Acid-responsive nanoparticles as a novel oxidative stress-inducing anticancer therapeutic agent for colon cancer

Article abstract of DOI:10.2147/IJN.S189923

Objective: Nanoparticles can efficiently carry and deliver anticancer agents to tumor sites. Mounting evidence indicates that many types of cancer cells, including colon cancer, have a weakly acidic microenvironment and increased levels of reactive oxygen species. The construction of nano drug delivery vehicles “activatable” in response to the tumor microenvironment is a new antitumor therapeutic strategy. Methods: Cinnamaldehyde (CA) was designed to link directly with dextran to form a polymer through an acid cleavable acetal bond. Herein, a novel pH-sensitive drug delivery system was constructed with co-encapsulated 10-hydroxy camptothecin (HCPT). Dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM) analysis, and release kinetics analysis of HCPT-CA-loaded nanoparticles (PCH) were conducted to investigate the physical and chemical properties. The cellular uptake signatures of the nanoparticles were observed by confocal microscopy and flow cytometry. Cell viability, cell scratch assay, apoptosis assay, and colony formation assay were performed to examine the potent antiproliferative and apoptotic effects of the PCH. The antitumor mechanism of the treatment with PCH was evaluated by Western blotting, flow cytometry, and TEM analysis. The pharmacokinetics of PCH were examined in healthy Sprague Dawley rats within 6 hours after sublingual vein injection. We lastly examined the biodistribution and the in vivo anticancer activity of PCH using the xenograft mouse models of HCT116 cells. Results: Both HCPT and CA were quickly released by PCH in an acidic microenvironment. PCH not only induced cancer cell death through the generation of intracellular reactive oxygen species in vitro but also facilitated the drug uptake, effectively prolonged drug circulation, and increased accumulation of drug in tumor sites. More attractively, PCH exhibited excellent therapeutic performance and better in vivo systemic safety. Conclusion: Overall, PCH not only utilized the tumor microenvironment to control drug release, improve drug pharmacokinetics, and passively target the drug to the tumor tissue, but also exerted a synergistic anticancer effect. The acid-responsive PCH has enormous potential as a novel anticancer therapeutic strategy.

Full text of DOI:10.2147/IJN.S189923

International Journal of Nanomedicine
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Open Access FullText Article
O r I g I N a l r e s e a r c h
acid-responsive nanoparticles as a novel oxidative
stress-inducing anticancer therapeutic agent for
colon cancer
This article was published in the following Dove Medical Press journal:
International Journal of Nanomedicine
chengwei Zhao1,*
Weilan cao2,*
hailun Zheng1,3,*
Zhongxiang Xiao1
Jie hu1,4
Objective: Nanoparticles can efficiently carry and deliver anticancer agents to tumor sites.
Mounting evidence indicates that many types of cancer cells, including colon cancer, have a
weakly acidic microenvironment and increased levels of reactive oxygen species. The construc-
tion of nano drug delivery vehicles “activatable” in response to the tumor microenvironment
is a new antitumor therapeutic strategy.
Methods: Cinnamaldehyde (CA) was designed to link directly with dextran to form a polymer
through an acid cleavable acetal bond. Herein, a novel pH-sensitive drug delivery system was
constructed with co-encapsulated 10-hydroxy camptothecin (HCPT). Dynamic light scattering
(DLS) analysis, transmission electron microscopy (TEM) analysis, and release kinetics analysis
of HCPT-CA-loaded nanoparticles (PCH) were conducted to investigate the physical and chemi-
cal properties. The cellular uptake signatures of the nanoparticles were observed by confocal
microscopy and flow cytometry. Cell viability, cell scratch assay, apoptosis assay, and colony
formation assay were performed to examine the potent antiproliferative and apoptotic effects
of the PCH. The antitumor mechanism of the treatment with PCH was evaluated by Western
blotting, flow cytometry, and TEM analysis. The pharmacokinetics of PCH were examined in
healthy Sprague Dawley rats within 6 hours after sublingual vein injection. We lastly exam-
ined the biodistribution and the in vivo anticancer activity of PCH using the xenograft mouse
models of HCT116 cells.
lehe Yang1
Min chen1
guang liang1
suqing Zheng1
chengguang Zhao1
1chemical Biology research center,
school of Pharmaceutical sciences,
Wenzhou Medical University,
Wenzhou, Zhejiang 325035, china;
2Department of coloproctology,The
second affiliated hospital and Yuying
children’s hospital,Wenzhou Medical
University,Wenzhou, Zhejiang 325035,
china; 3Department of Pharmacy,
The second affiliated hospital and
Yuying children’s hospital,Wenzhou
Medical University,Wenzhou, Zhejiang
325035, china; 4state Key laboratory
of Quality research in chinese
Results: Both HCPT and CA were quickly released by PCH in an acidic microenvironment.
PCH not only induced cancer cell death through the generation of intracellular reactive oxygen
species in vitro but also facilitated the drug uptake, effectively prolonged drug circulation,
and increased accumulation of drug in tumor sites. More attractively, PCH exhibited excellent
therapeutic performance and better in vivo systemic safety.
Medicine, Institute of chinese Medical
sciences, University of Macau,Taipa,
Macau 999078, china
Conclusion: Overall, PCH not only utilized the tumor microenvironment to control drug
release, improve drug pharmacokinetics, and passively target the drug to the tumor tissue, but
also exerted a synergistic anticancer effect. The acid-responsive PCH has enormous potential
as a novel anticancer therapeutic strategy.
*These authors contributed equally
to this work
Keywords: cinnamaldehyde, hydroxy camptothecin, ROS, pH-responsive nanoparticles, colon
cancer
correspondence: chengguang Zhao;
suqing Zheng
chemical Biology research center,
school of Pharmaceutical sciences,
Wenzhou Medical University,
Building 11, chashan street, University
Town, Wenzhou, Zhejiang 325035, china
Tel/fax +86 577 8669 9057
Introduction
In the last 2 decades, the use of stimulus-responsive nanoparticles has emerged as a
significant approach to selectively deliver antitumor drugs to cancerous sites within the
human body. External or physiological stimuli, such as light,1–3 temperature,4,5 ultra-
sound,6–8 magnetic force,9–11 enzymes,12,13 pH,14–17 reductive18 or oxidative stress,19,20 have
email zhaochengguang@wmu.edu.cn;
zsq_wmu@126.com
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hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission
for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
http://dx.doi.org/10.2147/IJN.s189923

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been used for triggering drug delivery and controlled release.
