Cell Permeating Nano-Complexes of Amphiphilic ... - ACS Publications

Subscriber access provided by University of Sussex Library

Article

Cell Permeating Nano-Complexes of Amphiphilic Polyelectrolytes Enhance Solubility, Stability and Anti-Cancer Efficacy of Curcumin Munazza Tamkeen Fatima, Abhishek Chanchal, Prabhu Srinivas Yavvari, Somnath Dharmaraj Bhagat, Mansi Gujrati, Ram Kumar Mishra, and Aasheesh Srivastava Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00417 • Publication Date (Web): 18 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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.

Biomacromolecules 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 34

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

Biomacromolecules

Amphiphilic Polyelectrolyte Complexes enhance aqueous solubility and anticancer efficacy of hydrophobic drug Curcumin 132x97mm (96 x 96 DPI)

ACS Paragon Plus Environment

Biomacromolecules

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 34

1

Cell Permeating Nano-Complexes of Amphiphilic

2

Polyelectrolytes Enhance Solubility, Stability and Anti-

3

Cancer Efficacy of Curcumin

4

Munazza T. Fatima#(a), Abhishek Chanchal#(a), Prabhu S. Yavvari#(a), Somnath D. Bhagat(a), Mansi

5

Gujrati(b), Ram K. Mishra* (b), Aasheesh Srivastava* (a)

6 7

AUTHOR ADDRESS:

8

(a)

9

Indian Institute of Science Education and Research Bhopal, Bhauri, Indore By-pass Road, Bhopal, 462

Department of Chemistry and (b)Department of Biological Sciences,

10

066, India.

11

# These authors contributed equally to this work

12

*E-mail: [email protected], [email protected]

13

KEYWORDS

14

Amphiphilic Polyelectrolytes, Polyelectrolyte Complexes, Curcumin, Biodegradable polymers, Anti-

15

cancer 1


Page 3 of 34

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

Biomacromolecules

1

ABSTRACT:

2

Many hydrophobic drugs encounter severe bioavailability issues owing to their low aqueous solubility

3

and limited cellular uptake. We have designed a series of amphiphilic polyaspartamide polyelectrolytes

4

(PEs) that solubilize such hydrophobic drugs in aqueous medium and enhance their cellular uptake.

5

These PEs were synthesized through controlled (~20 mol %) derivatization of polysuccinimide (PSI)

6

precursor polymer with hydrophobic amines (of varying alkyl chain lengths viz. hexyl, octyl, dodecyl

7

and oleyl), while the remaining succinimide residues of PSI were opened using a protonable and

8

hydrophilic amine, 2-(2 amino-ethyl amino) ethanol (AE). Curcumin (Cur) was employed as a

9

representative hydrophobic drug to explore the drug-delivery potential of the resulting PEs.

10

Unprecedented enhancement in the aqueous solubility of Cur was achieved by employing these PEs

11

through a rather simple protocol. In case of PEs containing oleyl/dodecyl residues, upto >60000x

12

increment in the solubility of Cur in aqueous medium could be achieved without requiring any organic

13

solvent at all. The resulting suspensions were physically and chemically stable for atleast two weeks.

14

Stable nano-sized polyelectrolyte complexes (PECs) with average hydrodynamic diameters (DH) of

15

150-170 nm (without Cur) and 220-270 nm (after Cur loading) were obtained by using sub-molar

16

sodium polyaspartate (SPA) counter polyelectrolyte. Zeta potential of these PECs ranged from +36 to

17

+43 mV. The PEC-formation significantly improved the cytocompatibility of the PEs while affording

18

reconstitutable nano-formulations having upto 40 wt.% drug-loading. The Cur-loaded PECs were

19

readily internalized by mammalian cells (HEK-293T, MDA-MB-231 and U2OS), majorly through

20

clathrin mediated endocytosis (CME). Cellular uptake of Cur was directly correlated with the length of

21

alkyl chain present in the PECs. Further, the PECs significantly improved nuclear transport of Cur in

2


Biomacromolecules

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 34

1

cancer cells, resulting in their death by apoptosis. Non-cancerous cells were completely unaffected

2

under this treatment.

3

TOC

4

5 6

3


Page 5 of 34

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

Biomacromolecules

Introduction

2

About 40% of the drugs currently employed for clinical use, and up to 75% of the active

3

pharmaceutical ingredients (APIs) under development have poor water-solubility1. Many potential

4

APIs recognized as orphan drugs owing to their low solubility and bioavailability2. Delivery of such

5

hydrophobic APIs, while keeping their efficacy and therapeutic potential, is a key challenge in drug

6

delivery. Chemical modification of drugs, either by conversion into salt form, or through attachment of

7

hydrophilic appendages is exploited to improve the solubility of APIs that are amenable to such

8

modifications3. Nonetheless, many APIs are devoid of such amenable functionalities. Other concerns

9

associated with this approach include partial loss of the therapeutic effect of parent drug, or untoward

10

complications arising from the appended moieties. The second critical factor to be considered to

11

enhance the bioavailability as well as therapeutic effect of a drug is its internalization by the target cells

12

and reaching the site of action. Many adjuvants are added to drug formulations for this purpose,

13

although easy solubilization of hydrophobic drugs and their delivery to the intracellular target site in

14

active form still remains a formidable challenge.

15

Novel drug delivery systems (NDDS) that can improve the solubility as well as therapeutic efficacy

16

of hydrophobic APIs, would greatly aid in realizing the true pharmaceutical potential of APIs that

17

suffer from poor bioavailability. Researchers have developed a variety of NDDS based on micro- and

18

nano-particles,4-6 hydrogels,7-12 polymer-drug conjugates,13-15 liposomes,16 cyclodextrins,17 and

19

micelles18,19 towards this end. The unique physicochemical properties of polyelectrolyte complexes

20

(PECs) derived from natural (bio)polymers and proteins have attracted significant attention as NDDS.

21

Due to their nano-dimensionality, the PECs exhibit efficient and spontaneous accumulation at tumor 4


Biomacromolecules

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 34

1

site via the enhanced permeability and retention (EPR) effect of tumor tissues20. Chitosan-based PECs

2

with other biopolymers such as alginate, hyaluronic acid, pectin, carrageenan, xanthan gum, and

3

carboxymethyl cellulose etc. have been widely explored for delivery of mostly hydrophilic drugs like

4

insulin, isoniazid, budenoside etc21. However, the natural polymers and protein fragments utilized for

5

PEC formation can also lead to immunogenic responses and batch-to-batch variations in their

6

properties.

