Calcium Phosphate-Reinforced Reduction-Sensitive Hyaluronic Acid

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Calcium phosphate-reinforced reduction-sensitive hyaluronic acid micelles for delivering paclitaxel in cancer therapy Bing Deng, Mengxin Xia, Jin Qian, Rui Li, Lujia Li, Jianliang Shen, Guowen Li, and Yan Xie Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 10, 2017

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Molecular Pharmaceutics

1

Title: Calcium phosphate-reinforced reduction-sensitive hyaluronic acid micelles for

2

delivering paclitaxel in cancer therapy

3

Bing Denga,1, Mengxin Xiaa,1, Jin Qiana, Rui Lia, Lujia Lia,b, Jianliang Shenc, Guowen Lib, Yan

4

Xiea,*

5

a

6

Medicine, Shanghai 201203, China

7

b

8

Traditional Chinese Medicine, Shanghai 200082, China

9

c

Department of Nanomedicine, Houston Methodist Research Institute, Houston 77030, USA

11

1

These authors contributed equally to this work.

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* Corresponding author:

13

Yan Xie, Ph.D.

14

Professor

15

Research Center for Health and Nutrition

16

Shanghai University of Traditional Chinese Medicine

17

1200 Cailun Road

18

Shanghai, China 201203

19

E-mail: [email protected]; [email protected]

20

Phone: +86(21)51322440

21

Fax: +86(21)51322407

Research Center for Health and Nutrition, Shanghai University of Traditional Chinese

Pharmacy Department, Shanghai TCM-integrated Hospital, Shanghai University of

10

22

1

ACS Paragon Plus Environment


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ABSTRACT

2

To address the dilemma of in vivo stability and anti-tumor effects of micellar drugs, novel

3

organic and inorganic hybridized nanoparticle, that is, hyaluronic acid mineralized micelles,

4

are designed to deliver paclitaxel (PTX) efficiently. The resulting micelles exhibit excellent

5

drug loading (30.6%) and entrapment efficiency (87.8%) for PTX with a small size (134.8

6

nm). Notably, the dual-sensitive release of PTX-loaded mineralized micelles is obtained in the

7

condition of 40 mM GSH and pH 5.0, while release is slow in the physiological environment.

8

With favorable cell uptake, mineralized micelles show decent tumor accumulation, which

9

corresponds to their significant targeting capacity from the observed real-time images.

10

Compared with Taxol, PTX-loaded mineralized micelles show a lower half-maximal

11

inhibitory concentration (IC50) value (0.025 µg/mL), higher cell apoptosis rate (23.5%) in

12

MDA-MB-231 cells, lower systemic toxicity to nude mice, and more potent in vivo tumor

13

inhibition (1.57 times higher than Taxol (p < 0.05)).

14

Keywords:

15

pH and reduction sensitive; mineralized micelles; PTX; MDA-MB-231 cell; anti-tumor

16

efficacy

17

2


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1.

Introduction

2

Self-assembled micelles composed of amphiphilic polymers have emerged as promising

3

anti-cancer drug carriers because they can encapsulate large amounts of hydrophobic

4

anti-cancer agents, thereby enhancing drug solubility, prolonging biological half-lives, and

5

targeting the cancer site via the enhanced permeability and retention effect. 1-3 However, it is

6

worth noticing that the therapeutic effects of drugs delivered by traditional micelles are

7

generally limited because of their inefficient release at the tumor site. 4-6 Furthermore, poor

8

stability in circulation which would lead to the leakage of drug is another severe problem

9

remaining to be solved for the existing micellar drug formulations. 7

10

In this regard, efforts have been devoted to developing polymeric micelles that are stable in

11

circulation in the body but release drugs efficiently in cancer cells. Of the various approaches,

12

reinforcing the micelle shells through controlled deposition of inorganic components, such as

13

calcium phosphate (CaP), through ionic interactions has received increasing attention in

14

recent years.

15

biocompatibility and displays pH-dependent solubility. It is sparsely soluble in physiological

16

fluids (pH 7.4) but dissociates into non-toxic ionic species in mildly acidic environments, i.e.,

17

the intra-cellular compartments (endosomes or lysosomes) or the extra-cellular matrix of

18

tumor tissues. Because of these properties, CaP mineralized micelles have been investigated

19

for pH-sensitive drug delivery.

20

micelles were usually prepared by using the non-sensitive micelles as the soft template. These

21

micelles are only responsive to the mildly acidic environment in the endosome and/or

22

lysosome and are not able to achieve more precisely controlled drug release to achieve the

23

therapeutic efficacy of drugs in vivo.

8-13

CaP, the main mineral component of bone and teeth, exhibits high

14, 15

However, the previously reported shell-mineralized

24

Similar to shell-mineralized micelles, reduction-sensitive micelles can also be used for

25

intra-cellular drug delivery and trigger drug release in cytosol and cell nucleus, where many

26

anti-cancer drugs are effectively functional. 16 As reported, glutathione (GSH) at a higher

27

concentration of approximately 2-10 mM is determined in some specific organelles such as

28

the cytosol, mitochondria, and cell nucleus, compared to the extremely low GSH level of

29

approximately 2-20 µM in the physiological environment. 17, 18 The significant difference in

30

GSH level is the premise of designing reduction-sensitive micelles and also makes it possible 3



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1

to deliver drugs to cancer cells. 19, 20 In order to optimize the drug release characteristics at the

2

tumor site, various types of polymeric drug delivery systems, such as pH and reduction

3

dual-sensitive nanocarriers, 21 that respond to dual stimuli and can achieve more intricate and

4

precise drug release have been reported. However, as far as we know, there are no reports on

5

the preparation of pH and reduction dual-sensitive nanocarriers based on organic and

6

inorganic mineralized micelles. Thus, the present study is aimed at developing a pH and

7

reduction dual-sensitive intra-cellular drug delivery system based on reduction-sensitive

8

micelles and CaP, which is expected to further fine-tune the drug release in cancer cells and

9

enhance their therapeutic efficiency.

