A LAPONITEÂ®-polyethylenimine based... (PDF Download Available)

Subscriber access provided by GAZI UNIV

Article

A LAPONITE®-polyethylenimine based theranostic nanoplatform for tumor-targeting CT imaging and chemotherapy Ying Zhuang, Lingzhou Zhao, Linfeng Zheng, Yong Hu, Ling Ding, Xin Li, Changcun Liu, Jinhua Zhao, Xiangyang Shi, and Rui Guo ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00528 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on January 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 51

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

ACS Biomaterials Science & Engineering

A LAPONITE®-polyethylenimine based theranostic nanoplatform for tumor-targeting CT imaging and

2

chemotherapy

3

4

Ying Zhuanga, 1, Lingzhou Zhaob, 1, Linfeng Zhengb, Yong Hua, Ling Dinga, Xin Lia, Changcun

5

Liub, Jinhua Zhaob*, Xiangyang Shia, c* and Rui Guoa* a

6

North Renmin Road, Songjiang District, Shanghai 201620, P. R. China

7

b 8

Department of Radiology, First People's Hospital, Shanghai Jiaotong University, 100 Haining Road, Hongkou District, Shanghai 20080, P. R. China

9

c 10

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of

11

Materials Science and Engineering, Donghua University, 2999 North Renmin Road, Songjiang

12

District, Shanghai 201620, P. R. China.

13

______________________________________________________________________________

14

* To whom correspondence should be addressed. Email: [email protected] (J. Zhao),

15

[email protected] (X. Shi), [email protected] (R. Guo).

ACS Paragon Plus Environment

1


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

Page 2 of 51

1

ABSTRACT: In this study, laponite (LAP) nanodisks and polyethylenimine (PEI) were used to

2

build a hybrid theranostic nanoplatform for targeted computed tomography (CT) imaging and

3

chemotherapy of cancer cells overexpressing CD44 receptors. Firstly, amphiphilic copolymer

4

poly(lactic acid)-poly(ethylene glycol) (PLA-PEG-COOH) were assembled on the surface of

5

LAP nanodisks via hydrophobic interaction, and then PEI were conjugated by the formation of

6

amide groups via1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling chemistry.

7

The developed LAP-PLA-PEG-PEI nanoparticles were used as templates for the embedding of

8

gold nanoparticles (Au NPs), followed by modification with hyaluronic acid (HA) as a targeting

9

ligand for cancer cells overexpressing CD44 receptors. Finally, anticancer drug doxorubicin

10

(DOX) was loaded. The formed LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes display

11

good stability, a high drug loading efficiency as 91.0 ± 1.8%, and sustained drug release profile

12

with a pH-sensitive manner. In vitro cell viability assay, flow cytometric analysis and laser

13

scanning confocal microscopy observation demonstrate that the formed nanocomplexes can

14

specifically deliver and inhibit cancer cells overexpressing CD44 receptors. In vivo experiments

15

illustrate that LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes can not only significantly

16

inhibit the growth of tumors and decrease the side-effect of DOX, but also be used as a targeted

17

contrast agent for CT imaging of tumors. Therefore, the developed LAP-PLA-PEG-PEI-(Au0)50-

18

HA/DOX nanocomplexes can be used as a promising theranostic platform for targeted imaging

19

and chemotherapy of CD44-overexpressed tumors.

20

KEYWORDS: Laponite, Gold nanoparticles, Hyaluronic acid targeting, CT imaging,

21

Chemotherapy

22


2

Page 3 of 51

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

2

Theranostic nanomedicine has been attracted much attention in treating various diseases recently

3

due to its combination of therapeutic and imaging functions. Especially in the cancer treatment,

4

the capability of diagnosis, drug delivery and monitoring therapeutic effect bring convenience to

5

patients, offer improved prognosis, and make personalized medicine possible. Although with the

6

advent of nanoscience and nanotechnology, various kinds of nanomaterials have been developed

7

as drug delivery systems or nanoprobes for biomedical applications,1-8 the major challenge of

8

theranostic nanomedicine is to combine imaging contrast agents and antitumor drugs into a

9

tumor-targeting nanoplatform in order to deliver specifically to cancer cells, control the drug’s

10

pharmacokinetic profile for improved chemotherapeutic efficacy, and provide distinct tumor

11

images for diagnosis.

INTRODUCTION

12

Among conventional imaging modalities,9-15 computed tomography (CT) has been most

13

extensively used in the diagnosis of tumor16-18 due to its high spatial and density resolution, deep

14

penetration capability, and facile post image-processing technique. However, the conventionally

15

used iodinated small molecular CT agents (e.g., Omnipaque) have some drawbacks, such as

16

short imaging time, renal toxicity and nonspecificity, limiting their application in tumor CT

17

imaging.19-22 Recently Au nanoparticles have been used as contrast agents for CT imaging due to

18

their high X-ray attenuation coefficient.23-25 For example, polyethyleneimine (PEI), a kind of

19

hyperbranched polymer with abundant amines on periphery, could stabilize Au nanoparticles and

20

conjugate with polyethylene glycol (PEG) to improve the biocompatibility and colloidal stability

21

for a long blood circulation time.26-27 After modification with folic acid (FA) or hyaluronic acid

22

(HA) as targeting agents, the PEI entrapped gold nanoparticles can be used as nanoprobes for

23

targeted CT imaging of cancer cells over expressing FA receptors or CD44 receptors.28-29


3


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

Page 4 of 51

1

Moreover, PEI can also be used as nancarriers to encapsulate anticancer drug doxorubicin (DOX)

2

and release DOX in a pH-dependent manner.30-31 However, the loading of Au NPs may occupy

3

the internal space of PEI’s hyperbranched structure, and reduce the drug loading capacity, which

4

may ruin the possibility of using PEI alone as a theranostic nanoplatform. Therefore, introducing

5

an effective drug loading component and composing with PEI into one harmonious system are of

6

paramount importance in the design of theranostic nanoplatforms for CT imaging and

7

chemotherapy.

