nucleotide sequences are encapsulated in the PEI through
electrostatic interactions. In addition, the interesting
feature of PEI is that it condenses the anionic siRNA and
subsequently protects the siRNA from degradation by
]. Preceding research has indicated that PEI
can prevent exocytosis through the proton sponge effect,
which induces the flow of chloride ions and thereby
promotes the osmotic swelling of endosomes/lysosomes and
subsequently releases the APIs [
]. Despite its efficiency
in delivery, several factors of PEI significantly affect the
transfection efficiency and toxicity of genes such as the
molecular weight and structure of PEI. In addition,
advancements in the PEI-based design are still obligatory
to achieve the efficient delivery of genes by reducing the
adverse effects simultaneously.
Pharmaceutical carriers often use polymers,
dendrimers, micelles, liposomes, inorganic nanomaterials and so
], which can all be employed for drug
delivery system. Amongst inorganic nanomaterials, bacterial
magnetosomes (BMs) have shown a great potential as
a novel carrier due to their excellent biocompatibility,
high surface area to volume ratio,
superparamagnetism and abundant active sites on the membrane of BMs
]. More often, BMs are extracted from magnetotactic
bacteria with magnetic iron oxide or iron sulfide enclosed
by a natural phospholipid membrane [
endowed them with high biocompatibility. In a case, the
purified and sterilized BMs have shown that they were
non-toxic to mouse fibroblasts in vitro [
addition, the pyrogen test revealed that the administered
BMs (1 mg) resulted in no significant change in the body
temperature of rabbits. In another study, Sun et al.
evaluated the acute toxicity, immunotoxicity, and
cytotoxicity of BMs [
]. The blood examination results of BMs
have shown no significant effect compared to the control
group of rats. However, BMs showed a slight cytotoxicity
in H22, HL60, or EMT6 cell lines. In recent times, BMs
have gained an increasing interest for the delivery of
proteins, chemotherapy drugs and DNA [
Motivated by these facts, this study reports the
synthesis of gene delivery system based on BMs for the effective
delivery of siRNA by using PEI as a crosslinker (BMs–
PEI–siRNA). Furthermore, various techniques were used
to systematically characterize the nanocomposites such as
transmission electron microscope (TEM) for morphology,
DLS measurements for particle size distribution and
others. Furthermore, the stability and bioactivity studies were
performed to elucidate the integrity and the
anti-proliferative effects of the siRNA-loaded BMs, respectively.
Results and discussion
Characterization of BMs and its conjugates
From the TEM images (Fig. 1), it is evident that the
particle sizes and morphology of BMs are uniform and
the diameter was between 30 and 50 nm with the
hexagonal arrangement (Fig. 1a). It reflects the coated layer
over the BMs indicating that the distinct membrane was
composed of phospholipids and fatty acids [
Furthermore, the zeta potential value of BMs was measured by
adjusting the pH of the sample to physiological pH (7.4).
The surface charge of BMs was − 48.3 ± 2.6 mV, owing to
the existence of abundant lipids and amino groups [
However, the negative surface charge is countered with a
positively-charged PEI for the efficient loading of genes,
which condenses the anionic siRNA molecules, and
interacts with negatively-charged BMs via electrostatic
To optimize the formulation of BMs–PEI–siRNA, we
performed agarose gel electrophoresis of samples with
different N/P ratios concerning the nitrogen in PEI and
phosphates in siRNA respectively, and the results were
shown in Fig. 2a. The N/P ratio at which the undetected
fraction of free siRNA demonstrates that the siRNA
was successfully bound to PEI via electrostatic
interactions. The retardation efficiency was increased with the
increase in N/P ratio. Eventually, the optimized N/P ratio
was found above 8, and the samples of BMs–PEI–siRNA
composites were prepared at that ratio and systematically
characterized using various techniques.
