Mitochondria-targeting nanomedicine self-assembled from GSH-responsive paclitaxel-ss-berberine conjugate for synergetic cancer treatment with enhanced cytotoxicity

Article abstract of DOI:10.1016/j.jconrel.2019.12.011

Mitochondria play a fundamental role in plenty of cellular metabolic processes, and mitochondria-homing drug delivery is a promising and effective strategy for cancer treatment. Paclitaxel (PTX) is a broad-spectrum anticancer drug, but its therapeutic eff

Full text of DOI:10.1016/j.jconrel.2019.12.011

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Mitochondria-targeting nanomedicine self-assembled from GSH-
responsive paclitaxel-ss-berberine conjugate for synergetic cancer
treatment with enhanced cytotoxicity
Yu Cheng, Yuanhui Ji
PII:
S0168-3659(19)30729-1
DOI:
https://doi.org/10.1016/j.jconrel.2019.12.011
COREL 10054
Reference:
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
22 September 2019
3 December 2019
9 December 2019
Please cite this article as: Y. Cheng and Y. Ji, Mitochondria-targeting nanomedicine self-
assembled from GSH-responsive paclitaxel-ss-berberine conjugate for synergetic cancer
treatment with enhanced cytotoxicity, Journal of Controlled Release (2019),
https://doi.org/10.1016/j.jconrel.2019.12.011
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Mitochondria-targeting nanomedicine self-assembled from
GSH-responsive paclitaxel-ss-berberine conjugate for synergetic
cancer treatment with enhanced cytotoxicity
Yu Cheng, Yuanhui Ji*
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, School of Chemistry and Chemical Engineering, Southeast
University, Nanjing, 211189, P.R. China
* Corresponding author: Tel: +86-13951907361; Email: yuanhui.ji@seu.edu.cn; yuanhuijinj@163.com.
Abstract:
Mitochondria play a fundamental role in plenty of cellular metabolic processes, and
mitochondria-homing drug delivery is a promising and effective strategy for cancer treatment.
Paclitaxel (PTX) is a broad-spectrum anticancer drug, but its therapeutic effect is highly limited
due to the development of multidrug resistance. Berberine (BBR) can selectively accumulate in
tumor cell mitochondria and inhibit the growth of cancer cells with different biological action
mechanism from PTX. Here, these two “old” drug molecules, BBR and PTX were linked together
by a disulfide bond rope to construct GSH-responsible drug-drug conjugate (PTX-ss-BBR).
Molecular dynamics simulation results revealed that PTX-ss-BBR conjugate could be
self-assembled in water to form nanoparticles (PTX-ss-BBR NPs) forced by - stacking and
hydrophobic interactions and the average size of NPs was around 165 nm measured by DLS. The
better in vitro potency of PTX-ss-BBR NPs against A549 cells might be ascribed to the
simultaneous drug release and mitochondria-targeting delivery, which dissipated mitochondria
membrane potential, upregulated ROS levels in cancer cells, arrested cells in phase G2/M, elicited
apoptosis of cancer cells and inhibited the growth of tumors. Furthermore, PTX-ss-BBR NPs also
exerted efficacy better than or comparable to BBR on S. aureus and E. coli, which were closely
associated with the development of lung cancer. The synergistic effect of PTX and BBR enhanced
the treatment effect of conventional chemotherapy drugs against A549 cells.
Keywords: Paclitaxel, berberine, mitochondria-targeting, self-assembled nanomedicine
1. Introduction
Cancer is the primary cause of disease-related mortality all over the world. Therefore, scientists
and researchers spare no efforts to finding better approaches and solutions to fight against cancer
[1]. In the middle of varieties of treatment approaches of cancer, chemotherapeutics remains an
obbligato method. Paclitaxel (PTX), a broad-spectrum anticancer drug acting on microtubule and
many of its nano-formulations have been successfully employed in clinical therapy of ovarian
cancer, lung cancer, breast cancer, and so on [2,3]. However, the occurrence of multidrug

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resistance (MDR) has severely limited the clinical usage of PTX [4]. A leading cause of MDR
development is that therapeutic drugs are pumped out by power-dependent efflux pump from the
cancer cells, which is obligate in the lower accumulation of drug in the cells and failed treatment
outcome [5]. Cellular energy production is mainly derived from aerobic metabolism in the
mitochondria, which play critical roles in configuring the apoptosis pathway for cancer cells [6].
Some reports have also found that PTX exerts a direct effect on mitochondria of cancer cells,
rendering the apoptosis of cancer cells [7-9]. Therefore, homing delivery of therapeutic drugs to
the mitochondria from cancer cells is a hopeful solution to diminish MDR in tumor chemotherapy.
Among mitochondria-targeting approaches, delocalized lipophilic cations (DLCs) are the most
fashionable chemicals such as triphenylphosphonium, mitochondria-penetrating peptides which
are usually conjugated to small molecules or decorated on the surface of drug delivery platforms
[10,11]. Due to more negative charged mitochondrial membrane potentials, DLCs are more likely
to accumulate in the mitochondria of tumor cells than normal cells. These drug delivery platforms
offer preferential mitochondrial accumulation and enhanced drug uptake [12]. Berberine (BBR) is
an isoquinoline alkaloid with plenty of prominent pharmacological effects including antimicrobial
[13], antiviral [14], antidiabetic [15], anti-inflammatory[16] and anticancer [17] activities.
Strikingly, BBR can selectively accumulate into the mitochondria in cancer cells, which may be
put down to its amphiphilic structure and delocalized positive charge [18-21]. Moreover, cancer
cell apoptosis induced by BBR acting on mitochondria has been confirmed with the deep
mechanisms interpreted as dissipating the mitochondrial membrane potential, upregulating ROS
levels, inducing changes in permeability of mitochondrial membrane [17,18].
Nanomedicine delivery system such as micelles [22], inorganic materials [23,24], liposomes
[25,26], vesicles [27] and dendrimers [28], have been immensely investigated in order to elevate
the physio-chemical properties and efficacy of therapeutic drugs. In the midst of the nanomedicine
delivery platforms, nanodrugs formed by small molecule self-assembly have shown great potential
for their high drug loading and reduced side effects without usage of drug carriers [29,30]. Besides,
the nanosystems are able to deliver drugs to the tumor sites by making use of the enhanced
permeability and retention (EPR) effect [31]. Glutathione (GSH) is a strong biological reducing
tripeptide and is as commonly used stimuli factors for disulfide bond at certain concentration [32].
The mitochondria in cancer cells express a higher level of GSH than that in the normal tissue
(varying between 2 and 10 μmolL−1), enough to reduce the disulfide bond [33]. Therefore,
mitochondria-targeting GSH-sensitive drug delivery platform is expected to enhance the effect of
chemotherapy.
Taking above observations into account, considering the excellent mitochondria-targeting
feature and potential synergetic anticancer effect of BBR and the clinically outstanding
contribution of PTX, we described herein a new GSH-responsive conjugate, which was

