Curcumin-bortezomib loaded polymeric nanoparticles for synergistic cancer therapy

Accepted Manuscript Macromolecular Nanotechnology Curcumin-bortezomib loaded polymeric nanoparticles for synergistic cancer therapy Sandra Medel, Zdenka Syrova, Lubomir Kovacik, Jiri Hrdy, Matus Hornacek, Eliezer Jager, Martin Hruby, Reidar Lund, Dusan Cmarko, Petr Stepanek, Ivan Raska, Bo Nyström PII: DOI: Reference:

S0014-3057(17)30698-5 http://dx.doi.org/10.1016/j.eurpolymj.2017.05.036 EPJ 7892

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

20 April 2017 19 May 2017 21 May 2017

Please cite this article as: Medel, S., Syrova, Z., Kovacik, L., Hrdy, J., Hornacek, M., Jager, E., Hruby, M., Lund, R., Cmarko, D., Stepanek, P., Raska, I., Nyström, B., Curcumin-bortezomib loaded polymeric nanoparticles for synergistic cancer therapy, European Polymer Journal (2017), doi: http://dx.doi.org/10.1016/j.eurpolymj. 2017.05.036

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Curcumin-bortezomib loaded polymeric nanoparticles for synergistic cancer therapy

Sandra Medel

a,b

, Zdenka Syrova

b*

, Lubomir Kovacik

b‡

, Jiri Hrdy b, Matus

Hornacek b, Eliezer Jager c, Martin Hruby c, Reidar Lund a, Dusan Cmarko b, Petr Stepanek c, Ivan Raska b, Bo Nyström a* a

Department of Chemistry, University of Oslo, Blindern, Oslo, Norway

b

First Faculty of Medicine, Charles University, Albertov 4, Prague, Czech Republic

c

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,

Heyrovsky Sq. 2, Prague, Czech Republic *Corresponding authors: [email protected], [email protected] ‡

Current address: Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Mattenstrasse 26, 4058 Basel, Switzerland

1

ABSTRACT: A series of well-defined methoxy-poly(ethylene glycol)-block-polylactic acid (mPEG-b-PLA) diblock copolymers were successfully synthesized, characterized and used for the construction of anticancer nanoparticle delivery system. Nanoparticles (NPs) based on these polymers were prepared by employing the nanoprecipitation method, and they were noncovalently loaded with curcumin, curcumin-bortezomib model or curcumin-bortezomib complex (curc-BTZ). Both curcumin and bortezomib are rather hydrophobic and poorly water-soluble potent anticancer drugs with synergic effects forming together a pH-sensitive complex, stable at pH of blood plasma, yet hydrolytically labile at mildly acidic milieu typical for endosomes and interstitial space in solid tumors. PEG-Curcumin-loaded and curc-BTZ-loaded NPs with 100-150 nm size showed the maximum cellular uptake by HeLa, MCF-7 and MDA-MB 231 cells after 3h. The NPs were located in the cytoplasm of the cells but not inside the nucleus. Bare NPs did not induce any cytotoxicity in the same cell lines in in vitro experiments, even at very high concentrations (up to 800 µg/mL). NPs containing curcumin were cytotoxic with an IC50 of 25 µg/mL, which corresponds to 2.5 µg/mL of loaded curcumin. These results show that the efficacy of curcumin is significantly enhanced when using the NPs as carriers. The efficiency was further augmented through the complexation of BTZ with curcumin. When using free curcBTZ-complex, MCF-7 cells were more sensitive to the free complex 18.8 nM (IC50) than MDAMB-231 cells 122.4 nM (IC50). Nanoparticle formulations with these drugs caused significant cytotoxicity with 7.5 nM (IC50) and 59.2 nM (IC50) after 24 hours of the treatment. Keywords: polymeric nanoparticles, light scattering, flow cytometry, cellular uptake, drug delivery

2

1.

Introduction Cancer is one of the leading causes of death worldwide. According to the World Cancer

Report 2014, 14.1 million cancer cases were recorded in 2012, accounting for 8.2 million deaths, and the number of new cases is expected to rise by about 70% over the next 20 years [1]. Among women, breast cancer is one of the most common cancer type diagnosed, which is difficult to cure due to its high heterogeneity. Several molecular classes (e.g., luminal-A, luminal-B, basal or triple-negative, and human epidermal growth factor 2-positive) of breast cancer were identified through gene-expression profile studies. Depending on these molecular differences, specific treatment strategies are needed, from surgery and radiation therapy to systemic therapy (e.g., chemotherapy, hormone therapy, and targeted therapy), that refers to the drugs that can reach the cancer cells in any part of the body [2, 3]. Triple-negative breast cancer (TNBC) is an aggressive subtype that is associated with poor long-term outcomes, compared with other breast cancer subtypes. Chemotherapy is the major treatment, although it may cause problems such as toxicity of drugs to healthy tissues or long-term side effects to the brain, spinal cord and nerves [4]. One of the approaches to overcome these problems in the treatment of breast cancer is combination of several anti-cancer drugs with synergistic effect [5]. Here, nanoparticle formulations provide a more effective system [6,7] that is even capable of overcoming multidrug resistance [8]. Numerous delivery systems have been exploited for treatment of cancer to improve the properties of free drugs. Polymers have a high potential in the advancement of drug delivery technology as they offer interesting possibilities in the controlled release, designing and administration of the amount and duration of the dose, the site of disease, and the tunable release of drugs that are either hydrophilic or hydrophobic [9]. The different polymeric carriers applied 3