7

Synthetic polyaspartamides have emerged as unique cell permeating polymers with demonstrated

8

biocompatibility, biodegradability, synthetic ease and modular functionalization.22,

23

9

pendant group on the hydrolytic stability of polyaspartamide polymers at physiological conditions has

10

been done by Y. Lu et al.23, where the author shows that polyaspartamides undergo un-catalysed

11

degradation whose rate was sensitive to the nature of the pendant groups. Due to their excellent

12

biocompatibility and biodegradability, synthetic polyaspartamides have been utilized for intracellular

13

delivery of insulin,24,25 as multi-responsive polymers,26 and have also demonstrated their potent anti-

14

mycobacterial ability.27 Herein, we report a series of amphiphilic polyaspartamide polyelectrolytes

15

(PEs) that can enhance the solubility and bioavailability of hydrophobic drugs, exemplified by

16

Curcumin (Cur). Cur was chosen as a model hydrophobic drug as it has tremendous potential in cancer

17

chemo-prevention and treatment. Nonetheless, it presents significant formulation difficulties due to its

18

low aqueous solubility and chemical instability in aqueous medium. Various curcumin analogs and

19

derivatives have been synthesized in order to enhance its poor aqueous solubility and instability.28, 29

20

The challenge of delivering Cur is further enhanced due to its poor absorption, quick metabolization

21

and rapid clearance from systemic circulation. Further, loading efficiencies of Cur in various

22

formulations are poor, and usually range in 5 to 10 wt% drug even after multi-step formulation

The effect of

5


Page 7 of 34

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

Biomacromolecules

1

protocols that require the use (and subsequent removal) of organic solvents during formulation.30 In the

2

following sections, we discuss design and synthesis of amphiphilic polyaspartamide PEs that

3

successfully overcome these challenges. We explore the influence of amphiphilicity of the PEs and

4

corresponding PECs on the biocompatibility, drug-loading as well as drug-delivery ability, and the

5

consequent cellular-level changes in cancerous (breast and bone cancer) and non-cancerous

6

(transformed human embryonic kidney) cells. Notably, we demonstrate that by improved intracellular

7

delivery, especially inside cancer cells, these PECs enhance the efficacy of Cur, and achieve selective

8

killing of cancer cells while remaining benign to non-cancerous cells.

9

Materials:

10

All chemicals were procured from various commercial suppliers and were used directly without any

11

further purification. Cur was purchased from Alfa Aesar, UK. The alkyl amines were obtained from

12

Spectrochem Pvt. Ltd., Mumbai. 2-(2-Amino-ethylamino) ethanol (AE) were purchased from Merck

13

Specialities Pvt. Ltd., Chennai. MTT, DMSO-d6 and Triton-X 100 were obtained from Sigma

14

Chemical Company, St. Louis, USA. Propidium iodide and Ethylene diamine tetra acetic acid (EDTA)

15

were purchased from HiMedia Laboratories Pvt. Ltd, Mumbai. Hoechst 33258 were purchased from

16

Invitrogen and Vectashield were obtained from Vector Laboratories Inc.

17

Methods:

18

Synthesis of the PEs: The precursor polymer PSI was synthesized using the protocol given by Neri et

19

al.22 The resulting PSI was found to have a Mw of ~40 kDa. For the synthesis of alkyl amine

20

derivatives, 1 g of PSI was dissolved in 10 ml of dimethyl formamide (DMF) and 0.20 eqv. of the

21

respective amine (hexyl, octyl, dodecyl or oleyl) (dissolved in 5 ml of DMF) was added dropwise for 6


Biomacromolecules

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 34

1

30 min to this solution. The reaction was allowed to proceed under constant stirring at room

2

temperature till complete consumption of the alkylamine was noticed through thin layer

3

chromatography. In the second step, the remaining succinimide residues of PSI were derivatized with

4

AE. For this, 2.5 equivalents of AE in DMF was added to the alkylated PSI polymer, and the reaction

5

was allowed to stir for 6 h. Complete disappearance of PSI was confirmed by FTIR (data not included)

6

and 1H NMR. The resulting polyelectrolytes (PEs) were dissolved in water and dialyzed for 24 h

7

against 1% aqueous HCl solutions, and lyophilized subsequently. For the non-alkylated control

8

polymer, 1 g of PSI was dissolved in 5 ml of dry DMF and 1.2 eq. of the AE was added, the reaction

9

was stirred for 6 h and then dialyzed against 1% aq. HCl. H NMR of the PEs: The PEs were characterized by 1H NMR (Bruker AVANCE III Ultra shield (400

10

1

11

MHz) spectrometer) at 10 mg/ml in DMSO-d6 to quantify the degree of derivatization (DOD) of the

12

alkyl side-chains based on the equation shown below.

13 14

Solubilization and quantification of Cur in PEs: 3 mg of individual PE was dissolved in 3 ml of

15

distilled water to obtain clear solution. Different amounts (1.5, 3, 4.5 and 6 mg) of procured Cur

16

powder was added to glass vials containing the PE solutions to obtain a final concentration of 0.5, 1,

17

1.5 and 2 mg drug/ml of polymer solution. This mixture was stirred for 12 h and the resulting Cur-PE

18

suspensions were centrifuged at 1500 rpm for 10 min to remove the undissolved Cur. For

19

quantification, Cur entrapped suspensions of the polymers were diluted in water/ethanol (1:1) and

20

absorption at 430 nm was recorded using a PerkinElmer Lambda 25 UV-Vis spectrophotometer. The

7


Page 9 of 34

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

Biomacromolecules

1

purpose of Cur dilution in water/ethanol mixture was to get clear and transparent solution through

2

complete dissolution of both Cur and the PEs. Each measurement was performed in triplicates, and the

3

final values represent the mean ± SD of the measurements. Relative loading was quantified against a

4

standard curve of Cur dissolved in aqueous:ethanol (1:1 v/v) mixtures (the current solvent system was

5

opted to solubilize curcumin to maximum extent in aqueous conditions and minimize scattering due to

6

undissolved particulates of curcumin). The UV-vis spectra of Cur solubilized in the different PEs were

7

also recorded by simply diluting the centrifuged suspensions in distilled water. While the Cur-AE

8

suspension was diluted 2 times (owing to lower entrapment), rest of the Cur-suspensions were diluted

9

20 times and their UV-vis spectra were recorded between 300 to 700 nm.