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constructed through a disulfide bond spacer to connect paclitaxel with berberine. Scheme 1
illustrated the structure and synthetic routes of the mitochondria-targeting paclitaxel-ss-berberine
(PTX-ss-BBR) conjugate, which was able to form unanimous nanoparticles (PTX-ss-BBR NPs).
Based on the aforementioned advantages, the NPs announced better in vitro potency on A549 cells
due to the simultaneous drug release and mitochondria-targeting drug delivery.
2. Materials and methods
2.1. Materials
Berberine hydrochloride, paclitaxel, methanol (CH3OH), acetonitrile (CH3CN) and glutathione
(GSH) were provided by Shanghai Adamas Reagent Co., Ltd. (Shanghai, China).
4-Dimethylaminopyridine
(DMAP),
3,3-dithiodipropionic
acid
and
N,N-Dicyclohexylcarbodiimide (DCC) were obtained from Nanjing Juyou Scientific Equipment
Co., Ltd. (Nanjing, China). 3-(4,5-Dimethyl-2-tetrazolyl)-2,5-diphenyltetrazolium bromide (MTT)
chloroform (CH3Cl3) were purchased from Nanjing Wanqing Chemical Glassware Istrument Co.,
Ltd. (Nanjing, China). Mitotracker Red was purchased from Jiangsu KeyGEN Biotech Co., Ltd.
(Nanjing, China). Other reagents were provided by local commercial suppliers.
2.2. Chemistry synthesis of GSH-responsive PTX-ss-BBR conjugate
Synthesis of Compound 2: Berberine hydrochloride (5.0 g, 13.47 mmol) in vacuo was stirred at
190 oC for 60 minutes to obtain crude product, which was purified via column chromatography on
silica gel (eluent: chloroform/methanol = 9/1, V/V) to produce compound 2 (3.68 g) as red-brown
solid with 85% yield.
Synthesis of Compound 3: Intermediate 2 (4.22 g, 13.14 mmol) and K2CO3 (1.81 g, 13.14
o
mmol) in CH3CN (18 mL) were reacted at 50 C for 30 minutes. 2-Bromoethanol (1.65 g, 13.14
mmol) was added to the mixed system, and subsequently the reacted temperature was gradually
heated to 82 oC and went on for 24 h. After the end of the reaction, the temperature was decreased
o
to 25 C slowly, and the remaining CH3CN was evaporated by rotary evaporation method. The
condensed solution was purified via column chromatography on silica gel (eluent:
chloroform/methanol = 9/1, V/V) to produce compound 3 (5.03 g) as yellow solid with 86% yield.
1H NMR (600 MHz, MeOD+CDCl3) δ 9.95 (s, 1H), 8.62 (s, 1H), 8.05 (d, J = 9.1 Hz, 1H), 8.00 (d,
J = 9.0 Hz, 1H), 7.59 (s, 1H), 6.93 (s, 1H), 6.13 (s, 2H), 4.99–4.93 (m, 2H), 4.54–4.50 (m, 2H),
4.12 (s, 3H), 4.00–3.96 (m, 2H), 3.31–3.26 (m, 2H). HRMS (ESI) calcd. for C21H20NO5 [M-Br]+,
366.1336; found, 366.13315.
Synthesis of Compound 4: Berberine derivative 3 (4.48 g, 10.11 mmol) was refluxed in CH3OH
(35 mL) for a quarter, then sodium borohydride (1.15 g, 30.33 mmol) was added to the solution

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o
little by little accompanied by vigorous stirring at 0 C. Twelve hours later, the CH3OH was
evaporated by rotary evaporation method. The condensed solution was purified via column
chromatography on silica gel (eluent: methanol/chloroform = 1/50, V/V) to obtain compound 4
(3.06 g) as light yellow solid with 57% yield. 1H NMR (600 MHz, CDCl3) δ 6.88 (d, J = 8.4 Hz,
1H), 6.79 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H), 6.59 (s, 1H), 5.91 (s, 2H), 4.26 (d, J = 15.5 Hz, 1H),
4.16–4.12 (m, 1H), 4.04 (dd, J = 10.3, 5.8 Hz, 1H), 3.88–3.81 (m, 5H), 3.57–3.51 (m, 2H), 3.22
(dd, J = 15.9, 3.3 Hz, 1H), 3.17 (dd, J = 10.4, 4.7 Hz, 1H), 3.10 (d, J = 11.2 Hz, 1H), 2.82 (dd, J =
15.5, 11.6 Hz, 1H), 2.63 (dd, J = 21.8, 10.6 Hz, 2H).; HRMS (ESI) calcd. for C21H23NO5 [M+H]+,
370.1654; found, 370.16589.
Synthesis of Compound 5: The 20 mL pyridine liquid including tetrahydroberberine 4 (3 g, 8.13
mmol) was added to the mixture system of DMAP (98.95 mg, 0.81 mmol), DCC (2.52 g, 12.14
mmol), 3,3’-dithiodipropionic acid (1.71 g, 8.13 mmol) in pyridine under dry N2, and the mixted
system was reacted at 0 °C for 48 h. Then, residual pyridine was evaporated by rotary evaporation
method. The condensed solution was purified by column chromatography on silica gel (eluent:
methanol/chloroform = 1/20, V/V) and dried under vacuo, obtaining the pure compound 5 (2.87 g)
as light yellow solid with 63% yield. 1H NMR (600 MHz, MeOD) δ 7.03 (s, 2H), 6.91 (s, 1H),
6.71 (s, 1H), 5.96 (s, 2H), 4.47 (s, 1H), 4.42–4.37 (m, 2H), 4.35–4.30 (m, 2H), 3.86 (d, J = 3.7 Hz,
3H), 3.72 (s, 1H), 3.64 (dd, J = 17.0, 4.3 Hz, 1H), 3.37 (s, 1H), 3.22 (s, 1H), 3.11–3.09 (m, 1H),
3.07–2.99 (m, 2H), 2.96 (dd, J = 13.5, 6.2 Hz, 4H), 2.82 (t, J = 6.9 Hz, 2H), 2.66 (t, J = 7.0 Hz,
2H), 2.59 (t, J = 6.1 Hz, 1H).; HRMS (ESI) calcd. for C27H31NO8S2 [M+H]+, 562.1569; found,
562.15688.
Synthesis of Compound 6: The 20 mL pyridine liquid including paclitaxel (2.0 g, 2.34 mmol)
was added little by little to the mixture system of DCC (724.22 mg, 3.51 mmol), compound 5
(1.31 g, 2.34 mmol), DMAP (28.09 mg, 0.23 mmol) in pyridine under dry N2, and the mixted
system was reacted at 0 °C for 48 h. Then, the residual pyridine was evaporated by rotary
evaporation method. The condensed solution was purified via column chromatography on silica
gel (eluent: methanol/chloroform = 1/50, V/V) and dried under vacuo, obtaining the pure
compound 6 (2.78 g) as light yellow solid with 85% yield. IR (KBr) : 3448, 2944, 1727, 1666,
1484, 1452, 1373, 1240, 1074, 979, 707. 1H NMR (600 MHz, CDCl3) δ 8.14 (d, J = 7.6 Hz, 2H),
7.75 (d, J = 7.2 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.55–7.48 (m, 3H), 7.44–7.37 (m, 6H), 7.34 (d, J
= 7.5 Hz, 1H), 7.06 (s, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 6.72 (s, 1H), 6.58 (d,
J = 6.5 Hz, 1H), 6.29 (s, 1H), 6.24 (t, J = 8.7 Hz, 1H), 5.98 (d, J = 9.0 Hz, 1H), 5.91 (s, 2H), 5.68
(d, J = 7.1 Hz, 1H), 5.53 (s, 1H), 4.97 (d, J = 9.0 Hz, 1H), 4.44 (t, J = 8.5 Hz, 1H), 4.38 (t, J = 9.4
Hz, 2H), 4.32 (d, J = 8.5 Hz, 1H), 4.26 (dd, J = 17.7, 14.3 Hz, 2H), 4.22–4.16 (m, 2H), 3.81 (d, J
= 10.5 Hz, 4H), 3.51 (s, 2H), 3.22 (d, J = 15.0 Hz, 1H), 3.12 (d, J = 24.4 Hz, 2H), 2.91 (t, J = 7.0