for anti-cancer targeted drug delivery encompass systems such as micelles, nanoparticles, polymersomes, dendrimers, and polymer-drug conjugates [10]. To be a good candidate for cancer therapy, these polymers must be biodegradable, biocompatible, and non-toxic. The polylactic acid (PLA) is a synthetic biodegradable polymer that meets all these criteria, being recognized as safe and approved by the US Food and Drug Administration in 2002. Therefore, it is used for biomedical applications such as medical materials and drug carriers [11]. In spite of its many advantages, medical applications of PLA as carrier are limited because of its high hydrophobicity and thereby great sticking probability. This is the main reason for ongoing attempts to PEGylate this polymer with poly(ethylene glycol) (PEG) [12,13]. In this work, we synthetized biodegradable and biocompatible methoxy-poly(ethylene glycol)-block-polylactic acid (mPEG-b-PLA) diblock copolymers, and loaded curcumin and bortezomib drug into the self-assembled nanoparticles, which were characterized by a number of physico-chemical methods and cryo-TEM. This approach allowed us to combine advantages of the amphiphilic character of the mPEG-b-PLA nanoparticles with the synergistic effect of inhibition of cancer cell growth by curcumin and bortezomib treatment [14-17]. As discussed in this paper, both curcumin and bortezomib are rather hydrophobic and poorly water-soluble potent anticancer drugs with synergic effects forming together a pH-sensitive complex, stable at pH of blood plasma, yet hydrolytically labile at mildly acidic milieu, typical for endosomes and interstitial space in solid tumors. In addition to that, fluorescent properties of curcumin enable to follow intracellular fate without the necessity of another fluorescent probe. Thanks to their amphiphilicity, PEG-b-PLA copolymers have great potential in drug delivery applications as tumor carriers [18] as they can form nanoparticles composed of a hydrophobic core and a

4

hydrophilic corona, which allow incorporation of hydrophobic drugs inside the core through hydrophobic interactions. Bortezomib, a highly specific 26S proteasome inhibitor, has in recent years successfully been used in treatment of patients with multiple myeloma [19,20] and several studies have been done on molecular level to evaluate its efficacy in breast cancer treatment, both in its free form and also encapsulated in nanoparticles [21,22]. It was observed [22] that bortezomib encapsulated nanoparticles (NPBTZ) are efficient to deliver the drug into both cancer stem cells (CSCs) and non-CSCs and the effects are proliferation inhibition and apoptosis induction. It was also noted that NPBTZ carriers affect the stemness of CSCs more efficiently than with free bortezomib. Curcumin possesses antioxidant and anti-inflammatory activities, and therefore it has been used as therapeutic agent for many chronic diseases and tested for cancer treatment under pre-clinical and clinical conditions [23,24]. The aim of this work is to study the efficiency of the newly developed (PEG-b-PLA)curcumin-bortezomib delivery system to treat breast cancer. For this purpose, we have selected two breast cancer cell lines (MCF-7, luminal, and MDA-MB-231, basal) [25] and the classical HeLa cell line and performed cytotoxicity and cellular uptake studies of the developed delivery systems. Our results show that the (PEG-b-PLA)-curcumin-bortezomib nanoparticles are effectively internalized in the cells and possess an anti-tumor efficiency, indicating that once the loaded NPs have been taken up by the breast cancer cells, the drug is released into the tumor mass.

2.

Experimental section

2.1.

Materials

5

Methoxy-terminated (mPEG5000, with only one hydroxyl group, Mn = 5000 g/mol), 3,6dimethyl-1,4-dioxane-2,5-dione (D,L-lactide), and stannous octoate were all acquired from Sigma-Aldrich. mPEG was dried by an azeotropic distillation in toluene prior to use. D,L-lactide was recrystallized twice from ethyl acetate and dried under vacuum at 40 ºC. Curcumin (≥80.0%) and 4-methoxyphenylboronic acid (≥95.0%) were both purchased from Sigma-Aldrich. Bortezomib (BTZ) was obtained from Tinib-Tools. Dichloromethane (DCM, 99.5%, dried over 4Å molecular sieves), ethyl acetate (≥99.5%), diethyl ether (≥99.8%), acetone (CHROMASOLV for HPLC, ≥99.8%), and phosphate buffered saline (PBS) from Sigma-Aldrich were all used as received, unless specified otherwise. Milli-Q water from the water purification facility (Millipore Milli-U10) was used. Toluene (100%) and tetrahydrofuran (THF, >99.7% for HPLC) were both purchased from VWR. 2.2. 1

Methods H NMR and

13

C NMR spectra were recorded on a Bruker 400 MHz spectrometer with

samples dissolved in CDCl3, dmso-d6 or DCl/D2 O/NaOD at room temperature. Chemical shifts were assigned using the residual non-deuterated solvent signal as an internal reference. Gel permeation chromatography (GPC) was performed on a Tosoh EcoSEC dual detection system (refractive index and UV) coupled to an external Wyatt Technologies miniDAWN TREOS multi-angle light scattering (MALS) detector for the analysis of polymer weight-average molecular weight (Mw), number-average molecular weight (Mn) and polydispersity index (PDI). The mobile phase was HPLC grade THF at a flow rate of 0.5 mL/min at 35 ºC. The column set was one MZ-Gel SDplus linear 5 µm (4.6x300 mm) and one Tosoh TSK-gel SuperH-RC (6.0x150 mm). Absolute molecular weights and molecular weight distributions were calculated

6

using the Astra software package. Polystyrene standard (dn/dc = 0.185 cm-3/g, Mn = 30 kDa) supplied by Wyatt was used for calibration. 2.3.

