Biomaterials 35 (2014) 5414e5424

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

pH-Responsive polymer-liposomes for intracellular drug delivery and tumor extracellular matrix switched-on targeted cancer therapy Yi-Ting Chiang a, Chun-Liang Lo a, b, c, * a

Department of Biomedical Engineering, National Yang Ming University, Taipei 112, Taiwan, ROC Biophotonics & Molecular Imaging Research Center (BMIRC), National Yang Ming University, Taipei 112, Taiwan, ROC c Biomedical Engineering Research Center, National Yang Ming University, Taipei 112, Taiwan, ROC b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 February 2014 Accepted 18 March 2014 Available online 4 April 2014

This study presents a tumor-extracellular matrix pH-induced targeting liposome (ECM-targeting liposomes), crosslinked from methoxy-poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide-cohistidine)-cholesterol copolymers and biotin2-polyethylene glycol crosslinkers by hydrogen bonds to overcome the defects of liposomes. In this study, ECM-targeting liposomes were completely investigated their pH-responsibility, drug releasing behaviors, anticancer efficiencies and the time-dependent organ distribution and toxic effects. Experimental results indicate that ECM-targeting liposomes showed rapid drug releasing profiles in acidic conditions. Because the ECM-targeting liposomes accumulated preferentially in tumor, the ECM-targeting liposomes exhibited exceptional anticancer activity in vivo and lower hepatic and renal toxicity. The ECM-targeting liposomes which are switched on the targeting ability in tumor ECM possess potential for future application in anticancer therapy. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Liposomes Polymers Tumor ECM-induced targeting Intracellular drug delivery Cancer therapy

1. Introduction Liposome, an important drug carrier, has low toxicity and biocompatibility and is widely used in pharmaceutical formulations [1e3]. However, liposomes have three main defects: low stability, slow drug release and large liver/spleen accumulation. To improve liposomal stability, a hydrophilic polymer, polyethylene glycol (PEG), has been coated on the surface of liposomes to form what are called stealth liposomes. Stealth liposomes can reduce the recognition by reticuloendothelial system (RES) and then prolong the blood circulation time [3e5]. However, the side effects of stealth liposomes are slow drug release, leading to low bioavailability and drug resistance of cancer cells [3]. To overcome the slow drug release problem, a polymer-based liposome with high pH sensitivity has been developed [6e10]. However, polymer detachment from liposomes leads to structural instability, a noticeable problem of this strategy [11,12]. Therefore, a polymer-caged liposome has been proposed to solve the problem of polymer detachment [13]. Another serious problem of liposomes is their large accumulations in the liver and spleen. Although liposomes can accumulate in tumors by the enhanced permeability and retention effect, many

* Corresponding author. Department of Biomedical Engineering, National Yang Ming University, Taipei 112, Taiwan, ROC. Fax: þ886 2 2821 0847. E-mail address: [email protected] (C.-L. Lo). http://dx.doi.org/10.1016/j.biomaterials.2014.03.046 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

studies have reported that accumulation of liposomes in the liver or spleen is preferential to that in a tumor, causing macrophage toxicity [14e17]. A simple method to enhance tumor-targeted specificity is to use conjugated targeting ligands on the surface of liposomes [18e20]. Small molecules, such as folic acid or other vitamin molecules, help reduce the accumulation of liposomes in the liver and spleen [21e23]. However, the accumulation of liposomes in the liver or spleen is higher than that in tumors because ligandereceptors interaction between liposomes and liver or spleen cells still occur. Otherwise, antibody liposomes, called immunoliposomes, could be limited in tumors. However, immunoliposomes can be applied only to a few specific tumor cells, and their immunogenicity is a major concern [24]. To overcome stability and organ distribution defects of liposomes, we have recently shown that a sophisticated tumorextracellular matrix (ECM) switched-on targeting liposome (ECMtargeting liposomes) composed of dipalmitoylphosphatidylcholine (DPPC), methoxy-poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide-co-histidine)-cholesterol (mPEG-P(HPMA-g-His)cholesterol) copolymers and biotin-polyethylene glycol-biotin (biotin2-PEG) crosslinkers [25]. This ECM-targeting liposome has several advantageous characteristics include (1) the ability to prevent protein adsorption and drug leakage from liposomes owing to biotin2-PEG crosslinker located on the interface layer of liposomes, (2) an enabled dissociating crosslinker to improve cancer cell uptake by hydrogen-bond breaking to expose biotin molecules in

Y.-T. Chiang, C.-L. Lo / Biomaterials 35 (2014) 5414e5424

tumor extracellular matrix (the average pH value of tumor ECM is about 6.5e6.8), and (3) an active ability to target tumor in tumor ECM to increase tumor accumulation. In addition to preventing protein adsorption and to improving tumor accumulation, ECM-targeting liposomes also exhibited unique properties for cancer therapy. In the present study, we identified the pH-sensitivity, intracellular drug delivery ability and anticancer efficiency in which ECM-targeting liposomes overcome the slow drug release problem of liposomes (Fig. 1). Additionally, to further evaluate the practicalities of the ECM-targeting liposomes in anticancer therapy, the time-dependent organ accumulation (tumor, liver, and spleen) and time-dependent hepatic and renal toxicities were monitored. 2. Materials and methods 2.1. Preparation of liposomes The mPEG-P(HPMA-g-His)-cholesterol (1 mmol) and DPPC (34 mmol) were dissolved in DCM/methanol (1/1 v/v). A polymer-incorporated lipid thin film was formed by rotary evaporation at room temperature. Then phosphate buffered saline (PBS) at pH 7.4 was added to rehydrate the thin film, and the solution was subjected to sonication for 6 min. To prepare polymer-incorporated liposomes, the solution was extruded by a 0.22-mm PVDF filter twice and by a 0.1-mm PVDF filter five times. To prepare ECM-targeting liposomes, cross-linking agents (12.5 mmol) were added to the polymer-incorporated liposomes solution, shaken for 5 min, and the solution

