Abstract

Curcumin (CM) has anticancer potential for several cancers and blocks several steps in the carcinogenesis process. However, the clinical application of CM is greatly limited due to its low effects in vivo resulted from its poor solubility and pharmacokinetics. This raises the possibility of taking CM as a novel model drug in a new nanoparticle-based delivery system. In this study, CM-loaded nanoparticles were prepared from three kinds of amphilic methoxy poly(ethylene glycol) (mPEG)–polycaprolactone (PCL) block copolymers. It was noted that CM-loaded nanoparticles prepared from mPEG10k–PCL30k showed not only the highest loading efficiency, but also the most sustained release pattern. In vitro studies showed that CM was effectively transported into A549 cells by nanoparticles and localized around the nuclei in the cytoplasm. In addition, the cytotoxicity of CM-loaded nanoparticles with mEPG10k–PCL30k as a drug carrier was in a dose- and time-dependent manner in A549 cells. Further apoptotic staining results demonstrated the superior pro-apoptotic effect of CM-loaded nanoparticles over free drug. Data in this study not only confirmed the potential of CM in treating lung cancer, but also offered an effective way to improve the anticancer efficiency of CM through the nano-drug delivery system.

Introduction

Lung cancer is an aggressive and progressive deadly disease with few treatment options and poor overall survival in non-surgical stages. Despite recent advances in the treatments for other cancers, the 5-year survival rate of patients suffering from lung cancer of all stages is still only 16% [1]. Systemic chemotherapy, one of the main cancer treatment methods, has unavoidable toxicity without satisfactory treatment effect. Therefore, it is important to identify potential drugs and explore more efficient therapeutic strategies for the treatment of lung cancer.

Curcumin (CM) is a biphenyl compound in the herb Curcuma longa and possesses anti-inflammatory, anticancer, antioxidant, wound healing, and antimicrobial activities [2,3]. CM has anticancer potential for several cancers and blocks several steps in the carcinogenesis process [4–7]. Despite these extraordinary properties, CM has limited application in the treatment of cancer due to its considerable hydrophobicity, instability, and poor pharmacokinetics, which greatly hampers its efficacy in vivo [8–10]. Thus, it is desirable to explore novel formulations of CM that overcome the limitations mentioned above.

Recent progress in drug delivery studies has focused on improving the delivery system for malignant diseases by nanomedicine and polymer techniques [11]. These drug carriers have the following advantages: (i) enhancing the solubility of hydrophobic agents; (ii) releasing the antitumor agents in a sustainable pattern; and (iii) having preferable biocompatibility and biodegradability [12–14]. Polycaprolactone (PCL) was chosen as the hydrophobic segment of block copolymer because of its good drug encapsulation ability. In addition, the slow degradation of PCL-based particles allows the extended release of the drug [15]. Polyethylene glycol (PEG) possesses a number of outstanding physiochemical and biological properties. Owing to its good biocompatibility, hydrophilicity, and the absence of antigenicity and immunogenicity, the core–shell structure with PEG as an outer shell enables nanoparticles to escape from the scavenging of reticuloendothelial systems [16].

In the present study, we prepared biodegradable methoxy poly(ethylene glycol) (mPEG)–PCL nanoparticles incorporating CM, which provides a feasible way to overcome the limitations of CM in clinical applications. The mPEG–PCL copolymers were prepared with different PEG/PCL ratios, which influenced the properties of copolymers and the effectiveness of nanoparticles. To determine which composition was most suitable for the delivery of CM, three kinds of mPEG–PCL copolymers were synthesized. In addition, we evaluated the feasibility of this mPEG–PCL system by testing its in vitro cytotoxicity against human lung cancer cell line A549.

Materials and Methods

Materials and cell culture

CM, ε-caprolactone (ε-CL), mPEG (2, 4, or 10 kDa) were purchased from Sigma (St Louis, USA). PEG samples were dehydrated by azeotropic distillation with toluene and vacuum-dried at 50°C for 12 h before use. ε-CL was purified by drying CaH2 at room temperature and distillation under reduced pressure. Stannous octoate, 4′,6-diamidino-2-phenylindole (DAPI), and 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. All other chemicals were of analytical grade and used without further purification. Human lung cancer cell line A549 was obtained from the Institute of Biochemistry and Cell Biology, CAS (Shanghai, China). Cells were maintained in RPMI-1640 medium (Life Technologies, Inc., Gaithersburg, USA) containing 10% fetal bovine serum (Life Technologies, Inc.), 100 U/ml penicillin, and 100 U/ml streptomycin in a humidified atmosphere with 5% CO2 at 37°C.