10

Hyaluronic acid (HA), a naturally formed linear polysaccharide, has been widely applied as

11

the component of drug delivery vehicles for tumor-targeted therapy, since HA binding

12

receptors like CD44 and RHAMM are reported to be over-expressed in many tumor cells. 22-24

13

Furthermore, HA has also been investigated as a soft material to prepare pH-sensitive

14

shell-mineralized nanoparticles due to the ability of carboxylate groups on HA to pre-organize

15

Ca2+ ions and initiate the nucleation of CaP. 13, 25 Furthermore, unlike other template materials,

16

HA is biodegradable, free of immunogenicity or toxicity, and can be easily chemically

17

modified. Therefore, HA has been used in the delivery systems of anti-cancer drugs in many

18

studies. 26-28

19

In this paper, a novel pH and reduction dual-sensitive hyaluronic acid mineralized micelle

20

was designed for efficient intra-cellular drug delivery. Paclitaxel (PTX) was selected as a

21

model hydrophobic drug due to its well-known anti-cancer activity against tumors such as

22

ovarian carcinoma, breast cancer, and acute leukemia. 29 At first, the amphiphilic conjugation

23

based on HA that can form self-assembled reduction-sensitive micelles was synthesized as the

24

carrier of PTX. Thereafter, the mineralized shells of reduction-sensitive micelles were

25

prepared by sequentially adding calcium nitrate and ammonium phosphate to obtain the pH

26

and reduction dual-sensitive hyaluronic acid mineralized micelles (Scheme 1). Finally, the

27

intra-cellular uptake and cytotoxicity of PTX-loaded mineralized micelles were evaluated in

28

the human breast cancer cell line MDA-MB-231, and a xenograft MDA-MB-231

29

tumor-bearing nude mouse model was used to investigate their in vivo biodistribution,

30

anti-tumor efficacy, and systemic toxicity. This research will provide new ideas about 4


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1

addressing the dilemma between stability in the extra-cellular environment and intra-cellular

2

drug release of ordinary micellar drugs.

3

2.

4

2.1. Materials

Experimental section

5

HA-SS-DOCA was synthesized as described in the supplementary information. GSH was

6

purchased from Aladdin Reagent Inc. (Shanghai, China). PTX was purchased from Natural

7

Field Bio-technique Co., Ltd. (Xi’an, China). Lyso-Tracker Red, Arsenazo III, and Coumarin

8

6

9

4’,6-Diamidino-2-phenylindole (DAPI) was purchased from Dojindo Molecular Technologies,

10

Inc. (Kumamoto, Japan). The near-infrared dye DiR was obtained from Beijing Fanbo

11

Science and Technology Co., Ltd. (Beijing, China). Dulbecco’s Modified Eagle’s Medium

12

(DMEM) and Minimum Essential Medium (MEM) were purchased from Thermo-Fisher

13

Biochemical Product (Waltham, MA, USA). Fetal bovine serum (FBS) and HEPES-buffered

14

saline

15

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

16

(MTS) was obtained from Promega biological products Co., Ltd. (Shanghai, China). FITC

17

Annexin V Apoptosis Detection Kit Ⅰ was purchased from BD Bioscience (San Jose, CA,

18

USA). The human breast cancer (MDA-MB-231) cell line and human hepatoma (HepG2) cell

19

line were purchased from Institute of Biochemistry and Cell Biology, SIBS, CAS (Shanghai,

20

China). All other chemicals were of analytical grade.

21

2.2. Preparation and characterization of PTX-loaded mineralized micelles

22

2.2.1. Preparation of PTX-loaded mineralized micelles

(C6)

were

were

obtained

purchased

from

from

Gibco

Sigma-Aldrich

Laboratory

(Grand

(Shanghai,

Island,

NY,

China).

USA).

23

PTX-loaded mineralized micelles were prepared by a sonication dialysis method, followed

24

by mineralization. Briefly, 36 mg HA-SS-DOCA conjugate was dissolved in 10 mL distilled

25

deionized water. Then, 20 mg of PTX dissolved in about 6.7 mL ethanol was added slowly to

26

the conjugate solution and stirred for 30 minutes at room temperature. The final mixed

27

solution was sonicated for 30 minutes at 100 W in an ice bath and dialyzed against excess

28

distilled deionized water overnight, followed by centrifuged at 3500 rpm for 10 minutes and

29

filtered through a 0.45 µm pore-sized membrane. Then, the mineralization of PTX-loaded

30

micelles was accomplished using a previously reported procedure with some modifications. 12, 5



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1

13

2

solution and equilibrated for 30 minutes, and then an ammonium phosphate solution (0.1 M)

3

was slowly dropped in the reaction mixture under stirring. This process was repeated 12 times

4

to obtain PTX mineralized micelles. The molar ratio of calcium ions [Ca2+] to [COO-] of HA,

5

and calcium ions [Ca2+] to phosphate was adjusted to 1:2 and 1.6:1, respectively. Following the

6

last addition of ammonium phosphate solution, the solution was dialyzed to discard unreacted

7

ionic species, and the dialysate was filtered through a 0.45 µm pore-sized membrane to obtain

8

the PTX-loaded mineralized micelles.

9

2.2.2. Characterization of PTX-loaded mineralized micelles

Briefly, an aqueous calcium nitrate solution (0.1 M) was firstly added to the stirred micelles

10

The particle sizes and zeta potential were determined by dynamic light scattering (DLS)

11

with a Zetasizer Nano Series (Malvern Instruments Ltd., Worcestershire, UK) at 25°C and a

12

scattering angle of 173°. The morphology of micelles was obtained using a transmission

13

electron microscope (TEM) (JEM-2010F, Tokyo, Japan) at an accelerating voltage of 200 kV.

14

Non-mineralized micelles were observed with the negative staining using a droplet of 2%

15

(w/v) uranyl acetate solution, while mineralized micelles were observed without this process.

16

Energy-dispersive X-ray photoelectron spectroscopy (EDS) measurements were conducted by

17

using a JEM-2010F equipped with an EDX Genesis Series γ-TEM at 200 kV.

18

For measuring the drug loading (DL) and encapsulation efficiency (EE), a PTX-loaded

19

mineralized micelle solution was mixed with methanol and disrupted by ultrasound in a water

20

bath for 15 min. After filtration, PTX concentrations were determined by high performance

21

liquid chromatography (HPLC, Agilent 1260 system, CA, USA) equipped with a Kromasil

22

C18 column (5 µm particle size, 250 mm × 4.6 mm), with the column temperature at 30°C

23

and mobile phase consisting of acetonitrile and water (55:45, v/v) at a flow rate of 1.0

24

mL/min. The detection wavelength was of 227 nm, and the injected volume of each sample

25

was 20 µL. The EE and DL of PTX were calculated with the following equations:

26

EE (%) = (weight of PTX in micelles) / (weight of PTX fed initially) × 100

27

DL (%) = (weight of PTX in micelles) / (weight of PTX in micelles + weight of conjugates) ×

28

100

29

2.2.3. Calcium disintegration of mineralized micelles

30

(1)

(2)

The blank mineralized micelles were obtained by mineralizing blank micelles (see the 6


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1

supporting information in Section 3.1. for more details) according to the same mineralization

2

method as described in Section 2.2.1, which were further used to investigate the calcium

3

disintegration. In brief, the blank mineralized micelles were sealed in a dialysis tube (MWCO