HCPT-CA-loaded nanoparticles (PCH) can release HCPT
Stimulus-responsive nanoparticles could provide a platform to and CA in an acidic microenvironment quickly. In addition,
reduce the side effects of free drugs, high toxicity, nonspecific PCH not only induced cancer cell death through the enhance-
biodistribution, and even multidrug resistance.21–23 Due to the ment of ROS level in vitro but also facilitated the drug
wide variation of the pH value in the human body, the pH value uptake, effectively prolonged drug circulation, and increased
has been extensively exploited for stimuli-responsive drug accumulation drug in tumor sites. Notably, the PCH showed
delivery. As the pH value has been found to be decreased in excellent therapeutic performance and better in vivo systemic
most solid tumors, drug delivery systems that respond to the safety. It is anticipated that the acid-responsive PCH have
slightly acidic extracellular environment of solid tumors have enormous potential as a novel anticancer therapeutic strategy.
been developed as a powerful strategy for tumor targeting.24
Materials and methods
Reactive oxygen species (ROS), such as H2O2, oxygen
radicals (O2.-) and hydroxyl radicals, are normal byproducts Materials
of a normal cellular metabolism in all aerobic organisms.25,26 Dextran (Mw =20,000) was obtained from Leuconostoc spp.;
Moderate increase of ROS can promote cell proliferation and HCPT, CA, CPT, pyrene, and trimethoxymethane were pur-
differentiation,27,28 while excessive ROS production can lead chased from Energy Chemical (Shanghai, China). Pyridinium
to oxidative damage of lipids, proteins, and DNA.29 There- p-toluenesulfonate, indocyanine green (ICG), DMSO, metha-
fore, the maintenance of ROS homeostasis is essential for nol, and other organic solvents, unless otherwise specified,
cell growth and survival. Compared to normal cells, cancer were purchased from Aladdin (Shanghai, China).
cells are usually in an uncontrolled state and have higher
ROS levels and antioxidant activity. However, cancer cells cell lines and antibodies
cannot tolerate additional oxidative stress and are susceptible Human colon cancer cell lines SW620 and HCT116 (3–4
to excessive ROS.30,31 Therefore, the regulation of ROS levels generations) were purchased from the Institute of Biochemistry
is a strategy that selectively kills cancer cells without causing and Cell Biology, Chinese Academy of Sciences (Shanghai,
serious toxicity to surrounding normal cells.
China). HCT116 cells were routinely cultured in McCoy’s
Cinnamaldehyde (CA) is a major component in the bark 5A medium containing 10% heat-inactivated FBS. SW620
of cinnamon trees, which has been widely used as dietary cells were routinely cultured in DMEM containing 10%
factor and food additive. Notably, CA and its analogs have heat-inactivated FBS. The cells were cultured in a humidified
been reported to inhibit the growth of various human cancer cell incubator with an atmosphere of 5% CO2 at 37°C. Antibod-
cells and induce cancer cell death through ROS generation, ies including anti-cleaved PARP and anti-P53 were purchased
ROS-mediated mitochondrial permeability transition, and from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
caspase activation.32–34
Antibodies including anti-mouse IgG-HRP (7076S), anti-
Although CA shows potent anticancer activity, its rabbit IgG-HRP (7074S), anti-BCL-2, anti-BAX, anti-CHOP,
clinical application is still limited by the ease with which anti-ATF4, anti-p-elf2α, and anti-GAPDH were purchased
aldehyde is oxidized in vivo and its lack of specificity for from Cell Signaling Technology (Danvers, MA, USA). The
pathological tissues.35,36 In this study, CA was directly linked annexin fluorescein isothiocyanate-labeled annexin V (Annexin
with dextran, biocompatible polymer of glucose, through V-FITC) apoptosis detection kit I and propidium iodide
acetal linkage. The polymer can spontaneously form acid- (PI) were purchased from BD (Franklin Lakes, NJ, USA).
responsive nanoparticles in water. Camptothecin (CPT) is
synthesis of ph-responsive dextran-ca
a kind of quinoline alkaloid with cancer cell cytotoxicity,
which can inhibit the activity of the DNA enzyme topo I.37 acetal conjugates polymers (Pc)
In clinical trials, CPT showed significant anticancer activity, CA (1 g, 6.17 mmol), trimethoxymethane (2.94 g,
but its solubility is low and adverse drug reactions occur. 27.75 mmol), and pyridinium p-toluenesulfonate (0.307 g,
Additionally, CPT has been reported to induce apoptosis 1.23 mmol) were dissolved in a suitable amount of methanol.
by promoting ROS production.37 Moreover, its derivative, Then the mixture was stirred for 3 hours at 60°C and moni-
10-hydroxy camptothecin (HCPT), has been shown to be tored by thin layer chromatography, with the mobile phase:
more potent and less toxic than CPT. In this study, HCPT petroleum ether/ethyl acetate =20:1. After completion, the
was chosen as model drug to play a synergistic role with CA mixture was quenched with NaHCO3 saturated solution, the
in promoting ROS production.
reaction mixture was extracted with ethyl acetate twice and
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Zhao et al
the organic layers were collected, dried over Na2SO4, and then and pH 6.5) under stirring. At appropriate time intervals, the
evaporated using a rotary evaporator. The reaction residue size and zeta potential of nanoparticles (resuspended in PBS,
was used in the next step without further purification.
0.25 mg/mL) were measured by DLS (Malvern Zetasizer
To synthesize dextran-CA acetal conjugates polymers Nano ZS) with 2-minute data acquisition time.
(PC), dextran (100 mg) was dissolved in anhydrous DMSO
Preparation of Pch and determination
(2 mL), then CA acetal (0.75 g, 2eq per hydroxyl group),
of the encapsulation efficiency (EE) of
molecular sieves (5 Å, 130 mg), and pyridinium p-toluene
sulfonate (25 mg, 5% mol per hydroxyl group) were added nanoparticles
to the mixture. The mixture was stirred for 72 hours at 60°C First, we mixed the 0.5 mL PC polymer solution (DMSO,
under N2 atmosphere. After completion, the molecular sieves 10 mg/mL) with 0.5 mL HCPT solution (DMSO, 6 mg/mL),
were removed by filter under vacuum. The product obtained vortexed it for 2 minutes, then added the solution to the
(PC) was precipitated in cold ethanol several times. Finally, 20 mL PBS (pH 7.4) under stirring and stirred the mixture for
the dextran derivative was dissolved in deionized (DI) water 0.5 hour. The mixture was dialyzed in DI water (MWCO:
and dialyzed for 48 hours in DI water (MWCO: 3.5 kDa) upon 3.5 kDa) upon stirring to remove DMSO and unencapsu-
stirring to remove DMSO and other little molecular compound. lated HCPT for 24 hours. The PCH were lyophilized from
Pure PC were obtained after freeze-drying and verified by pro- water. After measuring the absorption intensity, the amount
ton nuclear magnetic resonance (1H-NMR) spectra (AVANCE of HCPT loaded in the nanoparticles was calculated against
500 spectrometer, Bruker Corporation, Billerica, MA, USA). the calibration curve. The EE is calculated with the follow-
ing equation: EE (%) = (weight of loaded drug)/(weight of
Preparation and characterization
initially added drug)×100.
