Synergistic antitumor efficacy of hybrid micelles with mitochondrial targeting and stimuli-responsive drug release behavior

Article abstract of DOI:10.1039/c8tb02843e

The term synergism means that the overall therapeutic benefits should be greater than the sum of the effects of individual agents and that the optimal therapeutic efficacy can be achieved at reduced doses. Micellar systems usually fail to deliver multiple drugs to target sites at synergistic doses and thus are not able to maximize the antitumor efficacy. In the current study, we demonstrate a strategy to coordinate the release of camptothecin (CPT) and α-tocopheryl succinate (TOS) from hybrid micelles for nucleus and mitochondrion interferences. TOS is decorated with cationic triphenylphosphonium (TPP) to promote the targeting capability of TOS-TPP to mitochondria. The combination of CPT and TOS-TPP shows strong synergistism with a combination index of 0.186. Hyaluronic acid (HA) is conjugated with CPT or TOS-TPP via disulfide linkages for tumor cell targeting and intracellular reduction-triggered release. Both conjugates either separately self-assemble into M_C and M_T micelles, or are blended at different ratios to form M_C-T hybrid micelles. In response to elevated intracellular glutathione levels, the coordinated release of CPT and TOS-TPP from M_C-T results in a combination index of 0.26 and the dose-reduction indexes of CPT and TOS are 7.7 and 3.4, respectively. Compared with M_C and M_T, M_C-T micelles with 5 fold lower doses exhibit higher intracellular reactive oxygen species (ROS) levels, comparable tumor growth inhibition and animal survival, indicating no hematologic and intestinal toxicities. Moreover, the HA conjugates of M_C-T are linked to polylactide via acid-labile linkages and electrospun into short fibers (M_C-T@SF) as an injectable depot to release M_C-T in response to the acidic tumor microenvironment. At a predetermined synergistic ratio, M_C-T@SF with 5 fold lower doses achieves antitumor profiles comparable to those of individual micelle-loaded short fibers. Therefore, the hybrid micelles and micelle-releasing short fibers represent a feasible strategy to synergistically enhance the therapeutic efficacy and enable significant reduction in effective doses of chemotherapeutic agents.

Full text of DOI:10.1039/c8tb02843e

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ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

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DOI: 10.1039/C8TB02843E
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ARTICLE
Synergistic antitumor efficacy of hybrid micelles with
mitochondrial targeting and stimuli-responsive drug release
Zhoujiang Chen,a,b Zhanlin Zhang,a Maohua Chen,a Songzhi Xie,a Tao Wang,a Xiaohong Lia,*
Received 00th January 20xx,
Accepted 00th January 20xx
The term synergism means that the overall therapeutic benefits should be greater than the sum of the effects of individual
agents and that the optimal therapeutic efficacy can be achieved at reduced doses. Micellar systems usually fail to deliver
multiple drugs to target sites at synergistic doses and thus are not able to maximize the antitumor efficacy. In the current
study, we demonstrate a strategy to coordinate the release of camptothecin (CPT) and α-tocopherylsuccinate (TOS) from
hybrid micelles for nucleus and mitochondrion interferences. TOS is decorated with cationic triphenylphosphonium (TPP)
to promote the targeting capability of TOS-TPP to mitochondria. The combination of CPT and TOS-TPP shows strong
synergistism with a combination index of 0.186. Hyaluronic acid (HA) is conjugated with CPT or TOS-TPP via disulfide
linkages for tumor cell targeting and intracellular reduction-triggered release. Both conjugates either separately self-
assemble into MC and MT micelles, or are blended at different ratios to form MC-T hybrid micelles. In response to elevated
intracellular glutathione levels, the coordinate release of CPT and TOS-TPP from MC-T results in a combination index of 0.26
and the dose-reduction indexes of CPT and TOS are 7.7 and 3.4, respectively. Compared with MC and MT, MC-T micelles with
5 folds lower doses produce higher intracellular reactive oxygen species (ROS) levels, comparable tumor growth inhibitions
and animal survivals while indicating no hematologic and intestinal toxicities. Moreover, HA conjugates of MC-T are linked
with polylactide via acid-labile linkages and electrospun into short fibers (MC-T@SF) as an injectable depot to release MC-T
in response to the acidic tumor microenvironment. At the predetermined synergistic ratio, MC-T@SF with 5 folds lower
doses achieve antitumor profiles comparable to those of individual micelles-loaded short fibers. Therefore, the hybrid
micelles and micelles-releasing short fibers represent a feasible strategy to synergistically enhance the therapeutic efficacy
and enable significant reductions in effective doses of chemotherapeutic agents.
DOI: 10.1039/x0xx00000x
www.rsc.org/
copolymers are in general more stable than liposomes and
emulsions constructed from conventional surfactants, and the
self-assembling structure leads to higher stimuli-
1. Introduction
Cancer therapy is confronted with great challenges in
improving therapeutic efficacy while alleviating systemic
toxicity. Drug combinations have become a standard regimen
in clinical practice to treat multi-factorial diseases and address
the limitations of single-drug therapy.1 Drugs that are typically
used in combination act through different therapeutic
mechanisms to produce complementary and synergistic effects,
thus strengthening the treatment outcome.2 In addition,
liposomes, nanoparticles and micelles have been developed to
enhance drug accumulation in tumors and retard the drug
resistance. Micelles self-assembled from amphiphilic
responsiveness than nanoparticles.3 The hydrophobic inner
core of a micelle contributes to high loadings of poorly water-
soluble drugs, while the hydrophilic outer shell as well as their
nanoscale dimensions can reduce reticuloendothelial system
clearances and increase tumor-specific accumulations.4
Moreover, stimuli-responsiveness has been incorporated into
micelles to release the payload in response to certain triggers
like pH, light, redox potentials or tumor-specific proteases.5
Recently micelles have shown considerable promise as
multidrug delivery carriers for enhancing tumor accumulation
and synergism between anticancer drugs.6 Kutty et al.
fabricated micelles for the co-delivery of suberoylanilide
hydroxamic acid and paclitaxel, leading to significantly higher
cytotoxicities than the mere combination of free drugs.7
However, it is still considered rather challenging to
simultaneously load multiple drugs at an optimal dose ratio
and achieve controlled release at the targeted sites for a
synergistic efficacy, owing to the differences in the hydrophilic
and pharmacokinetic properties of individual drugs.8 For
instance, paclitaxel and doxorubicin were co-loaded into
recombinant high-density lipoprotein nanoparticles at the
a.Key Laboratory of Advanced Technologies of Materials, Ministry of Education,
School of Materials Science and Engineering, Southwest Jiaotong University,
Chengdu 610031, P.R. China. E-mail: xhli@swjtu.edu.cn; Fax: +86 28-87634649;
Tel: +86 28-87634068
b.College of Chemical Engineering, Huaqiao University, Xiamen 361021, P.R. China.