10

Preparation and characterization of void and Cur-loaded PECs: The void as well as Cur-loaded

11

PEs were first dissolved in distilled water. Varying millimolar ratios of polymer:SPA (ranging from

12

1.0:0.25 to 1.0:2.0) were added to test for a positive and stable zeta potential value. In all the cases, the

13

PECs formed were characterized for their hydrodynamic diameters (DH) using dynamic light scattering

14

(DLS). The void as well as Cur-loaded PEs (0.5 mg/ml) were prepared in water and equilibrated for 1

15

h at 25 °C. This solution was filtered through a 0.45 µm syringe filter. Hydrodynamic diameters (DH)

16

were calculated for this solution in the 25 °C temperature on a Delsa™ Nano (Beckman Coulter)

17

instrument using the CONTIN algorithm. The PECs were bath-sonicated for 10 min prior to measuring

18

these parameters. Each measurement was performed in triplicates and the reported values represent the

19

mean ± SD of the determinations.

20

In-vitro Drug-Release Kinetics: The release kinetic of curcumin was studied by Sample and Separate

21

Methods using PBS pH 7.4 as media of selection.31 In brief, Cur-loaded PECs were incubated with 20

22

mM phosphate buffered saline (PBS), pH 7.4. Different volumes of the PECs containing of 100 µg of 8


Biomacromolecules

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 34

1

the loaded drug were added to vial (in triplicate) and diluted in 1.0 ml of PBS (the volume variation

2

was necessitate due to different loadings of Cur in different PECs). An aliquot of samples (1.0 ml) were

3

taken in micro-centrifuge tube and centrifuged at 10,000 rpm for 10 min. The upper clear supernatant

4

were collected, mixed with equal volume of ethanol and the read by spectrophotometry at absorbance

5

at 425 nm recorded. The Cur release was analyzed by different release kinetic models like zero order

6

plot, first order plot, Higuchi plot, Korsmeyer-Peppas equation, Hixson-Crowell32.

7

Hemolysis Assay: Human RBCs were harvested by centrifuging whole blood at 2000 rpm for 10 min.

8

The plasma and buffy coat removed and the cells were further washed with 20 mM phosphate buffered

9

saline (PBS, pH 7.4) at 2000 rpm for 10 minute. 100 µl of the packed blood was then incubated with

10

same volume of PECs formulations of varying concentrations. 800 µl of PBS was then added to each

11

vial to make a final volume of 1 ml. The samples were incubated at 37 °C for 2 h. The samples were

12

centrifuged at 10,000 rpm for 10 min and the supernatant was read at 540 nm. 100 µl of packed cells

13

lysed in 1% Triton X-100 was taken as a measure of complete (100%) hemolysis.

14

MTT Assay: Cell-viabilities upon exposure to PEs was determined by MTT assay on HEK-293T,

15

MDA-MB-231 and U2OS cells.33 Briefly, in 96-well plate 15.6 x 103 cells/cm2 were seeded and

16

incubated for 24 to 48 h prior to treatment at 37 °C and 5 % CO2 atmosphere. Cell treatment was done

17

with and without PEs for 24 h. After the treatment time, 20 µl (stock 5 mg/ml in PBS) of 3-(4, 5-

18

Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was added to the wells and incubated

19

for 4 h at 37 °C. After careful removal of the supernatant, 150 µl of DMSO was added and incubated at

20

37 °C for 15 min. The absorbance was read using a micro-plate reader (Biotek, SynergyTM) at 540 nm.

21

The percentage of cytotoxicity was calculated using the background-corrected absorbance as per the

22

following equation: 9


Page 11 of 34

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

Biomacromolecules

1 2 3

Trypan Blue Exclusion Assay: Cells (15.6 x 103 cells/cm2) were seeded in a 6-well culture plates for

4

24 h at 37 °C and 5% CO2 atmosphere. Cells were then exposed to different formulations i.e. PECs

5

(with and without the entrapped drug) for 18 h. Concentrations of the Cur and PECs used for the study

6

were 50 µM and 50 µg/ml, respectively. After treatment, the cells were first washed by PBS and

7

removed from the plates by trypsinization. The cells were washed twice (1500 rpm for 5 min), diluted

8

with 1 ml media and further stained with equal volume of trypan blue (10 µl of cells were stained with

9

10 µl of 4% trypan blue solution). The populations of live and dead cells were then counted in a

10

hemocytometer with Leica DMIL-LED Inverted Fluorescence Microscope.

11

Cellular Uptake: 2.5 x 104 Cells/well were seeded onto glass cover slip in 6 well culture plate. After

12

24 h incubation at 37 °C and 5% CO2 atmosphere, the cells were exposed to each of the samples

13

including Cur per se (Cur dissolved in 10% v/v ethanol in water), Cur loaded Hex-, Oct-, Dod- and

14

Ole-PECs. The concentration of Cur was same i.e., 20 µM. The cells were incubated for 4 h, after

15

which they were washed with the PBS and fixed in 3.4 % formaldehyde since this protocol is known to

16

minimize artefacts encountered during imaging.34 Staining was done with Hoechst-33258 for 10 min

17

and the coverslip containing cells were washed and mounted on a glass slide with mounting medium

18

Vectashield. Imaging was done at 40X magnifications in Multiphoton Confocal system from Carl

19

Zeiss. Also, the mean fluorescent intensity of Cur and Cur-loaded PECs, in individual cells was

20

calculated using ZEN 2012 software.

21

Cell cycle distribution and Apoptosis by Flow Cytometry: Cells (1 x 106) were seeded in a 60 mm

22

cell culture dish and treated with Cur per se as well as Cur loaded PECs (50 µM) for 18 h, processed 10


Biomacromolecules

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 34

1

and washed twice in PBS (1500 rpm for 5 min). Cells were finally stained with propidium iodide (5

2

µg/ml) for 10 min, washed and suspended in PBS (20 mM pH 7.4) and analyzed by BD FACS ARIA

3

III.

