nir-ii fluorescence image-guided and ph-responsive nanocapsules for cocktail drug delivery · 2014....
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Nano Res
1
NIR-II fluorescence image-guided and pH-responsive
nanocapsules for cocktail drug delivery
Sheng Huang, Shan Peng, Yuanbao Li, Jiabin Cui, Hongli Chen, and Leyu Wang*()
Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-015-0702-5
http://www.thenanoresearch.com on December 23 2014
© Tsinghua University Press 2014
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Nano Research
DOI 10.1007/s12274-015-0702-5
TABLE OF CONTENTS (TOC)
NIR-II Fluorescence Image-Guided and
pH-Responsive Nanocapsules for Cocktail Drug
Delivery
Sheng Huang, Shan Peng, Yuanbao Li, Jiabin Cui, Hongli
Chen, and Leyu Wang*
Beijing University of Chemical Technology, China
A versatile hydrogen bond-based pH-responsive (pH 5.0) nanocapsule
with small sizes (< 100 nm), good ability of real-time NIR-II
fluorescence tracking of the drug pharmacokinetics and
biodistribution, tumor targeting, and sustained release (up to 14 days)
for general hydrophobic anti-cancer drugs were successfully fabricated
and applied for the targeting and pH-controlled release of therapeutic
agents.
NIR-II Fluorescence Image-Guided and pH-Responsive
Nanocapsules for Cocktail Drug Delivery
Sheng Huang, Shan Peng, Yuanbao Li, Jiabin Cui, Hongli Chen, and Leyu Wang*()
Received: day month year
Revised: day month year
Accepted: day month year
(automatically inserted by
the publisher)
© Tsinghua University Press
and Springer-Verlag Berlin
Heidelberg 2014
KEYWORDS
near IR fluorescence, pH
responsive, cocktail drug
delivery
ABSTRACT
Nanocapsule-based targeted delivery and stimuli-responsive release can
increase drug effectiveness while reducing side effects. However, difficulties in
the scaling-up synthesis, fast burst release, and low degradability are likely to
hamper the translation of drug nanocapsules from lab to clinic. Here we
controllably functionalize the biodegradable and widely available
polysuccinimide to get the amphiphilic poly(amino acid). By using this
polymer, we design the nanocapsules (<100 nm) for hydrophobic drug delivery
that can provide tumor targeting, hydrogen-bond-based pH-responsive release,
and real-time fluorescence tracking in the second near-infrared region. This
method is versatile, green, and easy to scale up at low cost for cocktail drug by
loading multiple anticancer drugs. Our nanocapsules are stable in blood vessel
(pH 7.4) and the pH-responsive release (pH 5.0 in lysosome) is sustained. The
chemotherapy results in tumor xenografted mice suggest that our nanocapsule
is safe and efficient and may be a useful tool for drug delivery.
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2 Nano Res.
1 Introduction
Nanocapsules have been successfully introduced
to the drug delivery owing to the ability to target
the delivery of drug more precisely, improve the
solubility of hydrophobic drugs, extend their
half-life, reduce the side-effect and improve their
theraputic effecacy.[1-3] The nanocapsule size,[4, 5]
stimuli-responsive,[6-8] controlled and sustained
drug release[9] play pivotal roles in the application
of nanocapsules during delivery processes. The
facile intravenous injectability of the nanocapsules
is another advantage which makes them attractive
in a variety of clinical applications. For longer
circulation time, the size of the injectable
nanocapsules should be in the range of 10-200 nm
to escape kidney filtration (< 5.5 nm)[10, 11] and
removal by resident macrophages in the
reticuloendothelial system,[12] including the liver
and the spleen.[4, 13] Some small-molecule
anti-cancer drugs such as paclitaxel (PTX)[14] and
camptothecine (CPT) have poor bioavailability and
suboptimal pharmacokinetics due to their
hydrophobicity and low molecular weight. In
addition to rapid clearance, another challenge is
the fast burst release of the chemotherapeutic
drugs from the nanocapsules. Immense progress in
materials chemistry and drug delivery has led to
the design of smart stimuli-responsive
nanocapsules to improve the water-dispersibility,
bioavailability, and controlled and sustained drug
release of anticancer drugs in response to specific
cellular signals.[15-19] Unfortunately, many available
stimuli-responsive systems have limited chances of
reaching the clinic because of poor degradability or
insufficient biocompatibility, the complexity of the
nanocapsule design, and difficulties for large-scale
production.[20]
Considering the pH gradients present in tumor
tissue and cancer cells, different kinds of
acid-sensitive covalent bonds such as disulfide,[21]
acyl hydrazone,[22] boronic acid[23] and acetal[24]
bonds have been adopted to develop pH-sensitive
nanocapsules. Despite the difficulties in
characterization,[25, 26] noncovalent bonds, including
hydrophobic interaction and hydrogen bonds
(H-bonds), play a key role in the interaction
between the drug and targets.[27, 28] Moreover, many
kinds of hydrogel for drug delivery are formulated
through H-bonds whose swelling-shrinking
processes are usually controlled by pH or
temperature.[29-31] However, H-bonded networks
will dilute and disperse in vivo due to water influx,
which leads to undesirable premature drug release.
