In Vivo Antitumor Activity of Folate-Conjugated Cholic Acid

Subscriber access provided by University of British Columbia Library

Article

The in vivo anti-tumor activity of folate-conjugated cholic acid-polyethylenimine micelles for the co-delivery of doxorubicin and siRNA to colorectal adenocarcinomas Muhammad Wahab Amjad, Mohd Cairul Iqbal Mohd Amin, Haliza Katas, Adeel Masood Butt, PRASHANT KESHARWANI, and Arun K Iyer Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00827 • Publication Date (Web): 14 Nov 2015 Downloaded from http://pubs.acs.org on November 20, 2015

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.

Molecular Pharmaceutics 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 40

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

Molecular Pharmaceutics

1

The in vivo anti-tumor activity of folate-conjugated cholic acid-polyethylenimine micelles

2

for the co-delivery of doxorubicin and siRNA to colorectal adenocarcinomas

3

Muhammad Wahab Amjada, Mohd Cairul Iqbal Mohd Amina*, Haliza Katasa, Adeel Masood

4

Butta, Prashant Kesharwanib, Arun K. Iyerb

5

a

6

Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia

7

b

8

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health

9

Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA

Center for Drug Delivery Research, Faculty of Pharmacy, Universiti Kebangsaan Malaysia,

Use-inspired Biomaterials & Integrated Nano Delivery (U-BiND) Systems Laboratory,

10

11

12

13

14

15

*Corresponding author: Center for Drug Delivery Research, Faculty of Pharmacy, Universiti

16

Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia.

17

E-mail address: [email protected];

18

Tel: +603-9289 7690;

19

Fax: +603-2698 3271

ACS Paragon Plus Environment


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

Abstract

2

Multidrug resistance poses a great challenge to cancer treatment. In order to improve the

3

targeting and co-delivery of small interfering RNA (siRNA) and doxorubicin, and to overcome

4

multidrug resistance, we conjugated cholic acid-polyethylenimine polymer with folic acid,

5

forming CA-PEI-FA micelles. CA-PEI-FA exhibited a low critical micelle concentration (80

6

µM), small average particle size (150 nm), and positive zeta potential (+ 12 mV). They showed

7

high entrapment efficiency for doxorubicin (61.2 ± 1.7%, w/w), forming D-CA-PEI-FA, and for

8

siRNA, forming D-CA-PEI-FA-S. X-ray photoelectron spectroscopic analysis revealed the

9

presence of external FA on D-CA-PEI-FA micelles. About 25% doxorubicin was released within

10

24 h at pH 7.4, while more than 30% release was observed at pH 5. The presence of FA

11

enhanced micelle anti-tumor activity. The D-CA-PEI-FA and D-CA-PEI-FA-S micelles inhibited

12

tumor growth in vivo. No significant differences between their in vitro cytotoxic activities or

13

their in vivo anti-tumor effects were observed, indicating that the siRNA co-loading did not

14

significantly increase the anti-tumor activity. Histological analysis revealed that tumor tissues

15

from mice treated with D-CA-PEI-FA or D-CA-PEI-FA-S showed the lowest cancer cell density

16

and the highest levels of apoptosis and necrosis. Similarly, the livers of these mice exhibited the

17

lowest level of dihydropyrimidine dehydrogenase among all treated groups. The lowest serum

18

vascular endothelial growth factor level (VEGF) (24.4 pg/mL) was observed in mice treated with

19

D-CA-PEI-FA-S micelles using siRNA targeting VEGF. These findings indicated that the

20

developed CA-PEI-FA nanoconjugate has the potential to achieve targeted co-delivery of drugs

21

and siRNA.

22

Keywords: Micelle, nanoparticle, in vivo tumor targeting, electron microscopy, drug delivery,

23

cytotoxicity


Page 2 of 40

Page 3 of 40

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

Introduction

2

Numerous studies have exhibited the therapeutic potential of RNAi in the treatment of several

3

diseases including viral infection and cancer.1,2 Apart from this prospective, an effective delivery

4

is vital for the applications of RNAi.3 Presently, non-viral delivery systems such as lipids (e.g.

5

lipofectamine) and cationic polymers, which were also used to carry plasmid DNA (pDNA), are

6

commonly employed as siRNA delivery agents.4 In contrast to cationic lipids, non-viral

7

polymeric vectors possess numerous benefits regarding physiological stability, easy to produce

8

on large scale and safety. So far, a range of natural and synthetic polymers have been explored as

9

siRNA or gene delivery vectors such as poly(ethyleneimine) (PEI)5, poly(L-lysine)6, chitosan7,

10

and polyamidoamine dendrimers.8 PEI has been recognized as the most useful carrier among the

11

natural and synthetic polymers due to its properties of exhibiting stable complexation with

12

siRNA and its exceptional ‘proton sponge effect’ for nanocomplexes.9

13

Although a huge number of drugs e.g doxorubicin and paclitaxel have showed their

14

anticancer strength, but these drugs are facing enormous challenges such as low solubility in

15

water, multi-drug resistance, and off-target effects, in their clinical trials. To overcome these

16

challenges polymeric micellar system composed of amphiphilic block copolymers have been

17

widely studied to enter in clinical trials10,11 or attaining clinical approval (i.e., Genexol in Korea).

18

The development of multifunctional carriers for the co-delivery of gene (siRNA), and

19

anti-cancer drugs has led to the current progress in nanomedicine-derived cancer therapy.12 Thus

20

far, several promising co-delivery systems have been developed using polymeric13, liposomal14,

21

and silica-based15 cationic nanoparticles. Amongst many findings, co-delivery of reporter gene

22

and an anticancer drug has been developed successfully in vitro by using copolymers of



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

amphiphilic nature e.g. PEI and poly(e-caprolactone).16 Inspired by these reports, in this study, a

2

versatile micellar system of PEI and cholic acid (CA) was established for the co-delivery of

3

siRNA and doxorubicin. CA, a bile acid synthesized from cholesterol, is an amphiphilic steroid

4

molecule that self-assembles into micelles above critical micelle concentration (CMC). Bile

5

acids, along with the phospholipids alter the permeability of cell membranes. A synthetic vector,

6

PEI, is commonly used in the delivery of genes, because of its ability to localize in the nucleus,

7

worth mentioning transfection efficiency, potential to condense nucleic acid and capacity to

8

escape endosomes. These factors encouraged us to synthesize CA-PEI polymeric micelles. The

9

ability of CA-PEI micelles to co-deliver siRNA targeting the multidrug resistance (MDR) gene

10

and doxorubicin was investigated. MDR is the main obstacle to the successful delivery of

11

chemotherapeutic agents because multidrug resistance can be associated with increased drug

12

efflux, activation of the detoxification system, DNA repair, and blockage of apoptosis.17

13

Although several attempts have been made to reverse MDR using nanotechnology18,19 and

14

RNAi-based20 approaches, complete prevention of MDR remains challenging.

15

Folic acid (FA) was attached to CA-PEI micelles in order to assist their internalization in

16

cancer cells. The expression of folate receptor was shown to be elevated in numerous tumor

17

types and that the FA bound to this receptor on the surface of cancer cell with strong affinity21;

18

this ability makes FA an ideal option to be used for the delivery of micelles to target tumor cells.

19

In this study, we assessed the anti-tumor potential of CA-PEI-FA in cell cultures and in nude

20

mice bearing human colorectal adenocarcinoma (DLD-1), in order to investigate in vivo co-

21

delivery efficiency and tumor targeting.

22


Page 4 of 40

Page 5 of 40

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

Experimental section

2

Materials, cell culture, and animals

3

CA, PEI (average molecular weight [MW] approximately 1300), N-hydroxysuccinimide (NHS),

4

FA, N,N′-dicyclohexylcarbodiimide (DCC), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

5

(EDC), hydrochloric acid (HCl), tetrahydrofuran, triethylamine, chloroform, methanol, dimethyl

6

sulfoxide (DMSO), phosphate-buffered saline (PBS), hematoxylin for microscopy (Hist.), eosin

7

Yand dichloromethane were purchased by Sigma-Aldrich (MO, St. Louis, USA). Doxorubicin

8

hydrochloride was obtained from Calbiochem (Darmstadt, Merck KGaA, Germany). Silencer

9

Select Negative Control No. 1 and siRNA targeting the ATP-binding cassette sub-family B

10

member 1 (ABCB1) gene (sense sequence: 5ʹ-GUUUGUCUACAGUUCGUAAtt-3ʹ) were

11

procured from Life Technologies (Carlsbad, California, USA). siRNA targeting the vascular

12

endothelial growth factor (VEGF) gene (sense; 5′-GGAGUACCCUGA UGAGAUCdTdT-3′,

13

antisense;

14

(Singapore). The dialysis membrane (Spectra/Por™, molecular weight cut-off (MWCO) = 1000

15

g/mol) was supplied by Spectrum Labs (Rancho Dominguez, CA, USA). American Type Culture

16

Collection (Manassas, ATCC, VA) supplied human colorectal adenocarcinoma (DLD-1) cell

17

line. Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and

18

penicillin-streptomycin, were purchased from Life Technologies (Carlsbad, California, USA). 6-

19

8 weeks old female Nu/Nu nude mice were purchased from Charles River laboratories (Taipei,

20

Taiwan). All animal procedures were performed according to an animal care protocol approved

21

by Universiti Kebangsaan Malaysia animal ethical committee. Approximately 1x 106 DLD-1

22

cells suspended in 50 µl saline were subcutaneously inoculated into the right flanks of Nu/Nu

23

mice. The tumor was monitored for length (l) and width (w) by Vernier caliper.

