Unravelling the Excellent Chemical Stability and Bioavailability of

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Unravelling the Excellent Chemical Stability and Bioavailability of Solvent Responsive Curcumin-loaded 2-ethyl-2-oxazoline-grad-2-(4dodecyloxyphenyl)-2-oxazoline Copolymer Nanoparticles for Drug Delivery Shubhashis Datta, Annamaria Jutkova, Petra Sramkova, Lenka Lenkavska, Veronika Huntosova, Dusan Chorvat, Pavol Miskovsky, Daniel Jancura, and Juraj Kronek Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00057 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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Biomacromolecules

Unravelling the Excellent Chemical Stability and Bioavailability of Solvent Responsive Curcuminloaded 2-ethyl-2-oxazoline-grad-2-(4dodecyloxyphenyl)-2-oxazoline Copolymer Nanoparticles for Drug Delivery Shubhashis Dattaa, Annamária Jutkováb, Petra Šrámkovác, Lenka Lenkavskáb, Veronika Huntošováa, Dušan Chorvátd, Pavol Miškovskýa,b, Daniel Jancura*a,b, Juraj Kronek*c

a

Center for Interdisciplinary Biosciences, Technology and Innovation Park, P. J. Šafárik University in Košice, Jesenná 5, 041 54 Košice, Slovak Republic

b

Department of Biophysics, Faculty of Science, P. J. Šafárik University in Košice, Jesenná 5, 041 54 Košice, Slovak Republic

c

Department for Biomaterials Research, Polymer Institute of the Slovak Academy of Sciences, Dúbravská cesta 9, 845 41 Bratislava, Slovak Republic d

Laboratory of Laser Microscopy and Spectroscopy, International Laser Centre, Iľkovičova 3, 841 04 Bratislava 4, Slovak Republic

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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

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Abstract: A new gradient copolymer has been synthesized by the living cationic ring-opening

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polymerization of hydrophilic 2-ethyl-2-oxazoline with lipophilic 2-(4-dodecyloxyphenyl)-2-oxazoline

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(EtOx-grad-DPOx). The prepared copolymer is capable of assembling in water to yield polymeric

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nanoparticles (NPs) that were successfully loaded with an anticancer agent, curcumin. Self-assembly of

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the copolymer was found to be tuned by the polarity as well as the hydrogen bonding ability of solvents.

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Solvent took distinctive role in the preparation of unloaded and curcumin-loaded nanoparticles. The

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stability of the nanoparticles was increased by curcumin loading promoted by curcumin-polymer

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interactions. Further, the chemical stability of curcumin in water is largely enhanced inside the

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polymeric nanoparticles. Curcumin-loaded (EtOx-grad-DPOx) copolymer nanoparticles showed excellent

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stability in the biological medium, low cytotoxicity and concentration dependent uptake by U87MG and

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HeLa cells which indicates the possibility of their efficient application in drug delivery.

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KEYWORDS: lipophilic poly(2-oxazoline); nanoparticles; drug delivery; curcumin-polymer interaction;

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cancer cells

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Introduction:

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Curcumin (CM), a naturally-occurring polyphenol derived from the turmeric plants, has received

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immense attention over the past decades because of its diverse biological activity, including anticancer,

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antioxidant, anti-amyloid, anti-inflammatory, antidiabetic, antibiotic, and antiviral properties.1-3 Hence,

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CM is recognized as a promising drug candidate for many diseases such as cancer, neurodegenerative

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diseases, infectious diseases, and diabetes. The inhibition of angio-genesis is attributed to the anticancer

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activity of CM.4 However, the application of CM in the therapeutic treatment has been hindered due to

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its extremely low aqueous solubility (maximum water solubility is about 30 nM) and chemical instability.

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Curcumin is rapidly hydrolyzed at physiological pH 7.4 in phosphate buffer with a half-life of only 20 min.

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Also due to the poor adsorption in blood and fast metabolism, CM is considered as the pan-assay

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interference compound (PAIN) or invalid metabolic panaceas (IMPS) candidate.5 Therefore, to increase

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the bioavailability of CM, various approaches like conjugating CM with a polymer, or encapsulating CM

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inside various nanomaterials, including polymeric nanoparticles (NPs), have been considered.6-12

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Polymeric NPs showing biomedical applications are generally formed from the self-assembly of the

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asymmetric amphiphilic block copolymers.13-16 The insoluble part of the block copolymer constitutes the

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dense core and the soluble block forms the corona which prevents the aggregation of the NPs. Factors

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like polymer composition17 and block order18 can strongly influence the self-assembled structures.

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Besides changing those factors, self-assembly of copolymer can also be influenced by monomer

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distribution.17 The NPs structure formed from the self-assembly of gradient/block copolymers is highly

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variable.19-20 Nanoparticles formed by the self-assembly of gradient copolymers were found to be

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smaller in size than nanoparticles formed by block copolymers and had higher critical micellar

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concentration (CMC) values.20 Block and gradient copolymers showed solvent responsive distinctive NPs

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structures.17 However, there are very few reports on the application of gradient copolymer

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nanoparticles in drug encapsulation and control release.21 The self-assembly structures of block


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copolymer were affected due to encapsulation of hydrophobic drugs inside the core of NPs.22 Therefore,

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it will be interesting to investigate the effect of drug loading on the structure and stability of NPs formed

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by the self-assembly of gradient copolymer. Also the effect of solvent polarity and hydrogen bonding

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ability on the self-assembly behavior of gradient copolymer in the absence and presence of hydrophobic

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molecules have never been investigated till now.

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Poly(2-oxazolines) (POx) can be used in the preparation of amphiphilic macromolecules appropriate

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for the formation of polymeric NPs. These polymers may be considered as the next generation of the

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polymers for drug delivery.22-28 POx are considered to be pseudo-peptides with structural elements

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similar to natural peptide backbones and may be thermoresponsive depending on the polymer

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composition.29-30 POx have been successfully applied in vivo to design, for example, drug24 or DNA31

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delivery systems as well as to modify peptides.32 Among POx-based copolymers, the gradient copolymer

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of 2-methyl- and 2-phenyl-2-oxazoline is one of the most popular and has been synthesized by various

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groups.17, 20, 33-34

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In this work, we present a synthesis of a new gradient copolymer using the living cationic ring-

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opening copolymerization of hydrophilic 2-ethyl-2-oxazoline with lipophilic 2-(4-dodecyloxyphenyl)-2-

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oxazoline.35 2-(4-Dodecyloxyphenyl)-2-oxazoline has higher hydrophobicity compared to other lipophilic

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monomers, like 2-propyl-2-oxazoline, 2-butyl-2-oxazoline or 2-phenyl-2-oxazoline.