of Pc nanoparticles
To 20 mL of PBS (pH 7.4) was added 1.0 mL of polymer PC release kinetics of Pch
solution in DMSO (5 mg/mL) dropwise under stirring. The In order to determine the release characteristics of HCPT and
resulting mixture was stirred for 5 minutes. The size and zeta CA in vitro environment, we transferred 3 mL of PCH solu-
potential of nanoparticles (resuspended in PBS, 0.25 mg/mL) tion (0.25 mg/mL) into a dialysis bag (MWCO =1,000) and
were measured by dynamic light scattering (DLS) (Malvern soaked in 50 mL of PBS (pH 5.0 or 7.4) at 37°C. The medium
Zetasizer Nano ZS, Malvern Instruments, Malvern, UK) with was collected at different intervals and the same amount of
2-minute data acquisition time. Transmission electron micros- fresh medium was added. The release amount of HCPT and
copy (TEM) samples were prepared by resuspension of nanopar- CA was calculated respectively according to the calibration
ticles in DI water with a concentration of 0.125 mg/mL. The curve.
samples were dropped onto the copper grid, dried in the air, and
then observed with TEM (H-7500, Hitachi Ltd., Tokyo, Japan). cellular uptake assay
Cells were treated with PCH (including ICG) for 1 hour,
Determination of critical micelle
2 hours, and 4 hours in McCoy’s 5A medium (pH 7.4 or 6.5),
concentration (cMc) of Pc nanoparticles washed twice with PBS, and stained with Hoechst 33258
First, 1 mL of nanoparticles solution with different concen- staining solution and then we captured images by using a con-
trations was added to a 1.5 mL tube which contained the focal laser scanning microscope (Nikon Corporation, Tokyo,
same concentration and excessive pyrene (6×10-7 M), kept Japan) with 40× amplification. Three microscopic fields were
at room temperature for 24 hours. The fluorescence emission selected randomly in each group. Quantitative data of ICG
spectra of pyrene-loaded nanoparticles were recorded using a uptake were measured by flow cytometry (FACSCalibur flow
fluorospectrometer with emission at 390 nm. The excitation cytometer, BD Biosciences, San Jose, CA, USA).
intensity ratio (I339/I336) was plotted against the logarithm of
the polymer concentration.
MTT cell viability assay
Cells were plated at a density of 8×103 cells per well into
96-well plates and then allowed to attach overnight in
McCoy’s 5A medium containing 10% heat-inactivated FBS.
The stability and responsiveness
of Pc micelles
An amount of 1.0 mL of polymer PC solution (DMSO, Cells were incubated with various concentrations of CA
5 mg/mL) was added dropwise to 20 mL of PBS (pH 7.4 (3.1µg/mL), HCPT(10µg/mL), CA+HCPT(3.1+10µg/mL),
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PC (36.2 µg/mL), PC + HCPT (36.2+10 µg/mL), and PCH protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA,
(46.2 µg/mL) (10 µg/mL equivalent HCPT). The cell viability USA). After adding the sample to loading buffer, the pro-
was determined by MTT assay after 48 hours.
tein sample was transferred to PVDF transfer membrane by
electrophoresis and then blocked for 1.5 hours with fresh
5% non-fat milk in tris-buffered saline with Tween 20 buf-
colony formation assay
We plated 1,000 cells on six-well plates overnight and then fer at room temperature. Membranes were incubated with a
incubated with drugs (equivalent to 10 µg/mL HCPT) or 1:1,000 dilution of specific primary antibody and 1:10,000
DMSO for 24 hours. Then cells were washed twice with PBS HRP conjugated secondary antibody. Membranes were inves-
and allowed to grow in McCoy’s 5A medium for 1 week. tigated using ImageJ computer software (National Institute
Cells were washed twice with PBS and fixed with parafor- of Health, Bethesda, MD, USA).
maldehyde for 10 minutes. The cells were washed twice with
PBS and stained with 1% crystal violet (25% methanol) at electron microscopy
room temperature for 15 minutes, washed, and dried.
HCT116 cells were incubated with DMSO, NAC (5 mM),
PCH (46.2 µg/mL) or NAC + PCH (5 mM +46.2 µg/mL) for
6 hours. Then the cells were harvested and soaked in 2.5%
glutaraldehyde overnight at 4°C. The cells were post-fixed
cell migration assay (in vitro wound-healing
assay)
HCT116 cells were plated on six-well plates overnight, and in 1% glutaraldehyde at room temperature for 60 minutes,
the white tip was used to scratch the cells with the same stained with 1% OsO4, dehydrated through graded acetone
width, then washed twice with PBS. After washing, cells solutions, and embedded in Epon. The areas containing cells
were incubated with CA (3.1 µg/mL), HCPT (10 µg/mL), were collected and cut into 70 nm slices, and detected by
CA + HCPT (3.1+10 µg/mL), PC (36.2 µg/mL), PC + HCPT electron microscopy (H-7500, Hitachi Ltd.).
(36.2+10 µg/mL), PCH (46.2 µg/mL) or DMSO for 12 hours.
It was then washed twice with PBS and allowed to grow in Measurement of rOs generation
McCoy’s 5A medium for 48 hours. The microscopic camera Flow cytometry (FACSCalibur flow cytometer) was used
system (Nikon Corporation) was used to capture images. The to measure the generation of ROS in cells. HCT116 cells
wound healing rate was calculated with the formula: (percent were plated on six-well plates, incubated overnight, and
wound healing) = average of [(gap area: 0 hour)-(gap area: treated with PCH for 4 hours, HCPT for 1 hour, and CA for
48 hours)]/[gap area (DMSO): 0 hours].
6 hours, then, ROS probe 2, 7,-dichlorofluorescein diacetate
(DCFH-DA) (10 µM) was added to fresh medium and incu-
bated in darkness for 30 minutes. Cells were harvested and
cell apoptosis analysis
HCT116 cells were plated on six-well plates for 24 hours, analyzed for fluorescence intensity using a flow cytometer
and then incubated with different concentrations of PCH (BD Biosciences). Before the cells were exposed to the com-
(23.1, 46.2 or 92.4 µg/mL), N-acetyl-cysteine (NAC) pound, they were pretreated with 5 mm NAC for 0.5 hour
(5 mM), NAC + 2PCH (5 mM +92.4 µg/mL) or DMSO and then the amount of ROS was analyzed. The statistics
for 12 hours. Cells were collected, washed twice with ice- were analyzed using FlowJo VX.
cold PBS, and stained with FITC conjugated Annexin V
and PI for 30 minutes in the binding buffer. Apoptosis was In vivo imaging and biodistribution
analyzed by flow cytometry (FACSCalibur flow cytometer; All animal experiments complied with the Wenzhou Medical
BD Biosciences).