In addition, most of the hydrogels are
noninjectable via vein due to their high viscosity
and thus large sizes, which often limits the in vivo
application through blood circulation.
Nevertheless chemical modification of the drug or
polymer is necessary and the developed
nanocapsules are just suitable for the specific
therapeutic agent in most cases. However, the drug
cocktail[32] nanocapsules in which drugs used in
combination strengthen each other's efficacy due to
the synergistic effect, were more effective than the
chemotherapy drug traditionally used alone. So, it
is highly desirable to formulate the versatile
pH-responsive cocktail nanocapsules with low-cost
and large-scale production, appropriate sizes and
good water dispersibility for multiple drugs based
on water-tolerate H-bonds with more precise pH
dependence. Moreover, the real-time tracking[33-35]
of the therapeutic agents without relying on radio
labelling in live animals is highly desirable as an
important tool in understanding and monitoring
tumour responses and tumour growth in vivo.
In this study, we design a facile and versatile
cocktail nanocapsule via the assembly of
biodegradable and amphiphilic poly(amino acid)
[polysuccinimide (PSI)[36] functionalized with
oleylamine (OAm), termed PSIOAm]. It should be
mentioned that the polysuccinimide is
biodegradable, widely available, and cheap
(<3000$/ton), which is highly suitable for
large-scale production. Chemotherapy drugs such
as paclitaxel (PTX) and camptothecine (CPT) are
successfully encapsulated in the nanocapsules via
hydrophobic interaction between hydrophobic
combteeth (OAm) of PSIOAm and hydrophobic parts
of PTX and hydrogen bonds between hydroxyls of
PTX and hydrophilic backbone (PSI) of PSIOAm.
Due to the hydrogen-bond breaking at low pH, the
drugs are released after endocytosis with the guide
of surface bioconjugated Arg-Gly-Asp (RGD) and
the enhanced permeability and retention (EPR)
effect because of the appropriate nanocapsule size
(<100nm). Our nanocapsules are stable at pH 7.4
(blood vessel pH) and the H-bond-based
pH-responsive release (pH 5.0 in lysosome) is
sustained up to 14 days. Moreover, compared to
the upconversion nanocrystals with emission in the
range of (450-800 nm) via 980 nm excitation,[37-42]
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3 Nano Res.
the co-encapsulated Ag2S QDs, emitting in the
second near-infrared region (NIR-II, 1000-1400 nm),
are more suitable for the real-time NIR-II
fluorescence tracking of the drugs in live animals
with negligible autofluorescence. The fabrication of
this versatile, biocompatible and biodegradable
nanocarrier is simple, green (water as solvent and
no surfactants needed), reproducible and
low-costing, and can be easily scaled-up to 6.12
g (1.1 L) from 59.1 mg (11.0 mL) in our Lab, which
will lead to the short period of bench-to-bedside
translation. The chemotherapy results in tumor
xenografted mice suggest that our nanocapsule is
more safe and efficient than the clinical Taxol® and
may be a useful tool for drug delivery. 2 Experimental
Preparation of amphiphilic comb-like PSIOAm.[43]
For PSIOAm-40%, 1.6 g of polysuccinimide (PSI) was
dissolved in 32 mL of N, N-Dimethylformamide
(DMF) at 90 C under magnetic stirring followed
by the addition of oleylamine (OAm, 2.17 mL). The
mixture was treated at 100 ºC for 5 h before cooling
to room temperature. Then methanol (80 mL) was
added to precipitate the product (PSIOAm). Finally,
the PSIOAm was redispersed into chloroform to get a
stock solution with concentration of 200 mg/mL
after centrifugation and then evaporating the trace
amount of residual methanol. For PSIOAm-30%, 1.63
mL of oleylamine was used.