5′-GAUCUCAUCAGGGUACUCCdTdT-3′)

was


purchased

from

1st

Base


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

Synthesis of the CA-PEI-FA polymer

2

The last amine group of PEI was conjugated to the side chain carboxyl group at the C-24

3

position of CA. Briefly, CA was dissolved in THF and DCC and NHS were added to activate it

4

for 8 h at 25 °C. Ice-cold n-hexane was added in the mixture to precipitate the activated CA and

5

dried for 2 h at 40°C in an oven. Incubation of PEI and activated CA in dichloromethane for 15 h

6

resulted in the formation of CA-PEI conjugate. Rotary evaporator was used to dry the conjugate.

7

Dilute HCl was used to dissolve the conjugate, which was subsequently precipitated with ice-

8

cold acetone. Afterwards, deionized water was used to mix the conjugate, followed by filtration

9

and freeze-drying to obtain CA-PEI conjugate. Three CA:PEI molar ratios (1:1, 1:3, and 3:1)

10

were used to synthesize the conjugates. Carbodiimide reaction facilitated the attachment of FA to

11

amino groups of PEI on the surface of the conjugate.22

12

Briefly, FA (40 mg) was dispersed into a mixture of triethylamine (TEA, 0.5 mL), anhydrous

13

DMSO (5 mL) and activated under anhydrous nitrogen environment by equivalent amounts of

14

NHS and EDC at room temperature for 2h. CA-PEI (3:1, 100 mg) was added in 25 mL of

15

distilled water, followed by dilution with 25 mL methanol and stirred until the optically

16

transparent solutions were obtained, subsequent to the addition of activated FA drop wise to CA-

17

PEI solution. The mixture was stirred under nitrogen environment for 24h at room temperature to

18

bind FA onto PEI molecules, and reaction was terminated by titrating it with 0.1M sodium

19

hydroxide (NaOH) at pH 9.0. The final mixture was first dialyzed for 3 days against phosphate

20

buffer saline (PBS) at pH 7.4 to eliminate surplus unreacted substrates and later dialyzed for 3

21

days against distilled water. The polymer was isolated by lyophilization. All processes were

22

performed in the dark and the number of moles of FA was kept constant for all molar ratios (1:1,

23

1:3 and 3:1) of CA:PEI.


Page 6 of 40

Page 7 of 40

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

Determination of the CMC of CA-PEI-FA

2

The CMC of surfactants can be determined using various techniques such as conductivity,

3

surface tension, and fluorescence measurements.23-25 Dynamic light scattering (DLS) is a

4

technique well suited for CMC determination. CA-PEI-FA (1 mg/mL in deionized water) was

5

10-fold serial diluted in separate Eppendorf tubes. All dilutions were analyzed on a Zetasizer

6

Nano ZS (Malvern Instruments, Malvern, UK) using a scattering angle of 90° at 37°C. The CA-

7

PEI-FA concentration was plotted against the intensity of scattered light (kilo counts/s). This

8

method was used for all molar ratios of CA-PEI-FA and all measurements were conducted in

9

triplicate.

10

Preparation of the CA-PEI-FA polymeric micelles

11

CA-PEI-FA micelles were prepared by probe sonication for 3 min. The tip of the sonicator was

12

placed directly into the micelle dispersion, which was immersed in an ice bath.

13

Fourier transform infrared (FTIR) spectroscopy

14

To assess the functional groups, CA-PEI-FA was characterized using the potassium bromide

15

(KBr) pellet method on a FTIR spectrophotometer (Spectrum 100; PerkinElmer, Waltham, MA,

16

USA). Prior to the experiment, KBr pellets were dried for 1 h at 50°C in an oven. CA-PEI-FA (1

17

mg) was mixed with 100 mg dried KBr and ground in a mortar to homogenize the powder. A

18

thin transparent pellet was then prepared using a stainless steel disc and plunger set (die). Each

19

sample pellet was placed in the FTIR spectrophotometer and the instrument was run for 16 scans

20

at a resolution of 4 cm-1. The spectra were recorded in the range of 3700-700 cm-1.

21

Nuclear magnetic resonance (1H-NMR) and ultraviolet-visible (UV-vis) spectroscopy



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

Two milligrams of CA-PEI-FA were dissolved in 0.6-0.7 mL deuterium oxide (D2O) in a small

2

beaker. The solution was subsequently filtered through a Pasteur pipette equipped with a glass

3

wool plug, and ejected into an NMR tube to a depth of approximately 4 cm prior to analysis in an

4

NMR spectrophotometer (Bruker Avance III, FT-NMR 600 MHz equipped with a cryoprobe,

5

Billerica, MA, USA). The resultant spectrum was analyzed to confirm the presence of CA-PEI-

6

FA. The degree of substitution was determined by dividing the total integral values of five FA

7

aromatic protons in the region of 6.5–9.0 ppm by the protons of the PEI hydrocarbon backbone

8

at 2.6 ppm.

9

The degree of conjugation of FA was determined by UV-vis spectroscopy. FA solutions

10

of known concentrations were prepared in D2O. Their respective UV absorbance was recorded

11

and plotted against the FA concentration to form a calibration curve. D2O was used to dissolve

12

ten milligrams of each sample and the UV absorbance was recorded at 363 nm to calculate the

13

level of FA from the calibration curve. The degree of conjugation of FA on a weight to weight

14

basis was calculated using the following equation:

Conjugation of folic acid wt% =

Weight of FA in conjugate × 100 Total weight of conjugate

15

Preparation of doxorubicin-loaded CA-PEI-FA (D-CA-PEI-FA) micelles

16

D-CA-PEI-FA micelles were prepared as follows: Doxorubicin hydrochloride (2.5 mg) was

17

dissolved in chloroform and mixed with 2 µL of TEA. CA-PEI-FA was dissolved in methanol. In

18

a glass beaker, the CA-PEI-FA and doxorubicin solutions were mixed and placed in dark for 1

19

day. The mixture was added dropwise to deionized water under ultrasonic agitation (using a

20

Sonifier®, Branson Ultrasonics Co., Danbury, CT, USA) for 10 min at power level 3. Solvents


Page 8 of 40

Page 9 of 40

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

such as methanol and chloroform were subsequently removed with the help of a rotary

2

evaporator. To eliminate unloaded doxorubicin, D-CA-PEI-FA was dialyzed for 24 h at 20°C

3

against 1 L deionized water using a dialysis membrane bag. For the first 12 h, the water was

4

changed after every 2 h and later every 6 h. D-CA-PEI-FA was freeze-dried immediately after

5

dialysis was completed. A calibration curve was constructed using pure doxorubicin. Based on

6

the curve, the amount of doxorubicin present in D-CA-PEI-FA micelles was calculated by

7

dissolving them in 4 mL co-solvent of a methanol and DMSO (1:1) and recording the absorbance

8

at 482.5 nm on spectrophotometer. 2 mg D-CA-PEI-FA was dissolved in D2O (0.6-0.7 mL) and

9

ejected into an NMR tube. The samples were run on an NMR spectrophotometer.

10

Preparation of doxorubicin-loaded and siRNA complexed CA-PEI-FA (D-CA-PEI-FA-S)

11

micelles

12

D-CA-PEI-FA polymer was added to PBS to form micelles, with subsequent addition of siRNA.

13

For 15 s, the mixture was vortexed and placed in dark for 30 min at room temperature to

14

facilitate the complexation among the micelles and siRNA. For D-CA-PEI-FA-S molar ratios

15

1:1, 3:1 and 1:3, the nitrogen to phosphate ratios were 5, 5, and 15, correspondingly. The siRNA

16

loading efficiency (%) was calculated by determining the concentration of free siRNA in

17

supernatant after centrifugation (35,000 × g, 15 min). Using a UV-vis spectrophotometer, the

18

concentration of siRNA was determined at a wavelength of 260 nm. The supernatant taken from

19

the D-CA-PEI-FA micelles served as reference.