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In the present work we have also investigated the self-assembly of the synthesized gradient

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copolymer of 2-ethyl-2-oxazoline and 2-(4-dodecyloxyphenyl)-2-oxazoline in aqueous media using

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dialysis method in the absence and presence of CM. The lipophilic monomer was chosen taking into

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account its miscibility with CM and high hydrophobicity predicted by the Flory-Huggins parameters36-37

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to achieve maximal loading of CM molecules. Block and gradient copolymers of 2-methyl- and 2-phenyl-

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2-oxazoline have been reported to form solvent responsive NPs structure.17 Hence in the present case,

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the synthesized copolymer is dissolved in solvents of different polarity and hydrogen bonding ability and


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Biomacromolecules

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their self-assembly in aqueous medium has been investigated. The chemical stability of CM

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encapsulated inside polymeric NPs has been investigated by monitoring its ground -state and excited-

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state photo-physical properties. The stability of polymeric NPs in biological medium is a crucial

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parameter for their efficient use as a delivery system in vivo.38 We have investigated the stability of

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unloaded and CM-loaded NPs in the presence of serum albumin. Moreover, cell cytotoxicity and uptake

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of these polymeric NPs by two different cancerous cell lines (U87 MG and HeLa) have been investigated

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to reveal a potential biomedical application of these NPs.

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2. Experimental Section

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2.1 Materials

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1-Bromododecane, 2-ethyl-2-oxazoline, calcium hydride, methyl-4-nitrobenzenesulfonate, and

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benzonitrile (distilled prior to use under reduced pressure) were purchased from Sigma Aldrich,

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(Weinheim, Germany). Potassium hydroxide and methanol (p.a. purity) were purchased from

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Microchem (Bratislava, Slovakia). 2-Ethyl-2-oxazoline (EtOx) was dried 48 h over KOH and distilled over

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calcium hydride under reduced pressure. 2-(4-Hydroxyphenyl)-2-oxazoline (HPOx) was prepared under

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procedure described elsewhere.39

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Curcumin (≥94 % purity from Curcuma longa), pyrene (GC, ≥99 %), human serum albumin (HSA,

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≥94 %), dimethyl sulfoxide (HPLC, ≥99 %), ethanol (HPLC, ≥97 %), acetone (HPLC, ≥96 %) were purchased

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from Sigma Aldrich, (Weinheim, Germany). Dialysis membrane (3.5 kDa- MWCO) and syringe filter (0.22

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µm pore size) were purchased from Merck-Millipore (Germany).

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Isopropanol (HPLC, ≥99.5%), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide),

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Dulbecco’s modified Eagle medium (DMEM), containing L-glutamine (862 mg/L), high glucose (4500

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mg/L), penicilin streptomycin (50 µg/mL), and 10 % fetal bovine serum (FBS), were purchased from

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Sigma-Aldrich, Germany. Glass coverslip bottom Petri dishes (35 mm) were purchased from MatTek,


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USA. MitoTracker OrangeTM CMTMRos and LysoTrackerTM Green DND-26 fluorescence probes were

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purchased from ThermoFisher Scientific (Slovakia).

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2.2 Instruments and methods

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The 1H and 13C NMR spectra were recorded in CDCl3 at room temperature on a Varian VXR-400 using

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tetramethylsilane (TMS) as an internal standard. Elemental analyses of 2-(4-dodecyloxyphenyl)-2-

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oxazoline were performed on FLASH 2000 Organic elemental analyzer (CHNS-O) (Thermo Fisher

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Scientific, USA) in triplicates for each sample. All samples were dried under reduced pressure and stored

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in argon atmosphere prior to measurement. The UV-VIS absorption spectra were recorded using a

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Shimadzu UV-2401 spectrophotometer and the steady-state fluorescence emission spectra were

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recorded using a spectrofluorimeter from Shimadzu (SHIMADZU RF-5301, Japan). DLS measurements

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were carried out on a Zetasizer Nano ZS (Malvern Instrument, Malvern, U.K.) using a He/Ne-laser (λex=

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633 nm) at a scattering angle of 173°. The morphology of the NPs was studied using scanning electron

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microscope JEOL 7500 F (JEOL, Japan) equipped with cryo-mode system from Quorum, UK (See

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Supporting information for details). Fluorescence lifetimes were measured using time-correlated single-

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photon counting apparatus (all components from Becker-Hickl GmBH, Berlin, Germany) (for details see

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Supporting information). Instrumentation which was used in size exclusion chromatography (SEC)

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characterization consists of the pumping system type P102 (Watrex, Czech Republic) and evaporative

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light scattering detector ELS - 1000 (PL-Agilent Technologies, Stretton, UK) (see Supporting information

20

for details). Circular dichroism (CD) spectra were recorded on a Jasco-810 automatic recording

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spectropolarimeter at 25ᵒC under constant nitrogen flush over a wavelength range of 190–260 nm with

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a scan speed 50 nm/min.

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Synthesis of 2-(4-dodecyloxyphenyl)-2-oxazoline (DPOx)


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2-(4-Hydroxyphenyl)-2-oxazoline (HPOx, 4.90 g, 0.03 mol) was dissolved in 10 mL of DMSO and mixed

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with powdered KOH (4.94g, 0.0075 mol). Then, 1-bromododecane (8.225 g, 0.033 mol) in 5 mL DMSO

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was dropwise added at room temperature and heated for 5 h at 60°C. After cooling suspension was

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poured into 300 mL of cold water, white precipitate was filtered off and washed with water to pH=7.

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Obtained white solid was dried under reduced pressure. Yield: 6.63 g (67%). M.p. 61.9±0.7°C (DSC,

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Mettler Toledo DSC821 instrument, METTLER-Toledo GmBh Analytical, Schwerzembach, Switzerland, a

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temperature ranges from 25 to 250°C, a heating rate of 10°C/min, nitrogen atmosphere, 1st heating in

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triplicate). 1H NMR (CDCl3): 7.86 (d, 2H, ar), 6.89 (d, 2H, ar), 4.41 (t, 2H, CH2O-ox), 4.03 (t, 2H, CH2O-Ph),

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3.99 (t, 2H, CH2N), 1.86-1.73 (m, 2H, CH2), 1.54-1.39 (m, 2H, CH2), 1.36-1.20 (bp. 16H, CH2), 0.88 (t, 3H,

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CH3, J=6.9 Hz).

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13

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67.5 (CH2O), 68.1 (CH2O), 114.2 (ar), 120.0 (ar), 129.8 (ar), 161.6 (ar), 164.5 (C=N).

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CHN elemental analysis for DPOx (C21H33NO2) in %: 76.13 C, 9.97 H, 4.23 N; found: 75.73 C, 9.99 H, 4.26

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N.

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Synthesis of gradient copolymers (EtOx-grad-DPOx)

C NMR (CDCl3) δ in ppm: 14.1 (CH3), 22.7 (CH2), 26.0 (CH2), 29.2-29.7 (7CH2), 31.9 (CH2), 54.9 (CH2N),

16

DPOx (0.50 g, 0.0015 mol) and methyl 4-nitrobenzenesulfonate (0.022 g, 0.0001 mol) were dried in a

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flask for 3h. Then, 2-ethyl-2-oxazoline (EtOx) (0.86 mL, 0.0085 mol) and benzonitrile (2 mL) were added

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(c-3 mol dm-3, DP=100). Copolymerization was performed for 24 h at 110 °C under argon and terminated

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with methanolic KOH (0.1 mol dm-3, 1.2 eq) at room temperature for 2h. Polymerization mixture was

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precipitated in 400 mL of diethylether, dried in vacuum oven at 40 °C for 24 h and purified by dialysis

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against distilled water. Yield: 1.03 g (77 %). Composition of copolymer was determined by 1H NMR

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spectroscopy performed in CDCl3. Conversions of both comonomers after 24 h determined by 1H NMR in

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CDCl3 were almost quantitative. Kinetic study of cationic copolymerization of EtOx and DPOx initiated by

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methyl 4-nitrobenzenesulfonate was performed in benzonitrile with the monomer concentration of 3


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mol dm-3 at 110°C using theoretical DP of 100 and the feeding ratio EtOx/DPOx of 80/20. The solution of

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the initiator, both monomers and benzonitrile was prepared in glove box (MBRAUN Inertgas Systems

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GMBH, Germany) in nitrogen atmosphere and divided to 11 vials. All vials were placed in pre-heated oil

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bath for certain time intervals (15, 30, 60, 120, 180, 240, 360, 480, and 1440 min). Then, each vial was

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cooled by liquid nitrogen. The conversion of copolymerization for all samples was determined by 1H

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NMR measured in CDCl3. Signals of benzonitrile at 7.3-7.5 ppm were used as an internal standard.