4

= 3500), which was immersed in a 10 mL release medium of PBS buffer (pH 7.4), PBS buffer

5

(pH 6.5), or acetate buffer (pH 5.0) and shaken in a water bath at 120 rpm at 37°C. Then, 100

6

µL samples of media were withdrawn and replaced with fresh media at predetermined time

7

points. The calcium ion concentration in the sample was determined based on a standard

8

absorption curve by mixing 100 µL of medium with 2 mL of Arsenazo III solution (0.02 mM)

9

in HEPES-buffered saline at 656 nm. 12

10

2.2.4. Stability of PTX-loaded non- and mineralized micelles

11

The stability of PTX-loaded non-mineralized and mineralized micelles was estimated by

12

DLS analysis in serum containing solutions. Briefly, FBS was added to the non-mineralized

13

and mineralized micelles to attain its concentration of 10% and then incubated at 37°C with

14

120 rpm vibration. At predetermined time points, the mean diameter and the scattered light

15

intensity of the micelles was monitored and compared to the initial value.

16

2.2.5. In vitro release of PTX from mineralized micelles

17

The in vitro release profiles of PTX from mineralized micelles were studied using a dialysis

18

method in four different media: PBS buffer (pH 7.4) and acetate buffer (pH 5.0) with or

19

without 40 mM GSH. 1 mL PTX-loaded mineralized micelles was transferred into a dialysis

20

membrane bag (MWCO = 3500). The dialysis bag was placed in 60 mL of release media,

21

which was shaken at 120 rpm in the water bath at 37°C. At the predetermined time points, 1

22

mL of the sample was taken and replaced with an equal volume of fresh medium. The

23

concentration of PTX in each sample was determined by HPLC as described in Section 2.2.2.

24

2.2.6. Hemolysis test of mineralized HA-SS-DOCA micelles

25

The hemolysis test was carried out as described previously. 30 0.5 mL of blank micelle and

26

PTX-loaded mineralized micelle solution was added into 0.5 mL of 2% rat red blood cell

27

(RBC) suspension. The final concentrations of micelles ranged from 0.05 to 1 mg/mL. The

28

mixture was incubated at 37°C for 1 h and then centrifuged at 3000 rpm for 10 minutes to

29

remove non-lysed RBCs. The positive control (100% hemolysis) was obtained by mixing 0.5

30

mL of water with 0.5 mL of 2% RBC suspension, and the negative control (0% hemolysis) 7



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was obtained by mixing 0.5 mL of 5% glucose injection solution with 0.5 mL of 2% RBC

2

suspension.

3

2.3. In vitro cellular studies

4

2.3.1. Cell culture

5

MDA-MB-231 and HepG2 cells were cultured in DMEM and MEM, respectively, both

6

supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a

7

humidified atmosphere containing 5% CO2.

8

2.3.2. Cellular uptake of micelles

9

The cellular uptake was observed by confocal laser scanning microscopy (CLSM, Leica,

10

Wetzler, Germany). C6, a fluorescence probe, was encapsulated into mineralized micelles, in

11

the same manner as described in Section 2.2.1. A total of 5×104 MDA-MB-231 cells and

12

HepG2 cells were seeded in glass bottom culture dishes and cultured in DMEM and MEM,

13

respectively, with 10% FBS for 24 h. Free C6 or C6 loaded micelles (0.l µg/mL of C6) in

14

cultured medium were added and incubated for 4 h and 8 h, respectively. After incubation,

15

cells were washed with cold PBS three times and stained with Lyso-Tracker Red according to

16

the manufacturer’s instructions. Then, cells were fixed with 4% paraformaldehyde and stained

17

by DAPI (10 µM, 3 min). Next, all reagents were removed and cells were examined under an

18

inverted confocal laser scanning microscope.

19

The cellular uptake of micelles was also analyzed quantitatively using a FACS Calibur flow

20

cytometer (BD Bioscience, Boston, MA, USA). MDA-MB-231 cells were seeded at a density

21

of 5×104 cells per well in 6-well plates and incubated for 24 h. After incubating with free C6

22

and C6-loaded micelles for 8 h, the cells were washed three times with cold PBS (pH 7.4),

23

then harvested and subsequently resuspended in 0.5 mL PBS for flow cytometry.

24

2.3.3. In vitro cytotoxicity studies

25

In vitro cytotoxicity of PTX-loaded and PTX-free micelles was evaluated by MTS assay

26

with MDA-MB-231 cells. Taxol was prepared by dissolving 12 mg of PTX in 1.0 mL ethanol

27

and 1.0 mL Cremophor EL, followed by sonication for 30 min.

28

non-mineralized and mineralized micelles, Taxol, and free PTX were diluted with culture

29

medium to a series of samples (equal to PTX concentration of 0.001-10 µg/mL).

30

MDA-MB-231 cells were seeded in a 96-well plate at a density of 8×103 cells/well, and then 8


31

The PTX-loaded

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1

cells were treated with different PTX formulations. After incubation for 48 h, MTS was added

2

to the medium and further incubated for 1 h. The absorbance at 490 nm was recorded with a

3

Multiskan Spectrum microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA).

4

Cell viability (%) was calculated as (OD of test group / OD of control group) ×100 with saline

5

used as control.

6

2.3.4. Cell apoptosis detection

7

The apoptosis of PTX-loaded mineralized micelles was further quantified by flow

8

cytometry using saline and Taxol as the control. Briefly, 1×105 of MDA-MB-231 cells were

9

seeded in each well of 6-well plates and subsequently treated with different formulations for

10

48 h which contained 0.1 mg/mL of PTX. After treatment, the cells were harvested and

11

washed twice with cold PBS buffer, followed by centrifugation at 1,000 rpm for 3 min at 4°C.

12

The cells were resuspended and stained with 5 µL of Annexin V-FITC and 5 µL of propidium

13

iodide (PI) for 15 min in the dark, and then samples were immediately analyzed using a flow

14

cytometer.

15

2.4. In vivo animal experiments

16

2.4.1. Animals

17

Nude mice (20-25 g, female) were purchased by Super-B&K laboratory animal Corp. Ltd.

18

(Shanghai, China). Mice were pathogen free and allowed free access to food and water. All

19

animal experiments complied with the Animal Research: Reporting of In Vivo Experiments

20

(ARRIVE) guidelines and were carried out in accordance with animal care protocols

21

approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine

22

for the use of experimental animals.

