Dual-pH sensitive charge-reversal polypeptide micelles for tumor-triggered targeting uptake and nuclear drug delivery

Article abstract of DOI:10.1002/smll.201402865

A novel dual-pH sensitive charge-reversal strategy is designed to deliver antitumor drugs targeting to tumor cells and to further promote the nuclei internalization by a stepwise response to the mildly acidic extracellular pH (≈6.5) of a tumor and endo/lysosome pH (≈5.0). Poly(l-lysine)-block-poly(l-leucine) diblock copolymer is synthesized and the lysine amino residues are amidated by 2,3-dimethylmaleic anhydride to form β-carboxylic amide, making the polypeptides self-assemble into negatively charged micelles. The amide can be hydrolyzed when exposed to the mildly acidic tumor extracellular environment, which makes the micelles switch to positively charged and they are then readily internalized by tumor cells. A nuclear targeting Tat peptide is further conjugated to the polypeptide via a click reaction. The Tat is amidated by succinyl chloride to mask its positive charge and cell-penetrating function and thus to inhibit nonspecific cellular uptake. After the nanoparticles are internalized into the more acidic intracellular endo/lysosomes, the Tat succinyl amide is hydrolyzed to reactivate the Tat nuclear targeting function, promoting nanoparticle delivery into cell nuclei. This polypeptide nanocarrier facilitates tumor targeting and nuclear delivery simultaneously by simply modifying the lysine amino residues of polylysine and Tat into two different pH-sensitive β-carboxylic amides.

Full text of DOI:10.1002/smll.201402865

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Drug Delivery
Dual-pH Sensitive Charge-Reversal Polypeptide Micelles
for Tumor-Triggered Targeting Uptake and Nuclear
Drug Delivery
Shi-Song Han, Ze-Yong Li, Jing-Yi Zhu, Kai Han, Zheng-Yang Zeng, Wei Hong,
Wen-Xin Li, Hui-Zhen Jia, Yun Liu, Ren-Xi Zhuo, and Xian-Zheng Zhang*
A novel dual-pH sensitive charge-reversal strategy is designed to deliver antitumor
drugs targeting to tumor cells and to further promote the nuclei internalization by
a stepwise response to the mildly acidic extracellular pH (≈6.5) of a tumor and
endo/lysosome pH (≈5.0). Poly(L-lysine)-block–poly(L-leucine) diblock copolymer
is synthesized and the lysine amino residues are amidated by 2,3-dimethylmaleic
anhydride to form β-carboxylic amide, making the polypeptides self-assemble into
negatively charged micelles. The amide can be hydrolyzed when exposed to the
mildly acidic tumor extracellular environment, which makes the micelles switch to
positively charged and they are then readily internalized by tumor cells. A nuclear
targeting Tat peptide is further conjugated to the polypeptide via a click reaction. The
Tat is amidated by succinyl chloride to mask its positive charge and cell-penetrating
function and thus to inhibit nonspecific cellular uptake. After the nanoparticles are
internalized into the more acidic intracellular endo/lysosomes, the Tat succinyl amide
is hydrolyzed to reactivate the Tat nuclear targeting function, promoting nanoparticle
delivery into cell nuclei. This polypeptide nanocarrier facilitates tumor targeting and
nuclear delivery simultaneously by simply modifying the lysine amino residues of
polylysine and Tat into two different pH-sensitive β-carboxylic amides.
1. Introduction
obstacles such as indistinguishable from healthy and tumor
Chemotherapy acts a significant role in tumor therapy and tissues and nonspecific drug internalization all over the
has received great attention in the past decades. But tra- body, which cause disappointing low therapeutic efficacy and
ditional antitumor drug systems still suffer from severe fatal side effects.[1,2] Targeting delivery of drugs emerges as
a significant method to specifically transport the antitumor
drugs to tumors and decrease the normal tissues internaliza-
tion, thus reducing the side effects and improving the clin-
ical efficiency. Nowadays various targeting delivery systems
have been developed and some have been already in clinical
trials.[3] It is found that nanoparticles less than certain size
S.-S. Han, Z.-Y. Li, J.-Y. Zhu, K. Han, H.-Z. Jia, Y. Liu,
Prof. R.-X. Zhuo, Prof. X.-Z. Zhang
Key Laboratory of Biomedical Polymers
of Ministry of Education and Department of Chemistry
Wuhan University
Wuhan 430072, P.R. China
E-mail: xz-zhang@whu.edu.cn
(≈200 nm) can passively accumulate in tumor tissues after
intravenous administration due to the enhanced permeability
and retention effect.[4] The targeting efficiency can be fur-
ther enhanced by actively conjugating targeting ligands such
as antibody, peptide, folate, and biotin to drug delivery sys-
tems, which can be selectively recognized by overexpressed
receptors on tumor cells.[5] However, normal tissues usually
Z.-Y. Zeng, W. Hong, Prof. W.-X. Li
State Key Laboratory of Virology and College of Life Sciences
Wuhan University
Wuhan 430072, P.R. China
DOI: 10.1002/smll.201402865
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also express the same receptors that recognize the targeting When the nanoparticles were internalized and arrived at the
ligands just at a lower level,[6] which definitely compromise acidic endo/lysosomes (pH ≈ 5.0), the β-carboxylic amide
the targeting efficiency and therapeutic effects.
was hydrolyzed and the nuclear localization function of Tat
Stimuli-responsive delivery systems, which can undergo recovered and then the nanocarriers delivered their cargos
chemical or physical transformations once exposed to tumor into cell nuclei. This strategy inhibits the nonspecific inter-
microenvironments or external stimuli, have emerged as a nalization of Tat peptide in bloodstream while preserves its
smart and powerful strategy for targeting delivery and con- nuclear targeting capacity once internalized in tumor cells.
trolled release. Among the various stimuli such as pH, tem-
In this study, a dual-pH sensitive charge reversal nanocar-
perature, redox potential, enzymes, light, magnetic field and rier was designed for tumor triggered targeting internalization
specific bioactive molecules,[2,8] pH responsivity is one of the and nuclear delivery of antitumor drugs as shown in Scheme 1.
most extensively exploited since pH values vary in different Polypeptides which possess excellent biocompatibility and
tissues and cellular compartments. It is found that tumor biodegradability were utilized here to form basic micelles to
extracellular environment is more acidic (pH 6.2–6.9) than packagehydrophobicdrugs.[29]Amphiphilicdiblockcopolymer
blood and normal tissues (pH 7.3–7.4),[9] while the pH value in poly(l-lysine)-block–poly(l-leucine) (PLL–PLLeu) was
cellular endo/lysosomes is even lower (4.0–6.0).[10] Recently, synthesized by ring opening polymerization (ROP) of N-car-
pH dependent charge reversal delivery systems, which can boxyanhydrides (NCA) to assemble into positively charged
reverse their negative charge to positive charge upon pH micelles. Then the Tat peptide amidated by succinyl chlo-
stimuli, have been developed and exhibit great superiority ride (Tat(SA)) was conjugated via click reaction to provide
in controlled release and targeted delivery of drugs. Noted the polypeptide the ability of endo/lysosome pH sensitive
that the surface charge of nanoparticles is proved to play a nuclear targeting function. Afterward, the lysine amino resi-
momentous role in the cell internalization and the blood sta- dues of the polylysine were amidated by 2,3-dimethylmaleic
bility.[11] The charge reversal nanocarriers can maintain their anhydride (DMA) into another β-carboxylic amide to form
original negative charged status under neutral conditions negative charged PLLeu–PLL(DMA)–Tat(SA) (PPDTS)
in bloodstream thus inhibiting nonspecific interactions with micelles. The amides would be hydrolyzed once the nano-
serum proteins and normal tissues, but quickly convert into particles reach the mildly acidic tumor extracellular environ-
positively charged status once arriving at the tumor tissues or ment (pH ≈ 6.5) and the micelles would switch to positive
endo/lysosomes thus enhancing the tumor cell internalization charged again and be quickly internalized by tumor cells. By
and achieving the controlled release. Kataoka group have simply modifying the lysine amino residues of polylysine and
developed the endo/lysosome pH triggered charge reversal Tat with two different amidating reagents, respectively, this
delivery systems for cytoplasm drug release,[12,13] while Shen nanocarrier will facilitate the tumor targeting and nuclear
group[14,15] and Wang group[16–18] further developed the delivery simultaneously.