Synthesis and characterization of mPEG and PCL block copolymers

mPEG–PCL block copolymers were synthesized according to the methods reported previously [17]. Briefly, a predetermined amount of ε-CL was added into a polymerization tube containing mPEG and a small amount of stannous octoate (0.1%, w/w). The tube was then connected to a vacuum system, sealed off, and placed in an oil bath at 130°C for 48 h. At the end of the polymerization, the crude copolymers were dissolved with dichloromethane and precipitated into an excess amount of cold methanol to remove the un-reacted monomer and oligomer. The precipitates were then filtered and washed with water several times before being thoroughly dried at reduced pressure. Gel permeation chromatography was used to characterize the copolymers.

Preparation of CM-loaded nanoparticles

CM-loaded nanoparticles were prepared by a nano-precipitation method as described previously with minor modifications [17]. Briefly, 10 mg of each copolymer and 2 mg of CM were dissolved in 0.3 ml of hot acetone. The obtained organic solution was added dropwise into 10-time volumes of distilled water under gentle stirring at room temperature. The solution was dialyzed in a dialysis bag (molecular weight cut-off 4 kDa; Sigma) to remove acetone thoroughly. The obtained bluish aqueous solution was filtered through a 0.22-μm filter membrane to remove non-incorporated drugs and copolymer aggregates. Coumarin-6, C20H18N2O2S, is a hydrophobic dye exhibiting green fluorescence under excitation with a molecular weight of 350.43 Da. Coumarin-6-loaded nanoparticles and blank nanoparticles were produced in a similar manner without adding drugs. These prepared nanoparticles were lyophilized for further use.

Size and zeta potential analysis of nanoparticles

The mean diameter and size distribution of nanoparticles were measured by dynamic light scattering (DLS) with a Brookheaven BI9000AT system (Brookhaven Instruments Corporation, New York, USA). Zeta potential was measured by using a laser Doppler anemometry (Zeta Plus, Zeta Potential Analyzer; Brookhaven Instruments Corporation). Measurements were performed at least in triplicate.

Morphology studies

Morphological examination of nanoparticles was carried out by using a JEM-100S transmission electron microscope (TEM) (Olympus Company Ltd., Tokyo, Japan). One drop of the nanoparticle solution was negatively stained with 1% (w/v) phosphotungstic sodium solution and placed on a copper grid covered with nitrocellulose membrane.

Drug loading content and encapsulation efficiency detection of CM-loaded nanoparticles

The concentration of CM was measured by using a LC-10AD HPLC system (Shimadzu, Tokyo, Japan) equipped with a Shimadzu UV detector and an Agilent C-18 RP-HPLC analytical column (Ø 5 μm, 250 mm × 4.6 mm). The mobile phase was composed of acetonitrile−monosodium phosphate (10 mM, pH 3.5 adjusted by orthophosphoric acid) (50 : 50, v/v) at a flow rate of 1.0 ml/min. The run time for analysis was 27 min and the detection wavelength was set at 425 nm. Each sample injection volume was 20 μl. The drug loading content (DLC) and encapsulation efficiency (EE) were calculated by Equations (1) and (2), respectively:  

(1)
formula
 
(2)
formula

In vitro release assay of CM-loaded nanoparticles

In vitro release of CM from the mPEG–PCL nanoparticles was evaluated using a dialysis bag diffusion technique after the preparation of CM-loaded nanoparticles as described previously with minor modifications [17]. Briefly, 10 mg of each kind of CM-loaded nanoparticles were suspended in 1 ml of 0.1 M phosphate-buffered saline (PBS, pH 7.4). The solution was placed into a pre-swelled dialysis bag with a 4-kDa molecular weight cut-off (Sigma) and immersed into 20 ml of 0.1 M PBS, pH 7.4, at 37°C with gentle agitation. At different times, 1 ml of incubation medium was analyzed to measure CM concentration as described above. Then, the other incubation medium was immediately supplemented with 1 ml of fresh PBS to maintain the volume. The concentration of CM released from nanoparticles was expressed as a percentage of total CM in the nanoparticles and plotted as a function of time.