Synthesis of mPEG-b-PLA mPEG-b-PLA diblock copolymers were synthesized by ring opening polymerization

(ROP) of lactide in the presence of mPEG5000 and stannous octoate (0.3% w/w) as described previously [26,27]. Briefly, lactide and mPEG were dissolved in 20 mL dry toluene under an atmosphere of nitrogen. Stannous octoate (0.3 wt% of the lactide weight) was added into the monomer solution; the reaction mixture was heated to 140 ºC and refluxed for 24h under a dry nitrogen atmosphere. The solvent was removed under vacuum and the product dissolved in DCM was precipitated in cold diethyl ether twice. The resultant precipitate was filtered and dried under vacuum at room temperature overnight. Yield: 75%. This procedure was repeated with three different ratios of mPEG/lactide, 1:1, 1:1.5 and 1:2. 1

H NMR (400 MHz, CDl3) δ ppm: 5.13 (m, CH, PLA), 3.61 (s, CH2CH2O, mPEG), 3.38 (s,

CH3O, mPEG), 1.53 (t, CH3, PLA). 13

C NMR (100 MHz, CDl3) δ ppm: 169.3 (CO, PLA), 70.5 (CH2, PEG), 69.0 (CH, PLA), 16.6

(CH3, PLA). Further characterization of the samples is given in Table 1.below. 2.4.

Preparation of mPEG-b-PLA nanoparticles

NPs were prepared according to the nanoprecipitation method specified by Fessi et al [28]. The copolymer mPEG-b-PLA (5 mg) was dissolved in acetone (1 mL) at room temperature. The resulting polymer solution was added dropwise into distilled water at different ratios (organic solvent/water, 1:1, 1:5 or 1:10) under magnetic stirring at 500 rpm for 24h, allowing evaporation 7

of acetone at room temperature. Nitrogen was purged through the solution with vigorous stirring to improve the evaporation of the remaining acetone. The final concentrations of the formulations were 5.0, 1.0 and 0.5 mg/L, respectively. Curcumin-loaded NPs and curc-BTZ-loaded NPs were prepared by the same protocol as for the bare NPs mentioned above. Curcumin or curc-BTZ complex and polymer were dissolved separately in acetone and then the two fractions were mixed together before nanoprecipitation. The ratio polymer/curcumin was 10:1 w/w. The produced NPs were centrifuged at 17,500 rpm for 20 min at 4 ºC; then washed with distilled water to remove non-encapsulated curcumin, freeze dried, and stored at 4 ºC in the dark. Drug incorporation efficiency was expressed both as drug loading content (DLC, %) and drug loading efficiency (DLE, %), calculated according to Eq. (1) and (2), respectively [29].
 (%) =

          

× 100

     


 (%) =        × 100,

(1)

(2)

where the weight of loaded drug is the weight of drug in feed minus the weight of the unloaded drug in the supernatant after centrifugation. The weight of the drug loaded nanoparticles was determined by the gravimetric method, while the weight of the unloaded drug was determined by UV-Vis spectroscopy. For this determination, an aliquot (200 µL) of the supernatant was diluted in 3 mL of ethanol. Absorbance measurements (λabs = 425 nm) were carried out on a Thermo Scientific Spectrophotometer Helios Gamma. The weight of the free drug in each sample was obtained by Lambert-Beer’s Law using the extinction coefficient of curcumin in ethanol (Ɛ = 46000 L/mol·cm) and the molecular weight of curcumin (Mw = 368.38 g/mol). 8

2.5.

Characterization of nanoparticles

For characterization of both bare and curcumin-loaded NPs, the samples were diluted with distilled water or PBS at 7.4 to the final concentrations of 0.5, 1.0 and 5.0 g/L. 2.5.1.

Dynamic light scattering (DLS) DLS measurements were performed to determine the size of the polymer nanoparticles.

The experiments were conducted using an ALV/CGS-8F multi-detector compact goniometer system with eight off fiber-optical detection units (ALV-GmbH, Langen, Germany), equipped with a laser (He–Ne, λ = 632.5 nm) and the beam was focused in the sample cell (10 mm NMR tube). The intensity of scattered light was measured simultaneously at eight scattering angles in a range of 22° to 141°. By measuring at different scattering angles we can establish whether the relaxation process is diffusive (q2-dependent) or not (cf. the discussion below). The temperature in the measuring cell was controlled with a heating/cooling circulator, circulating water around a cylindrical quartz container filled with a refractive index-matching liquid (cis-decalin). Prior to measurements, the solutions were filtered with a 5.0 µm PVDF filter (Millipore) into precleaned NMR tubes in a dust-free atmosphere of filtered air. In this study, the experimentally recorded intensity autocorrelation function g(2) (t), measured for different solutions, is directly related to the theoretically amenable first-order electric field correlation function g(1)(t), through the Siegert relation [30] g(2)(t) = 1 + B·|g(1) (t)|2, where B≤1 is usually treated as an empirical factor and the wave vector q is defined as q = (4πn/λ) sin(θ/2), where n is the refractive index of the solvent, λ is the wavelength of the incident light in the vacuum, and θ is the scattering angle [31].