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was extruded with a 0.1-mm PVDF filter twice. The particle size of each liposome was determined by dynamic laser scattering (DLS, Malvern zetasizer 3000), and morphology was observed by TEM (JEM-2000 EXII) with 2% uranyl acetate staining. 2.2. Stability tests and pH-responsive behaviors Samples (liposomes) were dissolved in pH 7.4 and pH 5.0 PBS, respectively, and shaken at 200 rpm at 37  C (by TAITEC constant temperature incubator shaker). At various intervals, samples were analyzed by DLS. 2.3. Preparation of DOX-loaded liposomes The mPEG-P(HPMA-g-His)-cholesterol (1 mmol) and DPPC (34 mmol) were dissolved in DCM/methanol (1/1 v/v). A polymer-incorporated lipid thin film was formed by rotary evaporation at room temperature. Then ammonium sulfate solution (250 mM) was added to rehydrate the thin film, and the solution was subjected to sonication for 6 min. Doxorubicin-HCl (Dox) in PBS (10 mg/mL) was mixed with the liposomes solution at 60  C for 2 h. To prepare Dox-loaded polymer-incorporated liposomes, the solution was extruded by a 0.22-mm PVDF filter twice and by a 0.1-mm PVDF filter five times. Biotin2-PEG crosslinkers were then added into the solution to prepare Dox-loaded ECM-targeting liposomes. Excess Dox was removed by a Sephadex G-50 with the PBS mobile phase. 2.4. Drug release assay The release behavior of Dox from liposomes in pH 5 and pH 7.4 PBS (around 0.1 mg/ml) at 37  C was measured by HPLC (Shimadzu LC with RF-20A FL detector) with FL at 480 nm for excitation and 560 nm for emission in a time-course procedure. Dox was isolated by dialysis bag (MWCO 10000; Millipore). Chromatographic

Fig. 1. (A) Schematic representation of the concept for ECM-targeting liposomes based on hydrogen bond crosslinking and their targeting ability and drug release after tumor accumulation. (B) Chemical structures of mPEG-P(HPMA-co-His)-Chol copolymer and biotin2-PEG crosslinker.

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separation was performed on an InertsilÒ ODS-3 column (200  4.6 mm i.d., 5 mm; GL Sciences, Japan). The mobile phase consisted of acetonitrile (A)e50 mM phosphoric acid (B) and programmed in a gradient manner as follows: A/B: 35/65(0e10 min), 10/ 90 (10e15 min), and 35/65 (15e20 min). Flow rate was 1.0 ml/min. Additionally, Doxloaded liposomes were fully dissolved in DMSO to determine loading efficiency and drug content by HPLC-FL. The accumulative release was calculated as: Cumulative release (%) ¼ (Dox conc. in buffer solution)/(total Dox conc. in each sample)  100%. 2.5. Cell cytotoxicity McCoy’s 5a medium containing 10% fetal bovine serum (FBS) at pH 7.4 and pH 6.5 (adjusting using HCl) were used. First, HCT116 cells (2  104 cells/mL) were seeded on a 96-well plate and incubated in a logarithmic growth phase under 5% CO2 and 37  C. Dox-loaded ECM-targeting liposomes, Dox-loaded polymer-incorporated liposomes, and free Dox in pH 7.4 or pH 6.5 medium were independently treated with HCT116 cells for 2 h by incubation at 37  C or 4  C. The HCT116 cells were washed twice with PBS solution, and fresh pH 7.4 medium was added for 24 h incubation. At experiment end, cell viability was analyzed by MTT assay [26]. The same procedure combined with excess of biotin molecules (75 mM) was repeated in pH 7.4 or pH 6.5 medium at 37  C for competitive study. Fluorescein isothiocyanate (FITC)-labeled liposomes were used in order to analyze by flow cytometer. Each sample was analyzed by BD FACSCalibur flow cytometer using the 488 nm argon/krypton laser line and a 520 nm band pass FL1-H emission filter. Additionally, Dox-loaded ECM-targeting liposomes, Dox-loaded polymer-incorporated liposomes, and free Dox with various concentrations of Dox were added for 24 h and 48 h at pH 7.4 and 37  C of co-culturing to obtain inhibitory concentration 50 (IC50).

approximately 500 mm3, free Dox, Cy5.5-conjugated Dox-loaded polymer-incorporated liposomes, and Cy5.5-conjugated Dox-loaded ECM-targeting liposomes (adjusted to the Dox dose of 10 mg/kg/mL) were administered via the tail vein. At days 1, 3 and 7 post-injection, the mice were sacrificed and the harvested tissues (including tumor, liver and spleen) were collected. Tissues were then frozen and embedded in O.C.T. medium. The frozen sections (4 mm-thick) of the tissues were cut using a microtome-cryostat (CM3050, Leica) and mounted with DAPI-containing mounting medium. The frozen sections were observed by Olympus FV1000 confocal laser scanning microscopy (CLSM) under the following conditions: at 405 nm for excitation and at 461 nm for DAPI emission, at 488 nm for excitation and at 520 nm for Dox detection, and at 633 nm for excitation and at 693 nm for Cy5.5 detection. 2.10. Time dependent biochemistry index monitoring At days 1, 3 and 7 post-injection of the free Dox, Dox-loaded polymer-incorporated liposomes, and ECM-targeting liposomes, the blood for each mouse was collected and centrifuged at 3000 rpm for 10 min to obtain the serum sample. The serum samples (10 mL) were carried out monitoring the time dependent hepatic and renal function by Fuji DRI-chem 4000i automated clinical chemistry analyzer. To measure the hepatic and renal function of the mice, GOT (or AST), GPT (or ALT), BUN and CRE were chosen to be the optimizations. 2.11. Statistical analysis All data were presented with an average values and its standard deviation, shown as mean  S.D. Comparison between groups was analysis with the two-tailed Student’s t-test (Excel, 2007). Differences were considered statistically significant when the p values were less than 0.05 (p < 0.05).