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Preparation of nanoparticles

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Gradient copolymer (4 mg) alone or copolymer (4 mg) with curcumin was dissolved in three different

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organic solvents, acetone (AC), dimethyl sulfoxide (DMSO) and ethanol (EtOH) individually. These

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solutions were then dialyzed against 3.5 kDa MWCO membrane in pH 7.4 for 48 h. Initially, up to 18 h,

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the solvent was changed at an interval of 3 h. After formation of NPs they were filtered through syringe

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filter of pore size 0.22 µM before any measurements.

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Calculation of drug loading capacity

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After 48 h of dialysis in deionized water, the sample was removed from the dialysis membrane and

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passed through a 0.22 μm (Millipore) filter to remove the precipitated drug that would provide false

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encapsulation efficiency. After freeze drying, respective organic solvents were added to re-dissolve the

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sample. UV-Vis Spectroscopy was undertaken, absorbance of CM was compared to the calibration curve,

19

and the concentration was back-calculated to determine the amount of CM inside the NPs. The

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encapsulation efficiency and drug loading capacity were then calculated according to Equations 1 and 2,

21

respectively.

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% = '() *+ ) , % =

!" " "#

× 100 …………… (1)

!" " "# ( ."/)

× 100 ………… (2)

Protein adsorption study


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Since albumin is the most abundant protein constituent present in the blood plasma, the protein

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adsorption study was carried out using human serum albumin, HSA. We have taken into account the

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surface area of NPs before performing this measurement.40 A solution of HSA (4 g/L) was prepared in a

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phosphate buffer solution (0.5× 10−2 M, pH 7.4) and added to a NPs suspension to get the desired

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concentration of HSA. The concentration of copolymers was kept at 4 g/L. These mixtures were stored at

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37 °C in a thermostatic bath for more than one hour. The samples were ultracentrifuged at 14 000 rpm

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for 30 min, forming a pellet after recovery of the supernatant. This pellet was then redispersed in water.

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Curcumin release study

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Release of curcumin from CM-loaded (EtOx-grad-DPOx) copolymer NPs was performed in triplicate in

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PBS (pH 7.4, 5 mM). 10 mL of (EtOx-grad-DPOx) copolymer NPs suspension was dialyzed against PBS at

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37ᵒC in the dark for 72hr. After certain time, 0.5 ml of solution was extracted from the dialysis bag which

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was used to calculate the drug loading capacity. Hence curcumin release was measured by monitoring

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the drug loading capacity at certain time of points.

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2.3 Cell Culture Studies

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Cell culture and curcumin administration protocol

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U87 MG (human glioma cells, Cells Lines Services, Germany) and HeLa (human cervix epithelial

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adenocarcinoma, a gift from the Prof. Fedorocko laboratory, P.J. Safarik University in Kosice, Slovakia)

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cells were grown in Dulbecco’s modified Eagle medium (D-MEM) containing L-glutamine (862 mg/L),

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glucose (4500 mg/L), penicilin/streptomycin (1% w/w) and supplemented with 10 % fetal bovine serum

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(FBS), in the presence of 5 % CO2 humidified atmosphere at 37 °C in the dark. Cells were used in

21

experiments after reaching 80 % confluence. Stock solution of curcumin was prepared in DMSO. Prior

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administration 100 µL of curcumin (DMSO content was under 1%) in PBS (30 min stabilization) or CM-

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loaded NPs were mixed with 900 µL cultivation medium at final CM concentration 5 µM, 10 µM, 15 µM

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and 20 µM. Cells were incubated 4 h with the mixture, washed, and the medium was replaced with fresh


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curcumin free medium, and cells were then used for confocal fluorescence microscopy measurements.

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Cells were incubated 24 h with the polymeric NPs when used for cell cytotoxicity test.

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Confocal fluorescence microscopy

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The cells were seeded into glass coverslip bottom Petri dishes. Cell mitochondria were stained with

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200nM MitoTrackerTM Orange CMTMRos and lysosomes were stained with 200nM LysoTrackerTM

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Green DND-26 for 30 min. LSM700 confocal fluorescence microscope (Zeiss, Germany) was used to

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obtain fluorescence images. This system was equipped with 63X oil objective (NA = 1.46) and a CCD

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camera (AxioCam HRm, Zeiss, Germany). LysoTracker green was excited by 488 nm CW laser light, and

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the emission was filtered in the range 510-560 nm. MitoTracker Orange CMTMRos was excited by 555

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nm CW laser light and the emission was collected in the range 590-630 nm. Fluorescence signals were

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analyzed by the Zen 2011 software (Zeiss, Germany).

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Cytotoxicity via MTT-assay

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Cytotoxicity of the unloaded and CM-loaded NPs was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-

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2,5-diphenyltetrazolium bromide) assay detected using 96 well plate absorption reader (GloMaxTM-

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Multiple detection system with Instinct Software, USA) at 560 nm and 750 nm. The principle of this

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method is that in the living cells the yellow tetrazolium dye can be reduced with the active NAD(P)H-

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dependent oxidoreductase enzymes to purple formazan. A standard protocol for MTT assay was

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performed: 10 µL of 5mg/mL MTT dissolved in phosphate saline buffer were added to each well filled

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with 100 µL medium and plate was 2 h incubated in the dark at 37 °C; after 2 h of the incubation, 50 µL

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medium were taken out, and 150 µL of acidic iso-propanol were added to each well to dissolved crystals

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of formazan. The formazan absorption was detected from the control cells and cells subjected to 5 µM,

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10 µM, 15 µM and 20 µM CM in PBS or nanoparticles (24 h in the dark). Absorption of formazan was

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detected 24 h after CM administration. The error bars represent standard deviation or standard error of

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the mean values of experimental data (performed in triplicates).


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3. Results and Discussion

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3.1 Monomer and copolymer synthesis

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2-(4-Dodecyloxyphenyl)-2-oxazoline (DPOx) is a strongly lipophilic monomer suitable for preparation

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of amphiphilic copolymers with properties highly dependent on the composition of copolymers. DPOx

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was prepared from 2-(4-hydroxyphenyl)-2-oxazoline by reaction with 1-bromoalkane in 67% yield

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(Scheme 1). 1H NMR and 13C NMR spectra of the synthesized DPOx are shown in Figure S1 and Figure S2,

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Supporting information.