8

Laponite (LAP) is a kind of synthetic and biodegradable nanoclay with a typical layered

9

structure as natural hectorite.32 The high specific surface area and strong cationic exchange

10

capability make LAP an ideal carrier system for the delivery of different kinds of drugs, such as

11

strontium ranelate,33 of loxacin34 and doxorubicin35. In our previous study, LAP can encapsulate

12

antitumor drug DOX with a ultrahigh loading efficiency as 98.3%, and the formed

13

nanocomplexes may significantly inhibit the growth of cancer and dramatically prolong the

14

survival time in comparison with free DOX.35 And LAP can be easily modified with polylactic

15

acid-polyethylene glycol (PLA-PEG) through hydrophobic interaction to prolong the blood

16

circulation time and improve anti-cancer efficacy.36 Moreover, after modification with targeting

17

agents,37 LAP can specifically deliver drugs to tumor cells and display targeted inhibition

18

efficacy.38 These studies in LAP nanodisks lead us to hypothesize that the union of PEI and LAP

19

may take the advantage of the ultrahigh drug loading efficiency of LAP and the abundant amine

20

groups on PEI for stabilizing Au NPs as CT imaging contrasts and conjugating targeting agents,

21

thereby constituting a hybrid theranostic nanoplatform for tumor-targeting CT imaging and

22

chemotherapy.


4

Page 5 of 51

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

In this study, LAP nanodisks were firstly modified with amphiphilic copolymer PLA-PEG-

2

COOH to provide additional stability and active carboxyl groups on surface, and then PEI were

3

conjugated by the formation of amide groups via 1-ethyl-3-(3-dimethylaminopropyl)

4

carbodiimide (EDC) coupling chemistry. The formed LAP-PLA-PEG-PEI nanodisks were used

5

as templates for embedding of gold nanoparticles, followed by modification with a targeting

6

ligand hyaluronic acid. Finally, anticancer drug DOX was loaded into the nanocomplexes. The

7

formed LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes were thoroughly characterized

8

via thermogravimetric analysis (TGA), FTIR spectrometry, UV-vis spectra spectrometry, zeta-

9

potential measurements, dynamic light scattering (DLS), and transmission electron microscope

10

(TEM). DOX release kinetics was assessed under different pH buffer solutions (pH = 5.0 and

11

7.4). The inhibition efficacy and targeting ability of the nanocomplexes to cancer cells

12

overexpressing CD44 receptors in vitro was evaluated by cell viability assay, flow cytometry

13

analysis, and confocal microscopy. Finally, a HeLa xenografted tumor model was established to

14

evaluate the targeted therapeutic effect and CT imaging of the LAP-PLA-PEG-PEI-(Au0)50-

15

HA/DOX nanocomplexes. To our knowledge, it is the first report about LAP-PEI based

16

nanocomplexes for targeted CT imaging and chemotherapy of tumor overexpressing CD44

17

receptors.

18

EXPERIMENTAL SECTION

19

Materials. All materials we used were offered by commercial companies in the Supporting

20

Information, and the purified water used in the experiment was similar to that described in our

21

previous report. 29, 36-37

22

Synthesis of LAP-PLA-PEG-PEI-(Au0)50-HA. LAP solution (10 mL, 10 mg•mL-1) was

23

dropwise added into PLA-PEG-COOH (50 mL, 6 mg•mL-1) solution. After stirring for 1 h, the


5


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

Page 6 of 51

1

above mixture was firstly dialyzed against phosphate buffered saline (PBS) and subsequently

2

water (2 L each time, 3 times each day) for 3 days with a 14,000 MWCO dialysis membrane.

3

The concentration of the LAP-PLA-PEG-COOH was calculated by lyophilizing a part of the

4

LAP-PLA-PEG-COOH solution with a specific volume.

5

Then, PEI was modified on the LAP-PLA-PEG-COOH via EDC coupling chemistry as our

6

previous work.29 In brief, an aqueous LAP-PLA-PEG-COOH (110 mL, 2.54 mg•mL-1) solution

7

was activated by EDC (66.95 mg) and NHS (40.15 mg) by magnetically stirring for 3 h. Then,

8

the activated LAP-PLA-PEG-COOH was dropwise added into PEI solution (30.98 mL, 1mg•mL1

9

) with magnetically stirring for 3 days. After that, the mixture was dialyzed with a 50,000

10

MWCO dialysis membrane and the concentration of the LAP-PLA-PEG-PEI was calculated by

11

the same method described above.