9

In the analyses of the correlation functions in suspensions of the bare polymer nanoparticles, it was found that the decays can be well-described by a single stretched exponential [32]

  t g (t) = exp -    τ fe (1)

  

β

  

(3)

in which t denotes the time, τfe is some effective relaxation time, and β (0 < β ≤ 1) is a measure of the width of the distribution of relaxation times [33]. The mean relaxation time is given by

τ =

τse  1  1 Γ  = 2 , β  β  q Dapp

(3a)

where Γ(1/β) is the gamma function of β-1, <τ > is the average relaxation time, and Dapp is the apparent mutual diffusion coefficient. This relaxation mode was always found to be diffusive. In the case of curcumin-loaded and curc-BTZ-loaded NPs in the suspension, together with some bare particles, we encounter a situation where we have two populations of NPs and two relaxation modes; a fast mode for the unloaded particles and a slow relaxation mode for the loaded particles. For this situation, the correlation functions can be described well by the sum of a single exponential and a stretched exponential: g(1)(t) = Af exp[-(t/τfe)] + As exp[-(t/τse)γ]

(4)

with Af + As = 1. The parameters Af and As are the amplitudes for the fast and the slow relaxation mode, respectively. The variables τfe and τse are the relaxation times characterizing the fast and the slow process, respectively. The slow mean relaxation time is given by

10

τs =

τ se  1  Γ  γ  γ 

(4a)

The correlation functions were analyzed with a nonlinear fitting algorithm to obtain best-fit values of the parameters τfe and β in Eq. (3) and the parameters Af, τfe, τse, and γ appearing on the right-hand side of Eq. (4). Continuous controls of both the fast and slow relaxation times always revealed that both modes were diffusive (τ-1 ∝ q2). Since all the relaxation modes are diffusive, the apparent hydrodynamic radii Rh of the unloaded and loaded particles can be calculated through the Stokes-Einstein relationship if we assume the entities to be spherical Rh =

k BT 6πηDapp

(5)

where Rh is the hydrodynamic radius of the particle, kB is the Boltzmann’s constant, T is the absolute temperature, η is viscosity of the solution, and Dapp is the apparent mutual diffusion coefficient [34]. 2.5.2.

Zeta potential

To determine the zeta-potential of the systems, the solutions of polymeric nanoparticles were analyzed by Laser Doppler Micro-electrophoresis with a Zeta-sizer Nano ZS instrument, MAL1049741 (Malvern instruments Ltd., United Kingdom). The measurements were conducted on aqueous polymer solutions with concentrations of 0.05, 0.1, 0.2, and 0.5 at 25 ºC. A dip cell was used as sample cell, including palladium electrodes with 2 mm spacing, and disposable cuvettes. Five measurements were registered for each sample and the average values were

11

reported. Malvern Zeta potential transfer standard DTS1235 (−42mV± 4.2mV) was used to check the instrument calibration. The zeta potential is calculated from the measurements on the basis of the Henry equation that relates the zeta potential, ζ, to the electrophoretic mobility, ???? (Eq. (6)) [35]. !" =

2$ζ (()*) 3'

(6)

where η and ε are the solvent viscosity and the dielectric constant, respectively, at a given temperature. The Smoluchowski approximation to Henry’s function (f(Ka)=1.5) was applied. To avoid disturbing aggregation in the characterization of the particles, zeta potential and DLS measurements were carried out in water suspensions and not in PBS medium. 2.5.3.

Electron microscopy

Cryo-TEM measurements were carried out on a Tecnai G2Sphera 20 electron microscope (FEI Company, Hillsboro, OR, USA) equipped with a Gatan 626 cryo-specimen holder (Gatan, Pleasanton, CA, USA) and a LaB6 gun. The samples for cryo-TEM were prepared by plungefreezing [36]. Briefly, 3 µL of the sample solution were applied to a copper electron microscopy grid covered with a perforated carbon film forming woven-mesh-like openings of different sizes and shapes (the lacey carbon grids #LC-200 Cu, Electron Microscopy Sciences, Hatfield, PA, USA), which was glow discharged for 40 s with 5 mA current prior to specimen application. Most of the sample was removed by blotting (Whatman no. 1 filter paper) for approximately 1 s, and the grid was immediately plunged into liquid ethane held at –183 ºC. The grid was then transferred without rewarming into the microscope. Images were recorded at the accelerating voltage of 120 kV and with magnifications ranging from 11,500× to 29,000× using a 12

GatanUltraScan 1000 slow scan CCD camera in the low-dose imaging mode, with the electron dose not exceeding 1,500 electrons per nm2. The magnifications resulted in final pixel size ranging from 0.3 to 0.9 nm, the typical value of applied under-focus ranged between 0.5 to 2.5 µm. The applied blotting conditions resulted in the specimen thickness varying between 100 to ca. 200 nm. All cryo-TEM pictures were carefully inspected for possible artefacts such as radiation damage and ice crystals, and high-quality images were band-pass filtered in order to suppress both ice thickness variations and noise below 1 nm detail size. 2.6.