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Scheme 1. Synthesis of 2-(4-dodecyloxyphenyl)-2-oxazoline. i) 1-bromododecane, DMSO, KOH, 5h, 60 °C.

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Copolymer of lipophilic 2-(4-dodecyloxyphenyl)-2-oxazoline with hydrophilic 2-ethyl-2-oxazoline

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(EtOx) was prepared via living cationic ring-opening polymerization (LCROP) (Scheme 2).

13

Copolymerization was carried out at 110 °C for 24h in 3 mol dm-3 benzonitrile solutions using 4-methyl

14

nitrobenzenesulfonate (MeONs) as an initiator. MeONs was recently reported as a very efficient cationic

15

initiator with a higher rate of cationic polymerization of 2-alkyl-2-oxazolines compared to methyl

16

tosylate.41

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Scheme 2. Ring-opening polymerization of 2-ethyl-2-oxazoline (EtOx) with 2-(4-dodecyloxyphenyl)-2-

2

oxazoline (DPOx) in 3 mol dm-3 benzonitrile initiated by methyl 4-nitrobenzenesulfonate, i) methyl 4-

3

nitrobenzenesulfonate, benzonitrile (3 mol dm-3 ), [M]/[I]=100, 24 h, 110 °C.

4

Distribution of lipophilic and hydrophilic units was determined by kinetic study of both co-monomer

5

consumptions monitored by 1H NMR spectroscopy (Figures 1a and 1b). The rate constant of EtOx

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consumption determined from 1H NMR measurements equals to 5.3×10-4 s-1 while the rate constant of

7

DPOx consumption is 1.4 ×10-4 s-1. Hence the rate of EtOx consumption is almost 4 times higher than

8

the rate of DPOx consumption (Figure 1a). Similar difference between kp values of both comonomers

9

was observed in the case of copolymerization of 2-ethyl-2-oxazoline with 2-phenyl-2-oxazoline, although

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the absolute values of kp is different due to different copolymerization conditions (type of initiator,

11

temperature).34 Similarly, the ratio of residual EtOx and DPOx monomers in the polymerization system

12

gradually decreased from 3.9 at the beginning of the copolymerization to 0.19 after 3 h of the

13

polymerization (Figure 1b). Both dependences demonstrate the gradient character of forming the

14

copolymer chain. A similar distribution of lipophilic and hydrophilic units was observed for the cationic

15

copolymerization of 2-phenyl-2-oxazoline and 2-ethyl-2-oxazoline 17, where gradient distribution of both

16

monomers along the polymer chain was also demonstrated.

17 18

Figure 1. Living cationic copolymerization of 2-ethyl-2-oxazoline and 2-(4-dodecyloxyphenyl)-2-oxazoline

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initiated by methyl 4-nintrobenzenesulfonate at 110°C, DP=100 and c=3 mol dm-3. a) Kinetics of both


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Biomacromolecules

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monomers consumption based on 1H NMR measurements, b) Plot of the ratio of [EtOx]/[DPOx] vs. time

2

during cationic copolymerization. Monomer consumption was calculated from 1H NMR spectra

3

measured in different times of copolymerization.

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Gradient copolymer composed of 2-methyl-2-oxazoline and 2-phenyl-2-oxazoline having 17.6 mol% of

5

2-phenyl-2-oxazoline was found effective for the formation of stable nanoparticles.33 The composition of

6

the synthesized copolymer was determined from 1H NMR spectrum (see Figure 2) using the ratio of

7

integrals of the signals at 3.87 ppm (CH2O in DPOx) and 1.05 ppm (CH3 in EtOx). Content of lipophilic

8

units from DPOx was 12.5 mol%, what is close to the initial feeding content 15 mol%. After 24 hr,

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conversions of both comonomers determined from 1H NMR spectrum were close to 100%.

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Figure 2. 1H NMR spectrum of poly[2-ethyl-2-oxazoline-grad-2-(4-dodecyloxyphenyl)-2-oxazoline] (EtOx-

12

grad-DPOx) measured in CDCl3. TMS was used as an internal standard.

13

Molecular characteristics of the prepared copolymer were determined using size exclusion

14

chromatography (SEC) (see Figure S3, Supporting information). Molar mass was equal to 12600 g/mol

15

which corresponds to theoretical molar mass for the degree of polymerization DP=100 (Mtheor=13400


13

Biomacromolecules

1

g/mol) and feeding ratio EtOx/DPOx equals to 85/15. Dispersity of the prepared copolymer (Đ=1.26)

2

demonstrates living process of the copolymerization.

3

3.2 Characterization of size and morphology of (EtOx-grad-DPOx) copolymer nanoparticles

4

3.2.1 Dynamic light scattering measurements

5

The size of polymeric nanoparticles in the sub-100 nm range is optimal for cancer targeting by EPR

6

effect.42-43 Lipophilic drugs are entrapped physically inside the NPs mainly by hydrophobic interactions

7

with the lipophilic polymeric block forming the core. Loading of lipophilic drugs in the hydrophobic

8

cavity may affect the size, morphology and stability of a nanocarrier.22, 44 Therefore, we have performed

9

DLS measurements to investigate how interactions between CM and lipophilic part of the polymer

10

(DPOx) influence the size and stability of (EtOx-grad-DPOx) copolymer NPs. The DLS spectra of unloaded

11

and CM-loaded (EtOx-grad-DPOx) copolymer NPs prepared by dialysis method45-46 are shown in Figure 3.

12

Hydrodynamic

diameters

evaluated

from

(a)

DLS

diagram,

are

presented

in

Table

1.

(b) Unloaded NPs(EtOH) Unloaded NPs(AC) Unloaded NPs(DMSO)

10

12 10

Intensity [%]

8

Intensity [%]

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 40

6 4 2

CM-loaded NPs(AC) CM-loaded NPs(DMSO) CM-loaded NPs(EtOH)

8 6 4 2

0

0

1

10

100

Size [nm]

1000

10

100

1000

Size [nm]

13 14

Figure 3. DLS spectra of (a) unloaded and (b) CM-loaded NPs in PBS of pH 7.4 at 25 °C. Size plots were

15

calculated from the autocorrelation function analyzed by constrained regularization (CONTIN) method.

16

From the DLS histogram (Figure 3a) and Table 1, large differences in the hydrodynamic diameters of

17

the unloaded NPs prepared from different initial polymer solutions can be observed. The dependence of

18

DLS intensity vs size for unloaded (EtOx-grad-DPOx) copolymer NPs shows bimodal distribution, while


14

Page 15 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

the same dependence for CM-loaded (EtOx-grad-DPOx) copolymer NPs shows single distribution except

2

for CM-loaded NPs (AC). Two populations of the unloaded NPs prepared from the polymer solution in

3

acetone (AC) have hydrodynamic diameters around 70 and 338 nm, while the diameters of those NPs

4

prepared from polymer solution in ethanol (EtOH) and DMSO are around (13 and 104) nm and (63 and

5

104) nm, respectively. By increasing the polarity of the solvent, the size of NPs produced by dialysis

6

method can be reduced.47 In the present case, the polarity (expressed by the permittivity constant, Ɛ) of

7

the used solvents are in the order: ƐAC < ƐEtOH < ƐDMSO. It means that the trend of the size (R) of NPs should

8

be in the order: RAC >REtOH >RDMSO. But the actual order observed in the present case is: RAC >RDMSO> REtOH.