Preparation of the PTX/NPs@PSIOAm
nanocapsules.[43] For PTX/ Ag2S@PSIOAm-30% NWs,
into 10 mL of NaOH (5.0 mM) aqueous solution,
1.0 mL of mixture chloroform solution of PSIOAm-30%
(120 mg), polyethylene-block-poly(ethylene
glycol)(PE-PEG, 5.0 mg), PTX (1.0 mg) and Ag2S
(0.4 mg) was added and followed by
ultrasonication (350 W, 6 min). Subsequently, the
chloroform was removed by evaporating at 58 ºC
for 30 min. The nanocomposites were collected and
purified by centrifugation at 11000 rpm for 10 min
and redispersed into PBS (1 mL). For the
PTX/Ag2S@PSIOAm-40% NSs, into 10 mL of NaOH (0.5
mM) aqueous solution, 1.0 mL of chloroform
colloidal dispersion containing PSIOAm-40% (60 mg)
instead of PSIOAm-30%, was added and followed by
ultrasonication (350 W, 6 min). Then the obtained
NSs was purified and collected as above. This
protocol also enabled the successful fabrication of
drug nanocapsules of PTX (1.0 mg), CPT (1.0 mg),
and PTX (0.5 mg)-CPT (0.5 mg) cocktail in the
absence of Ag2S nanoparticles for the small scale
production, respectively.
Large-scale production of PTX nanocapsules
and PTX-CPT cocktail nanocapsules. The
nanocapsule fabrication can be easily scaled up for
100 times in our lab. In brief, into 1000 mL of
NaOH (0.5 mM) aqueous solution, 100 mL of
mixture chloroform solution of PSIOAm-40% (6.0 g),
polyethylene-block-poly(ethylene glycol)(PE-PEG,
500 mg), PTX (100 mg) and Ag2S (40 mg) was
added and followed by ultrasonication (1000 W, 15
min). The purification process was identical to that
mentioned above. For PTX-CPT cocktail
nanocapsules, 50 mg of PTX and 50 mg of CPT
were added simultaneously.
PTX release. 1.5 mL of the stored
nanocomposites was diluted to 3.0 mL with PBS
and dialyzed against 60 mL of pH 5.0 or pH 7.4
PBS containing Sodium salicylate (to help
solubilizing PTX in water). 10 mL of the dialyzed
liquid outside the dialysis bag (molecule weight
cut 14000) was taken at certain time point and
extracted with 2 mL of octanol at 37 °C for 1 h. 10
mL of pH 5.0 or pH 7.4 PBS containing sodium
salicylate was supplemented into the dialysis
solution. Quantification of PTX solution in octanol
was performed on HPLC. Previous taken PTX
should be considered and carefully calculated.
Mobile phase of gradient elution was 90/100/100
(water/methanol) for 060min and then 0:10090:10
(water/methanol) for 6063min. The flow rate was
0.5 mL/min.
Bioconjugation with RGD peptide. 1 mL
phosphate buffer solution (PBS) containing 1 mg of
RGD and 1 mL PBS (pH=7.4, 0.1 M) containing
EDC (0.7 mg) and NHS (0.1 mg) were mixed with 1
mL hydrophilic nanospheres or nanowires,
followed by incubating at 25 °C for 4 h. Finally, the
RGD-nanocomposite bioconjugates were collected
by centrifugation (11000 rpm, 10 min) and washed
with PBS, and this purification process was
repeated for three times. The obtained RGD
conjugated nanocomposites were redispersed in
PBS (pH = 7.4, 1 mL) and stocked at 4 C for later
use. These nanocapsules are highly stable in not
only PBS buffer but also culture media. After being
dispersed in culture media containing serum for 7
days, no aggregate was observed, which is highly
desirable for in vivo applications especially via
intravenous injection.
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4 Nano Res.
In vitro anti-cancer effect estimate. Briefly,
HeLa cells (5104 cells/well) attached to the
bottom of the 96-well cell culture plate were
incubated with different amounts (0–200 µg
PTX/mL) of sterilized Taxol, NWs, NW@RGD, NSs,
or NS@RGD in each well at 37 °C for 24 or 48 h,
respectively. Thereafter, the cytotoxicity was
evaluated via the methyl thiazolyltetrazolium
(MTT) assay.
Cell imaging. HeLa cells were seeded on a
sterilized glass cover slide and cultured in a
12-well cell culture plate overnight under
recommended conditions at 37 °C in 5%
CO2−humidified incubator. Then the RGD
bioconjugated nanomaterial stock solution was
added into the cell culture well with a final
concentration of 20 g PTX/mL. The HeLa cells
were incubated with nanocapsules for another 4 h,
8 h, 24 h, and 48 h, respectively. As a control, the
bare nanomaterials without RGD in place of
nanomaterials conjugated with RGD, were
incubated with the HeLa cells under the same
conditions. Thereafter, the cells on the glass slide
were washed with phosphate buffer solution (PBS,
pH 7.4, 10 mM) and fixed in 4% paraformaldehyde
solution for 15 min. The luminescence imaging was
conducted on a TCS SP5 two-photon confocal
microscopes (Leica) equipped with a Mai Tai near
infrared (NIR) diode laser.