23

2.4.2. In vivo imaging analysis

24

MDA-MB-231 cells (3×106) were inoculated subcutaneously in the second breast region of

25

nude mice, and the tumor size was measured by a digital caliper. For in vivo imaging analysis,

26

DiR was loaded into the micelles according to the protocol described in Section 2.2.1. When

27

the tumor volume was approximately 200 mm3, free DiR, DiR loaded micelles, and DiR

28

loaded mineralized micelles were injected into the tail vein of the tumor-bearing mice at a

29

dose of 150 µg/kg. Imaging was performed at 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, and 48 h after tail

30

intravenous injection by in vivo Imaging System (Cold spring Biotech. Corp., Taipei, Taiwan, 9



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1

China). Finally, the mice were sacrificed, and the tumors and certain organs including heart,

2

liver, spleen, lung, kidney, and brain, were excised for imaging and analysis by the Living

3

Image® 4.3.1 software (Caliper Life Sciences, Hopkinton, MA, USA).

4

2.4.3. In vivo anti-tumor efficacy

5

A subcutaneous tumor model was established as according to the description in Section

6

2.4.2. The treatment was started when the tumor volume reached in a range of 50-70 mm3.

7

Mice were randomly divided into the following 4 groups (n = 6): (1) 5% glucose solution (the

8

control group), (2) Taxol, (3) PTX-loaded micelles, and (4) PTX-loaded mineralized micelles.

9

All mice were injected via tail vein every 2 days for 13 days, and the dose of PTX was fixed

10

at 10 mg/kg. After treatment, tumor size and body weight of each mouse was measured at

11

regular intervals up to 21 days. Tumor volumes were calculated by the equation of a×b2/2,

12

where a and b represent the largest and smallest diameter of the tumor, respectively. To further

13

evaluate the anti-tumor efficacy of Taxol, PTX-loaded micelles, and PTX-loaded mineralized

14

micelles, the tumors were histologically evaluated with hematoxylin and eosin (H&E)

15

staining.

16

2.4.4. Toxicity evaluation of PTX formulations

17

To investigate the potential toxicity of PTX formulations on mice and relative weight loss

18

throughout the in vivo therapeutic experiment were monitored. Organs of heart, liver, spleen,

19

lung, and kidney, were collected after the 21-day observation. The tissues excised from nude

20

mice were also evaluated with H&E staining.

21

2.5. Statistical analysis Statistical analysis was performed using Student’s t-test. Differences were considered

22 23

statistically significant at p < 0.05 (*) and p < 0.01 (**).

24

3.

25

3.1. Preparation and characterization of PTX-loaded mineralized micelles

Results and discussion

26

The successful mineralization of PTX-loaded micelles was mainly confirmed by DLS,

27

TEM, and EDS. After mineralization, the hydrodynamic mean size of mineralized micelles

28

(134.8 nm) was slightly larger than that of non-mineralized micelles (129.0 nm); the

29

polydispersity index of micelles size was slightly increased from 0.08 to 0.11 (Table 1),

30

indicating the homogeneity of both mineralized and non-mineralized micelles; the zeta 10


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1

potential values of PTX-loaded micelles were increased from -29.9 mV to -16.9 mV (Fig. 1A

2

and 1B), which was probably due to the formation of mineral CaP on the surface of micelles

3

caused by the electrostatic interactions between calcium ions and carboxylate ions, which

4

subsequently resulted in the partially shielded carboxylate anionic charges of HA by mineral

5

CaP. The formation of mineral CaP on the surface of micelles was also confirmed by TEM.

6

All the micelles were spherical in shape and homogeneous in particle sizes, which was

7

consistent with DLS results (Table 1). However, the particle size determined from TEM was

8

smaller than that estimated by DLS, which is probably due to their different sample treatment,

9

where the TEM drying process may induce shrinkage of hydrophilic part of micelles, and was

10

even intensified by super-hydrophilic (HA) based conjugatess25, compared to the DLS

11

samples which were prepared in aqueous condition. Notably, the mineralized micelles could

12

be visualized without a staining process, whereas non-mineralized micelles were only visible

13

after being negatively stained, which directly indicates the formation of CaP in PTX-loaded

14

micelles (Fig. 1C and 1D). This unique feature was also reported in other CaP mineralized

15

polymeric micelles. 9, 10, 32 As expected, the major components of mineralized micelles were

16

calcium and phosphorus (Fig. 1E), which further demonstrated that CaP was formed on the

17

surfaces of micelles. From the selected area electron diffraction pattern of the mineralized

18

micelles (inset in Fig. 1E), no sharp electron diffraction rings were observed, which also

19

provided the evidence that an amorphous CaP phase formed on the surface of micelles.

20

3.2. Calcium disintegration of mineralized micelles

21

The water solubility of CaP minerals is very poor in physiological environment but

22

significantly increased in an acidic pH environment. To investigate the pH-responsive

23

behavior of the mineralized micelles, a calcium dissolution assay according to the Arsenazo

24

III method was performed in the buffer solutions with different pH values. As shown in Fig. 2,

25

in the acidic environment in lysosomes (pH 5.0), 72% of the calcium ions were released

26

within 12 h, and approximately 37% calcium ions were released in pH 6.5 within the same

27

period of time. However, under the physiological condition (pH 7.4), only approximately 20.7%

28

of the calcium ions were released from the mineralized micelles after 72 h. These results

29

demonstrated that the CaP on the shell of micelles might be sensitive to pH. The calcium ion

30

release rate was accelerated as the pH value of the buffer solution decreased, which is 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

probably related to the fact that the dissolving of CaP coatings are much easier in acidic

2

environment (pH 5.0) than in the physiological condition (pH 7.4). 33

3

3.3. Stability of PTX-loaded non- and mineralized micelles

4

Structural stability in the bloodstream is one of the major requirements of nanocarriers,

5

allowing minimal premature release of drugs caused by interactions between drug carriers and

6

blood molecules such as serum proteins. 34, 35 As shown in Fig. 3A, the particle size of

7

mineralized micelles monitored in the presence of 10% FBS by DLS showed a slight increase

8

in 120 h compared with the beginning state. On the contrary, non-mineralized micelles

9

showed a large size variation in which the sizes increased from 145.5 nm to 215.2 nm under

10

the same conditions within 120 h. Furthermore, the scattering intensities for both

11

non-mineralized and mineralized micelles gradually decreased after incubation with FBS for

12

120 h (Fig. 3B). Specifically, the mineralized micelles maintained 84.4% of the initial

13

intensity, but the non-mineralized micelles decreased to 64.4% of the initial intensity,

14

demonstrating that a dramatic loss of scattered light intensity was observed in

15

non-mineralized micelles. From the particle size and intensity change results, it can be

16

inferred that the mineralized micelles can maintain their structural integrity in the blood

17

circulation. This ability is probably ascribed to the reinforcement of micelles by CaP. Apart

18

from the solidifying effect on the micelle surface, CaP may also cover part of the negative

19

potential on the surface, which could diminish the ability of micelles to combine with

20

substances in the blood, leading to the enhanced stability.