tumor mildly acidic microenvironment triggered tumor tar-
geting drug delivery, which exhibited obvious advantages and
great potential in tumor targeting drug delivery.
2. Results and Discussion
Most antitumor drugs such as doxorubicin (DOX), camp-
tothecin, and cisplatin exert their function by reacting with 2.1. Synthesis and Characterization of PPDTS
the DNA or inhibiting DNA topoisomerase to induce cell
death.[19,20] These processes mainly occur in the cell nuclei, The detailed synthesis protocol of PPDTS was shown in
where the drugs have to reach to exert their therapeutic Scheme 2. At first ε-benzyloxycarbonyl-l-lysine N-car-
effect. Multidrug resistance (MDR) is nowadays one of the boxyanhydride (LLZ–NCA) and l-leucine N-carboxyan-
most challenging and complicated barriers in chemotherapy, hydride (LLeu–NCA) were prepared by Fuchs-Farthing
since the therapeutic drugs are constantly pumped out from method through reacting ε-benzyloxycarbonyl-l-lysine
cytoplasm by the usually overexpressed P-glycoproteins (LLZ) and l-leucine (LLeu) with recrystallized triphosgene
which lead to extensively reduced therapeutic efficacy.[21,22] (Scheme 2A).[30,31] The amphiphilic diblock polypeptide PLL–
Therefore, delivery of antitumor drugs directly into cell PLLeu was then synthesized by two-step ROP of the NCA
nuclei will be of great significance to bypass the MDR effect and subsequent deprotection, while propargyl amine was used
and induce cell apoptosis more directly and efficiently.[23]
as the initiator to provide the polypeptide a terminal alkynyl
Tat peptide, derived from the protein transduction group for further click reaction (Scheme 2B). The NMR
domain of human immunodeficiency virus (HIV) transacti- spectra of LLeu–NCA, LLZ–NCA, poly(ε-benzyloxycarbonyl-
vator of transcription protein,[24] has been proved to have the l-lysine) (PLLZ), PLLZ–PLLeu, and PLL–PLLeu were
capacity of actively transporting cargos into cell nuclei and shown in Figure S1, Supporting Information. Dimethyl sulph-
thus has been exploited in many nuclear targeting delivery oxide (DMSO) -d6 was used as solvent, and all characteristic
systems.[25,26] However, the cationic Tat peptide is also char- peaks were assigned to the protons, which were in agreement
acterized by its cell penetrating capacity that quickly inter- with literature reports,[29,32] indicating the successful synthesis
nalized by all cells, which inhibits its application in targeting of every step.The feed ratios of LLZ–NCA to propargyl amine
delivery and in vivo studies.[27] Recently, a pH sensitive in polymerization were varied from 80:1, 54:1 to 36:1, and that
molecular modification strategy for Tat peptide was proposed of LLeu–NCA to LLZ–NCA were varied from 1:2.2, 1:3 to
to settle this problem.[28] The cell penetrating function of 1:6, and finally the polypeptide with the feed ratio of 1:54:25
Tat peptide was shielded via amidating by succinyl chloride. was selected for the following experiments in consideration of
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Scheme 1. Schematic illustration of A) the self-assembled polypeptide micelles stepwise responding to mildly acidic tumor tissues (pH ≈ 6.5) by
undergoing a charge conversion process and more acidic endosomes (pH ≈ 5.0) that the nuclear location function of Tat peptide was reactivated;
B) the tumor triggered cellular uptake of nanoparticles and nuclear delivery of drugs.
their solubility, assembly manner and cytotoxicity (data not into acid-labile β-carboxylic amides (Scheme S1, Supporting
shown). From the integration ratios of the characteristic reso- Information) which would be hydrolyzed at pH 5.0 environ-
nance signals, the degree of polymerization (DP) ratio of leu- ments.[28] The N3–Tat(SA) was also analyzed by MALDI–
cine to lysine was calculated to be 1:2.3. The weight-average TOF. As shown in Figure S2B, Supporting Information, an
mole mass of PLLZ–PLL determined by gel permeation increase of 200 or 300 of the molecular mass occurred com-
chromatography (GPC) in N,N′-dimethylformamide (DMF) pared to Tat peptide, implying that two lysine residues and a
was 17 370, so the DP of lysine and leucine were calculated to part of the glutamine residues were reacted. The two or three
be 55 and 24, respectively.
amino acids modification was reported sufficient to shield the
The azido-terminated N3–Tat peptide was synthesized positive charge of Tat and inhibit the nonspecific interaction
manually by standard Fmoc solid phase peptide synthesis with normal cells.[28]
(SPPS) method as demonstrated in Scheme S1, Supporting
Afterward the N3–Tat(SA) was conjugated to the amphi-
Information and characterized by matrix-assisted laser des- philic diblock polypeptide PLL–PLLeu via copper (Ι) cata-
orption/ionization time of flight (MALDI–TOF) as shown in lyzed azide–alkyne cycloaddition (CuAAC) click reaction
Figure S2A, Supporting Information.[33] The molecular mass (Scheme 2C).[35] The Fourier transform-infrared spectroscopy
of N3–Tat was calculated to be 1641.9 and that measured (FTIR) spectra of PLL–PLLeu, N3–Tat(SA), and PLLeu–
was around 1640.9, indicating the successful synthesis. The PLL–Tat(SA) (PPTS) were shown in Figure 1A. The peak at
weaker peak around m/z 1614 corresponded to a fraction of around 2100 cm−1 that derived from alkynyl groups or azide
byproduct that some azide groups were reduced to amino groups disappeared in PPTS, indicating the successful reaction.
group during peptide cleavage from the resin,[34] which could The click reaction was further confirmed by the UV–vis spectra
not be thoroughly avoided in the presence of thioscavenger, (Figure 1B) that the adsorption at 275 nm from tyrosine phenol
but it did not matter because the byproduct could not par- group of Tat emerged for PPTS, while PLL–PLLeu did not.
