This may be the author’s version of a work that was submitted/acceptedfor publication in the following source:

Raveendran, Radhika, Mullen, Kathleen, Wellard, Robert, Sharma, Chan-dra, Hoogenboom, Richard, & Dargaville, Tim(2017)Poly(2-oxazoline) block copolymer nanoparticles for curcumin loading anddelivery to cancer cells.European Polymer Journal, 93, pp. 682-694.

This file was downloaded from: https://eprints.qut.edu.au/104804/

c© Consult author(s) regarding copyright matters

This work is covered by copyright. Unless the document is being made available under aCreative Commons Licence, you must assume that re-use is limited to personal use andthat permission from the copyright owner must be obtained for all other uses. If the docu-ment is available under a Creative Commons License (or other specified license) then referto the Licence for details of permitted re-use. It is a condition of access that users recog-nise and abide by the legal requirements associated with these rights. If you believe thatthis work infringes copyright please provide details by email to qut.copyright@qut.edu.au

License: Creative Commons: Attribution-Noncommercial-No DerivativeWorks 2.5

Notice: Please note that this document may not be the Version of Record(i.e. published version) of the work. Author manuscript versions (as Sub-mitted for peer review or as Accepted for publication after peer review) canbe identified by an absence of publisher branding and/or typeset appear-ance. If there is any doubt, please refer to the published source.

https://doi.org/10.1016/j.eurpolymj.2017.02.043

1

Poly(2-oxazoline) Block Copolymer Micelles for Curcumin Loading and Delivery to Cancer Cells Radhika Raveendran1,2, Kathleen M. Mullen2, R. Mark Wellard2, Chandra P. Sharma,1

Richard Hoogenboom3, Tim R. Dargaville2* 1 Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Science and Technology, Poojappura, Thiruvananthapuram 695012, Kerala, India 2 Nanotechnology and Molecular Science Discipline, Science and Engineering Faculty Queensland University of Technology, Queensland 4001, Australia 3 Supramolecular Chemistry Group, Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4, B-9000 Ghent, Belgium * Corresponding author address A/Prof. Tim R Dargaville Nanotechnology and Molecular Science Discipline Science and Engineering Faculty Queensland University of Technology Brisbane 4001, Australia E-mail: t.dargaville@qut.edu.au

2

Abstract

Curcumin, obtained from spice turmeric, is renowned for its anti-cancer activity,

however, its use as a viable drug is impeded by its low aqueous solubility. To address

this, we have investigated the potential of amphiphilic block copolymers, based on

poly(2-alkyl-2-oxazoline)s (PAOx), as feasible nano-carriers for efficiently delivering

curcumin to cancer cells. The block copolymers comprising hydrophobic and hydrophilic

PAOx units in two different ratios were synthesized by cationic ring opening

polymerization (CROP). The micelles, ensuing from the self-assembly of the block

copolymers in aqueous media, could encapsulate curcumin in their hydrophobic core.

These curcumin loaded PAOx micelles, of size around 100 nm, exhibited excellent

stability and enhancement of aqueous solubility of curcumin. In vitro release studies

demonstrated a pH-sensitive release of curcumin from the PAOx micelles. Profound

cytotoxicity on C6 glioma cells, along with enhanced internalization of curcumin in these

cells was achieved by the micelle-mediated delivery. It was also revealed that the length

of the hydrophobic block of the PAOx copolymers was functionally important. These

systems offer potential clinical relevance as promising nano-carriers capable of

circumventing the clinical limitations of curcumin.

3

Introduction Cancer chemotherapy has witnessed the efficacy of many natural products in destroying

tumours [1]. One such natural product is curcumin which is extracted from the popular

spice, turmeric. This polyphenolic compound, chemically identified as (E,E)-1,7-bis(4-

hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is known to possess a plethora of

diverse therapeutic properties [2, 3]. Extensive research over the last few decades has

established the anti-cancer potential of curcumin to induce selective apoptosis in cancer

cells [4]. Moreover, curcumin also possesses the ability to reverse multi-drug resistance

by decreasing P-glycoprotein expression and promoting caspase-3 activation in cells [5].

Despite its promising potential, curcumin faces obstacles when it comes to clinical

administration, mainly due to its low aqueous solubility, owing to its hydrophobic nature,

and instability at physiological pH [6].

Recent research has exploited polymeric micelles for curcumin delivery as an

effective strategy to bypass these limitations for enhancing its clinical efficacy [7].

Polymeric micelles are self-assembled nano-constructs based on amphiphilic

macromolecules that have distinct hydrophobic and hydrophilic blocks. Such block

copolymer micelles are endowed with a host of favourable properties which offer great

prospects in the realm of drug delivery, including solubilization of hydrophobic drugs,

stealth properties leading to enhanced systemic circulation, preserving the integrity of the

encapsulated drugs, and the ability to be targeted to cancer cells [8, 9]. A typical polymer

micelle is designed by incorporating hydrophobic moieties, most commonly fatty acids

[10, 11] or hydrophobic polymers [8], to solubilize the poorly water soluble drugs. The

hydrophilic corona that offers stealth properties is usually constituted by polyethylene

4

glycol (PEG) blocks [12]. PEG is extensively used in biomedical applications, however it

lacks the ability to be functionalized along the backbone, meaning its physical properties

cannot be easily altered. Poly(2-alkyl-2-oxazoline)s (PAOx), prepared by cationic ring

opening polymerization (CROP) of 2-oxazolines are garnering attention due to their

similarities to PEG regarding biocompatibility and stealth behavior. The possibilities of

achieving tailor-made properties, by manipulation of the functional groups on the side

chains of 2-oxazoline monomers [13-15], make PAOx an attractive alternative to PEG, as

is evident from its broad application to various biomedical challenges in the recent years

[16-19].

This study is focused on exploring amphiphilic block copolymers based on PAOx

for solubilizing curcumin in aqueous medium. Surprisingly, there is a paucity of

information on curcumin-polymer miscibility parameters aside from a study based on

compatibility of curcumin with poly-ε-caprolactone (PCL) [20]. This has prompted us to

assess the curcumin solubility and compatibility with the PAOx micelle core using Flory-

Huggins interaction parameters. This qualitative technique can help in the screening

processes of polymers that are suitable for delivery of specific drugs to achieve highly

compatible drug-polymer pairs [21]. It is also noted that most of the investigated

polymeric micelles for curcumin delivery have low loading capacity; and micelles that do

have favourable loading are accompanied by the release of considerable amount of

curcumin at physiological pH which exacerbates the overall in vivo therapeutic efficacy

[22-24]. Therefore, it is imperative that with favourable drug loading, pH-sensitive drug

release is also attained at lower pH conditions, which could be exploited for efficient

drug delivery at the acidic tumour micro-environment and/or during cell uptake through

5

endocytosis. Curcumin delivery employing PAOx micelles has not been previously

reported and curcumin loaded PAOx micelles with their promising characteristics for in

vivo application could be a valuable addition to the repertoire of polymeric micelle-

mediated curcumin delivery systems.