4

Comet Assay: The Comet assay, or Single-Cell Gel Electrophoresis (SCGE) technique, was applied

5

for measuring DNA damage in individual cells.35 In brief, 2.5 x 104 cells were seeded in 24 wells cell

6

culture plate and treated with Cur and Cur-loaded PECs for 18 h. After 18 h, cells were harvested by

7

trypsinization followed by PBS washing. The 10,000 cells (treated and untreated) per 10 µl were mixed

8

with 75 µl of 1% low melting point agarose (LMPA) and poured on a base slide prepared by 1%

9

normal melting agarose (NMA), and a coverslip was placed on it immediately before the LMPA

10

hardened. The cover slip was gently removed and the area was exposed to a cold, freshly made lysis

11

solution (2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base-pH 10, 1% triton X-100 and 10% DMSO),

12

and kept in dark and cold for 2 h. The power supply was adjusted to 24 volts (~0.74 V/cm) and the

13

current to 300 mA by raising or lowering the buffer level and electrophoresis of the slides was allowed

14

for 30 min. The slides were further coated with neutralization buffer (400 mM Tris pH-7.5), for 5-10

15

minutes, repeated the same step twice. Slides were later stained with ethidium bromide (1 mg/ml in

16

doubled distilled water), and washed with chilled distilled water to remove excess stain. The slides

17

were visualized in an Apotome microscopy from Carl Zeiss to score the DNA damage.

18

Results and Discussion

19

Design and Physico-Chemical Characterization of PEs

20

The key advantages of using polyaspartamides derivatives for biomedical applications are their

21

excellent biocompatibility, biodegradability, easy and modular derivatization, and finally, their ability 11


Page 13 of 34

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

Biomacromolecules

1

to permeate through cell membranes. The heterochiral nature of the polymer backbone further

2

improves their stability against proteases. In this work, we focused our attention on utilizing

3

polyaspartamides to improve solubility and bioavailability of hydrophobic drug Cur that has extremely

4

poor water solubility, undergoes rapid degradation in aqueous milieu, and has low cellular uptake.

5

Keeping the favorable characteristics of polyaspartamides in mind, we designed amphiphilic

6

polyaspartamide PEs to address the above problems associated with Cur. Towards this end, we

7

synthesized five different amphiphilic PEs from the PSI precursor. The key design parameter that was

8

varied across the polymers was the length of alkyl tail present in the hydrophobic pendants. Thus,

9

polymers containing 20 mol% hexyl (Hex), octyl (Oct), dodecyl (Dod) and oleyl (Ole) residues were

10

prepared, and finally hydrophilic AE residues were incorporated into the polymers. The AE residues

11

contain a protonable secondary amine group that can impart polycationic character to the PEs, and the

12

hydroxyl moiety in AE can also provide additional H-bonding interactions with hydrophobic drugs like

13

Cur. Hence, the final PEs were designated as Hex-AE, Oct-AE, Dod-AE and Ole-AE respectively,

14

depending on the hydrophobic unit present in them (Figure 1a and S1a-S1b, ESI). A control PE with

15

100 mol% AE residues was also prepared. In an analogous approach, Civiale et al. had reported

16

amphiphilic poly(hydroxyethylaspartamide) (PHEA) copolymers bearing poly(ethylene glycol) and/or

17

hexadecylamine pendants to enhance permeability of these polymers across cell membranes and for in-

18

vivo bioavailability studies.36

19

All the PEs prepared herein had excellent water-solubility. We tested the ability of these PEs to

20

solubilize Cur. We noticed that these amphiphilic PEs were very effective in solubilizing Cur in water,

21

even when commercial Cur powder was directly added to the PE solution and stirred. The un-entrapped

22

Cur was simply removed by centrifugation. The digital image of resulting uniform aqueous suspensions 12


Biomacromolecules

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 34

1

is shown in Figure 1b. Micelle formation by these amphiphilic PEs seems to significantly enhance the

2

solubilization of Cur above 10-3 - 10-4 mg/mL PE concentration for all the PEs (Figure 1c). Further,

3

while low amount of Cur was solubilized by the control AE polymer that lacked hydrophobic residues,

4

in Hex-AE there was an increase in the amount of Cur entrapped, where up to 360 µg Cur/mg of PE,

5

(accounting to 36 wt% with respect to weight of PE employed) could be solubilized (Figure 1d). In

6

case of Oct-AE, Dod-AE and Ole-AE, the Cur solubilization capacity increased to 505, 652 and 720

7

µg/mg of the respective PE. In other words, Dod-AE and Ole-AE PEs were able to solubilize > 65 wt%

8

of Cur, and > 65000x increase in solubility of Cur was thus achieved compared to the same in water.

9

The solubilization efficiency of Cur was found to be directly correlated with the length of alkyl chain

10

present in the PEs. Hydrophobic interactions, thus, turned out to be the major driving force for the

11

efficient solubilization of Cur (Figure 1e). In all the samples, bimodal broad UV-Vis profiles were

12

obtained for Cur. However, the intensity of the two peaks (at 446 and 490 nm) in the UV-Vis spectra

13

varied between the samples. While Cur entrapped in Hex-AE and Oct-AE showed maximum

14

absorption at 490 nm, the Dod-AE and Ole-AE based samples had the same at 446 nm. The ratio of

15

absorbance values at 446 to 490 nm (Figure 1f) increased gradually as the hydrophobic chain length

16

increased (from 0.82 for AE to 0.91, 0.97, 1.06 and 1.12, respectively, in Hex-AE, Oct-AE, Dod-AE

17

and Ole-AE). Thus, Cur experienced significantly greater hydrophobic environment in Ole-AE

18

compared to the same in Hex-AE. The physico-chemical stability of Cur solubilized in water was also

19

significantly improved by the use of these amphiphilic PEs. For example, the suspension obtained

20

using Ole-AE was found to be stable for more than 10 days while the aqueous-ethanolic solution of Cur

21

decomposed within a day (Figure 1g).

13


Page 15 of 34

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

Biomacromolecules

1 2

Figure 1. a) Chemical structures of PEs employed in this study. b) Digital image of vials containing

3

Cur solubilized in water using the polymers. From Left to right: Water without any polymer, Hex-AE,

4

Oct-AE, Dod-AE and Ole-AE. (For details, refer to Experimental Section.) c) Cur solubilization

5

efficiency of the PEs at different concentrations. The highlighted area shows the PE-concentration

6

regime at which sudden increase in Cur-solubilization was noticed. d) Solubilization efficiency of the

7

polymers measured by UV-vis at different initial loadings of Cur. Data represents mean ± SD of three

8

independent determinants. e) UV-vis spectra of various Cur-suspensions. Numbers indicate extent of

9

dilution with PBS. Refer to Experimental Section for details. f) Ratio of absorbance values at 490 to

10

446 nm in the various Cur-suspensions shown in panel (e). g) Time variation in concentration of Cur

11

solubilized using Ole-AE (red symbols) compared to that using ethanol-water mixture (blue symbols). 14


Biomacromolecules

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 34

1

h) Digital and i) SEM image of cottony solid obtained on lyophilizing Cur-entrapping Ole-PECs. The

2

reconstituted formulation obtained on adding water to the solid mass is also shown in (h).