Electronic Supplementary Information (ESI) available: The preparation of TOS-TPP,
HA-ss-TOS-TPP, PLA-cdm-MC and PLA-cdm-MT; the CMC determination and cellular
uptake images of hybrid micelles, the ROS production and cell apoptosis, typical
H&E and IHC staining images of tumors and systemic toxicities after micelle
treatment; the characterization and micelles release from short fibers, typical H&E
and IHC staining images of tumors retrieved after short fiber treatment are
included in the Supporting Information. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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designed ratios for efficacious combination chemotherapy, but poly(DL-lactide)
Journal Name
(PLA)
via
2-propionic-3-methylmaleic
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DOI: 10.1039/C8TB02843E
their release rates were quite diverse and the leakage of anhydride (CDM) linkages to obtain PLA-cdm-MC-T, followed by
physically encapsulated drugs during blood circulation was configuration into short fibers for intratumoral injection. As
inevitable.9 To minimize the premature drug release during shown in Scheme 1b, in response to the acidic
blood circulation, camptothecin and doxorubicin were microenvironment of tumors,18 the acid-labile degradation of
covalently conjugated to polymers and released by hydrolysis fiber matrices leads to the release of HA conjugates and
of ester and amide bonds, and the different drug release rates simultaneous self-assembly into MC-T. In response to elevated
affected the treatment efficacy of the combination.10 It should glutathione (GSH) levels in tumor cells,19 MC-T micelles release
be noted that the term synergistic effect means that the free drugs after HA-mediated cell internalization. The
overall therapeutic benefits of micelle combinations are much synchronous breakage of disulfide linkages leads to
greater than the sum of the effects of individual micelles, and coordinated release of CPT and TOS-TPP at the predetermined
that the therapeutic efficacy can be achieved at reduced synergistic ratio. Thus, hybrid micelle-releasing short fibers can
doses.11 Though the above reports demonstrated that multi- overcome barriers in the drug delivery pathway to tumor cells
drug loaded micelles significantly improved the therapeutic and the synergistic drug combinations can improve the
efficacy compared to individual-drug loaded ones, few studies therapeutic efficacy with dose-reducing superiority.
have confirmed the synergistic effect of micelles and enabled
significant reductions in effective doses of chemotherapeutic
agents. Therefore, it remains great challenges for the micellar
2. Experimental
system to achieve a synergistic dose by synchronizing the drug
loading, blood circulation, cellular uptake and intracellular
release in target tumor cells.
2.1 Materials
HA (Mw: 9.8 kDa) was purchased from the Freda Biochem Co.,
Ltd. (Shandong, China), and CPT was from Knowshine
Pharmachemicals Inc. (Shanghai, China). Cystamine, lysine,
TOS, TPP and GSH were used as received from Aladdin (Beijing,
China). Triton X-100, propidium iodide, trypsin, pyrene, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and
dialysis bags were procured from Sigma (St. Louis, MO). Rabbit
anti-mouse antibodies of caspase-3 and Ki-67, goat anti-rabbit
Another challenge for micellar system is the limited drug
accumulation in tumors after intravenous administration. Up
to now few micelles have progressed through clinical trials,
mainly due to their removal from the blood compartment and
premature drug release in the systemic circulation.12 Localized
chemotherapy is an effective means to maintain therapeutic
levels of anticancer agents at the tumor site and relieve
systemic toxicities.13 The intratumoral injection of hydrogels is
initially used to locally deliver free drugs and nanoparticles,
but suffers from extensive burst release after injection into
tumors, due to the lag time between the injection and
precipitation/gelation process.14 Electrospun fibers represent a
superior alternative for localized chemotherapy, due to the
extremely large surface area, high porosity, and great flexibility
in matrix polymer selection.15 Previously, we constructed an
injectable and biodegradable short fibers for local and
sustained release of micelles in response to tumor pH,
enhancing drug accumulation and retention in tumor tissues.16
In the current study, camptothecin (CPT) and α-
tocopherylsuccinate (TOS) are combined into hybrid micelles
to interfere with nuclear and mitochondrial functions,
respectively. CPT is an inhibitor of the nuclear enzyme
topoisomerase-I and TOS is one of the most important agents
to trigger mitochondrial apoptosis. Their combination is
supposed to improve cancer treatment efficacy, as
mitochondria maintain a central role in cellular homeostasis,
energy production and apoptosis managing.17 In addition,
mitochondria show an excessively negative potential (-160 to -
180 mV) across the inner membrane,17 thus TOS is decorated
with cationic triphenylphosphonium (TPP) to promote the
mitochondrial targeting ability of TOS-TPP. To alleviate
premature drug release before cellular internalization of
micelles, CPT or TOS-TPP is conjugated to hyaluronic acid (HA)
via disulfide linkages. Both conjugates are mixed to prepare
MC-T hybrid micelles (Scheme 1a). After defining the synergistic
ratios of MC and MT, HA conjugates of MC-T are linked with
IgG–horseradish peroxidase (HRP) and 3,3-diaminobenzidine
(DAB) developer were purchased from Biosynthesis
Biotechnology Co., Ltd. (Beijing, China). All other chemicals
were of analytical grade and obtained from Changzheng
Regents Company (Chengdu, China), unless otherwise
indicated.
2.2 Preparation and characterization of hybrid micelles
TOS-TPP was synthesized by coupling of TOS with TPP-COOH
via lysine linkers, and CPT and TOS-TPP were conjugated with
HA using cystamine as disulfide linkers to obtain HA-ss-CPT and
HA-ss-TOS-TPP, respectively (Scheme S1). The preparation and
characterizaiton results are included in the Supporting
Information. Blends of HA-ss-CPT with HA-ss-TOS-TPP self-
assembled into MC-T via ultrasonication as described
previously.20 Briefly, different mass ratios of HA-ss-TOS-TPP
and HA-ss-CPT were dispersed in distilled water with a total
concentration 1 mg/mL, followed by sonication for 10 min
using a probe-type ultrasonicator. The resulting micelles were
filtrated through 0.45 μm pore-sized membranes. Similarly,
HA-ss-CPT and HA-ss-TOS-TPP were used to prepare MC and
MT micelles, respectively. The micelle size and zeta potential
were measured by dynamic light scattering (DLS, Nano-ZS90,
Malvern Ltd., UK). The morphology was observed by
transmission electron microscopy (TEM, Tecnai G2 F20 S-Twin,
FEI, USA). The critical micellar concentrations (CMCs) of MC
and MT micelles were determined by fluorescence
spectroscopy using a pyrene probe as described previously.21
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DOI: 10.1039/C8TB02843E
Scheme 1. (a) Synthesis routes of PLA-cdm-MT and PLA-cdm-MC copolymers by conjugation of MT and MC carriers with PLA-cdm. MT
and MC self-assemble into MC-T hybrid micelles, and PLA-cdm-MT and PLA-cdm-MC are blended and configured into MC-T@SF short
fibers. (b) Schematic drawing of the release of HA-drug conjugates from short fibers in response to the slightly acidic tumor matrix
(1), followed by self-assembly into hybrid micelles (2); The hybrid micelles are endocytosed into tumor cells via HA mediation (3); In
response to intracellular GSH, CPT and TOS-TPP are released to target nuclei and mitochondria (4) for induction of cell-cycle arrest (5)
and reactive oxygen species (ROS) production (6).