Mouse handling. 6-week-old female Balb/c mice
were obtained from Suzhou Belda
Bio-Pharmaceutical Co. and raised in an animal
facility under filtered air (22±2 °C). Animal studies
were performed under the guidelines approved by
Soochow University Laboratory Animal Center.
The mice were fed with standard pellet diet and
pure water. The study was performed with the
Guidelines for the Care and Use of Research
Animals. 20 (4×5) mice were inoculated with 4T1
tumor cells on the right hindlimb. Tumors grew for
7 days; when they reached 5-20 mm3 in volume,
the mice were completely shaven.
In vivo biodistribution study.
PTX/Ag2S@PSIOAm@RGD NSs and
PTX/Ag2S@PSIOAm NSs at 0.67 mg PTX/mL and 0.4
mg Ag2S/mL concentration was injected
intravenously, respectively. During injection, the
mice exposed to 2 L/min oxygen flow with 3%
Isoflurane for anesthesia. For tumor imaging,
prone animals were mounted on the imaging stage
beneath the laser. NIR-II fluorescence images were
collected using a liquid-nitrogen-cooled, 320256
pixel two-dimensional InGaAs array (Princeton
Instruments) for collecting photons in NIR-II. The
excitation light was provided by an 808-nm diode
laser (RMPC lasers) coupled to a 4.5-mm focal
length collimator (Thorlabs) and filtered by an
850-nm short-pass filter and a 1000-nm short-pass
filter (Thorlabs). The excitation power density at
the imaging plane was 140 mW/cm2, The NIR-II
fluorescence emitted from the animal was detected
with the InGaAs camera coupled with a 900-nm
long-pass filter and an 1100 nm long-pass filter
(Thorlabs).
In vivo anticancer effect estimate. For the
PTX/Ag2S@PSIOAm in vivo anti-tumor experiments,
there were 4 mice per group at each time point for
statistical analysis. PTX/Ag2S@PSIOAm@RGD NSs at
0.8 mg PTX/mL concentration was injected
intravenously. The mice were intravenously
injected every three days (Day 1, Day 4, Day 7, Day
10, Day 13) with 400 μL of (a) PBS, (b) Taxol® , (c)
PTX/Ag2S@PSIOAm-40% NSs, (d)
PTX/Ag2S@PSIOAm-30%@RGD NWs, and (e)
PTX/Ag2S@PSIOAm-40%@RGD NSs at 0.67 mg
PTX/mL concentration, respectively. We collected
the mouse body weights every three days for 17
days. The female Balb/c mice were sacrificed and
the organs were collected at 17 days post injection. 3 Results and discussion
Development of hydrogen-bond-based
pH-responsive nanocapsules. Due to the good
biocompatibility, drug carriers such as NK911
based on poly(aspartic acid) have already been
under clinical evaluation[44], but the micelle sizes
are very large, which will result in the low target
delivery. Herein, we chose the biodegradable,
cheap and widely available polysuccinimide (PSI)
as the coating materials for nanocapsule fabrication.
To fabricate the versatile drug nanocapsule, we
synthesized the amphiphilic and biodegradable
polyaspartic acid derivative, a comb-like
poly(amino acid) (PSIOAm), by functionalizing
polysuccinimide with oleylamine (OAm) ( Figure
S1). As shown in Scheme 1a, a versatile and
H-bond-based pH-responsive cocktail nanocapsule
for hydrophobic anti-cancer drugs was successfully
developed. PTX was captured in the nanocapsule
via hydrophobic interaction between combteeth
(OAm) of PSIOAm and hydrophobic parts of PTX
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5 Nano Res.
and hydrogen bonds between hydroxyls of PTX
and hydrophilic backbone (PSI) of PSIOAm (Scheme
1b). As shown in Scheme 1b, the H-bonds were
shielded in the hydrophobic cavity to eliminate the
water influx induced breaking. To demonstrate the
real-time fluorescence monitoring of
pharmacokinetics and bio-distribution of the
nanocapsules, hydrophobic Ag2S QDs (shown in
blue sphere in Scheme 1a) with fluorescence in the
second near-infrared region (NIR-II, 1000-1400
nm)[45-48], were co-encapsulated in PSIOAm[43, 49]
nanocapsules. To efficiently eliminate nonspecific
accumulation in RES organs, the shape and size of
the nanocapsule were deliberately tuned from
nanowires (NWs, tens of micrometres long) to
nanospheres (NSs, d < 100 nm) (Scheme 1a) by
replacing PSIOAm-30% with PSIOAm-40% and increasing
NaOH dose. Where the OAm-30% and OAm-40%
mean the molar ratio (ROAm/lactam) of oleylamine to
original lactam rings of PSI is 30% and 40%,
respectively. To provide targeted drug delivery, the
nanocapsule surface was further functionalized
with Arg-Gly-Asp (RGD)[50, 51], a tumor targeting
peptide (Scheme 1). It’s worth noting that in the
blood vessel of a normal tissue (pH 7.4), this
nanocapsule is pretty stable and has little drug
leakage, reducing the side effects of anti-cancer
drugs. However, after entering the lysosome (pH
5.0) via RGD targeting, the nanosphere capsule is
disrupted and the drugs are released due to the
H-bonds between PTX and PSIOAm, which are
highly sensitive to acidic pH (Scheme 1c).