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Hz, 2H), 2.87 (d, J = 5.1 Hz, 2H), 2.85–2.75 (m, 4H), 2.65 (d, J = 14.5 Hz, 2H), 2.54 (d, J = 14.7
Hz, 2H), 2.45 (s, 3H), 2.40–2.33 (m, 1H), 2.22 (s, 3H), 2.19–2.13 (m, 1H), 1.94 (s, 3H), 1.91–1.81
(m, 2H), 1.68 (s, 3H), 1.23 (s, 3H), 1.13 (s, 3H).; HRMS (ESI) calcd. for C74H80N2O21S2 [M+H]+,
1397.4773; found, 1397.47749.
Synthesis of PTX-ss-BBR conjugate 7: NBS (86.49 mg, 0.48 mmol) was added little by little to
the solution of compound 6 (450 mg, 0.32 mmol) in CHCl3 (10 mL), and reacted at 50 oC for 6 h.
o
The reaction solution was decreased to 25 C and then filtered. The condensed solution was
purified via column chromatography on silica gel (eluent: chloroform/methanol = 9/1, V/V) and
dried under vacuum, obtaining the pure compound 7 (133.57 mg) as yellow solid with 45% yield.
1
IR (KBr) : 3421, 2935, 1739, 1637, 1600, 1502, 1450, 1390, 1336, 1240, 1074, 927, 711. H
NMR (600 MHz, CD3OD, ppm): δ 9.75 (s, 1H), 8.70 (s, 1H), 8.10 (d, J = 8.4 Hz, 3H), 8.00 (d, J =
9.1 Hz, 1H), 7.79 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.6 Hz, 1H), 7.65 (s, 1H), 7.60 (d, J = 7.9 Hz,
2H), 7.52 (d, J = 7.5 Hz, 1H), 7.47–7.43 (m, 6H), 7.26 (t, J = 7.2 Hz, 1H), 6.94 (s, 1H), 6.40 (s,
1H), 6.10 (s, 2H), 6.01 (d, J = 10.1 Hz, 1H), 5.78 (d, J = 6.8 Hz, 1H), 5.61 (d, J = 7.2 Hz, 1H),
5.46 (d, J = 6.7 Hz, 1H), 4.97 (d, J = 7.9 Hz, 1H), 4.94–4.91 (m, 2H), 4.67 (t, J = 4.0 Hz, 2H),
4.56 (d, J = 8.2 Hz, 2H), 4.29 (d, J = 11.0 Hz, 1H), 4.16 (s, 2H), 4.10 (s, 3H), 3.76 (d, J = 7.2 Hz,
1H), 3.27 – 3.24 (m, 2H), 2.86 (d, J = 6.3 Hz, 2H), 2.81 (dd, J = 14.5, 8.1 Hz, 4H), 2.67 (t, J = 6.8
Hz, 2H), 2.37 (s, 3H), 2.13 (s, 3H), 1.87 (s, 2H), 1.76 (dd, J = 14.6, 4.6 Hz, 2H), 1.63 (s, 3H), 1.28
(s, 3H), 1.12 (s, 3H), 1.09 (s, 3H), 0.90 (t, J = 7.0 Hz, 1H).; HRMS (ESI) calcd. for C74H77N2O21S2
[M-Br]+, 1393.4455; found, 1393.44253.
1H NMR spectra were recorded on a Bruker AVANCE III HD NMR spectrometer operating at
600 MHz and using deuterated chloroform (CDCl3) or methanol (CD3OD) as solvent. The high
resolution mass spectra (HRMS) were recorded on a Bruker solanX 70 FT-MS with ESI resource.
FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer (Bio-Rad, Cambridge,
MA, USA) using KBr pellets in the 400-4000 cm-1 range.
2.3. Preparation and characterization of PTX-ss-BBR NPs
PTX-ss-BBR NPs were prepared from the nano-precipitation method. The PTX-ss-BBR
conjugate (10 mg) was dissolved in DMSO (0.5 mL), followed by ultrasound about 5 min and the
solution was injected gradually into the 20 mL deionized water to form the nanomedicine. After
that, the resulting nanoparticles were dialyzed (MWCO = 2000 Da) against deionized water for 48
h for complete removal of the DMSO and unbond compounds.
The morphological observation of the PTX-ss-BBR NPs was checked by the TEM (JEM-2100F,
JEOL). The size and size distribution of the NPs was detected by DLS (Malvern Zetasizer
Nano-ZS90, Malvern). The light scattering intensities (Kcps) of acquous solutions containing