ticipate in the following click reaction and would be cleared
Finally the PPDTS was obtained by reacting PPTS
out in the dialysis step. The Tat peptide was further reacted with DMA in basic aqueous solution (Scheme 2D).[17] The
with succinyl chloride to amidate the lysine amino residues lysine amino groups of polylysine were modified to another
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O
O
O
Cl3CO OCCl3
OH
O
HN
NH2
O
L-Leu
LLeu-NCA
O
A
B
O
O
O
O
O
Cl3CO OCCl3
HO
H2N
O
N
N O
N
O
H
H
H
L-Lys(Z)
LLZ-NCA
O
H
N
H
L-Leu-NCA HBr/CF COOH
n
N
L-Lys(Z)-NCA
3
N
m
NH2
H
H
O
NH2
PLL-PLLeu
2-Propynylamine
O OH
OH
O
HN NH2
NH
O
NH
HN NH2
NH
HN
O
O
O
N3-Tat(SA)
O
O
O
O
O
O
H
H
H
H
H
H
N
H
C
N
N
N
N
N
N
H2N
N
H
N
N
N
H
N
N
N
NH2
m
n
O
NN
O
H O
O
H
O
H O
H
O
CuAAC
HN
H2N NH
HN
HN
HN
NH2
O
NH
HO
H2N NH H2N NH H2N NH
HO O
PLLeu-PLL-Tat(SA)
O OH
OH
O
HN NH2
NH
O
NH
HN NH2
NH
HN
O
O
O
O
O
H
N
O
O
H
N
O
O
H O
H
N
O
H O
N
H
N
O
H O
N
D
O
N
H2N
N
H
N
N
N
H
N
N
N
NH2
n
m
NN
H O
H
O
H O
H
O
pH 9
HN
H2N NH
HN
HN
HN
O NH
HO O
O
NH
HO
H2N NH H2N NH H2N NH
HO O
PLLeu-PLL(DMA)-Tat(SA)
Scheme 2. Synthesis route of PPDTS. A) Synthesis of the NCA of leucine and lysine. B) Stepwise ROP of LLZ–NCA and LLeu–NCA and the subsequent
deprotection. C) CuAAC click reaction conjugating the diblock polypeptide with Tat(SA). D) Amidating of the lysine amino residues of polylysine
into β-carboxylic amide.
acid-labile β-carboxylic amide which was more sensitive to constitute the inner core and polylysine blocks form the
acid that would hydrolyze at mildly acidic conditions such hydrophilic shell, while Tat(SA) peptides act as the outer
1
as pH 6.5. The H NMR spectrum of PPDTS was shown in corona. To ensure it the critical micelle concentration (CMC)
Figure 2. The main resonance signals were well signed to the of PPDTS was determined by pyrene fluorescent probe
corresponding protons, indicating the successful synthesis. method. As demonstrated in Figure S3, Supporting Informa-
The facile amidating reaction between lysine amino groups tion, PPDTS could assemble into micelles at both pH 9.0 and
1
and DMA was further confirmed by the H NMR spectrum pH 5.0, which, respectively, represent the status before and
of PLLeu–PLL(DMA) (PPD) (Figure S3, Supporting Infor- after the amide hydrolyzation. The CMC values were deter-
mation), which demonstrated that most (approximately mined to be 5.04 and 19.45 mg L−1, respectively. The higher
82.4%) lysine amino residues were converted to the acid- CMC value at pH 5.0 indicated that the micelles probably
labile β-carboxylic amide. The successful amidating reaction were less stable after the hydrolyzation of β-carboxylic amide.
was also confirmed by their negative charge of PPDTS nano-
A widely used antitumor drug DOX was used as the
particles determined by zeta potential measurement (data hydrophobic model drug and loaded in PPDTS micelles by
shown and discussed below).
a dialysis method. The drug loading content (weight per-
centage of drug) was determined to be 15.6%. DOX loaded
PPD micelle was also fabricated as a negative control
without additional Tat peptide, and its DOX loading con-
tent was 14.8%. The morphology and size of PPDTS micelles
2.2. Characterization of PPDTS Micelle
The PPDTS is designed to be amphiphilic to self-assemble and DOX loaded PPDTS (PPDTS/DOX) was studied by
into micellar structure. The hydrophobic polyleucine blocks transmission electronic microscopy (TEM) and dynamic
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2.3. Charge Reversal Behavior of PPDTS Nanoparticles
A
To demonstrate the charge reversal behavior of PPDTS
nanoparticles at mild acidic tumor extracellular environ-
ments, the zeta potentials of PPDTS micelles at pH 7.4 and
6.5 were measured as a function of incubation time. As illus-
trated in Figure 3, the original zeta potentials of PPDTS
nanoparticles were both negative in pH 7.4 and 6.5. How-
ever, when incubated at pH 6.5, the zeta potential increased
quite rapidly that it became positive within just 10 min. It
was because the DMA amide could be rapidly hydrolyzed at
mildly acidic environment as illustrated by the NMR experi-
ment of PPDTS in pH 6.5 (Figure S6, Supporting Informa-
tion), which in result transformed the negatively charged
β-carboxyl groups to positively charged amino groups. When
incubated in pH 7.4, the zeta potential also increased but in
a much slower speed and remained negative in more than
1 h. These phenomena were also in good consistent with lit-
erature report.[15,17] Thus the DMA modified polypeptide
nanoparticles displayed a charge reversal behavior when
exposed to mildly acidic environment.
alkyne or
azido groups
PLLeu-PLL
N -Tat(SA)
3
PLLeu-PLL-Tat(SA)
4000
3000
2000
1000
0
Wavenumber (cm-1)
B
PLL-PLLeu
N3-Tat(SA)
1.2
0.6
0.0
PLLeu-PLL-Tat(SA)
phenol
groups of Tat
210
280
Wavelength (nm)
350
2.4. Cellular Uptake and Protein Adsorption
of PPDTS Nanoparticles
Figure 1. FTIR spetra A) and UV–vis spectra B) of PLLeu–PLL, N3–Tat(SA),
and PPTS.
Due to the significant role of surface charge of nanoparticles
in cell internalization and interaction with the blood pro-
light scattering (DLS), respectively. The TEM images teins, this charge reversal property was expected to endow
(Figure S4A, Supporting Information) showed that both the nanoparticles with a relative selectivity in cellular uptake
micelles had regular spherical shapes with diameter of about between normal tissues (pH 7.3–7.4) and tumor tissues (pH
20 nm, which were in consistent with the results determined 6.2–6.9). Herein, culture mediums of two pHs were used to
by DLS (Figure S4B, Supporting Information).
simulate the neutral normal tissue and mildly acidic tumor
O
d
OH
d
OH
d
O
d
HN NH2
O
O
HN NH2
HN
O
a
d
e
d
NH
O
NH
H
NH
c
a
b
b
c
b
b
c
c
b
c
f
O
O
O
O
c
O
O
c
O
H
H
N
H
N
H
H
H
f
n
g
g
g
g
g
g
g
N
N
N
N
N
H2N
d
N
N
N
H
N
N g
H
N
H
N g
NH2
g
g
g
g
c
m
c
H
c
c H
H
f
c
N N
O
O
O
O
O
O
b
b
d
b
b
b
d
b
d
b
d
e
e
c
HN
HN
HN
HN
O
NH
O
NH
d
HO
H2N NH
H2N NH H2N NH
NH
H2N
d
d
HO
O
HO O
Figure 2. The 1H NMR spectrum of PPDTS in D2O.
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8
to PPDTS nanoparticles at pH 7.4, but approximately 60%
BSA were adsorbed to PPDTS nanoparticles when incubated
at pH 6.5. These BSA adsorption results further demon-
strated the charge reversal property of PPDTS nanoparticles
at slightly acidic environments and displayed an additional
benefit in bloodstream stability.