9

Therefore, although the size of the NPs is largest in the solvent of the lowest polarity, it is not the

10

smallest in the solvent of the highest polarity. One explanation of this observation can be the different

11

hydrogen bonding capacity of the solvents used in the present study. Acetone is a non-hydrogen

12

bonding solvent. DMSO is a hydrogen-bond acceptor. However, EtOH can act as both hydrogen-bond

13

donor and acceptor. Considering the structure of (EtOx-grad-DPOx) copolymer we can say that out of

14

the three solvents used in the NPs preparation, EtOH is the only one able to form a hydrogen bond with

15

the copolymer. Therefore, the nature of the interaction between polymer and solvent can be different

16

in the solvents forming hydrogen bonds (like EtOH) in comparison with the solvents not forming

17

hydrogen bonds (like AC and DMSO) with the polymer.

18

Table 1. Size of unloaded and CM-loaded NPs from DLS and Cryo-SEM measurements. Hydrodynamic

19

diameters of unloaded and CM-loaded NPs obtained from the correlation function using the Cumulants

20

analysis of DLS measurements

Samples

Diameters (nm) Unloaded

NPs(AC) NPs(EtOH) NPs(DMSO)

DLS (70±3.4); (338±12.3) (PDI = 0.47)a (13±2.5); (104±8.9) (PDI = 0.29) (63±1.1); (314±6.8) (PDI = 0.47)

CM-loaded Cryo-SEM (99±72) (69±25) (92±65)

DLS (26±16.8); (160±5.6) (PDI = 0.29) (271±10.8) (PDI = 0.27) (176 ± 3.4) (PDI = 0.29)

Cryo-SEM (138±81) (117±71) (201±118)


15


1

a

Page 16 of 40

PDI -Polydispersity index

2

We have also measured the DLS spectra of CM-loaded (EtOx-grad-DPOx) copolymer NPs (see Figure

3

3b). It is interesting to note that the hydrodynamic diameter of CM-loaded NPs is different compared to

4

unloaded NPs. The size of CM-loaded NPs is significantly smaller compared to unloaded NPs when either

5

acetone or DMSO were used as initial solvents. On the other hand, the size of CM-loaded NPs is

6

significantly larger compared to unloaded NPs when ethanol was used as an initial solvent.

7

The aggregation behavior of amphiphilic polymers has been previously reported to be altered upon

8

drug encapsulation.48-49 Depending on the nature of a drug, the NPs increased48 or decreased50 in size.

9

More interestingly, drugs also induced sphere-to-rod transitions, where the incorporated drug shifted

10

the balance from spherical toward wormlike micelles.49, 51 Size and morphology of NPs prepared from

11

poly(2-oxazoline) triblock copolymer was also found to be changed due to the incorporation of a highly

12

hydrophobic drug, paclitaxel.22 Similarly in the present case, the size of (EtOx-grad-DPOx) copolymer NPs

13

increased or decreased due to incorporation of CM promoted by the interaction between CM and the

14

lipophilic 2-(4-dodecyloxyphenyl)-2-oxazoline part of the polymer.

15

We checked the stability of (EtOx-grad-DPOx) copolymer NPs by measuring their DLS spectra at

16

certain interval of time after their preparation. Although unloaded (EtOx-grad-DPOx) copolymer NPs

17

shows tendency to aggregate, no significant change in the hydrodynamic diameters of CM-loaded (EtOx-

18

grad-DPOx) copolymer NPs was observed even one month after the NPs preparation. It means that

19

these CM-loaded NPs are highly stable for the long-term storage. In order to investigate the possible

20

reasons behind this, we have determined the CMC values of unloaded and CM-loaded NPs using the

21

fluorescence intensity of pyrene described in the literature.44 It was found that the CMC of CM-loaded

22

NPs (0.009 mg/ml) is smaller than the CMC of unloaded NPs (0.015 mg/ml) which indicates that loading

23

of CM in the hydrophobic core increased the thermodynamic stability of NPs. Similar observations have


16

Page 17 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

been reported in the literature.44 Thus the enhanced thermodynamic stability of NPs upon lipophilic

2

drug loading is possibly due to the increase of the fraction of hydrophobic moieties in the NPs.12

3

3.2.2 Cryo-SEM measurements

4

Although DLS studies provide us information about the size of the synthesized polymeric NPs,

5

their shape and morphology are not determined by these measurements. Among different shapes,

6

wormlike and spherical shape NPs showed longer blood circulation time in vivo and increased drug

7

loading capacities.52-53 In this study we have analyzed (EtOx-grad-DPOx) copolymer NPs using cryo-SEM

8

micrographs (Figure 4). In all cases, NPs (CM-loaded or unloaded) of spherical shape were formed.

9

Unloaded NPs have diameter around 69-100 nm, while nanoparticles loaded with CM have diameter

10

around 117-200 nm (see Table 1). Similarly, to DLS measurements, in cryo-SEM we observed

11

populations of NPs with different diameters leading to higher values of standard deviations. From Table

12

1 it is observed that average size of unloaded NPs is different for NPs(DMSO), NPs(EtOH) and NPs(AC).

13

Also the size of CM-loaded NPs is greater than unloaded NPs.

14

Unloaded NPs(EtOH)

CM-loaded NPs(EtOH)

15

Unloaded NPs(AC)

Unloaded NPs(DMSO)

CM-loaded NPs(AC)

CM-loaded NPs(DMSO)

16

Figure 4. Cryo-SEM images of unloaded (upper panel) and CM-loaded NPs (lower panel) in PBS of pH 7.4

17

at 25 °C.


17


Page 18 of 40

1 2

3.3 Drug loading capacity

3

Apart from size and morphology, another important parameter is the drug loading capacity of a

4

nanocarrier. Most carriers suffer from their relatively low drug loading capacity.42,54 POx based

5

nanocarrier has been reported to have a very high payload capacity compared to other conventional

6

carriers.22, 54 In the present study, the efficiency of (EtOx-grad-DPOx) copolymer NPs to encapsulate CM

7

was determined.

8 9 10 11 12

Table 2. Encapsulation capacity and drug loading capacity of (EtOx-grad-DPOx) copolymer NPs

13

NPs

[Polymer] mg/ml

[CM] (M)

CM-loaded NPs(DMSO) CM-loaded NPs(EtOH) CM-loaded NPs(AC) CM-loaded NPs(DMSO) CM-loaded NPs(DMSO)

4 4 4 4 4

1×10-4 1×10-4 1×10-4 2×10-4 4×10-4

14 15 16 17

Encapsulation Capacity (%) (62±1.05) (69±2.31) (73±1.9) (75±0.9) (55±3.6)

Drug Loading Capacity (%) (15±1.25) (19±0.98) (21±1.36) (22±2.21) (12±2.9)

18

The drug loading and encapsulation capacity of (EtOx- grad-DPOx) copolymer NPs is shown in Table 2.

