Polymer Nanoparticles Modified with Photo- and pH-Dual-Responsive Polypeptides for Enhanced and Targeted Cancer Therapy

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00977

The cationic nature of cell penetrating peptides (CPPs) and their absence of cell selectivity restrains their applications in vivo. In this work, polymer nanoparticles (NPs) modified with photo- and pH-responsive polypeptides (PPPs) were successfully deve

Full text of DOI:10.1021/acs.molpharmaceut.5b00977

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Article
Polymer nanoparticles modified with photo- and pH-dual-
responsive polypeptides for enhanced and targeted cancer therapy
Yang Yang, Xiangyang Xie, Yanfang Yang, Zhiping Li, Fanglin Yu,
Wei Gong, Ying Li, Hui Zhang, Zhiyuan Wang, and XingGuo Mei
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00977 • Publication Date (Web): 04 Apr 2016
Downloaded from http://pubs.acs.org on April 5, 2016
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Molecular Pharmaceutics
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Polymer nanoparticles modified with photo- and pH-dual-responsive
polypeptides for enhanced and targeted cancer therapy
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a
a
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Yang Yang , Xiangyang Xie a,b, Yanfang Yang a,c, Zhiping Li , Fanglin Yu a, Wei Gong a, Ying Li a, Hui
Zhang a, Zhiyuan Wang a, Xingguo Mei a,*
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a State key Laboratory of Toxicology and Medical Countermeasure, Department of Pharmaceutics, Beijing
Institute of Pharmacology and Toxicology, Beijing 100850, China
b Wuhan General Hospital of Guangzhou Command
，Wuhan 430070, China
c
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Beijing Key Laboratory of Drug Delivery Technology and Novel Formulation, Institute of Materia
Medica, Chinese Academy of Medical Sciences & Peking Union Medical college, Beijing 100050, China
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Corresponding Author
* Xingguo Mei
Author Contributions
Yang Y. and X.X. contributed equally to this work.
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ABSTRACT:
The cationic nature of cell penetrating peptides (CPPs) and their absence of cell selectivity,
restrains their applications in vivo. In this work, polymer nanoparticles (NPs) modified with
photoꢀ and pHꢀresponsive polypeptides (PPPs) were successfully developed, which was
respond to nearꢀinfrared (NIR) light illumination at the tumor site and a lowered tumor
extracellular pH (pHe). In PPPs, the internalization function of CPPs (positively charged) is
quenched by a pHꢀsensitive inhibitory peptide (negatively charged), which is linked via a
photoꢀcleavable group. Small interfering RNA (siRNA) was loaded into NPs by a
doubleꢀemulsion technique. In vivo experiments included siRNA loading, cellular uptake, cell
apoptosis, siRNA transfection, tumor targeting delivery, and the in vivo antitumor efficacy
were carried out. Results showed that the prepared PPPꢀNPs could selectively accumulated at
the tumor sites, and internalized into the tumor cells by the NIR light illumination and the
lowered pHe at the tumor site. These studies demonstrated that PPPꢀNPs are a promising
carrier for future tumor gene delivery.
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KEYWORDS
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Photoꢀ and pHꢀresponsive polypeptides; Cellꢀpenetrating peptides; Small interfering RNA;
Tumor targeting nanoparticles
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ABBREVIATIONS
CPPs
ACPPs
PDT
Cellꢀpenetrating peptides
activable cellꢀpenetrating peptides
photodynamic therapy
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PTA
photothermal ablation therapy
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pHe
extracellular pH
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PPPs
EPR
photoꢀ and pHꢀresponsive polypeptides
enhanced permeability and retention
small interfering RNA
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siRNA
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INTRODUCTION
Cellꢀpenetrating peptides (CPPs), which efficiently facilitate the cell uptake of diverse
payloads with minor cytotoxicity, have been extensively studied for the delivery of
therapeutic agents to cancer cells.1 However, the cationic nature of CPP that generate both its
cell penetrating ability and absence of cell selectivity, hinders its applications in vivo. To
address this dilemma posed by conventional CPPs, a method using “activable cellꢀpenetrating
peptides (ACPPs)” for targeting cargos delivery is encouraging.2 In ACPPs, the
internalization feature generated by polyꢀcations of CPP was counteracted through covalently
attaching a polyanionic inhibitory peptide, which was linking via a divisible enzyme sensitive
molecule. Proteolysis of the enzyme sensitive linker that connected the polycationic CPP and
the polyanionic inhibitory peptide via certain special enzymes (e.g. matrix metalloproteaseꢀ2
and ꢀ9) was overꢀexpressed in the tumor site and afforded the separation of both sections and
enabled the dissociated CPPs to invade the cells. Although the ACPPꢀmediated nanocarriers
were able to transfer the pharmaceutical molecule into the targeted cells, the application of
ACPPs may be limited by the unreliable split of the enzymes and the inadequate departure of
the polyanionic inhibitory peptide from the polycationic CPP segment after cleavage.3, 4
Moreover, the efficient and controlled activity of CPPs via a singleꢀstimuli responsive
mechanism in a complex physiological environment without interference still poses a
considerable challenge.5 To overcome this challenge, it is favourable to build a proper
delivery system that is based on a dualꢀstimuli responsive mechanism, which may bring about
a feasible protocol for targeted cancer treatment.
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Because reliance on the aforementioned enzymeꢀsensitive cleaving mechanism may
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produce some disadvantages, which can be primarily attributed to the vast expression level
variations of certain specific enzymes between different individuals,6, 7 it is desirable to
develop a general and triggered cleaving methodology for the nanostructures. Among the
general triggers, nearꢀinfrared (NIR) light is a favourite triggering stimulus, which possesses
good spatial resolution, excellent controllability and nonꢀinjury due to its relatively low
energy nature. Over the past few decades, NIR lightꢀtriggered photodynamic therapy (PDT)
and photothermal ablation therapy (PTA) have been developed to treat cancer in a clinical
setting.8 The illumination of NIR light is not only performed at the superficial level of
exposure positions such as on the skin, but also at deepꢀseated tissue by employing a laser
head that connected to an endoscope through laparoscopy. Furthermore, more attention is
currently focused on the NIR irradiationꢀinduced bond cleavage reaction to establish a
platform to control the functions.9, 10 For example, the release of guest molecules in peptides
could be triggered by NIR irradiation using the 4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl group as
photoꢀcleavable molecular gates.11 Compared with enzymeꢀsensitive cleaving mechanisms,
the cleavable specificities and efficiency of a photoꢀcleavable group offer modular chemical
approaches for the rationally designing of selective cellꢀpenetrating nanostructures, which are
specifically and passively activated by the NIR irradiation in the tumor sites. Inspired by
these results, we developed a novel polypeptide that is composed of a polycationic CPP that
is attached to a polyanionic inhibitory peptide through a photoꢀcleavable group
(4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl group). When then tumor tissue was irradiated with the NIR
light, the photoꢀcleavable group would cleave and thus enabled the activated CPP to invade
the cells.