Experimental Section

1. Materials and methods

2-Ethyl-2-oxazoline (EtOx), methyl p-toluenesulfonate methyl tosylate, 1,6 diphenyl-

1,3,5 hexatriene (DPH), curcumin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) and Dulbeccos’s Modified Eagles Medium (DMEM) were obtained from

Sigma Aldrich. The 2-(but-3-enyl)-2-oxazoline (ButenOx) monomer was synthesized

according to [25] (the 1H NMR spectrum of ButenOx is shown in Figure S1). 4-Pentenoic

acid, N-hydroxysuccinimide and 2-chloroethylamine hydrochloride, used for synthesizing

this monomer, were also obtained from Sigma Aldrich. Fetal bovine serum (FBS) was

from GIBCO (USA). C6 glioma cells were obtained from National Centre for Cell

Science (Pune, India). All solvents were dried over molecular sieves before use.

2. Nuclear Magnetic Resonance (NMR) Spectroscopy

One-dimensional 1H NMR spectra were obtained using a Varian spectrometer operating

at 400 MHz. CDCl3 was used as solvent and an acquisition time of 5 s and a relaxation

delay of 8 s were employed for the collection of 1H NMR spectra of block polymers.

Deuterated DMSO was used as the solvent for the 1H NMR determination of the

ButenOx monomer. Diffusion ordered 2D NMR spectra were recorded in CDCl3 at 298K

6

with at QNP probe in a Bruker 400MHz Nanobay spectrometer using a standard pulse

sequence utilizing a double stimulated echo for convection compensation and LED, using

bipolar gradient pulses for diffusion and three spoiler gradients (DSTEBPGP3s) [26].

Acquisition parameters were relaxation delay 6 s; acquisition time 1.7 s; d20, 0.3s; p30,

1000 µs; 32k data points. Thirty-two averages were acquired for each of 32 increments

over the range of 5% to 95% gradient power. Spectra were processed with Topspin

software (Bruker).

3. Synthesis of P(EtOx-b-ButenOx) block copolymers

(a) Synthesis of P(EtOx33-b-ButenOx26): EtOx (0.25 g, 2.5 mmol) in acetonitrile (0.6 mL)

was reacted with methyl tosylate (0.018 g, 0.096 mmol) in a microwave vial. The vial

was capped and argon was bubbled through the solution for 20 mins. The reaction

solution was then heated in the microwave synthesizer (Biotage) for 15 mins at a

temperature of 140 oC. Aliquots of the reaction mixture were removed for the NMR

analysis of the first block. Then ButenOx (0.3 g, 2.4 mmol) was added to the vial under

argon atmosphere and further heated for 30 mins at 140 oC in the microwave reactor. The

formation of the second block was monitored by 1H NMR spectroscopy (Figure S2).

Finally, the reaction was quenched by adding a methanolic KOH solution (20 µL) and the

pale yellow viscous reaction mixture was dissolved in chloroform before washing with

sodium bicarbonate solution followed by a saturated solution of brine. The chloroform

layer was then dried over anhydrous sodium sulphate and concentrated in vacuo. The

concentrated solution was then added dropwise to ice-cold diethyl ether leading to the

formation of white precipitate. The solution was then filtered and the precipitate was

7

washed twice with ether and dried overnight under vacuum (0.34 g, 59% yield). 1H NMR

= 5.75-5.89 (m, 19H, CH2=CH-)); 4.96-5.12 (m, 37H, CH2=CH-)); 3.45 (br, 148H, (N-

CH2CH2)); 2.5-2.2 (m, 123H, CO-CH2-CH3, -CH2-CH2-CH=CH2); 1.11 (br, 71H, CO-

CH2-CH3), Mn= 6556 g/mol.

(b) Synthesis of P(EtOx17-b-ButenOx44): P(EtOx17-b-ButenOx44) was synthesized

accordingly using EtOx (0.15 g, 1.5 mmol), methyl tosylate (0.01 g, 0.06 mmol) in

acetonitrile (0.4 mL) and ButenOx (0.3g , 2.4 mmol). The same reaction and work-up

procedure as above were followed (0.3 g, 67% yield). 1H NMR = 5.75-5.89 (m, 60H,

CH2=CH-)); 4.96-5.12 (m, 117H, CH2=CH-)); 3.45 (br, 277H, (N-CH2CH2)); 2.5-2.2 (m,

285H, CO-CH2-CH3, -CH2-CH2-CH=CH2)); 1.11 (br, 67H, CO-CH2-CH3), Mn= 7225

g/mol.

4. Preparation of micelles

The micelles were prepared by a nano-precipitation method: P(EtOx-b-ButenOx) (6 mg)

and curcumin (1 mg) were dissolved in acetone (0.5 mL) and added dropwise from a

21G needle to de-ionized (DI) water (10 mL) stirring at 1300 rpm. The solution was

further diluted with DI water (10 mL) and allowed to stir for removal of acetone after

which it was filtered through a 0.22 µm pore syringe filter. Empty micelles of block

polymers were prepared by similar method without addition of curcumin. The micelle

solutions were lyophilized for further experimental studies.

5. Determination of Critical Micelle Concentration (CMC)

8

The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was employed to determine

the CMC of the two block copolymers [27]. A stock solution was prepared by dissolving

DPH (2.5 mg) in methanol (5 mL). The solution was diluted 20 times and 50 µL of the

diluted DPH solution was added to different concentrations of empty polymeric micelle

suspensions in the range of 3 × 10-4 to 0.65 mg/mL. The suspensions were vortexed and

kept overnight to equilibrate the DPH with the micelles. DPH fluorescence emission

spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer with λem

= 429 and λex = 358 nm. The CMC values for the micelles, indicated by the inflection

point of the curve in the graph of log [concentration] and fluorescence intensity, were

calculated from the point of intersection of the regression lines.