Scheme 1 Schematic illustration of the nanocapsule
fabrication and targeted pH-responsive drug release. (a)
Fabrication of PTX-PSIOam-Ag2S nanocapsules. (b)
Hydrophobic interaction and hydrogen bonds between PTX
and PSIOAm. (c) RGD-targeted delivery and pH-responsive
drug release. NIR FL NP: NIR-II fluorescence Ag2S QDs.
Fabrication and characterization of poly(amino
acid) nanocapsules. In order to fabricate the
general pH-responsive nanocapsule for PTX
delivery, we first encapsulated 1-aminopyrene in
PSIOAm-40% and poly(styrene95%-co- methylacrylic
acid5%) (PSMMA) NSs (Figure S2S5), respectively.
And then the 8-hydroxyquinoline was also
encapsulated in PSIOAm-40% NSs (Figure S6). As
expected, only the nanospheres (NSs) having
H-bonds between the encapsulated chemicals and
polymers were disrupted at pH 5.0, suggesting that
the pH-responsive delivery is attributed to the
H-bond disruption. Encouraged by these positive
results, we carried out the fabrication and
pH-responsive release of PTX/Ag2S@PSIOAm
nanocapsules.
Figure 1 TEM images of the nanocapsules at different pH. (a)
Nanowires (NWs) in pH 7.4 phosphate buffer solution (PBS).
(b) Magnification of image (a). (c) NWs in pH 5.0 PBS. (d)
Nanospheres (NSs) in pH 7.4 PBS. (e) Magnification of image
(d). (f) NSs in pH 5.0 PBS.
As shown in the transmission electron
microscope (TEM) images (Figure 1a), by using the
PSIOAm-30%, composite NWs containing both PTX
and Ag2S QDs were obtained. The Ag2S QDs were
clearly shown on the NWs (Figure 1b, pH 7.4), and
the NWs were disrupted into small fragments by
tuning the pH to 5.0 (Figure 1c). Although these
NWs can be applied for the encapsulation of PTX,
they are hampered for the in vivo delivery
application due to their large size which will result
in nonspecific accumulation in the liver and spleen
(see the in vivo experimental results). To address
this problem, small nanospheres (NSs) were
fabricated by using the PSIOAm-40% and increasing
the concentration of NaOH in the water (Figure 1d).
The average TEM size (Figure 1e, pH 7.4) and
hydrodynamic diameter (Figure 2a) of the NSs was
less than 100 nm, which is desirable for in vivo
delivery to tumor tissue, and the Ag2S QDs were
encapsulated in the NSs. Because of the coexistence
of both hydrophobic interaction and H-bonds
(Figure S7), the nanocapsules can be used for not
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6 Nano Res.
only the encapsulation but also the pH-controlled
release of hydrophobic therapeutic agents that
contain both hydrophobic parts and hydrophilic
moieties such as –OH and –NH2. When the pH was
adjusted to 5.0, the NSs disassembled and the PTX
and Ag2S QDs were released (Figure 1f). The
successful fabrication of PTX nanocapsules was
further characterized via the Fourier transform
infrared (FTIR) spectroscopy (Figure S8).
Figure 2 Characteristics of the nanocapsules. (a)
Hydrodynamic size distribution of PTX/Ag2S@PSIOAm-40%
NSs dispersed in pH 7.4 PBS. (b) Circular dichroism (CD)
spectra of PTX in CH3OH (black), PTX in CHCl3 [27], and
PTX@PSIOAm-40% in water (blue), PTX/Ag2S@PSIOAm-40% in
water (green), and PSIOAm-40% in CHCl3 (magenta). (c)
pH-sensitive release of PTX as a function of time for
PTX/Ag2S@PSIOAm-30% NWs, PTX/Ag2S@PSIOAm-40% NSs,
and PTX/Ag2S@PSMMA NSs, as detected by HPLC. (d)
HeLa cell viability following 48 h of exposure to Taxol and
nanocapsules containing PTX in cell culture media (pH 7.4),
respectively.
