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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.
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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
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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).
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