12

The formed LAP-PLA-PEG-PEI nanodisks were used as templates to synthesize Au NPs via

13

sodium borohydride reduction chemistry with the LAP-PLA-PEG-PEI/Au salt molar ratio of 1:

14

50, which is similar to our previous study.39 Briefly, an aqueous HAuCl4 solution (566 µL, 30

15

mg•mL-1) was added into an aqueous LAP-PLA-PEG-PEI solution (130 mL, 1.45 mg•mL-1) by

16

magnetically stirring for 10 min. After that, NaBH4 (8 mg) dissolving in ice water/ethanol

17

solution (1 mL) was rapidly added to the mixture solution with a molar ratio of NaBH4 and

18

HAuCl4 at 3:1. The reaction mixture became red after a few seconds and then stirred for 3 h in

19

ice water.

20

The activation process of HA is similar to the literature.28 Then, the activated HA solution (4.6

21

mL, 3 mg•mL-1) was dropped into the above LAP-PLA-PEG-PEI-(Au0)50 solution (91 mL, 1.45

22

mg•mL-1) by magnetically stirring at a high rate for 3 days. The excess HA was removed by


6

Page 7 of 51

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

centrifugation for 3 times, and the purified LAP-PLA-PEG-PEI-(Au0)50-HA nanodisks were

2

dispersed in PBS solution.

3

Encapsulation of DOX within LAP-PLA-PEG-PEI-(Au0)50-HA nanocomplexes. The

4

method for loading DOX into LAP-PLA-PEG-PEI-(Au0)50-HA nanodisks was adopted just as

5

our previous work described.36 Briefly, the DOX solution (8.78 mL, 1 mg•mL-1) was mixed with

6

the LAP-PLA-PEG-PEI-(Au0)50-HA (13 mL, 2.75 mg•mL-1) suspension under magnetic stirring

7

in the dark for 24 h. According to our previous work,37 we selected the mass ratio of LAP-PLA-

8

PEG-PEI-(Au0)50-HA to DOX is 3:1. The free DOX was removed by centrifugation (8500 rpm, 6

9

min) and then the LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes were collected and

10

stored in the dark. The concentrations of free DOX in the collected supernatants were measured

11

by a Lambda 25 UV-vis spectrophotometer (PerkinElmer, Waltham, MA) at 481 nm using a

12

standard DOX absorbance-concentration calibration curve. The DOX loading efficiency can be

13

calculated using eqn (1).

14

Loading efficiency = (Mo-Mu)/Mo×100%

(1)

15

where Mu and Mo stand for the mass of the unloaded DOX and the initial DOX, respectively.

16

Characterization techniques. The characterizations in this experiment were measured by

17

standard procedures reported in the literature.30, 38 The in vitro release kinetics of DOX were

18

measured by the same experimental condition as our previous work.37

19

Cell culture. HeLa cells were continuously cultured as our previous work. HeLa cells are

20

usually characterized by high levels of hyaluronan receptor (CD44) expression, and hence were

21

named as HeLa-HCD44 in this work. And HeLa cells expressing low-level CD44 receptors

22

(HeLa-LCD44) were also cultured by incubating in HA-containing medium (2 mM) for 2 h. 28


7


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

Page 8 of 51

1

In vitro cytotoxicity assay and cell morphology observation. CCK-8 assay and cell

2

morphology of HeLa cells (a human cervical cancer cell line) and L929 cells (a mouse fibroblast

3

cell line) were measured to assess the cytotoxicities of free DOX, LAP-PLA-PEG-PEI-(Au0)50-

4

HA, and LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes at different concentrations.30, 40, 41

5

6

Flow cytometry analysis. Flow cytometry was used to evaluate the targeting specificity of the

7

LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes at different DOX concentrations (2.5, 5.0,

8

7.5, 10.0 and 15.0 µg•mL-1, respectively) to HeLa cells overexpressing CD44 receptors.30

9

Confocal microscopy. Confocal microscopy was measured to evaluate the targeting

10

specificity of the LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes at DOX concentrations

11

of 15.0 µg•mL-1 to HeLa overexpressing CD44 receptors according to our previous work.30

12

In vivo targeted cancer cell inhibition. All animal experiments according to protocols were

13

approved by the institutional committee for animal care. Female 6 week old BALB/c nude mice

14

(22-26 g, Shanghai Slac Laboratory Animal Center, Shanghai, China) were subcutaneously

15

injected with 4×106 HeLa cells/mice in the right rear position. When the tumors reached a

16

volume of 0.5-1.2 cm3, these mice were randomly divided into four groups. For the saline group,

17

the saline was injected into tumor of mice (control). For the free DOX group, the PBS solution of

18

free DOX was also injected into tumor of mice. For the nanocomplexes group, the

19

nanocomplexes solution ([Au] = 0.01 M, [DOX] = 1.45 mg•mL-1, 100 µL PBS) was injected into

20

tumor of mice. For the HA+nanocomplexes group, free HA (2 mM, 100 µL PBS) were injected

21

into tumor of mice 2 h before the injection of the nanocomplexes solution. The above-mentioned

22

four groups were subjected to different intratumoral treatment once a week. The tumor size was

23

measured by calipers every week and the tumor volume was calculated according to the formula


8

Page 9 of 51

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 (tumor length × (tumor width)2)/2. The relative tumor volume (denoted as V/V0, where V0 and

2

V are the tumor volumes before and after different treatments at varying time points, respectively)

3

and body weight of each mouse was recorded at experimental time points.