3 4

The above Cur-suspensions could be lyophilized to yield a fibrous solid that contains >40% w/w of

5

active drug, and can be stored in this form for >6 months. The aqueous solubility of Cur with >65000x,

6

was much higher than that reported by Gao M et al.37 An additional advantage of our protocol was that

7

we did not employ any organic solvent at any stage of formulation. This is in sharp contrast to many of

8

the earlier reports that required the use of organic solvents during Cur formulation.38-40 The SEM image

9

of solid obtained by lyophilizing Cur-suspensions (Figure 1i) showed presence of microfibrous

10

morphologies that were markedly different from Cur microcrystals shown by Shipu Li et al.41 The dried

11

Cur formulation can be readily reconstituted when required by simple addition of water and manual

12

shaking in buffer of choice, further enhancing the solubility in water (Figure 1h). For example, 5 mg of

13

the lyophilized solid (containing ~2 mg of Cur) could be solubilized in 200 µl of water, thus increasing

14

the solubility of Cur to unprecedented 10 mg/mL!

15

Complexation of polyelectrolyte significantly reduces the toxicity of the amphiphilic PEs

16

The biocompatibility of the amphiphilic PEs was studied next by performing the cytotoxicity profiles

17

by MTT assay on mammalian cell lines (MDA-MB-231 and HEK-293T) as well as by probing lysis of

18

red blood cells (RBCs) upon exposure to these PEs. The cytoxicity of the polymers increased rapidly as

19

the polymethylene chain length increased. It was maximum for Ole-AE (ca. 23% at 0.5 mg/ml) and

20

lowest for Hex-AE (below 5% at the same concentration) in the breast cancer cell line, MDA-MB-231.

21

This is consistent with earlier reports, wherein the membrane-disrupting activity of amphiphilic 15


Page 17 of 34

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

Biomacromolecules

1

polymers has been reported to be mainly dependent on the charge as well as length of the alkyl tail and

2

hence hydrophobicity of the polymer.42,43 These PEs were even more toxic to non-cancerous cells,

3

HEK-293T when compared to breast cancer cell line, MDA-MB-231. For example, at 0.5 mg/ml the

4

most toxic Ole-AE induced >60% cytotoxicity in HEK-293T cells, almost 2.5 times more than that

5

observed with MDA-MB-231 cells at similar concentrations (Figure 2a). A similar pattern of toxicity

6

was observed in other PSI derivatives (Hex-AE, Oct-AE & Dod-AE) for the two cell lines, and the

7

effect was dose-dependent (Figure S2, ESI). The release of hemoglobin from RBCs by rupturing their

8

cell membrane (hemolysis) method was also employed to assess cytotoxic effects of the PEs. Within 2

9

h of incubation with 5 mg/ml of Dod-AE or Ole-AE polymers, upto 45% hemolysis was observed for

10

Ole-AE and Dod-AE (Figure 2b). The hemolysis was also found to occur in dose-dependent manner

11

(Figure S3, ESI).

16


Biomacromolecules

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 34

1 2

Figure 2. a) Cytotoxicity of the PEs (red bar) and their corresponding PECs (blue bar) towards MDA-

3

MB-231 and HEK-293T at 0.5 mg/ml PE concentration. b) Hemolytic activity of the PEs and the

4

corresponding PECs. c) The hydrodynamic diameters (DH or size) (square symbols) and zeta potential

5

values (triangle symbols) of the void PECs (unfilled symbols) and Cur-loaded PECs (filled symbols).

6

Fortunately, the above drawbacks of these amphiphilic PEs could be effectively circumvented by

7

complexing them with anionic sodium polyaspartate (SPA, obtained by hydrolyzing the PSI precursor

8

with NaOH) at 1:0.35 molar ratio of polymer:SPA. The use of SPA resulted in the formation of nano-

9

dimensional polyelectrolyte complexes (PECs) with average hydrodynamic diameters (DH) in the range

10

of 140-170 nm, and zeta potentials from +36 to +43 mV (Table ST1, ESI). Encapsulation of Cur in the 17


Page 19 of 34

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

Biomacromolecules

1

PEs resulted in increase of the DH values between 220 to 270 nm (Figure 2c and Table ST2, ESI). The

2

PECs exhibited good physical stability, and showed constant DH values for at least ten days (data not

3

shown). The biocompatibility of the void PECs including Hex-, Oct-, Dod- and Ole-PECs was

4

evaluated by MTT assay in the HEK-293T and MDA-MB-231 cell lines (Figure S2, ESI). In

5

significant contrast to the PEs constituting them, all the void PECs showed excellent biocompatibility,

6

and the viability of both normal cells and tumor cells was >90% even at the concentration of 0.5

7

mg/ml. Phase contrast optical microscopy images further confirmed the biocompatibility of void PECs

8

on these cells (Figure S4a and S4b, ESI) and showed the presence of completely confluent cells in all

9

the treated groups. Similarly, PECs also exhibited markedly improved hemo-compatibility (> Hex Cur PECs >>

13

drug per se (Cur) (Figure S7, ESI). Thus, upto 10x more Cur was delivered by employing Ole-Cur-

14

PECs or Dod-Cur-PECs compared to Cur administered as ethanolic solution (Figure 3d). When cellular

15

uptake of curcumin between tumor (MDA-MB-231) and non-tumor (HEK-293T) cells was compared

16

(Figure S7, ESI), the MDA-MB-231 showed upto 3x more uptake than the HEK-293T. Further, in case

17

of MDA-MB-231 cells, Cur delivered using Ole-Cur-PECs was found to have generously entered the

18

nuclei (Figure 4a-4c), thus allowing better target sites for its activity and enhancing its efficacy.