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Although these novel polypeptides were efficiently cleaved by NIR irradiation, the
polyanionic inhibitory peptide was difficult to separate from the polycationic CPP after
cleavage due to an electrostatic attraction.12 To solve this, we investigated the isoelectric
point conversion of an inhibitory peptide that has the potential to eliminate the electrostatic
attraction.13 With the development of a tumor tissue, a lowered tumor extracellular pH (pHe)
will emerge. Rapid tumor growth requires a high glycolytic activity, which leads to more
lactic acid production and brings a lowered pHe (pH 5.8ꢀ7.2) in almost every type of human
cancer.14, 15 Therefore, this point was utilized in our design to establish a pHꢀsensitive
delivery system, and a inhibitory peptide (EEEERRRR) with an isoelectric point of around
6.4 was chosen, which matches the low level pH of the tumor tissue and may reduce the
electrostatic attraction at this pH value. The peptide is negatively charged under the
physiological conditions (pH 7.4), but it will mostly uncharged or even positively charged at
a low pHe.
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In this work, a novel delivery tactic was applied in building a new therapeutic agent
delivery system by making full use of photoꢀ and pHꢀresponsive polypeptides (PPPs), and
this delivery system would transport cargos more selectively and efficiently to tumor cells.
The working scheme of the PPPꢀmodified PLGA nanoparticles (PPPꢀNPs) is shown in Fig. 1.
In this system, the PPP includes three elements: a cellꢀpenetrating peptide sequence
(CGRRMKWKK), a photoꢀdecomposable group (4, 5ꢀdimethoxyꢀ2ꢀnitrobenzyl group) and a
pHꢀsensitive inhibitory peptide (EEEERRRR). CGRRMKWKK is a CPP that is derived from
Penetratin, and it can enhance the membrane translocation efficiency.16 After systemic
administration, PPPꢀNPs may cumulate at the tumor sites through the enhanced permeability
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and retention (EPR) effects. The cell penetration ability of CPPs is effectively shielded by the
opposite electric charges within the pHꢀsensitive inhibitory peptides in circulation. Upon NIR
light irradiation at the tumor position, the photoꢀcleavable group is cleaved. Simultaneously,
the pHꢀsensitive inhibitory peptide eliminates the electrostatic attraction at a lowered pHe.
After cleaving the linker and eliminating the electrostatic attraction, the PPP could release its
inhibitory peptides to expose the CPPs. Then, with the help of revitalized CPPs, the NPs
efficiently enter into the cancer cells. Therefore, the photoꢀ and pHꢀ responsive strategy is
planned to amend the targeted delivery efficiency for tumor cells and overcome the
shortcomings of singleꢀstimuli responsive mechanism. As the specificity of siRNAꢀinduced
gene silencing, the siRNA can be employed as a model drug to evaluate the intracellular
delivery efficiency of PPPꢀNPs. Epidermal growth factor receptor (EGFR), is a cellꢀsurface
receptor that is overꢀexpressed in a number of solid tumors, such as anal cancer, lung cancer
and glioblastoma multiforme.17 Based on this, siRNAꢀloaded nanocarriers for EGFR target
was studied in this paper, and their downregulation effects on the target gene was assessed.
Here, the physicochemical features of the prepared PPPꢀNPs for antiꢀEGFR siRNA was
described, then their biological characterizations of were investigated at the cellular level, and
their in vivo antiꢀcancer effects was also explored.
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ꢀ
EXPERIMENTAL SECTION
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Materials.
1,2ꢀdistearoylꢀsnꢀglyceroꢀ3ꢀphosphoethanolamineꢀNꢀmaleimide
(polyethylene
glycol)
(DSPEꢀPEG2000ꢀMal)
and
1,2ꢀdistearoylꢀsnꢀglyceroꢀ3ꢀphosphoethanolamineꢀNꢀmethoxy
(polyethyleneglycol)
(ammonium salt) (DSPEꢀPEG2000) were obtained from Avanti Polar Lipids, Inc. (Alabaster,
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AL, USA). PLGA with lactic/glycolic acid ratio (50/50) and 0.55ꢀ0.75 dL/g inherent viscosity
was obtained from Shandong Institute of Medical Instruments (Shandong, China). Scramble
siRNA (sense strand: 5/ꢀUUC UCC GAA CGU GUC ACG UTTꢀ3/; antisense strand: 5/ꢀACG
UGA CAC GUU CGG AGA ATTꢀ3/. entitled as NCꢀsiRNA), antiꢀEGFR siRNA (sense strand:
5/ꢀAGG AAU UAA GAG AAG CAA CAU dTdTꢀ3/; antisense strand: 5/ꢀAUG UUG CUU
CUC UUA AUU CCU dTdTꢀ3/. entitled as EGFRꢀsiRNA) and fluoresceinꢀlabeled siRNA (5/
end of the sense strand, FAMꢀsiRNA or Cy5ꢀsiRNA) were obtained from GenePharma
(Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium
(DMEM) were purchased from GIBCO, Invitrogen Corp. (Carlsbad, USA). All chemicals
were of reagent grade and all purchased from SigmaꢀAldrich.
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Human breast adenocarcinoma cells (MCFꢀ7 cells) obtained from the Cell Resource
Centre of IBMS (Beijing, China) was kept in Dulbecco’s modified eagle’s medium (DMEM)
supplemented with 10% FBS, 100 mg/mL streptomycin, and 100 IU/mL penicillin. The cells
were incubated in a humidified incubator (37 °C) with a 5% CO2 atmosphere.
Female BALB/c nude mice (weighing 18ꢀ22 g) were provided by Vital River
Laboratories (Beijing, China). The mice were treated in accordance with the requirements of
ethics in research, training and testing of animals as laid down by the Animal Care and Use
Ethics Committee of Beijing Institute of Pharmacology and Toxicology. The MCFꢀ7 tumor
bearing nude mice model was developed as introduced in our previous paper.18 In short,
MCFꢀ7 cells (2 × 106) were injected subcutaneously in the right flank of the mice.
Synthesis of the PPP. Photoꢀsensitive amino acids (Psaa) were prepared as the previous
report of Shigenaga.11 Briefly, compound 1 and compound 2 were dissolved in DMF and
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added to K2CO3. Then, the resulting suspension was stirred overnight. An aqueous NH4Cl
solution was added in and kept stirring for 30 min, and deionized water was added in the
reaction bottle and extracted with diethyl ether. The diethyl ether phase was rinsed with H2O,
saturated aqueous NH4Cl solution and brine, dried in vacuum and purified via column
chromatography (SiO2, hexane/AcOEt=20/1) to obtain compound 3. Compound 3 was
dissolved in THF and added to glacial acetic acid and water, and it was stirred for 12 h. The
reactant mixture was extracted with AcOEt, dried and purified according to the above steps.