2.3 Preparation and characterization of short fibers with loaded
hybrid micelles
The degradation and micelle release from short fibers were
determined after incubation in pH 7.4 and pH 6.8 buffers as
described previously.16 Briefly, short fibers (around 20 mg)
were exactly weighed and placed into dialysis bags (3.5 kDa cut
Micelle carriers of MC and MT were conjugated with PLA via
acid-labile CDM linkers to obtain PLA-cdm-MC and PLA-cdm-MT
copolymers (Scheme 1a), and the preparation and
characterizaiton results are included in the Supporting
Information. PLA-cdm-MC, PLA-cdm-MT and their blends with
the weight ratio of 1/2 were used to prepare injectable short
fibers (MC@SF, MT@SF and MC-T@SF).16 Briefly, the matrix
polymers were dissolved in chloroform/dimethyl formamide
(5/1, v/v), and electrospun fibers were deposited on a
grounded rotating mandrel. Aligned fibers were cryosectioned
with a thickness of 20 μm, followed by ultrasonication of the
fiber bundles to harvest short fibers. The morphology of short
fibers were detected by scanning electron microscopy (SEM;
FEI Quanta 200, The Netherlands) equipped with a field-
emission gun (20 kV) and a Robinson detector after 2 min of
gold coating to minimize the charging effect. The diameter and
length of short fibers were measured from SEM images to
generate an average value by using the tool of Photoshop 10.0
edition.
off) and immersed into 30 ml of release media at 37 C. At
predetermined time intervals, short fibers were retrieved for
weight loss measurement. The molecular weight of matrix
polymers was detected by gel permeation chromatography
(GPC, Waters 2695 and 2414, Milford, MA) using polystyrene
as standard. The molecular structure was determined by
nuclear magnetic resonance (NMR) spectroscopy (Bruker AM
400 apparatus). In addition, 1.0 mL of the released media was
withdrawn from inside and outside the dialysis bag for analysis
of the released micelles and free drugs respectively, and equal
amount of fresh buffers was added back. The micelle and drug
concentrations were detected by
a
fluorescence
spectrophotometer (Hitachi F-7000, Japan) at the
excitation/emission wavelengths of 298/345 and 365/430 nm
for MT (or TOS-TPP) and MC (or CPT), respectively. In another
batch of experiment, MC-T@SF short fibers were incubated in
pH 6.8 buffers for 3 days and the released micelles were
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retrieved for morphological observation and reductive microplate spectrophotometer (Elx-800, Bio-Tek Instrument
View Article Online
sensitivity determination.
Inc., Winooski, VT). On the basis of IC50sDoOfIC: 1P0T.1,0T3O9/SC,8aTnBd02T8O43SE-
TPP, the mixtures of CPT/TOS and CPT/TOS-TPP were tested at
concentrations of 2 and 4 folds higher or lower than their
IC50s to assess of the synergistic cytotoxicities.23,24 In addition,
MC-T were detected under different ratios of HA-ss-CPT to HA-
ss-TOS-TPP. Based on the cell viability data after single and
combination treatment, the synergism of free drugs and
hybrid micelles was determined using CalcuSyn software as
described previously.25 Synergism, additivism, and antagonism
were indicated by the combination indexes less than 1, equal
to 1, and greater than 1, respectively.
The cytotoxicities of micelles released from short fibers
were evaluated on H22 cells after incubation in pH 7.4 and 6.8
media as described previously.16 The cell culture under pH 6.8
was performed by adjusting the pH of culture media with 0.1
M HCl.26 The cells were treated by short fibers with the equal
amount of micelles released in pH 6.8 buffers during 72 h. The
cytotoxicity was determined by alamar Blue assay as above.
2.4 Reductive sensitivity of hybrid micelles
The reductive sensitivity of micelles (MC, MT, and MC-T) and the
retrieved micelles after release from short fibers was
measured from the size change and drug release profiles in
response to 10 μM and 10 and 40 mM GSH in PBS. Briefly,
micelle suspensions were placed in dialysis bags (1 kDa cutoff),
which were immersed into 30 mL of release media to achieve a
sink condition and kept shaking at 37 °C. After incubation for
48 h, micelles were retrieved and the size changes were
monitored by DLS as above. At given time intervals, 1 mL of
release media were withdrawn and refreshed with PBS
containing GSH. The TOS-TPP and CPT in the retreived media
were detected by a fluorospectrophotometer as above.
2.5 Cellular uptake of hybrid micelles
The cellular uptake of MC, MT, and MC-T micelles was evaluated
on H22 cells as described previously.21 Briefly, H22 cells from
the American Type Culture Collection (Rockville, MD) were
cultured in RPMI 1640 (Invitrogen, Grand Island NY)
supplemented with 10% heat inactivated fetal bovine serum
(FBS, Gibco BRL, Grand Island, NY). H22 cells were seeded into
6-well tissue culture plates (TCPs) at a density of 1.0 × 106
cells/well and incubated for 24 h before treatment. Then the
cells were cultured in media containing free drug (CPT and
TOS-TPP) or micelles with a final drug concentration of 10.0
μg/mL. For comparison, media containing 5 mg/mL of HA were
tested and cells without treatment were used as control. After
2.7 ROS production, cell apoptosis and cell cycle analysis
TOS is known to be able to destabilize mitochondria in tumor
cells, which often involves the elevated formation and buildup
of intracellular ROS.27 The intracellar ROS levels and cell
apoptosis were detected after treatment of H22 cells with free
drugs (CPT, TOS, and TOS-TPP) or micelles (MC, MT, and MC-T),
using cells without treatment as control. Briefly, cells were
cultured on 6-well TCPs at 5 × 105 cells/well, followed by
exposure to micelles and free drugs at the final drug
concentration of 5 μg/mL. After treatment for 6 h, cells were
collected, suspended, and incubated with DCFH-DA (10 μM) at
37 °C for 20 min according to the manufacturer’s protocol,
followed by fluorescence measurement via flow cytometry (BD
Accuri C6, Franklin Lakes, NJ).28 In antoher batch of experiment,
the adherent and non-adherent cells were collected after 48 h
of incubation, suspended in the binding buffer and stained
using the Annexin V-FITC apoptosis detection kit (Beijing 4A
Biotech Co., Beijing, China).29
incubation for 6 h, cells were washed with PBS, lysed by 500
of 0.5% Triton X-100 for 2 h at 4 °C. The fluorescence intensity
of the cell lysate was detected by fluorescence
l
a
spectrophotometer as above, and the cellular uptake
efficiency was obtained by comparison with the fluorescence
intensity of drugs or micelles added. In another batch of
experiment, H22 cells were seeded into 24-well TCP at a
density of 1 × 105 cells/well. After treatment as above for 6 h,
the culture media were removed, and cells were washed with
PBS and fixed with 4% paraformaldehyde. The cell nuclei were
2.8 In vivo antitumor efficacy of hybrid micelles and short fibers
stained with propidium iodide and observed by confocal laser All animal procedures were performed in accordance with the
scanning microscopy (CLSM, Leica TCS SP2, Germany).