Cellular uptake analysis of particles

The cellular uptake analysis was carried out according to previous reports [18,19]. Coumarin-6 was used to detect the uptake efficiency of nanoparticles by tumor cells. Cells were seeded in six-well plates at a density of 4 × 104 cells per well. After incubation in a humidified atmosphere with 5% CO2 for 24 h at 37°C, cells were exposed to medium containing coumarin-6-loaded nanoparticles (12.5 μg/ml). After 2-h incubation, the cell monolayers were washed 3–4 times with PBS at 37°C. Then, the cells were examined under a fluorescence microscope.

In vitro cytotoxicity assay of CM-loaded nanoparticles

The half maximal inhibitory concentration (IC50) of CM-loaded nanoparticles on A549 cells were determined by MTT assay. Briefly, cells were seeded in 96-well plates at 1 × 104 cells per well. Twenty-four hours later, cells were exposed to a series of equivalent doses of free CM or CM-loaded nanoparticles, ranging from 20 to 70 μg/ml. After 24, 48, or 72 h, 20 μl of 5 mg/ml MTT solution was added to each well and the plate was incubated for 4 h. Then, the medium was removed and dimethylsulfoxide (150 μl) was added to each well. The optical density (OD) of each well was measured by using a microplate reader (Bio-Rad, Hercules, USA) at 560 nm. Cell viability was calculated by Equation (3):  

(3)
formula
All data obtained from MTT assay were confirmed by repeating the experiment for at least three times and by testing in triplicate each time.

DAPI staining assay

Cells were treated with blank nanoparticles (200 μM), free CM (40 μM), or CM-loaded nanoparticles (40 μM) for 48 h, washed once in PBS, and then fixed in cold methonal : acetone (1 : 1) for 5 min. After washing three times with PBS, cells were treated with 4 μg/ml DAPI for 10 min at room temperature. Cells with morphological changes of apoptosis were observed with an original magnification of ×200.

Statistical analysis

Data were expressed as the mean ± SD. Student's t-test and analysis of variance were used to analyze the data. P < 0.05 was considered statistically significant.

Results

Size, zeta potential, and morphology of CM-loaded nanoparticles

The particle size and size distribution of the CM-loaded nanoparticles in aqueous solution were determined by DLS and the results are displayed in Table 1. The mean diameter of mPEG10k–PCL30k is about 140 nm, the largest among the three nanoparticles prepared. All the nanoparticles are negatively charged. The particle size and zeta potential of the blank nanoparticles were not significantly different from those of CM-loaded nanoparticles (data not shown).

Table 1

Characterization of three kinds of nanoparticles prepared from different copolymers

Group Diameter (nm) Polydispersity Zeta potential (mV) 
mPEG4k–PCL20k 125.3 ± 8.3 0.17 ± 0.04 −4.7 ± 0.4 
mPEG2k–PCL4k 102.3 ± 11.3 0.17 ± 0.05 −6.9 ± 1.0 
mPEG10k–PCL30k 140.3 ± 14.2 0.16 ± 0.04 −7.8 ± 1.4 
Group Diameter (nm) Polydispersity Zeta potential (mV) 
mPEG4k–PCL20k 125.3 ± 8.3 0.17 ± 0.04 −4.7 ± 0.4 
mPEG2k–PCL4k 102.3 ± 11.3 0.17 ± 0.05 −6.9 ± 1.0 
mPEG10k–PCL30k 140.3 ± 14.2 0.16 ± 0.04 −7.8 ± 1.4 

Figure 1(A) shows a typical picture of the solution of CM-loaded nanoparticles (prepared from the copolymer mPEG10k–PCL30k), which looks yellow due to the encapsulated CM. CM-loaded nanoparticles were completely dispersed in aqueous media with no aggregates and free CM exhibited poor aqueous solubility (data not shown). Figure 1(B) shows the TEM pictures of nanoparticles prepared by mPEG10k–PCL30k. It was clearly shown that the shape was nearly spherical and the size was about 120 nm in diameter.

Figure 1

Characterization of copolymers and nanoparticles (A) Picture of CM-loaded nanoparticles (mPEG10k–PCL30k) (a) and free CM (b). (B) TEM image of CM-loaded nanoparticles (mPEG10k–PCL30k). Bar = 200 μm. (C) In vitro release curves of three kinds of CM-loaded nanoparticles.