The dynamic light scattering (DLS) measurements gave
the hydrodynamic mean diameters as well as zeta
potential values of BMs–PEI–siRNA composites. As shown
in Fig. 2c, e, the diameters of the synthesized
nanocomposites decreased with the increase of N/P ratios,
demonstrating that the addition of siRNA and PEI complex
resulted in the compact nanocomposites. Moreover, the
zeta potential values of respective nanocomposites were
in a positive range, which confirms the conjugation of
PEI. The hydrodynamic diameter and the positive
potential of BMs–PEI–siRNA formulation with N/P ratio 20
(200 nm) are optimum for the accumulation through
enhanced permeation and retention (EPR) effect and ease
of cellular internalization in the tumor [
Further, we investigated the concentration of BMs that
would be effective in the formation of BMs–PEI–siRNA
through agarose gel electrophoresis. The concentration of
BMs at which the gel devoid of free siRNA band indicates
that they have no significant effect on the
immobilization. Figure 2b shows the gel images of BMs–PEI–siRNA
composites with different BMs/siRNA weight ratios, at
which the bandwidth of BMs/siRNA weight ratio 1:2 was
found optimum. Moreover, the size was relatively small
(Fig. 2f ) and the potential was positive (Fig. 2d), at this
ratio, which was extremely beneficial for establishing the
interactions with the cell and subsequent internalization
siRNA is one of the most sensitive biomolecules in
the body, which suffer from certain limitations during
delivery such as short circulation times, reduced
therapeutic effects and others due to in vivo degradation [
In addition, a few factors such as serum proteins are
considered during the formulation of genes for cancer
theranostics. Herewith, we demonstrated the stability
of our design through various methods such as
decomplexation assay, enzyme stability assay, and others. In
heparin decomplexation assay, the siRNA dissociates
from the synthesized nanocomposites due to the stronger
interaction of the heparin with the nanocomposites. The
experiment was performed by mixing various
concentrations of heparin with the nanocomposites, and the
resultant siRNA in the supernatant was subjected to gel
electrophoresis. The results (Fig. 3a) indicated that the
siRNA was utterly dissociated from the nanocomposites
at a specific weight ratio of heparin to siRNA (10:1) after
incubating for 15 min. Figure 3b elucidates the stability
of siRNA in the presence of serum proteins. It is evident
that the naked siRNA was degraded rapidly in 50% FBS
for 60 min, while the fraction can be still observed in the
case of BMs–PEI–siRNA nanocomposites after 150 min
of exposure, demonstrating that the designed
nanocomposites offered significant protection to siRNA.
Eventually, the stability of siRNA in the presence of enzymes
was demonstrated by suspending the designed
nanocomposites in the presence of RNase A. The results showed
that the immobilization of siRNA in the PEI network on
BMs significantly reduced the degradation, which can
enhance the circulation time in vivo.
Cell viability assay
The anti-tumor efficacy of our novel BMs-based gene
delivery system was performed using CCK-8 assay in
the HeLa cell line. The experiments were designed such
that they represent both dose-dependent by treating
various doses and time-dependent cytotoxicity by
measuring the viability at different time points. In the
dosedependent assay, the viability of cells gradually decreased
with the increase in the concentration of
nanocomposites (Fig. 4a). Moreover, the inhibition rate of BMs–PEI–
siRNA (STAT 3) composites at a dose of 10 pmol was
significantly higher than that of siRNA alone
accounting for 70% of cell death. The inhibition effect of BMs–
PEI–siRNA (NC) was similar to that of BMs–PEI vector,
indicating that the cytotoxicity of siRNA was
sequencespecific. Based on these results, the nanoconjugates at
a concentration of 5 pmol were chosen as an optimized
dose for further investigations.