National Institutes of Health Guide for Care and Use of
Laboratory Animals of China and approved by the Animal Care
and Use Committee of Southwest Jiaotong University. The
antitumor efficacy was determined from the tumor growth
inhibition, animal survival and histological analysis of tumors
retrieved as described previously.16 Briefly, female Kunming
mice weighing 22 ± 2 g were supplied by Sichuan Dashuo
Biotech Inc. (Chengdu, China). H22 cells were maintained in
the peritoneal cavities of mice for around 1 week, and the
ascites was withdrawn and diluted with PBS at l × 107 cells/mL.
The cell suspension was subcutaneously inoculated into the
right armpit region of each animal at a dose of 5 × 106 cells per
2.6 Cytotoxicity synergism of free drugs, hybrid micelles and short
fibers
The cytotoxicity of free drugs and micelles was detected by
alamar Blue assay as described previously.22 Briefly, H22 cells
were seeded in 96-well TCPs at a density of 1 × 104 cells/well
and incubated for 24 h. Media were then replaced with fresh
media containing various concentrations of free drugs (CPT,
TOS, and TOS-TPP) from 0.1 to 100 μM and micelles (MC and
MT) from 0.01 to 10 μM, using cells without treatment as
control. After treatment for 72 h, 10 µL of alamar Blue reagent
(Yeasen Biological Technology Co., Shanghai, China) was added
mouse, allowing the tumor growth to 50
100 mm3. The
established tumor-bearing mice were randomly divided into
eight groups with 8 mice per group. Free drugs (CPT and TOS)
into each well, allowing to incubate at 37 C for 1 h. The
absorbance of each well was measured using a
Quant
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and micelles (MC, MT, and MC-T) at equivalent doses of 4 mg TPP moieties to increase the target delivery to mitochondria.
View Article Online
CPT/kg and 8 mg TOS/kg were injected through the tail vein on Gogvadze et al. indicated that vitaminDEOIa: 1n0a.1lo03g9/wC8aTsB02208–4530E
days 0, 2, 4, and 6, using saline injection as control. MC-T hybrid folds more efficient in mitochondrial targeting than TOS.30 As
micelles with dilutions of 5 and 10 folds were also tested and shown in Scheme S1a, TPP containing a single amino group
defined as MC-T(1/5), and MC-T(1/10), respectively. In another was synthesized via sequential reaction with 6-bromohexanoic
batch of experiment, when tumors reached about 500 mm3 in acid and lysine and then coupled with TOS by DCC/NHS
volume after 10 days of growth, the tumor-bearing mice were chemistry to obtain TOS-TPP (Fig. S1).
randomly divided into five groups with 8 mice per group. The
The combination index analysis is an effective way to
mice were intratumorally injected with MC@SF, MT@SF and determine optimal drug combinations that work
MC-T@SF at equivalent doses of 4 mg CPT/kg and 8 mg TOS/kg, synergistically.11,31 Fig. 1a shows the cytotoxicities of CPT, TOS,
using PBS as control. The 5-flod lower dose of short fibers and TOS-TPP against H22 cells with IC50 values of about 1.19,
were also tested and named as MC-T@SF(1/5).
3.14 and 0.99 μg/mL, respectively. TOS-TPP possessed higher
The body weights, tumor volumes and survival rates of mice cytotoxicity, since the positive charge of TPP enhanced the
were measured every two days after treatment. To calculate cellular uptake and mitochondrial targeting. The cytotoxic
the tumor volume, the lengths of the major axis (longest activities of TOS or TOS-TPP used in combination with CPT
diameter) and minor axis (perpendicular to the major axis) of a were determined at 0.25, 0.5, 1, 2 and 4 folds of their IC50
tumor were measured with a vernier caliper.16 Survival curves concentrations.23,24 As shown in Fig. 1b, both combinations
were plotted at each time point, and the treatment efficacy exhibited strong growth inhibition at low drug concentrations.
was evaluated according to the 50% mean survival time. After The combination index of TOS and CPT was 0.238, while that of
treatment for 21 days, mice were sacrificed to remove tumors, TOS-TPP and CPT was 0.186. Both combinations showed strong
followed by embedding in paraffin and cutting with a synergism by combination indexes below 0.3.32 The
microtome at a thickness of 4
m. Tumor sections were combination of CPT with TOS-TPP exhibited higher synergistic
stained with hematoxylin and eosin (H&E) to evaluate the cell effect, suggesting that the conjugation of TPP to TOS not only
morphology and tissue necrosis. Immunohistochemical (IHC) enhanced the cytotoxicity of TOS, but also improved the
staining of Ki-67 and caspase-3 was performed on tumor combined effect with CPT.
sections for investigating the proliferation and apoptosis. The
slides were observed with a light microscope (Nikon Eclipse 3.2 Characterization of hybrid micelles
E400, Japan) and the positively stained cells of Ki-67 or
Cystamine was conjugated onto HA to introduce disulfide
caspase-3 were counted in five randomly areas in IHC staining
linkages and terminal amino groups, followed by TOS-TPP
images. The ratios of proliferation or apoptosis were obtained
coupling via a condensation reacton. The substitution degree
of cystamine on HA was around 30%, which was defined as the
by comparing with the total number of cells in the same areas.
number of cystamine molecules per 100 sugar residues of HA.