4
0
-4
-8
pH 7.4
pH 6.5
-12
-16
2.5. Nuclear Delivery of PPDTS Nanoparticles
0
20 40 60 80 100 120
Time (min)
Nuclear delivery of antitumor drugs is of great potential which
is expected to kill tumor cell more directly and help bypass
the MDR effect. The decoration of amidated Tat peptide
is expected to provide the nanoparticles a nuclear delivery
function, while inhibiting its nonspecific cell penetrating
Figure 3. Zeta potential of PPDTS nanoparticles (0.5 mg mL−1) at pH 6.5
or 7.4 in 20 × 10−3 M PBS as a function of incubation time.
tissue extracellular environment, respectively. Hela cells behavior before being internalized by tumor cells. When
were cultured with fluorescein isothiocyanate (FITC) labeled the nanoparticles were internalized and arrived at the acidic
PPDTS at pH 7.4 or 6.5 for 1 h, and their cellular uptake cell endo/lysosomes, the succinyl amides of Tat(SA) were
were studied by confocal laser scanning microscopy (CLSM) reported to be readily hydrolyzed and subsequently recover
and flow cytometry. FITC labeled negatively charged PLLeu– their nuclear localization function.[28] The nuclear delivery
PLL(SA)–Tat(SA) (PPSTS) and positively charged PPTS of PPDTS nanoparticles was investigated by CLSM analysis.
were used as controls. The cellular uptake of FITC–PPDTS Hela cells were cultured with DOX loaded PPDTS nanopar-
in pH 7.4 and 6.5 were also investigated in Cos7 cells as a cell ticles for 6 h, and DOX loaded PPD nanoparticles without
control.
Tat peptide were used as control. As shown in Figure 7,
As shown in Figure 4, the PPTS–FITC nanoparticles for Hela cells incubated with non-Tat PPD/DOX there was
which were positively charged at both pH 7.4 and 6.5 were little red fluorescence from DOX in cell nuclei, but it was
internalized intensively in both pHs, while PPSTS–FITC obviously enhanced when Hela cells were incubated with
nanoparticles which could not hydrolyze their amide bonds PPDTS/DOX. These results demonstrated that the PPDTS
and maintain negatively charged in pH 6.5, were rarely nanoparticles could promote the delivery of cargoes to cell
internalized in both pH 7.4 and 6.5. The significantly dif- nuclei after cell internalization. To further verify the nuclear
ferent uptake degree confirmed the important role of surface delivery behavior of PPDTS nanoparticles, FITC labeled
charge in cellular uptake and implied the great significance of PPDTS and PPD nanoparticles were incubated in Hela cells
charge reversal in selective cell internalization. As expected, and the intracellular distribution were studied. As shown in
the PPDTS–FITC nanoparticles were remarkably internal- Figure S8, Supporting Information, after internalization by
ized by Hela cells at pH 6.5 and distributed extensively in Hela cells, most PPDTS–FITC nanoparticles were distributed
cytoplasm, while far less nanoparticles were internalized at in the nucleus or very close to the nuclear membrane. In con-
pH 7.4. Very similar results were gotten when FITC–PPDTS trast, the non-Tat PPD–FITC nanoparticles were completely
were incubated in noncancerous cell line Cos7 cells at pH 7.4 in the cytoplasm and no FITC could be found in nucleus.
and 6.5 as shown in Figure S7, Supporting Information. Thus These distinct results gave a direct evidence of the nuclear
the nanoparticles exhibited obvious selectivity in cellular targeted delivery of PPDTS nanoparticles.
uptake between different pH environments.
The nuclear delivery behavior was further studied by
This different cellular uptake capacities induced by the quantitative study of DOX accumulation in cell nuclei. As
charge reversal property was further confirmed by flow shown in Figure 8, after incubation for 8 or 24 h, the DOX
cytometry analysis. As shown in Figure 5, FITC positive cells quantity in cell nuclei for PPDTS/DOX incubation was obvi-
was only 12.2% at pH 7.4, but it increased to 45.9% at pH ously larger than that for PPD/DOX incubation, indicating
6.5, exhibiting a remarkable enhancement in cellular uptake. the enhanced nuclear uptake by PPDTS nanoparticles. The
The rarely cell internalization of PPDTS at pH 7.4 also con- results above demonstrated that PPDTS could definitely
firmed that the cell penetrating function of Tat peptide was enhance the nuclear uptake of nanoparticles.
inactivated by the succinyl amidation.
Serum proteins in blood mostly possess negative charges
and readily bind onto positively charged nanoparticles via 2.6. In Vitro Drug Release
electrostatic interaction which leads to their aggregation.[36]
Keeping drug nanocarriers negatively charged is beneficial The drug release behavior of DOX loaded PPDTS nano-
for their stabilization in bloodstream. Here, bovine serum particles at pH 7.4, 6.5, and 5.0 were investigated, respec-
albumin (BSA) was used as a model serum protein to inves- tively. As shown in Figure S9, Supporting Information, the
tigate the adsorption of PPDTS and PPSTS nanoparticles DOX release was slow at pH 7.4 that 30% of drugs were
at pH 7.4 and 6.5. As shown in Figure 6, few BSA were released over 12 h and 42% for 48 h, and it was similar at
adsorbed to PPSTS nanoparticles at either pH 7.4 or 6.5 due pH 6.5 with just a slight increase. Incubation of nanoparticles
to their negative charge. Less than 20% BSA were adsorbed at pH 5.0 resulted in faster DOX release that 41% of drugs
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Figure 4. CLSM images of HeLa cells incubated with positively charged PPTS, negatively charged PPSTS, and charge reversal PPDTS samples at
pH 6.5 or 7.4 for 1 h. PPTS, PPSTS, and PPDTS samples were labeled with green fluorescent FITC and nuclei were stained blue by Hoechst 33342.
Merged 1: FITC field plus nuclei field; merged 2: FITC field and nuclei field plus bright field. Scale bar = 20 µm.
were released in 12 h and 60% for 48 h. These results were in cytotoxicity with the cell viability higher than 85% at a con-
consistent with the CMC values of PPDTS which was larger centration of 110 µg mL−1, indicating the good biocompat-
at pH 5.0 and implied a more unstable micellar structure. The ibility, while the non-Tat control PPD nanoparticles showed
faster release at pH 5.0 may also resulted from the improved a slightly higher cytotoxicity. The cytotoxicity of DOX loaded
solubility of DOX at acidic environment.