21

3.4. In vitro release of PTX from mineralized micelles

22

The most attractive function of PTX-loaded mineralized micelles was their ability to

23

rapidly release PTX in a reducing and acidic environment. We have verified that PTX-loaded

24

micelles showed a reduction-sensitive drug release profile (Fig. S4). To further ascertain these

25

characteristics, the release of PTX from its mineralized micelles was investigated in

26

environments simulating physiological reducing or acidic conditions. The results

27

demonstrated that the release profile of PTX from the PTX-loaded mineralized micelles was

28

dependent on the reducibility and acidity of the release medium (Fig. 4). At pH 7.4, the

29

release of PTX from the mineralized micelles was rather slow. Only 32.8% of PTX was

30

released in 144 h, which was probably because of the release barrier formed by CaP minerals 12


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1

on the shell of the micelles. However, the release of PTX was accelerated at pH 5.0 with 67.5%

2

of PTX released at the same time, likely due to the acid-induced dissolution of the CaP

3

coating. Similarly, approximately 58% of PTX was released within 144 h under reducing

4

conditions (pH 7.4, 40 mM GSH), which was more rapid than the release in a physiological

5

environment (pH 7.4), confirming the reduction-sensitive property of PTX-loaded

6

mineralized micelles. The release of PTX was clearly slower in reducing conditions (pH 7.4,

7

40 mM GSH) compared to that in an acidic environment (pH 5.0). This slower release was

8

likely attributed to the slowly dissolved CaP shield hindering the released drug induced by the

9

cleavage of disulfide bond. Notably, the fastest drug release was obtained under 40 mM GSH

10

and pH 5.0 conditions, in which 75.1% of PTX was released in 144 h and 91.9% of PTX was

11

released in 216 h. This result indicates that the mineralized micelles are sensitive to both pH

12

and reducing conditions. Overall, the above results suggest that the PTX-loaded mineralized

13

micelles are relatively stable in a physiological environment, but could quickly release drug in

14

the intra-cellular reducing and acid environment due to their dual sensitivity.

15

3.5. Hemolysis test of mineralized micelles

16

The adaptability of nanoparticles for intravenous administration should be seriously

17

considered. 36 Therefore, the hemolysis of blank and PTX-loaded mineralized micelles was

18

investigated to evaluate the feasibility for parenteral administration. The hemolysis of both

19

blank and PTX-loaded mineralized micelles increased with their increasing concentration (Fig.

20

5). PTX-loaded mineralized micelles exhibited negligible hemolytic activity, with a hemolysis

21

rate of only 3.01% at 0.8 mg/mL. Regarding the maximum concentration (1.0 mg/mL), the

22

hemolysis rate of blank micelles is 4.02%, indicating that the blank micelles would not result

23

in hemolysis activities. Although the peak hemolysis rate of PTX-loaded mineralized micelles

24

reaches 5.34%, the continuous dilution effect of blood will inhibit the hemolysis activity.

25

Thus, it can be inferred that PTX-loaded mineralized micelles would be non-toxic towards

26

erythrocytes after intravenous injection.

27

3.6. Cellular uptake behavior of C6 loaded mineralized micelles

28

The over-expressing CD44 on MDA-MB-231 cells was confirmed and the results are

29

summarized in the supporting information (Fig. S5). The cellular uptake of C6 loaded

30

mineralized micelles was observed in MDA-MB-231 cells using CLSM and quantitatively 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

analyzed by flow cytometry analysis. As a fluorescence probe, C6 has been extensively

2

applied in cellular uptake studies of nanocarriers for observation by CLSM. 37, 38 After 4 h of

3

incubation, the fluorescence of C6 could be observed throughout the cellular cytosol in the

4

three groups (Fig. S6), and the fluorescence intensity was reinforced after the incubation for 8

5

h (Fig. 6A). The fluorescence intensity was rarely observed in MDA-MB-231 cells treated

6

with free C6 after 8 h incubation, which indicated that free C6 was hardly taken up by these

7

tumor cells. However, the fluorescence intensity of both C6 loaded micelles and C6 loaded

8

mineralized micelles is higher than that of free C6, indicating that C6 loaded micelles or C6

9

loaded mineralized micelles are readily taken up by MDA-MB-231 cells (Fig. 6A).

10

Meanwhile, the similar fluorescence intensity of C6 was observed in CD44-negative HepG2

11

cells after treated with free C6, C6 loaded micelles, and C6 loaded mineralized micelles,

12

which confirmed the specific CD44 targeting of these two types of micelles (Fig. 6B).

13

Additionally, the flow cytometry results showed that the mean C6 fluorescence intensity of

14

C6 loaded micelles and C6 loaded mineralized micelles was 1.78 and 2.15 times higher than

15

that of free C6, respectively (Fig. 6C and 6D). This inconformity was mainly ascribed to the

16

HA-CD44 interaction, which led to C6 loaded micelles or C6 loaded mineralized micelles

17

selectively binding to CD44 and being internalized into MDA-MB-231 cells via

18

receptor-mediated endocytosis. 39 It should be noted that the fluorescence intensity of cells

19

after treatment with C6 loaded mineralized micelles was relatively higher when compared

20

with C6 loaded micelle incubated cells. This result is likely due to the rapid release of C6

21

from C6 loaded mineralized micelles after internalization into a reductive and acid

22

intra-cellular environment.

23

3.7. In vitro cytotoxicity studies

24

The cell viability of blank micelles and blank mineralized micelles against MDA-MB-231

25

cells was evaluated by the MTS assay. On one hand, despite that the concentrations of

26

micelles were ranged from 0.02 to 100 µg/mL, the viability of blank micelles and blank

27

mineralized micelles was approximately 100% under all concentrations at 48 h (Fig. 7A),

28

indicating that blank micelles and blank mineralized micelles are non-toxic and biocompatible

29

with drug delivery. On the other hand, the cytotoxicity of PTX, Taxol, PTX-loaded micelles,

30

and PTX-loaded mineralized micelles toward the MDA-MB-231 cells was obviously depend 14


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1

on their concentration (Fig. 7B). The half-maximal inhibitory concentration (IC50) value of

2

PTX-loaded mineralized micelles was 0.0248 µg/mL, while the IC50 of Taxol and PTX-loaded

3

micelles were 4.44- and 1.44-fold higher than the IC50 of PTX-loaded mineralized micelles.