6. Determination of Micelle Size by Dynamic Light Scattering (DLS) and

Transmission Electron Microscopy (TEM)

The size of the micelles was assessed by DLS (Nano-ZS, Malvern Instruments). The

temperature was maintained at 25 oC and measurements were recorded as the average of

three test runs. DLS measurements were also performed to study the stability of micelles

prepared in de-ionized water. For the observation of size and morphology of the micelles,

micelle solutions were administered onto 200 mesh carbon coated copper grids and

negatively stained using 5% uranyl acetate solution. Samples were air-dried prior to

imaging. TEM measurements were performed on a JEOL 1400 at an acceleration voltage

of 120 kV.

Calculation of Aggregation Number: The aggregation number of a micelle was calculated

according to:

9

0MMNag = (1)

where M and M0 denote the molar mass of the micelle and the polymer, respectively [28].

The following equation was used to calculate the molar mass of a micelle

2

=ν

π3N4 3ARM

(2)

where NA is Avogadro’s number, R is the radius of the micelle and ν2 (= 0.88 cm3/g [29])

is the partial specific volume of the polymer.

7. Determination of Curcumin Loading in the Micelles

Calculated amounts of lyophilized curcumin loaded PAOx were dissolved in methanol

and the absorbance was recorded on a UV-Vis spectrophotometer at 420 nm. A

calibration curve was based on methanolic curcumin solutions with concentrations

ranging from 0.5 µg/mL to 25 µg/mL. The absorbance of these solutions was recorded

and expressed as function of curcumin concentration, which was used for the

determination of curcumin loading in the micelles. Drug loading capacity and

encapsulation efficiency were calculated as follows:

Drug loading capacity w/w% = Amount of curcumin    

(Amount of curcumin loaded PAOx micelles)×100

Encapsulation efficiency % = Encapsulated amount of curcumin    

(Initial amount of curcumin )×100

10

8. Drug-Polymer Compatibility Calculations

The compatibility between curcumin and the micelle core forming block (ButenOx) was

calculated by employing the Hildebrand-Scatchard equation [20]:

( )RTVpolymerdrugpolymerdrug2δδχ −− = (3)

where χdrug-polymer is the Flory-Huggins interaction parameter, δdrug is the solubility

parameter for the drug (curcumin), δpolymer is the solubility parameter for the core forming

block, R is the gas constant , T is the temperature and V is the molar volume of the drug

(253.1 cm3/mol for curcumin) which is calculated from the group contributions method

derived by Fedors [30]. Calculation of δdrug and δpolymer was obtained from Van

Krevelen’s additive group contribution method according to which the solubility

parameter is the sum of dispersion [δd] , polar [δp] and hydrogen bonding components

[δh] [31].

2222 hpddrug δδδδ ++= (4)

and 2222 hpdpolymer δδδδ ++= (5)

The individual components were calculated by the following equations:

VFdi∑= dδ (6)

( )

VFpi

2/12

p∑=δ (7)

2/1

⎟⎠

⎞⎜⎝

⎛= ∑ V

Ehihδ (8)

where Fdi , Fpi and Ehi are the molar dispersion, polar attraction constants and hydrogen

bonding energy, respectively, for each structural group in the molecules .

9. Release Study

Lyophilized curcumin loaded PAOx was redispersed in 10 mL PBS (pH 7.4) or acetate

buffer (pH 4.3) at a concentration of 0.5 mg/mL and was divided into 1 mL aliquots in

11

Eppendorf tubes and put in a shaker table maintained at 37 oC. At pre-determined time

intervals, the aliquots were centrifuged to separate the released curcumin from the

micelles, which gathered at the bottom of the tubes. The pelleted curcumin was extracted

in methanol and quantified at 420 nm using a UV-vis spectrophotometer. The released

amount was expressed as a percentage of the total curcumin loaded.

10. Spectral Characterization

UV-Visible spectroscopic analysis was performed using a Shimadzu UV-1800 UV

spectrometer over the range 400-700 nm. The fluorescence emission spectra of free

curcumin and curcumin loaded micelles were recorded from 450-700nm with an

excitation wavelength of 425 nm on a Varian Cary Eclipse fluorescence

spectrophotometer.

11. Cell Culture Studies

(a) In vitro cytotoxicity

(i) MTT Assay

The cytotoxicity of empty and curcumin loaded PAOX micelles was assessed on C6

glioma cells using the MTT assay. DMEM medium along with 10% FBS were utilized

for seeding the cells prior to their incubation for 24 h at 37 °C in 5% CO2 and 95%

humidity. The cells were trypsinized and transferred to a 24-well cell culture plate at a

density of 2×104 cells/well and were exposed to different concentrations of empty and

loaded PAOx micelles for 24 h, followed by addition of 100 µL MTT solution (0.5

mg/mL) and incubated for 3 h at 37 oC. The resulting formazan crystals were dissolved

12

with the addition of 300 µL of DMSO per well. The absorbance was measured at 570 nm

using a plate reader (Finstruments Microplate Reader). Cell viability was evaluated by

measuring the mitochondrial-dependent conversion of the yellow tetrazolium MTT salt to

purple formazan crystals by metabolically active cells. A concentration range of 1-5

mg/mL empty PAOx micelles in PBS (0.01M, pH 7.4) was tested to determine their

cytotoxicity. Cytotoxicity of the loaded micelles was assessed where the concentration of

curcumin in the micelles was in the range of 5 µM-20 µM. In the case of free curcumin,

as it is insoluble in PBS, DMSO was employed to aid the solubility but it was ensured

that the final concentration of DMSO in the cell culture medium was less than 0.05 %.

The cells treated with medium were used as a negative control. The percentage cell

viability (CV) was calculated as:

CV   % =NtNC

×100  

where Nt is the absorbance of the cells treated with free curcumin or curcumin loaded

micelles and Nc was the absorbance of the untreated cells.

(ii) Live/Dead Assay

A live/dead assay was performed to further qualitatively assess the cytotoxicity of the

curcumin loaded PAOx micelles on cancer cells. C6 glioma cells were seeded into

24 well plates at a seeding density of 4×104 cells/well and allowed to adhere overnight in

an atmosphere of 5% CO2 at 37 °C. The cells were then incubated with 100 µL of the

samples for 24 h where an equivalent concentration of 20 µM of curcumin was

maintained in the free and the loaded forms. Cells were washed three times with PBS

prior to the assay and stained with 2 µM calcein AM (acetomethoxy derivate of calcein)

and 4 µM EthD-1 (ethidium homodimer) assay reagents, followed by 30 min of

13

incubation at 37 °C. The cells were then visualized and imaged using a fluorescence

microscope (Leica, DMI3000).