Cell culture

MDA-MB-231 cell lines (ATCC), HeLa cells (kindly provided from Max-Planck Institute in Dresden), and MCF-7 cells (kindly provided from the Institute of Macromolecular Chemistry, ASCR, in Prague) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, 1% penicillin, 1% streptomycin, and 4mM glutamine. Cells were cultivated in an incubator at 37 ºC and 5% carbon dioxide (CO2). 2.7.

In vitro cellular uptake

For live cell microscopy, cells were cultured in glass bottom Petri dishes (In Vitro Scientific). Live cell imaging was performed using a spinning disc confocal system based on Olympus IX81 microscope equipped with Olympus UPlanSApo 60×/1.4NA oil immersion objective, CSU-X spinning disc module (Yokogawa) and Ixon Ultra EMCCD camera (Andor). Cells were maintained at 37 °C and 5% CO2 with a microscope incubator (Okolab). Image analysis was performed in ImageJ. For flow cytometry, cells were cultured at a cell density of 100,000 cells/well in a 12-well plate. After 24 h of incubation at 37 °C and 5% CO2, the cell culture medium was replaced by 13

1000 µL of fresh culture medium containing tested nanoparticles or free curcumin. Cells were incubated with nanoparticles at a final concentration of 20 µg/mL or 40 µg/mL. The cellular uptake of NPs was measured at 45, 90, and 180 min, as well as 6, 12, and 24 hours after addition of the nanoparticles. Samples were acquired by BD FACS Canto II (Becton Dickinson) using BD FACS Diva 6 Software and analyzed by FlowJo (TreeStar). 2.8.

In vitro cytotoxicity

The MTT cytotoxicity assays were performed when cells became confluent. For the metabolic activity MTT assay, cells were cultured at a cell density of 5,000 for HeLa cells, 7,500 for MDA-MB-231 cells and 10,000 for MCF-7 cells/well in a 96-well microtiter plate. After 24 h or 48 h of incubation at 37 ºC in 5% CO2, the cell culture medium was replaced by 100 µL of fresh culture medium containing tested NPs or free curcumin (curcumin-bortezomib). After further 24 h of incubation at 37 ºC and 5% CO2, the medium was aspirated, and cells were incubated with 50 µL of MTT solution (1 mg/mL in PBS) at 37 ºC in 5% CO2 for 2 h. Thereafter, MTT solution was aspirated and 100µL isopropanol was added. Epoch Microplate Reader instrument (BioTek, USA) was used to assess cell viability by spectrophotometry at a wavelength of 570 nm (reference wavelength 690 nm). Results of MTT assay were expressed as percentage of controls (cells in control medium), which was considered as 100%. The tests were performed on at least three separate experiments. Cell viability is calculated from the following expression: +,, -.*/.,.0 (%) = (1/2 3( 2*45,+⁄1/2 3( 63783,) 9 100

(7)

where “Abs of sample” denotes the absorbance obtained from the wells containing treated cells and “Abs of control” is the absorbance of untreated cells. 14

3.

Results and discussion

3.1.

Synthesis and characterization of mPEG-b-PLA

The diblock copolymer mPEG-b-PLA was synthesized by ROP of lactide using mPEGOH as a macroinitiator and stannous octoate as a catalyst in toluene solution (Scheme 1). By variation of the ratio of reactants used in the polymerization reaction, the ratio of the PLA to PEG block composition of the copolymer was controlled.

Scheme 1. Synthesis of mPEG-b-PLA block copolymers.

The average molecular weight of the mPEG-b-PLA was calculated by comparing the integrated area of the peaks at 5.15 and 1.56 ppm (=CH- and -CH3 in PLA, respectively) with that of the peak at 3.64 ppm (-CH2-CH2-O- in PEG) as shown in Fig. 1 for mPEG113-b-PLA115 copolymer as an example. The integral intensity of the methylene group (Ib) of the PEG block was determined to be 452, based on the theoretical number of protons; the number-averaged molecular weight (Mn,NMR) values of the copolymers were determined according to Eq. (8): ;,=>? (@/43, ) = BCD GF CH 9 44 + C31CK E

L"M

+ BCD N G P CH 9 72K E OE

LRS



(8)

15

Fig. 1. 1H and 13C NMR spectra of mPEG113-b-PLA115 copolymer (12k) in CDCl3.

The GPC analysis of the copolymers showed a single and sharp peak. The molecular weight distributions were narrow and therefore low polydispersity indices (Mw/Mn) were obtained. Moreover, the number-average molecular weight (Mn,GPC) increased coherently with increasing ratio of lactide monomer in the feed. NMR and GPC data are summarized in Table 1. Table 1

Number and weight average molecular weights (Mn and Mw, respectively), refractive index increment values (dn/dc) and polydispersity index (Mw/Mn) of mPEG113-b-PLAy block copolymers. Mn,NMR y

Sample

mPEG/lactide

Mn,GPC

Mw,GPC

Mw/

(g/mol)

(g/mol)

Mn

dn/dc (g/mol)

mPEG113-PLA35

35

8k

1:1

7500

0.064

8500

9000

1.1

mPEG113-PLA78

78

10k

1:1.5

10680

0.061

11000

16000

1.5

mPEG113-PLA115

115

12k

1:2

13240

0.058

14000

16800

1.2

16

Molar mass (g/mol)

100000

Molar mass LS RI

10000

1000

100

10

5.5

6.0

6.5

7.0

Time (min)

Fig. 2. Typical GPC analysis of mPEG113-b-PLA35 copolymer (8k) in THF as an example.