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varying concentration PTX-ss-BBR conjugates were measured on DLS analyzer, and then plot
over the concentration (log scale) of PTX-ss-BBR to determine the critical micelle concentration.
2.4. In vitro drug release
The in vitro drug release activity of PTX-ss-BBR NPs was evaluated by using a typical dialysis
strategy. Briefly, 1 mL of NPs solution was placed in the dialysis bag (MWCO = 2000 Da) and
immersed in 30 mL buffer solution (pH = 7.4, with or without 10 mM GSH) containing 1% (v/v)
Tween 80 with continuously shaking at a ratio of 100 rpm and keeping at 37 °C. At set time points,
2 mL release medium were taken out for fluorescence measurement and an equal amount of fresh
PBS was used to replace the release medium. A fluoromax 4 spectrofluorometer (HORIBA, USA)
(excitation at 370 nm) was employed for measuring the amount of released BBR.
2.5. Cell culture.
A549 cell is a human lung adenocarcinoma cell line, HeLa cell is a cervical cancer cell line and
HepG2 cell is a human hepatoma cell line, which is received from China Pharmaceutical
University (Nanjing, China). All cells were cultured in RPMI 1640 or DMEM medium
complemented with 100 μg/mL streptomycin, 10% FBS, 100 U/mL penicillin and 100 μg/mL
streptomycin in a humidified incubator at 5% CO2, 37 °C.
2.6. Cell viability assay.
The MTT assay was employed to detect the in vitro cytotoxicity of PTX-ss-BBR NPs and PTX
against A549, HeLa and HepG2 cells. The 96-well plates (1×104 cells per well) were used to seed
various cells and kept overnight. Following the attachment of cells, fresh culture medium
containing PTX-ss-BBR NPs and PTX were added at tested concentrations of 0.625, 1.25, 2.5, 5,
10, 20 and 40 μM and incubation time was set 24 h and 48 h. After washing twice, 25 μL 5% MTT
stock solution was added and continued to incubate for another 4 h. The 150 μL dimethyl
sulfoxide solvent was used to dissolve generated formazan precipitate and the absorbance intensity
at 570 nm of each sample measured by microplate-680 reader (Bio-Rad, CA). Cell viability (%) of
experimental groups against the different cells was determined and untreated cells as the control
group. Each group was performed in parallel three times and GraphPad Prism software was
employed for calculating the half-maximal inhibitory concentration (IC50) value.
2.7. Mitochondrial targeting
Laser confocal scanning microscope (CLSM) was used to observe the mitochondrial
localization of BBR and PTX-ss-BBR NPs in A549 cells. The glass-bottomed dish (1×104 cells
per dish) were used to seed A549 cells for 12 h at 5% CO2 and 37 oC and cultured with 5 M BBR
or PTX-ss-BBR NPs for another 60 or 120 minutes under the same culture conditions. After

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treatment, the A549 cells were washed 3 times with 2 ml of buffer solution in dark conditions, and
then the A549 cells were stained with mitochondrial localization reagent Mitotracker Red (1 μM)
and cultured for 25 minutes under the same culture conditions. After the completion of the culture,
the cells were washed again by using 2 ml of the buffer solution three times, and then the
fluorescence of the cells was observed by CLSM.
2.8. Mitochondrial membrance potential
The JC-1 (Jiangsu KeyGEN, Nanjing, China), a fluorescent dye was used to measure the
change of mitochondrial membrance potential (Δψm). The 6-well plates (1×105 cells per dish)
o
were used to seed A549 cells for 12 h at 5% CO2 and 37 C and cultured with 5 M BBR or
PTX-ss-BBR NPs for another 48 h under the same culture conditions. After treatment, the A549
cells were washed 3 times with 2 ml of buffer solution in dark conditions, and then the A549 cells
were stained with 0.5 mL of 5 g/mL JC-1 and cultured for a quarter under the same culture
conditions. After the completion of the culture, the cells were washed again by using 2 ml of the
buffer solution three times before analysis by FACScan flow cytometer.
2.9. ROS detection assay
The intracellular production of ROS induced by different groups was measured using a
2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) kit (Jiangsu KeyGEN, Nanjing, China).
The 6-well plates (1×105 cells per dish) were used to seed A549 cells for 12 h at 5% CO2 and 37
oC and cultured with 5 M BBR or PTX-ss-BBR NPs for another 48 h under the same culture
conditions. After treatment, the A549 cells were washed 3 times with 2 ml of buffer solution in
dark conditions, and then the A549 cells were stained with 0.5 mL of 20 μM DCFH-DA and
cultured for 60 minutes under the same culture conditions. After the completion of the culture, the
cells were washed again by using 2 ml of the buffer solution three times before detection of
dichlorofluorescein (DCF) by FACScan flow cytometer.
2.10. Apoptosis effect in vitro
The cell apoptosis effect of tested groups was measured using Annexin V-FITC/PI apoptosis
detection kit (Jiangsu KeyGEN, Nanjing, China). The 6-well plates (1×105 cells per dish) were
o
used to seed A549 cells for 12 h at 5% CO2 and 37 C and cultured with 5 M BBR or
PTX-ss-BBR NPs for another 48 h under the same culture conditions. After treatment, the A549
cells were washed 3 times with 2 ml of buffer solution in dark conditions, and then the A549 cells
were stained with 5 μL iodide and 5 μL Annexin V-FITC and cultured for 30 minutes under the
same culture conditions. After the completion of the culture, the cells were washed again by using
2 ml of the buffer solution three times before analysis by FACScan flow cytometer.

