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

Just Accepted

This is a “Just Accepted” manuscript, which has been examined by the peer-review process and has been

accepted for publication. A “Just Accepted” manuscript is published online shortly after its acceptance,

which is prior to technical editing and formatting and author proofing. Tsinghua University Press (TUP)

provides “Just Accepted” as an optional and free service which allows authors to make their results available

to the research community as soon as possible after acceptance. After a manuscript has been technically

edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP

article. Please note that technical editing may introduce minor changes to the manuscript text and/or

graphics which may affect the content, and all legal disclaimers that apply to the journal pertain. In no event

shall TUP be held responsible for errors or consequences arising from the use of any information contained

in these “Just Accepted” manuscripts. To cite this manuscript please use its Digital Object Identifier (DOI®),

which is identical for all formats of publication.

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.

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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]

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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.

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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

[1]. Sugano, K.; Kansy, M.; Artursson, P.; Avdeef, A.;

Bendels, S.; Di, L.; Ecker, G. F.; Faller, B.; Fischer, H.;

Gerebtzoff, G.; Lennernaes, H.; Senner, F. Coexistence

of passive and carrier-mediated processes in drug

transport. Nat. Rev. Drug Discov. 2010, 9, 597-614.

[2]. Petros, R. A.; DeSimone, J. M. Strategies in the

design of nanoparticles for therapeutic applications. Nat.

Rev. Drug Discov. 2010, 9, 615-627.

[3]. Fu, J. L.; Yan, H. Controlled drug release by a

nanorobot. Nat. Biotechnol. 2012, 30, 407-408.

[4]. MacKay, J. A.; Chen, M. N.; McDaniel, J. R.; Liu,

W. G.; Simnick, A. J.; Chilkoti, A. Self-assembling

chimeric polypeptide-doxorubicin conjugate

nanoparticles that abolish tumours after a single

injection. Nat. Mater. 2009, 8, 993-999.

[5]. Kong, G.; Braun, R. D.; Dewhirst, M. W.

Hyperthermia enables tumor-specific nanoparticle

delivery: effect of particle size. Cancer Res. 2000, 60,

4440-4445.

[6]. Mura, S.; Nicolas, J.; Couvreur, P.

Stimuli-responsive nanocarriers for drug delivery. Nat.

Mater. 2013, 12, 991-1003.

[7]. Kearney, C. J.; Mooney, D. J. Macroscale delivery

systems for molecular and cellular payloads. Nat. Mater.

2013, 12, 1004-1017.

[8]. Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Muller,

M.; Ober, C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.;

Tsukruk, V. V.; Urban, M.; Winnik, F.; Zauscher, S.;

Luzinov, I.; Minko, S. Emerging applications of

stimuli-responsive polymer materials. Nat. Mater. 2010,

9, 101-113.

[9]. Lee, T. K.; Sokoloski, T. D.; Royer, G. P. Serum

albumin beads: an injectable, biodegradable system for

the sustained release of drugs. Science 1981, 213,

233-235.

[10]. Zhou, C.; Hao, G. Y.; Thomas, P.; Liu, J. B.; Yu, M.

X.; Sun, S. S.; Oz, O. K.; Sun, X. K.; Zheng, J.

Near-infrared emitting radioactive gold nanoparticles

with molecular pharmacokinetics. Angew. Chem. Int.

Edit. 2012, 51, 10118-10122.

[11]. Choi, H. S.; Ipe, B. I.; Misra, P.; Lee, J. H.;

Bawendi, M. G.; Frangioni, J. V. Tissue- and

organ-selective biodistribution of NIR fluorescent

quantum dots. Nano Lett. 2009, 9, 2354-2359.

[12].Prescott, J. H.; Lipka, S.; Baldwin, S.; Sheppard, N.

F.; Maloney, J. M.; Coppeta, J.; Yomtov, B.; Staples, M.

A.; Santini, J. T. Chronic, programmed polypeptide

delivery from an implanted, multireservoir microchip

device. Nat. Biotechnol. 2006, 24, 437-438.