University’s Policy on the Care and Use of Laboratory
Animals. Protocols for animal studies were approved by the
Wenzhou Medical University Animal Policy and Welfare
Western blot analysis
The cells were harvested within 24–36 hours at drug or Committee. BALB/cA nu/nu female mice (18–22 g) bearing
DMSO concentration of approximately 80%, and then lysed HCT116 tumors with volume approximately 150 mm3 were
in a cold cell lysis buffer containing phosphatase inhibitors intravenously (iv) injected with free ICG (2 mg/kg) or ICG-
and protease inhibitors. Then the sample was disposed by loaded PCH nanoparticles (ICG/PCH) (2 mg/kg equivalent
centrifugation (12,000 rpm, 4°C) for 10 minutes. Protein ICG) (n=6). At 6 hours, 24 hours, and 48 hours after injection,
concentrations in all samples were detected by the Bradford the intensity of fluorescence signal in animals was observed
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using an in vivo imaging system (CRi maestro, Cambridge specified time point. At the end of the experiments, animals
Research & Instrumentation, Inc., Hopkinton, MA, USA) were sacrificed, and the tumors were excised and weighed
with the excitation wavelength of 704 nm and a filter of for use in the histology and Western blot analysis.
735 nm. Some mice in each group were sacrificed at 24 hours
after injection, and the organs such as heart, liver, spleen, lung, h&e staining
kidneys, and tumor were excised for fluorescent imaging.
The harvested heart, kidney, and liver tissues were soaked
in 4% formaldehyde, dehydrated with an ethanol gradient,
embedded in paraffin, and the paraffin tissue sections (5 µm)
were stained with H&E. Images of each slice were taken
Pharmacokinetics study of Pch
nanoparticles in vivo
All animal experiments complied with the Wenzhou Medical using an optical microscope, (kidney: 40× amplification,
University’s Policy on the Care and Use of Laboratory heart and liver: 20× amplification, Nikon Corporation).
Animals. Protocols for animal studies were approved by
statistical analysis
the Wenzhou Medical University Animal Policy and Welfare
Data were presented as mean standard error (SE). The Stu-
dent’s t-test or one-way ANOVA for multiple comparisons
in GraphPad Prism 7 (GraphPad Software, Inc., La Jolla,
CA, USA) were used to determine the statistical differ-
ences between groups. P,0.05 was considered statistically
significant.
Committee. PCH (equivalent to 5 mg/kg HCPT) or free HCPT
(5 mg/kg) were iv injected into female Sprague Dawley rats
weighing 200–220 g to analyze pharmacokinetic characteris-
tics of PCH. Blood samples (200 µL) were collected from the
tail vein into heparinized tubes at appropriate intervals (5, 15,
30, 45, 60, 90, 120, 180, 240, 360 minutes) after administra-
tion (n=5 at each time point), centrifuged (10,000× rpm for
10 minutes at 4°C) to harvest the plasma, and stored at -25°C
until required for analysis. The samples were pre-incubated
Results
synthesis and characterization of Pc
with acetic acid (10%) for 24 hours, HCPT in plasma was nanoparticles
extracted with methanol. The HCPT concentrations in plasma PC can be easily synthesized from CA through a two-step
were detected using high performance liquid chromatography reaction. First, CA is reacted with trimethyl orthoformate in
(1260 VWD, Agilent Technologies, Santa Clara, CA, USA) methanol in the presence of pyridinium p-toluenesulfonate
with CPT as internal standard.
to produce CA acetal, which reacts with dextran to generate
the corresponding product dextran CA acetal copolymers
(Scheme S1). The successful connection between CA and
In vivo antitumor study
All animal experiments complied with the Wenzhou Medical dextran was confirmed by nuclear magnetic resonance. Based
University’s Policy on the Care and Use of Laboratory on the 1H-NMR spectrum of the copolymer, the calculated
Animals. Protocols for animal studies were approved by the conjugation ratio was 10%–11%. To further confirm the
Wenzhou Medical University Animal Policy and Welfare successful modification and clearly demonstrate the degra-
Committee. For in vivo experiments, 5-week old athymic dation behaviors of the polymer in an acidic environment,
BALB/cA nu/nu female mice (18–22 g) were purchased from acid-induced polymer decomposition was investigated in an
1
Vital River Laboratories (Beijing, China). Standard rodent acid solution by H-NMR spectroscopy (Figure 1). When
diet and water were fed to laboratory animals under standard the pH value was adjusted from 7.4 to 6.5, acetal protons
experimental animal conditions. The mice were randomized (~6.2 ppm) disappeared and aldehyde protons appeared at
to four groups of six per group. A subcutaneous tumor was 9.5 ppm. These changes demonstrated that CA was degraded
established by injecting HCT116 cells (5×106 cells in 0.1 mL from dextran CA acetal conjugates under acidic conditions.
PBS) into the right flank of nude mice. According to the
Amphiphilic dextran CA acetal copolymers have the
dosage of 5 mg/kg HCPT, HCPT + CA and PCH water- ability to self-assemble in aqueous solution to form stable
soluble preparations were intraperitoneally injected into the nanoparticles with a hydrophilic dextran shell and CA
nude mice when the tumor volume reached 100–200 mm3. segments in the hydrophobic core. The average diam-
For the control group, liposome carrier in PBS was injected. eter is approximately 182.3 nm (polydispersity index
The volume of the tumor (V=0.5×l×w2) was calculated by [PDI] ~2.25×10-2), as determined by DLS and TEM analysis
measuring the length (l) and width (w) of the tumor at the (Figure 2A). Studies have shown that when the nanoparticle
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Figure 1 1h-NMr of dextran-cinnamaldehyde acetal conjugates.
Note: 1h-NMr spectrum of dextran-cinnamaldehyde acetal conjugates at normal ph (7.4) and at acidic ph (6.5) in DMsO-d6, aldehyde protons were observed at 9.5 ppm.