The resulting product and PCC were added to dichloromethane and stirred for 6 h. The
mixture was filtered through Cerite 535, and the organic layer was handled with a saturated
NH4Cl aqueous solution, dried and purified to obtain compound 4. NaClO2,
2ꢀmethylꢀ2ꢀbutene and sodium dihydrogen phosphate were added to the mixture of
acetone/tertꢀBuOH/H2O (17/12/3 v/v/v, 12.8 mL) with compound 4. It was stirred for 5 h, a
saturated aqueous NH4Cl solution was added, and it was extracted with AcOEt, dried over
Na2SO4 and concentrated in vacuum. The AcOEt containing hydrogen chloride was putted
into the obtained powder and agitated for 2 h, dried in vacuum, dissolved in acetonitrile/
Na2CO3 aqueous solution, added to FmocOSu, stirred for 6 h, acidified with KHSO4 aqueous
solution, and extracted with diethyl ether, and the organic phase was rinsed with brine and
evaporated in vacuum. The resulting product was handled via column chromatography, and
the Fmoc derivative 5 was obtained.
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The PPP was synthesized in a similar method via the standard solid phase
Fmocꢀprotocol on a peptide synthesizer (CEM, Matthews, North Carolina, USA) with the use
of Nankai Hecheng Rink amide resin at a 0.44 mmol/g concentration (Tianjin, China).19 The
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crude peptides were purified using Waters PrepLC 4000 highꢀperformance liquid
chromatography with greater than 95% purity. MALDIꢀTOFꢀMS (Autoflex III; Bruker
Daltonics Inc., Billerica, Massachusetts, USA) was employed to analysis the molecular
weight of the obtained peptides.
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Photo-triggered cleavage and zeta-potential of the PPP. To study the sensitivity of the
PPP cleavage to NIR light, a solution of PPP in 50% v/v acetonitrile/phosphate buffer (pH
7.6, 20 mM) was illuminated with a NIR light (λ=740 nm, 0ꢀ50 J·cmꢀ2, 0ꢀ83 mW, 2 min
interval after 1 min irradiation), and the concentration of PPP at different time was measured
using HPLC (Agilent 1211, Agilent Technologies, USA). The chromatographic conditions
were as follows: a C18 column (250 mm×4.6 mm, 5 ꢁm) (Agilent Technologies Inc., Avondale,
PA, USA) and detection at 220 nm. To study the sensitivity of the pH, the zetaꢀpotential of
the PPP at pH 7.4 and 6.0 (with or without NIR illumination) was determined using a Marvin
Zetasizer Nano analyser (Marvin Instruments Ltd., UK).
Synthesis of DSPE-PEG2000-PPP. DSPEꢀPEG2000ꢀPPP and DSPEꢀPEG2000ꢀCPP were
synthesized by conjugating DSPEꢀPEG2000ꢀMal to the cysteine residue on PPP and CPP,
respectively. DSPEꢀPEG2000ꢀCPP was one of the photoproducts of DSPEꢀPEG2000ꢀPPP,
which was applied to indicate the penetrating capacity changes of DSPEꢀPEG2000ꢀPPP before
and after the light activation. PPP and CPP were coupled with DSPEꢀPEG2000ꢀMal (1:1 molar
ratio) in chloroform that included triethylamine (TEA, 5 eq.) around 20 °C with stirring for
24 h. The resulting mix was putted in dialysis bag (its molecular weight cutoff was 3.5 kDa)
with distilled water and dialyzed for 48 h to dispel the chloroform and unreacted materials.
The liquid in dialysis bag was dried by a rotary evaporator and kept at ꢀ20 °C. The formation
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of the conjugations was verified by a MALDIꢀTOF mass spectrometry (MALDIꢀTOF MS).
Preparation of the NPs. A double emulsion (w/o/w) technique was used to prepare the
normal NPs (NꢀNPs) as described in a previous report.20 Briefly, EGFRꢀsiRNA
(FAMꢀsiRNA, NCꢀsiRNA) and spermidine were added in TrisꢀEDTA buffer (10 mM Tris, 1
mM EDTA, pH 7.4) (TE buffer). After 30 min, the formed siRNAꢀspermidine complexes
were dripping to a PLGAꢀ (50 mg/ml) methylene chloride (DCM) solution at a volume ratio
of 1:10 under a vortex. This mixture was emulsified via sonication using an ultrasonic
processor (SCIENTZꢀIID, Scientz Biotechnology Co., China) into the first emulsion (w/o).
This emulsion was then poured into a TrisꢀEDTA buffered 5% (w/v) PVA (at a volume ratio
of 1:3), which contained DSPEꢀPEG2000 as the conjugate to PLGA at a molar ratio of 1:100.
The resultant mix was treated by ultrasound to generate the second emulsion (w/o/w). The
emulsion was quickly putted into TrisꢀEDTA buffered 0.5 % (w/v) PVA at a volume ratio of
1:25, and the NPs were kept stirring for 4 h to evaporate the DCM and solidify the particles.
To prepare the PPPꢀmodified NPs (PPPꢀNPs), similar operations were carried out except the
amount of DSPEꢀPEG2000ꢀPPPs, which were used to displace the equimolar lipids(5%, 10%
and 20% molar ratio). The preparation of the CPPꢀmodified NPs (CPPꢀNPs) followed the
procedure of the PPPꢀNPs.
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Characterization of NPs. Transmission electron microscopy (TEM, HITACHI, Hꢀ7650,
Japan) and atomic force microscopy (AFM, NanoWizarc, JPK Ltd., Germany) were used to
analysis the morphology of the prepared PPPꢀNPs. The diameter of the PPPꢀNPs was
measured by a Marvin Zetasizer Nano analyzer (Marvin Instruments Ltd., UK). All assays
were executed in triplicate.
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Cellular uptake study. For flow cytometry, MCFꢀ7 cells were cultured in 6ꢀwell culture
plates overnight and each well contained 2×105 cells. After achieved 80ꢀ90% confluence, the
cells were cultured in serumꢀfree medium (pH of 7.4 or 6.0), which contained the different
formulations including 75 nM FAMꢀsiRNA. Among the samples, the PPPꢀNPs were
pretreated with or without NIR illumination (λ=740 nm, 50 J·cmꢀ2, 83 mW, 2 min interval
after 1 min irradiation) for 30 min prior to their addition to the cells. After 4 h, the cells were
digested with trypsin, rinsed with cold PBS, and then quickly analysed by a flow cytometry
(BD FACSCalibur, USA). The mean fluorescence intensity is calculated by subtracting the
fluorescence value of cells without any formulations.