2.6. Internalization Accumulated liposomes and released Dox behavior in HCT116 cells were observed using a Olympus FV1000 confocal laser scanning microscope (CLSM). The HCT116 cells were seeded on coverslides for 16 h at 37  C and then treated with Dox or liposomes for 0.5 h at pH 6.5 and 4  C. Cells were washed twice with PBS to remove untrapped Dox or liposomes and then were cultured under pH 7.4 medium. After an interval, the cells were washed twice with PBS and mounted on a slide with 4% paraformaldehyde for CLSM observation. Fluorescence observation was carried out with a confocal microscope at 488 nm for excitation and an LP filter of 590 nm for Dox detection. Liposome observation was carried out with a confocal microscope at 405 nm for excitation and an appropriate filter for FITC detection. 2.7. pH-responsive capability study (biodistribution) The HCT116 cells were transplanted subcutaneously into the rear of 4-week female Balb-c/nude mice (1  106 cells/0.1 mL). When the transplanted tumor volume reached approximately 500 mm3, mice were pre-treated of saline or 75 mM biotin solution by tail vein injection. After 1 h, mice were injected with the Cy5.5-conjugated Dox-loaded polymeric-incorporated liposomes or Cy5.5-conjugated Dox-loaded ECM-targeting liposomes through the tail vein. Saline or biotin solution was administrated into mice every 6 h. After treatment for 24 h, the tumor-bearing mice were sacrificed. Tumor tissues and other organs of interest (i.e., heart, lung, liver, spleen, kidney, pancreas and brain) were excised and observed by IVIS 50 image system. 2.8. In vivo antitumor activity The HCT116 cells were transplanted subcutaneously into the rear of 4-week female Balb-c/nude mice (1  106 cells/0.1 mL). At 4e6 weeks posttransplantation, HCT116 tumor-bearing mice (tumor volume: approximately 500 mm3) were used in the antitumor activity study. Mice were treated i.v. via the tail vein on days 0 and 7. Animals were then injected with a drug PBS solution (free Dox, Dox-loaded polymer-incorporated liposomes, and Dox-loaded ECM-targeting liposomes at a dose of 10 mg/kg). Tumor size was measured three weekly using a Vernier’s caliper. Tumor volume was calculated as V ¼ (ab2)/2, where a and b indicate are the major and minor axes of the tumor, respectively. To evaluate animal wellness and clinical status, the body weight of mice was recorded. 2.9. Time dependent organ accumulation HCT116 cells (1 106 cells/0.1 mL) were subcutaneously transplanted to the rear of 4-week female Balb-c/nude mice. When the tumor volume grew and reached

3. Results and discussion 3.1. Preparation and characterization of liposomes In this study, biotin2-PEG crosslinker and two compositions of functional linear copolymers, mPEG-b-P(HPMA-co-His)-Chol ([mPEG]: [HPMA]: [His] : Chol ¼ 1:44:14:1 and 1:47:9:1, abbreviated as Poly_HP44His14 and Poly_HP47His9, respectively), were synthesized as reported elsewhere [25]. Table 1 summarizes the composition and characterization of mPEG-b-P(HPMA-co-His)Chol copolymers. For the preparation of liposomes, the polymerincorporated liposomes composed of DPPC and mPEG-b-P(HPMAco-His)-Chol were prepared in a stepwise process of rehydration, sonication, and filtration. The ECM-targeting liposomes were prepared by directly mixing biotin2-PEG and a polymer incorporating liposome solution. For the characterization of liposomes, each liposome structure was shown in Table 2. The sizes of DPPC liposomes, polymer-incorporated liposomes, and ECM-targeting liposomes were all around 90e110 nm by dynamic light scattering (DLS). The polydispersity index (PDI) of DPPC liposomes was 0.182, larger than those of polymer-incorporated liposomes (ca. 0.1) and ECM-targeting liposomes (ca. 0.1) because the low phase transition temperature value of DPPC molecules (fluid state) formed a related instable liposome structure. 3.2. pH-responsive and drug release behaviors of liposomes As mentioned, slow drug release is a major problem of liposomes in clinic application. Reports have been shown that the commercial product Doxil may produce new side effects, including palmar-plantar erythrodysesthesia syndrome and mucositis, which might be consequences of instability and slow drug release [2,27].

Table 1 Composition and characterization of mPEG-P(HPMA-co-His)-Chol copolymers. In copolymer (molar ratio)a

Code

In feed (molar ratio) mPEG

HPMA

His

Chol

mPEG

HPMA

His

Chol

Poly_HP47His9 Poly_HP44His14

1 1

40 40

8 16

1 1

1 1

47 44

9 14

1 1

a b

Composition and molecular weight of copolymers were determined by 1H NMR. PDI (polydispersity index) was determined by GPC.

MWa

PDIb

12,500 12,300

1.05 1.06

Y.-T. Chiang, C.-L. Lo / Biomaterials 35 (2014) 5414e5424 Table 2 Characterizations of DPPC liposomes, polymer-incorporated liposomes, and ECMtargeting liposomes. Means  SD (n ¼ 3). Composition (wt %) DPPC

Biotin2-PEG

0 33 33 25 25

100 67 67 50 50

0 0 0 25 25

96.7 108.4 103.9 90.6 96.2

    

3.7 0.6 1.5 1.9 2.1

0.18 0.13 0.08 0.14 0.11

    

0.05 0.02 0.02 0.07 0.03

a His9 polymer-incorporated liposomes and His9 ECM-targeting liposomes were prepared from Poly_HP47-His9 copolymers. b His14 polymer-incorporated liposomes andHis14 ECM-targeting liposomes were prepared from Poly_HP44-His14 copolymers. c Particle sizes and PDIs were determined by DLS.

Because the imidazole ring of histidine in mPEG-b-P(HPMA-coHis)-Chol could be mostly protonated below pH 6.0 such as intracellular endosomes and secondary lysosomes, the positive charges of mPEG-b-P(HPMA-co-His)-Chol were expected to generate