3.2.

pH dependent interactions between curcumin and bortezomib

The anticancer drug bortezomib or its model substance can be conjugated to the curcumin by interaction between the boronic acid motive in bortezomib and the acetylacetone tautomeric motive in curcumin to generate a boronate ester compound. This facile conjugation is formed through dynamic covalent chemistry and it is reversible in a pH-sensitive way. Curcumin and boronic acid structure in bortezomib form a stable, covalently-bonded complex at alkaline or neutral pH typical for blood plasma (pH 7.4). However, in slightly acidic milieu typical for endosomes (pH below 5) and tumor interstitial space (pH ca 6.5) this complex dissociates to release the free active bortezomib drug and the free curcumin (Scheme 2).

17

Scheme 2. Chemical structures of curcumin, model and bortezomib, and their complex formation governed by pH. This complexation process and the effect of pH on the sensitivity of curcuminbortezomib dissociation were investigated using 1H NMR spectroscopy, following a strategy similar to the characterization of the pH-dependent reversible binding between bortezomib and catechol compounds [37]. First, curcumin and bortezomib model were dissolved in deuterated dmso-d6 separately, and later mixed together at the same concentration of 0.01 M (Fig. 3a). The prepared curc-BTZ conjugate was diluted in deuterated aqueous solution at different pH values in the range of 4.5-8.4 and analyzed by NMR spectroscopy (Fig. 3b).

18

Fig. 3. 1H NMR spectra of a) curcumin and bortezomib model in deuterated dmso where the

combination of their proton signals are assigned, and b) curcumin-bortezomib model conjugate in deuterated aqueous solution at different pH values. In curc-BTZ conjugates, and below pH 6, the structure dissociates to release the free BTZ and the free curcumin. It was observed by the disappearance of Hs curcumin signals and at this pH the curcumin precipitated. 3.3.

Preparation and characterization of mPEG-b-PLA and loaded nanoparticles

The use of curcumin as an anticancer drug is limited due to its extremely poor watersolubility (11 ng/mL) [38]. In the present work, curcumin-loaded and curc-BTZ-loaded NPs

19

were prepared according to the nanoprecipitation method using a curcumin/polymer ratio of 1:10 w/w and a curcumin/bortezomib equimolar mixture when the complex is used for loading. The drug loading content (DLC) and drug loading efficiency (DLE) of NPs were determined by UV spectroscopy. As shown in Fig. 4, the DLC is around 2.5% while DLE is around 20-26% depending on the copolymer composition of the NPs sample. Two tendencies are clearly observed, the drug loading rises slightly as the length of the hydrophobic block increases; the efficiency of curcumin loading is higher when it is forming a complex with the bortezomib model. By this loading and dispersion method, the solubility of curcumin in water was increased from 11 to 30,000 ng/mL.

a)

10

b)

5 20

0

DLC (%)

DLE (%)

40

curc-NPs, DLE (%) curcBTZ-NPs, DLE (%) curc-NPs, DLC (%) curcBTZ-NPs, DLC (%)

0

PLA35

PLA78

PLA115

LoadedNPs

Fig. 4. a) Properties of curcumin and curc-BTZ-loaded NPs determined by UV absorbance. Data represent mean values ± SD of experiments measured for 3 different batches of NPs. b)

20

Curcumin (left) forming sediment at the bottom of the vial because of its poor solubility in water and curcumin-loaded NPs (right) suspended in water and the yellow curcumin color is evident. 3.3.1. Dynamic light scattering

The size of NPs in aqueous solution was determined by multi-angle dynamic light scattering. Concentration dependence of the apparent hydrodynamic radius was studied for bare NPs samples prepared at different water-organic solvent ratios, with final concentrations of 0.05 wt%, 0.1 wt%, and 0.5 wt%, at 25 ºC. It was found that the nanoprecipitation of copolymers with a mixing ratio (organic solvent/water) of 1:1 formed larger NPs (around 80 nm) than the same copolymers at a mixing ratio of 1:10, which formed particles of ca. 30 nm. Fig. 5 illustrates the correlation functions at various scattering angles and their corresponding fits for 0.1 wt% mPEG113-PLA35 bare NPs (Fig. 5a) and curc-BTZ NPs (Fig. 5b) in water, as an example. The inset plot shows that the relaxation process is diffusive or q2-dependent. All the studied relaxation processes were found to be diffusive. Blank and loaded NPs displayed narrow size distributions with a value of β higher than 0.9.

a)

b)