(b) In vitro Cellular Uptake Studies by Fluorescence Microscopy

Cells were seeded at a density of 1×104 cells/well and were treated with free curcumin

and curcumin loaded PAOx micelles prior to incubation at 37 oC for 4 h. An equivalent

concentration of 5 µM of curcumin as a free and loaded form was used for the uptake

study. After subjecting to PBS washing, the cells were fixed in 3.7% paraformaldehyde

solution before viewing under the fluorescence microscope.

Results and Discussion

The ability of amphiphilic PAOx block copolymers to act as efficient polymeric

carriers has been previously reported for a number of drug and gene delivery systems [18,

32-35]. Extending on this potential of PAOx-based nano-carriers, this study elucidated

PAOx micelles for enhanced dissolution of curcumin and subsequent delivery to cancer

cells. Curcumin loading in polymer micelles is determined by the miscibility and

hydrophobicity of the core forming block and can be predicted using Flory-Huggins

parameters (χdp). Lower values of χdp (ideally equal to zero) reflect the effectiveness of a

polymer as a thermodynamically good solvent for the drug, therefore indicating that there

are favourable interactions enhancing the miscibility between the polymer and the drug,

in addition to hydrophobic interactions, which contributes to the drug solubilization.

When comparing poly(2-butenyl-2-oxazoline) (χdp = 0.05) to four other commonly used

PAOx homopolymers, namely, those with butyl, pentyl, nonyl and phenyl pendant

groups, only the phenyl functional PAOx (χdp = 0.0045) had a lower χdp value (Table S1).

14

In the context of non-PAOx polymers, even the poorest performing PAOx (nonyl χdp =

2.03) is superior to PCL (χdp= 2.91) in regard to curcumin miscibility [20].

While phenyl PAOx afforded the most favourable solubility parameter of the

polymers surveyed, the high glass transition temperature of this polymer (Tg ~ 100 oC)

may pose limitations in its use. Furthermore, the vinyl functional group of ButenOx is

attractive for future modification (e.g. drug-polymer conjugates of degradable cross-

links) and so was chosen as the hydrophobic block component. Hence, we prepared

diblock PAOx copolymers containing the hydrophilic EtOx and hydrophobic ButenOx

segments (Figure 1). The EtOx is commercially available, while the ButenOx was

synthesized according to the modified Wenker method reported by Gress et al. [25]. This

method has recently been improved upon via α-deprotonation of the cheap starting

material 2-methyl-2-oxazoline [36]. However, for the small scale synthesis used in this

study the modified Wenker method was sufficient. Two copolymers each with similar

degrees of polymerization, namely, P(EtOx33-b-ButenOx26) and P(EtOx17-b-ButenOx44),

were synthesized by the CROP of EtOx before adding the second monomer (ButenOx)

and further polymerization. The disappearance of the monomer peaks and appearance of

the backbone protons of the polymer at 3.45 ppm by 1H NMR spectroscopy (Figure S2),

confirmed the polymerization of both monomers. To check that bot blocks were

incorporated into the same polymer chains 2D DOSY experiments were performed

confirming that all the polymer signals had the same relative diffusion coefficients

(Figure 2).

15

Figure 1. Schematic representation depicting the synthesis of the P(EtOx-b-ButenOx) block copolymers and the formation of curcumin loaded P(EtOx-b-ButenOx) micelles.

Figure 2. 2D DOSY spectra of P(EtOx33-b-ButenOx26) (left) and P(EtOx17-b-ButenOx44)

(right) demonstrating the same diffusion coefficients for the EtOx and ButenOx blocks.

16

Critical micelle concentration (CMC) is of paramount importance in dictating the

stability of polymer micelles in systemic circulation. The CMCs of the PAOx micelles

were determined by using DPH as a fluorescent probe. DPH is strongly hydrophobic and

thus can readily incorporate itself into the hydrophobic core of micelles. The transition

from the aqueous environment of the probe to the hydrophobic core of the micelles

results in significant increase in fluorescence intensity [37, 38]. The CMCs of P(EtOx33-

b-ButenOx26) and P(EtOx17-b-ButenOx44) were calculated to be 0.12 mg/mL and 0.07

mg/mL, respectively (Figure 3b). The wt% of ButenOx in

P(EtOx17-b-ButenOx44) and P(EtOx33-b-ButenOx26) was 33.5% and 21.8%, respectively.

The lower relative CMC value of P(EtOx17-b-ButenOx44) was attributed to the higher

proportion of hydrophobic ButenOx units. This trend of decreasing CMC values with

increasing hydrophobic PAOx units is in accordance with the literature [34, 39]. Micelle

dissociation is related to the composition and the cohesion of the hydrophobic core. An

increment in the hydrophobic units resulted in the enhanced cohesion of the core leading

to lower CMC [40-42].

17

Figure 3. Dependence of fluorescence intensity of DPH on the concentration of P(EtOx-

ButenOx) micelles

Empty and loaded micelles of the P(EtOx-b-ButenOx) polymers were prepared by

the nano-precipitation method based on the displacement of a solvent with non-solvent.

In contrast to the emulsion/solvent diffusion technique, which uses immiscible solvents,

nano-precipitation utilizes a non-solvent for the drug that is miscible with the solvent

used during initial mixing of the drug with the polymer. Furthermore, the

emulsion/solvent diffusion technique might include addition of surfactants that could

have toxic effects. Pure nanoparticle suspensions can be prepared by dropwise addition

by using volatile solvents that permit easy evaporation. The size of the particles formed

by this method is strongly influenced by the diffusion process [43]. In our preparation, we

dissolved curcumin and P(EtOx-b-ButenOx) in acetone and added the polymer/drug

solution dropwise to stirring water, followed by dilution and removal of acetone. This can

18

favour the decrease of free energy of the system by rapid removal of hydrophobic

fragments from the aqueous environment to form the drug loaded micellar core (Figure

4a) [44]. Encapsulation of curcumin in the micelles rendered it soluble in aqueous media

(Figure 4 b-d), while micelle size in the range of 70-110 nm was confirmed by DLS

measurements (Table 1 and Figure S3).

Figure 4. (a) Schematic diagram showing the preparation of curcumin loaded micelles by

the nano-precipitation method; photographs showing: (b) that curcumin is insoluble in

water, (c) curcumin loaded P(EtOx33-b-ButenOx26) micelles in water, and (d) curcumin

loaded P(EtOx17-b-ButenOx44) micelles in water; TEM images of: (e) curcumin loaded

P(EtOx33-b-ButenOx26) micelles, and (f) curcumin loaded P(EtOx17-b-ButenOx44)

micelles.