As shown in Fig. 4b, BMs–PEI, and BMs–PEI–siRNA
(NC) showed no apparent growth inhibition of cells
within the incubation times, indicating that the BMs–PEI
composites resulted in low toxicity. Cytotoxicity of BMs–
PEI–siRNA (STAT 3) sample showed the time-dependent
Fig. 2 Physical characterization of BMs–PEI–siRNA nanocomposites including the agarose gel electrophoresis results for optimization of
formulation. Images showing the gel electrophoresis results of BMs–PEI–siRNA composites a at different N/P ratios, 1–8: 0, 1, 2, 4, 8, 10, 16, 20; and b at
different BMs/siRNA weight ratios, 1–5: 1:5, 1:2, 1:1, 2:1, 5:1 along with free siRNA; zeta potential values of BMs–PEI–siRNA composites c at different
N/P ratios; and d at different BMs/siRNA mass ratios; hydrodynamic diameters of BMs–PEI–siRNA composites e at different N/P ratios, 1–4: 8, 10, 16,
20; f at different BMs/siRNA ratios, 1–5: 1:5, 1:2, 1:1, 2:1, 5:1
inhibitory effect on HeLa cells about 40% after 72 h of
incubation and was significantly higher compared to that
of siRNA treatment group of cells.
Cell apoptosis assay
To further assess the anti-tumor effect of BMs–PEI–
siRNA composites, the cell apoptosis of designed
sample was examined using acridine orange/ethidium
bromide (AO/EB) dual stain. As shown in Fig. 5, cells in
the negative control group were green in color
elucidating no apparent cell apoptosis. Comparatively, the cells
in siRNA, as well as BMs–PEI–siRNA treated groups
indicated more orange stained cells demonstrating that
the cells underwent early and late apoptosis. The results
were consistent with that of anti-tumor efficacy. Overall,
siRNA-loaded BMs–PEI delivery system not only
efficiently expressed the silencing effect of siRNA but also
induced the apoptosis compared to the naked siRNA.
Cellular uptake of BMs–PEI–siRNA nanocomposites
Indeed, cellular uptake of nanoparticles plays a crucial
role during the formulation of nano-delivery systems for
the efficient delivery of therapeutic cargo. However, the
positively-charged BMs–PEI nanoparticles are highly
suitable for delivering siRNA as they interact with the
negatively-charged cell membrane. To validate the
internalization of designed nanocarriers, we labeled FAM
to siRNA and tracked the presence of nanocomposites
using confocal laser scanning microscope (CLSM) in
HeLa cells after incubation for 0.5 and 6 h (Fig. 6).
Interestingly, the BMs–PEI–siRNA nanocomposites were at
the proximity of the nucleus in cells. In addition, some
characteristic changes associated with the apoptosis
such as chromatin condensation and nucleus shrinkage
were observed in the cells treated with BMs–PEI–siRNA
nanocomposites, indicating that the cells underwent
apoptosis. This phenomenon was different from the
results of cell uptake in most previous studies [
Preceding reports indicated that the RNA interference
(RNAi) occurs in the cytoplasm, while other studies have
revealed that potent RNAi expressed in the nucleus of
human cells . The reason for these contrast findings
might be the altered dynamics and distribution of siRNA
due to the presence of BMs, which promoted their
delivery close to the nucleus.
In summary, we designed a novel delivery system
based on siRNA-loaded BMs using cationic PEI as a
crosslinker. After improving the synthetic conditions,
the optimal BMs–PEI–siRNA nanocomposites have
shown an enhanced cellular uptake and exhibited serum
stability as well as enzymatic hydrolysis. These stable
nanocomposites resulted in more significant inhibitory
effects on HeLa cells. This delivery system takes
advantage of efficient delivery of siRNA into cancer cells, and
also provides an opportunity for the development of
various novel therapeutic strategies.