The substitution degree of TOS-TPP on HA was 15.1%, and the
2.9 Systemic toxicity of hybrid micelle treatment
Except the body weight, the blood and intestinal toxicity are drug loading was aroud 13.0% as determined from the 1H-NMR
important indicators for examining the toxicity of CPT. Mice of spectrum (Fig. S2). HA-ss-TOS-TPP copolymers were self-
each group were randomly chosen and sacrificed on day 21 assembled into MT micelles with an average hydrodynamic
after treatment. Blood was taken from the eyes, and white diameter of 98 nm, while MC micelles had an average size of
blood cells (WBC), red blood cells (RBC) and lymphocytes were around 160 nm (Fig. 1c). As shown in Fig. S4, the CMCs of MT
counted by an auto hematology analyzer (TEK-II MINI, Tecom and MC micelles were around 2.7 and 3.1 μg/mL. Fig. 1d shows
Science Co., China). The jejunums were removed and washed the size and zeta potential of MC-T hybrid micelles self-
with PBS, fixed in 4% formaldehyde, embedded in paraffin, and assembled from blends of HA-ss-CPT and HA-ss-TOS-TPP with
cut into sections for H&E staining.
different ratios. The avergage size of MC-T micelles gradually
decreased with increasing HA-ss-TOS-TPP contents in the
mixtures. In addition, the DLS analysis revealed a single peak,
suggesting that MC-T micelles were in a hybrid not a mixed
state. Fig. 1e shows TEM images of MT and MC-T micelles,
displaying spherical morphologies with average sizes of 46 and
58 nm, respectively.
2.10 Statistical analysis
Data are expressed as mean ± standard deviation. Whenever
appropriate, comparisons among multiple groups were
performed by analysis of variance, while a two-tailed Student’s
t-test was used to discern the statistical difference between
two groups. A probability value (p) of less than 0.05 was
considered to be statistically significant.
3.3 Reductive sensitivity of hybrid micelles
The reductive sensitivities of micelles were monitored with
respect to the varations in size and drug release after
incubation in PBS containing 10 μM and 10 and 40 mM GSH.
As shown in Fig. 2a, the average sizes of MC, MT, and MC-T
micelles were around 167, 98, and 126 nm, respectively, after
3. Results and discussion
3.1 Synergistic effect of CPT and TOS-TPP
Owing to the large potential from -160 to -180 mV across
mitochondrial membrane,17 TOS was modified with cationic
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Fig. 1. Characterization of synergistic cytotoxicities of CPT and TOS-TPP and the formation of hybrid micelles (n = 3). (a) Cytotoxicity to
H22 cells after incubation for 72 h with CPT, TOS and TOS-TPP. (b) Cytotoxicity of CPT combined with TOS or TOS-TPP at 0.25, 0.5, 1, 2
and 4 folds of their IC50 concentrations. (c) Size distributions of MT, MC, and MC-T micelles (MC/MT ratio: 1/2) determined by DLS
measurement. (d) Average sizes and zeta potentials of hybrid micelles with different ratios of MC and MT. (e) Typical TEM images of
MT and MC-T micelles.
3.4 Synergistic cytotoxicity of hybrid micelles
incubation at the plasma GSH level (i.e. 10 μM), indicating no
significant difference from those of prinstine micelles (Fig. 1c). MC and MT micelles had IC50 values of 0.92 and 0.73 μg/mL
In contrast, MC, MT, and MC-T micelles indicated rapid size against H22 cells (Fig. 3a). MC-T hybrid micelles self-assembled
increases to around 985, 451 and 586 nm, respectively, after from HA-ss-CPT and HA-ss-TOS-TPP with different ratios
incubation with 40 mM GSH, corresponding to the GSH showed dose-dependent growth-inhibitory effect (Fig. 3b). Fig.
concentration in cytoplasma of tumor cells.33 It was indicated 3c summarizes combination indexes of MC-T at different molar
that the micelles disintegrated once internalized into tumor ratios of HA-ss-CPT and HA-ss-TOS-TPP. The lowest
cells.
combination index was 0.26 at the Mc/MT ratio of 1/2, which
Fig. 2b,c summarizes the drug release behaviors of MC and was used in the following study. In addition, the dose-
MT micelles, indicating simialr profiles in response to GSH reduction index is a measure of the dose for each drug alone
concentrations. Around 6% of drugs were relased from at a given effect level compared with the dose of each drug in
micelles after 24 h of incubation with 10 μM GSH. The drug a synergistic combination.35 In clinical situations the dose
release from micelles was remarkably accelerated in the reduction alleviated the side effects while retaining the
presence of 10 and 40 mM GSH due to thiol-disulfide exchange therapeutic efficacy. Fig. 3d shows dose-reduction indexes for
reactions. For instance, about 13.6% of TOS-TPP was released various molar ratios of CPT and TOS-TPP. At the synergistic
from MT micelles in 4 h and near 46.5% in 24 h after incubation ratio of CPT to TOS-TPP at 1/2, the dose-reduction indexes of
with 10 mM GSH. As shown in Fig. 2d, there were around 60% CPT and TOS-TPP were 7.7 and 3.4, respectively.
of CPT and TOS-TPP released from MC-T after 24 h of incubation
The cellular uptake of micelles was determined on H22 cells
with 40 mM GSH, while less than 5% in PBS, indicaitng redox- from the intracellular CPT and TOS contents after cell lysis. As
dependent drug release like MC and MT. The release of CPT or shown in Fig. 3e, the cell uptake efficiency was around 34.9%
TOS-TPP from MC-T showed a similar profile, thus, the ratios of and 36.7% for MC and MT micelles, respectively. After
HA-ss-CPT and HA-ss-TOS-TPP used for hybrid micelle incubation with MC-T, the uptake efficiency of CPT is about
preparation should represent the drug ratios after the GSH- 35.2%, similar to that of TOS at about 35.0%. The addition of
responsive release. In addition, the thiolated compounds free HA into the culture media led to over 3-fold decrease in
released after the breakage of disulfide linkages indicated the uptake efficiency to around 10% (p < 0.05), due to
cytotoxicities and cytotoxic mechanisms similar to those of competitive blocking of the receptors.36 In addition, the uptake
free drugs.34 Therefore, the synchronous release of TOS-TPP efficiency of free drug (CPT or TOS) was around 11%, and the
and CPT from MC-T was beneficial to control the ratio of drugs increased uptake efficiency of micelles indicated that the
reaching the cell nuclei and mitochondria.