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For the confocal imaging, cells were seeded in 24ꢀwell culture plates with 2×105 cells
each well. The following incubation operation was the same as above except that the
FAMꢀsiRNA of in every well was 225 nM. Afterwards, Hoechst 33258 was used to execute
the nuclear staining (10 min). Then, the cells were analysed by a UltraVIEW Vox confocal
laser scanning microscopy (CLSM) (UltraVIEW Vox, PerkinElmer, USA).
Evaluation of endosomal escape. After a 4 h cultivation with FAMꢀsiRNAꢀloaded
PPPꢀNPs (the samples were pretreated with NIR light illumination (λ=740 nm, 50 J·cmꢀ2, 83
mW, 2 min interval after 1 min irradiation, total for 30 min) in the serum free medium under
pH 6.0), MCFꢀ7 cells were rinsed with cold PBS for three times and incubated in complete
medium for an extra time (0.5 h or 2 h). LysoTracker Red (Invitrogen/Molecular Probes, CA,
USA) was empoyed to executed the endosome/lysosome labeling for 0.5 h (500 nM).
Thereafter, the cells were washed with cold PBS and assayed by the CLSM.
In vitro siRNA transfection and gene expression. Small dishes (35ꢀmm) were uesed to
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culture the MCFꢀ7 cells with 2.0×106 cells per well. After a 24 h incubation (37 °C with 5%
CO2), fresh and serumꢀfree medium contained different siRNAꢀloaded NPs was used to
replaced the former medium. Certain medium included with PPPꢀNPs (pH 7.4 or 6.0) would
be irradiated with the NIR light (λ=740 nm, 50 J·cmꢀ2, 83 mW, 2 min interval after 1 min
irradiation, total for 30 min). After 5 h, the serumꢀfree medium were removed and the
complete medium were added for an extra 48 h (mRNA assays) or 72 h (protein
quantification) in the incubator. After that, EGFR mRNA and its related proteins were
respectively measured by a quantitative realꢀtime polymerase chain reaction (qRTꢀPCR) and
western blot analysis. The details of the two methods were described in our previous study.21
Cell apoptosis analysis. MCFꢀ7 cells (2.0×106 per flask) in 6 mL of complete DMEM
medium were plated on each tissue culture flask (25 cm2). After a incubation of 24 h, the cells
were rinsed with PBS (0.1 M, pH 7.4) and then added with serumꢀfree medium including
tested NPs. Certain medium included with PPPꢀNPs (pH 7.4 or 6.0) would be irradiated with
the NIR light (λ=740 nm, 50 J·cmꢀ2, 83 mW, 2 min interval after 1 min irradiation, total for
30 min). After a incubation of 6 h, the cells were gathered and handled with an Annexin
VꢀFITC apoptosis detection kit (Beyotime Institute of Biotechnology, Jiangsu, China) based
on the product’s instructions, and were quickly measured by a FACScan flow cytometer
(10,000 events per sample).
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Animal model. Female BALB/c nude mice with the weight of 18ꢀ22 g were provided by
Vital River Laboratories (Beijing, China). An xenotransplantation model was created by
subcutaneously injecting MCFꢀ7 cells as previously reported.19 All treatments relating to the
animals were authorized by the Animal Care and Use Ethics Committee of Beijing Institute
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of Pharmacology and Toxicology.
In vivo distribution. 200 ꢁL of 5% glucose (control) and different formulations of NPs
including Cy5ꢀsiRNA at 20 ꢁg/kg were respectively injected to the MCFꢀ7 tumor bearing
nude mice by tail intravenous. After 4 h, the tumorꢀxenografted mice were fixed and the
surfaces of the tumor sites (PPPꢀNPs group) was illuminated with the NIR light (λ=740 nm,
50 J·cmꢀ2, 83 mW, 2 min interval after 1 min irradiation) at an exposure area of 1.0 cm2 for
30 min. 6 h and 24 h after the injection, a IVIS® Lumina II in vivo imaging system (Caliper
life sciences, USA) was used to obverse the fluorescence. After that, the animals were killed
by anesthesia, and the tumor and major organs were removed and assayed.
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In vivo antitumor efficacy. The MCFꢀ7 tumorꢀbearing nude mice were injected by tail
intravenous with 5% glucose (control), free siRNA, and various formulations of NPs (20
ꢁg/kg siRNA, corresponding to 1.2 mg/kg of NPs). The injection was performed once every
other day for a total of 5 times. 4 h after the final injection, half of the mice in the PPPꢀNPs
group were illuminated with the NIR light (λ=740 nm, 50 J·cmꢀ2, 83 mW, 2 min interval after
1 min irradiation) for 30 min. The animals were weighed and their tumor volumes were
metered by a Vernier caliper every day during the whole experiment period. The relative
tumor volume (RTV) was computed as RTV = (tumor volume at day n)/(tumor volume at day
0)×100%.
Gene expression in tumor tissues. The in vivo EGFR expression analysis was performed
24 h after the final injection. The animals (n=3) were killed by anesthesia and their tumor
were removed for analysis. Tumor pieces were obtained for the total mRNA (qRTꢀPCR)22 or
protein extraction (Western blot assay) 23 as described in the previous reports, respectively.
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Immunohistochemical analysis. The apoptosis of the tumor tissues was performed 24 h
after the final injection. The animals (n=3) were killed by anesthesia and their tumor were
removed for analysis. For the immunohistochemical analysis, the obtained tumor tissues were
first frozen and then cut into thin slices (4ꢀmm). For apoptosis assay, TRITC staining in situ
Apoptosis Detection Kit (KeyGEN, Nanjing, China) was used to performed the terminal
deoxynucleotidyl transferase (TdT)ꢀmediated dUTP nick end labeling (TUNEL) staining. The
operation was carried out on the instructions of the kit. After the nuclei stained with PI (37 °C)
for 25 min, the samples were then analysed by the CLSM.
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Statistical analysis. Data were expressed as means ± standard deviation (SD). ANOVA
analysis was used to calculate the difference between any two groups. The P value less than
0.05 was considered to be statistically significant.
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ꢀ
RESULTS AND DISCUSSION
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Synthesis and characterization of the PPP. The peptide was synthesized as shown in Fig.
2 A. Compound 1 was alkylated with photoꢀsensitive compound 2 in the presence of K2CO3
to generate compound 3. Then, the TBS group of compound 3 was removed under the acidic
condition, and PDC was added to oxidize the resulting hydroxyl group and afford compound
4. After treatment with NaClO2, acid treatment, and FmocOSu, the Fmocꢀprotected amino
acid 5 was obtained. The total yield of the amino acid was approximately 70%. For the last
step, amino acid 5 was incorporated into the designed peptide sequence via Fmoc solidꢀphase
peptide synthesis (Fmoc SPPS). The observed mass of the PPP was 2976.03 Da (Fig. 2B,
labeled with an arrow), which is equal to its theoretical molecular mass of 2975.49 Da.