21


Page 23 of 34

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

Biomacromolecules

1 2

Figure 4. Fluorescence micrographs at 40x magnification of (a) HEK-293T, and (b) MDA-MB-231

3

cells, treated with 20 µM of Ole-Cur-PECs nanoformulations for 4 h indicating differential localization

4

of Cur in the non-cancerous and cancerous cells. (c) Pearson’s overlap coefficients derived from the

5

micrographs of blue (Hoechst) and green (Cur) channels in HEK-293T (blue) and MDA-MB-231 (red)

6

cells. (d) Relative cellular uptake of Cur through Ole-Cur-PECs in MDA-MB-231cell lines at low

7

temperature (blue symbols) and in presence of 4 mM sucrose (red symbols).

22


Biomacromolecules

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 34

1

Interestingly in the case of HEK-293T cells, Cur seems to be largely localized in the cytoplasm.

2

While Cur is known to inherently reach the nuclei of cancer cells,44 our formulations significantly

3

expedited and enhanced this process. So, the increased cellular uptake along with significant presence

4

of Cur in nucleus of cancer cells compared to that in the non-cancerous cells, seems to be contributing

5

to the observed enhancement in selectivity and cytotoxicity for tumour cells. The cause of enhanced

6

nuclear delivery of Cur using PECs is hitherto not explored in detail by us but can be attributed to

7

increased nuclear membrane pores as characteristic of cancer cell.45

8

Cur-loaded PECs follows clathrin-mediated endocytosis

9

The cellular uptake pathway of the Cur-loaded PECs was investigated in detail in the MDA-MB-231

10

cells. Subsequent to their adherence to the cell membranes (Fig. 3b), the uptake within the cells may

11

occur via several possible mechanisms such as pinocytosis, nonspecific or receptor-mediated

12

endocytosis, or phagocytosis.46 The rapid endocytosis of cationic nanoparticles via clathrin mediated

13

endocytosis (CME) is known.47 We investigated the role of both clathrin dependent and independent

14

endocytosis in MDA-MB-231 by investigating the uptake of PECs either at lower temperature (4 °C) or

15

upon pre-treatment with sucrose to block the clathrin assembly and dependent receptors.48 Upon

16

pretreatment with sucrose, relatively similar reduction (~75%) in Cur uptake occurred for all

17

formulations (Figure 4d, red symbols). This indicates CME indeed was the major pathway involved in

18

PEC-mediated Cur-delivery. However, uptake at 4 °C was affected to a greater extent (~85% decrease)

19

in the case of Dod- and Ole-Cur-PECs (Figure 4d, blue symbols), while the other formulations

20

exhibited reduction in uptake similar to that observed upon sucrose pre-treatment. Thus, while CME

21

seems to play a major role in the internalization of all the Cur-loaded PECs, additional internalization

22

processes may also be involved in the case of Dod- /Ole-Cur-PECs. These processes could be in the 23


Page 25 of 34

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

Biomacromolecules

1

form of non-clathrin mediated endocytosis or enhanced uptake due to greater membrane perturbations

2

that occur with formulations containing longer hydrocarbon chains.

3

A common problem associated with endocytotic uptake is the endosomal entrapment of the material

4

internalized. However, fluorescence microscopy images showed that Cur was distributed throughout

5

the cells (Figure 4a and 4b). This implies that subsequent to its cellular uptake, the drug does not

6

remain entrapped in endo-lysosomal compartments. The basic AE residues present in these polymers

7

probably exert the proton-sponge like action required to disrupt the endosomes and release Cur in the

8

cytoplasm.

9

Cur loaded PECs induces G0/G1-phase cell cycle arrest and apoptosis

10

The mechanism of cell-death in cancer cells was further probed in detail. Optical micrograph images

11

showed the presence of apoptotic blebs in MDA-MB-231 cells (Figure 5 and S8, ESI) in the treatment

12

groups, indicating apoptosis as the possible mechanism of selective killing of tumor cells by various

13

Cur formulations. While the live cell population had almost no apoptotic blebs, their numbers increased

14

progressively in cells treated with drug per se, Hex- to Ole-Cur-PECs (Figure S8, ESI). The induction

15

of apoptosis subsequent to Cur-PECs treatments was further confirmed by single cell gel

16

electrophoresis (Comet assay).35 In this assay, progressively longer tailing of the nuclear material was

17

observed for cells treated with drug per se, Hex- to Ole-Cur-PECs. The extent of tailing correlates with

18

a progressive increase in the DNA damage (corresponding right panel of Figure 5 and S8, ESI). Thus,

19

this study further endorses an increase in the alkyl tail of PEs involved in PEC formation dictates the

20

efficacy of the entrapped drug. This observation was further replicated in the case of U2OS cells

21

undergoing similar treatment (Figure S9, ESI). There was a progressive increase in the apoptotic

24


Biomacromolecules

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 34

1

population in cells treated with Cur to Hex- to Ole-Cur-PECs, indicating delivery through PECs was

2

highly effective, and the effectiveness improved with the length of alkyl tail present in the PE.

3 4

Figure 5. Left panels: Phase contrast optical micrographs of MDA-MB-231 cells treated with either

5

Cur only (panel a) or with Ole-Cur-PECs (panel b) at 50 µM Cur for 18 h. Arrows indicate the

6

presence of apoptotic blebs. Right panels are single cell gel electrophoresis (Comet assay) images

7

highlighting the extent of apoptosis in form of nucleotide tailing as well as numbers.

8

Cur is also known to arrest cell-cycle progression in the G0/G1 and/or G2/M phases in cancer cells,

9

which in turn induces apoptosis in these cells.49,50 We investigated the effect of enhanced cellular

10

uptake of delivered Cur via PECs on cell-cycle progression in MDA-MB-231 cells (Figure 6 and S10,

11

ESI) through flow cytometry using propidium iodide. We observed that enhanced Cur delivery resulted

12

in significant apoptosis in the treatment groups. While only 12 to 18% cells exhibited apoptotic

13

signatures in Cur per se, Hex-Cur-PECs and Oct-Cur-PECs, this percentage rose significantly to 35 and 25


Page 27 of 34

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

Biomacromolecules

1

55% in case of Dod- and Ole-Cur-PECs treated MDA-MB-231 cells. Further, in cells treated with Dod-

2

and Ole-Cur-PECs, significant G0/G1-phase arrest was also evident.