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Fig. 2. Characterization of GSH responsiveness of MC-T hybrid micelles with MC/MT ratio of 1/2 (n = 3). (a) Typical DLS images of MC-T
,
MT and MC micelles after incubation at 37 °C in PBS and PBS containing 10 μΜ and 40 mM GSH for 48 h. (b) Percent drug release from
MC, and (c) MT micelles after incubation with different GSH concentrations. (d) Percent release of TOS-TPP and CPT from MC-T hybrid
micelles after incubation with different GSH concentrations.
micelles would be good nanocarriers for combinational drug summarizes the percentage of cells in the early and late
delivery. Fig. S5 shows CLSM images of H22 cells after apoptosis stages after treatment with free drug and micelles.
incubaiton with MC and MC-T micelles, indicating similar uptake MC-T induced the highest cell apoptosis of 83.4%, and the
profiles. The internalization of MC was more effective than that treatment with MC-T(1/5) led to about 54.1% of apoptosis rate,
of free CPT due to the CD44 receptor-mediated endocytosis. which was close to those of MC (57.8%) and MT micelles
The addition of HA interfered the endocytosis of MC but did (61.6%).
not affect the internalization of free CPT.
Mitochondrial ROS production is an important mechanism of 3.5 In vivo antitumor efficacy of hybrid micelles
the mitocan-induced activation of cancer cell apoptosis.37 The
intracellular ROS levels were detected on H22 cells after
The in vivo antitumor performance of micelles was
investigated from the tumor growth inhibition, animal survival,
treatment with micelles (MC, MT and MC-T) and free drug (CPT
histological and IHC staining of retrieved tumors. Fig. 4a shows
and TOS), as well as the reduced micelle doses (MC-T(1/5) and
the tumor growth status since the first administration of free
MC-T(1/10)). Fig. S6 shows the flow cytometry analysis of DCF
drugs and micelles, using saline treatment as a control. Mice in
fluorescence, and Fig. 3f summarizes the ROS levels after each
the control group showed a significant increase in tumor
volume to around 3320 mm3 after 21 days. The CPT and TOS
treatment. CPT slightly increased ROS production at 158%
higher than that of controls (p < 0.05), due to inhibition of
treatment indicated around 40% and 32% of tumor growth
oxygen consumption and ROS formation in cells.38 TOS was
inhibition, respectively. The inhibition of MC and MT micelles
subjected to interfere with the ubiquinone-binding site of the
on the tumor progressing was significantly stronger than the
mitochondrial complex II impairing electron transfer flowing
corresponding free drugs. Among the testing groups, MC-T
along the redox chain.39 The TOS treatment resulted in 2.7
showed the superior tumor inhibition (86%) after 21 days of
folds higher ROS levels than controls (p < 0.05). The HA-
treatment (p < 0.05). MC-T(1/5) showed an apparent anticancer
effect (around 1160 mm3), which was close to those of MT and
mediated cellular uptake and GSH-responsive release from MC
Mc treatment (around 1100 mm3). Fig. 4b summarizes the
and MT micelles caused 1.4 and 2.2 folds higher levels than
those of CPT and TOS, respectively (p < 0.05). As shown in Fig.
survival rates of tumor-bearing mice in a KaplanMeier
3f, MC-T induced significantly higher ROS levels than MT, even
MC-T(1/5) showed stronger ROS production than MT micelles
and free drugs (p < 0.05).
The apoptosis induction by micelles was determined by flow
cytometry after Annexin V-FITC and propidium iodide staining.
Fig. S7 shows flow cytometry images of tumor cells and Fig. 3g
plotting. The median survival time was 17, 22 and 23 days after
treatment with PBS, free CPT and TOS. The MC and MT
treatment prolonged animal survivals, and MC-T treatment
displayed an extended median survival time of 34 days. In
addition, 50% of mice survived after treatment with MC-T(1/5)
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Fig. 3. Characterization of synergistic cytotoxicities of hybrid micelles (n = 3). (a) Cytotoxicity to H22 cells after incubation for 72 h with
MC and MT. (b) Cytotoxicities, (c) combination indexes and (d) dose-reduction indexes of hybrid micelles with different ratios of MC and
MT. (e) H22 uptake efficiency of free CPT,TOS, MC, MT and MC-T. (f) Mean DCF fluorescent intensities in H22 cells and (g) cell apoptosis
rate after incubation with PBS, free CPT, TOS, MC, MT, MC-T, MC-T(1/5) and MC-T(1/10).
for 32 days, which was slightly longer than those of MC (29 role in the apoptosis execution, in which caspase-3 plays a
days) and MT (30 days).
central role.42 As revealed in IHC staining images and cell
Necrosis within tumors represents a significant prognostic counting results (Fig. 4e,f and Fig. S8c), 97.4% of cells were
factor of tumor growth and survival of patients after apoptotic after MC-T treatment, which were significantly higher
chemotherapy.40 As shown in Fig. 4c and Fig. S8a, H&E staining than others (p < 0.05). It should be noted that both the cell
images of tumors after micelle treatments led to larger proliferation inhibition and apoptosis induction by MC-T(1/5)
necrotic regions (pink area) than those of free drugs. A large were comparable to those by MC and MT (p > 0.05).
amount of living cells (blue area) were detected in tumors in
the control group, and tumor cells showed obvious nucleolus 3.6 In vivo toxicity after micelle treatment
cleavage and high extent of malignancy. It should be noted
Changes in body weight are one of the safety markers in tumor
that the MC-T(1/5) treatment showed larger area of necrosis
chemotherapy. As shown in Fig. S9a, there was no apparent
comparable to MC and MT. The cell proliferation and apoptosis
weight loss in the groups of TOS and MT micelles due to few
in tumor tissues were evaluated by IHC staining of Ki-67 and
toxicities of TOS to normal tissue. Free CPT treatment led to
caspase-3, respectively. Fig. 4d and Fig. S8b show IHC staining
greater weight reduction than MC and MC-T. There was no
images of Ki-67, a nuclear marker of active cells in the G1, G2,
apparent weight loss when reducing the dose of hybrid
and S phases of the cell cycle.41 As shown in Fig. 4f, the MC-T
micelles like MC-T(1/5) and MC-T(1/10). CPT-associated
treatment led to around 11.2% of proliferative cells in tumors,
leukopenia and gastrointestinal toxicity, especially severe
diarrhea, limit its clinical applicability.43 Fig. S9b summarizes
which was significantly lower than those of MC (39.6%) and MT
(35.2%) micelles (p < 0.05). The caspase family plays a crucial
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Fig. 4. In vivo antitumor efficacy of hybrid micelles. (a) Tumor growth, (b) overall survival of tumor-bearing mice, (c) typical H&E staining
images (“N” represents necrotic area, “T” represents tumor mass), (d) IHC staining images of Ki-67 and (e) caspase-3, (f) the number of
caspase-3 and Ki-67-positive cells compared with the total number of cells in IHC staining images of tumors retrieved on day 21 after
intravenous administration of free CPT, TOS, MC, MT, MC-T, MC-T(1/5) and MC-T(1/10), using saline injection as control (n = 8).