BMs extracted from Magnetospirillum gryphiswaldense
MSR-1 were presented kindly by professor Li Ying and
Jiang Wei (Department of Microbiology, China
Agricultural University). STAT 3 siRNA and siRNA (NC)
were purchased from GenePharma Co., Ltd. (Shanghai,
China), siRNA (NC) was used as the negative control of
STAT 3 siRNA without homology. Branched PEI (MW:
25 KD) was purchased from Sigma Aldrich (USA). The
cervical carcinoma cell line (HeLa cells), was obtained
from China Academy Typical Culture Preservation
Committee Cell Library (Shanghai, China). Cell culture
medium was composed of Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 10% fetal bovine
serum (FBS). The cells were incubated in humidified air
maintained at 37 °C with 5% CO2.
Preparation and characterization of BMs
The extraction process of BMs was performed by
following our reported procedure given below [
cells of M. gryphiswaldense MSR-1 were suspended in
phosphate buffered saline (PBS, 0.1 M, pH 7.4) and then
the cell membrane was disrupted by ultrasonication. The
cell debris was removed by magnetic adsorption, and
the process was repeated for about 20 times. The
resultant suspension of BMs was treated with DNase I for 2 h
at 37 °C. The BMs were then washed for about 20 times
and conserved at − 20 °C after being freeze-dried.
Further, the suspension of BMs was subjected to physical
The morphology of BMs was confirmed by
capturing images using TEM. The zeta potential and particle
size distribution of BMs were measured by Zetasizer
(ZEN3600, Malvern Instruments Ltd, UK).
Preparation of BMs–PEI–siRNA nanocomposites
The nanocomposites with different nitrogen of
PEI/phosphate of siRNA (N/P) ratios (N/P ratios were set as 0,
1, 2, 4, 8, 10, 16 and 20) were prepared by mixing a
certain amount of siRNA with PEI in diethyl pyrocarbonate
(DEPC) water, and fixed amounts of BMs were added
(BMs/siRNA mass ratio was 1:2), followed by vortexing
for 2 min and incubated for 25 min at room temperature.
The synthetic process of the composites was
demonstrated in Fig. 7.
Mixed with siRNA
Fig. 7 Schematic illustration showing the synthetic outline of BMs–
To obtain the optimal weight ratio of BMs to siRNA,
various amounts of BMs were added to the PEI/siRNA
(N/P = 20) complexes in DEPC water (weight ratios of
BMs to siRNA were set as 1:5, 1:2, 1:1, 2:1 and 5:1), and
incubated for 25 min to obtain BMs–PEI–siRNA
nanocomposites. The binding ability was estimated by agarose
gel retardation assay.
The agarose gel electrophoresis assay was performed to
estimate the encapsulation efficiency of siRNA in BMs–
PEI–siRNA nanocomposites. The resultant
nanocomposites were loaded on 0.8% (w/v) agarose gel containing
1% (v/v) Gel Stain in tris acetate EDTA (TAE) buffer, and
the gel was run at 70 V for 20 min. The gel image was
captured using UV transilluminator and a digital imaging
system (GIS2008, Tanon Science & Technology Co., Ltd,
The stability of designed nanocomposites was
determined by incubating them in various conditions
provided, which mimic the physiological fluids. One of them
was the heparin decomplexation assay. Heparin (heparin/
siRNA weight ratios: 2, 10, 25 and 100) was mixed with
BMs–PEI–siRNA and incubated for another 15 min at
room temperature. The resultants were subjected to
agarose gel electrophoresis (DYY-6C, Liuyi Biological
Technology Co., Ltd, China).
To determine the serum stability assay, naked siRNA
and BMs–PEI–siRNA nanocomposites were treated with
50% fetal bovine serum and incubated for 60, 60, 90, 120
and 150 min. At predetermined time intervals, heparin
was added to nanocomposites group, followed by
incubation for 15 min. All samples were loaded on 0.8% agarose
gel electrophoresis for retardation analysis.