A

A 2.4

0 h, pH 7.4 6 h, pH 7.4 24h, pH 7.4

1.6

Change Ratio of Size

1.2 0.8 0.4

1.6 1.2 0.8

** TL 14 is H

0 h, pH 5.0 6 h, pH 5.0 24 h, pH 5.0

1.0

0.7

M

is

9E is H

1.2 0 h, pH 7.4 6 h, pH 7.4 24 h, pH 7.4

EC

14

M

PL

TL

L

H

D

B

1.0 0.8

C

PP

C

TL EC

H

H

is

is

14

H

9E

is

C

M

PL 14

TL M

9P is H

D

PP

C

L

0.0

0.9

0.8 PDI

0.6 PDI

2.0

0.4

0.0

B

0h, pH 5.0 6h, pH 5.0 24h, pH 5.0

9P

Change Ratio of Size

2.0

is

DPPC His9PLa His14 PLb His9ECMTLa His14ECMTLb

Copolymer

PDIc

Particle size (nm)c

repulsive force to destroy DPPC lipid bilayers of ECM-targeting liposomes to release loaded Doxorubicin-HCl (Dox). Therefore, the aim of this study was to evaluate the pH-sensitivity of our liposome system for further application in intracellular drug delivery. Firstly, DPPC liposome, polymer-incorporated liposomes, and ECMtargeting liposomes in pH 7.4 phosphate buffer salines (PBSs) were shaken under 200 rpm at 37  C to understand the stability of liposomes in neutral surroundings. The size and PDI of the DPPC liposome increased more than those of polymer-incorporated liposomes and ECM-targeting liposomes, demonstrating that the DPPC liposome was unstable physiologically (Fig. 2A and B). For pHresponsive analysis, liposomes were suspended in pH 5.0 PBS solutions and then shaken under 200 rpm at 37  C. The ECM-targeting liposomes with high histidine content responded rapidly to pH in both size and PDI observation, unlike DPPC liposomes, polymerincorporated liposomes and low histidine ECM-targeting liposomes (Fig. 3A and B). Experimental results indicate that high histidine content in liposomes has high pH sensitivity because of the large repulsive force from protonated histidine.

H

Code

5417

0.5 0.4

0.6 0.4

0.3 0.2

0.2 0.1

M EC

is H

is

14

H

C 9E is

TL

PL 14

TL M

9P is H

PP

H

14 H

is

D

M EC

is H

C 9E is H

TL

PL 14

TL M

9P is H

D

PP

C

L

C

L

0.0

0.0

Fig. 2. (A) The changes of particle sizes and (B) particle size distribution for DPPC liposomes, polymer-incorporated liposomes, and ECM-targeting liposomes under pH 7.4 and 37  C surroundings for evaluating liposome stabilities. Means  SD (n ¼ 3).

Fig. 3. (A) The changes of particle sizes and (B) particle size distribution for these particles under pH 5.0 and 37  C surroundings for evaluating pH responsibility. (An asterisk means liposomes aggregation without determined by DLS.) Means  SD (n ¼ 3).

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A

pH 5.0, 4 h

ECM-targeting liposomes

Polymerincorporated liposomes

pH 7.4, 4 h

Cummulative release (wt%)

120

PILs, pH 7.4 ECMLs, pH 7.4 CLs, pH 7.4

100

*** 80 *** *** 60

*** *

A

PILs, pH 5.0 ECMLs, pH 5.0 CLs, pH 5.0

** **

*** *

* ** **

40

120 100

Cell viability(%)

B

method [28]. The drug contents for polymer-incorporated liposomes and ECM-targeting liposomes were 16.9  3.69 wt % and 18.2  1.35 wt %, respectively, whereas the drug encapsulation efficiencies were approximately 84.7  12.06% and 81.3  15.14%. The Dox release behavior of liposomes in pH 5.0 and pH 7.4 PBS solution (around 0.1 mg/ml) at 37  C was then determined by highperformance liquid chromatography (HPLC), with the fluorescence (FL) detector measurement at 480 nm for excitation and 560 nm for emission [29], in a time-course procedure. Dox was isolated by dialysis bag (MWCO 10000; Millipore, country). Commercial stealth liposomes were used for comparison. Both polymerincorporated liposomes and ECM-targeting liposomes had a small initial burst around 20 wt% of Dox release, and then remained stable at pH 7.4 (Fig. 4B). Additionally, they also had drug release behaviors based on pH changes because of histidine protonation. At a low pH, the drug release rate for ECM-targeting liposomes was faster than that for polymer-incorporated liposomes. Otherwise, commercial stealth liposomes had a much lower initial burst than polymer-incorporated liposomes and ECM-targeting liposomes though; the pH-responded drug release of commercial stealth liposomes was not significant. Notably, ECM-targeting liposomes exhibited rapid drug release rates for around 75 wt% of Dox in the

20

80 60 40 free Dox PILs ECMLs

20

0 0

5

10 15 Time (h)

20

25

Fig. 4. (A) TEM images of polymer-incorporated liposomes and ECM-targeting liposomes under different pH values (pH 7.4 and 5.0) and 37  C surroundings. (B) Dox release behavior of commercial stealth liposomes (CLs), Dox-loaded polymer-incorporated liposomes (PILs) and Dox-loaded ECM-targeting liposomes (ECMLs) in pH 7.4 and 5.0 PBS solutions at 37  C. Asterisk indicate statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.005). Means  SD (n ¼ 3).

0 0.1

1

10

100

Concentration(µg/mL)

B

120

The TEM images were utilized as further evidence for the pHresponsive study. Fig. 4A shows TEM images of high histidine contents of polymer-incorporated liposomes and ECM-targeting liposomes after treatment with neutral and acid buffers. The TEM micrographs clearly demonstrate that the structure of ECMtargeting liposomes collapsed at pH 5.0 after treatment for 4 h. Nevertheless, the structure of polymer-incorporated liposomes did not change under the same treatment. These analytical results also provide evidence that crosslinking dissociation destroyed most of the liposome structure. Based on stability and pH-sensitivity results, high histidine contents of polymer-incorporated liposomes and ECM-targeting liposomes were chosen for the following experiments due to their rapid responses to stimuli. To investigate drug-release behavior, polymer-incorporated liposomes and ECM-targeting liposomes were encapsulated doxorubicin hydrochloride (Dox) by the ammonium sulfate gradient

Cell viability(%)

100 80 60 40 free Dox PILs ECMLs

20 0 0.1

1

10

100

Concentration(µg/mL) Fig. 5. The cytotoxicities of free Dox, Dox-loaded polymer-incorporated liposomes (PILs), and Dox-loaded ECM-targeting liposomes (ECMLs) after incubation with HCT116 cells for (A) 24 h and (B) 48 h.