19

Table 1. Size, CMC and loading characteristics of the micelles obtained from the P(EtOx-b-

ButenOx) copolymers.

Polymer

CMC

(mg/ml) a

Size (nm) b

Loading

capacity

(%) c

Encapsulation

efficiency

(%) d

Empty

Micelles

Loaded

micelles

P(EtOx33ButenOx26)

P(EtOx17ButenOx44)

0.12

0.07

97.7 ± 8.5

69.4 ± 1.1

107.5 ± 2.1

79.5 ± 1.4

7.4 ± 1.7

11.8 ± 2.2

51.9 ± 1.9

82.7 ± 4.7

a Determined from the point of inflection of the graphs in Figure 2; b Determined from DLS measurements; c,d Calculated using equations 1-2.

Particle size is paramount in determining the bio-distribution, cellular uptake and

in vivo stability of polymer micelles [45]. Polymeric micelles due to their small size, in

the range of 10-200 nm, can accumulate in tumour cells due to the enhanced permeation

and retention (EPR) effect [46]. When measuring the size of micelles prepared by the

nano-precipitation method care needs to be exercised as variabilities in how the polymer

is added to the water can influence particle size. For these experiments the same gauge

needle was used when adding the polymer/drug solution to water (stirred at a constant

speed) to help keep the droplet size constant. Despite the possibility of variation, the

P(EtOx17-b-ButenOx44) micelles consistently exhibited smaller size than P(EtOx33-b-

ButenOx26) for multiple replicates (Table 1), which could be explained by the lower

space demands of the hydrophilic block in aqueous media [47]. The shorter and less

20

extended PAOx hydrophilic chains contributed to smaller hydrodynamic volume leading

to reduction in overall particle size.

The size of micelles obtained by TEM was much smaller compared to the DLS

measurements due to the drying of the hydrophilic chains of micelles during sample

preparation (Figure 4e,f and S4). It is perceived that longer hydrophobic and shorter

hydrophilic chains may cause formation of non-spherical aggregates [28]. Interestingly,

from the TEM images it was seen that P(EtOx17-b-ButenOx44) did not undergo any

transformation and exhibited similar spherical morphology to P(EtOx33-b-ButenOx26).

We believe that this could be related to similar micelle aggregation numbers, namely

1.69×104 and 5.14×104 for P(EtOx17-b-ButenOx44) and P(EtOx33-b-ButenOx26),

respectively (calculated using equations 1 and 2).

It is understood that curcumin loading content in polymeric micelles is a factor

which is constantly compromised despite its enhanced solubility in aqueous media.

Curcumin loading efficiencies as low as 1-4% w/w have been reported in polymeric

micelles [22-24, 48] including curcumin conjugated to water soluble polymers that were

capable of self- assembling into micelles [49, 50]. It has always been a challenge to

sprepare micelles with size less than 200 nm while maintaining high curcumin loading.

Therefore, there is continued research aimed at attaining higher cargo capacity, in tandem

with favourable physico-chemical properties for curcumin delivery systems. Most of the

curcumin loading content in polymeric micelles has been reported in the range of 1-4%

w/w [22, 23] thus implying a need for polymers with greater cargo capacity for curcumin.

Curcumin loadings of 7.5 ± 1.7 and 11.6 ± 2.2 % w/w were achieved with P(EtOx33-b-

ButenOx26) and P(EtOx17-b-ButenOx44) micelles, respectively (Table 1).

21

Spectral characterization studies confirmed the encapsulation of curcumin in the

micelles (Figure 5). Curcumin absorption peak in the loaded micelles was evident at 425

nm with enhanced intensity. In the fluorescence spectra, curcumin showed a weak broad

peak at 560 nm and the curcumin-loaded P(EtOx33-b-ButenOx26) and P(EtOx17-b-

ButenOx44) micelles showed a well-defined high intensity blue shifted peak at 548 and

538 nm, respectively. This blue shift in the emission spectrum is indicative of the

transition of curcumin to the hydrophobic interior of a micellar environment [51]. The

encapsulation of curcumin in the hydrophobic core of the micelles is facilitated through

the hydrogen bond interaction between the hydroxyl groups of curcumin and ButenOx

core. The shift towards shorter wavelength, indicating increased hydrophobicity, was

evident with the higher number of ButenOx units.

Figure 5. (A) UV-visible spectrum, (B) fluorescence spectrum of: (a) curcumin loaded

P(EtOx17-b-ButenOx44) micelles, (b) curcumin loaded P(EtOx33-b-ButenOx26) micelles

and, (c) curcumin.

22

Stability is an important parameter for drug loaded micelles in systemic

circulation. Micelles designed for drug delivery should remain intact to prevent release of

the drug payload before reaching the target cells. The size of the P(EtOx17-b-ButenOx44)

micelles with and without curcumin was almost constant for over a period of 30 days and

was evidently more kinetically stable than P(EtOx33-b-ButenOx26) (Figure 6a). Increased

hydrophobic chain length which determined lower CMC values correlated to increased

stability of micelles. Empty micelles of P(EtOx33-b-ButenOx26) exhibited an increase in

size after 8 days, however the corresponding curcumin loaded micelles did not follow a

similar trend. Additional hydrophobic interactions of an encapsulated drug are known to

stabilize the core of micelles [28]. It is evident that curcumin loaded in the core of the

micelles enhanced the hydrophobicity of the core and could enhance the stability of the

micelles [52] .

Figure 6. (a) Change in size of the curcumin loaded and empty P(EtOx-ButenOx)

micelles with time, (b) release of curcumin from the loaded P(EtOx-ButenOx) micelles

at pH 4.5 and 7.4 with time.