the numbers of WBCs, RBCs and lymphocytes in mice after of TOS showed a peak at 0.9 ppm, and the drug loading was
treatment with free drug and micelles. Free CPT treatment about 5.4%, determined from the integral ratios of peaks at
showed significant decreases in the WBC and lymphocyte 0.9 (-CH3) and 1.87 ppm (-CH3) or 5.13 ppm (-CH(-C)-). GPC
numbers compared with PBS, and the inoculation of CPT in MC- analysis showed that the weight-average molecular weights
T and MC micelles reduced the toxicity. The lower doses of MC- (Mw) of PLA-NH2 and PLA-cdm-MT were 26.3 and 36.8 kDa,
T(1/5) and MC-T(1/10) indicated few toxicities to blood cells. respectively (Fig. S3c). MC@SF, MT@SF, and MC-T@SF were
The gastrointestinal toxicity was examined via H&E staining of obtained by electrospinning of PLA-cdm-MC, PLA-cdm-MT, and
small intestines retrieved after 24 h from the last injection. As their blends at the weight ratio of 1/2, respectively. Fig. 5a
shown in Fig. S9c, epithelial cells and villi were tightly arrayed shows typical SEM image of MC-T@SF, indicating uniform
in the control, MC-T(1/5) and MC-T(1/10) groups, but irregular morphologies with smooth surface. MC@SF, MT@SF, andMC-
villi, crypt shrink or number decrease were obviously observed T@SF showed lengths of 21.4 ± 1.3, 19.5 ± 1.1 and 20.7 ± 1.6
after free CPT treatment. It was indicated that the micelles μm, respectively (p > 0.05), and the average diameter was
could reduce the toxicity of CPT, and the dose-reduced hybrid around 1.62 ± 0.16 μm (Fig. S10a,b).
micelles showed no hematologic and intestinal toxicities.
β-carboxylate amide groups show significant sensitivities to
weakly acidic microenvironment in tumor tissues (e.g. pH
6.8),44 thus, PLA-cdm-MT and PLA-cdm-MC copolymers were
degraded to release micelles from short fibers. Fig. S10c shows
the mass residual of MC-T@SF after incubation in pH 7.4 and pH
6.8 buffers. There was a quick mass loss (around 30%) during
the initial 2 weeks of incubation in pH 6.8 buffers, while
3.7 Characterization of short fibers with loaded hybrid micelles
Scheme 1a shows the synthetic process of PLA-cdm-MT by
conjugation of HA-ss-TOS-TPP with PLA-cdm. As shown in the
1H NMR spectrum of PLA-cdm-MT (Fig. S3a), the methyl group
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Fig. 5. Characterization of short fibers with loaded hybrid micelles. (a) SEM image of MC-T@SF short fibers. (b) Typical GPC elution
profiles of MC-T@SF after incubation at 37 °C in pH 7.4 and 6.8 buffers for 7, 14, 21 and 28 days. (c) Percent release of micelles and (d)
free drugs from MC-T@SF after incubation in pH 7.4 and 6.8 buffers at 37 °C (n = 3). (e) Typical TEM images of MC-T micelles released
from MC-T@SF. (f) Cytotoxicity to H22 cells after incubation for 72 h with MC@SF, MT@SF and MC-T@SF in pH 7.4 and 6.8 media (n =
6). (g) Tumor growth, (h) overall survival of tumor-bearing mice, (i) the number of caspase-3 and Ki-67-positive cells compared with
the total number of cells in IHC staining images of tumors retrieved on day 21 after intratumoral administration of MC@SF, MT@SF,
MC-T@SF and MC-T@SF(1/5), using saline injection as control (n = 8).
around 90% of mass residual was detected after 4 weeks in pH
3.8 Characterization of hybrid micelle release from short fibers
7.4. Fig. 5b shows GPC elution profiles of the retrieved MC-
The acid-labile degradation of MC-T@SF short fibers led to the
T@SF during the degradation process. A shoulder peak
release of amphiphilic copolymers of HA-ss-CPT and HA-ss-
appeared at the eluting time of 14.2 min (26 kDa) after
TOS-TPP. When both amphiphilic polymers were dispersed in
incubation for 1 week in pH 6.8 buffers, and the shoulder peak
an aqueous solution, hybrid micelles were formed with
gradually rose during the following weeks. There was almost
hydrophilic HA shells and hydrophobic cores of CPT and TOS-
no shoulder peaks after incubation in pH 7.4 buffers. It was
TPP.20 Fig. 5c,d shows the percentage of micelles and free
indicated that HA-ss-CPT and HA-ss-TOS-TPP copolymers were
drugs released from MC-T@SF after incubation in pH 7.4 and pH
released into the degradation media after the breakage of
1
6.8 buffers. Compared with around 10% of release in pH 7.4
buffers, around 90% of micelles were released from MC-T@SF
CDM linkers. As shown in the H NMR spectrum, there were
almost no characteristic peaks of HA after 4 weeks of
after 4 weeks of incubation in pH 6.8 buffers due to the acid-
incubation in pH 6.8 buffers (Fig. S10d).
labile breakage of fiber matrix. As indicated above, the similar
degradation profiles of MC@SF and MT@SF led to the
simultaneous release of HA-ss-CPT and HA-ss-TOS-TPP. The
polymer mixtures were self-assembled into MC-T hybrid
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micelles containing the same molar ratio of CPT and TOS-TPP staining. The cell proliferation and apoptosis in tVuiemwoArrtictleisOsnulienes
DOI: 10.1039/C8TB02843E
as the blend ratio in the fiber matrix. There was less than 10% were evaluated by IHC staining of Ki-67 and caspase-3 (Fig.
of drug release after incubation in pH 7.4 and pH 6.8 buffers S12b,c), respectively, and Fig. 5i summarizes the percentage of
for 4 weeks, due to the minimal hydrolysis of HA-ss-CPT and positively stained cells. It was obvious that the MC-T@SF
HA-ss-TOS-TPP (Fig. 5d). In addition, MC@SF and MT@SF treatment induced significantly higher cell apoptosis and lower
showed similar release profiles of micelles (MC and MT) and proliferation compared with the other groups. There were
free drug (CPT and TOS-TPP) to those of MC-T@SF (Fig. S11a,b). around 94.2% of caspase-3-positive cells after MC-T@SF
Fig. 5e shows the typical TEM image of the retrieved MC-T
,
treatment, which was more effective in apoptosis induction
indicating a spherical morphology with an average diameter of than MC@SF and MT@SF at around 66.8% and 73.6%,
57 nm. The retrieved MC and MT micelles had average sizes of respectively (p < 0.05). Compared with around 32% (MC@SF)
68 and 45 nm, which were close to those prepared via and 24% (MT@SF) of proliferative cells, the Ki-67-positive cells
ultrasonication (Fig. S11c,d).
in tumors after MC-T@SF treatment were around 9% (p < 0.05).