6

0.8

4

β=1

3

0.8

2

0.4 0.2 0.0 10-4

0

22° 39° 56° 73° 90° 107° 124° 141° 10-2

0

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10-1

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21

Fig. 5. Plot of g2(t) versus time at different scattering angles and their corresponding fits of 0.1

wt.% water solution of a) Bare mPEG113-PLA115 NPs and b) Bare mPEG113-PLA35, curcuminloaded and curc-BTZ-loaded NPs at 25 ºC (scattering angle of 90º). The inset shows a plot of the inverse relaxation time versus q2 for different scattering angles, demonstrating the diffusive behavior of the systems. From the Stokes-Einstein, Eq. (5), the values of the apparent hydrodynamic radii can be calculated, and the values of Rh as a function of hydrophobic block length of the copolymer for 0.1 wt% NPs suspensions are shown in Fig. 6. A characteristic feature at all PLA-lengths is that the size of the species rises intensely as the NPs are loaded with curcumin and an additional size–increase is observed upon further loading with BTZ. This trend is expected, because when the NPs take up the additives, and to some extent are decorated with additives, they are expected to expand. A more challenging issue is to survey the effect of PLA block length on the size of the NPs. The picture that emerges is that the size of the particles increases initially as the PLA-block length becomes longer,[39-41] but for the longest PLA block, Rh drops and this trend is more pronounced for the loaded particles. This finding can be rationalized in the following scenario. Initially when the PLA block length increases, the nanoparticle size increases because the longer PLA chains occupy more space. But for sufficiently long PLA chains, the chains will be close-packed inside the core to avoid contact with the aqueous surroundings and this will reduce the size of the particles. For the loaded and swelled nanoparticles, the effect of the intramolecular association and contraction should be stronger because more is gained by close-packing of the chains. In a previous study on dilute aqueous solutions of hydroxy(ethyl cellulose) that was hydrophobically modified with

22

alkyl chains of different lengths (C8 or C16) [42] the contraction of the molecules increased strongly with increasing length of the alkyl chain. This lends support to our observation.

200

Curc-BTZ (Rh,s) Curcumin Bare Curc-BTZ (Rh,f)

Rh (nm)

150

100

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PLA78

PLA115

Fig. 6. Hydrodynamic radii of bare particles, curcumin-loaded NPs, and curc-BTZ-loaded NPs

for solutions of 0.1 wt.% at 25 ºC. Data represent mean values ± SD (n=3) of experiments measured for 2 different batches of NPs. The error bars are of the same size as the size of the symbols. 3.3.2. Zeta Potential

The resulting bare and loaded NPs were characterized in terms of zeta potential. The zeta-potential results of blank NPs (0.5 wt%, 0.1 wt% and 0.05 wt%) and curcumin-loaded NPs (0.2, 0.1 and 0.05 wt%) in aqueous media at 25 ºC are shown in Fig. 7.

23

0

NP 8k NP 10k NP 12k

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-5

Curc-NP 8k Curc-NP 10k Curc-NP 12k

-10 -15 -20 -25 -30 -35

0.0

0.2

0.4

0.6

Concentration (% wt)

Fig. 7. Zeta potential for different concentrations of blank NPs and curcumin-loaded NPs (see

Table 1) at 25 ºC. Data represent mean values ± SD of experiments measured 5 times. The zeta potential in 0.5 wt% suspensions of the unloaded particles changes from approximately -13 mV to -30 mV as the length of the charged PLAy block varies from y=35 to y=115. This is expected because the charge density increases when the PLA block length becomes longer. A similar trend, but less prominent, is also detected at the other particle concentrations. Besides the electrostatic stabilization from the PCL-block, the long hydrophilic mPEG block may also contribute to the stabilization of the particles. For the curcumin loaded NPs, a more intricate picture emerges. In this case the value of the zeta potential will be governed by factors such as the thickness of the curcumin layer decorating the NPs, the number of non-decorated NPs, and aggregates. The results seem to indicate that the 24

lowest particle concentration and particles prepared from the copolymer with the shortest PLAlength promote the highest absolute value (-27 mV) of the zeta potential and these NPs can be considered to be electrostatically stabilized. The highest particle concentration of NPs prepared from the copolymer with the longest PLA-block yields an approximate zeta potential of -13 mV, which is the lowest value and likely too low to provide electrostatic stabilization of the particles. This finding may suggest that particles with a long PLA block may be more efficient to adsorb curcumin on the particle surface. Similar trends in the zeta potential have recently been reported by Hu et al. for micelles of curcumin-loaded PEGMA-PLLA-PEGMA triblock copolymers [43]. These values indicate a moderate stability behavior of the colloidal dispersion. 3.3.3. Electron microscopy

Cryo-electron microscopy was utilized to visualize the morphology of both unloaded and loaded nanoparticles in suspension, and some typical images are depicted in Fig. 8.

a)

b)

Fig. 8. a) Cryo-TEM image of bare mPEG113-b-PLA78 NPs (scale bar 100 nm), and b) cryo-TEM

image of curc-BTZ-NPs mPEG113-b-PLA78 NPs (scale bar 50 nm).

25

The amphiphilic diblock copolymers formed particles with diameters in the range of 10-20 nm (Fig. 8a) in aqueous suspensions (the concentration of the formulations was 1.0 mg/mL in PBS pH 7.4). The image indicates that the particles are spherical and rather monodisperse and this is confirmed by the DLS measurements. In the presence of curc-BTZ complexes, larger particles (40-50 nm in diameter) are observed (see Fig. 8b) and these particles are decorated and loaded with the curc-BTZ complexes. These particles coexist with small particles that probably are not forming complexes. This picture is consistent with the DLS results (Fig. 6) for NPs in the presence of curc-BTZ complexes, where two relaxation modes were observed; the fast relaxation mode was assigned to bare particles or particles with a small amount of loading, and the slow mode to particles loaded with curc-BTZ. 3.4.