20

Determination of siRNA complexation by the gel retardation assay

21

The binding efficiency of siRNA to CA-PEI-FA and D-CA-PEI-FA was determined by gel

22

electrophoresis. 20 µL each of CA-PEI-FA-S and D-CA-PEI-FA-S were added in the wells of



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

pre-casted agarose gel (4% w/v) plate with SYBR Green. CA-PEI-FA and D-CA-PEI-FA

2

micelles were used as negative while free unloaded siRNA was used as a positive control. The

3

duration of electrophoresis was 26 min, as per supplier’s procedure (2005-2006, Invitrogen,

4

Waltham, MA, USA). After electrophoresis, UV transilluminator (Invitrogen) was used to

5

visualize the bands of siRNA.

6

Particle size and zeta potential measurements

7

The particle size and zeta potential of the micelles were measured by dynamic light scattering

8

technique using a Zetasizer Nano ZS (Malvern Instruments) at 25°C. Five measurements of

9

particle size and zeta potential each were recorded for all samples and their mean were calculated

Page 10 of 40

10

to document the final results.

11

Surface chemistry

12

X-ray photoelectron spectroscopy (XPS; AXIS His-165 Ultra, Kratos Analytical, Shimadzu

13

Corp.) was used to confirm the presence of FA on the surface of D-CA-PEI-FA. In a fixed

14

transmission mode and a pass energy of 80 eV, the spectrum of binding energy was recorded

15

from 0-1100 eV.

16

In vitro release of doxorubicin

17

Freeze-dried samples (15 mg) of D-CA-PEI-FA and D-CA-PEI-FA-S were resuspended in 5 mL

18

of sodium acetate buffer solution (pH 5.0) or PBS (pH 7.4) and placed in a dialysis membrane

19

bag. The dialysis bag was transferred to a beaker containing the same 40 mL of buffer solution,

20

placed on a magnetic stirrer at 37oC. At pre-determined time intervals, the solution around the

21

dialysis bag was removed and analysed on UV-vis spectrophotometer at a wavelength of 482.5


Page 11 of 40

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

nm to determine the concentration of doxorubicin, and was substituted with fresh solution. The

2

cumulative amount of released drug was calculated, and the percentage of drug released from the

3

micelles was plotted against time.

4

In vitro cell viability analysis using CA-PEI-FA and D-CA-PEI-FA-S micelles

5

ATCC supplied the Chinese hamster lung fibroblast (V79) cell lines which were propagated in

6

DMEM supplemented with 1% penicillin-streptomycin and 10% FBS, and maintained in a

7

humidified 5% CO2:95% air atmosphere at 37 °C in an incubator. RPMI-1640 medium

8

supplemented with 1% penicillin-streptomycin and 10% FBS was used to culture DLD-1 cells.

9

The cells were maintained under a humidified 5% CO2/95% air atmosphere at 37°C.

10

The effect of CA-PEI-FA micelles on cell viability was assessed using V79 cells. The V79 cells

11

were incubated for 24 h at seeding density of 4 × 104 cells per well in a 96-well culture plate and

12

treated with increasing concentrations of CA-PEI-FA micelles starting from 31.25 to 500

13

µgmL−1 and incubated for an additional 24 h in a 5% CO2: 95% air environment at 37 °C.

14

Finally, alamarBlue® reagent (20 µL) was introduced to each well of culture plate containing

15

cells and the plate was incubated for another 4 h. The absorbance reading from each well was

16

recorded at 570 nm using a microplate reader. The following equation was used to calculate the

17

cell viability (%):

18

Cell viability (%) = A570 of treated cells/A570 of control cells × 100

19 20

The cytotoxicity of D-CA-PEI-FA-S micelles was investigated using alamarBlue® assay. DLD-1

21

cells were incubated for 48 h at a seeding density of 2 × 104 cells/well in 96-well culture plates at

22

5% CO2/95% air environment at 37 °C. The cells were treated with 200 µL of D-CA-PEI-FA-S



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 40

1

micelles containing doxorubicin at concentrations of 50, 25, 12.5, 6.25, 1.56, 0.19, and 0.09

2

µgmL−1 respectively. The plates were incubated for 24 h, followed by the introduction of

3

alamarBlue® (20 µL) to each well of the plate, and were further incubated for 4 h. The

4

absorbance reading from each well was recorded at 570 nm using a microplate reader.

5

In vivo anti-tumor effect

6

The Nu/Nu mice were housed in individually ventilated cages and maintained in an animal

7

facility that fulfilled the guidelines and requirements for biosafety level 2 at the Universiti

8

Kebangsaan, Malaysia. Mice bearing a visible DLD-1 tumor were randomly divided into saline,

9

CA-PEI, doxorubicin, D-CA-PEI, D-CA-PEI-S, D-CA-PEI-FA, and D-CA-PEI-FA-S groups,

10

which were administrated the respective treatment intravenously on day 0 and day 7 at dose of 8

11

mg/kg body weight. Body weight and tumor volumes were monitored and recorded twice weekly

12

for 20 days. Subsequently, the mice were euthanized and their tumors were removed.

13

Histology

14

For metastasis examination, tumor, lung, spleen, kidney, heart, and liver were removed, followed

15

by fixation in 10% solution of formalin and were later embedded in paraffin for staining with H

16

& E. The micrographs of these organs were taken using a microscope.

17

Enzyme-linked immunosorbent assay (ELISA)

18

ELISA was performed to measure the dihydropyrimidine dehydrogenase (DPD) concentration in

19

the liver, as per manufacturer’s protocol (Uscn, Wuhan, China). Using the recombinant DPD

20

standard curve, the DPD concentrations in liver were calculated as ng/mg of protein. An ELISA

21

was also used to detect vascular endothelial growth factor (VEGF) in the mouse sera (Life

22

Technologies), in accordance with the manufacturer’s instructions. For this experiment, two

23

separate groups of mice were treated with micellar formulations containing siRNA-targeting


Page 13 of 40

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

VEGF (D-CA-PEI-SV and D-CA-PEI-FA-SV, where SV denotes VEGF siRNA), in addition to

2

the treatment groups described above.

3

Statistical Analysis

4

All data are expressed as the mean ± the standard deviation of three readings. GraphPad Prism 5

5

(GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analyses (one-way

6

analyses of variance, ANOVA, followed by post hoc Tukey’s test for multiple comparisons) of

7

all data. The significance level was 0.05 and the data were indicated with (∗) for p < 0.05, (∗∗)

8

for p < 0.01, and (∗∗∗) for p < 0.001, correspondingly.

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

1

Results and discussion

2

FA was introduced into CA-PEI to produce CA-PEI-FA polymeric micelles for use as nano-

3

carriers to co-deliver doxorubicin and siRNA. Under anhydrous nitrogen atmosphere, the

4

carboxyl moiety of FA was activated using NHS/EDC chemistry, followed by the amide linkage-

5

coupling with amine group of PEI to yield CA-PEI-FA.

Page 14 of 40

6 7

Scheme 1. Complete synthesis scheme for CA-PEI-FA.

8

Determination of the CMC of the CA-PEI-FA polymer

9

The CMCs of CA-PEI-FA solutions containing various molar ratios of CA to PEI are shown in

10

Figure 1. CA-PEI-FA micelles possessing CA to PEI molar ratio 3:1 had the lowest CMC (80

11

µM), while the CMC was calculated as 100 µM for both 1:1 and 1:3 molar ratios of CA to PEI.


Page 15 of 40

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

As CA possesses hydrophobic steroidal nucleus, an increased level of CA might increase the

2

hydrophobic interactions among the polymer chains in the core of micelle, hence stabilizing the

3

assembly. Furthermore, the CMC of the CA-PEI-FA micelles was lower than that of the CA-PEI

4

micelles. The CMC decreases with the increase in the size of hydrophobic block, that is, micelles

5

formation takes place at lower concentration of polymer. This phenomenon is important for the

6

entrapment efficiency (EE) and drug solubilization of micelles.26 CA-PEI micelles with low

7

CMCs would be beneficial, as they are stable against precipitation in blood owing to dilution and

8

also against dissociation. Additionally, the occurrence of embolisms, which can result from high

9

polymer concentrations, could be avoided.27

10 11

Figure 1. Critical micelle concentrations of CA-PEI-FA micelles (CA:PEI=1:1, 1:3 and 3:1).