3 4

Figure 6. Flow cytometric analysis of cell cycle phase distribution (apoptotic or Ap, G0/G1, S and

5

G2/M phase of the cell cycle) in MDA-MB-231 cells treated with different Cur-loaded-PECs nano-

6

formulations (50 µM Cur for 18 h).

7

Conclusions

8

In conclusion, we have reported a series of amphiphilic biodegradable PEs in which length of

9

polymethylene chains of the hydrophobic residues (ca. 20 mol%) was varied. These PEs are also

10

endowed with protonable hydroxyethylamine pendants (ca. 80 mol%). These amphiphilic PEs were

11

effective in solubilizing and stabilizing hydrophobic drug Cur in aqueous medium. The length of

12

polymethylene chains present in these PEs seems to dictate the most important properties of these

13

polymers such as their efficiency to solubilize Cur, as well as their cytotoxicity and hemotoxicity. The

14

latter two, which could have significantly limited the biomedical applicability of these polymers, were 26


Biomacromolecules

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 34

1

readily tamed by the formation of nano-sized polyelectrolyte complexes (PECs) using sodium

2

polyaspartate. The resulting Cur-loaded PECs could be lyophilized to yield reconstitutable nano-

3

formulations of Cur. The Cur-loaded PECs were taken up more efficiently by cancerous cells than their

4

non-cancerous counterparts, mainly through endocytosis. Cur delivered through PECs was found

5

distributed throughout cytoplasm, indicating its ability to overcome endosomal entrapment. In

6

cancerous cells, significant amounts of Cur were present even inside nuclei of cancer cells. Enhanced

7

uptake as well as nuclear delivery of Cur in cancer cells achieved selective killing of cancerous cells

8

over non-cancerous cells, through arrest of cell cycle and induction of apopotosis. The efficiency of

9

Cur-delivery within the cancer cells, and their consequent apoptosis, were intimately controlled by the

10

polymethylene chain length of the amphiphilic PEs. These findings are summarized in Fig. 7.

11 12

Figure 7. Schematic representation summarizing the findings of this work. Amphiphilic

13

polyelectrolytes (PEs) allow efficient entrapment of the hydrophobic drug Cur. The inherent

14

cytotoxicity of these PEs can be significantly reduced through preparation of polyelectrolyte complexes

27


Page 29 of 34

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

Biomacromolecules

1

(PECs). The drug-loaded PECs achieve intracellular delivery of Cur, resulting in selective toxicity to

2

the breast and bone cancer cells while non-cancerous cell showing high viability under this treatment.

3

28


Biomacromolecules

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 34

1

ASSOCIATED CONTENT

2

Supporting Information. Includes details synthetic scheme, characterization (1H NMR, DLS, Zeta

3

potential) of polymers and PECS, drug entrapment, drug release kinetics, microscopy studies done for

4

cellular uptake of drug loaded PECs and FACS analysis.

5

AUTHOR INFORMATION

6

Corresponding Author

7

*E-mail: [email protected], [email protected]

8

Present Addresses: NA

9

Author Contributions

10

The manuscript was written through contributions of all authors. All authors have given approval to the

11

final version of the manuscript. #These authors contributed equally.

12

Funding Sources

13

AS acknowledges Bioengineering research grant (No. BT/PR12361/MED/32/361/2014) from the Dept.

14

of Biotechnology for the research funds.

15

Notes

16

Any additional relevant notes should be placed here.

17

ACKNOWLEDGMENT

18

MTF and AC acknowledges IISER Bhopal for the institute postdoctoral fellowship. PSY, SDB and

19

MG thanks UGC for SRF. 29


Page 31 of 34

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

Biomacromolecules

REFERENCES (1)

3

Williams, H. D.; Trevaskis, N. L.; Charman, S. A.; Shanker, R. M.; Charman, W. N.; Pouton, C. W.; Porter, C. J. H. Pharmacol. Rev. 2013, 65, 315-499.

4

(2)

Kalepua, S.; Nekkantib, V. Acta Pharmaceutica Sinica B 2015, 5, 442–453.

5

(3)

Acharya, G.; Lee, J.; Lee, S. C.; Park, K. Pharmaceutical applications of hydrotropic agents,

6

polymers thereof, and hydrogels thereof. WO2002030466 A2, April 18, 2002.

7

(4)

Li, F.; Zhu, A.; Song, X.; Ji, L. Colloids Surf. B 2014, 115, 377-383.

8

(5)

Licciardi, M.; Di Stefano, M.; Craparo, E. F.; Amato, G.; Fontana, G.; Cavallaro, G.;

9 10

Giammona, G. Int. J. Pharm. 2012, 433, 16-24. (6)

11 12

3712-3722. (7)

13 14

Wongpinyochit, T.; Uhlmann, P.; Urquhart, A. J.; Seib, F. P. Biomacromolecules 2015, 16,

Ham, T. R.; Lee, T. R; Han, S.; Haque, S.; Vodovotz, Y.; Gu, J.; Burnett, L. R.; Tomblyn, S.; Saul, J. M. Biomacromolecules 2016, 17, 225-236.

(8)

15

Pitarresi, G.; Tripodo, G.; Calabrese, R.; Craparo, E. F.; Licciardi, M.; Giarnmona, G. Macromol. Biosci. 2008, 8, 891-902.

16

(9)

17

(10) Tripodo, G.; Pitarresi, G.; Palumbo, F. S.; Craparo, E. F.; Giammona, G. Macromol. Biosci.

18 19 20 21 22 23

Pradal, C.; Grondahl, L.; Cooper-White, J. J. Biomacromolecules 2015, 16, 389-403.