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2.11. Cell-cycle analysis
The 6-well plates (1×105 cells per dish) were used to seed A549 cells for 12 h at 5% CO2 and 37
oC and cultured with 5 M BBR or PTX-ss-BBR NPs for another 48 h under the same culture
conditions. After treatment, the A549 cells were washed 3 times with 2 ml of buffer solution in
dark conditions, and then the A549 cells were resuspended in buffer solution with 50 g/mL PI
and 50 μg/mL RNase A. and cultured for 30 minutes under the same culture conditions. After the
completion of the culture, the cells were washed again by using 2 ml of the buffer solution three
times before analysis by FACScan flow cytometer.
2.12. Statistical analysis
All data are presented as the average ± standard deviation (SD, n = 3). One-way ANOVA
analysis by GraphPad Prism software was employed for the analysis of statistical significance.
3. Results
3.1. Chemistry synthesis of GSH-responsive PTX-ss-BBR conjugate
The target PTX-ss-BBR conjugate 7 was synthesized via multi-step reactions from
commercially available berberine, bromoethanol, 3,3´-dithiodipropionic acid, paclitaxel. The
synthetic process was presented in Scheme 1. Compound 2 was easily obtained by selective
o
9-position demethylation of berberine at 190 C under vacuo for 60 minutes. Berberine
intermediate 2 was ulteriorly reacted with bromoethanol through nucleophilic substitution in
CH3CN under reflux to gain compound 3 in 86 % yield, which was reduced by NaBH4 in
methanol to produce compound 4 in 57 % yield. The key intermediate 5 was obtained via the
condensation reaction of compound 4 and 3,3´-dithiodipropionic acid in pyridine with DMAP and
DCC. Compound 6 was obtained by a condensation reaction of compound 5 and PTX in pyridine
with DMAP and DCC. The target paclitaxel-berberine conjugate 7 was obtained in 28 % yield by
o
the oxidation of compound 6 with NBS in CHCl3 at 50 C for 6 h. Targeted conjugate
PTX-ss-BBR and intermediates were confirmed by 1H NMR, and HRMS spectra.
Scheme 1
Reagents and conditions: i) 190 oC/vacuo; ii) K2CO3, CH3CN, bromoethanol, reflux, 24 h; iii) NaBH4, CH3OH, 0 oC; iv)
3,3´-dithiodipropionic acid, pyridine, DMAP, DCC, 0 oC, 48 h; v) PTX, pyridine, DCC, 0 oC; vi) NBS, CHCl3, 50 oC.
Scheme 1. Synthetic route of GSH-responsive PTX-ss-BBR conjugate.
3.2. Characterization of PTX-ss-BBR NPs
The TEM and DLS were used to observe and characterize the morphology and size distribution
of PTX-ss-BBR NPs. TEM image demonstrated that the PTX-ss-BBR NPs showed a spherical
structure with the size of approximately 150 nm (Fig. 1A). The DLS analysis suggested that

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PTX-ss-BBR NPs exhibited a mean diameter of 165.8 ± 2.597 nm (Fig. 1C) with a narrow
polydispersity index (PDI = 0.115) and showed a small critical micelle concentration (CMC =
1.26 μM) of PTX-ss-BBR conjugates (Fig. 1F). The diameter measured by DLS remained
unvaried over 3 days, suggesting excellent stability of PTX-ss-BBR NPs in PBS and in PBS
including 10% fetal bovine serum (FBS, Fig. 1D). The reducing agent-induced dissociation of
disulfide bonds in PTX-ss-BBR NPs was monitored by TEM in the presence of 10 mM GSH for 6
h (Fig. 1B).
Figure 1
Fig. 1. (A) TEM image of PTX-ss-BBR NPs. (B) TEM image of PTX-ss-BBR NPs treated with
10 mM GSH for 6h. (C) Particle size of PTX-ss-BBR NPs. (D) Changes of diameter of
PTX-ss-BBR nanoparticles in PBS or in PBS and FBS for 3 days. (E) In virto release kinetics of
drug from NPs in pH 7.4 PBS with or without GSH. (F) The light scattering intensities (Kcps) of
acquous solutions containing varying concentration PTX-ss-BBR conjugates.
3.3. Self-assembly mechanism of PTX-ss-BBR conjugate
Computer simulation provides another way to evaluate the self-assembly process of complex
systems [34]. To unravel the self-assembly principle of the PTX-ss-BBR conjugate, molecular
dynamics (MD) simulations for conjugates were performed in water environment. As shown in
Fig. 2, ten PTX-ss-BBR conjugates gathered quickly and uniformly to form a cluster of multimer.
After the end of self-assembly, the whole nanocluster’s position varied, but remained aggregated
together. Noncovalent hydrophobic interactions and π-π stacking accounted for the self-assembly
of the ten PTX-ss-BBR conjugates because PTX moieties with multiple phenyl fragments (green
color) were in the internal part of the cluster and the more polar BBR fragments (pink color)
surrounded the internal part and were exposed to the water media. Furthermore, it was observed
that in the first 50ns of the simulation the solvent accessible surface area and the number of
hydrogen bonds (Fig. 3A) between the cluster and water were reduced gradually, which
announced that the complex system was formed gradually. After longer simulation times, the
cluster was apt to keep stable. When the solvation free energy is a negative value, the solvation
will be thermodynamically favorable [35]. In this research (Fig. 3B), the solvation free energy was
always a negative value and it gradually increased in the first 50 ns. After then, it remained almost
unchanged, which indicated that the intermolecular and intramolecular interactions of conjugates
contributed more to the formation of nanosystems than spontaneous solvation.
Figure 2
Fig. 2. The supramolecular assembly process of PTX-ss-BBR conjugate in water by MD
simulations.
Figure 3

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Fig. 3. (A) The solvent accessible surface areas of the assembled nanostructures and the number of
hydrogen bonds between the cluster and water. (B) Solvation free energy of assembled
nanostructures.
3.4. In vitro drug release of PTX-ss-BBR NPs
The mitochondria in cancer cells express a drastically higher content of GSH than that in the
normal environment, enough to reduce the disulfide bond [36]. BBR and PTX were linked
together by a disulfide bond rope, which made PTX-ss-BBR NPs liable to disassembly by
reducing agent and the nanosystems could be employed as a GSH-responsible drug delivery
platform for stimulating release of the drugs in cancer cells. To estimate the GSH sensitivity, the in
vitro release kinetics of the drugs from PTX-ss-BBR NPs was checked by a typical dialysis
method in PBS (pH = 7.4, with 10 mM GSH) or PBS (pH = 7.4, without GSH), simulating the
reductive environment of cytoplasm of tumor cells and normal physiological environment,
respectively. Here, the accumulative amount of BBR released from PTX-ss-BBR NPs was studied
since both BBR and PTX will release simultaneously. According to Fig. 1E, the PTX-ss-BBR NPs
was GSH-sensitive and cumulative release amount of BBR was ≈ 55% after 10 h treatment at
o
37 C. Only approximately 7% BBR released from PTX-ss-BBR NPs in normal physiological
environment over a period of 55 h, which was due to dynamic thermodynamic equilibrium
between free PTX-ss-BBR monomer and self-assembled NPs. These release results suggested that
the PTX-ss-BBR NPs were as-expectedly minimal drug release in blood circulation and could
instantly disassembly and release drugs in a highly reductive microenvironment.
3.5. In vitro anti-tumor efficacy of PTX-ss-BBR NPs
In vitro anticancer activity of PTX-ss-BBR NPs against A549, HeLa and HepG2 cells were
studied using MTT assays and the cell viability details and IC50 values were presented in Table 1
and Fig. 4, respectively. Free PTX and BBR were chosen as positive control. As shown in Fig. 4,
anticancer activity was enhanced with increasing PTX-ss-BBR NPs concentrations and extending
treatment time, suggesting that the anticancer efficacy of PTX-ss-BBR NPs was concentration-
and time-dependent. BBR exhibited moderate anticancer activity on all cells tested after 48 h
treatment. Fig. 4A and B disclosed that self-assembled NPs showed considerably higher anticancer
activity on A549 cells than the free PTX, especially after 48 h treatment, the inhibition activity of
PTX-ss-BBR NPs (IC50 = 0.243 M) increased by approximately 22% compared to PTX (IC50 =
0.31 M), which might be reasonably interpreted by the fast disassembly of the NPs under
reductive tumor microenvironment and the promotion of mitochondria-targeting of drug delivery.
While the inhibition efficacy of NPs against HeLa cells was weaker than PTX after 24 h
incubation, the anti-tumor activity of NPs (IC50 = 1.512 M) was better than PTX (IC50 = 1.653
M) after 48 h incubation, which might be due to the effective release of the PTX-ss-BBR NPs