Y.-T. Chiang, C.-L. Lo / Biomaterials 35 (2014) 5414e5424

A

O

80 Viability (%)

initial 2 h at pH 5.0. The reason is likely due to the rapid pH response and structural deformation of ECM-targeting liposomes. Otherwise, ECM-targeting liposomes could prevent protein adsorption effectively and maintain their structure under neutral environment. Dox leakage from ECM-targeting liposomes in 10% fetal bovine serum PBS solution was lower than that from polymerincorporated liposomes (see Supporting Information). Twenty-five wt % of Dox was released from ECM-targeting liposomes at either 24 or 72 h, while the Dox leakage from polymer-incorporated liposomes was increased with time. Compared to other pH-sensitive liposomes from literature [6e10,30e32], this system can rapidly release a drug under low pH and stabilize under protein-rich surroundings, while others cannot. The fast release of drug from liposome systems may increase the bioavailability and prevent drug resistance [4].

100 37 C, pH 7.4 O 37 C, pH 6.5 O 4 C, pH 6.5

* *

60 40 20 0

B

6

Fluorescence Intensity

Free Dox

5

PILs

5419

ECMLs 3.3. Tumor ECM-targeting induced in vitro cytotoxicity

PILs w/o free biotin PILs with free biotin ECMLs w/o free biotin ECMLs with free biotin

**

4 3 2 1 0 pH 7.4

pH 6.5

Fig. 6. (A) The cytotoxicities of free Dox, Dox-loaded polymer-incorporated liposomes (PILs), and Dox-loaded ECM-targeting liposomes (ECMLs) after incubation with HCT116 cells at pH 6.5 to evaluate tumor ECM-targeting induced cytotoxicity. Dox dosage was 50 mg/mL. Means  SD (n ¼ 6). (B) The binding of Dox-loaded polymer-incorporated liposomes (PILs) and Dox-loaded ECM-targeting liposomes (ECMLs) on cell membrane after incubation with HCT116 cells for 24 h in the presence of 75 mM biotin molecules for competition test. Dox dosage was also 50 mg/mL. Means  SD (n ¼ 6). Asterisk indicate statistically significant differences (*p < 0.05; **p < 0.01).

1 h Incubation Dox

Superimpose

Cy5.5

3 h Incubation Dox

Superimpose

ECMtargeting liposomes

Polymerincorporated liposomes

Free Dox

Cy5.5

The cytotoxic effects of Dox-loaded polymer-incorporated liposomes and Dox-loaded ECM-targeting liposomes were compared with Dox alone using the MTT assay. HCT116 colon rectal cancer cells were treated with free Dox, Dox-loaded polymer-incorporated liposomes, and Dox-loaded ECM-targeting liposomes in pH 7.4 McCoy’s 5a medium. Cell viabilities of all materials (i.e., copolymers and crosslinkers) were around 100% under the same dosage (see Supporting Information). When incubated at pH 7.4 and 37  C (Fig. 5), the IC50 value of Dox-loaded ECM-targeting liposomes for HCT116 colon rectal cancer cells at 48 h was 19.3 mg/ml, which was similar to those of Dox-loaded polymer-incorporated liposomes and free Dox. To evaluate tumor ECM-targeting induced cytotoxicity, HCT116 cells were treated with free Dox, Dox-loaded polymer-incorporated liposomes, and Dox-loaded ECM-targeting liposomes in pH 7.4 and pH 6.5 McCoy’s 5a medium. When incubated at pH 7.4 and 37  C (Fig. 6A), the cell viability of Dox-loaded ECM-targeting liposomes and Dox-loaded polymer-incorporated liposomes at a fixed concentration of Dox did not differ. Conversely, Dox-loaded ECM-targeting liposomes had higher cytotoxicity than Doxloaded polymer-incorporated liposomes at pH 6.5 and 37  C, demonstrating that low pH surrounding can improve targeting and cytotoxicity. Additionally, HCT116 cells were incubated at pH

Fig. 7. Confocal images of HCT116 cells incubated with Dox, Dox-loaded polymer-incorporated liposomes, and Dox-loaded ECM-targeting liposomes for 1 and 3 h. Green fluorescence represents FITC-labeled particles, and red fluorescence represents Dox or released Dox.

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Heart

Pancreas

Kidney

Lung

Spleen

Tumor

Liver

PILs

Brain

2.0

ECMLs + biotin

1.8 1.6 1.4 x108 1.2

ECMLs

1.0 0.8 p/sec/cm^2/sr

Fig. 8. pH-responsive capability study of HCT116 tumor xenografts in Balb-C/nude mice at 24 h post-injection of Cy5.5-labeled Dox-loaded polymer-incorporated liposomes (PILs), Cy5.5-labeled Dox-loaded ECM-targeting liposomes (ECMLs), and Cy5.5-labeled Dox-loaded ECM-targeting liposomes combined with excess of free biotin molecules (ECMLs þ biotin). The color revealed the relative fluorescence. All experiments were repeated twice with similar results.

A

10

Relative Tumor Volume

9 8

Control Free Dox PILs ECMLs

7 6 5 4 3 2

**

* ** * * * ** * ** *** **

1 0 0 2 4 6 8 10 12 14 16 18 20 22 24

Time (day)