12

FTIR spectroscopy

13

The presence of fine peaks in the region of 3700-3000 cm-1 represented OH stretching,

14

confirming the presence of CA (Figure 2a). OH stretching vibrations were observed at 3524 and

15

3324 cm-1 and CH vibrations were observed at 2968, 2935, and 2872 cm-1. A sharp peak at 1715

16

cm-1 confirmed the presence of C=O of the COOH stretching. Moreover, some characteristic CA



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 40

1

characteristic peaks were observed at 1375-1401, 1329, 1288, 1242, 1121, 1092, 1078, and 1045

2

cm-1. The peaks at 3323, 3417 and 3545 cm-1 in the FA spectrum corresponded to the secondary

3

NH, NH2 and ring O-H groups. A peak with less intensity appearing at 3110 cm-1 in the FA

4

spectrum indicated O-H stretching of COOH. The most characteristic FA bands (amide I and II)

5

appeared at 1695 cm-1 and 1572 cm-1 respectively. In the CA-PEI spectrum, The peaks for N-H

6

bending, C=O absorbance, and C-H and N-H stretching appeared in the CA-PEI spectrum at

7

1574 cm-1, 1625 cm-1, 2850–2930 cm-1, and 3326 cm-1, correspondingly. The N-H bending (1574

8

cm-1) and C=O absorbance bands (1625 cm-1) overlapped, appearing as a doublet in spectrum of

9

CA-PEI. This doublet confirmed the formation of amide bond among PEI and CA.28 In the CA-

10

PEI-FA spectrum, a small shoulder peak was observed at 1700 cm-1, indicating the presence of

11

FA.29 Moreover, most FA peaks (from 2500-3600 cm-1) disappeared from the CA-PEI-FA

12

spectrum. Recently, this phenomenon was also observed by other researchers studying FA-

13

targeted micelles.29

14 15 16

Figure 2. FTIR spectra of CA, PEI, FA, CA-PEI, and CA-PEI-FA (a), HNMR spectra of CAPEI-FA (b), and D-CA-PEI-FA (c).

17

1

18

In the CA-PEI-FA 1H-NMR spectrum (Figure 2b), proton shifts appeared in the range of 1-2

19

ppm, corresponding to the characteristic CA peaks. Doublet, triplet, and multiplet peaks

H-NMR and UV-vis spectroscopy


Page 17 of 40

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

appeared between 1-2 ppm, corresponding to the characteristic CA peaks. The number of protons

2

in CA was designated by the integration values between 0.8-2 ppm. The presence of PEI was

3

indicated by the proton shifts in the region of 2.2-3.52 ppm. The occurrence of weak signals

4

(corresponding to aromatic protons of FA) between 6.6-8.7 ppm confirmed the presence of FA in

5

CA-PEI-FA.32 The concentration of FA in the CA-PEI-FA was 26%, whereas the degree of

6

substitution of FA onto CA-PEI was 26.6%, as determined by UV spectroscopy. Moreover, the

7

spectrum of D-CA-PEI-FA (Figure 2c) showed peaks that were characteristic of doxorubicin.

8

These peaks were not present in the CA-PEI-FA spectrum and were in the regions of 4.1-5.5 and

9

7.2-7.6 ppm. Similar peaks have also been observed by other researchers studying doxorubicin-

10

loaded nanoparticles.33

11

Preparation of D-CA-PEI-FA micelles

12

Hydrophobic doxorubicin was loaded in the core (CA) of CA-PEI-FA micelles. Doxorubicin’s

13

affinity for the polymeric micelles can be enhanced by including a suitable hydrophobic block in

14

copolymer. Yokoyama et al. prepared PEO-poly(L-aspartate) micelles which involved both

15

physical encapsulation and chemical binding of doxorubicin with the polymer.30 Micelles can be

16

fabricated using AB-type copolymers if one fragment of block copolymer provides adequate

17

interchain cohesive interactions in solvent. The drug-core cohesive interaction includes hydrogen

18

bonding, π-π interactions, hydrophobic and electrostatic attraction; however, hydrophobic

19

interaction is the main driving force behind the encapsulation of majority of anticancer drugs

20

because of their poor water-solubility.31 The EE of doxorubicin in the D-CA-PEI-FA micelles

21

was 61.2% (w/w).

22

Preparation of D-CA-PEI-FA-S micelles and determination of siRNA complexation by the

23

gel retardation assay



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 40

1

siRNA was complexed with CA-PEI-FA and D-CA-PEI-FA micelles. The zeta potential of CA-

2

PEI-FA and D-CA-PEI-FA micelles decreased after siRNA complexation. Greater siRNA

3

complexation was observed with CA-PEI-FA micelles than with D-CA-PEI-FA micelles, since

4

their zeta potential was more positive and siRNA complexation predominantly involves

5

electrostatic attraction. The amount of free siRNA detected was very low, signifying that

6

majority of siRNA was encapsulated onto micelles. High loading efficiency of siRNA could be

7

attributed to strong electrostatic attraction between PEI and siRNA.34

8

Agarose gel electrophoresis was used to investigate siRNA complexation with CA-PEI-FA and

9

D-CA-PEI-FA micelles (Figure 3). The mobility of siRNA in an electric field is lost due to the

10

complexation of cationic polymers with siRNA. Thus, a polymer’s ability to complex with

11

siRNA is reflected by the retardation of siRNA’s mobility in electrophoresis. The migration of

12

siRNA was completely retarded with both CA-PEI-FA-S and D-CA-PEI-FA-S, indicating

13

complete neutralization of the siRNA’s negative charge.35 Prior to siRNA complexation, CA-

14

PEI-FA micelles exhibited an average hydrodynamic size of 125 nm and a zeta potential of +7.5

15

mV. A reduction in the zeta potential (decreased by 3.5 mV to +4.02 mV) of CA-PEI-FA was

16

observed after complexation with siRNA, reflecting the neutralization of cationic CA-PEI-FA by

17

anionic siRNA. Similarly, D-CA-PEI-FA micelles exhibited an average particle size of 130 nm

18

and a zeta potential of +5.5 mV before siRNA complexation. A reduction in the zeta potential

19

(decreased by 3.36 mV to +2.14 mV) of D-CA-PEI-FA was observed after complexation with

20

siRNA. The decrease in micelle size after siRNA complexation can be attributed to electrostatic

21

attraction among PEI (highly cationic) and siRNA (highly anionic). A small positive charge and

22

size are important for electrostatic assembly and cell internalization.


Page 19 of 40

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 3

Figure 3. Gel retardation assay. Well 1 (DNA ladder), 2 (free siRNA), 3 (CA-PEI-FA), 4 (CAPEI-FA-S), and 5 (D-CA-PEI-FA-S).

4

Particle size and zeta potential analysis

5

Zeta potential36, particle size37, and receptor-mediated38 internalization are three crucial

6

determinants of cellular micelle uptake. Generally, a positive zeta potential enables the linking of

7

micelles to cells through electrostatic interactions, although a high density of cations in direct

8

contact with the cell membrane can result in cytotoxicity. The endocytosis of the nanoparticles is

9

facilitated by small particle size. The cell internalization and delivery specificity of nanoparticles

10

can be enhanced by attaching a targeting ligand to nanoparticles. The mean particle diameter of

11

freshly prepared CA-PEI-FA micelles in this study was determined by DLS (Table 1). The self-

12

aggregation behavior of CA-PEI-FA in an aqueous medium was influenced by numerous forces

13

such as hydrogen bonding between hydrophilic segments, hydrogen bonding of water molecules

14

and hydrophilic segments and the hydrophobic-hydrophobic associations between CA segments.

15

Apart from governing self-aggregation of the amphiphilic CA-PEI-FA polymer, these

16

interactions also determine the aggregate size. The micellar CA-PEI-FA solution was obtained



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 40

1

by sonication and the mean particle size obtained at all molar CA:PEI ratios was less than 200

2

nm, with a uniform distribution. The nanoparticle size between 10-100 nm is considered to be

3

ideal for passive tumor targeting.39

4

The zeta potential of CA-PEI micelles of all molar ratios was positive. The molar ratio (1:3) of

5

CA-PEI micelles containing maximum PEI content exhibited highest (positive) zeta potential

6

(+18.7 ± 1.5 mV) whereas a 3:1 molar ratio produced the lowest zeta potential. FA conjugation

7

decreased the zeta potential. Zeta potential is a vital determinant of the cellular uptake

8

mechanism and efficiency, as well as the in vivo fate of nanoparticles.40,41 Though, the optimal

9

zeta potential (e.g., negative, neutral or positive) and charge densities were documented to differ

10

between nanoparticles, as determined by their abilities to avert unspecific accumulation in

11

undesired sites, lessen non-specific clearance of nanoparticles and extend blood circulation time.