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after a longer incubation period in HeLa cells. In contrast, HepG2 cells seemed to be less sensitive
to PTX-ss-BBR NPs, the IC50 values of NPs on HepG2 were higher than PTX after either 24 or 48
h incubation.
Figure 4
Fig. 4. In vitro anticancer activity of self-assembled PTX-ss-BBR NPs, PTX and BBR against
A549 cells for 24h (A) or 48h (B), HeLa cells for 24h (C) or 48h (D), HepG2 cells for 24h (E) or
48h (F). Data are presented as the mean ± SD (n=3). *P < 0.05 vs. PTX; #P < 0.05 vs. BBR; ##P <
0.01 vs. BBR; ###P < 0.001 vs. BBR.
Table 1 IC50 values of free BBR, PTX and PTX-SS-BBR NPs on A549, HeLa and HepG2 cells at
different times.
Table 1
3.6. In vitro antibacterial efficacy of PTX-ss-BBR NPs
The formation and development of cancer, especially for lung cancer is closely related to
bacterial infections, which occurs in up to 70% lung cancer patients and markedly influence the
clinical outcome and patient survival [37]. In particular, Escherichia coli (E. coli), Staphylococcus
aureus (S. aureus) and Streptococcus pneumoniae (S. pneumoniae) are the most common in lung
cancer [38]. Therefore, we evaluated the efficacy of PTX-ss-BBR NPs above three bacteria and
berberine as the control group (Table 2). Albeit the NPs exhibited weak activity against S.
pneumoniae than BBR, it could effectively repress the growth of S. aureus (minimal inhibitory
concentration (MIC) = 4 M) and the inhibition ability was twice that of BBR. Moreover,
PTX-ss-BBR NPs exhibited an anti-E. coli activity comparable to that of BBR with the MIC value
of 8 M.
Table 2 In vitro antibacterial activity data as MIC (M) for PTX-ss-BBR NPs and BBR against S.
aureus, S. pneumonia and E. coli.
Table 2
3.7. Apoptosis-inducing effect in vitro by PTX-ss-BBR NPs
In order to check the apoptosis effect of PTX-ss-BBR NPs, a quantitative annexin V-FITC/PI
staining experiment was performed. In Fig. 5, the total apoptotic ratio (early and late apoptosis) in
A549 cells after incubation by PTX-ss-BBR NPs was 48.46%, which was much higher than free
BBR with 22.07% apoptotic ratio and free PTX with 32.23% apoptotic ratio, respectively. The
substantially elevated apoptosis-inducing effect of the PTX-ss-BBR NPs could be a result of the
accumulated amount of intracellular BBR and PTX, and the activation of mitochondria-based
apoptotic pathway.
Figure 5

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Fig. 5. (A) In vitro apoptosis analysis against A549 cells treated by BBR, PTX-ss-BBR NPs, and
PTX and analyzed by Annexin-v-FITC/PI double staining method. (B) Quantitative ratio of
apoptosis. Data are presented as the mean ± SD (n=3). ###P < 0.001 vs. Control;
BBR; ***P < 0.001 vs. PTX.
P < 0.001 vs.
3.8. Cell cycle analysis of A549
A549 cells were harvested with BBR, PTX-ss-BBR NPs and PTX and the untreated cells served
as control group. As seen in Fig. 6, BBR, PTX-ss-BBR NPs and PTX induced notable cell-cycle
interference in A549. A remarkable decrease in the phase of G0/G1 cells was noticed from 42.90 %
(control) to 37.56 %, 29.81 % (lower 30.5% than control), and 32.02 %, while a great increase in
G2/M phase of cells was observed from 16.76 % (control) to 23.24 %, 30.84 % (higher 84.1%
than control) and 26.07 %, after 48 h exposure to BBR, PTX-ss-BBR NPs and PTX. The cell
apoptosis and cell cycle assays indicated that PTX-ss-BBR NPs were conducive to the entry of
BBR and PTX into A549 cells and the enhancement of apoptosis effects.
Figure 6
Fig. 6. (A) In vitro cell cycle distribution on A549 cells after applying BBR, PTX-ss-BBR NPs
and PTX by flow cytometry. (B) G2/M phase rates of A549 cells. Data are presented as the mean
#
Δ
± SD (n=3). P < 0.05 vs. Control; ###P < 0.001 vs. Control; **P < 0.01 vs. BBR; P < 0.05 vs.
PTX-ss-BBR NPs.
3.9. Co-localization in the mitochondria of PTX-ss-BBR NPs
Mitotracker Red was used as a fluorescent probe to evaluate the capacity of the mitochondria
target of PTX-ss-BBR NPs. CLSM was employed to observed the mitochondria localization of
o
NPs using A549 cells cultured at 37 C for 1h and 2h, respectively. The yellow fluorescence
produced by the overlap of green fluorescence and red fluorescence suggested that the chemical
achieved mitochondria targeted localization. As shown in Fig. 7, both BBR and NPs were
observed with significant green fluorescence that predominantly localized in mitochondria stained
as red fluorescence by Mitotracker Red and the fluorescence intensity increased in a
time-dependent manner for PTX-ss-BBR NPs with a high Pearson’s coefficient (0.91 for Fig. 8A4,
0.94 for Fig. 8B4, 0.98 for Fig. 8C4), suggesting its efficient co-localization in the mitochondria
of A549 cells.
Figure 7
Fig. 7. Mitochondria targeting of the BBR and PTX-ss-BBR NPs in A549 cells. The cells in (A1,
B1, C1) the green channel, (A2, B2, C2) the red channel and (A3, B3, C3) the merged image were
co-loaded with BBR or PTX-ss-BBR NPs with Mitotracker. (A4) The intensity profile of the
linear regions of interest across the cell (white line in (A3)). (B4) The intensity profile of the linear
regions of interest across the cell (white line in (B3)). (C4) The intensity profile of the linear
regions of interest across the cell (white line in (C3)). Green channel: λEX = 405 nm, λEM
=