PPDTS at pH 7.4 and 6.5 to Hela cells after treating for
1 h and subsequent 48 h incubation were demonstrated in
Figure 9A. The viability of Hela cells at pH 6.5 was obviously
lower than that at pH 7.4 because of the promoted cell inter-
nalization proved above. The viability of Cos7 cells treated
2.7. In Vitro Cytotoxicity
The in vitro cytotoxicity of PPDTS and DOX loaded PPDTS with PPDTS/DOX at pH 7.4 and 6.5 showed the same trend
nanoparticles were estimated by 3-[4,5-dimethylthiazol-2-yl]- (Figure S10B, Supporting Information). These results were
2,5-diphenyltetrazolium-bromide (MTT) assay at various consistent with the selective cellular uptake at different pH
nanoparticles/DOX concentrations.As shown in Figure S10A, induced by charge reversal behavior. The cytotoxicity of
Supporting Information, PPDTS nanoparticles showed little PPD/DOX as a non-Tat control was also investigated. Hela
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Figure 5. Flow cytometry analysis of Hela cells incubated with PPDTS–FITC at pH 7.4 B) or 6.5 C) for 1 h. Hela cells incubated without PPDTS–FITC
was used as control A).
cells were treated with PPDTS/DOX or PPD/DOX at var- amidation charge reversal strategy helps to realize the tumor
ious DOX concentrations for 8 h and further incubated for targeting uptake and nuclear delivery simultaneously and
48 h before MTT analysis. As presented in Figure 9B, the thus provides a promising method for drug delivery in tumor
cytotoxicity of PPDTS/DOX was larger than the non-Tat chemotherapy.
control because of the promoted delivery of DOX to cellular
nuclei and more direct killing cells. These cytotoxicity results
further demonstrated the advantages of charge reversal tar-
geting uptake and nuclear delivery of drugs and indicated the
4. Experimental Section
Materials: Rink Amide-AM resin (100–200 mesh, loading:
0.59 mmol g−1), N-diisopropylethylamine (DIEA), piperidine,
1-hydroxybenzotriazole (HOBt), o-benzotriazole-N,N,N′,N′-tetr
amethyluroniumhexafluophosphate (HBTU), L-leucine (LLeu),
LLZ and N-Fluorenyl-9-methoxycarbonyl protected amino acids
(Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Gly-OH, Fmoc-Tyr(tBu)-OH) were purchased from GL Biochem. Ltd.
(Shanghai, China). Succinyl chloride and DMA were purchased
from Aladdin Reagent Co. Ltd. (Shanghai, China). Triphosgene was
purchased from J&K Scientific (Beijing, China) and recrystallized
by diethyl ether prior to use. Tris(3-hydroxypropyltriazolylmethyl)
amine (THPTA) and CuBr were synthesized in prior work of our
group.[37] Propargyl amine, trifluoroacetic acid (TFA), DMF and tet-
rahydrofuran (THF) were of analytical grade and distilled before
use. DOX hydrochloride was provided by Zhejiang Hisun Pharma-
ceutical Co. (China). Dulbecco’s Modified Eagle’s Medium (DMEM),
fetal bovine serum (FBS), penicillin, streptomycin, were purchased
from Invitrogen (CA, USA). MTT and Hoechst 33342 were pur-
chased from Lonza Co. (USA). All other reagents and solvents were
obtained from Shanghai Reagent Chemical Co. and used directly.
Synthesis of LLZ–NCA and LLeu–NCA: LLZ–NCA and LLeu–
NCA were prepared by Fuchs-Farthing method using triphosgene
according to literature reports.[30,31] For LLeu–NCA synthesis, L-leu-
cine (2.6 g, 20 × 10−3 M) was dissolved in freshly prepared dry THF
(90 mL) in a three-necked flask. Then the flask was charged with
steady flow of N2 to ensure anhydrous environment and triphos-
gene (2.5 g, 8.4 × 10−3 M) solution in THF (10 mL) was added drop-
wise under vigorous stirring at 50 °C. The reaction was stopped
when the mixture turned to a clear solution. Subsequently the
solution was poured into excess petroleum ether to obtain crude
crystals of LLeu–NCA, which was further recrystallized from THF/n-
hexane twice, filtrated, and dried in vacuum. LLZ–NCA was synthe-
sized with the similar procedure above.
promising application of PPDTS micelle in drug delivery for
tumor therapy.
3. Conclusions
In summary, we designed a polylysine-block–polyleucine
diblock polypeptide micelle decorated with Tat peptide,
and the lysine amino residues of Tat and polylysine were,
respectively, modified into two different β-carboxylic amides.
The amides were demonstrated to stepwise hydrolyzed
responding to acidic tumor extracellular environments (pH ≈
6.5) and more acidic cell endo/lysosomes (pH ≈ 5.0), respec-
tively, by which the amino groups and their original functions
were recovered.The zeta potential measurements and in vitro
cellular experiments proved that the PPDTS nanoparticles
exhibited a dual-pH sensitive charge reversal behavior that
they were specifically internalized by cells at tumor mildly
acidic environment and directly transported into cell nuclei.
Further investigations might be needed to depress the pre-
disassembly before reaching cell nuclei so as to signifi-
cantly enhance the therapeutic efficiency. Overall, this double
60
pH 6.5
pH 7.4
40
20
0
PPDTS
PPSTS
Synthesis of Propargyl-Terminated Diblock Copolymer PLL–
PLLeu: PLL–PLLeu was synthesized by stepwise ROP of LLZ–NCA
and LLeu–NCA using propargyl amine as initiator. First, propargyl-
Figure 6. BSA adsorption of charge reversal PPDTS or negatively
charged PPSTS at pH 6.5 or 7.4 after incubation at 37 °C for 2 h. Data
are shown as mean SD (n = 3).
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chains were grown on Rink Amide AM resin in
a stepwise manner at room temperature with
distilled anhydrous DMF as solvent. Twenty
percent piperidine in DMF (v/v) was employed
to remove the Fmoc protecting group, while
amino acid coupling was carried out with
4 equiv of Fmoc amino acid, HBTU, HOBt and
8 equiv of DIEA in DMF solution for 2 h, and
the ninhydrin assay was used to monitor the
coupling efficiency. The azide group was con-
jugated by reaction with 2-azidoacetic acid
at the end with the same method. After that
the resin was washed with DMF, methanol,
and dichloromethane three times each and
dried under vacuum for 4 h. The peptide was
gotten by cleavage from the resin and fully
deprotected simultaneously using a cleavage
cocktail of TFA, thioanisole, H2O, phenol and
1,2-ethanedithiol in a volume ratio of 83:
4.3: 4.3: 6.3: 2.1 for 2 h. The filtrate was col-
lected, concentrated by rotary evaporation,
and then precipitated into excess cold ether.
Figure 7. Confocal images of Hela cells incubated with PPD/DOX or PPDTS/DOX for 6 h . The
concentration of DOX was 5.0 µg mL−1. Merged 1: DOX field plus nuclei field; merged 2: DOX
field and nuclei field plus bright field. Scale bar = 10 µm.
The obtained precipitation was further washed with cold ether four
times to remove the cleavage residuals and impurities. After that
the precipitation was dissolved in distilled water and freeze dried
to obtain the final peptide.
terminated PLLZ was synthesized by ROP of LLZ–NCA initiated by
propargyl amine in dried DMF with certain ratio. The solution was
stirred at 40 °C under N2 atmosphere for 3 days and then precipi-
tated by diethyl ether twice and dried under vacuum to acquire
PLLZ white powders. Second the PLLZ–PLLeu copolymer was pre-
pared by further ROP of LLeu–NCA using PLLZ as initiator. Given
amount of PLLZ and LLeu–NCA were dissolved in dried DMF and
stirred under N2 atmosphere for 3 days. The product was purified
by repeated precipitation in excess diethyl ether and dried under
vacuum.
PLL–PLLeu was obtained by the deprotection of PLLZ–PLLeu.