[13]. Gref, R.; Minamitake, Y.; Peracchia, M. T.;

Trubetskoy, V.; Torchilin, V.; Langer, R. Biodegradable

long-circulating polymeric nanospheres. Science 1994,

263, 1600-1603.

[14]. Namgung, R.; Mi Lee, Y.; Kim, J.; Jang, Y.; Lee,

B.-H.; Kim, I.-S.; Sokkar, P.; Rhee, Y. M.; Hoffman, A.

S.; Kim, W. J. Poly-cyclodextrin and poly-paclitaxel

nano-assembly for anticancer therapy. 2014, 5,

4702-4713.

[15]. Cui, J. W.; Yan, Y.; Wang, Y. J.; Caruso, F.

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

10 Nano Res.

Templated sssembly of pH-labile polymer-drug particles

for intracellular drug delivery. Adv. Funct. Mater. 2012,

22, 4718-4723.

[16]. Yan, Y.; Wang, Y. J.; Heath, J. K.; Nice, E. C.;

Caruso, F. Cellular association and cargo release of

redox-responsive polymer capsules mediated by

exofacial thiols. Adv. Mater. 2011, 23, 3916-3921.

[17]. Liu, Z.; Fan, A. C.; Rakhra, K.; Sherlock, S.;

Goodwin, A.; Chen, X. Y.; Yang, Q. W.; Felsher, D. W.;

Dai, H. J. Supramolecular stacking of doxorubicin on

carbon nanotubes for in vivo cancer therapy. Angew.

Chem. Int. Edit. 2009, 48, 7668-7672.

[18]. Peng, F.; Su, Y. Y.; Wei, X. P.; Lu, Y. M.; Zhou, Y.

F.; Zhong, Y. L.; Lee, S. T.; He, Y.

Silicon-nanowire-based nanocarriers with ultrahigh

drug-loading capacity for in vitro and in vivo cancer

therapy. Angew. Chem. Int. Edit. 2013, 52, 1457-1461.

[19]. Wang, Y.; Xu, Z.; Guo, S.; Zhang, L.; Sharma, A.;

Robertson, G. P.; Huang, L. Intravenous delivery of

siRNA targeting CD47 effectively inhibits melanoma

tumor growth and lung metastasis. Mol. Ther. 2013, 21,

1919-1929.

[20]. Wang, Q. L.; Zhuang, X. Y.; Mu, J. Y.; Deng, Z. B.;

Jiang, H.; Xiang, X. Y.; Wang, B. M.; Yan, J.; Miller, D.;

Zhang, H. G. Delivery of therapeutic agents by

nanoparticles made of grapefruit-derived lipids. Nat.

Commun. 2013, 4, 2886-2896.

[21]. Santra, S.; Kaittanis, C.; Santiesteban, O. J.; Perez,

J. M. Cell-specific, activatable, and theranostic prodrug

for dual-targeted cancer imaging and therapy. J. Am.

Chem. Soc. 2011, 133, 16680-16688.

[22]. Du, J. Z.; Du, X. J.; Mao, C. Q.; Wang, J.

Tailor-made dual pH-sensitive polymer-doxorubicin

nanoparticles for efficient anticancer drug delivery. J.

Am. Chem. Soc. 2011, 133, 17560-17563.

[23]. Ellis, G. A.; Palte, M. J.; Raines, R. T.

Boronate-mediated biologic delivery. J. Am. Chem. Soc.

2012, 134, 3631-3634.

[24]. Chen, W.; Meng, F. H.; Li, F.; Ji, S. J.; Zhong, Z. Y.

pH-responsive biodegradable micelles based on

acid-labile polycarbonate hydrophobe: synthesis and

triggered drug release. Biomacromolecules 2009, 10,

1727-1735.

[25]. Kolano, C.; Helbing, J.; Kozinski, M.; Sander, W.;

Hamm, P. Watching hydrogen-bond dynamics in a

beta-turn by transient two-dimensional infrared

spectroscopy. Nature 2006, 444, 469-472.