4

The enhanced cytotoxicity of PTX mineralized micelles was likely attributable to the

5

enhanced cellular uptake of micelles by MDA-MB-231 cancer cells via HA-receptor

6

mediated endocytosis (Fig. 6). Furthermore, the quick intra-cellular PTX release through

7

rapid disassembly of micelles in response to intra-cellular low pH cytoplasm and high

8

reductive GSH (Fig. 4) was also responsible for the cytotoxicity enhancement of PTX

9

mineralized micelles.

10

3.8. Detections of apoptosis

11

The pro-apoptosis was quantitatively studied using flow cytometry on MDA-MB-231 cells

12

after treatment with Taxol, PTX-loaded micelles, and PTX-loaded mineralized micelles, with

13

the corresponding PTX concentration for 48 h, and saline was used as the control group. Cells

14

in different status, such as early apoptosis, late apoptosis and necrosis, were observed in each

15

group treated with above formulations (Fig. 8A). The total apoptosis percentages of

16

MDA-MB-231 cells treated with Taxol, PTX-loaded micelles, and PTX-loaded mineralized

17

micelles were 14.1%, 18.5%, and 23.5%, respectively (Fig. 8B). Clearly, the PTX-loaded

18

mineralized micelles induced a higher apoptotic rate compared to other groups (1.66 times

19

and 1.26 times higher than that induced by Taxol and PTX-loaded micelles (p < 0.05)),

20

indicating that mineralized micelles could enhance the apoptotic effect of PTX on

21

MDA-MB-231 cells. Furthermore, the total number of early apoptotic cells was much larger

22

than that of late apoptotic cells, which demonstrated that PTX-loaded mineralized micelles

23

primarily function in accelerating the early apoptosis. The apoptosis results were agree with

24

the cytotoxicity assay, which is likely related to the specific uptake of MDA-MB-231 cancer

25

cells and relatively rapid intra-cellular drug release.

26

3.9. In vivo tumor targeting assays

27

The real-time images of MDA-MB-231 tumor-bearing mice after intravenous injection of

28

DiR loaded non- and mineralized micelles were shown in Fig. 9A. At two hours after

29

intravenous administration of the DiR formulations, the distinct signals of DiR were clearly

30

observed in the liver and tumors from the two micellar formulation groups. The distribution 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

of micelles in the liver seems reasonable given that the liver sinusoidal endothelial cell has

2

been proved to express the specific receptors of HA in the previous reports. 40-42 Afterwards,

3

the fluorescence signals gradually increased at the tumor site, and the maximum signal

4

intensity was observed at 10 h post-injection. Clearly, the fluorescence in the DiR micellar

5

treated group was much higher than that in group treated with free DiR in tumors at the same

6

time points, which demonstrated that both non- and mineralized micelles are available for

7

tumor-specific drug delivery. Compared with DiR micelles, DiR mineralized micelles showed

8

preferential accumulation at the tumor site, which might result from the targeting release due

9

to the dual sensitivity of mineralized micelles. Furthermore, the low pH and reduction

10

initiated intracellular disassembly of micelles also play an important role in reducing the

11

elimination of mineralized micelles and prolonging the retention time of DiR in tumor site.

12

The ex vivo fluorescent images of excised organs and tumors further confirmed that the

13

DiR formulations mainly accumulated in the liver, spleen, and tumors (Fig. 9B). In excised

14

organs of free DiR group, DiR was distributed in liver and only a small tumor accumulation

15

was observed. In contrast, the highest fluorescence intensity of C6 per weight on tumor was

16

obtained in mineralized micelles group, which was 1.66- and 4.29-fold (p < 0.05) of that in

17

DiR loaded micelles group and free DiR group (Fig. 9C), respectively. The preferential

18

accumulation of mineralized micelles at tumor site was consistent to the results in the

19

real-time images. Overall, these results indicate that mineralized micelles are expected to be a

20

highly efficient drug delivery vehicle to achieve targeted intracellular delivery of anti-cancer

21

drugs.

22

3.10. In vivo anti-tumor effect

23

As shown in Fig. 10A, the control group, which showed a rapid increase of tumor size,

24

exhibited negligible anti-tumor activity, whereas the Taxol group showed slightly inhibited

25

tumor growth. However, compared to the Taxol group, the anti-tumor efficacy was much

26

higher in PTX-loaded micelles (p < 0.05) and PTX-loaded mineralized micelles (p < 0.01),

27

which was primarily ascribed to high tumor targetability of nanoparticles. Furthermore,

28

PTX-loaded mineralized micelles exhibited the highest anti-tumor efficacy of the three PTX

29

formulations, suggesting a promising effect of mineralization on tumor targeting. At the end

30

of the treatment, tumors were excised and their images were shown in Fig. 10D. The tumor 16


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1

inhibitory rate of PTX-loaded mineralized micelles was 73.06%, which is 1.57 times that of

2

the Taxol group (p < 0.05) and 1.40 times more than PTX-loaded micelles (p < 0.05) (Fig.

3

10C). This may be partially attributed to the prolonged retention of PTX in blood circulation

4

because the drug encapsulated in micelles is probably protected from metabolism, while free

5

PTX was highly hydrophobic and had a strong affinity with plasma protein. 43 Meanwhile, the

6

dual sensitivity of mineralized micelles could also induce targeting therapy.

7

In addition to the anti-tumor activity, safety profiles of PTX formulations were evaluated

8

by measuring the changes in body weight as a function of time (Fig. 10B). Body weight did

9

not decrease in the control group or the PTX-loaded non-mineralized and mineralized micelle

10

treated group, suggesting that PTX-loaded micellar groups had a good biocompatibility. On

11

the contrary, there was an obvious loss of body weight after the tumor-bearing mice were

12

treated with Taxol. Furthermore, histological analysis of organs through H&E staining was

13

also performed. As shown in Fig. S7, no obvious organ toxicity was observed in non- and

14

mineralized micelles treatment group in comparison with the control group. Combining these

15

results with the results of body weight, it can be inferred that PTX-loaded non- and

16

mineralized micelles are well tolerated in vivo. The failure to observe toxicity of PTX-loaded

17

non-mineralized and mineralized micelles might be attributable to the reduced drug release in

18

normal tissues, the enhanced tumor targeting of drugs by micelles, and the pH- and

19

reduction-dependent drug release behavior of micelles. However, the Taxol group caused

20

systemic toxicity as characterized by the vacuolization in the cardiomyocytes (Fig. S7

21

Taxol-Heart), which might be related to the inherent toxicity of the solvent in Taxol and the

22

non-selected distribution of Taxol. Taken together, the mineralized micelle treated group

23

showed significant anti-tumor activity against MDA-MB-231 xenografts with minimal

24

toxicity.

25

4.