2005, 5, 1074-1084. (11) Castelli, F.; Sarpietro, M. G.; Micieli, D.; Ottimo, S.; Pitarresi, G.; Tripodo, G.; Carlisi, B.; Giammona, G. Eur. J. Pharm. Sci. 2008, 35, 76-85. (12) LoPresti, C.; Vetri, V.; Ricca, M.; Fodera, V.; Tripodo, G.; Spadaro, G.; Dispenza, C. React. Funct. Polym. 2011, 71, 155-167. (13) Tripodo, G.; Mandracchia, D.; Collina, S.; Rui, M.; Rossi, D. Med. Chem. 2014, S1-004. 30


Biomacromolecules

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 5 6 7 8 9 10 11 12

Page 32 of 34

(14) Yu, Y.; Chen, C. K.; Law, W. C.; Weinheimer, E.; Sengupta, S.; Prasad, P. N.; Cheng, C. Biomacromolecules 2014, 15, 524-532. (15) Clementi, C.; Miller, K.; Mero, A.; Satchi-Fainaro, R.; Pasut, G. Mol. Pharmaceutics 2011, 8, 1063-1072. (16) Celia, C.; Calvagno, M. G.; Paolino, D.; Bulotta, S.; Ventura, C. A.; Russo, D.; Fresta, M. J. Nanosci. Nanotechnol. 2008, 8, 2102-2113. (17) Trapani, A.; Laquintana, V.; Lopedota, A.; Franco, M.; Latrofa, A.; Talani, G.; Sanna, E.; Trapani, G.; Liso, G. Int. J. Pharm. 2004, 278, 91-98. (18) Craparo, E. F.; Ognibene, M. C.; Casaletto, M. P.; Pitarresi, G.; Teresi, G.; Giammona, G. Nanotechnology 2008, 19, 485603. (19) Licciardi, M.; Craparo, E. F.; Giammona, G.; Armes, S. P.; Tang, Y.; Lewis, A. L. Macromol. Biosci. 2008, 8, 615-626.

13

(20) Butsele, K. V.; Jerome, R.; Jerome, C. Polymer 2007, 48, 7431-7443

14

(21) Luo, Y.; Wang, Q. Int. J. Bio Macromol. 2014, 64, 353-367.

15

(22) Neri, P.; Antoni, G.; Benvenuti, F.; Cocola, F.; Gazzei, G. J. Med. Chem. 1973, 16, 893-897.

16

(23) Lu, Y.; Chau, M.; Boyle, A. J.; Liu, P.; Niehoff, A.; Weinrich, D.; Reilly, R. M.; Winnik, M.;

17 18 19 20 21 22

A. Biomacromolecules 2012, 14, 1296-306. (24) Sharma, A.; Kundu, S.; Reddy, M. A.; Bajaj, A.; Srivastava, A. Macromol. Biosci. 2013, 13, 927-937. (25) Licciardi, M.; Pitarresi, G.; Cavallaro, G.; Giammona, G. Mol. Pharmaceutics 2013, 10, 16441654. (26) Sharma, A. and Srivastava, A. Polym. Chem. 2013, 4, 5119-5128.

31


Page 33 of 34

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

Biomacromolecules

(27) Sharma, A.; Pohane, A. A.; Bansal, S.; Bajaj, A.; Jain, V.; Srivastava, A. Chem. Eur. J. 2015, 21, 3540-3545.

3

(28) Fang, X.; Fang, L.; Gou, S.; Cheng, L. S. Bioorg. Med. Chem. Lett. 2013, 23, 1297-301.

4

(29) Zambre, A. P.; Kulkarni, V. M.; Padhye, S.; Sandur, S. K.; Aggarwal, B. B. Bioorg. Med.

5

Chem. 2006, 14, 7196-7204

6

(30) Fang X.; Fang L.; Gou, S.; Cheng, L. Bioorg. Med. Chem. 2013, 23, 1297–1301.

7

(31) D’Souza, S. S.; DeLuca, P. P. Pharm. Res. 2006, 23, 460-74.

8

(32) Costa, P.; Lobo, J. M. S. Eur. J. Pharm. Sci. 2001, 13, 123-33.

9

(33) Mosmann, T. J. Immunol. Methods. 1983, 65, 55-63.

10 11

(34) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl Cancer Inst. 1990, 82, 1107-12.

12

(35) Dhawan, A.; Bajpayee, M.; Parmar, D. Cell Biol. Toxicol. 2009, 25, 5-32.

13

(36) Civiale, C.; Licciardi, M.; Cavallaro, G.; Giammona, G.; Mazzone, M. G. Int. J. Pharma. 2009,

14 15 16 17 18

378, 177–186 (37) Gao, M.; Chen, C.; Fan, A.; Zhang, J.; Kong, D.; Wang, Z.; Zhao, Y. Nanotechnology 2015, 26, 275101. (38) Li, J.; Wang, Y.; Yang, C.; Wang, P.; Oelschlager, D. K.; Zheng, Y.; Tian, D. A.; Grizzle, W. E.; Buchsbaum, D.; J.; Wan, M. Mol Pharmacol. 2009, 76, 81-90.

19

(39) Wu, X.; Xu, J.; Huang, X.; Wen, C. Drug Dev Ind Pharm. 2011, 37, 15-23.

20

(40) Yallapu, M. M.; Jaggi, M.; Chauhan, S. C. Drug Discov Today. 2012, 17, 71-80.

21

(41) Zhao, Z.; Xie, M.; Li Y.; Chen, A.; Li, G.; Zhang, J.; Hu, H.; Wang, X.; Li, S.; Int. J.

22 23

Nanomedicine 2015, 10, 3171-3181. (42) Sambhy, V.; Peterson, B. R.; Ayusman S. Angew. Chem. Int. Ed. Engl. 2008, 47, 1250-1254. 32


Biomacromolecules

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 34 of 34

1

(43) Kuroda, K.; DeGrado, W. F. J. Am. Chem. Soc. 2005, 127, 4128-4129.

2

(44) Kunwar, A.; Barik, A.; Mishra, B.; Rathinasamy, K.; Pandey, R.; Priyadarsini, K. I. Biochim

3

Biophys Acta. 2008, 1780, 673-679

4

(45) Kau, T. R.; Way, J. C.; Silver, P. A. Nature Reviews Cancer 2004, 4, 106-117.

5

(46) Sahay, G.; Alakhova, D. Y.; Kabanov, A. V. J. Control. Release 2010, 145, 182-95.

6

(47) Harush-Frenkel, O.; Debotton, N.; Benita, S.; Altschuler, Y. Biochem. Biophys. Res. Commun.

7

2007, 353, 26-32.

8

(48) Heuser, J. E.; Anderson, R. G. J. Cell Biol. 1989, 108, 389-400.

9

(49) Shishodia, S.; Amin, H. M.; Lai, R.; Aggarwal, B. B. Biochem. Pharmacol. 2005, 70, 700-713.

10

(50) Srivastava, R. K.; Chen, Q.; Siddiqui, I.; Sarva, K.; Shnkar, S. Cell Cycle 2007, 6, 2953-2961.

11

33


Cell Permeating Nano-Complexes of Amphiphilic ... - ACS Publications

Recommend Documents