4

X-ray attenuation measurements. The LAP-PLA-PEG-PEI-(Au0)50-HA/DOX were imaged

5

at the Au concentration range of 0-20 mM by a GE Discovery STE PET/CT system with a tube

6

voltage of 100 kV, an electrical current of 220 mA, and a slice thickness of 0.625 mm.28

7

Targeted CT imaging of cancer cells in vitro. HeLa-HCD44 and HeLa-LCD44 cells were

8

separately seeded and incubated with LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexe at

9

different concentrations ([Au] = 0, 20, 40, 80, and 150 µM, respectively) by the similar method

10

in previous work.30 After professional treated and gathered in 1.5 mL Eppendorf tubes,42 the

11

cells were scanned by the same CT system to collect CT images and values (quantitative

12

attenuation intensity, HU).

13

Targeted CT imaging of xenograft tumor model in vivo. When the tumors reached a volume

14

of 0.5-1.2 cm3, these mice were divided into two groups. For the nanocomplexes group, the PBS

15

solution of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX ([Au] = 0.01 M, [DOX] = 1.45 mg•mL-1, 100

16

µL PBS) was injected into mice. For the HA+nanocomplexes group, the mice were pre-injected

17

with free HA (2 mM, 100 µL PBS) for 2 h and then injected with the nanocomplexes solution

18

(HA + Nanocomplexes). The mice in above-mentioned two groups were subjected to

19

intratumoral injection route once a week. For in vivo CT imaging, the scans were performed

20

before (baseline) and 10 min post-injection of the particles for three times using the same system.

21

Histology examinations. The mice treated with saline group, free DOX group, LAP-PLA-

22

PEG-PEI-(Au0)50-HA/DOX nanocomplexes group, and a HA-block group (HA + LAP-PLA-


9


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

Page 10 of 51

1

PEG-PEI-(Au0)50-HA/DOX nanocomplexes) for 21 days were euthanized. Then, the tumors and

2

other organs were treated just as our previous work described.37

3

In vivo biodistribution study. The tumor-bearing BALB/c nude mice were anesthetized by

4

intraperitoneal injection of pentobarbital sodium (40 mg•kg-1). After intratumoral injection of the

5

LAP-PLA-PEG-PEI-(Au0)50-HA/DOX ([Au] = 0.01 M, [DOX] = 1.45 mg•mL-1, 100 µL PBS for

6

each mouse), the mice were euthanized on day 7 and day 14. Then the main organs (heart, liver,

7

spleen, lung, kidney, and tumor) were extracted and weighed. The organs were digested by aqua

8

regia for 2 days, and the Au content in different organs was quantified by ICP-OES. More

9

experimental details are provided in the Supporting Information.

10

11

12

RESULTS AND DISCUSSION Synthesis

and

characterization

of

the

LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

13

nanocomplexes. In this work, amphiphilic PLA-PEG-COOH were self-assembled on the surface

14

of LAP to provide additional colloidal stability and active carboxyl groups, and then

15

hyperbranched PEI were modified by the formation of amide groups via EDC chemistry. The

16

developed LAP-PLA-PEG-PEI nanoparticles were used as templates for the embedding gold

17

nanoparticles, and HA were conjugated on the surface as targeting agents for cancer cells

18

overexpressing CD44 receptors. Finally, the anticancer drug DOX was loaded to form the LAP-

19

PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes (Scheme 1).


10

Page 11 of 51

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

2

Scheme 1.Schematic illustration of the synthesis of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

3

nanocomplexes.

4

To confirm the stepwise synthesis of LAP-PLA-PEG-PEI-(Au0)50-HA nanoparticles, TGA was

5

applied to measure the weight loss of intermediate products in the range of room temperature to

6

700 oC (Figure 1a). The pristine LAP showed only 10.9% loss before 200 oC, which can be

7

attributed to the weakly bonded water molecules absorbed within the interlayers. Compared with

8

LAP, LAP-PLA-PEG-COOH and LAP-PLA-PEG-PEI exhibited approximately 74.3% and 84.0%

9

weight loss from 200 oC to 700 oC, which corresponds to the thermal decomposition of the

10

organic molecules. Those results indicate that 74.3% PLA-PEG-COOH and 9.7% PEI have been

11

modified onto the surface of LAP. After the formation of gold nanoparticles, the weight loss of

12

LAP-PLA-PEG-PEI-(Au0)50 decreased to 80.8%, demonstrating that about 3.2% of Au are

13

loaded in the nanoparticles. For LAP-PLA-PEG-PEI-(Au0)50-HA, the weight loss of is 82.6%,

14

indicating that 1.8% HA was conjugated and the targeted LAP-PLA-PEG-PEI-(Au0)50-HA


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

Page 12 of 51

1

nanocomplexes were successfully synthesized as design. In addition, the formation of LAP-PLA-

2

PEG-COOH and LAP-PLA-PEG-PEI was further characterized by FTIR spectroscopy (Figure

3

S1, Supporting Information).

4

5

Figure 1.(a) TGA curves of LAP, LAP-PLA-PEG-COOH, LAP-PLA-PEG-PEI, LAP-PLA-

6

PEG-PEI-(Au0)50, and LAP-PLA-PEG-PEI-(Au0)50-HA nanoparticles; (b) UV-vis spectra of

7

LAP-PLA-PEG-PEI, LAP-PLA-PEG-PEI-(Au0)50, LAP-PLA-PEG-PEI-(Au0)50-HA, and LAP-

8

PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes solution.