12

On the other hand, PEG-PDLLA micelles of negative and neutral charge showed no significant

13

difference in the kinetics of blood clearance. Still, micelles with negative charge remarkably

14

decreased the non-specific uptake by spleen and liver in comparison with micelles of neutral

15

charge, which was due to the presence of electrostatic repulsion between micelles (negatively

16

charged) and cell surface.42

17

Table 1. The zeta potential and average particle size of CA-PEI micelles at different molar ratios

18

and of CA-PEI-FA, CA-PEI-FA-S, D-CA-PEI-FA, and D-CA-PEI-FA-S micelles. CA-PEI 3:1

19

was used to synthesize CA-PEI-FA. Micelle formulations

Zeta potential Particle size Polydispersity index (mV)

CA-PEI 1:1

+12.2 ± 1.2

(nm) 172 ± 5.2


0.326±0.04

Page 21 of 40

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


CA-PEI 1:3

+18.7 ± 1.5

148 ± 6.7

0.287±0.04

CA-PEI 3:1

+9.2 ± 1.1

164 ± 8.6

0.296±0.02

CA-PEI-FA

+7.5 + 1.0

125 ± 7.1

0.316±0.03

CA-PEI-FA-S

+4.0 ± 1.5

120 ± 6.7

0.226±0.02

D-CA-PEI-FA

+5.5 ± 1.1

130 ± 8.6

0.357±0.04

D-CA-PEI-FA-S

+2.1 ± 1.0

118 ± 7.1

0.334±0.03

1

2

On the basis of aforementioned information, we believed that the optimal positive surface charge

3

and smaller size of the CA-PEI-FA-S and D-CA-PEI-FA-S micelles (Table 1) may be ideal for

4

adequate ligand-targeted co-delivery and cellular uptake of doxorubicin and siRNA. In addition,

5

a slight positive zeta potential might improve the in vivo stability of nanoparticle, although a high

6

positive charge may increase nanoparticle interactions with negatively charged proteins.

7

Surface chemistry

8

The elements on the surface of micelles were recognized by measuring specific binding energy

9

(eV) using XPS. The occurrence of FA on the surface of micelles can be investigated using N 1s

10

region of the XPS spectrum. Figure 4 exhibits the characteristic FA peak at 400 eV in the wide

11

scan of CA-PEI-FA and D-CA-PEI-FA. The characteristic N peak intensity was greater in CA-

12

PEI-FA than in D-CA-PEI-FA. This might be due to the interaction of some FA with

13

doxorubicin. In general, the characteristic N peaks increased in sharpness and intensity as the

14

degree of FA substitution increased. The N-containing aromatic polymer, PEI, exhibited weak π-

15

π satellite features shifted several eV from the main N peak.



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 40

1 2 3

Figure 4. The XPS wide scan spectra of the CA-PEI-FA and D-CA-PEI-FA on the left. The spectra on the right show the relative nitrogen signals at high resolution of both formulations.

4

5

In vitro release of doxorubicin

6

Doxorubicin release behaviors from D-CA-PEI-FA and D-CA-PEI-FA-S were compared at 37°C

7

in aqueous solutions of pH 5.0 and pH 7.4. The pH 5.0 was selected to simulate the acidic

8

lysosomal atmosphere. As per Figure 5, a characteristic release pattern containing two stages was

9

witnessed in all four tests i.e., an early fast release followed by a slow sustained release for up to

10

several days. Furthermore, the release of doxorubicin from both micelles was quicker at pH 5.0.

11

The quicker doxorubicin release under acidic conditions can be attributed to accelerated

12

degradation of CA core and re-protonation of doxorubicin’s amino group. A similar pH-reliant

13

release of doxorubicin was reported in other doxorubicin-loaded micelles43 and this was

14

supposed to improve intracellular release of druge following endocytic entry into tumor cells and

15

subsequent entrapment within endosomes/lysosomes. Additionally, both FA conjugation and

16

siRNA complexation were reported to delay the release of doxorubicin, as compared to that

17

observed previously using CA-PEI micelles.44


Page 23 of 40

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 3 4

Figure 5. The cumulative release of doxorubicin (%) from D-CA-PEI-FA and D-CA-PEI-FA-S micelles at pH 5 and 7.4. The release of doxorubicin from each formulation at pH 5 and 7.4 was statistically analyzed.

5

In vitro cell viability in the presence of CA-PEI-FA micelles

6

Biocompatibility is a major concern for biomaterials and we therefore performed a preliminary

7

evaluation of micellar cytotoxicity using the Alamar Blue® assay. A cell viability assay (Figure

8

6a) indicated that the tested concentrations of CA-PEI-FA micelles were non-toxic to V79 cells.

9

No marked reduction in cell viability was observed, even at a high concentration of 500 µg/mL

10

CA-PEI-FA. These results suggest that CA-PEI-FA micelles are non-toxic to normal cells.



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 40

1 2 3 4 5

Figure 6. In vitro cell viability assays after the exposure of V79 cells to CA-PEI-FA micelles (a), in vitro cytotoxicity of D-CA-PEI-FA and D-CA-PEI-FA-S micelles against DLD-1 cells after 24 h (b). In Figure (b), at each doxorubicin concentration, inhibition (%) of D-CA-PEI, D-CAPEI-FA, D-CA-PEI-FA-S were compared to doxorubin for statistical analysis.

6

In vitro cytotoxicity of D-CA-PEI-FA-S micelles

7

As per Figure 6b, the inhibition (%) of cancer cell was higher in the presence of D-CA-PEI-FA-S

8

micelles than in the presence of D-CA-PEI-FA micelles. The incorporation of doxorubicin into

9

the D-CA-PEI-FA and D-CA-PEI-FA-S micelles improved their cytotoxic potential against

10

cancer cells. The IC50 values for D-CA-PEI-FA (4.88 µg/mL) and D-CA-PEI-FA-S (3.88

11

µg/mL) micelles were lower than those determined previously for D-CA-PEI (5.85 µg/mL) or

12

free doxorubicin (10.58 µg/mL). The high IC50 of doxorubicin and low inhibition (%) of cells, in

13

contrast to the corresponding values for D-CA-PEI-FA and D-CA-PEI-FA-S micelles, may

14

reflect the removal of doxorubicin by drug efflux pumps from tumor interstitium,45 and high

15

retention and permeability of micelles in tumor. Additionally, the improved permeation of D-

16

CA-PEI-FA-S micelles increases drug delivery to the target site and hence increases the retention

17

time of doxorubicin at site of action. Both D-CA-PEI-FA and D-CA-PEI-FA-S caused the death

18

of cells in a concentration-dependent manner and there was no significant difference between


Page 25 of 40

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

their effects. This indicated that FA conjugation enhanced the activity of D-CA-PEI micelles,

2

while siRNA loading did not produce additional cytotoxicity. The in vivo cytotoxic effectiveness

3

of doxorubicin loaded onto micelles is expected to be further enhanced, owing to the EPR effect

4

of micelles. So, the adverse effects and toxicity of doxorubicin can be reduced by the selective

5

micellar uptake by cancer cells.

6

In vivo anti-tumor effect

7

Tumor growth in the saline group was rapid, with the mean tumor volume expanding from 30

8

mm3 to 1977 mm3 within 20 days. The CA-PEI micelle-treated mice showed a similar tumor

9

growth profile, reaching a tumor volume of 1937 mm3, which indicated that these micelles had

10

little physiological activity (Figure 7a). Doxorubicin and D-CA-PEI produced considerable

11

tumor inhibition. D-CA-PEI-S also inhibited tumor growth, suggesting that reducing expression

12

of the MDR gene suppressed tumor growth in vivo. D-CA-PEI-FA and D-CA-PEI-FA-S micelles

13

enhanced these tumor inhibitory effects because of FA-mediated tumor targeting. Consistent

14

with the in vitro cytotoxicity data, no significant variation among the effects of D-CA-PEI-FA

15

and D-CA-PEI-FA-S was found. This comprehensive in vivo study revealed that FA conjugation

16

enhanced the anti-tumor activity of CA-PEI micelles but showed that siRNA co-loading did not

17

significantly enhance this effect.



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 26 of 40

1 2 3 4 5

Figure 7. In vivo anti-tumor effect (Figure 7 a-b): the tumor inhibiting rate at day 21 (a), body weight of tumor-bearing mice (b). In (a), all groups were compared with doxorubicin for statistical analysis. In weight gain (%) of (b), all groups were compared with saline for statistical analysis.

6

Since body weight change is an indicator of systemic toxicity, this was measured in

7

parallel with the anti-tumor effect (Figure 7b). Mouse body weight in the CA-PEI-treated groups

8

was not significantly different from that of the saline-treated mice, indicating that these

9

nanoparticles did not cause significant systemic toxicity. The D-CA-PEI-FA- and D-CA-PEI-

10

FA-S-treated groups did not display obvious body weight fluctuations. However, their tumor

11

weights were much lower than those of the other groups, indicating that D-CA-PEI-FA and D-

12

CA-PEI-FA-S micelles did not negatively influence the health of the mice; this contrasted with

13

free doxorubicin-treated animals, which had larger tumors but showed decreased body weight.