PLLZ–PLLeu was dissolved in TFA in an ice bath and a 33% solu-
tion of HBr in acetic acid was added dropwise. The mixture was
stirred at 0 °C for 1 h and subsequently precipitated by diethyl
ether, centrifuged, and dried in vacuum. The crude product was
further purified by dialysis in a dialysis bag (molecular weight cut-
off (MWCO) = 3500) against distilled water for 48 h, during which
the water was refreshed every 6 h. PLL–PLLeu was obtained after
lyophilized.
Synthesis of Succinyl Amidated N3–Tat(SA): N3–Tat was ami-
dated by succinyl chloride according to a previous report.[28] N3–
Tat (0.2 g) was dissolved in 10 mL phosphate buffered solution
(PBS) (pH 8.5, 50 × 10−3 M) and cooled to 0 °C. Succinyl chloride
(0.5 mL) was added dropwise to the solution and about 4 equiv
of NaOH was added to maintain the pH around 8.5. After reacting
overnight at 0 °C, the mixture was loaded into a dialysis bag
(MWCO 1000) and dialyzed against distilled water of which the pH
was adjusted around 8.5 by 0.1 M NaOH solution for 24 h, and the
water was changed every 4 h. The following solution was lyophi-
lized to obtain the N3–Tat(SA).
Synthesis of PPTS by Click Reaction: PPTS was synthesized
by conjugating of PLL–PLLeu with N3–Tat(SA) by copper (Ι) cata-
lyzed azide-alkyne cycloaddition reaction. PLL–PLLeu (150 mg)
and N3–Tat(SA) (50 mg) were dissolved in distilled water (10 mL)
and charged with constant N2 under magnetic stirring. Then CuBr
(30 mg) and THPTA (40 mg) were added to get a celadon solution.
After reaction for 24 h, the solution was loaded into a dialysis bag
(MWCO 3500) and dialyzed against ethylenediaminetetraacetic
acid (EDTA) solution of which the pH was adjusted around 8.0 by
NaOH. The water was changed every 4 h until the solution became
colorless. The subsequent solution was freeze dried to obtain the
PPTS.
Synthesis of Azido-Terminated Tat Peptide N3YGRKKRRQRRR
(N3–Tat): N3–Tat was synthesized manually by the standard Fmoc
SPPS protocol using Rink Amide AM resin.[33] Briefly, peptide
400
PPD/DOX
PPDTS/DOX
300
Synthesis of PPDTS, PPD, and PPSTS: PPDTS was prepared by
modifying the lysine amino residues of PLL blocks into β-carboxylic
amide using DMA according to previous literature.[17] PPTS (40 mg)
and three equivalents (to amino groups) of DMA (80 mg) were
dissolved in distilled water and NaOH was added to the solution
to maintain the pH around 10.0. After reaction for 12 h, the solu-
tion was purified by dialysis (MWCO 3500) against slightly basic
water (pH 9–10) and freeze dried. The FITC labeled samples were
prepared directly reacting between its amino group and FITC in
dark for 6 h and subsequently dialysis and lyophilized. PPD was
200
100
0
8h
24h
Figure 8. Quantitative study of nuclear delivery behavior of PPDTS
nanocarrier: the nuclear accumulation of DOX for HeLa cells incubated
with PPD/DOX or PPDTS/DOX for 8 h or 24 h. Data are shown as mean
SD (n = 3).
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probe. Pyrene solution in acetone (100 µL,
6 × 10−6 M) was added into a series of cen-
trifugal tubes, and the acetone was allowed
to evaporate. Then 1.0 mL of PPDTS aqueous
solution of various concentrations was added
to the above tubes, shaked vigorously, and
kept overnight. The final concentration of
pyrene was 6 × 10−7 M, exceeding the satu-
ration solubility of pyrene in water at room
temperature. Emission wavelength was car-
ried out at 390 nm and excitation spectra
were recorded ranging from 300 to 360 nm.
Hela pH7.4
Hela pH6.5
PPDTS/DOX
PPD/DOX
100
75
50
25
0
100
75
50
B
A
25
0
0
5
10
15
20
0
5
10
15
20
Concentration (mg/L)
Concentration (mg/L)
Figure 9. Cell viability of Hela cells treated with A) PPDTS/DOX at pH 7.4 or 6.5 for 1 h and
further incubated of 48 h; B) PPDTS/DOX or PPD/DOX for 8 h and further incubated for 48 h.
Cell viability were determined by MTT assay at various DOX concentrations (mean SD, n = 4).
Both emission and excitation slit widths were
5 nm. A redshift of excitation peak arose
around 335 nm. The intensity ratios as a func-
tion of logarithm of the PPDTS concentrations
synthesized by PLL–PLLeu and DMA in the similar method. PPSTS
was synthesized by PPTS and succinyl choloride similar to that of
N3–Tat(SA).
were analyzed and plotted. The CMC value was determined from
the intersection of the tangent to the curve at the inflection with
the horizontal tangent through the points at low concentration.[3]
Changes of Zeta Potential of the PPDTS Micelles at Different
pHs: The PPDTS was dispersed in pH 7.4 and 6.5 PBS (20 × 10−3 M)
at the concentration of 0.5 mg mL−1 and incubated at 37 °C. Sam-
ples were taken at designated time intervals and the zeta poten-
tials were measured. Each measurement was performed for three
times and each time for 30 runs and the results presented are the
average data.
Hydrolysis of PPDTS in pH 6.5 Monitored by NMR: The PPDTS
was dispersed in PBS of pH 6.5 (20 × 10−3 M) and incubated. After
certain time (0, 10, 30, and 60 min), some PPDTS solution was
quickly taken out and freeze dried. The lyophilized samples were
characterized by 1H NMR in D2O recorded on a Varian Mercury VX
300 MHz spectrometer. The signals around 2.9 and 3.1 ppm in the
spectra were compared and analyzed.
Cellular Uptake at Different pHs Observed by CLSM: Hela cells
were seeded in a glass bottom dish at a density of 1 × 105 cells
per dish in 1 mL of DMEM with 10% FBS and 1% antibiotics (peni-
cillin–streptomycin, 10 000 U mL−1). The cells were incubated at
37 °C in a humidified atmosphere containing 5% CO2 for 24 h.
Then the culture medium was replaced by fresh DMEM containing
FITC–PPDTS, FITC–PPSTS, or FITC–PPTS at the equivalent sample
concentration of 30 µg mL−1 at pH 7.4 or 6.5, respectively. After
incubation for 1 h at 37 °C, the cells were washed five times with
PBS. Subsequently the cell nuclei were stained by Hoechst 33342
for 15 min and washed three times with PBS. The cellular uptake
of samples was visualized under a laser scanning confocal micro-
scope (C1-Si, Nikon, Japan). The measurements of cellular uptake
of FITC–PPDTS in Cos7 cells at pH 7.4 or 6.5 were also investigated
with the same method.
Cellular Uptake at Different pHs Measured by Flow Cytometry:
Hela cells were seeded onto a six-well plate at a density of 5 × 104
cells per well and cultured for 24 h. Then the culture medium was
removed and replaced with DMEM (pH 7.4 or 6.5) containing FITC–
PPDTS at concentration of 40 µg mL−1 and the cells were further
incubated for 1 h at 37 °C. The medium was removed and the cells
were washed thrice with PBS. Then 0.5 mL of trypsin was added
to each well to digest the cells. The cells were collected and cen-
trifuged at 1000 rpm for 3 min. The supernatant was removed and
the cells at the bottom were washed once, centrifuged again, and
resuspended in PBS. After been filtrated the cells were quickly
Preparation of DOX Loaded PPDTS and PPD: A widely used
antitumor drug DOX was chosen as the model drug. DOX hydro-
chloride (10 mg) was dissolved in 3 mL of DMSO and triethylamine
(0.15 mL) was added to remove the HCl of the DOX hydrochloride.