19

The encapsulation efficiency of the synthesized (EtOx- grad-DPOx) copolymer NPs was higher than 60%,

20

which is in accordance with the data reported in the literature for other POx NPs.48, 55 The efficiency to

21

encapsulate CM was 69 %, 62 % and 73 % in NPs prepared from polymer solutions in ethanol, DMSO and

22

acetone, respectively. Although the size of NPs prepared in acetone is smaller compared to NPs

23

prepared in other solvents, their encapsulation efficiency is the highest. Therefore, the smaller size of

24

NPs results in stronger packing of CM inside the NPs. We have also investigated whether variation of


18

Page 19 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

drug and polymer ratio has any influence on the loading capacity of these NPs. For this reason, we have

2

prepared NPs from polymer solutions in DMSO having different ratios of CM and polymer. The polymer

3

concentration was kept at 4 mg/mL in DMSO and the concentration of CM was varied. Initially, as the

4

drug and polymer ratio increases, the drug loading capacity increases. However, after a certain drug and

5

polymer ratio, the drug loading capacity decreased.

6

3.4 Photo-physical properties of curcumin inside polymeric NPs

7

UV-Vis absorption and fluorescence spectra of CM encapsulated inside different (EtOx-grad-

8

DPOx) copolymer NPs have been recorded. CM is poorly soluble in water and exists mainly in two

9

different tautomeric forms (keto and enol form). The absorption spectra of CM in water showed a

10

maximum at 433 nm and a shoulder around 353-355 nm with extremely low absorbance (see Supporting

11

information, Figure S4).The band around 433 nm corresponds to the enol form while the shoulder

12

around 355 nm represents the keto form.56-57 When encapsulated inside the NPs, the absorbance of CM

13

in water increased along with the shift of the absorption maxima. Increase in absorbance of CM inside

14

polymeric NPs compared to absorbance in water indicates that the solubility of CM is highly enhanced

15

inside NPs. The absorption spectra of CM-loaded (EtOx-grad-DPOx) copolymer NPs are shown in Figure

16

5a. It is observed that the main absorption peak of CM is shifted to 430 nm. Moreover, the shoulder

17

around 350 nm vanished and a new shoulder near 453 nm appeared.


19

Biomacromolecules

(a)

(b) Normalized Flurescence Intensity (a.u)

1,0

Normalized Absorbnace (a.u)

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

0,8

0,6

0,4

CM-loaded NPs(DMSO) CM-loaded NPs(AC) CM-loaded NPs(EtOH)

0,2

0,0 300

Page 20 of 40

350

400

450

500

550

509 nm 500 nm

1,00

520 nm

0,75

0,50

0,25

0,00

CM-loaded NPs(Ac) CM-loaded NPs(DMSO) CM-loaded NPs(EtOH) 450

Wavelength (nm)

500

550

600

Wavelength (nm)

1 2

Figure 5. Normalized UV-Vis absorption (a) and fluorescence emission (λex = 430 nm) (b) spectra of CM-

3

loaded NPs prepared by dialysis method using EtOH, acetone and DMSO as initial solvent of preparation.

4

Similar changes in the absorption spectrum of CM was observed when its spectra were recorded in

5

water-ethanol mixture with increasing alcoholic content.58 It was explained that with the increasing

6

ethanol content, the enol form is better stabilized by the hydrogen bonding and thus the keto-enol

7

tautomer equilibrium is shifted to the enol form. Therefore, it can be concluded that enol form of CM is

8

the major tautomeric form present inside (EtOx-grad-DPOx) NPs.

9

CM shows a very broad emission spectrum59 in water, and the fluorescence quantum yield of CM in

10

this solvent (pH 7.4) is very low (ϕ=0.001).59 Inside (EtOx-grad-DPOx) copolymer NPs, the fluorescence

11

intensity of CM sharply increased along with a blue shift of its emission maxima. A similar type of blue

12

shift of the emission maxima of CM has been previously reported in various micellar environments.57, 60

13

The maximum of the emission peak of CM is near 497 nm inside NPs. However, there exists a sharp

14

difference in the peak maximum position when NPs are prepared from solution of CM and polymer in

15

three different solvents, acetone (Ac), dimethyl sulfoxide (DMSO) and ethanol (EtOH). The emission

16

maximum inside NPs (Ac), NPs (EtOH) and NPs (DMSO) are at 520, 509 and 500 nm, respectively (Figure

17

5b). This difference in the position of emission maximum of CM should be due to facing different

18

hydrophobic environment inside the core of the NPs. Therefore, CM faced the highest hydrophobic

19

environment inside NPs(DMSO).


20

Page 21 of 40

1

3.5 Time-resolved fluorescence measurements

2

Fluorescence lifetime of a fluorophore is sensitive to the alteration of the fluorophore

3

microenvironment and also to the interactions taking place in the excited state of the fluorophore. In

4

order to the probe the microenvironment, the fluorescence lifetime of CM has been measured inside

5

different CM-loaded (EtOx-grad-DPOx) copolymer NPs. The samples were excited at 375 nm and the

6

fluorescence decays were collected in the range 480-500 nm. The fluorescence decay profile of CM

7

inside POx NPs prepared by dialysis method using different initial solvent is shown in Figure 6.

8

Curcumin has been reported to show a multi-exponential fluorescence decay in different micro-

9

heterogeneous environments.60-61 Two main components of the decay are attributed to two major

10

processes occurring in the excited state of CM.60-63 One is the excited state intramolecular proton

11

transfer (ESIPT) and another one is solvation of CM. In a nonpolar solvent, CM forms a six membered

12

chelate favoring ESIPT with a very short fluorescence lifetime component around few hundreds of

13

femtoseconds.63 In a polar solvent, the ESIPT is not favored, which results in the relatively longer

14

fluorescence lifetime of CM (comparing to a non-polar solvent) of around few hundreds of

15

picoseconds.63

1

Normalized log Count

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

CM-loaded NPs(AC) CM-loaded NPs(DMSO) CM-loaded NPs(EtOH) IRF

0,1

0,01

1E-3

1E-4

0

2

4

6

8

10

Time (ns)

16 17

Figure 6. Normalized fluorescence decay profile of CM inside NPs in PBS of pH 7.4 at 37ᵒC. The samples

18

have been excited at 375 nm and the emissions has been collected at 480-500 nm using variable band-

19

pass filter at magic angle.


21


Page 22 of 40

1

The decay profiles of CM fluorescence inside (EtOx-grad-DPOx) copolymer NPs (Figure 6) have been

2

fitted with bi-exponential equation. The major contribution comes from the very short lifetime which

3

indicates that the excited state lifetime of CM inside NPs is also influenced by ESIPT. The contribution

4

and the lifetime of this very short component of CM fluorescence inside (EtOx-grad-DPOx) copolymer

5

NPs prepared from different initial solvent is different (see Table T1, Supporting information). We have

6

calculated an average lifetime from the individual lifetime components using the procedure described in

7

the literature.64 The average lifetime of CM inside NPs (AC) is 223 ps with components 175 ps (82 %) and

8

459 ps (17%). The average lifetime of CM inside NPs (DMSO) is 357 ps with components 275 ps (81%)

9

and 711 ps (19 %). The average lifetime of CM inside NPs (EtOH) is 293 ps with components 226 ps (81

10

%) and 592 ps (19 %). Therefore, the average lifetime of CM inside NPs prepared from acetone or

11

ethanol as an initial solvent is relatively low compared to CM fluorescence lifetime in NPs prepared in

12

DMSO. The difference in the fluorescence lifetime values of CM inside different NPs can be explained by