Figure 1

Characterization of copolymers and nanoparticles (A) Picture of CM-loaded nanoparticles (mPEG10k–PCL30k) (a) and free CM (b). (B) TEM image of CM-loaded nanoparticles (mPEG10k–PCL30k). Bar = 200 μm. (C) In vitro release curves of three kinds of CM-loaded nanoparticles.

In vitro release pattern of CM-nanoparticles

All nanoparticles exhibited a fast release of CM at initial stage, and then a sustained one [Fig. 1(C)]. An initial burst of 35%–45% release was found at the fifth hour due to the affiliation of drug to nanoparticle surface. Afterwards, it was observed that CM was released from nanoparticles in a sustained manner. CM was released more rapidly from smaller nanoparticles. mPEG2k–PCL4k showed the most prominent burst release (∼45% at the 5th hour and 60% at the 24th hour), while the release pattern of mPEG10k–PCL30k was the most sustainable with the smallest initial burst rate (∼30% at the 5th hour and 45% at the 24th hour). Hence, CM-loaded nanoparticles formed by using mPEG10k–PCL30k were chosen for in vitro cytotoxicity assay due to its relatively higher DLC and EE and better sustained release pattern.

DLC and EE of nanoparticles

Table 2 shows the DLC and EE of three kinds of nanoparticles. mPEG10k–PCL30k possessed the highest DLC which was detected to be 12.3% ± 1.5%, as well as an EE > 80%. Meanwhile, the DLCs of CM in other two kinds of nanoparticles were <10%. All of them had an EE >75%, indicating that CM was encapsulated into mPEG–PCL nanoparticles with a relatively high DLC and EE, which might be due to the strong hydrophobic interaction between CM and PCL.

Table 2

DLC and EE of three kinds of nanoparticles

Group mPEG4k–PCL20k mPEG2k–PCL4k mPEG10k–PCL30k 
DLC (%) 7.2 ± 0.9 9.3 ± 1.2 12.3 ± 1.5 
EE (%) 79.2 ± 9.2 75.2 ± 6.3 83.1 ± 5.8 
Group mPEG4k–PCL20k mPEG2k–PCL4k mPEG10k–PCL30k 
DLC (%) 7.2 ± 0.9 9.3 ± 1.2 12.3 ± 1.5 
EE (%) 79.2 ± 9.2 75.2 ± 6.3 83.1 ± 5.8 

Cellular uptake studies

Figure 2 shows the cellular uptake of CM-loaded nanoparticles and it was obvious that 2-h incubation was sufficient for A549 cells to uptake nanoparticles which were localized in the cytoplasm.

Figure 2

Fluorescent microscopic images of A549 cells treated with coumarin-6-loaded nanoparticles (A) Lower-power fluorescent field. (B) High-power fluorescent field. (C) Lower-power bright field. (D) High-power bright field.

Figure 2

Fluorescent microscopic images of A549 cells treated with coumarin-6-loaded nanoparticles (A) Lower-power fluorescent field. (B) High-power fluorescent field. (C) Lower-power bright field. (D) High-power bright field.

In vitro cytotoxicity of CM-nanoparticles

The cytotoxicity of CM-loaded particles against A549 cells was evaluated by MTT assay. Both CM and CM-loaded nanoparticles showed cytotoxicity against the cells in similar dose- and time-dependent manners (Fig. 3). IC50 values of free CM and CM-loaded nanoparticles are shown in Table 3. It was obvious that CM-loaded nanoparticles, at equivalent dose, exhibited better cytotoxicity than free CM.

Table 3

IC50 values of CM and CM-loaded nanoparticles against A549 cells for different time periods

Group IC50s (µM)
 
24 h 48 h 72 h 
CM 81.1 ± 7.9 66.6 ± 8.8 53.0 ± 6.8 
CM-loaded nanoparticles 67.9 ± 9.1* 51.3 ± 5.4* 34.5 ± 4.3* 
Group IC50s (µM)
 
24 h 48 h 72 h 
CM 81.1 ± 7.9 66.6 ± 8.8 53.0 ± 6.8 
CM-loaded nanoparticles 67.9 ± 9.1* 51.3 ± 5.4* 34.5 ± 4.3* 

*P < 0.05 vs. CM at equivalent time.