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500–600 nm; red channel: λEX = 561 nm, λEM = 580–660 nm.
3.10. Mitochondrial membrane potential (Δψm)
The energy produced by respiration on the mitochondrial inner membrane drives protons
through the inner mitochondrial membrane while establishing mitochondrial membrane potential
(Δψm), which is essential for many biological functions of cells, and its alteration activates the
apoptotic pathway of the cell [39]. JC-1, a lipophilic cationic dye, is capable of selectively
accumulating in the matrix of mitochondria and it will shift from red J-aggregates to green
monomers when the mitochondrial electrochemical gradient is destructed. As indicated in Fig. 8,
compared to the control group, A549 cells treated with BBR, PTX-ss-BBR NPs and PTX
exhibited obviously decreased Δψm. In contrast, the loss of Δψm of PTX-ss-BBR NPs was the
most significant, which was almost 1.36-times and 2.14-times higher than that of the BBR and
PTX.
Figure 8
Fig. 8. (A) Changes in the mitochondrial membrane potential measured by flow cytometry in
A549 cells after treatment with BBR, PTX-ss-BBR NPs and PTX. (B) Quantitative ratio of
decreased mitochondrial membrane potential (Δ). Data are presented as the mean ± SD (n=3).
**P < 0.01 vs. BBR; ###P < 0.001 vs. PTX.
3.11. ROS production
ROS is obligate in maintaining redox homeostasis in cells. Cancer cells are more sensitive to
high contents of ROS than normal cells and enhanced intracellular ROS elicits cell death by
damaging mitochondrial membrane [40,41]. 2,7-Dichlorofluorescein diacetate (DCFH-DA) is a
cell-permeant fluorescent probe, which is employed for visualizing ROS level. As shown in Fig. 9,
BBR and PTX-ss-BBR NPs exerted pronounced increase in the fluorescence intensity of
2,7-dichlorofluorescein (DCF) after 48 h treatment with BBR and PTX-ss-BBR NPs in A549 cells
by comparison to the control groups and PTX, maybe due to its higher mitochondrial
accumulation.
Figure 9
Fig. 9. (A) Intracellular ROS generation measured by flow cytometry in A549 cells after treatment
with BBR, PTX-ss-BBR NPs and PTX. (B) Quantitative ratio of mean fluorescent intensity of
ROS. Data are presented as the mean ± SD (n=3). #P < 0.05 vs. Control; ###P < 0.001 vs. Control;
*P < 0.05 vs. BBR; ΔΔP < 0.01 vs. PTX-ss-BBR NPs.
4. discussion
In addition to providing the energy necessary for cellular metabolism, mitochondria are also
associated with mitochondria in the occurrence of multidrug resistance to many chemotherapeutic

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drugs, thus transporting the chemotherapeutic drug to mitochondria are expected to address the
resistance of chemotherapy drugs. Considering the excellent mitochondria-targeting feature,
antibacterial activity and potential synergetic anticancer effect of BBR and the clinically
outstanding contribution of PTX, our work described herein a new GSH-responsive conjugate,
which was constructed through a disulfide bond spacer to connect PTX with BBR.
The prepared PTX-ss-BBR NPs had a particle size of about 165 nm, which was relatively stable,
and had a prominent GSH-responsible characteristic in the environment of the tumor compared to
the normal environment, and contributed to the rapid release of the drug in cancer cells. Molecular
simulations revealed that the main driving forces for the formation of nanoparticles were
hydrophobic interactions and π-π stacking, which provided theoretical guidance for the
development of new self-assembling nanodrugs.
The results of cytotoxicity showed that the PTX-ss-BBR NPs had better anticancer activity,
especially against A549 cells. After 48 hours of treatment, the inhibition effect of cancer cells was
20% higher than that of PTX, which might be due to the rapid release of PTX by the PTX-ss-BBR
NPs in A549 cells and the synergistic anticancer effect of BBR. Therefore, the biological
evaluation by PTX-ss-BBR NPs is worthy of further study. Some researches had found that more
than 50% lung cancers are caused by bacterial infections [37, 38]. We also studied the ability of
the PTX-ss-BBR NPs to inhibit common lung bacteria, and it was found to have a better activity
than BBR against Staphylococcus aureus with an MIC value of 4 M, which provided a new
potential for the treatment of lung cancer with bacterial infection.
PTX-ss-BBR NPs could promote apoptosis of cancer cells, which might be due to the action of
BBR and PTX on mitochondria. To further investigate the possible mitochondrial mechanisms of
nanoparticles, we conducted mitochondrial-related studies. The experimental results found that
PTX-ss-BBR NPs could be targeted for delivery to mitochondria and was time dependent. The
nanoparticles promoted a decrease in mitochondrial membrane potential and production of
reactive oxygen species. In addition, the nanoparticles could also arrest cancer cells in the G2/M
phase.
5. Conclusion
In summary, we developed a mitochondria-targeting nanoparticles (PTX-ss-BBR NPs) which
were formed through hydrophobic interactions and π−π stacking, as being revealed and verified by
using MD simulations. The prepared nanoplatform was sensitive to tumor microenvironment and
was imparted the ability for controlled release of drugs. Moreover, PTX-ss-BBR NPs accumulated
in the mitochondria of A549 cells via charge and lipocationic properties at BBR moiety. The in
vitro anticancer potency of PTX-ss-BBR NPs on A549 cells was enhanced due to upregulated
ROS levels, dissipated mitochondria membrane potential. We also confirmed that PTX-ss-BBR
NPs exerted comparable efficacy to BBR against bacteria closely associated with the development