9

Furthermore, UV-vis spectra of LAP-PLA-PEG-PEI, LAP-PLA-PEG-PEI-(Au0)50, LAP-PLA-

10

PEG-PEI-(Au0)50-HA and LAP-PLA-PEG-PEI-(Au0)50-HA/DOX were measured in Figure 1b.

11

Compared with LAP-PLA-PEG-PEI, a strong surface plasmon resonance (SPR) peak at around

12

520 nm can be clearly seen at the spectrum of LAP-PLA-PEG-PEI-(Au0)50, indicating the

13

formation of gold nanoparticles. After the loading of DOX, the LAP-PLA-PEG-PEI-(Au0)50-

14

HA/DOX nanocomplexes displayed an obvious peak at 480 nm, which is attributed to the

15

characteristic absorption of DOX.43 The DOX loading efficiency was calculated to be 91.0 ±

16

1.8% by a standard calibration curve, which is higher than that of LAP modification with PEG-

17

PLA modified LAP (85%) in previous study.36 The increase of loading efficiency of LAP-PLA-


12

Page 13 of 51

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

PEG-PEI-(Au0)50-HA nanoparticles may be due to the drug loading capacity of the PEI

2

component modified on surface of LAP.30 These results demonstrated that DOX were

3

successfully loaded in the LAP-PLA-PEG-PEI-(Au0)50-HA nanoparticles with a high loading

4

efficiency.

5

The morphology and size of Au NPs in nanocomplexes were investigated by TEM (Figure 2).

6

Pristine LAP displayed as circle shadows with a mean diameter of 25 nm as shown in literature

7

(Figure 2a).44 After the synthesis of Au NPs, dark dots appeared both on the surface and

8

surroundings of LAP nanodisks (Figure 2b) and a broad view image of the nanocomplexes is

9

shown in Figure S2. In the inserted high-resolution TEM image, the lattices of Au crystals can be

10

clearly observed, indicating the crystal structure of gold nanoparticles in LAP-PLA-PEG-PEI-

11

(Au0)50-HA/DOX nanocomplexes. And the mean diameter of Au NPs is about 4.6±1.4 nm

12

(Figure 2c). Energy dispersive spectroscopy (EDS) analysis illustrated the existence of Au, Mg,

13

Si, C and O elements, which further confirms the formation of LAP-PLA-PEG-PEI-(Au0)50-

14

HA/DOX nanocomplexes. The existence of Cu element should be assigned to the copper grid

15

used for TEM sample preparation.


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

Page 14 of 51

1

2

Figure 2. TEM images of (a) LAP and (b) LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

3

nanocomplexes, (c) size distribution histogram of Au NPs, (d) EDS spectrum of LAP-PLA-PEG-

4

PEI-(Au0)50-HA/DOX nanocomplexes.

5

Dynamic light scattering was used to assess the hydrodynamic diameters and zeta potentials of

6

pristine LAP, LAP-PLA-PEG-COOH, LAP-PLA-PEG-PEI, LAP-PLA-PEG-PEI-(Au0)50, LAP-

7

PLA-PEG-PEI-(Au0)50-HA

8

assembling of PLA-PEG-COOH, the hydrodynamic diameter of LAP increased from 81.8 nm to

9

265.8 nm, and at the mean time the surface potential decreased from -33.7 mV to -15.6 mV due

and


(Table

1).

After


14

Page 15 of 51

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

to the shield of negative charge on LAP by PLA-PEG. When the hyperbranched PEI were

2

modified on surface, LAP-PLA-PEG-PEI displayed positive charged surface at 34.9 mV and a

3

slight increased size of 288.6 nm, indicating the successful conjugation of PEI. The formation of

4

gold nanoparticles has neglectable influence on the surface charge (33.7 mV) and size (324.8 nm)

5

of nanoparticles. After further conjugation of HA, the size of LAP-PLA-PEG-PEI-(Au0)50-HA

6

increased to 432.8 nm, and the surface potential becomes negative as -18.5 mV, confirming the

7

successful modification of HA. Finally, the drug-loaded LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

8

showed a mean diameter of 546.7 nm and -1.90 mV of surface charge. To verify the colloidal

9

stability of nanocomplexes, the UV-vis spectra of the LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

10

nanocomplexes under a pH range of 5-8, different temperatures (4-50 oC) and different solvents

11

(water, PBS, and FBS) were investigated(Figure S3, Supporting Information). The curves of

12

nanocomplexes under different conditions do not show obvious change, indicating that the LAP-

13

PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes have good colloidal stability at different pH,

14

temperature conditions and different solvents.42 This is essential for their applications as a

15

theranostic nanoplatform in tumor diagnosis and treatment.