It is worth noting that in the absence of PTX,
only NSs but not NWs can be obtained, regardless
of what ROAm/lactam is. On the other hand, the NSs
without PTX are not disrupted in pH 5.0
phosphate buffer solution (PBS) (Figure S9). We
can infer that, along with hydrophobic interaction,
the hydrogen-bonds (H-bonds) between PTX and
PSIOAm play an important role in nanocapsule
formation. More importantly, the low pH-induced
disruption of the nanocapsule and the resultant
drug release may originate from the pH-responsive
breaking of H-bonds between the drug and
polymer. As we know, the H-bond hidden in the
hydrophobic cavity can be hardly characterized
with 1H nuclear magnetic resonance (NMR)
spectroscopy because it is hard to stimulate the 1H
in hydrophobic environments[30]. So, circular
dichroism (CD) spectroscopy, as an alternative,
was widely used for H-bond characterization in
hydrophobic environments[25, 30], which is pretty
suitable for our case. As shown in Figure 2b, the
230 nm band of PTX in CH3OH red shifts to the 233
nm band of PTX in CHCl3 because the CHCl3 is
more apolar than CH3OH. The 233nm band of
blue spectra indicate that PTX was encapsulated in
the hydrophobic PSIOAm-40% cavity. The
disappearing of 298 nm band for the
nanostructures indicate the π-π* transition of the
aromatic rings in the PTX side chain is strongly
affected by the H-bond between PTX and
PSIOAm-40%[52]. The 264 nm shoulder may be
attributed to the C2-O-benzoyl group (Figure S5)[52].
The H-bond based pH-responsive release was
further identified by other nanospheres and this
facile method was successfully applied for the
pH-controlled release of camptothecine (CPT,
another widely used hydrophobic anti-cancer drug)
(Figure S10S13).
As aforementioned, the drug cocktail
nanocapsules would be more effective than the
chemotherapy drug traditionally used alone. As
shown in Figure S14, we successfully carried out
the fabrication of cocktail drug nanocapsules via
this versatile strategy by co-encapsulating PTX and
CPT simultaneously, suggesting our method can be
easily extended to the drug cocktail. Moreover, in
order to improve the bench-to-bedside translation,
a simple and straightforward preparation process
is required for practical large-scale generation of
nanocapsules that can be loaded with multiple
drugs. Our method allowed for large-scale
production of more customized therapeutic
delivery nanocapsules. Via this strategy, we
scaled-up the nanocapsule production from 59.1
mg (11.0 mL) to 6.12 g (1.1 L) with the highest
ultrasonication power available in our lab. TEM
images of both PTX@PSIOAm-40% and
PTX/Ag2S@PSIOAm-40% NSs prepared with large
scale production were shown in Figure S15,
suggesting that our method is easy to scale up for
large scale production of drug nanocarriers.
In vitro pH-responsive drug release and
cytotoxicity. In vitro release studies were carried
out by incubating the nanocapsule in pH 7.4 and
pH 5.0 PBS at 37 C, respectively. The released PTX
was quantified via high performance liquid
chromatography (HPLC). Due to lack of H-bonds,
when the PTX/Ag2S@PSMMA NSs were dispersed
in pH 5.0 PBS for 24 h, only a little of PTX leaked
from the polymer matrixes. Even by prolonging
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7 Nano Res.
the immersion time to 14 days, only about 21% of
PTX was released (Figure 2c). At pH 7.4, PTX
leakage was less than 14% after 336 h (14 days).
Meanwhile, both PTX/Ag2S@PSIOAm-30% NWs and
PTX/Ag2S@PSIOAm-40% NSs, at pH 7.4 release less
than 30% of PTX after 14 days. However, if the pH
was decreased to 5.0, the PTX release was much
faster. No burst release of PTX from the NSs and
NWs was observed. After 24 h, about 35% and 22%
were released from the NSs and NWs, respectively.
After 14 days, over 89% and 97% of PTX was
released from the NSs and NWs, respectively.
These results indicate that the pH-sensitive release
is sustained, which is highly desirable for
achieving prolonged therapeutic action over an
extended period of time.