23

The ability to ultimately release the drugs, in addition to sequestration and

solubilization of hydrophobic drugs, completes the profile of efficient drug delivery

systems. In our previously investigated matrices, despite better curcumin loading

capacity, there was a setback of high curcumin release at physiological pH from the

polymer micelles [53, 54]. It was interesting to note from our release experiments with

P(EtOx-b-ButenOx) that pH-dependent curcumin release was observed. Approximately

12.6 ± 4.5 % and 34 ± 5 % of curcumin were released from P(EtOx17-b-ButenOx44) and

P(EtOx33-b-ButenOx26) micelles, respectively over 168 hours at pH 7.4. This compares to

86 ± 3% and 87 ± 5% of released curcumin at pH 4.3, for P(EtOx17-b-ButenOx44) and

P(EtOx33-b-ButenOx26), respectively (Figure 6b). Similar pH-dependent release

characteristics were previously reported with PAOx [32]. The more acidic pH may

induce partial protonation of the tertiary amide groups on the PAOx backbone, which can

subsequently enable intra/intermolecular interactions between micelles leading to

aggregation and finally, micellar deformation. Moreover, the hydronium ions may

compete stronger with curcumin-PAOx hydrogen bonding, thereby destabilizing the

micellar core. Faster release in acidic pH is conducive to the tumour micro-environment

as it relates to the lower pH in the endocytic compartment of tumor cells (pH 4.5-6.5). It

was also observed that the release of curcumin from the micelles was dependent on the

hydrophobicity of the micelles. Enhanced hydrophobic cohesive interaction due to a

greater number of ButenOx units accounted for the relatively lower release of curcumin

from P(EtOx17-b-ButenOx44) micelles.

24

The favourable loading and release profile of curcumin loaded P(EtOx-b-

ButenOx) micelles encouraged us to further conduct cell culture studies. The empty

PAOx micelles were assessed for their cytotoxicity on C6 glioma cells. There was no

significant reduction in the cell viability on exposure to the unloaded PAOx micelles (up

to 5 mg/mL) indicating the non-toxicity of synthesized PAOx micelles (Figure 7a) and in

agreement with other PAOx-cell studies [55-57][58]. However, with the curcumin loaded

PAOx micelles, a dose dependent cytotoxicity was observed highlighting the anti-cancer

effect of curcumin on cancer cells [4]. Free curcumin was incapable of exerting the

desired cytotoxic effect owing to its poor aqueous solubility. Curcumin-loaded PAOx

micelles promoted cell death, compared to free curcumin (Figure 7b). At 20 µM of

equivalent curcumin concentration, 14 ± 3% , 24 ± 4 %, 72 ± 6 % cell viability was

observed with curcumin loaded P(EtOx17-b-ButenOx44), curcumin loaded P(EtOx33-b-

ButenOx26) and free curcumin, respectively. Curcumin loaded PAOx micelles induced

profound cell death compared to free curcumin, which may be ascribed to the enhanced

aqueous solubility of curcumin in the micelles in combination with accelerated release of

curcumin in an acidic environment, proposedly during endocytosis. The cytotoxic effects

were further corroborated by a live-dead assay (Figure 7). The glioma cells were treated

with free and micellar solutions of curcumin and then incubated with the live and dead

cell markers, calcein AM and ethidium bromide dye, respectively. Consistent with

cytotoxicity assay, enhanced cell death (red fluorescence) was prominent for the cells

exposed to curcumin loaded PAOx micelles. Free curcumin incubated cells remained

viable (green fluorescence) due to their low intracellular accessibility owing to the drug’s

insolubility.

25

Figure 7. Cytotoxicity study (a) empty P(EtOx-b-ButenOx) micelles, (b) curcumin

loaded P(EtOx-b-ButenOx) micelles. Live-dead assay on C6 glioma cells (c) control, (d)

curcumin, (e) curcumin loaded P(EtOx17-b-ButenOx44) micelles, (f) curcumin loaded

P(EtOx33-b-ButenOx26) micelles

The cellular internalization of curcumin loaded PAOx micelles was visualized by

exploiting the green intrinsic fluorescence of curcumin. From the fluorescence

microscopy images, it was observed that curcumin loaded P(EtOx-b-ButenOx) micelles

exhibited prominent green fluorescence indicating relatively better uptake than the free

26

curcumin (Figure 8). Relatively better uptake of curcumin loaded P(EtOx17-b-ButenOx44)

micelles was seen compared to P(EtOx33-b-ButenOx26) micelles due to the greater

hydrophobicity of P(EtOx17-b-ButenOx44)

Figure 8. Cellular uptake in C6 glioma cells (a) curcumin, (b) curcumin loaded

P(EtOx17-b-ButenOx44) micelles, (c) curcumin loaded P(EtOx33-b-ButenOx26) micelles.

Amphiphilic PAOx are known to enter cells efficiently by endocytosis [59] and it

is understood that hydrophobicity plays a decisive role in the cellular uptake of these

polymers. Our uptake results were consistent with previous reports that a reduction in the

hydrophobicity of the block polymer diminishes the cellular uptake of PAOx, while the

hydrophilic blocks play insignificant role in the cellular internalization [58]. Free

27

curcumin was not readily taken up by the cancer cells, which was evident from the

diminished green fluorescence. Encapsulation of curcumin in P(EtOx-b-ButenOx)

micelles exhibited prominent green fluorescence indicating relatively better cellular

uptake than the free curcumin.

Conclusion

In summary, amphiphilic PAOx block copolymers, capable of self-assembling in aqueous

media, were synthesized and the feasibility of employing these polymeric micelles to

encapsulate curcumin was demonstrated. Enhanced anti-cancer effects and significantly

higher cellular uptake were achieved with curcumin loaded P(EtOx-b-ButenOx) micelles.

It is apparent that of the two P(EtOx-b-ButenOx) copolymers, P(EtOx17-b-ButenOx44)

exhibited superior properties, relative to P(EtOx33-b-ButenOx26), in terms of loading,

release, cytotoxicity and enhanced cellular internalization in cancer cells. Altering the

amphiphilic architecture influenced the performance related parameters such as micelle

size, loading capacity, curcumin release pattern and uptake efficiency. P(EtOx17-b-

ButenOx44) micelles, hence, could be envisaged as a future drug delivery vehicle that has

the potential to overcome the current stagnation in the clinical administration of

curcumin.

Acknowledgements

We are grateful to the QUT-SCTIMST exchange program in the support of RR and to the

Institute of Future Environments and the Central Analytical Research Facility at QUT for

28

access to instrumentation. TD greatly acknowledges ARC Future Fellowship scheme

(FT150100408).