Abbreviations: ca, cinnamaldehyde; Dex, dextran; 1h-NMr, proton nuclear magnetic resonance.
size is ,200 nm, they preferentially accumulate in tumor probe encapsulated in the hydrophobic core forming ther-
sites due to enhanced permeability and retention effects. modynamically stable micelles was higher than 70.7 g/mL
The zeta potential of the nanoparticles was determined to (Figure 2B).
be ~-3.86 mV at pH 7.4.
acid-responsive degradation of Pc
nanoparticles in vitro
The acetal linkage is usually stable at physiological neutral
The cMc of dextran ca acetal
copolymers
Pyrene was selected as a hydrophobic fluorescent probe to pH and is rapidly hydrolyzed at acidic pH. Along with the
determine the CMC of the nanoparticles. The pyrene was hydrolysis of the CA acetal from dextran, the nanoparticles
combined with the different concentrations of polymer and are broken into water-soluble fragments, resulting in the
the fluorescence intensity of the pyrene-loaded micelles was rapid dissociation of nanoparticles. The pH-dependent
monitored at an excitation wavelength of 339 and 336 nm and stability of PC at pH 7.4 and 6.5 was examined by DLS
at an emission wavelength of 390 nm. Their ratio (I339/I336) analysis. As shown in Figure 2C and D, the average particle
was measured as an indicator of micelle hydrophobicity. The size increased from 182 nm to 600 nm over 24 hours at
plot of I339/I336 vs the logarithm of the polymers’ concentra- pH 6.5 (Figure 2C), whereas the average size remained stable
tions demonstrated that the concentration of fluorescent at pH 7.4 (Figure 2D) for 5 days.
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Notes: (A) a representative transmission electron microscopy (TeM) image and dynamic light scattering (Dls) analysis of nanoparticles in phosphate buffer (ph 7.4).
Dh ~182.3 nm, polydispersity index ~0.225, ζ ~-3.86 mV. (B) The critical micelle concentration of nanoparticles. (C, D) DLS profiles of nanoparticles recorded at different
ph values. (E) release kinetics of 10-hydroxy camptothecin (hcPT) from hcPT-ca-loaded nanoparticles (Pch) at ph 7.4 and 5.0. (F) release kinetics of cinnamaldehyde
(ca) from Pch at ph 7.4 and 5.0. Data presented as mean sD (n=3).
57.6%, respectively. The average nanoparticle size decreased
characterization of Pch and
acid-responsive drug release in vitro
The hydrophobic anticancer drug HCPT was chosen for
encapsulation in PC nanoparticles to determine whether
PC nanoparticles can effectively release the encapsulated
drugs in a weakly acidic environment. The drug loading
from ~182.3 nm to ~166.4 nm (PDI ~0.15) as a result of
increased hydrophobic core. The zeta potential of the nanopar-
ticles decreased to ~16.4 mV, which indicated that the drug
was effectively encapsulated. The release kinetics of HCPT
and CA from PCH were investigated at different pH values.
content and EE were calculated to be 0.216 mg/mg and As shown in Figure 2E, at pH 5.0, most of the HCPT was
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released within 10 hours and 90% of the HCPT was released weaken antioxidant defenses or increase ROS production
within 80 hours, while at pH 7.4 only a very small amount to levels or thresholds at which cells cannot survive. It has
(45%) of HCPT was released after 80 hours. The amount of been reported that both CA and HCPT can induce intracel-
CA released at pH 5.0 within 80 hours was more than twice lular ROS production.32–34,37 Accordingly, we wondered
that at physiological pH 7.4 (Figure 2F). These findings whether PCH could selectively kill malignant tumor cells
validated our hypothesis that PCH nanoparticles have good by increasing ROS levels without causing significant
pH-responsiveness and the ability to act as a pH-responsive toxicity to normal cells. The effect of PCH nanoparticles
drug delivery system.
on cell viability was assessed by the MTT assay in non-
malignant human lung epithelial BEAS-2B cells, human
vascular endothelial HUVEC cells, human cardiomyocytes
4910, and human colon cancer HCT116 cells at 48 hours.
Uptake of nanoparticles by cancer cells
in vitro at different ph values
Since free drugs are taken up into cells through a rapid dif- As shown in Figure 4A, PC and PCH showed negligible
fusion process, and drug carrier particles are internalized cytotoxicity against normal BEAS-2B cells, these results
into cells by endocytosis relying on energy, drug carriers suggested that PC or PCH nanoparticles had good cell
are usually taken up slower, and more easily affected by the biocompatibility compared with other free non-selective
microenvironment conditions. We chose ICG as a model therapeutic drugs, with significant cytotoxicity against
molecule to examine the cellular uptake of nanoparticles normal cells. HUVEC cells and 4910 cells showed similar
into the colon cancer HCT116 cells at different pH values. results (Figure 4B and C). In addition, cell viability of colon
The ICG uptake by HCT116 cells was examined by confocal cancer HCT116 cells was also investigated. CA and PC
microscopy at different pH values. As shown in Figure 3A, showed minimal cytotoxicity, due to the lower drug efficacy
after 2 hours’ incubation, a sharp near-infrared (NIR) fluo- of CA, which is consistent with previous studies.41 However,
rescence signal could be detected at a slightly more acidic HCPT and HCPT combined with CA or PC plus HCPT
condition (pH 6.5) than at a normal pH condition, indicating all exhibited significantly cytotoxic effects. Notably, PCH
that ICG is more effectively distributed into cells under acidic also showed significant cytotoxicity against colon cancer
conditions. More significantly, ICG encapsulated by PC can HCT116 cells (Figure 4D). These data confirmed that PCH,
be more effectively translocated into nucleus after internal- once absorbed by cancer cells, can effectively release the
ization. To further confirm the differences in the subcellular HCPT and CA, thereby making the encapsulated drug as
localization of ICG, quantitative data were collected by flow effective as the free drugs.
cytometry (Figure 3B and C). Compared to the impact on
To further evaluate the effects of PCH on cancer cell
uptake at different pH values, the uptake increased as the growth, colony formation assay was performed with SW620
pH value decreased. The results revealed that more ICG was colon cancer cells (Figure 4E). CA alone had no impact on
present in HCT116 cells incubated at pH 6.5, than in cells the growth and proliferation of colon cancer cells. PC alone
incubated at pH7.4 (95.74% fluorescence intensity of pH 6.5 exhibited moderate anti-proliferation activity against
group), which is consistent with the previously mentioned SW620 colon cancer cells. Similar to the free drugs, PCH
observation. These results indicate that the weakly acidic significantly inhibited the growth and proliferation of human
tumor microenvironment can accelerate the delivery of drugs colon cancer cells, leading to apoptosis.
carried by nanoparticles into HCT116 cells, particularly
into the nuclei.
As is widely known, malignant cancer cells have invasive
and migratory abilities. To determine whether PCH has any
effect on the migratory capacity of cancer cells, cell scratch
assays were conducted with HCT116 cells. As shown in
Figure 4F, CA and PC showed a negligible inhibitory effect
The anti-proliferation and anti-migratory
effects of Pch
Mounting evidence suggests that normal cells have lower on the migratory ability of HCT116 cells and even promoted
ROS levels and an appropriate increase of ROS levels it. In comparison, PCH exhibited a similar effect to that of the
can promote cell proliferation and differentiation without combination of HCPT with CA, leading to a potent inhibition
causing cell damage. In comparison, ROS levels in various of cell migration, which is one of the keys to cancer treat-
types of cancer cells have higher ROS levels than normal ment. Quantitative data are shown in Figure S1. However,
cells.38–40 Consequently, cancer cells are more susceptible to the mechanism remains unknown, thus further studies are
ROS damage induced by exogenous substances that either required to confirm our hypothesis.