14

This suggested that D-CA-PEI-FA-S, which possesses efficient anti-tumor activity together with

15

low systemic toxicity, can act as a promising nano-platform to co-deliver chemotherapeutic

16

drugs and nucleic acids into tumor interstitiums, resulting in a more effective cancer treatment.

17

Histology

18

The lungs, heart, liver, kidneys, spleen, and tumor of the animals were analyzed in all treatment

19

groups (Figure 7). The tumors from the saline- and CA-PEI-treated groups appeared more


Page 27 of 40

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

hypercellular and exhibited a higher level of nuclear polymorphism in the H & E-stained tissue

2

sections, as compared with those collected from the other therapeutic groups. The tumor cells

3

were polygonal, containing scanty to moderate cytoplasm and pleomorphic vesicular nuclei, with

4

a prominent nucleolus. The tumor cells were arranged in groups and islands, with occasional

5

glandular formation. The tumor could be classified as a poorly differentiated adenocarcinoma.

6

The lung sections showed intra-alveolar hemorrhages, with no tumor deposits. The myocardium

7

and endocardium sections appeared normal and no tumor was observed. The central vein of the

8

liver was normal. Moreover, the hepatocytes and portal triad were also normal, with no signs of

9

DLD-1 tumor. The spleen section showed extramedullary hematopoiesis without metastasis. The

10

kidney sections also had a normal morphology without metastasis.

11

The doxorubicin-treated group showed a tumor composed of polygonal cells containing scanty

12

cytoplasm and pleomorphic vesicular nuclei, with a prominent nucleolus. The tumor cells were

13

arranged in groups and islands with a glandular formation and there were areas of apoptosis and

14

necrosis. Increased mitosis was also observed. The tumor could be referred to as a poorly

15

differentiated adenocarcinoma. The lung section showed areas of congestion and septae with

16

edema and neutrophilic infiltration. No tumor deposit was present. The myocardium,

17

endocardium, and liver sections appeared normal, with no evidence of a tumor. The spleen

18

section showed extramedullary hematopoiesis without metastasis. The kidney had normal

19

glomeruli, tubules, and blood vessels. Apart from the tumor size, similar histology was seen in

20

the other groups, including those treated with the D-CA-PEI, D-CA-PEI-S, D-CA-PEI-FA, and

21

D-CA-PEI-FA-S micelles. D-CA-PEI-FA and D-CA-PEI-FA-S micelles showed the highest

22

anti-tumor activities and tumor tissues from these groups had the lowest cancer cell density and

23

the highest level of cell apoptosis and necrosis. This phenomenon was previously observed by



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 28 of 40

1

researchers investigating the co-delivery of anticancer drugs and siRNA with anti-tumor

2

activity.46 This indicated that the optimal size and zeta potential of these nanoparticles, FA-

3

mediated tumor targeting, and enhanced doxorubicin release in the acidic environment optimized

4

their in vivo cytotoxicity.

5 6 7

Figure 7. H & E-stained sections of organs collected from mice after treatment with the different micelle formulations.

8

Liver DPD level

9

An increase in the intra-tumoral DPD level is reported to be one of causes behind drug

10

resistance.47 DPD is extensively dispersed in various organs, particularly in liver.48 In the current

11

study, DPD protein expression was investigated in the liver tissues of the treated mice (Figure

12

8a). After induction of DLD-1 xenografts, an elevated level of DPD protein was observed in the

13

liver tissues. The level of liver DPD was decreased in doxorubicin-treated mice, in contrast to

14

saline- or CA-PEI-treated animals. This may indicate the cytotoxic potential of doxorubicin,

15

which reduced the tumor severity and hence, the DPD level decreased. However, the

16

administration of hydrophobic doxorubicin without a carrier only produced a moderate effect. D-


Page 29 of 40

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

CA-PEI showed an enhanced effect of doxorubicin on the DPD level. D-CA-PEI-S had an

2

additional synergistic effect on the DPD level, although this effect was not significantly better

3

than that of D-CA-PEI. The liver DPD levels in mice treated with D-CA-PEI-FA and D-CA-PEI-

4

FA-S were 1224.8 and 1174.6 ng/mg, respectively. As compared to saline, a significant

5

reduction in the level of DPD was seen in mice treated with D-CA-PEI-S and D-CA-PEI-FA-S

6

(Figure 8a). This could be attributed to multiple factors, such as doxorubicin, folate targeting,

7

particle size, and zeta potential.

8 9 10 11 12

Figure 8. Dihydropyrimidine dehydrogenase (DPD) expression in the liver of mice treated with saline, CA-PEI, doxorubicin, D-CA-PEI, D-CA-PEI-S, D-CA-PEI-FA, or D-CA-PEI-FA-S (a). The effect of the different micellar formulations on the expression of VEGF in serum (b). All the groups were compared with saline for statistical analysis.

13

Tumor progression and metastasis are greatly reliant on neoangiogenesis which may arise

14

either from bone marrow-derived endothelial precursor cells, circulating endothelial cells or from

15

preexisting blood vessels.49 VEGF is amongst one of the chief regulators of physiological and

16

pathological angiogenesis.50 Its expression is upregulated in several tumors, such as primary and

17

metastatic colorectal adenocarcinoma.51 VEGF via interaction with VEGFR-1 and VEGFR-2

18

was discovered to be a mitogen and survival factor for endothelial cells.52 The survival role is



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 30 of 40

1

facilitated via upregulation of the inhibitors of apoptosis family members, upregulation of Bcl-2

2

and activation of phosphatidylinositol 3-kinase-Akt signaling pathway.53,54 The expression of

3

VEGF is controlled in a complex manner at the transcriptional and translational levels via several

4

tumor and oncogenic suppressor pathways, by the estradiol receptor and by hypoxia.55 The tumor

5

growth and metastasis are highly reliant on angiogenesis, as observed from the preclinical and

6

clinical studies conducted on colon cancer. Moreover, VEGF was found to be the main

7

angiogenic factor.56

8

VEGF levels were analyzed in sera from the sacrificed mice in each study group (Figure 8b).

9

Two additional treatment groups (D-CA-PEI-SV and D-CA-PEI-FA-SV) were included in this

10

study, specifically for VEGF analysis. These groups were treated in the same way as the other

11

groups, except that siRNA targeting VEGF was used, instead of siRNA targeting ABCB1. The

12

serum VEGF level was significantly elevated by more than seven-fold in tumor-bearing mice

13

treated with saline (53.6 pg/mL), as compared with healthy mice (7.4 pg/mL). This showed that

14

VEGF is an important cancer biomarker. The effect of the CA-PEI micelles on VEGF (53.6

15

pg/mL) was comparable to that of saline. The level of VEGF in serum was decreased in

16

doxorubicin-treated mice. Although doxorubicin has no reported effect on VEGF, this slight

17

decrease in VEGF most likely reflected the cytotoxicity of doxorubicin. The D-CA-PEI micelles

18

produced similar results as those obtained with doxorubicin alone. The D-CA-PEI-SV micelles

19

considerably reduced the VEGF serum level. The most apparent reason for this is the SV-

20

mediated silencing of VEGF. The D-CA-PEI-S-treated group had a VEGF level that was similar

21

to that of the doxorubicin- or D-CA-PEI-treated mice. This may be because ABCB1, and not

22

VEGF, siRNA was present in these micelles. The combined effects of doxorubicin and ABCB1

23

siRNA were predominantly cytotoxic, causing a slight decrease in the VEGF level. FA targeting


Page 31 of 40

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

greatly enhanced the activity of the D-CA-PEI-FA micelles, producing a serum VEGF level of

2

37.8 pg/mL in these mice. The highest anti-VEGF activity was observed following

3

administration of the D-CA-PEI-FA-SV micelles (VEGF: 24.4 pg/mL). FA-mediated tumor

4

targeting of these micelles, and entrapment in the acidic tumor endosomal/lysosomal

5

compartments, caused rapid release of doxorubicin and VEGF siRNA, which directly inhibited

6

VEGF expression and thus lowered serum VEGF levels. The cytotoxic effect of doxorubicin also

7

decreased VEGF levels. Lastly, the D-CA-PEI-FA-S micelles produced a similar VEGF level

8

(36.4 pg/mL) as the D-CA-PEI-FA micelles.