References

[1] J. Mann, Natural products in cancer chemotherapy: past, present and future, Nat. Rev. Cancer. 2 (2002) 143-148. [2] B.B. Aggarwal, K.B. Harikumar, Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases, Int. J. Biochem. Cell Biol. 41 (2009) 40-59. [3] J. Epstein, I.R. Sanderson, T.T. MacDonald, Curcumin as a therapeutic agent: the evidence from in vitro, animal and human studies, Br. J. Nutr. 103 (2010) 1545-1557. [4] M. Salem, S. Rohani, E.R. Gillies, Curcumin, a promising anti-cancer therapeutic: a review of its chemical properties, bioactivity and approaches to cancer cell delivery, RSC Adv. 4 (2014) 10815-10829. [5] X.Q. Tang, H. Bi, J.Q. Feng, J.G. Cao, Effect of curcumin on multidrug resistance in resistant human gastric carcinoma cell line SGC7901/VCR, Acta. Pharmacol. Sin. 26 (2005) 1009-1016. [6] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807-818. [7] O. Naksuriya, S. Okonogi, R.M. Schiffelers, W.E. Hennink, Curcumin nanoformulations: a review of pharmaceutical properties and preclinical studies and clinical data related to cancer treatment, Biomaterials. 35 (2014) 3365-3383. [8] S. Croy, G. Kwon, Polymeric micelles for drug delivery, Curr. Pharm. Des. 12 (2006) 4669-4684. [9] C. Oerlemans, W. Bult, M. Bos, G. Storm, J.F.W. Nijsen, W.E. Hennink, Polymeric micelles in anticancer therapy: targeting, imaging and triggered release, Pharm. Res. 27 (2010) 2569-2589. [10] M. Bonferoni, G. Sandri, E. Dellera, S. Rossi, F. Ferrari, M. Mori, C. Caramella, Ionic polymeric micelles based on chitosan and fatty acids and intended for wound healing. Comparison of linoleic and oleic acid, Eur. J. Pharm. Biopharm. 87 (2014) 101-106. [11] R. Trivedi, U.B. Kompella, Nanomicellar formulations for sustained drug delivery: strategies and underlying principles, Nanomedicine. 5 (2010) 485-505. [12] K. Miyata, R.J. Christie, K. Kataoka, Polymeric micelles for nano-scale drug delivery, React. Funct. Polym. 71 (2011) 227-234. [13] B. Guillerm, S. Monge, V. Lapinte, J.J. Robin, How to modulate the chemical structure of polyoxazolines by appropriate functionalization, Macromol. Rapid.Comm. 33 (2012) 1600-1612. [14] K. Lava, B. Verbraeken, R. Hoogenboom, Poly (2-oxazoline) s and click chemistry: A versatile toolbox toward multi-functional polymers, Eur.Polym. J. 65 (2015) 98-111.

29

[15] E. Rossegger, V. Schenk, F. Wiesbrock, Design strategies for functionalized poly (2-oxazoline)s and derived materials, Polymers.5 (2013) 956-1011. [16] R. Hoogenboom, Poly (2�oxazoline)s: A Polymer Class with Numerous Potential Applications, Angew. Chem. Int. Ed. 48 (2009) 7978-7994. [17] M. Barz, R. Luxenhofer, R. Zentel, M.J. Vicent, Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure–property relationships to better defined therapeutics, Polym. Chem.2 (2011) 1900-1918. [18] R. Luxenhofer, Y. Han, A. Schulz, J. Tong, Z. He, A.V. Kabanov, R. Jordan, Poly (2�oxazoline) s as Polymer Therapeutics, Macromol.Rapid.Comm. 33 (2012) 1613-1631. [19] O. Sedlacek, B.D. Monnery, S.K. Filippov, R. Hoogenboom, M. Hruby, Poly (2�Oxazoline)s–Are They More Advantageous for Biomedical Applications Than Other Polymers?, Macromol.Rapid.Comm. 33 (2012) 1648-1662. [20] K. Letchford, R. Liggins, H. Burt, Solubilization of hydrophobic drugs by methoxy poly (ethylene glycol)�block�polycaprolactone diblock copolymer micelles: Theoretical and experimental data and correlations, J.Pharm.Sci. 97 (2008) 1179-1190. [21] A.S. Mikhail, C. Allen, Block copolymer micelles for delivery of cancer therapy: transport at the whole body, tissue and cellular levels, J. Control. Releas. 138 (2009) 214-223. [22] S. Podaralla, R. Averineni, M. Alqahtani, O. Perumal, Synthesis of novel biodegradable methoxy poly (ethylene glycol)–zein micelles for effective delivery of curcumin, Mol.Pharm. 9 (2012) 2778-2786. [23] A. Sahu, U. Bora, N. Kasoju, P. Goswami, Synthesis of novel biodegradable and self-assembling methoxy poly (ethylene glycol)–palmitate nanocarrier for curcumin delivery to cancer cells, Acta.Biomater. 4 (2008) 1752-1761. [24] C. Yang, H. Chen, J. Zhao, X. Pang, Y. Xi, G. Zhai, Development of a folate-modified curcumin loaded micelle delivery system for cancer targeting, Colloids. Surf B Biointerfaces. 121 (2014) 206-213. [25] A. Gress, A. Völkel, H. Schlaad, Thio-click modification of poly [2-(3-butenyl)-2-oxazoline], Macromolecules. 40 (2007) 7928-7933. [26] A. Jerschow, N. Müller, Suppression of convection artifacts in stimulated-echo diffusion experiments. Double-stimulated-echo experiments, J. Magn. Reson. 125 (1997) 372-375. [27] X. Zhang, J.K. Jackson, H.M. Burt, Determination of surfactant critical micelle concentration by a novel fluorescence depolarization technique, J. Biochem. Biophys. Methods. 31 (1996) 145-150. [28] S.C. Owen, D.P. Chan, M.S. Shoichet, Polymeric micelle stability, Nano.Today. 7 (2012) 53-65. [29] T.R. Dargaville, R. Forster, B.L. Farrugia, K. Kempe, L. Voorhaar, U.S. Schubert, R. Hoogenboom, Poly (2�oxazoline) Hydrogel Monoliths via Thiol�ene Coupling, Macromol.Rapid.Comm. 33 (2012) 1695-1700. [30] R.F. Fedors, A method for estimating both the solubility parameters and molar volumes of liquids, Polym. Eng. Sci. 14 (1974) 147-154. [31] D. Van Krevelen, Properties of polymers: their correlation and chemical structure: their numerical and prediction and additive group contributions, Elsevier, Amsterdam, 1997.