PPDTS (50 mg) was added to the solution and stirred in dark for
4 h. Then the solution was added dropwise to 5 mL PBS (pH 9.5)
and stirred for another 3 h. After that the solution was loaded into
a MWCO 3500 dialysis bag and dialyzed against pH 9–10 water
until there was almost no DOX in the dialyzed water monitored
by fluorescence spectroscopy. The obtained solution was mildly
centrifuged and the supernatant was stored in 0 °C which would
be used directly. The DOX loading content was determined by lyo-
philizing 1.0 mL of the above solution and dissolved the obtained
powder in DMSO. The DOX concentration was measured by fluores-
cence analysis (emission wavelength at 560 nm, excitation wave-
length at 488 nm). The DOX loaded PPD was fabricated with the
similar procedure.
Characterizations: The molecular mass of peptide N3–Tat and
N3–Tat(SA) was determined by the MALDI–TOF on an Axima TOF2
mass spectrometer (Shimadzu, Kyoto, Japan). Every step synthe-
sizing PLL–PLLeu and the final product PPDTS were monitored by
1H NMR recorded on a Varian Mercury VX 300 MHz spectrometer
using DMSO-d6 or D2O as the solvent and tetramethylsilane (TMS)
as the internal standard. Molecular weight of polypeptide was
determined by GPC system equipped with a Waters 2690D sepa-
rations module and a Waters 2410 refractive index detector. DMF
was used as the eluent solution and polystyrene as standard. The
success of the click reaction was characterized by FTIR collected on
a Perkin-Elmer spectrophotometer (USA) using potassium bromide
pellet, and UV–vis was also performed on a Lambda Bio 40 UV–vis
spectrometer (Perkin-Elmer, USA).
The zeta potential, hydrodynamic particle size, and size dis-
tribution were measured by DLS on a Malvern Zetasizer Nano-ZS
ZEM3600 apparatus (UK). Morphology observation of micelles was
performed by TEM with a JEM-100CX II microscopy at an accelera-
tion voltage of 100 kV and the samples were negatively stained by
phosphotungstic acid. Fluorescent measurement was performed
on a RF-530/PC fluorescence spectrometer.
Determination of CMC: The CMC value of PPDTS was determined
by fluorescence spectroscopy using pyrene as a hydrophobic
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examined by flow cytometry (BD FACS Aria Ш, USA). Cells cultured fresh medium. The buffer collected was subjected to fluorescence
at pH 7.4 DMEM without FITC–PPDTS was used as negative control. analysis to determine the DOX concentration. The fluorescence
Protein Adsorption of the Micelles: BSA was used as model spectroscopy was measured at emission wavelength of 560 nm
protein to determine the protein adsorption ability of the PPDTS and excited at 488 nm, using a standard calibration curve experi-
and PPSTS at different pHs.[18] The PPDTS/PPSTS were incubated mentally obtained. The experiments were carried out in triplicate
with 1 mL of BSA solution in PBS at pH 7.4 or 6.5 with the final and data were shown as mean standard deviation (SD).
concentration of micelles and protein at 0.2 and 0.4 mg mL−1
,
Cytotoxicity Assay: The cytotoxicity of PPDTS, PPDTS/DOX, PPD,
respectively. After incubation at 37 °C for 1 h, each sample was and PPD/DOX were evaluated by the MTT assay. Hela and Cos7
centrifuged at 8000 rpm for 15 min to precipitate the protein cells were seeded in 96-well plates at a density of 6000 cells per
adsorbed aggregates, and the supernatant was carefully collected. well in 100 µL DMEM containing 10% FBS and cultured for 24 h at
The protein concentration of supernatant was determined by UV– 37 °C. Then the cells were treated with 100 µL culture medium of
vis spectroscopy by measuring its characteristic UV absorbance at pH 7.4 or 6.5 containing fixed amount of nanoparticles for 1 or 8 h
280 nm, and compared to the standard curve obtained from BSA and further incubated for 48 h. After that the medium was replaced
solutions of known concentrations. Then, the adsorbed proteins with 200 µL of fresh DMEM and 20 µL MTT (5 mg mL−1 in PBS)
on the samples were calculated as the degree adsorbed.
and incubated for another 4 h, allowing live cells to change the
Nuclear Localization of DOX Measured by CLSM: Hela cells were yellow tetrazolium salt into dark blue formazan crystals. Then the
seeded in a glass bottom dish at a density of 1 × 105 cells per dish medium was removed and 200 µL DMSO was added. The absorb-
and incubated at 37 °C for 24 h. Then the culture medium was ance at 570 nm was collected using a micro plantreader (Bio-Rad,
replaced by fresh DMEM containing PPDTS/DOX or PPD/DOX at the Model 550, USA). The cytotoxicity was expressed as percentage of
equivalent DOX concentration of 5 µg mL−1 at pH 6.5, respectively. cell viability versus the control: cell viability = ODsample/ODcontrol
×
After incubation for 6 h at 37 °C, the cells were washed three times 100%, where ODsample was obtained in the presence of PPDTS or
with PBS. Subsequently the cell nuclei were stained by Hoechst PPDTS/DOX and ODcontrol was obtained in the absence of samples
33342 and washed with PBS. The nuclear uptakes of DOX were (OD means optical density). Data were shown as mean SD based
visualized under a confocal microscope.
on four independent experiments.
Nuclear Delivery of FITC Labeled Nanoparticles Measured by
CLSM: Hela cells were cultured as above and treated with PPDTS–
FITC and PPD–FITC for 6 h at 37 °C. Then the cells were washed
three times with PBS. Subsequently the cell nuclei were stained
by Hoechst 33342 and washed with PBS. The intracellular distribu-
tion of FITC labeled nanoparticles were observed under a confocal
microscope.
Supporting Information
Supporting Information is available from the Wiley Online Library
or from the author.
Quantitative Studies of DOX Accumulation in Cell Nuclei: Hela
cells were seeded onto six-well plates at a density of 1 × 105 cells
per well and cultured in DMEM with 10% FBS at 37 °C. After incu-
bation for 24 h, the cells were cultured in DMEM containing PPDTS/
DOX or PPD/DOX at DOX concentration of 10 µg mL−1 for 8 or
24 h. Then the cells were washed with PBS three times and trypsin
digested. After centrifugation at 1400 rpm for 3 min, the cells
were resuspended in PBS and washed twice. Then the concentra-
tions of cells were counted with a hemocytometer. Subsequently,
nuclei were isolated from cytoplasm by disrupting cell membranes
using surfactants and further centrifugation as follows. The cells
were suspended in lysis buffer of 100 × 10−3 M NaCl solution with
1 × 10−3 M EDTA, 1% Triton X-100 and 10 × 10−3 M Tris buffer (pH
7.4) at 4 °C for 10 min. The suspension was then centrifuged at
4000 rpm for 6 min to separate the cell nuclei from the plasma
membrane fragments. Then the precipitate of cell nuclei was col-
lected and suspended in lysis buffer again and disrupted by
ultrasound for 0.5 h. The amount of DOX in the cell nuclei was
determined by fluorescence measurement.