B

1.5 1.4 1.3

Relative Weight

6.5 and 4  C with Dox-loaded polymer-incorporated liposomes and Dox-loaded ECM-targeting liposomes to allow for cell membraneebiotin interaction. After 2 h incubation, HCT116 cells were replaced with fresh pH 7.4 medium and warmed to 37  C for cellular internalization. Analytical results show that Dox-loaded ECM-targeting liposomes had higher cytotoxicity than Doxloaded polymer-incorporated liposomes. The competition test with excess biotin molecules in medium co-incubated with drug carriers at either pH 7.4 and 6.5 and a temperature of 37  C was conducted to make biotin compete with the biotin receptor on the surface of HCT116 cells. FITC dye with NHS ester was reacted with the amino group in histidine to prepare fluorescently labeled liposomes. After incubation for 1 h, unbound liposomes and free biotin were removed and treated with cells under the same procedures as cytotoxicity test followed by analysis with flow cytometry measurements to quantitative study of cell binding. The results indicate that the interaction between the cell membrane and drug carriers was inhibited by excess biotin (Fig. 6B and Supporting Information). Confocal laser scanning microscopy (CLSM) was used to observe the fluorescence images of Dox-loaded polymer-incorporated liposomes and Dox-loaded ECM-targeting liposomes and released Dox behavior after HCT116 cancer cells uptake, to evidence tumor ECM-induced targeting ability and intracellular drug delivery of ECM-targeting liposomes. FITC dye with NHS ester was reacted with the amino group in histidine to prepare fluorescently labeled liposomes. HCT116 cancer cells were coincubated with samples for 0.5 h at pH 6.5 and 4  C, washed twice by pre-cooling PBS to remove medium’s samples, and then fresh pH 7.4 medium was added for 1 h and 3 h incubation at 37  C. Fig. 7 shows that both liposome systems were internalized into cells and were located in the cytoplasm after 1 h and Dox were released after 3 h, indicating that both liposomes systems were taken up from extracellular medium into cancer cells, and that the pH values of the endosomes were changed to release Dox. Although low temperature (4  C) could not inhibit the internalization process in HCT116 cancer cells, Dox-loaded ECMtargeting liposomes exhibited a larger accumulation than Doxloaded polymer-incorporated liposomes at pH 6.5, strongly indicating that ECM-targeting liposomes can improve the cancer therapeutic efficiency.

Control Free Dox PILs ECMLs liposomes

1.2 1.1 1.0 0.9 0.8 0.7 0.6 0 2 4 6 8 10 12 14 16 18 20 22 24

Time(day) Fig. 9. (A) In vivo tumor growth inhibition and (B) body weight changes in Balb-C/ nude mice bearing HCT116 tumors after intravenous administration of free Dox, Dox-loaded polymer-incorporated liposomes (PILs), and Dox-loaded ECM-targeting liposomes (ECMLs) at a 10 mg/kg Dox equivalent dose. The administrations were carried out twice with a seven day interval. Asterisk indicate statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.005). Means  SD (n ¼ 6).

Y.-T. Chiang, C.-L. Lo / Biomaterials 35 (2014) 5414e5424

Day 3

Day 7

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Spleen

Free Dox Dox-loaded polymerincorporated liposomes

Tumor

Day 1

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Fig. 10. Confocal images of the tumors, livers and spleens at day 1, 3 and 7 post-injection with free Dox, polymer-incorporated liposomes and ECM-targeting liposomes to observe the time-dependent organ distribution. Red fluorescence indicates Cy5.5-labeled particles. Green and blue fluorescence indicate Dox (free Dox or untrapped Dox) and DAPI, respectively. All experiments were repeated three times with similar results.

3.4. pH-responsive capability and anticancer efficiency of liposomes in animal Additionally, Cy5.5 labeled Dox-loaded polymer-incorporated liposomes and Cy5.5 labeled Dox-loaded ECM-targeting liposomes (Cy5.5 dye with NHS ester was reacted with the amino group of histidine in liposomes) were administrated into HCT116 tumorbearing mice by tail intravenous injection to directly evaluate the pH-responsive capability in animal. Especially, Cy5.5 labeled Doxloaded ECM-targeting liposomes with excess free biotin molecules were used for comparison. Free biotin molecules were injected into mice at 6-h intervals for competition study. All mice were sacrificed after 24 h post-injection and their major organs were harvested to observe the fluorescent intensities of liposomes by IVIS system. The results show that large amount of Dox-loaded ECM-targeting liposomes were accumulated in tumor (in Fig. 8). Additionally, the fluorescent intensities of Dox-loaded ECM-targeting liposomes combined with free biotin molecules injection in

organs were similar to those of Dox-loaded polymer-incorporated liposomes, indicating that free biotin molecules abolished partial ECM-targeting liposomes-cell binding and demonstrated the enhancement of the accumulation of liposomes in tumors by biotin2-PEG crosslinkers. Otherwise, the zeta-potential was used to further evidence that the liposomes-cell binding was contributed from biotin2-PEG crosslinkers. The zeta-potential values for Doxloaded polymer-incorporated liposomes and Dox-loaded ECMtargeting liposomes were similar and they were increased with pH decreasing; 6.7 mV for polymer-incorporated liposomes and 8.2 mV for ECM-targeting liposomes at pH 7.4, 4.5 mV for polymer-incorporated liposomes and 4.2 mV for ECM-targeting liposomes at pH 6.5, and 7.8 mV for polymer-incorporated liposomes and 6.8 mV for ECM-targeting liposomes at pH 6.0 (in Supporting Information). The zeta-potential results exclude the influence of charge differences on liposomes from histidine protonation at low pH. Our previous study has indicated that biotin2PEG crosslinkers could be exposed from ECM-targeting liposomes

Y.-T. Chiang, C.-L. Lo / Biomaterials 35 (2014) 5414e5424

Liver Spleen

Dox-loaded ECM-targeting liposomes

Tumor

Spleen

Liver

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Fig. 10. (continued).

to improve tumor accumulation under the acidic tumor ECM surroundings [25]. These results here provide more direct evidence for the pH-responsive capability on tumor targeting and tumor accumulation. To evaluate the therapeutic efficiency of liposomes, nude mice bearing HCT116 colon cancer cells were treated with free Dox, Doxloaded polymer-incorporated liposomes, and Dox-loaded ECMtargeting liposomes. Mice were injected with total 20 mg/kg doxorubicin at adjusted concentrations on days 0 and 7. Fig. 9 shows tumor inhibition and cytotoxicity of mice treated with drugs and drug carriers after 27 days. The tumors in the control group grew with time. Compared to control mice, free Dox, Doxloaded polymer-incorporated liposomes, and Dox-loaded ECM-

targeting liposomes inhibited tumor growth. Particularly, Doxloaded ECM-targeting liposomes had the best cancer therapy efficiency. Although free Dox and Dox-loaded polymer-incorporated liposomes also retarded the tumor growth rate, free Dox and Doxloaded polymer-incorporated liposomes resulted in obvious weight loss during the administration period. Notably, the body weight of mice injected with Dox-loaded ECM-targeting liposomes did not differ significantly. Thus, the ECM-targeting liposome system improved targeting, had an excellent therapeutic effect, and reduced the incidence of side effects. To further evaluate the relationships between ECM-targeting effect and anticancer efficiency, mice were treated with one dose of liposomes and their specific tissue sections were observed by