Conclusion

26

In this study, calcium phosphate-reinforced reduction-sensitive HA micelles were

27

successfully prepared by controlled deposition of calcium and phosphate ions via a sequential

28

addition method. Mineralized micelles could effectively encapsulate anti-cancer drug of PTX

29

with the DL up to 30.6% and an EE of 87.8%. PTX-loaded mineralized micelles

30

demonstrated pH- and reduction-dependent drug release characteristics, but was stable in the 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

normal physiological conditions (pH 7.4). Compared to Taxol, PTX-loaded mineralized

2

micelles displayed an excellent anti-tumor efficacy with the lower in vitro/in vivo toxicity and

3

better tumor accumulation, which resulted in a higher tumor inhibition of 73.06% and

4

considerable biocompatibility. Overall, the calcium phosphate-reinforced reduction-sensitive

5

micelles present promising potential as carriers of hydrophobic chemotherapeutic drugs.

6 7

Supporting Information

8

Synthesis of HA-SS-DOCA conjugates

9

Preparation and characterization of micelles

10

CD44 expression analysis

11

Fig. S1. 1H NMR spectra and FT-IR spectrum of HA, HA-CYS conjugate, and HA-SS-DOCA

12

conjugate

13

Fig. S2. CMC determination of HA-SS-DOCA conjugates

14

Fig. S3. TEM images of blank HA-SS-DOCA micelles. Time dependent changes of the mean

15

diameter of HA-SS-DOCA micelles with or without GSH addition. Size distribution of

16

HA-SS-DOCA micelles after incubated with or without GSH for 24 h

17

Fig. S4. Release behavior of PTX from HA-SS-DOCA micelles in PBS with or without GSH

18

Fig. S5. CLSM images of MDA-MB-231 cells and HepG2 cells after incubating anti-CD44

19

antibody overnight

20

Fig. S6. Confocal laser scanning microscopy (CLSM) images of MDA-MB-231 cancer cells

21

after 4 h incubation with C6, C6 loaded micelles, and C6 loaded mineralized micelles,

22

respectively

23

Fig. S7. Optical microscopy images of H&E staining major organs and tumor after treatment

24

of different PTX formulations

25

Table S1. Characteristics of HA-SS-DOCA conjugates. Related to Fig. S3

26 27

Acknowledgements

28

This work was sponsored by the Shanghai Talent Development Fund (201565), the “Shu

29

Guang” project supported by Shanghai Education Development Foundation and Shanghai

30

Municipal Education Commission (15SG39), the Shanghai Pujiang Program (16PJD044). 18


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1 2 3

Conflict of interest The authors declare that there is no conflict of interest.

4 5

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carbonate-mineralized polymer nanoparticles for pH-responsive robust nanocarriers of

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Well-defined, reversible disulfide cross-linked micelles for on-demand paclitaxel delivery.

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Q.

Redox-sensitive

micelles

self-assembled

from

amphiphilic

hyaluronic

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Polysaccharide-based nucleic acid nanoformulations. Adv. Drug Deliver. Rev. 2013, 65,

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1123-1147.

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hydrophilic AURKA specific siRNA by redox-sensitive micelles for effective treatment of

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24


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1

Figure Captions

2

Scheme 1. Fabrication of pH and reduction dual-sensitive hyaluronic acid mineralized

3

micelles: firstly, the micelles consist of amphiphilic conjugation based on HA and DOCA

4

connected by disulfide bond was synthesized as the carrier of PTX; then, the PTX-loaded

5

micelles were reinforced by CaP shields (the sequential addition of the calcium nitrate and the

6

ammonium phosphate) to obtain the pH and reduction dual sensitivity.

7

Fig. 1. (A) Size distribution and (B) zeta potential value of PTX-loaded micelles (green line)

8

and PTX-loaded mineralized micelles (red line); (C) TEM images of PTX-loaded micelles; (D)

9

TEM images of PTX-loaded mineralized micelles; (E) TEM-EDS analysis of PTX-loaded

10

mineralized micelles (the inset is for the selected-area diffraction pattern).

11

Fig. 2. The release pattern of calcium ions from mineralized micelles in different pH buffer

12

solutions. The error bars represent standard deviation (n = 3).

13

Fig. 3. Time dependent changes of the mean diameter (A) and the ratio of scattered light

14

intensities (B) of PTX-loaded micelles or PTX-loaded mineralized micelles incubation in the

15

presence of 10% FBS for 120 h. The error bars represent standard deviations (n = 3).

16

Fig. 4. PTX release profiles from PTX-loaded mineralized micelles under different pH values

17

with or without 40 mM GSH. The error bars in the graph represent standard deviations (n =

18

3).

19

Fig. 5. Hemolysis of blank mineralized micelles and PTX-loaded mineralized micelles. The

20

error bars represent standard deviations (n = 3).

21

Fig. 6. (A) Confocal laser scanning microscopy (CLSM) images of MDA-MB-231 cells and

22

(B) CLSM images of HepG2 cells after 8 h incubation with C6 (a), C6 loaded micelles (b),

23

and C6 loaded mineralized micelles (c), respectively. Images from left to right show the green

24

fluorescence of C6, the red fluorescence of LysoTrackerRed, the blue fluorescence of DAPI,

25

and the merged fluorescence of C6, LysoTrackerRed, and DAPI. Scale bars correspond to 25

26

µm in all the images. (C) Mean fluorescence intensity of C6 in MDA-MB-231 cells after

27

treated with C6 loaded mineralized micelles, C6 loaded micelles, and free C6 at 8 h (blank

28

cells as the control). (D) Flow cytometry graphs of C6 accumulation in MDA-MB-231 cells at

29

8 h (n = 3).

30

Fig. 7. (A) Cytotoxicity of blank micelles and blank mineralized micelles. (B) Viability of 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

cells treated with different concentration of PTX-loaded micelles, PTX-loaded mineralized

2

micelles, Taxol, and free PTX against MDA-MB-231 cells for 48 h. The results were

3

expressed as the mean ± SD from 6 independent experiments.

4

Fig. 8. (A) Flow cytometric analysis of cell apoptosis using fluorescein Annexin V-FITC and

5

PI double labeling. Cells were treated with saline (a), Taxol (b), PTX-loaded micelles (c), and

6

PTX-loaded mineralized micelles (d) for 48 h. (B) Total counts of apoptotic and necrotic cells

7

after treated with saline, Taxol, PTX-loaded micelles, and PTX-loaded mineralized micelles

8

(n = 3). * p < 0.05.