Figure 3

Cytotoxicity assays of CM-loaded nanoparticles and free CM against A549 cells (n = 3).

Figure 3

Cytotoxicity assays of CM-loaded nanoparticles and free CM against A549 cells (n = 3).

Apoptotic staining

A549 cells were treated with a series of concentrations of blank nanoparticles, free CM, and CM-loaded nanoparticles for 48 h, respectively. DAPI staining assay showed that cells in control or blank nanoparticle group had rounded and intact nuclei (Fig. 4), while cells treated with free CM or CM-loaded nanoparticles had nuclei that were smaller and brighter, showing crescent-shaped profiles around the periphery of the nucleus and separate globular structures (apoptotic bodies). The number of apoptotic cells treated with CM-loaded nanoparticles dramatically increased, when compared with those treated with free CM (P< 0.05). These data demonstrated that blank nanoparticles had no toxicity and that CM had an effect to induce A549 cells apoptosis. Quantitative analysis revealed that CM-loaded nanoparticles had a higher apoptosis rate, when compared with free CM [Fig. 4(E)].

Figure 4

Apoptosis analysis of A549 cells detected by DAPI staining (A) Non-treated cells. (B) Cells treated with 200 μM blank nanoparticles. (C) Cells treated with 40 μM free CM. (D) Cells treated with 40 μM CM-loaded nanoparticles. (E) Quantitative analysis of apoptotic rate of cells exposed to different agents. *P < 0.05 vs. control group and #P < 0.05 vs. free CM group, n = 3.

Figure 4

Apoptosis analysis of A549 cells detected by DAPI staining (A) Non-treated cells. (B) Cells treated with 200 μM blank nanoparticles. (C) Cells treated with 40 μM free CM. (D) Cells treated with 40 μM CM-loaded nanoparticles. (E) Quantitative analysis of apoptotic rate of cells exposed to different agents. *P < 0.05 vs. control group and #P < 0.05 vs. free CM group, n = 3.

Discussion

Plasmonic nanoparticles have received considerable attention due to their potential use in cancer diagnostics and therapeutic applications [20,21]. In the present study, three kinds of mPEG–PCL copolymers were chosen as drug carriers. It was found that mPEG10k–PCL30k block copolymers possessed the highest drug loading efficiency and the most sustained release pattern.

Fluorescent microscopy indicated that the uptake of CM by A549 cells was facilitated when delivered by nanoparticles. It was reported that the percentage of cellular uptake was influenced by the concentration of nanoparticles [22]. The cellular uptake of nanoparticles is important and mediated through endocytosis rather than passive diffusion [23], which might be a saturable process. When the concentration of nanoparticles is beyond the uptake capacity of endocytosis, the uptake efficiency will not increase in a concentration-dependent manner [24].

In in vitro cytotoxicity assay, CM-loaded nanoparticles led to higher cell death rates with lower IC50, when compared with free CM (Fig. 3). However, at doses >80 μM, CM-loaded nanoparticles induced similar cell inhibition. These results were in accordance with previous studies [22,25–27]. The possible mechanism is through the enhanced intracellular drug accumulation by nanoparticle uptake [28]. Data from cellular uptake experiments also confirmed the high cell affinity of nanoparticles (Fig. 2). However, the intracellular uptake of nanoparticles by cells still has limits, which presents a potential mechanism to explain why further increases in the dose cause similar cell inhibition in cells treated with free CM or CM-loaded nanoparticles [22].

Programmed cell death is characterized by apoptotic morphology, including chromatin condensation, membrane blabbing, internucleosome degradation of DNA, and apoptotic body formation [29,30]. In this present study, DAPI staining also demonstrated that CM-loaded nanoparticles induced more apoptosis than free CM, which was consistent with the results of cytotoxicity assay.

To enhance the targeting effect of the nano-drug system, active-targeting strategies of specific antibody conjugation with nanoparticles are under careful consideration in our lab. In our preliminary study, CM-loaded nanoparticles modified by epidermal growth factor receptor antibodies showed specific accumulation in tumor site. The current study confirmed the potential of CM in treating lung cancer and offered an effective way to improve the anticancer efficiency of CM by nano-drug delivery system. Our future research will focus on the in vivo evaluation in xenografts, which will promote the development of this system.

Acknowledgement

The authors would like to thank Ms Deni Jennings for proofreading the manuscript.