13

considering the ESIPT process which has been found to play a key role in determining the fluorescence

14

lifetime of CM. It has been previously reported that perturbation of the ESIPT process in CM localized

15

inside reverse micelles and other organized media increased lifetime of CM.60 We have already pointed

16

out that the interactions between drug and polymer, play an important role in the determination of the

17

size and morphology of (EtOx-grad-DPOx) copolymer NPs. It seems that inside NPs, the interaction

18

between CM and lipophilic 2-(4-dodecyloxyphenyl)-2-oxazoline part of the polymer perturbs the ESIPT

19

process in CM, but to a varying extent depending on the used solvent. ESIPT process of CM is perturbed

20

to a higher extent in NPs prepared from DMSO as an initial solvent compared to NPs prepared from

21

acetone or ethanol. Lipophilic 2-(4-dodecyloxyphenyl)-2-oxazoline part of the polymer can form a

22

hydrogen bond with CM and thereby can perturb the ESIPT process. The varying extent of the

23

perturbation inside different NPs is due to difference in the strength of interaction. Therefore, the

24

structure of NPs prepared from DMSO as initial solvents is such that it leads to the stronger interaction


22

Page 23 of 40

1

between CM and lipophilic 2-(4-dodecyloxyphenyl)-2-oxazoline part of the polymer resulting in relatively

2

longer fluorescence lifetime.

3

3.6 The stability of CM inside (EtOx-grad-DPOx) copolymer NPs and drug release

4

As discussed in the introduction, the main problem in using CM as a therapeutic drug is that CM

5

is rapidly hydrolyzed in aqueous solutions.65 CM is very unstable in aqueous solutions of neutral pH and

6

approximately 80 % of CM is hydrolyzed within 1 h.66 Consequently, our goal was to prepare a delivery

7

system which can protect CM from degradation. We have investigated the stability of CM inside (EtOx-

8

grad-DPOx) copolymer NPs using UV-Vis absorption spectroscopy at different time intervals after

9

preparation of CM-loaded (EtOx-grad-DPOx) copolymer NPs. The absorbance at 430 nm has been used

10 11

to calculate the stability parameter using the following equation (Equation 3): % Decreased Stability (S) = (A0-At)/A0×100

……………… (3)

12

where A0 and At refer to the absorbance of CM at the very beginning of CM-loaded (EtOx-grad-DPOx)

13

copolymer NPs preparation and after a certain time period, respectively. The comparison of the S value

14

for CM inside different NPs is shown in Figure 7.

40

Decreased Stability (S)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

CM-loaded NPs (AC) CM-loaded NPs (EtOH) CM-loaded NPs (DMSO)

32

24

16

8

0 0

5

10

15

20

25

Time (In days)

15 16

Figure 7. Decrease of CM stability inside different NPs in the time intervals up to 25 days analyzed by

17

UV-Vis absorption spectroscopy in pH 7.4 at 37ᵒC.


23


Page 24 of 40

1

The value S is more than 80% for CM in aqueous solutions of pH 7.4 after 1hr. However, CM inside

2

(EtOx-grad-DPOx) copolymer NPs is highly protected from the degradation. We have measured the

3

stability in CM- loaded NPs up to 25 days after complex formation. The value of S is 10%, 14% and 18%

4

for NPs (DMSO), NPs (AC) and NPs (EtOH), respectively, 25 days after immersion of CM into (EtOx-grad-

5

DPOx) copolymer NPs. It was reported that the OH- ion from water or any other base is responsible for

6

the hydrolysis of CM.66-68 Therefore, chemical stability of CM can be increased by protecting it from the

7

access of OH- ions. There are various reports in the literature about the rate of degradation of CM in

8

different micellar media and polymeric self-assemblies.62, 67 However, the chemical stability of CM inside

9

(EtOx-grad-DPOx) copolymer NPs is higher compared to those reports. Therefore, (EtOx-grad-DPOx)

10

copolymer NPs can be considered as an excellent stabilizer of CM in aqueous solutions. The reason

11

behind this is a large stability of the (EtOx-grad-DPOx) copolymer NPs and their ability to load CM

12

molecules into the highly hydrophobic environment to protect these molecules from an aqueous

13

environment. The difference in the value of S is a reflection of the hydrophobic environment faced by

14

CM inside NPs. Curcumin faces highly hydrophobic environment in NPs (DMSO) and lowest in NPs

15

(EtOH). This is also reflected by the emission spectra of CM-loaded (EtOx-grad-DPOx) copolymer NPs,

16

where maximum blue shift in emission maxima was observed in NPs (DMSO). Therefore, initial solvents

17

used for the preparation of (EtOx-grad-DPOx) copolymer NPs has an impact on the self-assembly of the

18

polymer molecules promoted by varying extent of interaction between lipophilic 2-(4-

19

dodecyloxyphenyl)-2-oxazoline part of the polymer and drug which results in difference in the CM

20

stability.


24

Page 25 of 40

12 NPs(DMSO) NPs(AC) NPs(EtOH)

10

Curcumin Release (%)

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

8 6 4 2 0

0

10

20

30

40

50

60

70

Time (In hour)

1 Figure 8. Release of CM from CM-loaded NPs in PBS (pH 7.4) at 37ᵒC.

2 3

In vitro release behavior of CM from CM-loaded NPs was investigated in PBS (pH 7.4) over a period of

4

72 h. Approximately 5%, 8% and 10% of CM were released from NPs(DMSO), NPs(AC) and NPs(EtOH),

5

respectively, after 72h (Figure 8). This low level of CM release from NPs indicates that CM is strongly

6

entrapped in the core of the NPs. It can be expected that the (EtOx-grad-DPOx) copolymer NPs can act

7

as excellent carrier to prevent premature CM leakage during long blood circulation. It is also observed

8

from Figure 8 that the release of CM from NPs depends on the hydrophobicity of the core of the NPS.

9

Fluorescence measurements and stability experiments indicated that CM faced the highest hydrophobic

10

environment inside NPs(DMSO). Curcumin release experiment in this section also conclude that due to

11

facing the highest hydrophobic environment inside NPs(DMSO), the rate of release of CM is the

12

slowest.12

13

3.7 Intracellular distribution and cytotoxicity of curcumin in U87 MG and HeLa cells

14

Intracellular distribution of CM was previously studied in several cancerous and non-cancerous cell

15

lines.11, 69-73 A higher CM uptake was demonstrated in tumor than in normal cells.70 The aim of this part

16

of our study was to demonstrate the efficacy of CM-loaded (EtOx-grad-DPOx) copolymer NPs transport

17

system for drug delivery to U87 MG and HeLa cells. We have mimicked the bloodstream aqueous

18

solution carrying the transport system to tumor tissue.


25


Page 26 of 40

1 2

Figure 9. Representative fluorescence images of A) U87 MG and B) HeLa cells subjected 4 h to 10 µM

3

curcumin (green color) in PBS (PBS labeled row) or in the NPs (DMSO) (nanoparticles labeled row) (b)

4

with and (a) without additional organelles labeling. Mitochondria were 30 min labeled with 300 nM

5

MitoTracker Orange CMTMRos (red color) and lysosomes with 300 nM LysoTracker green (cyan color).