[26]. Zhang, J.; Chen, P. C.; Yuan, B. K.; Ji, W.; Cheng,

Z. H.; Qiu, X. H. Real-space identification of

intermolecular bonding with atomic force microscopy.

Science 2013, 342, 611-614.

[27]. Takahara, P. M.; Rosenzweig, A. C.; Frederick, C.

A.; Lippard, S. J. Crystal structure of double-stranded

DNA containing the major adduct of the anticancer drug

cisplatin. Nature 1995, 377, 649-652.

[28].Prota, A. E.; Bargsten, K.; Zurwerra, D.; Field, J. J.;

Diaz, J. F.; Altmann, K. H.; Steinmetz, M. O. Molecular

mechanism of action of microtubule-stabilizing

anticancer agents. Science 2013, 339, 587-590.

[29]. Kim, B. S.; Park, S. W.; Hammond, P. T.

Hydrogen-bonding layer-by-layer assembled

biodegradable polymeric micelles as drug delivery

vehicles from surfaces. ACS Nano 2008, 2, 386-392.

[30]. Ma, M. L.; Kuang, Y.; Gao, Y.; Zhang, Y.; Gao, P.;

Xu, B. Aromatic-aromatic interactions induce the

self-assembly of pentapeptidic derivatives in water to

form nanofibers and supramolecular hydrogels. J. Am.

Chem. Soc. 2010, 132, 2719-2728.

[31]. Xing, B. G.; Yu, C. W.; Chow, K. H.; Ho, P. L.; Fu,

D. G.; Xu, B. Hydrophobic interaction and hydrogen

bonding cooperatively confer a vancomycin hydrogel: A

potential candidate for biomaterials. J. Am. Chem. Soc.

2002, 124, 14846-14847.

[32]. Pasparakis, G.; Manouras, T.; Vamvakaki, M.;

Argitis, P. Harnessing photochemical internalization

with dual degradable nanoparticles for combinatorial

photo-chemotherapy. Nat. Commun. 2014, 5,

4623-4631.

[33]. Mikhaylov, G.; Mikac, U.; Magaeva, A. A.; Itin, V.

I.; Naiden, E. P.; Psakhye, I.; Babes, L.; Reinheckel, T.;

Peters, C.; Zeiser, R.; Bogyo, M.; Turk, V.; Psakhye, S.

G.; Turk, B.; Vasiljeva, O. Ferri-liposomes as an

MRI-visible drug-delivery system for targeting tumours

and their microenvironment. Nat. Nanotechnol. 2011, 6,

594-602.

[34].Kaittanis, C.; Shaffer, T. M.; Ogirala, A.; Santra, S.;

Perez, J. M.; Chiosis, G.; Li, Y. M.; Josephson, L.;

Grimm, J. Environment-responsive nanophores for

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

11 Nano Res.

therapy and treatment monitoring via molecular MRI

quenching. Nat. Commun. 2014, 5, 4384-4394.

[35]. Horcajada, P.; Chalati, T.; Serre, C.; Gillet, B.;

Sebrie, C.; Baati, T.; Eubank, J. F.; Heurtaux, D.;

Clayette, P.; Kreuz, C.; Chang, J. S.; Hwang, Y. K.;

Marsaud, V.; Bories, P. N.; Cynober, L.; Gil, S.; Ferey,

G.; Couvreur, P.; Gref, R. Porous

metal-organic-framework nanoscale carriers as a

potential platform for drug delivery and imaging. Nat.

Mater. 2010, 9, 172-178.

[36]. Wang, H. J.; Wang, L. Y. One-pot syntheses and

cell imaging applications of poly(amino acid) coated

LaVO4:Eu3+ luminescent nanocrystals. Inorg. Chem.

2013, 52, 2439-2445.

[37]. Wang, L. Y.; Zhang, Y.; Zhu, Y. Y. One-Pot

synthesis and strong near-infrared upconversion

luminescence of poly(acrylic acid)-functionalized

YF3:Yb3+/Er3+ nanocrystals. Nano Res. 2010, 3,

317-325.