Further, the enzyme stability assay was performed by
incubating naked siRNA and BMs–PEI–siRNA
nanocomposites individually with RNase A for 60, 60, 90, 120
and 150 min. The samples were then subjected to agarose
Cell viability assay
The cytotoxicity of the designed nanoconjugates was
measured using CCK-8 assay at a different siRNA
concentration (dose-dependent) and incubation times
(timedependent). HeLa cells were seeded into 96-well plates at
2 × 104 cells/well and incubated for proper cell
attachment. After 24 h of incubation, the cells were subjected
to treatment with siRNA, BMs–PEI, BMs–PEI–siRNA
(STAT 3) and BMs–PEI–siRNA (NC) (the contents
of siRNA were set as 2.5, 5, 7.5 and 10 pmol) in 100 μL
of serum-free DMEM for 6 h. The medium was then
replaced with 200 μL of DMEM containing 10% FBS
and incubated for further 48 h. At the end of the
incubation, 20 μL of CCK-8 reagent was added to each well
and further incubated for 2 h. Finally, the absorbance was
recorded by using a microplate reader at 450 nm
(Multiskan GO, Thermo Scientific Co., Ltd, USA).
Time-dependent assessment of cell viability was
performed as described above by incubating the cells with
samples [siRNA, BMs–PEI, BMs–PEI–siRNA (STAT 3)
and BMs–PEI–siRNA (NC) (the content of siRNA was
5 pmol)] at a different time periods 24, 48 and 72 h.
Cell apoptosis assay
To observe the cell apoptosis induced by BMs–PEI–
siRNA nanocomposites, HeLa cells were seeded at a
density of 1 × 105 cells/well in 24-well plates and
incubated for 24 h. Later, cells were treated with siRNA and
BMs–PEI–siRNA (the concentration of siRNA was
50 nM) suspended in 0.5 mL serum-free DMEM for 6 h
and then replaced with 1 mL DMEM containing 10%
FBS and incubated for 48 h. Subsequently, cells were
harvested and washed three times with PBS, then 25 μL
of cell suspension was stained with 1 μL of AO/EB dual
stain reagent for 2–3 min in the dark according to the
manufacturer’s instructions. The apoptotic cells were
analyzed by observing them under CLSM (Leica TCS
Cellular uptake study
HeLa cells were cultured on 35 mm glass-bottom dishes
at a density of 4 × 105 cells/dish and incubated for 24 h
for proper cell attachment. Cells were then treated with
FAM-labeled BMs–PEI–siRNA nanocomposites (the
concentration of siRNA was 50 nM) for 0.5 and 6 h. After
pirating the medium, the cells were washed thrice with
cold PBS and then the cells were fixed with formaldehyde
(4%) for 10 min, then washed and stained with DAPI. The
dishes were eventually observed under CLSM (Leica TCS
AO/EB: acridine orange/ethidium bromide; N/P: nitrogen/phosphate; MDR:
multidrug resistance; siRNA: small interfering RNA; RNase: ribonuclease; PEI:
polyethyleneimine; APIs: active pharmaceutical agents; DLS: dynamic light
scattering; TEM: transmission electron microscope; EPR: enhanced permeation
and retention; RNAi: RNA interference; PBS: phosphate buffered saline; BMs:
bacterial magnetosomes; BMs–PEI–siRNA: siRNA loaded BMs delivery system
by using polyethyleneimine as a crosslinker; DEPC: diethyl pyrocarbonate; TAE:
tris acetate EDTA; DMEM: Dulbecco’s Modified Eagle’s Medium; CLSM: confocal
laser scanning microscope.
QD and YL conceived and designed the experiments. QD performed the
experiment and compiled the manuscript. JW and WJ kindly presented the
nanomaterials and gave helpful advice during the experiments and
compilation of manuscripts. RL, RKK, and SW contributed to the data analysis and
technical notes. All authors read and approved the final manuscript.
We would like to thank the editor and reviewers for taking lots of time and
giving constructive suggestions.
The authors declare that they have no competing interests.
Availability of data and materials
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economic innovation and development project (16PYY007SF17), the Science
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