16

Table 1. Zeta potentials and hydrodynamic sizes of LAP, LAP-PLA-PEG-COOH, LAP-PLA-

17

PEG-PEI, LAP-PLA-PEG-PEI-(Au0)50, LAP-PLA-PEG-PEI-(Au0)50-HA, and LAP-PLA-PEG-

18

PEI-(Au0)50-HA/DOX nanocomplexes. The data are expressed as mean ± S.D (n = 3). Materials

Zeta (mV)

potential Hydrodynamic size (nm)

Polydispersity index (PDI)

LAP

-33.7 ± 2.4

81.8 ± 2.8

0.557 ± 0.014

LAP-PLA-PEG-COOH

-15.6 ± 2.1

265.8 ± 1.4

0.309 ± 0.046

LAP-PLA-PEG-PEI

34.9 ± 0.8

288.6 ± 7.9

0.505 ± 0.038


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

Page 16 of 51

LAP-PLA-PEG-PEI-(Au0)50

33.7 ± 0.9

324.8 ± 9.6

0.451 ± 0.056

LAP-PLA-PEG-PEI-(Au0)50-HA

-18.5 ± 0.2

432.8 ± 11.7

0.471 ± 0.026


-1.9 ± 0.4

546.7 ± 20.1

0.430 ± 0.064

1

2

In vitro release kinetics. As a smart drug delivery system, the anticancer drug should be

3

released more rapidly in tumor site with slight acid than under physiological conditions in order

4

to inhibit the growth of tumors effectively and lower the side effect to normal tissues. To

5

evaluate the release performance of DOX from nanocomplexes, buffer solutions of pH = 5.0 and

6

pH = 7.4 were selected to imitate the environment of the tumor and normal tissue, respectively.45

7

Figure 3a shows the release profile of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes.

8

The release of DOX from nanocomplexes exhibited a similar process under different pH

9

conditions that began with an initial burst release in the first 4 h followed by a continued-release

10

pattern. After 72 h, about 35.1 ± 1.2% of DOX were released from LAP-PLA-PEG-PEI-(Au0)50-

11

HA/DOX nanocomplexes under pH = 5.0, while only about 11.8 ± 0.9% of DOX were released

12

at pH = 7.4. Therefore, LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes could release

13

DOX in a sustained and pH-sensitive release manner. The higher DOX release rate under acidic

14

condition (pH = 5.0) than under physiological condition (pH = 7.4) should be associated with the

15

pH-dependent water solubility of DOX, as illustrated in previous study.35,

16

LAP/DOX,37 this system displayed a fast release in the beginning period of 2 h (20% vs 5%).

17

This may be due to some DOX molecules loaded in hyperbranched PEI, which may be easier to

18

release in the solution than those encapsulated by LAP nanodisks.

38

Compared with


16

Page 17 of 51

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

2

Figure 3. (a) Cumulative release of DOX from the LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

3

nanocomplexes in PBS (pH = 7.4) and acetate buffer (pH = 5.0) at 37 oC. The data are expressed

4

as mean ± S.D (n = 3); (b) CCK-8 assay of HeLa cells treated with LAP-PLA-PEG-PEI-(Au0)50-

5

HA nanoparticles, LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes, and free DOX at

6

different DOX concentrations for 24 h, respectively. HeLa cells treated with PBS buffer were

7

used as control (n = 5).

8

In vitro antitumor efficacy. The in vitro therapeutic activity of the LAP-PLA-PEG-PEI-

9

(Au0)50-HA/DOX nanocomplexes was assessed by CCK-8 assay of HeLa cell treated with

10

nanocomplexes. As shown in Figure 3b, the viability of HeLa cells treated with the LAP-PLA-

11

PEG-PEI-(Au0)50-HA nanoparticles is about 85%-90% in the studied concentration range,

12

indicating the good biocompatibility of nanocarriers. In contrast, the viability of HeLa cells

13

treated with LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes decreased dramatically with

14

the increase of DOX concentration. This result suggests that the antitumor efficacy of LAP-PLA-

15

PEG-PEI-(Au0)50-HA/DOX nanocomplexes are solely associated with the encapsulated DOX.

16

Moreover, the half-maximal inhibitory concentration (IC50) of nanocomplexes (5.8 µg•mL-1) is

17

higher than that of free DOX (1.8 µg•mL-1). This is possibly due to the fact that the concentration


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

Page 18 of 51

1

of DOX released from nanocomplexes is lower than that of free DOX due to the slow drug

2

release rate.30 Meanwhile, the cytotoxicity of LAP-PLA-PEG-PEI-(Au0)50-HA and LAP-PLA-

3

PEG-PEI-(Au0)50-HA/DOX nanocomplexes were confirmed via morphology observation. As

4

shown in Figure S4, LAP-PLA-PEG-PEI-(Au0)50-HA nanoparticles do not display apparent

5

cytotoxicity at the studied concentration. In contrast, the number of HeLa cells gradually

6

decrease with the increase of DOX concentration, confirming the antitumor cytotoxicity of LAP-

7

PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes (Figure S5, Supporting Information). In

8

addition, normal cells (mouse L929 fibroblastic cell line) were treated with LAP-PLA-PEG-PEI-

9

(Au0)50-HA/DOX nanocomplexes under the same experimental condition, and limited inhibition

10

of cell proliferation was observed, demonstrating the specific targeting effect of LAP-PLA-PEG-

11

PEI-(Au0)50-HA/DOX nanocomplexes (Figure S6 and Figure S7, Supporting Information).