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blue; the fluorescence of ICG is red. (B) Flow cytometric analysis of hcT116 cells’ endocytosis incubated with Icg-loaded nanoparticles for 2 hours. (C) geometric mean
fluorescence of HCT116 cells after incubation at different pH values, for different time periods. *P,0.05, compared with the corresponding con group (n=3).
Abbreviations: ca, cinnamaldehyde; con, control; hcPT, 10-hydroxy camptothecin; Icg, indocyanine green; Pch, hcPT-ca-loaded nanoparticles.
inhibitor of ROS production, significantly alleviated PCH-
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induced apoptosis in colon cancer cells. Quantitative data
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cytometric analysis using Annexin V-FITC and PI staining
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treatment with PCH. After PCH treatment for 24 hours, the
colon cancer cells underwent apoptosis in a dose-dependent
apoptotic manner (Figure 5A). Noteworthy, NAC, a specific proteins PARP, P53 and BAX and decreased the expression
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Zhao et al
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Notes: (A) Normal Beas-2B cells’ survival rate determined by MTT assay. (B) cardiomyocytes 4910 cell survival rate determined by MTT assay. (C) human saphenous
vein endothelial cell hUVec survival rate determined by MTT assay. (D) colon cancer cell hcT116 survival rate determined by MTT assay. ***P,0.001, ****P,0.0001,
compared with DMsO (n=3). (E) The results of hcT116 cell scratch experiments. (F) The results of colony formation assay, quantitative data are shown in Figure s1.
(Pch equivalent to 10 µg/ml hcPT).
Abbreviations: ca, cinnamaldehyde; con, control; hcPT, 10-hydroxy camptothecin; Pch, hcPT-ca-loaded nanoparticles.
of anti-apoptotic protein BCL-2. However, these changes regulation of apoptosis. To further confirm whether PCH
were partially mitigated by pre-incubation with NAC. has an effect on mitochondria morphology, we performed
Besides being involved in apoptosis, studies have shown that electron microscopy analysis to examine the mitochondrial
BAX and BCL-2 play important roles in the mitochondrial morphology in HCT116 colon cancer cells exposed to PCH
pathways. Mitochondria are known to be important in the for 6 hours in the presence or absence of NAC. As shown
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Notes: (A) hcT116 cells were pre-incubated with or without 5 mM Nac for 0.5 hour before exposure to Pch at the indicated concentrations for 12 hours. The percentage of
cell apoptosis was determined by Annexin-V/PI staining and flow cytometry. (B) The percentage of apoptotic cells in the treatment groups was calculated. **P,0.01;
***P,0.001, compared with the con group (n=3). (C) The colon cancer cell lines were pre-incubated with or without 5 mM Nac for 0.5 hour before exposure to Pch
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section. The morphology of mitochondria in hcT116 cells was examined with a transmission electron microscope (×10,000 or ×50,000). results from a representative cell
sample out of three studied in each group are shown. The shape of the mitochondria is normal in the control group cells, the Pch + Nac (5 mM) group cells, and Nac
(5 mM) group cells (arrows indicate normal mitochondria). exposure to Pch for 6 hours caused mitochondrial dysfunction in the Pch group cells (arrows indicate swollen
mitochondria). (Pch equivalent to 10 µg/ml hcPT).
Abbreviations: Annexin V-FITC, annexin fluorescein isothiocyanate-labeled annexin V; CA, cinnamaldehyde; Con, control; HCPT, 10-hydroxy camptothecin; NAC,
N-acetyl-cysteine; Pch, hcPT-ca-loaded nanoparticles; PI, propidium iodide; se, standard error.
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in Figure 5D, the mitochondria in cells treated with PCH PCH can remarkably enhance intracellular ROS accumula-
were abnormally enlarged or swollen, showing shredded tion in HCT116 cells.
cristae and impaired outer membrane integrity. In addition,
Research has shown that oxidative stress can induce
the NAC pre-treatment reversed the PCH-induced mitochon- tumor cell apoptosis by activating ER stress.42 Accordingly,
drial swelling. Noteworthy, the NAC alone had no effect on we hypothesized that PCH might activate ER stress by
mitochondrial morphology. These results demonstrated that increasing ROS production. We confirmed our hypothesis
PCH may induce ROS production and cause cancer cells’ by Western blot analysis. PCH regulated the expression
mitochondrial dysfunction and apoptosis.
of ER-stress-related proteins (such as ATF4, p-elf2α, and
CHOP) in a time-dependent and dose-dependent manner
(Figures 6B and C, S3, S4). However, pre-treatment with
NAC clearly reversed these changes. Electronic microscopy
was used to examine the effect of PCH on the morphology
Pch-activated endoplasmic reticulum
(er) stress signaling by promoting the
accumulation of rOs
To further confirm our hypothesis, ROS levels in HCT116 of the ER in HCT116 cells. As shown in Figure 6D, the ER
colon cancer cells were determined by flow cytometry using in HCT116 cells treated with DMSO (arrow) had a smooth
the redox-sensitive fluorescent probe DCFH-DA. As shown appearance. Instead, the ER in HCT116 cells treated with
in Figure 6A, both CA and HCPT showed a moderate ability PCH for 6 hours appeared swollen (arrow), suggesting the
to increase the ROS level, while PCH caused a sharp increase accumulation of misfolded protein in the ER. However,
in ROS levels. NAC significantly inhibited PCH-induced in cells pretreated with NAC, the ER showed the familiar
increase of ROS production. These results indicated that smooth morphology, which is also shown in cells treated
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Figure 6 Pch increased the rOs level and induced the expression of mitochondrial and er-stress-related proteins.
Notes: (A) ROS generation in HCT116 cells treated with 100 mM of CA, HCPT or PCH by flow cytometric analysis. *P,0.05; **P,0.01, ***P,0.001 compared with
DMsO (n=3). hcT116 cells were pre-incubated with or without 5 mM Nac for 0.5 hour before exposure to Pch. Then, intracellular rOs generation was measured by
flow cytometric analysis. (B) hcT116 cells were treated with Pch for the indicated times, the protein levels of aTF4, p-elf2α, and chOP were determined by Western blot
analysis. gaPDh and elf2α were used as internal control. (C) hcT116 cells were pre-incubated with or without 5 mM Nac for 0.5 hour before exposure to Pch at the
indicated concentrations, then aTF4, p-elf2α, and chOP expressions were detected by Western blot analysis. gaPDh and elf2α were used as internal control. (D) effect
of Pch on the morphology of er in hcT116 cells. hcT116 cells were treated as previously described. The morphology of the er in hcT116 cells was examined using a
transmission electron microscope (×10,000 or ×50,000). The results from a representative cell sample out of three studied in each group are shown. The er morphology
is normal in the control group, the Pch + Nac (5 mM) group, and Nac (5 mM) group (arrows indicate normal er). exposure to Pch for 6 hours caused mitochondrial
dysfunction in Pch group (arrows indicate swollen er). (Pch equivalent to 10 µg/ml hcPT).