Y.-T. Chiang, C.-L. Lo / Biomaterials 35 (2014) 5414e5424

Blood Liver Index (U/L)

A

700 600

free Dox-GOT PILs-GOT ECMLs-GOT

free Dox-GPT PILs-GPT ECMLs-GPT

500 400 300 200 100 0

Blood Kidney Index (mg/dL)

B

50 40

1 day

3 day

7 day

free Dox-CRE PILs-CRE ECMLs-CRE

free Dox-BUN PILs-BUN ECMLs-BUN

30 20 10 0

1 day

3 day

7 day

Fig. 11. Changes in biochemical markers of liver and kidney function following 1, 3, and 7 days treatment course. (A) Hepatic damage (GOT and GPT values) and renal toxicity (BUN and CRE) were carried out with the aim of evaluating the potential instant toxicity of free Dox, Dox-loaded polymer-incorporated liposomes (PILs), and Dox-loaded ECM-targeting liposomes (ECMLs). Mean  SD (n ¼ 3).

CLSM. Cy5.5-NHS ester was used to label the Dox-loaded liposomes. The nude mice bearing HCT116 cancer cells were administered with free Dox, Cy5.5-labeled Dox-loaded polymerincorporated liposomes and Cy5.5-labeled Dox-loaded ECM-targeting liposomes. Fig. 10 shows the liposome accumulation and drug releasing behaviors for liver, spleen, and tumor at days 1, 3 and 7 post-injection. At 1 day post-injection, ECM-targeting liposomes accumulated largely in tumor tissue, while polymer-incorporated liposomes were less. Conversely, the liver and spleen exhibited high intensity of polymer-incorporated liposomes and untrapped (released) Dox. Two possible reasons can be interpreted. The first reasonable explanation is that the adsorbed proteins on the surface of polymer-incorporated liposomes induced liposome deformation and drug leakage and caused large accumulation of untrapped drug and liposomes in the liver and spleen [25,33]. Otherwise, ECMtargeting liposomes could prevent protein adsorption, leading to the preferential tumor accumulation. The second reason is that the biotin moieties could enhance the tumor targeting ability and cell uptake for the ECM-targeting liposomes, thereby preventing extravasation of liposomes into the blood circulation. Therefore, the accumulation of ECM-targeting liposomes in tumor was increased with time. Otherwise, the accumulated behavior for polymerincorporated liposomes in tumor was adverse. The reason is perhaps due to liposome deformation in blood circulation and extravasation of liposomes from tumor. Notably, free Dox was found in tumor ECM at day 1 post-injection and in the liver at day 3 post-injection, indicating that free Dox could not be remained in tumor and then be eliminated by hepatobiliary excretion. The time

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dependent organ accumulation of liposomes corresponds to the results of in vivo tumor inhibition. Free Dox and polymerincorporated liposomes showed decreasing accumulation in tumor with time, resulting in a related poor tumor inhibition. For the precise insight of the hepatic or renal toxicity from the Dox (frre Dox and untrapped Dox) and liposomes, the clinical biochemistry indexes were measured. GOT and GPT were used for evaluating the hepatic function; BUN and CRE were for the renal function. The normal values of GOT, GPT, BUN and CRE were 28e 132 U/L, 59e247 U/L, 18e29 mg/dL, and 0.2e0.8 mg/dL [34]. At day 1 post-injection, the GPT values for all samples were lifted (in Fig. 11A), especially in the groups of free Dox and Dox-loaded polymer-incorporated liposomes. Notably, GOT value for polymerincorporated liposomes was also abnormal. The GOT and GPT values for polymer-incorporated liposomes were correlated with the results from frozen tissue sections that large untrapped Dox and liposomes accumulated in liver due to protein adsorption and drug leakage in trafficking. Because polymer-incorporated liposomes still accumulated gradually in the liver and spleen by time, the remaining polymer-incorporated liposomes caused liver damage continuously. The hepatic damage also happened in the groups of free Dox. Therefore, the body weights of mice for free Dox and polymer-incorporated liposomes treatments were decreased in the initial 10 days. Conversely, ECM-targeting liposomes were prone to deposit in tumor, and the hepatic toxicity which was the adverse effect in Dox chemotherapy did not occur. Besides, the renal toxicity was not found in all groups because Dox was eliminated by hepatobiliary excretion (in Fig. 11B). Judging from the results of the biodistribution and the biochemistry indexes, ECM-targeting liposomes could be considered to minimize the undesired side effects and improve the chance of success for anticancer therapy. 4. Conclusions In this study, high levels of histidine in ECM-targeting liposomes provide excellent pH response for rapid drug release in acidic environments. In vitro studies suggest that ECM-targeting liposomes have tumor-ECM switched on targeting ability with intracellular drug delivery ability. Additionally, in vivo experiments clearly showed that animals treated with ECM-targeting liposomes provided a significantly higher anti-tumor activity and lower toxicity. It appears that ECM-targeting liposomes have great potential for use in cancer therapy. Acknowledgments The authors would like to thank the National Science Council of the Republic of China (Taiwan) (NSC 100-2320-B-010-007-MY3, 101-2221-E-010-002-MY2, and 102-2627-E-010-001) and Yen Tjing Ling Medical Foundation (CI-103-9) for financially supplementary this work. TEM images were supported in part by the Electron Microscopy Facility in NYMU. Dr. Yi-Chun Chen is appreciated for kindly providing experimental techniques for CLSM observations. We would also like to acknowledge confocal laser scanning microscopy of Imaging Core Facility of Nanotechnology of the UST-YMU and College of Life Science and Instrumentation Center at NTU (Taiwan) for the excellent technical assistant. Additionally, we thank the Taiwan Mouse Clinic which is funded by the National Research Program for Biopharmaceuticals (NRPB) at the NSC of Taiwan for technical support in IVIS experiment. Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.03.046.