The cleavage of the PPPs by NIR light stimulus is the critical stage to achieve the
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dualꢀstimuli responsive PPPꢀNPs that were developed in this study. The 4,
5ꢀdimethoxyꢀ2ꢀnitrobenzyl group is very sensitive to the 740 nm NIR irradiation, and it has
been recently applied as a photoꢀcleavable group to govern the behaviours of cells.24, 25 Due
to its good stability and reactivity, this group was introduced into the linking moiety of a
quasi amino acid. The NIR light would induce removal of the photoꢀcleavable group on the
phenolic hydroxyl group 26 as displayed in Fig. 3A. When irradiated with the NIR laser, the
photoꢀcleavable group would cleave, and then the pHꢀsensitive inhibitory peptide would
dissociate from the polycationic CPP. CPP is the photochemical product of the PPPs, which
was confirmed using an HPLC assay. After illumination, the PPPs (approximately 20 min)
were converted to corresponding CPPs (approximately 11 min), as displayed in Fig. 3 B and
C. These data suggest that the occurrence of a specific cleavage, as predicted. The rate of PPP
cleavage was performed by the exposure of PPP solutions (in buffer) to NIR laser irradiation
(Fig. 3D). The cleavage of PPP is related to the laser irradiation power. With an increase in
irradiation power, there was an improvement in the degree of cleavage. When the irradiation
power was 50 J·cmꢀ2, approximately 92% and 94% of the initial chemical structure of PPP
was split at 30 min and 60 min after the exposure, respectively, which was available for cell
binding and penetration. Furthermore, the amount of the PPP did not decrease without NIR
illumination. The PPP construct was highly susceptible to NIR light illumination, and thus,
further experiments were performed using 50 J·cmꢀ2 and 30 min of light illumination.
To enhance the separation efficiency, an inhibitory peptide (EEEERRRR) with an
isoelectric point of 6.4 was designed and synthesized. This inhibitory peptide was
pHꢀsensitive and it was negatively charged under physiological condition, but it would be
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electrically neutral or even positively charged at a lower pHe. The corresponding
zetaꢀpotential values of the PPPs were 1.2 ± 3.1 mV at pH 7.4 (physiological condition) in
the dark. Meanwhile, the zetaꢀpotential values of the peptides were 5.5 ± 2.2 mV in the dark
and 5.4 ± 1.9 mV after illumination at pH 6.0 (the mimetic tumor microenvironments). The
pHꢀsensitive inhibitory peptide would be negatively charged under physiological conditions
and thus cloak the CPP well, whereas this masking effect would be vanished in the tumor
microenvironments.
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Synthesis and identification of DSPE-PEG2000-PPP. The PPPꢀNPs were developed by
modifying the synthesized functional material, DSPEꢀPEG2000ꢀPPP. As shown in Fig. 4A, via
the reaction between the cysteine residue and maleimide, the PPP was covalently conjugated
to the terminus of DSPEꢀPEG2000ꢀMal. DSPEꢀPEG2000ꢀPPP was successfully formed and this
was verified by the results of MALDIꢀTOF MS. The synthesized products demonstrated a
mass/charge ratio of 5893.01 (Fig. 4B, noted with an arrow), which is equal to the theoretical
molecular mass of 5895.42. Additionally, DSPEꢀPEG2000ꢀCPP was the cleavage product of
DSPEꢀPEG2000ꢀPPP. Here, DSPEꢀPEG2000ꢀCPP was also synthesized by the same method
and was employed to measure the penetrating effect changes of DSPEꢀPEG2000ꢀPPP after its
activation. The final product was then used to prepare targeted PPPꢀNPs or CPPꢀNPs in the
experiments.
Preparation and characterization of the NPs. All of the functional materials that we
used were preꢀsynthesized. Thus, these NPs (NꢀNPs, CPPꢀNPs and PPPꢀNPs) could be
conveniently constructed via a singleꢀstep nanoprecipitation method.
Because the content of DSPEꢀPEG2000ꢀPPP in the NPs is a key factor that significantly
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influences the cellular uptake efficiency of the NPs, the cellular uptake of different
FAMꢀsiRNA loading NPs with various amounts of DSPEꢀPEG2000ꢀPPP were evaluated in the
MCFꢀ7 cells to screen the formulations. As displayed in Fig. 5A, the cellular uptake of the
prepared PPPꢀNPs was not changed in the dark by using various amounts of
DSPEꢀPEG2000ꢀPPP. However, the cellular uptake of the PPPꢀNPs was remarkably affected
by the addition of the DSPEꢀPEG2000ꢀPPP concentration after illumination. When the NPs
were modified with 10% of DSPEꢀPEG2000ꢀPPP, the illumination would generate a notable
increase of the cellular uptake. But when the DSPEꢀPEG2000ꢀPPP molar ratios continuously
increased (20%), the uptake level decreased, which might suggest that a NIR light irradiation
time of 30 min might be insufficient to photodegrade the added PPPs. Based on this, the
preferred molar ratio of DSPEꢀPEG2000ꢀPPP on the NPs was chosen to be 10%. Additionally,
the amount of DSPEꢀPEG2000ꢀCPP on the NPs was chosen to be 10% (molar ratio), and the
CPPꢀNPs were also prepared using the same procedure.
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The physiochemical characteristics of the three different NPs formulations are presented
in Table 1. The siRNA encapsulation efficiency of all the three formulations was
approximately 87%, and the siRNA in the PPPꢀNPs or PPPꢀNPs (with NIR) did not degrade
much even after 24 h as showed in the Supporting Information. The particle size of the
prepared NPs was in the range of 108 to 116 nm. This particle size was suitable for delivery
in circulation. Because this size was large enough to avoid kidney filtration (>10 nm) but also
small enough to cross over tissues, to approach cell surface receptors and aid the intracellular
transport.27 TEM and AFM observations confirmed the existence of the PPPꢀNPs (Fig. 5 B
and C). The mean particle size of the PPPꢀNPs given by the TEM images was near the values
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provided by the laser particle analyser (Fig. 5D). AFM was further used to describe the
particle surface morphology of the PPPꢀNPs.