30

[32] G.-H. Hsiue, H.-Z. Chiang, C.-H. Wang, T.-M. Juang, Nonviral gene carriers based on diblock copolymers of poly (2-ethyl-2-oxazoline) and linear polyethylenimine, Bioconjug. Chem.17 (2006) 781-786. [33] S.C. Lee, C. Kim, I.C. Kwon, H. Chung, S.Y. Jeong, Polymeric micelles of poly (2-ethyl-2-oxazoline)-block-poly (ε-caprolactone) copolymer as a carrier for paclitaxel, J. Control. Release.89 (2003) 437-446. [34] R. Luxenhofer, A. Schulz, C. Roques, S. Li, T.K. Bronich, E.V. Batrakova, R. Jordan, A.V. Kabanov, Doubly amphiphilic poly (2-oxazoline) s as high-capacity delivery systems for hydrophobic drugs, Biomaterials, 31 (2010) 4972-4979. [35] C.H. Wang, C.H. Wang, G.-H. Hsiue, Polymeric micelles with a pH-responsive structure as intracellular drug carriers, J. Control. Release. 108 (2005) 140-149. [36] T.R. Dargaville, K. Lava, B. Verbraeken, R. Hoogenboom, Unexpected switching of the photogelation chemistry when cross-linking poly (2-oxazoline) copolymers, Macromolecules. 49 (2016) 4774–4783. [37]N. Shaik, N. Giri, W.F. Elmquist, Investigation of the micellar effect of pluronic P85 on P�glycoprotein inhibition: Cell accumulation and equilibrium dialysis studies, J.Pharm.Sci. 98 (2009) 4170-4190. [38] Y. Zeng, W.G. Pitt, A polymeric micelle system with a hydrolysable segment for drug delivery, J.Biomater. Sci. Polym.17 (2006) 591-604. [39] M. Hruby, S.K. Filippov, J. Panek, M. Novakova, H. Mackova, J. Kucka, D. Vetvicka, K. Ulbrich, Polyoxazoline thermoresponsive micelles as radionuclide delivery systems, Macromol. Biosci. 10 (2010) 916-924. [40] G. Gaucher, M.-H. Dufresne, V.P. Sant, N. Kang, D. Maysinger, J.-C. Leroux, Block copolymer micelles: preparation, characterization and application in drug delivery, J. Control. Release. 109 (2005) 169-188. [41] J. Lee, E.C. Cho, K. Cho, Incorporation and release behavior of hydrophobic drug in functionalized poly (D, L-lactide)-block–poly (ethylene oxide) micelles, J. Control. Release.94 (2004) 323-335. [42] G.H. Van Domeselaar, G.S. Kwon, L.C. Andrew, D.S. Wishart, Application of solid phase peptide synthesis to engineering PEO–peptide block copolymers for drug delivery, Colloids.Surf B.Biointerfaces.30 (2003) 323-334. [43] S. Hornig, T. Heinze, Efficient approach to design stable water-dispersible nanoparticles of hydrophobic cellulose esters, Biomacromolecules, 9 (2008) 1487-1492. [44] V.P. Torchilin, Structure and design of polymeric surfactant-based drug delivery systems, J. Control. Release.73 (2001) 137-172. [45] S.S. Feng, Nanoparticles of biodegradable polymers for new-concept chemotherapy, Expert.Rev.Med. Devices. 1 (2004) 115-125. [46] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review, J. Control. Release. 65 (2000) 271-284. [47] B. Guillerm, S. Monge, V. Lapinte, J.J. Robin, Well�defined poly (oxazoline)�b�poly (acrylate) amphiphilic copolymers: From synthesis by polymer–polymer coupling to self�organization in water, J. Polym. Sci A. Polym. Chem. 51 (2013) 1118-1128. [48] A. Sahu, N. Kasoju, P. Goswami, U. Bora, Encapsulation of curcumin in Pluronic block copolymer micelles for drug delivery applications, J. Biomater.Appl. 25 (2011) 619-639.

31

[49] S. Dey, K. Sreenivasan, Conjugation of curcumin onto alginate enhances aqueous solubility and stability of curcumin, Carb.Polym. 99 (2014) 499-507. [50] S. Manju, K. Sreenivasan, Synthesis and characterization of a cytotoxic cationic polyvinylpyrrolidone–curcumin conjugate, J.Pharm.Sci. 100 (2011) 504-511. [51] A. Sahu, N. Kasoju, U. Bora, Fluorescence study of the curcumin− casein micelle complexation and its application as a drug nanocarrier to cancer cells, Biomacromolecules. 9 (2008) 2905-2912. [52] T. Chang, D. Trench, J. Putnam, M.H. Stenzel, M.S. Lord, Curcumin-Loading-Dependent Stability of PEGMEMA-Based Micelles Affects Endocytosis and Exocytosis in Colon Carcinoma Cells, Mol. Pharm. 13 (2016) 924-932 [53] R. Raveendran, G. Bhuvaneshwar, C.P. Sharma, In vitro cytotoxicity and cellular uptake of curcumin-loaded Pluronic/Polycaprolactone micelles in colorectal adenocarcinoma cells, J. Biomater.Appl. 27 (2013) 811-827. [54] R. Raveendran, C. Pillai, G. Bhuvaneshwar, C.P. Sharma, Enhanced Cytotoxicity and Cellular Internalization of Hemocompatible Curcumin Loaded Pluronic Linolenate Micelles in Cancer Cells, J. Nanopharmaceutics. Drug. Delivery. 2 (2014) 36-51. [55] P. Goddard, L.E. Hutchinson, J. Brown, L.J. Brookman, Soluble polymeric carriers for drug delivery. Part 2. Preparation and in vivo behaviour of N-acylethylenimine copolymers, J. Control. Release. 10 (1989) 5-16. [56] C. Maechling�Strasser, P. Déjardin, J. Galin, A. Schmitt, V. Housse�Ferrari, B. Sebille, J. Mulvihill, J. Cazenave, Synthesis and adsorption of a poly (n�acetylethyleneimine)�polyethyleneoxide�poly (n�acetylethyleneimine) triblock�copolymer at a silica/solution interface. influence of its preadsorption on platelet adhesion and fibrinogen adsorption, J.Biomed. Mater.Res. 23 (1989) 1395-1410. [57] S. Zalipsky, C.B. Hansen, J.M. Oaks, T.M. Allen, Evaluation of blood clearance rates and biodistribution of poly (2�oxazoline)�grafted liposomes, J. Pharm Sci. 85 (1996) 133-137. [58] R. Luxenhofer, G. Sahay, A. Schulz, D. Alakhova, T.K. Bronich, R. Jordan, A.V. Kabanov, Structure-property relationship in cytotoxicity and cell uptake of poly (2-oxazoline) amphiphiles, J. Control. Release. 153 (2011) 73-82. [59] J. Tong, R. Luxenhofer, X. Yi, R. Jordan, A.V. Kabanov, Protein modification with amphiphilic block copoly (2-oxazoline) s as a new platform for enhanced cellular delivery, Mol.Pharm.7 (2010) 984-992.