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References [1] Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 2005;4:145e60. [2] Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science 2004;303:1818e22. [3] Drummond DC, Meyer O, Hong K, Kirpotin DB, Papahadjopoulos D. Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev 1999;51:691e743. [4] Papahadjopoulos D, Allen TM, Gabizon A, Mayhew E, Matthay K, Huang SK, et al. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc Natl Acad Sci U S A 1991;88:11460e4. [5] Molineux G. Pegylation: engineering improved pharmaceuticals for enhanced therapy. Cancer Treat Rev 2002;28:13e6. [6] Yatvin MB, Kreutz W, Horwitz BA, Shinitzky M. pH-Sensitive liposomes: possible clinical implications. Science 1980;210:1253e4. [7] Lee ES, Gao Z, Bae YH. Recent progress in tumor pH targeting nanotechnology. J Control Release 2008;132:164e70. [8] Seki K, Tirrell DA. pH-Dependent complexation of poly(acrylic acid) derivatives with phospholipid vesicle membranes. Macromolecules 1984;17: 1692e8. [9] Sakaguchi N, Kojima C, Harada A, Kono K. Preparation of pH-sensitive poly(glycidol) derivatives with varying hydrophobicities: their ability to sensitize stable liposomes to pH. Bioconjug Chem 2008;19:1040e8. [10] Roux E, Lafleur M, Lataste E, Moreau P, Leroux JC. On the characterization of pHsensitive liposome/polymer complexes. Biomacromolecules 2003;4:240e8. [11] Adlakha-Hutcheon G, Bally MB, Shew CR, Madden TD. Controlled destabilization of a liposomal drug delivery system enhances mitoxantrone antitumor activity. Nat Biotechnol 1999;17:775e9. [12] Kirpotin D, Hong K, Mullah N, Papahadjopoulos D, Zalipsky S. Liposomes with detachable polymer coating: destabilization and fusion of dioleoylphosphatidylethanolamine vesicles triggered by cleavage of surfacegrafted poly(ethylene glycol). FEBS Lett 1996;388:115e8. [13] Lee SM, Chen H, Dettmer CM, O’Halloran TV, Nguyen ST. Polymer-caged liposomes: a pH-responsive delivery system with high stability. J Am Chem Soc 2007;129:15096e7. [14] Huang SK, Mayhew E, Gilani S, Lasic DD, Martin FJ, Papahadjopoulos D. Pharmacokinetics and therapeutics of stability of sterically stabilized liposomes in mice bearing C-26 colon carcinoma. Cancer Res 1992;52:6774e81. [15] Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated liposomal doxorubicin: review of animal and human studies. Clin Pharmacokinet 2003;42:419e36. [16] Daemen T, Hofstede G, Ten Kate MT, Bakker-Woudenberg IAJM, Scherphof GL. Liposomal doxorubicin-induced toxicity: depletion and impairment of phagocytic activity of liver macrophages. Int J Cancer 1995;61:716e21. [17] Daemen T, Regts J, Meesters M, Ten Kate MT, Bakker-Woudenberg IAJM, Scherphof GL. Toxicity of doxorubicin entrapped within long-circulating liposomes. J Control Release 1997;44:1e9. [18] Maruyama K, Ishida O, Takizawa T, Moribe K. Possibility of active targeting to tumor tissues with liposomes. Adv Drug Deliv Rev 1999;40:89e102.

[19] Torchilin VP. Affinity liposomes in vivo: factors influencing target accumulation. J Mol Recognit 1996;9:335e46. [20] Gabizon A, Horowitz AT, Goren D, Tzemach D, Shmeeda H, Zalipsky S. In vivo fate of folate-targeted polyethylene-glycol liposomes in tumor-bearing mice. Clin Cancer Res 2003;9:6551e9. [21] Russell-Jones G, McTavish K, McEwan J, Rice J, Nowotnik D. Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumors. J Inorg Biochem 2004;98:1625e33. [22] Lee ES, Na K, Bae YH. Super pH-sensitive multifunctional polymeric micelle. Nano Lett 2005;5:325e9. [23] Gabizon A, Shmeeda H, Horowitz AT, Zalipsky S. Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid-PEG conjugates. Adv Drug Deliv Rev 2004;56:1177e92. [24] Mastrobattista E, Koning GA, Storm G. Immunoliposomes for the targeted delivery of antitumor drugs. Adv Drug Deliv Rev 1999;40:103e27. [25] Chiang YT, Cheng YT, Lu CY, Yen YW, Yu LY, Yu KS, et al. Polymer-liposome complexes with a functional hydrogen-bond cross-linker for preventing protein adsorption and improving tumor accumulation. Chem Mater 2013;25: 4364e72. [26] Lin YH, Tsai SC, Lai CH, Lee CH, He ZS, Tseng GC. Genipin-cross-linked fucosechitosan/heparin nanoparticles for the eradication of Helicobacter pylori. Biomaterials 2013;34:4466e79. [27] Gordon KB, Tajuddin A, Guitart J, Kuzel TM, Eramo LR, VonRoenn J. Hand-foot syndrome associated with liposome-encapsulated doxorubicin therapy. Cancer 1995;75:2169e73. [28] Haran G, Cohen R, Bar LK, Barenholz Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta 1993;1151:201e15. [29] Fraier D, Frigerio E, Pianezzola E, Strolin Benedetti M, Cassidy J, Vasey P. A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl) methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection. J Pharm Biomed Anal 1995;13:625e33. [30] Kono K, Zenitani K, Takagishi T. Novel pH-sensitive liposomes: liposomes bearing a poly(ethylene glycol) derivative with carboxyl groups. Biochim Biophys Acta 1994;1193:1e9. [31] Zignani M, Drummond DC, Meyer O, Hong K, Leroux JC. In vitro characterization of a novel polymeric-based pH-sensitive liposome system. Biochim Biophys Acta 2000;1463:383e94. [32] Fujiwara M, Grubbs RH, Baldeschwieler JD. Characterization of pH-dependent poly(acrylic acid) complexation with phospholipid vesicles. J Colloid Interface Sci 1997;185:210e6. [33] Semple SC, Chonn A, Cullis PR. Interactions of liposomes and lipid-based carrier systems with blood proteins: relation to clearance behavior in vivo. Adv Drug Deliv Rev 1998;32:3e7. [34] Lu PL, Chen YC, Ou TW, Chen HH, Tsai HC, Wen CJ, et al. Multifunctional hollow nanoparticles based on graft-diblock copolymers for doxorubicin delivery. Biomaterials 2011;32:2213e21.