References

1
Rocks
N
Bekaert
S
Coia
I
Paulissen
G
Gueders
M
Evrard
B
Van Heugen
JC
, et al.  . 
Curcumin-cyclodextrin complexes potentiate gemcitabine effects in an orthotopic mouse model of lung cancer
Br J Cancer
 , 
2012
, vol. 
107
 (pg. 
1083
-
1092
)
2
Maheshwari
RK
Singh
AK
Gaddipati
J
Srimal
RC
Multiple biological activities of curcumin: a short review
Life Sci
 , 
2006
, vol. 
78
 (pg. 
2081
-
2087
)
3
Sintara
K
Thong-Ngam
D
Patumraj
S
Klaikeaw
N
Curcumin attenuates gastric cancer induced by N-methyl-N-nitrosourea and saturated sodium chloride in rats
J Biomed Biotechnol
 , 
2012
, vol. 
2012
 pg. 
915380
 
4
Huang
MT
Lou
YR
Ma
W
Newmark
HL
Reuhl
KR
Conney
AH
Inhibitory effects of dietary curcumin on forestomach, duodenal, and colon carcinogenesis in mice
Cancer Res
 , 
1994
, vol. 
54
 (pg. 
5841
-
5847
)
5
Dorai
T
Dutcher
JP
Dempster
DW
Wiernik
PH
Therapeutic potential of curcumin in prostate cancer−V: interference with the osteomimetic properties of hormone refractory C4–2B prostate cancer cells
Prostate
 , 
2004
, vol. 
60
 (pg. 
1
-
17
)
6
Duvoix
A
Blasius
R
Delhalle
S
Schnekenburger
M
Morceau
F
Henry
E
Dicato
M
Chemopreventive and therapeutic effects of curcumin
Cancer Lett
 , 
2005
, vol. 
223
 (pg. 
181
-
190
)
7
Wang
Z
Zhang
Y
Banerjee
S
Li
Y
Sarkar
FH
Notch-1 down-regulation by curcumin is associated with the inhibition of cell growth and the induction of apoptosis in pancreatic cancer cells
Cancer
 , 
2006
, vol. 
106
 (pg. 
2503
-
2513
)
8
Wang
YJ
Pan
MH
Cheng
AL
Lin
LI
Ho
YS
Hsieh
CY
Lin
JK
Stability of curcumin in buffer solutions and characterization of its degradation products
J Pharm Biomed Anal
 , 
1997
, vol. 
15
 (pg. 
1867
-
1876
)
9
Tonnesen
HH
Masson
M
Loftsson
T
Studies of curcumin and curcuminoids. XXVII. Cyclodextrin complexation: solubility, chemical and photochemical stability
Int J Pharm
 , 
2002
, vol. 
244
 (pg. 
127
-
135
)
10
Shoba
G
Joy
D
Joseph
T
Majeed
M
Rajendran
R
Srinivas
PS
Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers
Planta Med
 , 
1998
, vol. 
64
 (pg. 
353
-
356
)
11
Kataoka
K
Harada
A
Nagasaki
Y
Block copolymer micelles for drug delivery: design, characterization and biological significance
Adv Drug Deliv Rev
 , 
2001
, vol. 
47
 (pg. 
113
-
131
)
12
LaVan
DA
McGuire
T
Langer
R
Small-scale systems for in vivo drug delivery
Nat Biotechnol
 , 
2003
, vol. 
21
 (pg. 
1184
-
1191
)
13
Pridgen
EM
Langer
R
Farokhzad
OC
Biodegradable, polymeric nanoparticle delivery systems for cancer therapy
Nanomedicine
 , 
2007
, vol. 
2
 (pg. 
669
-
680
)
14
Cheng
J
Teply
BA
Sherifi
I
Luther
G
Gu
FX
Levy-Nissenbaum
E
Radovic-Moreno
AF
, et al.  . 
Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery
Biomaterials
 , 
2007
, vol. 
28
 (pg. 
869
-
876
)
15
Uhrich
KE
Cannizzaro
SM
Langer
RS
Shakesheff
KM
Polymeric systems for controlled drug release
Chem Rev
 , 
1999
, vol. 
99
 (pg. 
3181
-
3198
)
16
Herold
DA
Keil
K
Bruns