[38]. Deng, M. L.; Ma, Y. X.; Huang, S.; Hu, G. F.;

Wang, L. Y. Monodisperse upconversion NaYF4

nanocrystals: syntheses and bioapplications. Nano Res.

2011, 4, 685-694.

[39]. Deng, M. L.; Tu, N.; Bai, F.; Wang, L. Y. Surface

functionalization of hydrophobic nanocrystals with one

particle per micelle for bioapplications. Chem. Mat.

2012, 24, 2592-2597.

[40]. Deng, M. L.; Wang, L. Y. Unexpected

luminescence enhancement of upconverting

nanocrystals by cation exchange with well retained

small particle size. Nano Res. 2014, 7, 782-793.

[41]. Li, H.; Wang, L. Y. Controllable Multicolor

Upconversion Luminescence by Tuning the NaF Dosage.

Chem.-Asian J. 2014, 9, 153-157.

[42]. Li, H.; Wang, L. Y. Preparation and upconversion

luminescence cell imaging of O-carboxymethyl

chitosan-functionalized NaYF4:Yb3+/Tm3+/Er3+

nanoparticles. Chin. Sci. Bull. 2013, 58, 4051-4056.

[43]. Huang, S.; Bai, M.; Wang, L. Y. General and facile

surface functionalization of hydrophobic nanocrystals

with poly(amino acid) for cell luminescence imaging.

Sci. Rep. 2013, 3, 2023-2027.

[44]. Peer, D.; Karp, J. M.; Hong, S.; FaroKhzad, O. C.;

Margalit, R.; Langer, R. Nanocarriers as an emerging

platform for cancer therapy. Nat. Nanotechnol. 2007, 2,

751-760.

[45]. Hong, G. S.; Robinson, J. T.; Zhang, Y. J.; Diao, S.;

Antaris, A. L.; Wang, Q. B.; Dai, H. J. In vivo

fluorescence imaging with Ag2S quantum dots in the

second near-infrared region. Angew. Chem. Int. Edit.

2012, 51, 9818-9821.

[46]. Jiang, P.; Tian, Z. Q.; Zhu, C. N.; Zhang, Z. L.;

Pang, D. W. Emission-tunable near-infrared Ag2S

quantum dots. Chem. Mat. 2012, 24, 3-5.

[47]. Du, Y. P.; Xu, B.; Fu, T.; Cai, M.; Li, F.; Zhang, Y.;

Wang, Q. B. Near-infrared photoluminescent Ag2S

quantum dots from a single source precursor. J. Am.

Chem. Soc. 2010, 132, 1470-1471.

[48]. Yi, H. J.; Ghosh, D.; Ham, M. H.; Qi, J. F.; Barone,

P. W.; Strano, M. S.; Belcher, A. M. M13

phage-functionalized single-walled carbon nanotubes as

nanoprobes for second near-infrared window

fluorescence imaging of targeted tumors. Nano Lett.

2012, 12, 1176-1183.

[49]. Tu, N. N.; Wang, L. Y. Surface plasmon resonance

enhanced upconversion luminescence in aqueous media

for TNT selective detection. Chem. Commun. 2013, 49,

6319-6321.

[50]. Hu, S. H.; Chen, S. Y.; Gao, X. H. Multifunctional

nanocapsules for simultaneous encapsulation of

hydrophilic and hydrophobic compounds and

on-demand release. ACS Nano 2012, 6, 2558-2565.

[51]. Ruoslahti, E.; Pierschbacher, M. D. New

perspectives in cell adhesion: RGD and integrins.

Science 1987, 238, 491-497.

[52]. Balasubramanian, S. V.; Straubinger, R. M.

Taxol-lipid interactions: taxol-dependent effects on the

physical properties of model membranes. Biochemistry

1994, 33, 8941-8947.

[53]. Lv, P. P.; Ma, Y. F.; Yu, R.; Yue, H.; Ni, D. Z.; Wei,

W.; Ma, G. H. Targeted delivery of insoluble cargo

(paclitaxel) by PEGylated chitosan nanoparticles grafted

with Arg-Gly-Asp (RGD). Mol. Pharm. 2012, 9,

1736-1747.