Notably, cytotoxicity studies with HeLa cells
demonstrated that the PTX-loaded NSs and NWs
have no obvious cytotoxicity if the nanocapsule
surfaces were not bioconjugated with the targeting
peptide RGD[50] (Figure 2d and Figure S16). Even
after incubation with 200 μg PTX/mL of
nanocapsules for 48 h (pH 7.4), over 84% and 81%
of cells were still alive for the NSs and NWs,
respectively. This result suggests that our
nanosphere is a safe vehicle for in vivo drug
delivery because PTX will not be released in the
blood vessels of a normal tissue (pH 7.4). On the
other hand, the commercialized Taxol® (PTX with
Cremophor EL/ethanol) remains highly toxic to
cells (pH 7.4)[53], which means that Taxol® may
blindly cause harm to normal tissues. However,
after being bioconjugated with the targeting
peptide RGD, the PTX/Ag2S@PSIOAm-40% NSs were
easily captured and endocytosed by cancer cells,
thus killing the cells through the pH-sensitive
release of PTX. The RGD targeted pH-responsive
release is in line with the confocal laser scanning
microscopy (CLSM) results (Figure S17S19). As
shown in Figure 2d, 80% HeLa cells were dead
after being exposed to 3.0 μg PTX/mL of
PTX/Ag2S@PSIOAm-40%@RGD NSs for 48 h,
suggesting a very high drug efficacy. For
PTX/Ag2S@PSIOAm-30%@RGD NWs, however, when
the PTX concentration was 3.0 μg/mL, only 20% of
cells were killed, indicating that the NSs are more
suitable for drug delivery than the NWs, even for
in vitro therapy, because of their small particle sizes.
However, when the PTX concentration was
increased up to 200 μg/mL, almost 100% of the
cells were dead after incubation for 48 h (pH 7.4)
either with the NWs or NSs. It should be
mentioned that, especially at low PTX
concentration (3.0 μg/mL), the efficacy of
PTX/Ag2S@PSIOAm-40%@RGD NSs is far better than
that of Taxol® or the NWs. This suggests that to
reach a similar level of tumor drug uptake, a much
lower injected dose can be used with our
NSs system than with Taxol® or the NWs, which is
highly favorable for lowering toxic side effects to
normal organs and tissues. All of the results
indicate that the Ag2S QD containing NSs are
nontoxic and suitable for in vivo targeted drug
delivery.
Figure 3 NIR-II fluorescence imaging of the in-vivo 4T1
tumors. (a-f) and in-vitro organs (g and h) that are uptaking
PTX/Ag2S@PSIOAm-40% NSs (a-c and g) or
PTX/Ag2S@PSIOAm-40%@RGD NSs (d-f and h) at 0.5 h (a and
d), 5 h (b and e), 10 h (c and f), and 24 h (g and h) after
injection.
In vivo bio-distribution and therapeutic effects.
We investigated the bio-distribution of
PTX/Ag2S-loaded NSs that were injected into
tumor-bearing mice using non-invasive real-time
NIR-II fluorescence imaging. The NIR-II
(1000–1400 nm) fluorescence is more desirable for
in vivo imaging than visible (450-700 nm) and
traditional NIR-I (700-950 nm) fluorescence
because it provides negligible tissue
autofluorescence, greatly reduced photon
absorption and scattering by tissues, and
high-fidelity in vivo imaging with deeper
penetration depth (808-nm irradiation)13. The mice
were injected via the tail vein with 13 mg/kg of
PTX in either PTX/Ag2S@PSIOAm-40% NSs or
PTX/Ag2S@PSIOAm-40%@RGD NSs, both dispersed in
PBS. As shown in Figure 3, 30 min after injection,
the NSs were distributed all over the animal body
(Figure 3a, and d). After circulating in vivo for 5 h,
bright NIR-II fluorescence was observed in the
tumor (Figure 3b, and e). The tumor that uptook
PTX/Ag2S@PSIOAm-40%@RGD NSs (Figure 3e) is
brighter than the tumor uptaking
PTX/Ag2S@PSIOAm-40% NSs (Figure 3b), which can be
attributed to the targeting ability of RGD (Scheme
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8 Nano Res.
1c and Figure S20). Tumor uptake of PTX-loaded
NSs increased significantly at 10 h post
injection, indicating accumulation of NSs through
blood circulation during this period (Figure 3c, and
f). The accumulation of PTX/Ag2S@PSIOAm-40% NSs
in the tumor can be ascribed to the enhanced
permeability and retention (EPR) effect (Figure 3c).
For the mice treated with
PTX/Ag2S@PSIOAm-40%@RGD NSs (Figure 3f),
RGD-mediated accumulation (active targeting of
tumor cells and vessels) in the tumor is shown by
stronger fluorescence and larger fluorescent area.
Ex vivo imaging of the excised organs and tumors
was performed 24 h after injection. We observed
higher nanocapsule uptake in the liver and spleen
(RES organs) for the PTX/Ag2S@PSIOAm-40% NSs
without RGD guidance (Figure 3g) than for the
PTX/Ag2S@PSIOAm-40%@RGD NSs (Figure 3h).