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Table 1. Properties of the NPs (n = 3)
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Sample ID
NꢀNPs
Dimater (nm)
108.26 ± 0.11
113.68 ± 0.10
116.27 ± 0.12
Polydispersity index Entrapment efficiency (%)
0.10 ± 0.01
0.09 ± 0.01
0.10 ± 0.01
87.34 ± 2.02
86.87 ± 1.54
88.20 ± 1.92
CPPꢀNPs
PPPꢀNPs
PPPꢀNPs
115.56 ± 0.14
0.08 ± 0.01
87.05 ± 1.87
(NIRꢀpretreated)
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Cellular uptake and endosomal escape. In this experiment, flow cytometry and CLSM
was used to analysis the cellular uptake of the tested NPs, in which FAMꢀsiRNA was
entrapped in as a fluorescent marker. As shown in Fig. 6A, compared with FAMꢀsiRNA,
the cellular uptake of CPPꢀNPs was remarkably increased, which could be due to the
CPPꢀmediated translocation effect. Additionally, upon NIR light illumination at pH 6.0, the
mean fluorescence intensity of the PPPꢀNPs (with NIR, under pH 6.0) was 115.73, which was
at a similar level to the CPPꢀNPs, suggesting that the PPPs were surffiently revitalized to
execute CPP’s penetrating effect on the mimetic tumor. Meanwhile, at pH 7.4, the mean
fluorescence intensity of the PPPꢀNPs (with NIR at pH 7.4) declined to 92.42 even when
pretreated with NIR to cleave the linker, which could be explained by the insufficient
separation of the pHꢀsensitive inhibitory peptide with the CPP segment after cleavage. On the
contrary, the mean fluorescence intensity of the PPPꢀNPs (without NIR light) was not ideal,
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either at pH 7.4 (42.55) or 6.0 (44.87), they were similar as that of NꢀNPs (41.35). The results
suggest that the photoꢀcleavable group was not cleaved without the NIR irradiation. The CPP
was still shielded by the pHꢀsensitive inhibitory peptide, and the penetrating ability of the
PPP was not activated even at pH 6.0. All of these results indicate that both NIR light and a
low pH are indispensable to activating PPP. From the CLMS results, simiar trend was found
in the cellular uptake experiment. As displayed in Fig. 6B, the NꢀNPs and PPPꢀNPs (without
NIR) groups had a alike relatively low cellular uptake, either at pH 7.4 or 6.0. Whereas, the
CPPꢀNPs had a remarkble cellular uptake. After the treatment of NIR irradiation, compared
with the PPPꢀNPs (with NIR at pH 7.4), the PPPꢀNPs (with NIR at pH 6.0) showed a
remarkble increase of cellular uptake, implying the activation of PPP. This result might be
due to the synergism between the photo activation and pH sensitiveness. The above results
are consistent with that of flow cytometry analysis. Overall, the above cell uptake data
intensily support our previous anticipation, PPPs combined with NIR light, can enhance the
cancer cells recognition and uptake of prepared carriers, and thus reduce the nonspecific cell
uptake.
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After internalization by the cells via the activated CPP, the ability of the siRNAꢀloaded
PPPꢀNPs to surmount over the intracellular endolysosomal delivery barriers and efficiently
deliver siRNA into the cytoplasm is crucial for the efficient gene silencing activities of
siRNA. FAMꢀsiRNA that was formulated in the PPPꢀNPs (with NIR at pH 6.0) and then
incubated with cells stained with LysoTracker (red) to track endosomal escape by CLSM. As
shown in Fig. 6C, for the FAMꢀsiRNAꢀloaded PPPꢀNPs (with NIR at pH 6.0), the endosomal
escape is timeꢀdependent within 0.5 h of transfection, and the yellow fluorescence in the
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figure suggests the colocalization of the green fluorescent FAMꢀsiRNA and the LysoTracker
Redꢀlabelled endosomes/lysosomes. After transfecting for 2 h, most green fluorescence was
found in the cytosol but not coexisted with LysoTracker (red), suggested that much
FAMꢀsiRNA was left from the lysosomes and come into the cytoplasm. This phenomenon
could be explained as follows: the sequence of CCP (positively charged) can assemble ion
pairs with the lipids (negatively charged) in endosome membrane and therefore replace the
surfaceꢀbound water on endosome membrane, and consequently break the endosomal
membrane.28
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In vitro gene silencing and cell apoptosis assay. The in vitro gene silencing activity of
EGFRꢀsiRNA against the EGFR gene that was delivered by the PPPꢀNPs was further
determined using qRTꢀPCR and western blot analysis. As exhibited in Fig. 7 A and B, the
EGFR mRNA and protein expression of the MCFꢀ7 cells incubated with NCꢀsiRNAꢀloaded
PPPꢀNPs (with NIR, under pH 6.0) were not inhibited, while the cells treated to different NPs
loaded with EGFRꢀsiRNA displayed a decreased EGFR gene expression, suggesting a gene
silencing. However, the results display that free EGFRꢀsiRNA does not exhibit gene silence
activity for EGFR, indicating that free siRNA cannot easily cross the cell membrane for their
negative charge and large volume.29 Among all the groups, the PPPꢀNPs and CPPꢀNPs
groups in MCFꢀ7 cells showed the stronger silencing effect, which is also agreed with the cell
uptake results (Fig. 6 A and B). Compared with the no NIR irradiated PPPꢀNPs group (pH
6.0), NIR irradiated PPPꢀNPs (pH 6.0) group showed lowered EGFR mRNA and protein
expressions. This indicated that the NIR irradiation could increase cell uptake of siRNA by
the photoꢀdegradation of PPPs. Additionally, the PPPꢀNPs (with NIR at pH 6.0)
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demonstrated stronger gene silencing effect than the PPPꢀNPs (with NIR, under pH 7.4),
which could be due to the increased internalization of EGFRꢀsiRNA. This suggests an
adequately activated PPPꢀmediated cell uptake of carriers with PPP modification, as
exhibited in the cellular uptake experiment (Fig. 6 A and B).
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In the treated MCFꢀ7 cells, apoptosis, which was induced by various formulations that
carried NCꢀsiRNA and EGFRꢀsiRNA, were analysed via a flow cytometry. The MCFꢀ7 cells
that were exposed to the EGFRꢀsiRNA loaded formulations showed significant apoptosis
(33%ꢀ67%), suggesting that the apoptosis was primarily yielded from the low expression of
EGFR. Additionally, the delivery of the EGFRꢀsiRNAꢀloaded PPPꢀNPs (with NIR, under pH
7.4) (48%) produced a higher level of apoptosis than that of NꢀNPs (33%), PPPꢀNPs (without
NIR, under pH 6.0) (32%) and PPPꢀNPs (without NIR, under pH 7.4) (34%). As expected,
compared with the PPPꢀNPs (with NIR, under pH 7.4), cells exposed to the PPPꢀNPs (with
NIR, under pH 6.0) that were loaded with EGFRꢀsiRNA presented a higher percentage (59%)
of apoptotic cells, which is in accordance with the aforementioned mRNA and protein
expression analysis results (Fig. 7 A and B). Whereas, a weak cell apoptosis was found in the
control and NCꢀsiRNAꢀloaded PPPꢀNP (with NIR at pH 6.0) groups (3%), implying that the
HTꢀ1080 cells’ apoptosis were also generated from the down regulation of EGFR mRNA.