Abbreviations: ca, cinnamaldehyde; con, ; er, endoplasmic reticulum; hcPT, 10-hydroxy camptothecin; Nac, N-acetyl-cysteine; Pch, hcPT-ca-loaded nanoparticles.
with NAC alone. In summary, PCH activated the ER stress clearance rate (0.437 mL/h). These data showed that PCH
pathway by increasing ROS level.
nanoparticles prolonged the circulation time, which would
potentially enhance the efficacy of chemotherapy.
Pch effectively enhanced the
accumulation of drug in tumor tissues
The in vivo biodistribution of PCH nanoparticles was
further investigated. ICG, an NIR fluorescent dye, was
Prolonging the drug circulation time and enhancing drug simultaneously encapsulated in PCH to produce ICG/PCH.
accumulation in cancerous sites are the main advantages of ICG/PCH or free ICG was injected into tumor-bearing nude
nanoparticle drug delivery systems. In this study, the pharma- mice, and an in vivo imaging system was used to monitor
cokinetics of HCPT and PCH were evaluated within 6 hours the biodistribution of the ICG (Figure 7C). At 24 hours after
after sublingual vein injection in healthy Sprague Dawley rats injection, free ICG only resulted in a weak fluorescent signal,
(Figure 7A and B). In contrast to free HCPT, PCH showed a and rapidly declined afterward. In contrast, ICG/PCH gener-
significantly extended half-life (t1/2=7.042 h) and decreased ated an intense fluorescent signal at 6 hours after injection
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A
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8
HCPT
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Figure 7 The pharmacokinetic parameters and bio-distribution of Pch nanoparticles in vivo.
Notes: sprague Dawley rats were intravenously injected with Pch (equivalent to 5 mg/kg hcPT) or free hcPT (5 mg/kg). (A) Blood samples (200 µl) were collected
at appropriate intervals after administration (n=5) at each time point and the plasma hcPT concentration was analyzed by high performance liquid chromatography.
(B) Pharmacokinetic parameters of hcPT and Pch micelles. Data show mean se (n=5). Tumor-bearing BalB/ca nu/nu female mice were intravenously injected with free
Icg (2 mg/kg) or Icg-loaded Pch nanoparticles (Pch/Icg). (C) The biodistribution of Icg recorded by an in vivo imaging system at 6 hours, 24 hours, and 48 hours. red
circles: tumor sites (n=6). (D) The bio-distribution of Icg in major organs and tumor tissues at 24 hours.
Abbreviations: ca, cinnamaldehyde; hcPT, 10-hydroxy camptothecin; Icg, indocyanine green; Pch, hcPT-ca-loaded nanoparticles; se, standard error.
at the tumor site, which peaked at 24 hours and remained the major organs and tumors excised from the tumor-bearing
detectable until 48 hours after injection. These results clearly nude mice (Figure 7D). The fluorescent images confirmed that
demonstrated the increased accumulation of PCH at tumor PCH mostly accumulated in tumors, weakly accumulated in
sites, indicating its passive tumor-targeting ability. They also kidneys, and was hardly detectable in other organs. In com-
clearly showed that the PCH has a strong ability to accumu- parison, the free ICG dye mostly accumulated in liver and
late at tumor sites, passively targeting the tumor.
partially in kidneys. In general, the systemic administration
In order to further study the biodistribution of PCH of PCH nanoparticles improved the efficacy of chemotherapy
nanoparticles, the in vivo imaging system was used to examine by effectively promoting drug accumulation in tumors.
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Zhao et al
The in vivo anticancer effect and
biosafety of Pch
The change in body weight was simultaneously measured
after administration of the drugs, which serves as an indicator
of general toxicity. The results shown in Figure 8D reveal
that the body weight of the group treated with free drugs was
significantly decreased compared with the negligible changes
detected for the PCH-treated group, indicating the remarkable
in vivo systemic safety of the PCH. In addition, to further
confirm the potential toxicity of PCH, histologic sections
of the major organs including kidneys, liver, and heart were
stained with H&E and examined by microscopy. As shown in
Figure 9A, livers and hearts in all groups showed no notice-
able abnormality. However, both HCPT alone and HCPT with
CA caused kidney damage, including structural disturbance
in areas of the kidney. In comparison, PCH treatment only
We further investigated the in vivo anticancer effects of
PCH using xenograft HCT116 cells tumor models. Female
nude mice bearing HCT116 cell-induced tumors (tumor
volume ~100 mm3) were randomly divided into several
groups and administered various formulations at the equiva-
lent amounts of HCPT (5 mg/kg) every 3 days. As shown
in Figure 8A–C, the blank control group administered PBS
showed gradual and continuous tumor growth. The tumor
volume was successfully suppressed by treatment with
HCPT or HCPT combined with CA. Notably, PCH showed
the strongest ability to suppress tumor growth and the tumor
volume remained steady, which was better than mechani-
cally mixed drugs. Overall, PCH exhibited effective in vivo caused negligible tissue damage or lesions in the kidney. These
inhibitory effects on human colon cancer cells.
results clearly indicated the safety of PCH as cancer treatment.
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Figure 8 In vivo anticancer effects of Pch.
Notes: hcT116 tumor-bearing nude mice (tumor volume .100 mm3) were randomly divided into four groups (six/group) and intravenously injected with saline, free hcPT
(5 mg/kg), HCPT and CA, and PCH alone every 3 days, five times. The tumor volume (A) and body weight (B) were monitored every 2 days. The experiment was repeated
three times. Bars shown are mean se. (C) Images of tumor-bearing mice treated with various formulations. (D) Weight of tumor-bearing mice treated with various
formulations. **P,0.01, ***P,0.001 compared with con group (n=6).
Abbreviations: ca, cinnamaldehyde; con, control; hcPT, 10-hydroxy camptothecin; Pch, hcPT-ca-loaded nanoparticles.
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Figure 9 h&e images and Western blot analysis of tumor tissue lysates.
Notes: (A) Kidney, liver, and heart tissue from four groups of mice were sectioned (5 µm) and the slides were stained with h&e, (n=3 in each group). all images were
obtained with a microscope with 20× magnification (scale bar, 50 µm) or 40× magnification (scale bar, 100 µm). Pch administration did not cause histological abnormalities in
kidneys, livers, and hearts. green arrows indicate structural disturbance in areas of the kidney. (B) Western blot analysis of the expression of mitochondrial pathway-related
and er-stress-related proteins from respective tumor tissue lysates. gaPDh was used as protein loading control.