9

Fig. 9. (A) In vivo imaging of tumor bearing mice after administration of free DiR (a), DiR

10

loaded micelles (b), and DiR loaded mineralized micelles (c), at 2 h, 4 h, 6 h, 8 h, 10 h , 24 h,

11

and 48 h under DiR channel (720 nm for excitation and 790 nm for emission). (B) Ex vivo

12

fluorescence images of heart, liver, spleen, lung, kidney, brain, and tumors collected at 48 h

13

post injection of free DiR, DiR loaded micelles, and DiR loaded mineralized micelles under

14

DiR channel (720 nm for excitation and 790 nm for emission). (C) Percentage of fluorescence

15

intensity of DiR per weight on liver, spleen, and tumor (n = 3). * p < 0.05.

16

Fig. 10. In vivo anti-tumor activity in MDA-MB-231-bearing female nude mice treated with

17

various PTX formulations. Tumor growth curve (A) and changes of body weight of mice (B)

18

after treated with three PTX formulations, tumor inhibitory rate calculated with excised tumor

19

weight (C) and the images of excised tumor (D). The results were expressed as the mean ± SD

20

(n = 6). * p < 0.05, ** p < 0.01.

21

26


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1

Table 1

2

Properties characterization of various micelles (n = 3).

Micelles

Size (nm)a

PDIb

Zeta (mV)

EEc (%)

DLd (%)

Blank micelles

140.9 ± 1.04

0.09 ± 0.010

-33.4 ± 0.44

--

--

PTX-loaded micelles

129.0 ± 0.86

0.08 ± 0.014

-29.9 ± 0.42

90.2 ± 0.98

32.2 ± 0.32

134.8 ± 0.50

0.11 ± 0.008

-16.9 ± 1.17

87.8 ± 0.69

30.6 ± 0.81

PTX-loaded mineralized micelles 3

a

Mean diameters of micelles.

4

b

Polydispersity index of micelles size.

5

c

Drug encapsulated efficiency.

6

d

Drug loading efficiency.

7 8

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Fabrication of pH and reduction dual-sensitive hyaluronic acid mineralized micelles: firstly, the micelles consist of amphiphilic conjugation based on HA and DOCA connected by disulfide bond was synthesized as the carrier of PTX; then, the PTX-loaded micelles were reinforced by CaP shields (the sequential addition of the calcium nitrate and the ammonium phosphate) to obtain the pH and reduction dual sensitivity. 2418x1068mm (96 x 96 DPI)


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Fig. 1. (A) Size distribution and (B) zeta potential value of PTX-loaded micelles (green line) and PTX-loaded mineralized micelles (red line); (C) TEM images of PTX-loaded micelles; (D) TEM images of PTX-loaded mineralized micelles; (E) TEM-EDS analysis of PTX-loaded mineralized micelles (the inset is for the selectedarea diffraction pattern). 981x539mm (144 x 144 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 2. The release pattern of calcium ions from mineralized micelles in different pH buffer solutions. The error bars represent standard deviation (n = 3). 777x696mm (96 x 96 DPI)


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Fig. 3. Time dependent changes of the mean diameter (A) and the ratio of scattered light intensities (B) of PTX-loaded micelles or PTX-loaded mineralized micelles incubation in the presence of 10% FBS for 120 h. The error bars represent standard deviations (n = 3). 975x497mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 4. PTX release profiles from PTX-loaded mineralized micelles under different pH values with or without 40 mM GSH. The error bars in the graph represent standard deviations (n = 3). 673x578mm (96 x 96 DPI)


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Fig. 5. Hemolysis of blank mineralized micelles and PTX-loaded mineralized micelles. The error bars represent standard deviations (n = 3). 615x466mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 6. (A) Confocal laser scanning microscopy (CLSM) images of MDA-MB-231 cells and (B) CLSM images of HepG2 cells after 8 h incubation with C6 (a), C6 loaded micelles (b), and C6 loaded mineralized micelles (c), respectively. Images from left to right show the green fluorescence of C6, the red fluorescence of LysoTrackerRed, the blue fluorescence of DAPI, and the merged fluorescence of C6, LysoTrackerRed, and DAPI. Scale bars correspond to 25 µm in all the images. (C) Mean fluorescence intensity of C6 in MDA-MB231 cells after treated with C6 loaded mineralized micelles, C6 loaded micelles, and free C6 at 8 h (blank cells as the control). (D) Flow cytometry graphs of C6 accumulation in MDA-MB-231 cells at 8 h (n = 3). 1199x890mm (144 x 144 DPI)


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Fig. 7. (A) Cytotoxicity of blank micelles and blank mineralized micelles. (B) Viability of cells treated with different concentration of PTX-loaded micelles, PTX-loaded mineralized micelles, Taxol, and free PTX against MDA-MB-231 cells for 48 h. The results were expressed as the mean ± SD from 6 independent experiments. 524x727mm (144 x 144 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 8. (A) Flow cytometric analysis of cell apoptosis using fluorescein Annexin V-FITC and PI double labeling. Cells were treated with saline (a), Taxol (b), PTX-loaded micelles (c), and PTX-loaded mineralized micelles (d) for 48 h. (B) Total counts of apoptotic and necrotic cells after treated with saline, Taxol, PTXloaded micelles, and PTX-loaded mineralized micelles (n = 3). * p < 0.05. 1579x789mm (96 x 96 DPI)


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Fig. 9. (A) In vivo imaging of tumor bearing mice after administration of free DiR (a), DiR loaded micelles (b), and DiR loaded mineralized micelles (c), at 2 h, 4 h, 6 h, 8 h, 10 h , 24 h, and 48 h under DiR channel (720 nm for excitation and 790 nm for emission). (B) Ex vivo fluorescence images of heart, liver, spleen, lung, kidney, brain, and tumors collected at 48 h post injection of free DiR, DiR loaded micelles, and DiR loaded mineralized micelles under DiR channel (720 nm for excitation and 790 nm for emission). (C) Percentage of fluorescence intensity of DiR per weight on liver, spleen, and tumor (n = 3). * p < 0.05. 1392x725mm (144 x 144 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 10. In vivo anti-tumor activity in MDA-MB-231-bearing female nude mice treated with various PTX formulations. Tumor growth curve (A) and changes of body weight of mice (B) after treated with three PTX formulations, tumor inhibitory rate calculated with excised tumor weight (C) and the images of excised tumor (D). The results were expressed as the mean ± SD (n = 6). * p < 0.05, ** p < 0.01. 1423x1212mm (96 x 96 DPI)


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Title: Calcium phosphate-reinforced reduction-sensitive hyaluronic acid micelles for delivering paclitaxel in cancer therapy Bing Denga,1, Mengxin Xiaa,1, Jin Qiana, Rui Lia, Lujia Lia,b, Jianliang Shenc, Guowen Lib, Yan Xiea,*


Calcium Phosphate-Reinforced Reduction-Sensitive Hyaluronic Acid

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