9

Conclusion

10

D-CA-PEI-FA micelles were successfully prepared, characterized and subsequently siRNA was

11

complexed with these micelles to produce D-CA-PEI-FA-S with a high siRNA loading

12

efficiency. The zeta potential of D-CA-PEI-FA (+5.5 mV) micelles decreased after siRNA

13

complexation (+2.14 mV) while XPS analysis revealed the presence of FA outside D-CA-PEI-

14

FA micelles. Doxorubicin showed a two-phase release from D-CA-PEI-FA-S micelles, with an

15

immediate release and followed by a sustained-release profile. Furthermore, doxorubicin was

16

released much faster at pH 5.0 as compared at pH 7.4. The D-CA-PEI-FA and D-CA-PEI-FA-S

17

micelles were found to inhibit in vivo tumor growth, however, no significant difference in their

18

in vitro cytotoxic activity or in vivo anti-tumor effect was observed. It can be concluded that the

19

FA presence enhanced the micellar activity, whereas the presence of siRNA did not significantly

20

increase their anti-tumor activity. The body weight of the mice in the D-CA-PEI-FA-S-treated

21

group did not obviously fluctuate, indicating that the micelles did not exhibit significant systemic

22

toxicity. Histological analysis revealed that tumor tissue from mice treated with D-CA-PEI-FA

23

or D-CA-PEI-FA-S had the lowest cancer cell densities, and the highest level of apoptosis and



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 32 of 40

1

necrosis. Moreover, other vital organs were not affected by these treatments. The liver DPD

2

levels were the lowest in mice treated with D-CA-PEI-FA or D-CA-PEI-FA-S, as shown by

3

ELISA. A group of mice treated with micelles containing siRNA-targeting VEGF instead of

4

ABCB1 (D-CA-PEI-FA-SV) showed the lowest serum VEGF levels. These results indicated that

5

the developed nanoconjugate, CA-PEI-FA, has the potential to achieve targeted co-delivery of

6

doxorubicin and siRNA.

7

Acknowledgements

8

This

9

(ERGS/1/2013/skk02/UKM/02/4) from Ministry of Higher Education Malaysia.

project

was

funded

by

an

Exploratory

10


Research

Grant

Scheme

Page 33 of 40

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

References

2

(1) D.M. Dykxhoorn, C.D. Novina, P.A. Sharp, Killing the messenger: short RNAs that silence

3

gene expression, Nat. Rev. Mol. Cell. Biol. 4 2003 457-467.

4

(2) B. Li, Q. Tang, D. Cheng, C. Qin, Y. Xie, Q. Wei, Using siRNA in prophylactic and

5

therapeutic regimens against SARS coronavirus in Rhesus macaque, Nat. Med. 11 2005 944-951.

6

(3) T.S. Zimmermann, A.C.H. Lee, A Akinc, B Bramlage, D Bumcrot, M.N. Fedoruk, RNAi

7

mediated gene silencing in non-human primates, Nature 441 2006 111-114.

8

(4) A. Akinc, A. Zumbuehl, M. Goldberg, E. Leshchiner, V. Busini, N. Hossain, A combinatorial

9

library of lipid-like materials for delivery of RNAi therapeutics, Nat. Biotechnol. 26 2008 561-

10

569.

11

(5) T. Merdan, J. Kopecek, T. Kissel, Prospects for cationic polymers in gene and

12

oligonucleotide therapy against cancer, Adv. Drug. Deliv. Rev. 54 2002 715-758.

13

(6) K. Miyata, Y. Kakizawa, N. Nishiyama, Y. Yamasaki, T. Watanabe, M. Kohara, Freeze-dried

14

formulations for in vivo gene delivery of PEGylated polyplex micelles with disulfide crosslinked

15

cores to the liver, J. Control. Release. 109 2005 15-23.

16

(7) F. Maclaughlin, R. Mumper, J. Wang, J. Tagliaferri, I. Gill, M. Hichicliffe, Chitosan and

17

depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery, J.

18

Control. Release. 56 1998 259-272.

19

(8) K. Wood, S. Little, R. Langer, P Hammond, A family of hierarchically selfassembling linear-

20

dendritic hybrid polymers for highly efficient targeted gene delivery, Angew. Chem. Int. Ed.

21

Engl. 44 2005 6407-6408.



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

(9) O. Boussif, F. Lezoualc’h, M. Zanta, M. Mergny, D. Scherman, B. Demeneix, A versatile

2

vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine,

3

Proc. Natl. Acad. Sci. USA. 92 1995 7297-7301.

4

(10) T. Yap, C. Carden, S. Kaye, Beyond chemotherapy: targeted therapies in ovarian cancer,

5

Nat. Rev. Cancer. 9 2009 167-181.

6

(11) V. Moebus, C. Jackisch, H. Lueck, A. Bois, C. Thomssen, C. Kurbacher, Intense dose-dense

7

sequential chemotherapy with epirubicin, paclitaxel, and cyclophosphamide compared with

8

conventionally scheduled chemotherapy in high-risk primary breast cancer: mature results of an

9

AGO phase III study, J. Clin. Oncol. 28 2010 2874-2880.

Page 34 of 40

10

(12) Y. Wang, S. Gao, W. Ye, H. Yoon, Y. Yang, Co-delivery of drugs and DNA from cationic

11

core-shell nanoparticles self-assembled from a biodegradable copolymer, Nat. Mater. 5 2006

12

791-796.

13

(13) C. Zhu, S. Jung, S. Luo, F. Meng, X. Zhu, T. Park, Co-delivery of siRNA and paclitaxel into

14

cancer cells by biodegradable cationic micelles based on PDMAEMA-PCL-PDMAEMA triblock

15

copolymers, Biomaterials 31 2010 2408-2416.

16

(14) Z. Xu, Z. Zhang, Y. Chen, L. Chen, L. Lin, Y. Li, The characteristics and performance of a

17

multifunctional nanoassembly system for the co-delivery of docetaxel and iSur-pDNA in a

18

mouse hepatocellular carcinoma model, Biomaterials 31 2010 916-922.

19

(15) H. Meng, M. Liong, T. Xia, Z. Li, Z. Ji, J Zink, Engineered design of mesoporous silica

20

nanoparticles to deliver doxorubicin and P-glycoprotein siRNA to overcome drug resistance in a

21

cancer cell line, ACS. Nano. 4 2010 4539-4550.

22

(16) L.Y. Qiu, Y.H. Bae, Self-assembled polyethylenimine-graft-poly(e-caprolactone) micelles

23

as potential dual carriers of genes and anticancer drugs, Biomaterials 28 2007 4132-4142.


Page 35 of 40

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

(17) G. Szakacs, J.K. Paterson, J.A. Ludwig, C. Booth-Genthe, M.M. Gottesman, Targeting

2

multidrug resistance in cancer, Nat. Rev. Drug. Discov. 5 2006 219-234.

3

(18) D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nanocarriers as an

4

emerging platform for cancer therapy, Nat. Nanotechnol. 2 2007 751-760.

5

(19) S.M. Nie, Y. Xing, G.J. Kim, J.W. Simons, Nanotechnology applications in cancer, Annu.

6

Rev. Biomed. Eng. 9 2007 257-288.

7

(20) M. Izquierdo, Short interfering RNAs as a tool for cancer gene therapy, Cancer. Gene. Ther.

8

12 2005 217-227.

9

(21) P. Low, W. Henne, D. Doorneweerd, Discovery and development of folic-acid based

10

receptor targeting for imaging and therapy of cancer and inflammatory diseases, Acc. Chem.

11

Res. 41 2008 120-129.

12

(22) J.M. Li, Y.Y. Wang, W. Zhang, H. Su , L.N. Ji, Z.W. Mao, Low-weight polyethylenimine

13

cross linked 2-hydroxypopyl-β-cyclodextrin and folic acid as an efficient and nontoxic siRNA

14

carrier for gene silencing and tumor inhibition by VEGF siRNA, Int. J. Nanomedicine. 8 2013

15

2101-2117.

16

(23) K.S. Birdi, Handbook of Surface and Colloid Chemistry, CRC Press, Boca Raton, New

17

York, 1997.

18

(24) A. Dominguez, A. Fernandez, N. Gonzalez, E. Iglesias, L.J. Montenegro, Determination of

19

critical micelle concentration of some surfactants by three techniques, J. Chem. Ed. 74 1997

20

1227-1231.

21

(25) Y. Nakahara, T. Kida, Y. Nakatsuji, M. Akashi, New fluorescence method for the

22

determination of the critical micelle concentration by photosensitive monoazacryptand

23

derivatives, Langmuir 21 2005 6688-6695.



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 36 of 40

1

(26) K. Letchford, B. Helen, A review of the formation and classification of amphiphilic block

2

copolymer nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes,

3

Eur. J. Pharm. Biopharm. 65 2007 259-269.