Figure 4 Intravenouslyly injected nanocapsules deliver
chemotherapeutic drugs to xenografted tumor. Inhibition of
4T1 xenografts tumor growth in nude mice treated with (a)
PBS, (b) Taxol®, (c) PTX/Ag2S@PSIOAm-40% NSs, (d)
PTX/Ag2S@PSIOAm-30%@RGD NWs, or (e)
PTX/Ag2S@PSIOAm-40%@RGD NSs. (f) Images of excised
tumors and average tumor weight (grams) from each group
(Lines 1-5 correspond to treatments (a)-(e)). (g) Body weight
change of the mice during the different treatments. Each group
has 4 mice.
To demonstrate the drug efficacy of our
nanocapsules, we carried out a pilot toxicity study
by treating tumor-bearing mice with PBS (Figure
4a), Taxol (Figure 4b), PTX/Ag2S@PSIOAm-40% NSs
(Figure 4c), PTX/Ag2S@PSIOAm-30%@RGD NWs
(Figure 4d), and PTX/Ag2S@PSIOAm-40%@RGD NSs
(Figure 4e). The mice were treated once every three
days for 13 days (Day 1, Day 4, Day 7, Day 10, Day
13) at a constant PTX dosage (13 mg/kg). The
organs of the female Balb/c mice were collected at
17 days post injection. As shown in Figure 4 and
Table S1, the tumors treated with
PTX/Ag2S@PSIOAm-40%@RGD NSs were the smallest
(Figure 4f, line e, average weight 0.037 g), and were
16-fold smaller than the tumors treated with PBS
(Figure 4f, line a, average weight 0.59 g).
Meanwhile, the tumors treated with Taxol (Figure
4b and 5f, line b), PTX/Ag2S@PSIOAm-40% NSs (Figure
4c and 5f, line c), and PTX/Ag2S@PSIOAm-30%@RGD
NWs (Figure 4d and 5f, line d) were almost the
same size, but 4-fold larger than the tumors treated
with PTX/Ag2S@PSIOAm-40%@RGD NSs (Figure 4e
and 5f, line e). These results indicate that RGD
active targeting and the EPR effect synergistically
improve PTX/Ag2S@PSIOAm-40%@RGD NS
accumulation in the tumors. As a result, PTX was
successfully released from the nanocapsules in the
low pH environment of lysosomes. In addition,
due to their much larger axial size (Figure 1a), the
RGD-bioconjugated NWs were more easily
uptaken by macrophage abundant organs such as
the liver and spleen (Figure S21). Thus the drug
efficacy of the NWs was not as good as that of the
RGD-bioconjugated NSs. We also collected the
mice’s body weights every three days for 17 days
(Figure 4g). We observed neither mortality nor
weight loss in mice treated with any of the five
media. 4 Conclusions
We have designed and prepared a versatile
H-bond-based pH-responsive nanocapsule for the
hydrophobic anti-cancer drugs with small size
(<100 nm), safe delivery through blood vessels (no
disruption at pH 7.4), and sustained release (up to
14 days). This nanocapsule fabrication method can
potentially be used to achieve excellent control
over drug loading and release for numerous
hydrophobic therapeutic agents that can form
hydrogen-bonds with a biodegradable,
amphiphilic comb-like coating poly(amino acid).
These multifunctional cocktail nanocapsules are
nontoxic to cells until they undergo cellular uptake
into acidic lysosomes (pH 5.0) with the help of
RGD targeting, at which point they disrupt due to
the breaking of hydrogen bonds, thus making
them a great candidate for pH-responsive drug
delivery systems. Moreover, the co-encapsulated
Ag2S QDs facilitate real-time NIR-II fluorescence
tracking of the therapeutic agents in live animals
with negligible autofluorescence, which is highly
desirable as an important tool for understanding
tumor responses to anti-cancer drugs and
monitoring tumor growth in vivo without radio
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research
9 Nano Res.
labellings. The fabrication of this versatile,
biocompatible and biodegradable cocktail drug
nanocapsule is simple, green, reproducible and
low-costing, and can be easily scaled-up, which
could lead to the short period of bench-to-bedside
translation. We believe that this combination of
features makes the multifunctional nanocapsule
formulation uniquely attractive for the
development of advanced drug nanocarriers.
Acknowledgements
The authors gratefully acknowledge for the
financial support by the National Natural Science
Foundation of China (21475007 and 21275015), the
State Key Project of Fundamental Research of
China (2011CB932403 and 2011CBA00503), the
Fundamental Research Funds for the Central
Universities (YS1406 and ZZ1321), the Scientific
Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry, and the
Program for Changjiang Scholars and Innovative
Research Team in University (IRT1205). We also
thank the support from the “Public Hatching
Platform for Recruited Talents of Beijing University
of Chemical Technology”.
Electronic Supplementary Material: Figures
S1-S21 and Table S1 are available in the online
version of this article at
http://dx.doi.org/10.1007/s12274-***-****-*. References
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