Among all of the tested formulations, the CPPꢀNPs containing EGFRꢀsiRNA demonstrated
the most significant apoptosis at 67%.
Therefore, these results demonstrate that the PPP ligand that was introduced into the
NPs significantly helped the RNAi induced gene silencing and growth inhibition, when the
PPPs were split by the NIR irradiation to form dissociative CPPs by the lowered pHe.
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In vivo imaging. The in vivo distribution and tumorꢀtargeting characteristics of the
payload formulated in the NPs was investigated in a MCFꢀ7 nude mice model by using
Cy5ꢀsiRNA. For NIR imaging, illumination of a cyanine dye was not exactly at the
absorption wavelength, this may cause some loss of fluorescence due to photoꢀbleaching. As
shown in Fig. 8A, in the mice treated with PBS, no fluorescence was observed during the
experimental period. As the fluorescence intensity of free Cy5ꢀsiRNA rapidly decreased after
intravenous injection, tumor accumulation did not occur. The reason for this phenomenon is
related with the inherent problems of siRNA, such as its instant degradation by Rnase (A type
of nucleases) and its rapid renal excretion after administration. After illumination, the
PPPꢀNP (with NIR)ꢀtreated mice exhibited the most intense tumor distribution during the
entire period. Moreover, the result was further verified by the strongest fluorescence signals
found in the isolated tumor tissues (Fig. 8B). This accordance indicated that the nanocarriers
modified with PPPs could enhance their accumulation in the tumors, which benefited from
the dual stimuli of the NIR light illumination and lowered pHe. In contrast, for the CPPꢀNP
group, fluorescence signal from the tumor site almost disappeared after 24 h. Due to the no
specific cell penetrating ability of CPP, the CPPꢀNPs had a lower in vivo tumor selectivity
than the PPPꢀNPs (with NIR). However, the higher cellular uptake of FAMꢀsiRNA in the
CPPꢀNP group (Fig. 6 A and B) could be attributed to the assisted entry of the NPs into the
cells via CPP. For the NꢀNPs, the fluorescence in the tumor sits lasted for 24 h but its
intensity was notably decreased. The nonꢀmodified NPs provided an impressive
selectiveꢀdrugꢀtransport via the EPR effect,30 which is due to the suitable particle size of the
NPs. Additionally, the mice injected with the PPPꢀNPs (without NIR) exhibited a lower
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fluorescence intensity in the tumor similar to the NꢀNPs, suggesting the cancer cells were
recognized and the penetrating ability of the PPPs was not activated. Although the
electrostatic attraction of the CPP and the pHꢀsensitive inhibitory peptide was eliminated at
the in vivo lowered pHe, the photoꢀcleavable group was not cleaved without NIR light
illumination, and the CPP was still shielded by the pHꢀsensitive inhibitory peptide.
The isolated organs and tumor tissues were further observed by sacrificing the mice 24 h
after administration. As shown in Fig. 8B, the tumors from the PPPꢀNP (with NIR)ꢀtreated
group displayed the strongest fluorescence signals, whereas less fluorescence was observed in
other organs. The results suggest that the PPPꢀNPs (with NIR) can efficiently and selectively
delivery the siRNA to the tumor sites of nude mice model, and thus reduce the nonꢀspecific
cumulation in the healthy tissues. Meanwhile, the PPPꢀNP (with NIR)ꢀtreated group
exhibited a comparable fluorescence intensity in liver and kidneys, which is consistent with
another recent study.31 This demonstrates that nanocarriers may target tumors to some extent
via PPP mediation. Meanwhile, the nanocarriers could also be subject to reticuloendothelial
system (RES) uptake and renal excretion. However, the related strong liver and kidney
uptake mechanisms of the PPPꢀmediated nanocarriers in vivo require further study.
Overall, these results demonstrated that the PPPꢀNPs (with NIR) have the latent capacity
to realize the selective delivery of siRNA to a tumor in vivo.
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In vivo antitumor efficacy. Inspired by the satisfactory tumor target results, we further
explored the in vivo antitumor activity of the PPPꢀNPs that carry EGFRꢀsiRNA in MCFꢀ7
xenografted nude mice.
According to the results of the preliminary experiment, the max fluorescence would
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appear in the tumor sit about 4 h after the injection, which indicated that there would be a
great of nanoparticles accumulated in the tumor 4 h after the administration. Therefore, to
gain a high siRNA concentration in the tumor site, the irradiation was operated at 4 h after the
injection. This point in time for light illumination was near to Park et al. reported (6 h)32. The
irradiation power was selected by the in vitro results (Figure 3D). The irradiation time was
mainly chosen based on the thermo influence of the light. To avoid the NIR light to burn the
exposed tumor tissue, 30 min exposure (2 min interval after 1 min irradiation) was chosen.
Because at this time setting (under 50 J·cmꢀ1), only a slight increase of the tumor temperature
was observed (less than 1 °C).
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As shown in Fig. 9A, due to the inherent shortfalls of siRNA, mice in the free
EGFRꢀsiRNA groups (RTV = 543 ± 44%) exhibited no significant tumor growth inhibition
compared to the glucose group (RTV = 571 ± 42%) at day 18, which displayed almost no
growth inhibition. Additionally, the administration of NCꢀsiRNAꢀcontained PPPꢀNPs (with
NIR, RTV = 531 ± 41%) showed no tumor inhibition effect, suggesting that the anticancer
activity of NPs that carried siRNA was specially associated with EGFRꢀsiRNA. However,
compared to the control group, the groups contained EGFRꢀsiRNA as NꢀNPs (RTV = 349 ±
41%) and PPPꢀNPs (without NIR, RTV = 333 ± 58%) exhibited a mild increase of tumor
inhibition effect. These results demonstrated that the introducing of EGFRꢀsiRNA into the
nanocarrier could improve the in vivo tumor inhibition effect. However, as expected, the
PPPꢀNP (with NIR) group containing with EGFRꢀsiRNA (RTV = 235 ± 49%) displayed a
superior antitumor effect. A reason for this may be the improved EGFRꢀsiRNA transfection
efficiency by PPPꢀNPs (with NIR) with the dual stimulus of photons and pH. This result was
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consistent with the aforesaid results, which revealed the superiority of PPPꢀNPs (with NIR)
than the other tested nanocarriers evaluated in in vivo imaging (Fig. 8 A and B). In contrast,
for the EGFRꢀsiRNAꢀloaded CPPꢀNPꢀtreated group (RTV = 411 ± 40%), which was
attributed to the CPPs' nonꢀselective penetrating ability.33 The modification of the nanocarrier
surfaces with CPPs leads to a specific interaction and uptake in nonꢀtargeted cells. This
drawback limits the in vivo antitumor efficacy of CPPꢀNPs. With respect to evaluation of the
safety, the body weight of the tumorꢀbearing mice was recorded through the testing period.