4

(27) V.P. Torchilin, PEG-based micelles as carriers of contrast agents for different imaging

5

modalities, Adv. Drug. Deliver. Rev. 54 2002 235-252.

6

(28) D.L. Pavia, G.M. Lampman, G.S. Kriz, Infrared spectroscopy: Survey of the important

7

functional groups with examples. Introduction to spectroscopy. 2nd ed. Philadelphia: Saunders

8

College Publishing; 1996:69-70.

9

(29) J. Varshosaz, F. Hassanzadeh, A.H. Sadeghi, M. Nayebsadrian, M. Banitalebi, Synthesis

10

and characterization of folate-targeted dextran/retinoic acid micelles for doxorubicin delivery in

11

acute leukemia, BioMed. Res. Int. 2014;2014:14 pages.

12

(30) M. Yokoyama, T. Okano, Y. Sakurai, K. Kataoka, Improved synthesis of Adriamycin

13

conjugated poly(ethylene oxide)-poly(aspartic acid) block copolymer and formation of unimodal

14

micellar structure with controlled amount of physically entrapped Adriamycin, J. Control.

15

Release. 32 1994 269-277.

16

(31) Y. Wang, L. Yu, L. Han, X. Sha, X. Fang, Difunctional Pluronic copolymer micelles for

17

paclitaxel delivery: synergistic effect of folate mediated targeting and Pluronic-mediated

18

overcoming multidrug resistance in tumor cell lines, Int. J. Pharm. 337 2007 63-73.

19

(32) M. Prabaharan, J.J. Grailer, S. Pilla, D.A. Steeber, S. Gong, Folate-conjugated amphiphilic

20

hyperbranched block copolymers based on Boltorn H40, poly(L-lactide) and poly(ethylene

21

glycol) for tumor targeted drug delivery, Biomaterials 30 2009 3009-3019.


Page 37 of 40

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

(33) B. Karagoz, L. Esser, H.T. Duong, J.S. Basuki, C. Boyer, T.P. Davis, Polymerization-

2

Induced Self-Assembly (PISA)-control over the morphology of nanoparticles for drug delivery

3

applications, Polymer. Chem. 5 2014 350-355.

4

(34) J. Manosroi, W. Lohcharoenkal, F. Gotz, R.G. Werner, W. Manosroi, A. Manosroi,

5

Transdermal absorption and stability enhancement of salmon calcitonin by Tat peptide, Drug.

6

Dev. Ind. Pharm. 39 2013 520-525.

7

(35) G. Chen, W. Chen, Z. Wu, R. Yuan, H. Li, J. Gao, MRI-visible polymeric vector bearing

8

CD3 single chain antibody for gene delivery to T cells for immunosuppression, Biomaterials 30

9

2009 1962-1970.

10

(36) M. Thomas, A.M. Klibanov, Non-viral gene therapy: polycation-mediated DNA delivery,

11

Appl. Microbiol. Biotechnol. 62 2003 27-34.

12

(37) J. Green, E. Chiu, E. Leshchiner, J. Shi, R. Langer, D. Anderson, Electrostatic ligand

13

coatings of nanoparticles enable ligand-specific gene delivery to human primary cells, Nano.

14

Lett. 7 2007 874-879.

15

(38) R. Arote, S.K. Hwang, H.T. Lim, T.H. Kim, D. Jere, H.L. Jiang, The therapeutic efficiency

16

of FP-PEA/TAM67 gene complexes via folate receptor-mediated endocytosis in a xenograft

17

mice model, Biomaterials 31 2010 2435-2445.

18

(39) M.E. Davis, Z.G. Chen, D.M. Shin, Nanoparticle therapeutics: an emerging treatment

19

modality for cancer, Nat. Rev. Drug. Discov. 7 2008 771-782.

20

(40) M.L. Schipper, G. Iyer, A.L. Koh, Z. Cheng, Y. Ebenstein, A. Aharoni, Particle size, surface

21

coating, and PEGylation influence the biodistribution of quantum dots in living mice, Small 5

22

2009 126-134.



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 38 of 40

1

(41) R.L. Juliano, D. Stamp, The effect of particle size and charge on the clearance rates of

2

liposomes and liposome encapsulated drugs, Biochem. Biophys. Res. Commun. 63 1975 651-

3

658.

4

(42) Y. Yamamoto, Y. Nagasaki, Y. Kato, Y. Sugiyama, K. Kataoka, Long-circulating

5

poly(ethylene glycol)-poly(D, L-lactide) block copolymer micelles with modulated surface

6

charge, J. Control. Release. 77 2001 27-38.

7

(43) K. Kataoka, T. Matsumoto, M. Yokoyama, T. Okano, Y. Sakurai, S. Fukushima,

8

Doxorubicin-loaded poly(ethylene glycol)-poly(b-benzyl-L-aspartate) copolymer micelles: their

9

pharmaceutical characteristics and biological significance, J. Control. Release. 64 2000 143-153.

10

(44) M.W. Amjad, M.C. Amin, H. Katas, A.M. Butt, Doxorubicin-loaded cholic acid-

11

polyethyleneimine micelles for targeted delivery of antitumor drugs: synthesis, characterization,

12

and evaluation of their in vitro cytotoxicity, Nanoscale Res Lett 7: (1)687 2012

13

doi:10.1186/1556-276X-7-687.

14

(45) A.M. Butt, M.C.I.M. Amin, H. Katas, N. Sarisuta, W. Witoonsaridsilp, R. Benjakul, In vitro

15

characterization of Pluronic® F127 and D-α-Tocopherol polyethylene glycol 1000 succinate

16

mixed micelles as nanocarriers for targeted anticancer-drug delivery, J. Nanomater. 2012;

17

2012:11 pages.

18

(46) T. Yin, P. Wang, J. Li, Y. Wang, B. Zheng, R. Zheng, Tumor-penetrating codelivery of

19

siRNA and paclitaxel with ultrasound-responsive nanobubbles hetero-assembled from polymeric

20

micelles and liposomes, Biomaterials 35 2014 5932-5943.

21

(47) R.B. Diasio, M.R. Johnson, Dihydropyrimidine dehydrogenase: its role in 5-fluorouracil

22

clinical toxicity and tumor resistance, Clin. Cancer. Res. 5 1999 2672-2683.


Page 39 of 40

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

(48) S. Naito, M. Eto, N. Shinohara, Y. Tomita, M. Fujisawa. M. Namiki, Multicenter phase II

2

trial of S-1 in patients with cytokine-refractory metastatic renal cell carcinoma, J. Clin. Oncol. 28

3

2010 5022-5029.

4

(49) S. Rafii, D. Lyden, R. Benezra, K. Hattori, B. Heissig, Vascular and haematopoietic stem

5

cells: novel targets for anti-angiogenesis therapy?, Nat. Rev. Cancer. 2 2002 826-835.

6

(50) E.M. Conway, D. Collen, P. Carmeliet, Molecular mechanisms of blood vessel growth,

7

Cardiovasc. Res. 49 2001 507-521.

8

(51) M. Detmar, Tumor angiogenesis, J. Investig. Dermatol. Symp. Proc. 5 2000 20-23.

9

(52) H.F. Dvorak, J.A. Nagy, D. Feng, L.F. Brown, A.M. Dvorak, Vascular permeability

10

factor/vascular

endothelial

growth

factor

and

the

significance

11

hyperpermeability in angiogenesis, Curr. Top. Microbiol. Immunol. 237 1999 97-132.

12

(53) P. Carmeliet, M.G. Lampugnani, L. Moons, F. Breviario, V. Compernolle, F. Bono,

13

Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-

14

mediated endothelial survival and angiogenesis, Cell 98 1999 147-157.

15

(54) J. Tran, J. Rak, C. Sheehan, S.D. Saibil, E. LaCasse, R.G. Korneluk, Marked induction of

16

the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells,

17

Biochem. Biophys. Res. Commun. 264 1999 781-788.

18

(55) J. Rak, Y. Mitsuhashi, C. Sheehan, A. Tamir, A. Viloria-Petit, J. Filmus, Oncogenes and

19

tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in

20

ras-transformed epithelial cells and fibroblasts, Cancer. Res. 60 2000 490-498.

21

(56) R.S. Warren, H. Yuan, M.R. Matli, N.A. Gillett, N. Ferrara, Regulation by vascular

22

endothelial growth factor of human colon cancer tumorigenesis in a mouse model of

23

experimental liver metastasis, J. Clin. Investig. 95 1995 1789-1797.


of

microvascular


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

127x91mm (300 x 300 DPI)


Page 40 of 40

In Vivo Antitumor Activity of Folate-Conjugated Cholic Acid

Recommend Documents