12

Specific targeting of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes to CD44

13

receptor-overexpressing cancer cells. HA was generally considered as an attractive targeting

14

ligand due to its specific targeting to CD44 receptor-overexpressing cancer cells.46 In this study,

15

the targeting efficiency of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes were verified

16

by flow cytometry (Figure 4 and Figure S8, Supporting Information). HeLa-HCD44 and HeLa-

17

LCD44 cells were treated with nanocomplexes at different DOX concentrations for 4 h, then

18

washed, suspended in PBS solution, and finally subjected to flow cytometric analysis. For

19

comparison, the cells treated with PBS are used as control. It is clear that with the increase of

20

DOX concentration, the mean fluorescence of HeLa-HCD44 cells and HeLa-LCD44 cells

21

display an obvious enhancement. More importantly, the mean fluorescence of HeLa-HCD44

22

cells is higher than that of HeLa-LCD44 cells (p < 0.001) at the same DOX concentration,

23

indicating the higher uptake of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes by HeLa-


18

Page 19 of 51

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

HCD44 cells. This could be due to the HA-mediated targeting to cancer cells overexpressing

2

CD44 receptors, in agreement with previous study.30

3

4

Figure 4. Flow cytometric analysis of the HeLa-LCD44 or HeLa-HCD44 cells incubated with

5

LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes at different DOX concentrations for 4 h,

6

respectively. The data are expressed as mean ± S.D.(n = 3).

7

The confocal microscope was further used to confirm the specific cellular uptake of the LAP-

8

PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes by CD44-overexpressed cancer cells (Figure

9

5). It is clear that control cells treated with PBS only display DAPI-counterstained blue

10

fluorescence of cell nuclei. At the same DOX concentration, HeLa-LCD44 cells display quite

11

weak red fluorescence signals, while HeLa-HCD44 cells treated with nanocomplexes are able to

12

exhibit the strong red fluorescence signals in the cytoplasm and cell nuclei. These results

13

demonstrate that LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes are able to specifically

14

deliver DOX to CD44-overexpressed cancer cells, thereby exerting enhanced therapeutic

15

efficacy to the targeted cancer cells.


19


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

Page 20 of 51

1

2

Figure 5. Confocal microscopy images of the (b) HeLa-LCD44 or (c) HeLa-HCD44 cells

3

incubated with LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes at the DOX concentration

4

of 15 µg•mL-1 for 4 h, respectively. HeLa cells treated with (a) PBS were used as control.

5

In vivo antitumor efficacy. A xenografted HeLa tumor model was established to investigate

6

the in vivo therapeutic efficacy of the LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes.

7

The concentration of DOX in nanocomplexes group and free DOX group was 1.45 mg•mL-1, and

8

saline group was set as control. In order to verify the targeted therapy effect of nanocomplexes,

9

HA+nanocomplexes (HA-block) group was set by intratumoral injection of HA solution 2 h

10

before nanocomplexes in order to block the specific interaction between HA on nanocomplexes

11

and the CD44 receptors on HeLa cells. The treatment protocol was designed as intratumoral

12

injection three times once a week (on day 0, 7 and 14), and the tumor size of mice were


20

Page 21 of 51

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

measured (Figure 6a). After 21 days treatment, the volume of tumors treated with saline solution

2

is about 7 times higher than before treatment, while the tumors treated with nanocomplexes and

3

HA+nanocomplexes groups displayed only about 2 times and 4 times respectively, indicating the

4

effective inhibition of tumor growth by the formed nanocomplexes. And the better therapeutic

5

effect of nanocomplexes group over HA-block group may be due to that HA modified on the

6

surface of nanocomplexes may mediate the efficient targeting and uptake of nanocomplexes to

7

cancer cells overexpressing CD44 receptors. It is interesting that at the same dose of DOX, free

8

DOX group caused a decrease in tumor size and exhibited the best therapeutic effect. This may

9

take advantage of intratumoral injection, which may focus drug molecules in tumor site and

10

improve the inhibition effect of DOX. And the less therapeutic effect of the LAP-PLA-PEG-PEI-

11

(Au0)50-HA/DOX nanocomplexes may be due to the gradual release of DOX and the lower

12

practical drug concentration in tumor in the short time.

13

14

Figure 6.(a) The relative tumor volume, (b) body weight, and (c) survival rate of mice bearing

15

HeLa xenografted tumors after the intratumoral injection of saline, free DOX, LAP-PLA-PEG-

16

PEI-(Au0)50-HA/DOX ([Au] = 10 mM, [DOX] = 1.45 mg•mL-1, 0.1 mL PBS) with the tumor

17

region pre-injection of free HA (2 mM, 0.1 mL PBS) for 2 h (HA + Nanocomplexes), and LAP-

18

PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes ([Au] = 10 mM, [DOX] = 1.45 mg•mL-1, 0.1


21


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

Page 22 of 51

1

mL PBS). The relative tumor volumes and body weight was normalized according to their initial

2

weights (Mean ± SD, n = 6).

3

Moreover, the body weights of the mice were measured once a week and the survival rates

4

were monitored in order to assess the in vivo therapeutic efficacy and toxicity of various

5

treatments to HeLa-tumor bearing mice (Figure 6b and 6c). Although the body weights for all

6

four groups have no obvious change during 21 days treatment, no mouse is alive in the saline

7

group after 80 days. And only 50% of mice in free DOX group are survived, which are much

8

lower than the nanocomplexes group (83.3%) and the HA+nanocomplexes group (66.7%).

9

Therefore, compared with free DOX, the LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes

10

may not inhibit cancer growth immediately after administration, but they could suppress the

11

growth in a sustained manner and prolong the survival time due to its targeting modification and

12

controlled release property.