As shown in Fig. 9B, there was no remarkable body weight changes among the tested groups
during the whole experiment, indicating the safety of our formulations.
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To test whether the antitumor effect (as displayed above) was related to the EGFR gene
silencing, the mRNA and protein levels of EGFR in tumors was respectively evaluated using
qRTꢀPCR and western blot analysis, after sacrificing the animals at the completion of the
study. For the PPPꢀNPs containing EGFRꢀsiRNA (with NIR), both the EGFR mRNA (Fig.
9C) and the protein (Fig. 9D) levels exhibited obvious alterations compared with other
formulations. The results of molecular analysis were in coincidence with the antitumor
effects previously described (Fig. 9A), providing the proofs for the correlation between the
tumor inhibition and EGFR gene silencing.
Terminal deoxynucleotidyl transferase (TdT)ꢀmediated dUTP nick end labeling
(TUNEL) technique was widely applied in the estimate of tumor cell apoptosis via detecting
the breaks in DNA strand.34 As shown in Fig. 9E, neither control (5% glucose) nor free
siRNA groups displayed no green fluorescence (usually appeared in TUNELꢀpositive cells),
indicating no remarkable cell apoptosis was detected. In contrast, the EGFRꢀsiRNAꢀloaded
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PPPꢀNPs (with NIR) group demonstrated remarkable cell apoptosis (with stronger green
fluorescence) as compared with the other groups containing siRNAs. This pattern found in
the apoptotic experiment was in coincidence with the results exhibited in the study of in vivo
antitumor efficacy.
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Therefore, the aforementioned results suggested that the antiꢀtumor effect of the
EGFRꢀsiRNAꢀentrapped PPPꢀNPs combined with NIR irradiation is remarkably better than
that of free and other NPs in the nude mice tumor models, indicating the combined
mechanisms of photon and pH activation. However, the restraint of the NIR light method is
that the used external stimulus is limited to the surface tissues, notwithstanding a deep
organization may be approached via the aid of laparoscopy in the clinic. As a proof of
principle study, the long term toxicity effects of the prepared NPs was not studied in this
report, and this test will be performed when the NPs are pushed into the new drug research
process.
ꢀ
CONCLUSION
Here, we presented an effective dualꢀstimuli responsive nanoparticle system, PPPꢀNPs,
which can efficiently transport entire siRNA into the target cells for in vivo siRNA
transfection. After intravenous injection, the PPPꢀNPs can cumulate at the tumor sites
through the EPR effect, and then they will penetrate into the target cells via the activation of
CPP, which is triggered by NIR light illumination at the tumor site and lowered the pHe.
Although in preliminary, this study provided a novel strategy for experimental and clinical
oncotherapy and encourages further investigations in the exploration of CPPs.
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SUPPORTING INFORMATION
The siRNA encapsulation efficiency (EE) of prepared formulations (1) and stability of
siRNAꢀloaded NPs (2).
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ꢀ
ACKNOWLEDGMENTS
The authors gratefully acknowledge funding from the National Natural Science
Foundation of China (Grant No. 81202466 and 81402874) and the Important National
Science & Technology Specific Projects (Grant No. 2012ZX09301003ꢀ001ꢀ009) of China,
which supported this research.
ꢀ
REFERENCES
(1) Vive`s, E. Present and future of cellꢀpenetrating peptide mediated delivery systems: ‘‘is
the Trojan horse to wild to go only to Troy?’’. J. Control. Release. 2005, 109, 77ꢀ85.
(2) Jiang, T; Olson, E. S.; Nguyen, Q. T.; Roy. M.; Jennings, P. A.; Tsien, R. Y. Tumor
Imaging by Means of proteolytic activation of cellꢀpenetrating peptides. Proc. Natl.
Acad.Sci. U.S.A. 2004, 101, 17867ꢀ17872.
(3) Shi, N. Q.; Gao, W.; Bai, X.; Qi, X. R. Ehancing cellular uptake of activable
cellꢀpenetrating peptideꢀdoxorubicin conjugate by enzymatic cleavage. Int. J.
Nanomedinine. 2012, 7, 1613ꢀ1621.
(4) Koren, E.; Torchilin, V. P. Cellꢀpenetrating peptides: breaking through to the other side.
Trends Mol. Med. 2012, 18, 385ꢀ393.
(5) Jiang, T.; Zhang, Z.; Zhang, Y.; Lv, H.; Zhou, J.; Li, C.; Hou, L.; Zhang, Q.
Dualꢀfunctional liposomes based on pHꢀresponsive cellꢀpenetrating peptide and
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Molecular Pharmaceutics
1
2
3
4
5
580
hyaluronic acid for tumorꢀtargeted anticancer drug delivery. Biomaterials. 2012, 33,
9246ꢀ9258.
6
7
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
8
9
(6) Krate, F.; Abu Ajaj, K.; Warnecke, A. Anticancer carrierꢀlinked prodrugs in clinincal
trials. Expert Opin. Investig. Drugs. 2007, 16, 1037ꢀ1058.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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35
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40
41
42
43
44
45
46
47
48
49
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51
52
53
54
55
56
57
58
59
60
(7) Li, L.; ten Hagen T. L.; Schipper, D.; Wijnberg T. M.; van Rhoon, G. C.; Eggermont,
A.M.; Lindner, L.H.; Koning, G.A. Triggered content release from optimizedstealth
thermosensitive liposomes using mild hyperthermia. J. Control. Release. 2010, 143,
274ꢀ279.
(8) Hambley, T. W. Is anticancer drug development heading in the right direction? Cancer
Res. 2009, 69, 1259ꢀ1261.
(9) Zhang, J.; Chen, H. Y.; Xu, L.; Gu, Y. Q. The targeted behavior of thermally
responsivenanohydrogel evaluated by NIR system in mouse model. J. Control. Release.
2008, 131, 34ꢀ40.
(10) Melancon, M. P.; Elliott, A. M.; Shetty, A.; Huang, Q.; Stafford, R.J.; Li, C.;
Nearꢀinfrared light modulated photothermal effect increases vascular perfusion and
enhances polymeric drug delivery. J. Control. Release. 2011, 156, 265ꢀ272.
(11) Shigenaga, A.; Yamamoto, J.; Sumikawa, Y.; Furuta, T.; Otaka, A. Development and
photoꢀresponsive peptide bond cleavage reaction of twoꢀphoton nearꢀinfrared
excitationꢀresponsive peptide. Tetrahedron. Lett. 2010, 51, 2868ꢀ2871.
(12) Huang, S.; Shao, K.; Kuang, Y.; Liu, Y.; Li, J.; An, S.; Guo, Y.; Ma, H.; He, X.; Jiang, C.
Tumor targeting and microenvironmentꢀresponsive nanoparticles for gene delivery.
Biomaterials. 2013, 34, 5294ꢀ5302.
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