In Vitro Release of DOX from PPDTS/DOX: The release behavior
of DOX from PPDTS/DOX was investigated in three different
mediums, namely, pH 7.4 phosphate buffer (20 × 10−3 M), pH
6.5 phosphate buffer (20 × 10−3 M), and pH 5.0 acetate buffer
(20 × 10−3 M). PPDTS/DOX aqueous solution (0.5 mL) was added to
each dialysis bag (MWCO 3500). The dialysis bags were immersed
in 10 mL of the above three buffers, respectively, and kept in
37 °C bath with constant shaking (200 rpm). At predetermined
time the whole medium was collected and resupplied with 10 mL
Acknowledgements
We acknowledge the financial support from the National Natural
Science Foundation of China (51125014 and 51233003), the
Ministry of Science and Technology of China (2011CB606202),
and Natural Science Foundation of Hubei Province of China
(2013CFA003).
[1] R. Langer, Nature (London) 1998, 392, 5.
[2] Z. S. Ge, S. Y. Liu, Chem. Soc. Rev. 2013, 42, 7289.
[3] I. K. Kwon, S. C. Lee, B. Han, K. Park, J. Controlled Release 2012,
164, 108.
[4] K. Kim, J. H. Kim, H. Park, Y. S. Kim, K. Park, H. Nam, S. Lee,
J. H. Park, R.-W. Park, I. S. Kim, K. Choi, S. Y. Kim, K. Park,
I. C. Kwon, J. Controlled Release 2010, 146, 219.
[5] Y. Li, G. H. Gao, D. S. Lee, Adv. Healthcare Mater. 2013, 2, 388.
[6] N. Parker, M. J. Turk, E. Westrick, J. D. Lewis, P. S. Low,
C. P. Leamon, Anal. Biochem. 2005, 338, 284.
[7] J. Y. Rao, A. Khan, J. Am. Chem. Soc. 2013, 135, 14056.
[8] J. Y. Rao, C. Hottinger, A. Khan, J. Am. Chem. Soc. 2014, 136,
5872.
[9] R. A. Cardone, V. Casavola, S. J. Reshkin, Nat. Rev. Cancer 2005,
5, 786.
[10] G. F. Luo, W. H. Chen, Y. Liu, J. Zhang, S. X. Cheng, R. X. Zhuo,
X. Z. Zhang, J. Mater. Chem. B 2013, 1, 5723.
small 2015,
DOI: 10.1002/smll.201402865
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.small-journal.com
11

full papers
www.MaterialsViews.com
[11] S. E. A. Gratton, P. A. Ropp, P. D. Pohlhaus, J. C. Luft, V. J. Madden, [25] S. S. Patel, B. J. Belmont, J. M. Sante, M. F. Rexach, Cell 2007,
M. E. Napier, J. M. DeSimone, Proc. Natl. Acad. Sci. USA 2008,
105, 11613.
[12] Y. Lee, T. Ishii, H. Cabral, H. J. Kim, J. H. Seo, N. Nishiyama,
129, 83.
[26] L. M. Pan, Q. J. He, J. N. Liu, Y. Chen, M. Ma, L. L. Zhang, J. L. Shi,
J. Am. Chem. Soc. 2012, 134, 5722.
H. Oshima, K. Osada, K. Kataoka, Angew. Chem. Int. Ed. 2009, 48, [27] J. Hoyer, I. Neundorf, Acc. Chem. Res. 2012, 45, 1048.
5309.
[28] E. L. Jin, B. Zhang, X. R. Sun, Z. X. Zhou, X. P. Ma, Q. H. Sun,
J. B. Tang, Y. Q. Shen, E. V. Kirk, W. J. Murdoch, M. Radosz, J. Am.
Chem. Soc. 2013, 135, 933.
[13] Y. Lee, T. Ishii, H. J. Kim, N. Nishiyama, Y. Hayakawa, K. Itaka,
K. Kataoka, Angew. Chem. Int. Ed. 2010, 122, 2606.
[14] P. S. Xu, E. A. V. Kirk, Y. H. Zhan, W. J. Murdoch, M. Radosz, [29] Z. Y. Li, H. Y. Wang, C. Li, X. L. Zhang, X. J. Wu, S. Y. Qin,
Y. Q. Shen, Angew. Chem. Int. Ed. 2007, 46, 4999. X. Z. Zhang, R. X. Zhuo, J. Polym. Sci. Pol. Chem. 2011, 49, 286.
[15] Z. X. Zhou, Y. Q. Shen, J. B. Tang, M. H. Fan, E. A. V. Kirk, [30] A. C. Farthing, R. J. W. Reynolds, Nature 2014. 65, 647.
W. J. Murdoch, M. Radosz, Adv. Funct. Mater. 2009, 19, [31] M. Kar, P. S. Vijayakumar, B. L. V. Prasad, S. S. Gupta, Langmuir
3580.
2010, 26, 5772.
[16] J. Z. Du, T. M. Sun, W. J. Song, J. Wu, J. Wang, Angew. Chem. Int. [32] A. J. Qin, J. W. Y. Lam, B. Z. Tang, Chem. Soc. Rev. 2010, 39, 2522.
Ed. 2010, 122, 3703.
[17] J. Z. Du, X. J. Du, C. Q. Mao, J. Wang, J. Am. Chem. Soc. 2011, 133,
17560.
[18] Y. Y. Yuan, C. Q. Mao, X. J. Du, J. Z. Du, F. Wang, J. Wang, Adv.
Mater. 2012, 24, 5476.
[19] D. L. Crowe, Recent Res. Dev. Cancer 2002, 4, 65.
[33] W. C. Chan, P. D. White, Fmoc Solid Phase Peptide Synthesis: A
Practical Approach, Oxford, Oxford, UK 2000.
[34] X. H. Luo, F. W. Huang, S. Y. Qin, H. F. Wang, J. Feng, X. Z. Zhang,
R. X. Zhuo, Biomaterials, 2011, 32, 9925.
[35] P. E. Schneggenburger, B. Worbs, U. Diederichsen, J. Pept. Sci.
2010, 16, 10.
[20] M. A. Fuertes, J. Castilla, C. Alonso, J. M. Perez, Recent Res. Dev. [36] L. Rong, L. H. Liu, S. Chen, H. Cheng, C. S. Chen, Z. Y. Li,
Mol. Pharmacol. 2002, 1, 37. S. Y. Qin, X. Z. Zhang, Chem. Commun. 2014, 50, 667.
[21] G. Szakacs, J. K. Paterson, J. A. Ludwig, C. B. Genthe, [37] C. Deng, J. T. Wu, R. Cheng, F. H. Meng, H. A. Klok, Z. Y. Zhong,
M. M. Gottesman, Nat. Rev. Drug Discovery 2006, 5, 219.
[22] H. Lage, Cell Mol. Life Sci. 2008, 65, 3145.
Prog. Polym. Sci. 2014, 39, 330.
[23] L. M. Pan, J. N. Liu, Q. J. He, L. J. Wang, J. L. Shi, Biomaterials
2013, 34, 2719.
[24] E. Vives, P. Brodin, B. Lebleu, J. Biol. Chem. 1997, 272, 16010.
Received: September 25, 2014
Revised: December 26, 2014
Published online:
www.small-journal.com
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2015,
DOI: 10.1002/smll.201402865
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