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of lung cancer. The synergistic effect of PTX and BBR improved the treatment effect of
conventional chemotherapy drugs against A549 cells.
Acknowledgement
The authors gratefully acknowledge the financial support for this research from the National
Natural Science Foundation of China (Grant No.: 21606043, 21776046), the Six Talent Peaks
Project in Jiangsu Province (Grant No.: XCL-079), the Fundamental Research Funds for the
Central Universities (Grant No.: 2242019K40145), and the Recruitment Program for Young
Professionals (the Thousand Youth Talents Plan).
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Lists of tables, figures and schemes captions
Scheme 1 Synthetic route of GSH-responsive PTX-ss-BBR conjugate.
Fig. 1. (A) TEM image of PTX-ss-BBR NPs. (B) TEM image of PTX-ss-BBR NPs treated with
10 mM GSH for 6h. (C) Particle size of PTX-ss-BBR NPs. (D) Changes of diameter of
PTX-ss-BBR nanoparticles in PBS or in PBS and FBS for 3 days. (E) In virto release kinetics of
drug from NPs in pH 7.4 PBS with or without GSH. (F) The light scattering intensities (Kcps) of
acquous solutions containing varying concentration PTX-ss-BBR conjugates.
Fig. 2. The supramolecular assembly process of PTX-ss-BBR conjugate in water by MD
simulations.
Fig. 3. (A) The solvent accessible surface areas of the assembled nanostructures and the number
of hydrogen bonds between the cluster and water. (B) Solvation free energy of assembled
nanostructures.
Fig. 4. In vitro anticancer activity of self-assembled PTX-ss-BBR NPs, PTX and BBR against
A549 cells for 24h (A) or 48h (B), HeLa cells for 24h (C) or 48h (D), HepG2 cells for 24h (E) or
48h (F). Data are presented as the mean ± SD (n=3). *P < 0.05 vs. PTX; #P < 0.05 vs. BBR; ##P <
0.01 vs. BBR; ###P < 0.001 vs. BBR.
Table 1 IC50 values of free BBR, PTX and PTX-SS-BBR NPs on A549, HeLa and HepG2 cells at
different times.
Table 2 In vitro antibacterial activity data as MIC (M) for PTX-ss-BBR NPs and BBR against S.
aureus, S. pneumonia and E. coli.
Fig. 5. (A) In vitro apoptosis analysis against A549 cells treated by BBR, PTX-ss-BBR NPs, and
PTX and analyzed by Annexin-v-FITC/PI double staining method. (B) Quantitative ratio of
apoptosis. Data are presented as the mean ± SD (n=3). ###P < 0.001 vs. Control; ...P < 0.001 vs.
BBR; ***P < 0.001 vs. PTX.
Fig. 6. (A) In vitro cell cycle distribution on A549 cells after applying BBR, PTX-ss-BBR NPs
and PTX by flow cytometry. (B) G2/M phase rates of A549 cells. Data are presented as the mean
#
Δ
± SD (n=3). P < 0.05 vs. Control; ###P < 0.001 vs. Control; **P < 0.01 vs. BBR; P < 0.05 vs.
PTX-ss-BBR NPs.
Fig. 7. Mitochondria targeting of the BBR and PTX-ss-BBR NPs in A549 cells. The cells in (A1,
B1, C1) the green channel, (A2, B2, C2) the red channel and (A3, B3, C3) the merged image were
co-loaded with BBR or PTX-ss-BBR NPs with Mitotracker. (A4) The intensity profile of the
linear regions of interest across the cell (white line in (A3)). (B4) The intensity profile of the linear
regions of interest across the cell (white line in (B3)). (C4) The intensity profile of the linear
regions of interest across the cell (white line in (C3)). Green channel: λEX = 405 nm, λEM
=
500–600 nm; red channel: λEX = 561 nm, λEM = 580–660 nm.

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Fig. 8. (A) Changes in the mitochondrial membrane potential measured by flow cytometry in
A549 cells after treatment with BBR, PTX-ss-BBR NPs and PTX. (B) Quantitative ratio of
decreased mitochondrial membrane potential (Δ). Data are presented as the mean ± SD (n=3).
**P < 0.01 vs. BBR; ###P < 0.001 vs. PTX.
Fig. 9. (A) Intracellular ROS generation measured by flow cytometry in A549 cells after treatment
with BBR, PTX-ss-BBR NPs and PTX. (B) Quantitative ratio of mean fluorescent intensity of
ROS. Data are presented as the mean ± SD (n=3). #P < 0.05 vs. Control; ###P < 0.001 vs. Control;
*P < 0.05 vs. BBR; ΔΔP < 0.01 vs. PTX-ss-BBR NPs.

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Table 1 IC50 values of free BBR, PTX and PTX-SS-BBR NPs on A549, HeLa and HepG2 cells at
different times.
IC50 (M) on A549 cells
24 h 48 h
1.545±0.232 0.31±0.021
28.59±0.352
IC50 (M) on HeLa cells
24 h 48 h
8.876±0.147 1.653±0.117 8.471±0.027 1.936±0.085
40.18±0.537 37.72±0.489
IC50 (M) on HepG2 cells
Formulation
24 h 48 h
PTX
BBR



PTX-SS-BBR
NPs
1.295±0.132 0.243±0.014 10.573±0.231 1.512±0.201 12.402±0.134 2.515±0.148

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Table 2 In vitro antibacterial activity data as MIC (M) for PTX-ss-BBR NPs and BBR against S.
aureus, S. pneumonia and E. coli.
Formulation
BBR
S. pneumoniae S. aureus E. coli
16
8
8
PTX-ss-BBR
NPs
32
4
8

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Mitochondria-targeting
nanomedicine
self-assembled
from
GSH-responsive
paclitaxel-ss-berberine conjugate for synergetic cancer treatment with enhanced cytotoxicity
Yu Cheng, Yuanhui Ji*
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, School of Chemistry and Chemical
Engineering, Southeast University, Nanjing, 211189, P.R. China.

Corresponding author: Tel: +86-13951907361; Email: yuanhui.ji@seu.edu.cn;
yuanhuijinj@163.com.
Mitochondria-targeting GSH-sensitive nanoparticles (PTX-ss-BBR NPs) were formed through
hydrophobic interactions and π-π stacking. The prepared nanoplatform was sensitive to tumor
microenvironment and was imparted the ability for controlled release of drugs. The in vitro
anticancer potency of PTX-ss-BBR NPs on A549 cells was enhanced due to upregulated ROS levels,
dissipated mitochondria membrane potential.

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►New mitochondria-targeting PTX-ss-BBR conjugate was developed.
►Computer simulation studies rationalized the self-assembly process of PTX-ss-BBR.
►PTX-ss-BBR NPs exhibited better activity against A549 cells and S. aureus.
►PTX-ss-BBR NPs were GSH-sensitive and mitochondria-targeting.
►PTX-ss-BBR NPs can effectively dissipate mitochondrial membrane potential, upregulate ROS
levels, arreste cells in phase G2/M.