DE
Oxidation of polyethylene glycol by alcohol dehydrogenase
Biochem Pharmacol
 , 
1989
, vol. 
38
 (pg. 
73
-
76
)
17
Li
XL
Li
RT
Qian
X
Ding
Y
Tu
Y
Guo
R
Hu
Y
, et al.  . 
Superior antitumor efficiency of cisplatin-loaded nanoparticles by intratumoral delivery with decreased tumor metabolism rate
Eur J Pharm Biopharm
 , 
2008
, vol. 
70
 (pg. 
726
-
734
)
18
Hu
Y
Xie
J
Tong
Y
Wang
C
Effect of PEG conformation and particle size on the cellular uptake efficiency of nanoparticles with the HepG2 cells
J Control Release
 , 
2007
, vol. 
118
 (pg. 
7
-
17
)
19
Kim
JH
Kim
YS
Park
K
Kang
E
Lee
S
Nam
HY
Kim
K
, et al.  . 
Self-assembled glycol chitosan nanoparticles for the sustained and prolonged delivery of antiangiogenic small peptide drugs in cancer therapy
Biomaterials
 , 
2008
, vol. 
29
 (pg. 
1920
-
1930
)
20
Liu
L
Ni
F
Zhang
J
Jiang
X
Lu
X
Guo
Z
Xu
R
Silver nanocrystals sensitize magnetic-nanoparticle-mediated thermo-induced killing of cancer cells
Acta Biochim Biophys Sin
 , 
2011
, vol. 
43
 (pg. 
316
-
323
)
21
Zhao
D
Sun
X
Tong
J
Ma
J
Bu
X
Xu
R
Fan
R
A novel multifunctional nanocomposite C225-conjugated Fe3O4/Ag enhances the sensitivity of nasopharyngeal carcinoma cells to radiotherapy
Acta Biochim Biophys Sin
 , 
2012
, vol. 
44
 (pg. 
678
-
684
)
22
Davda
J
Labhasetwar
V
Characterization of nanoparticle uptake by endothelial cells
Int J Pharm
 , 
2002
, vol. 
233
 (pg. 
51
-
59
)
23
Rosen
H
Abribat
T
The rise and rise of drug delivery
Nat Rev Drug Discov
 , 
2005
, vol. 
4
 (pg. 
381
-
385
)
24
Li
XL
Zhen
DH
Lu
XW
Xu
H
Shao
Y
Xue
QP
Hu
Y
, et al.  . 
Enhanced cytotoxicity and activation of ROS-dependent c-Jun NH2-terminal kinase and caspase-3 by low doses of tetrandrine-loaded nanoparticles in Lovo cells—a possible Trojan strategy against cancer
Eur J Pharm Biopharm
 , 
2010
, vol. 
75
 (pg. 
334
-
340
)
25
Van
VLE
Duan
ZF
Seiden
MV
Amiji
MM
Modulation of intracellular ceramide using polymeric nanoparticles to overcome multidrug resistance in cancer
Cancer Res
 , 
2007
, vol. 
67
 (pg. 
4843
-
4850
)
26
Zhang
L
Yang
M
Wang
Q
Li
Y
Guo
R
Jiang
X
Yang
C
, et al.  . 
10-Hydroxycamptothecin loaded nanoparticles: preparation and antitumor activity in mice
J Control Release
 , 
2007
, vol. 
119
 (pg. 
153
-
162
)
27
Zhang
L
Hu
Y
Jiang
X
Yang
C
Lu
W
Yang
Y
Camptothecin derivative-loaded poly(caprolactone-co-lactide)-b-PEG-b-poly(capro-lactone-co-lactide) nanoparticles and their biodistribution in mice
J Control Release
 , 
2004
, vol. 
96
 (pg. 
135
-
148
)
28
Shenoy
D
Little
S
Langer
R
Amiji
M
Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs. 1. In vitro evaluations
Mol Pharm
 , 
2005
, vol. 
2
 (pg. 
357
-
366
)
29
Rechsteiner
M
Rogers
SW
PEST sequences and regulation by proteolysis
Trends Biochem Sci
 , 
1996
, vol. 
21
 (pg. 
267
-
271
)
30
Wyllie
AH
Kerr
JF
Currie
AR
Cell death: the significance of apoptosis
Int Rev Cytol
 , 
1980
, vol. 
68
 (pg. 
251
-
306
)