13

The in vivo therapeutic efficacy of different groups can be further confirmed via H&E staining

14

(Figure 7a). Compared with the whole blue stained tissue in saline treated tumor section, part of

15

tumor sections treated with free DOX, the nanocomplexes and HA+nanocomplexes are pink

16

stained, indicating that portion of the tumor undergo necrosis. More importantly, the pink stained

17

part in the nanocomplexes group treated section (necrosis) is similar to that of free DOX group

18

and larger than that of HA+nanocomplexes group. This result indicates that the nanocomplexes

19

group could inhibit the growth of cancer cells overexpressing CD44 receptors more effectively

20

than HA-block group due to the targeting agent HA modified on the surface. Moreover, TUNEL

21

staining of the tumors were applied to detect DNA fragments of cell apoptosis via labeling the

22

terminal ends of nucleic acids(Figure 7b).47 Brown staining cells indicate TUNEL-positive cells,

23

and the cell apoptosis rate of tumor is calculated. The saline group shows the lowest apoptosis


22

Page 23 of 51

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

rate of 23.9% among all groups, indicating the active proliferation of the tumor. And the cell

2

apoptosis rate of HA+nanocomplexes group (58.9%) is much lower than nanocomplexes group

3

(73.7%) and free DOX (80.1%). This result demonstrated the in vivo targeted therapeutic

4

efficacy of the LAP-PLA-PEG-PEI-(Au0)50-HA/DOX, consistent with the H&E staining result. It

5

is worth noting that free DOX group shows a neglectable difference in comparison with the

6

nanocomplexes group, partially owing to its high practical concentration of DOX in tumor by

7

intratumoral injection. In conclusion, considering their sustained therapeutic effect, less toxic

8

side effect and longer survival rate, the LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes

9

may be developed as a potential targeted drug delivery system in tumor therapy.

10

11

Figure 7. Representative H&E staining images, TUNEL assay images and apoptosis rate of

12

xenografted HeLa tumors treated with saline, free DOX, LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

13

([Au] = 10 mM, [DOX] = 1.45 mg•mL-1, 0.1 mL PBS) with the tumor region pre-injection of

14

free HA (2 mM, 0.1 mL PBS) for 2 h (HA + Nanocomplexes), and LAP-PLA-PEG-PEI-(Au0)50-

15

HA/DOX nanocomplexes ([Au] = 10 mM, [DOX] = 1.45 mg•mL-1, 0.1 mL PBS). The data of

16

apoptosis rate is the TUNEL picture from the same slice. The scale bars of H&E staining images

17

and TUNEL assay images represent 200 µm.


23


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

Page 24 of 51

1

In vitro and in vivo targeted CT imaging of cancer. Gold NPs have been used as CT

2

imaging agents in recent studies due to their better X-ray attenuation property and longer

3

circulation time than conventional iodine-based CT contrast agent.27 In this work, the potential

4

application of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes in CT imaging was also

5

investigated. The CT images and CT values of nanocomplexes were measured by a GE

6

Discovery STE PET/CT system (Figure S9, Supporting Information), and the Au concentration

7

in nanocomplexes solution was identified by ICP. With the increase of Au concentration, the CT

8

images of nanocomplexes solution become brighter, indicating the stronger signal in CT imaging.

9

And the CT value of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes solution increases

10

linearly with the Au concentration as demonstrated in previous studies,27,

11

potential applications as contrast agents in CT imaging.

48

illustrating its

12

Considering the specific targeting property of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

13

nanocomplexes to CD44-overexpressed cancer cells, the possibility of nanocomoplexes as

14

targeted CT contrast agents is investigated. Firstly, CT images of HeLa-HCD44 and HeLa-

15

LCD44 cells treated with the nanocomplexes for 4 h were collected. It is clear that the CT

16

images of cell pallets become brighter with the increase of Au concentration (Figure 8a),

17

indicating the high uptake of nanocomplexes by cancer cells. Furthermore, the quantitative CT

18

values of different cells were also measured (Figure 8b). The CT values of both HeLa-HCD44

19

cells and HeLa-LCD44 cells treated with the nanocomplexes are much higher than those of HeLa

20

cells treated with PBS solution. It is worth to mention that the CT value of HeLa-HCD44 cells is

21

significantly higher than that of HeLa-LCD44 cells at the same Au concentration. This result

22

confirms that the modification of HA renders the developed nanocomplexes with targeting

23

specificity to CD44-overexpresed cancer cells.


24

Page 25 of 51

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

2

Figure 8. (a) CT images and (b) CT value of the HeLa-LCD44 or HeLa-HCD44 cells incubated

3

with LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes at different Au concentrations (0, 10,

4

20, 40, 80, and 150µM, respectively) for 4 h.

5

During the evaluation of in vivo antitumor efficacy of LAP-PLA-PEG-PEI-(Au0)50-HA/DOX

6

nanocomplexes, CT images of mice before and 10 min after each treatment were also collected

7

(Figure 9a). The saline group was used as control. After each treatment, both the nanocomplexes

8

group and HA+nanocomplexes (HA-block) group show brighter CT images and higher CT

9

values in tumor due to the immediate intratumoral injection of LAP-PLA-PEG-PEI-(Au0)50-

10

HA/DOX nanocomplexes. It is worth noting that on day 7 and day 14 before injection, the tumor

11

CT value of the nanocomplexes group is always significantly higher than the HA-block group

12

(p

A LAPONITEÂ®-polyethylenimine based... (PDF Download Available)

Recommend Documents