Abbreviations: ca, cinnamaldehyde; con, control; er, endoplasmic reticulum; hcPT, 10-hydroxy camptothecin; Pch, hcPT-ca-loaded nanoparticles.
To further confirm our hypothesis that PCH can activate cancer cells. In conclusion, the PCH-induced apoptosis in
the ER-stress pathway and cause mitochondrial dysfunction HCT116 cells is closely related to the activation of ER-stress
by increasing ROS level, the mouse tumors were analyzed by and resulting mitochondrial dysfunction in vivo.
Western blot analysis. The results revealed that PCH treat-
ment regulated the expression of proteins associated with the Discussion
ER-stress pathway, including XBP-1S, p-elf2α, and CHOP It has been well-established that ROS has double-edged
(Figure 9B), and mitochondrial dysfunction, including BAX effects on living cells, depending on their concentration
and BCL-2 (Figure 9B), which increased apoptosis in colon and duration, which can determine the fate of cancer cells.31
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Zhao et al
Based on these properties, many efforts have been made to hydroxyl radicals that preferentially kill cancer cells.43
induce oxidative stress and thus preferentially kill cancer cells. In addition, nanoparticles designed to incorporate CA in its
CA is the main ingredient of cinnamon, and it turns out that it pH-sensitive hydrophobic backbone via acid-cleavable acetal
can increase the ROS levels in tumor cells and cause cancer linkages and encapsulate CPT, which induced apoptotic
cell death.32–34 Although CPT has been considered as a drug cell death through the generation of intracellular ROS and
with a unique antitumor activity spectrum which is mediated their apoptotic activities were significantly enhanced with a
through the selective inhibition of eukaryotic DNA topo-I, payload of CPT and have been used in in vitro and in vivo
its analog HCPT has also been reported to induce ROS.37 model of SW 620 colon tumor-bearing mice.44 However, their
There are a few studies which found that CA combined with uptake by cells, biodistribution, safety and toxicity pharma-
HCPT induces ROS production, especially when delivered cokinetics in normal tissues and organs were not thoroughly
as an acidic microenvironment-responsive nanoparticle.
investigated. Also, although the nanoparticles induced ROS
The Fenton reaction-performing polymer (PolyCAFe) production to cause cancer cell apoptosis, the related down-
micelle is a new ROS manipulation anticancer therapeu- stream mechanism is still unknown. In particular, these are
tic agent. Amphiphilic PolyCAFe conjugates benzoyloxy important potential targets that need to be evaluated for the
CA and iron-containing compounds in its skeleton and development of new therapeutics for cancer, and thus have
self-assembles to form micelles which generate cytotoxic great significance for further research.
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Figure 10 schematic illustration of the underlying mechanism of the anticancer activity of Pch.
Abbreviations: ca, cinnamaldehyde; hcPT, 10-hydroxy camptothecin; Pch, hcPT-ca-loaded nanoparticles.
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In this work, we developed pH-responsive nanoparticles More importantly, PCH showed excellent therapeutic
by linking CA with biocompatible dextran, which was then performance and better systemic safety in vivo, which
allowed to self-assemble to form the nanoparticles (PC), significantly alleviated the renal damage caused by HCPT.
which were further used to encapsulate HCPT (PCH) for It is anticipated that acid-responsive PCH holds great
the purpose of developing a synergistic therapeutic for use promise for the treatment of cancer.
against colon cancer. PCH nanoparticles dissociated rapidly
and showed a significantly faster release kinetics in vitro in Acknowledgments
acidic environments. The results showed that PCH nanopar- This work was financially supported by the National Key
ticles have good pH-responsiveness and have the ability to R&D Program of China (2017YFA0506000), National
serve as a drug delivery system in response to a decrease Natural Science Funding of China (21502144, 81502912, and
in the pH value in tumor environments. Cellular uptake 81622043), Zhejiang Provincial Natural Science Foundation
experiment verified that PCH can be internalized into cells of China (LR16H310001), and Medical Scientific Research
more easily under acidic conditions (Figure 3), and further Fund of Zhejiang Province (2019322308).
accumulated in the nuclei of cancer cells. As the structure of
PCH nanoparticles was designed to target the tumor sites, the Disclosure
in vivo imaging system also revealed that different from free The authors report no conflicts of interest in this work.
ICG, the PCH nanoparticles preferentially accumulated in the
tumor sites. In summary, PCH nanoparticles have been shown References
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Supplementary materials
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Abbreviations: ca, cinnamaldehyde; con, control; hcPT, 10-hydroxy camptothecin; Pch, hcPT-ca-loaded nanoparticles.
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Zhao et al
C
BCL-2 (density gradient)
D
Cle-PARP (density gradient)
30,000
*
30,000
20,000
10,000
0
*
*
20,000
10,000
0
*
Figure S2 Quantitative data of Figure 5c.
Notes: hcT116 cells were pre-incubated with or without 5 mM Nac for 0.5 hour before exposure to Pch at the indicated concentrations, then the expressions of
apoptosis-related proteins (A) BaX, (B) P53, (C) Bcl-2, and (D) cle-ParP were detected by Western blot analysis. *P,0.05; **P,0.01.
Abbreviations: ca, cinnamaldehyde; hcPT, 10-hydroxy camptothecin; Nac, N-acetyl-cysteine; Pch, hcPT-ca-loaded nanoparticles.
A
ATF4 (timing point)
B
CHOP (timing point)
50,000
40,000
30,000
20,000
10,000
0
50,000
40,000
30,000
20,000
10,000
0
**
**
*
*
Con
1 h
3 h
6 h
Con
30 min
45 min
60 min
C
elf2α (timing point)
p-elf2α (timing point)
D
25,000
20,000
15,000
10,000
5,000
0
40,000
30,000
20,000
10,000
0
***
**
Con
1 h
3 h
6 h
Con
1 h
3 h
6 h
Figure S3 Quantitative data of Figure 6B.
Notes: hcT116 cells were treated with Pch for the indicated times, the protein levels of (A) aTF4, (B) chOP, (C) elf2α, and (D) p-elf2α were determined by Western
blot analysis. *P,0.05, **P,0.01, ***P,0.001.
Abbreviations: con, control; er, endoplasmic reticulum.
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Zhao et al
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Notes: hcT116 cells were pre-incubated with or without 5 mM Nac for 0.5 hour before exposure to Pch at the indicated concentrations, then (A) aTF4, (B)chOP,
(C) elf2α and (D) p-elf2α expressions were detected by Western blot analysis.*P,0.05, **P,0.01, ***P,0.001.
Abbreviations: ca, cinnamaldehyde; er, endoplasmic reticulum; hcPT, 10-hydroxy camptothecin; Nac, N-acetyl-cysteine; Pch, hcPT-ca-loaded nanoparticles.
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