The reductive sensitivity of the retrieved micelles was In addition, the treatment with MC-T@SF(1/5) led to around
determined with respect to the drug release and size changes 69.6% and 30.9% of caspase-3 and ki-67-positive cells in
in response to 10
M, 10 and 40 mM of GSH. As shown in Fig. tumors, respectively, indicating similar treatment efficacy to
S11e,f the retrieved MC-T micelles had similar release profiles those of MT@SF and MC@SF.
of CPT and TOS-TPP in response to the GSH levels. There was
Increase of tumor accumulation as well as co-loading and
less than 10% of drug release after 96 h of incubation in PBS coordinate release of multiple drugs represent a challenging
with or without 10 M GSH, while over 85% of release in the task for micelles to achieve a synergistic dose. To address
presence of 10 and 40 mM of GSH. As shown in Fig. S11g, the these challenges, we confirmed the synergy effect of hybrid
incubation with 40 mM GSH led to a significant increase in MC-T micelles, and then developed injectable short fibers as an
size from 132 to 328 nm. In addition, there was no difference implantable depot for micelle releasing to ensure the
in the redox-responsive drug release and size change among synergistic ratio of drugs in the targeting site. The acid-labile
the retrieved MC-T, MT and MC micelles (Fig. S11h,i), which degradation of short fibers led to the release of HA conjugates,
were also similar to those of fresh micelles prepared via followed by self-assembly into hybrid micelles (Fig. 5). The
ultrasonication (Fig. 2).
blend ratio of PLA-cdm-MC and PLA-cdm-MT in short fibers
The cytotoxicities of micelles were determined on H22 cells defined the ratio of MC to MT in MC-T micelles. After HA-
after incubation with short fibers in pH 7.4 and 6.8 buffers. Fig. mediated cellular uptake, the cleavage of disulfide bonds
5f shows the dose-dependent cytotoxicities of short fibers, resulted in effective and synchronous releases of TOS and CPT
indicating significantly higher cytotoxicities in pH 6.8 buffers to maintain the synergistic drug ratio (Fig. 2). Next, we
than in pH 7.4. The IC50 values of MC@SF, MT@SF and MC-T@SF determined the combination index and dose-reduction index
in pH 6.8 buffers were expressed by the free drug of hybrid micelles and confirmed the synergistic therapeutic
concentrations and determined to be 1.41, 1.37, and 0.52 efficacy with dose-reducing superiority. The 5-fold lower dose
μg/mL, respectively, revealing the synergistic performance of of MC-T produced higher ROS levels and induced similar cell
hybrid micelles released from short fibers.
apoptosis rates compared with MC and MT (Fig. 3). In vivo
treatment with MC-T(1/5) showed comparable tumor growth
inhibition, slightly extended animal survival, and higher tumor
necrosis than those of MC and MT (Fig. 4). It is worth to note
that the dose reduction of MC-T significantly alleviated the side
effect of drugs, indicating no hematologic and intestinal
toxicities (Fig. S9). In addition, compared with MT@SF and
MC@SF short fibers, the MC-T@SF(1/5) treatment achieved
similar antitumor profiles with respect to the tumor growth
inhibition, animal survival, histological and IHC staining of
tumor slices (Fig. 5g-i). Therefore, hybrid micelles or micelles-
releasing short fibers with 5-fold reduced doses could maintain
or increase the therapeutic efficacy while minimizing the side
effects, offering high potential for synergistic cancer therapy.
3.9 Antitumor efficacy after short fiber treatment
Fig. 5g summarizes the tumor growth in mice after treatment
with MC@SF, MT@SF and MC-T@SF. The tumors without
treatment grew from around 500 to 3980 mm3 after 21 days,
and the treatment with MT@SF and MC@SF indicated around
68% and 61% of tumor growth inhibition, respectively. The
most significant inhibition of tumor growth (80%) was
detected for MC-T@SF, reaching around 790 mm3 in tumor
volume after 21 days (p < 0.05). Moreover, the treatment with
MC-T@SF(1/5) showed considerable therapeutic effect
comparable to those of MT@SF and MC@SF. The enhanced
treatment efficacy of MC-T@SF was also reflected in the
survival rates of tumor-bearing mice (Fig. 5h), showing an
extended median death time of 30 days. The treatments with
MT@SF,MC@SF and MC-T@SF(1/5) led to 50% dead mice after
25, 22, and 24 days, respectively.
4. Conclusions
Hybrid micelles are developed to coordinately release CPT and
TOS for nucleus and mitochondrion interferences, followed by
electrospinning into injectable short fibers for local tumor
therapy. The synergistic effect of free CTP and TOS-TPP is
confirmed, and the combination index and dose-reduction
index of MC-T micelles are determined. Hybrid micelles
Fig. S12a shows H&E staining images of tumors retrieved
after treatment for 21 days. Intratumoral injection of short
fibers led to obvious necrotic regions in tumors, and MC-T@SF
treatment showed the largest necrotic areas with overall pink
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14 R. R. Thakur, H. L. McMillan and D. S. JoneVsi,ewJA. rtCicolenOtnrloinle.
precisely deliver the drug combination at a synergistic ratio to
the target site by simultaneous intracellular release of the
combinations via synchronous disulfide cleavage, thus
exhibiting significantly higher cytotoxicities. Compared with MC
and MT micelles, MC-T with 5-fold lower doses produce higher
intracellular ROS levels, comparable tumor growth inhibition
and slightly higher animal survival, while indicating no
hematologic and intestinal toxicities. The acid-labile
degradation of MC-T@SF short fibers leads to a sustained
release of MC-T micelles with similar cellular uptake and
cytotoxicity behaviors to those of micelles prepared via
ultrasonication. MC-T@SF with 5-fold lower doses achieve
tumor growth inhibitions comparable to MT@SF and MC@SF
and prolong animal survivals. Therefore, this study validates
that the hybrid micelles or micelles-releasing short fibers could
achieve enhanced therapeutic efficacy with dose-reducing
superiority through drug synergy, highlighting their potential
for synergistic chemotherapy.
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Acknowledgements
This work was supported by National Natural Science
Foundation of China (31771034 and 31470922), and the Key
Research and Development Program of Sichuan Province
(2018SZ0348).
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DOI: 10.1039/C8TB02843E