6

The biocompatibility of C) U87 MG and D) HeLa cells with curcumin (5-20 µM) in PBS or encapsulated in

7

(EtOx-grad-DPOx) copolymer NPs(DMSO) incubated 24 h in the dark was evaluated by MTT-assay.

8

Fluorescence images of U87 MG (Figure 9A) and HeLa (Figure 9B) cells in the presence of CM

9

administered in PBS solution and inside (EtOx-grad-DPOx) copolymer NPs(DMSO) were collected 4 h

10

post administration. One can recognize green fluorescence of CM inside U87 MG cells that is

11

significantly more intense when CM is delivered in (EtOx-grad-DPOx) copolymer NPs(DMSO) (Figure 9Aa,

12

Ba). Observed differences in fluorescence intensities can be explained by CM degradation occurring in

13

PBS. We expect such an effect to take place in blood-stream before spontaneous CM binding with serum


26

Page 27 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

proteins. CM administered in (EtOx-grad-DPOx) copolymer NPs was clearly found to be distributed

2

homogeneously within the cells.

3

The Petit group

71

demonstrated the intracellular distribution of CM in endoplasmic reticulum,

4

partially in lysosomes, and the punctuated pattern was found close to mitochondria. We have observed

5

weak fluorescence of punctuated pattern of CM administered in PBS that only partially colocalized with

6

lysosomes (Figure 9Aa, Ba). CM was not found in mitochondria and nuclei of U87 MG or HeLa cells.

7

Furthermore, CM administered in (EtOx-grad-DPOx) copolymer NPs was distributed homogeneously in

8

the cytoplasm without preferential specificity towards lysosomes or mitochondria (Figure 9Aa, Ba).

9

Indeed, regarding fluorescence images, lysosomes, mitochondria and other subcellular organelles,

10

except the nucleus, can be saturated by CM. A similar type of distribution in the cytoplasm of HepG2

11

cells was reported for CM-loaded polymeric nanoparticles having diameter around 185 nm.72

12

The cytotoxicity of CM administered in PBS and (EtOx-grad-DPOx) copolymer NPs (DMSO) and

13

incubated 24 h with U87 MG and HeLa cells was determined via MTT assay (Figure 9C, D). The level of

14

cytotoxicity was found to be similar between CM delivered in PBS and in the nanoparticles. Interestingly,

15

the cells treated 24 h with 20 µM CM maintained the level of viability compared to non-treated control.

16

In several other studies, 20 µM concentration of curcumin led to remarkable apoptotic changes71 and

17

inhibition of cancer cells proliferation.74 This differences in CM anti-proliferative effect observed in vitro

18

could be caused by a decrease in CM intracellular concentration.

19

3.8 Stability of NPs presence of protein

20

The stability of (EtOx-grad-DPOx) copolymer NPs in biological media was investigated using the

21

protocols described by Rouzes75 and El Fagui 76. The interactions between nanoparticles and cells can be

22

interpreted by characterizing the adsorption of protein coronas to nanoparticles.77 There are several

23

techniques to characterize the NPs protein corona.77 In the present case, we have used fluorescence


27


Page 28 of 40

1

spectroscopy, DLS measurements and CD spectroscopy to characterize NPs protein corona using human

2

serum albumin (HSA) as a protein mimicking the biological fluids.

3

In the presence of (EtOx-grad-DPOx) copolymer NPs, the intrinsic tryptophan fluorescence intensity of

4

HSA remains almost unaffected even after long time of complexation (see Figure S5, Supporting

5

information). The secondary structure of HSA recoded by CD spectra is altered insignificantly in the

6

presence of NPs (see Figure S6, Supporting information). We have compared the size of NPs before and

7

after the protein adsorption. The size of unloaded NPs is increased only by (13.3±0.2) nm and size of CM

8

loaded NPs is increased only by (12.8±0.2) nm. The increase in particle size is less than 10 % of their

9

original size which is significantly lower as compared with the data shown in the literature. The increase

10

in the particle size of CM-loaded NPs is even less. These observations confirm the fact that these (EtOx-

11

grad-DPOx) copolymer NPs show much less protein adsorption like other poly (2-oxazoline) NPs and

12

hence can be better alternative to hydrophilic PEG based polymeric NPs.

13 14

Conclusions

15

The (EtOx-grad-DPOx) copolymer synthesized in this work was capable to self-assemble in water to

16

yield polymeric NPs that were successfully loaded with CM. Unloaded and CM-loaded polymeric NPs

17

produced by self-aggregation present a spherical shape typically between 50 and 350 nm. Although

18

unloaded NPs showed little tendency to aggregate, CM loaded NPs did not show any tendency to

19

aggregate and are stable for long term storage. The enhanced stability of CM-loaded NPs compared to

20

unloaded NPs is due to incorporation of hydrophobic molecule, CM inside the core of NPs which

21

decreased the CMC of copolymer. The chemical stability and solubility of CM is highly enhanced due to

22

encapsulation inside NPs. The average fluorescence lifetime of CM inside NPs is increased due to

23

perturbation in the rate of ESIPT. The present study demonstrates that self-assembly of (EtOx-grad-

24

DPOx) copolymer is finely tuned by the polarity and hydrogen bonding ability of solvent. More


28

Page 29 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

importantly, the effect of solvent polarity on the self-assembly of copolymer is different in the absence

2

or presence of CM. The size of the unloaded NPs is largest in the solvent of the lowest polarity, but it is

3

not the smallest in the solvent of the highest polarity. The drug loading and encapsulation efficiency is

4

highest for (EtOx-grad-DPOx) NPs(AC). The highest chemical stability and the slowest release rate CM

5

from NPs(DMSO) is because of facing the highest hydrophobic environment inside the core of NPs. A

6

high cytocompatibility of the gradient-copolymer NPs is indicated by the in vitro experiments. CM

7

loaded gradient-copolymer NPs showed a concentration dependent accumulation into U87MG and HeLa

8

cells. These results suggest that (EtOx-grad-DPOx) copolymer NPs efficiently encapsulate bioactive

9

compounds and deliver them into the cellular environments. In the future, the cellular uptake

10

mechanism and the intracellular fate of the designed NPs should be investigated in more detail. Also it

11

will be interesting to investigate the excretion profile in future of these NPs because of their permanent

12

hydrophobic block.

13 14

Supporting Information

15

Experimental details of size exclusion chromatography (SEC), Cryo-scanning electron microscope

16

(Cryo-SEM) and time resolved fluorescence decay measurements; 1H NMR and 13C NMR of DPOx; GPC of

17

(EtOx-grad-DPOx); UV-Vis absorption spectra of CM; Fluorescence emission spectra of tryptophan of

18

HSA in the presence of NPs; CD spectra of HSA in the presence of NPs; Table presenting fluorescence

19

lifetime of CM inside different NPs;

20

Acknowledgments

21

The authors are thankful to the Slovak Grant Agency VEGA for the financial support in the project No.

22

2/0124/18 and the Slovak Research and Development Agency for financial support in the project No.


29


Page 30 of 40

1

APVV-15-0485. We would like to thank Kurt Magsamen for his helpful comments and assistance in

2

preparing the manuscript.

3

Corresponding Authors

4

Email: [email protected] [email protected]

5 6 7

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Unravelling the Excellent Chemical Stability and Bioavailability of

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