In vitro cellular uptake of nanoparticles

Time evolution of NPs loaded with curcumin in the HeLa cell line was monitored using spinning disc confocal microscopy and flow cytometry. Fig. 9(A-C) shows the cellular uptake of curcumin bortezomib model-loaded NPs (10k, see Table 1) in HeLa cells after 3 hours of incubation with a concentration of 20 µg/mL. To achieve better cellular localization of NPs, DIC (differential interference contrast) images (A) were captured and merged (C) with FITC filter (fitting fluorescent spectral properties of curcumin) image (B). In contrast to control (data not shown), cells exhibit the strongest FITC signal in the perinuclear area of cells, whereas in the cytoplasm the intensity of the green fluorescence is weaker; no fluorescence intensity can be traced in the nucleus of the cells. This is consistent with the nuclear pore channel size, which is in the range of 3-5 nm in humans [44]. Therefore, the NPs can pass through the cell membrane but not through the nuclear pore complex. Z. Wang et al. also presented cellular location of PEG-PLA micelles loaded with curcumin in cytoplasm with the most pronounced fluorescence 26

intensity around nucleus of HeLa cells [45]. During 24 hours of incubation HeLa cells in the presence of curcumin-bortezomib model-loaded NPs (12k) at a particle concentration of 20 µg/mL (see Fig. 9(D-F)), FITC positive signal was observed in the cytoplasm already after 45 min. After additional 3 hours, the perinuclear zone of the cells had higher green fluorescence intensity in comparison to the rest of the cell. After 24 hours there was a significant decrease of the fluorescence intensity in perinuclear area with a disperse signal in the cytoplasm. The cellular uptake of NPs analyzed by flow cytometry revealed that the concentrationdependent pattern corresponds to patterns already known in other tested NPs of different kinds (data not shown), e.g., in the case of Raw cells incubated with PEG-PCL NPs with diameters between 100 nm to 125 nm [46]. Significant differences were observed during 24 hours of incubation with the highest percentage of positive signal after 3 hours (Fig. 9G) for all tested NPs (8k, 10k, and 12k). At longer times, the positive signal gradually decreased. While the pattern of the curves is very similar for all the tested NPs, there is a clear difference between weaker positive signals of curcumin loaded NPs and curcumin-BTZ model-loaded NPs, in the range 2-4 fold, with the weakest positivity for 8k NPs compared to 10k and 12k samples. This trend can be rationalized in the following way. It is reasonable to assume that nanoparticle size can influence cell uptake as well as the magnitude of the zeta potential. The nanoparticles with the shortest lactide chain are expected to be the least stable; this may lead to curcumin leakage. Curcumin in aqueous milieu may have lower fluorescence quantum yield because water is more polar than the interior of the nanoparticle core. In addition, curcumin can be bound to blood plasma proteins present in the medium [47], which may reduce the cell uptake because less free drug is present in media and most is bound to blood proteins.

27

A

B

C

E

F

C PrN

N

D

G) Cellular uptake HeLa cells 40

curc-NPs 8k curcBTZ-NPs 8k curc-NPs 10k curcBTZ-NPs 10k curc-NPs 12k curcBTZ-NPs 12k

Positive cells %

30

20

10

0 0

300

600

900

1200

1500

Time of incubation (min)

Fig. 9. Confocal microscopy images of HeLa cells. (A-C) The cellular uptake of curc-BTZ

model-loaded NPs (10k) (20 µg/mL) after 3 hours of incubation, (A) DIC (N - nucleus, C cytoplasm, PrN - perinuclear area), (B) FITC, (C) Merged DIC and FITC images. (D-F) Time evolution of the cellular uptake of curc-BTZ model-loaded NPs (12k) at 45min (D), 180min (E), and 24 hours (F), (G) Flow cytometric analysis of the cellular uptake in HeLa cells of NPs with curcumin and curcumin-bortezomib model. The cellular uptake and intracellular distribution of drug-loaded NPs in MCF-7 and MDA-MB-231 breast cancer cell lines were tested and compared with HeLa cancer cells. MCF-7 represent a luminal-like model that expresses estrogen receptor (ER+) and progesterone receptor 28

(PR+) with a lower metastatic behavior compared to the MDA-MB-231 cells, which is basal Blike and triple-negative (ER-, PR-, HER2-) model with highly metastatic behavior.[25] Accordingly, these two cell lines may serve as models for breast cancer and its therapy, where MCF-7 cells act as a model for hormone therapy, whereas MDA-MB 231 cells may stand as model for chemotherapy. Fig. 10 shows the cellular uptake (at a concentration of 40 µg/mL) of curcumin-loaded NPs, curc-BTZ model-loaded NPs, and free drugs after 3 hours of incubation. Fig. 10(A) presents histograms of flow cytometric analysis on the cellular uptake of curc-BTZ model-loaded NPs (8k) and curc-BTZ model-loaded NPs (10k) in HeLa, MCF-7, and MDA-MB231 cell lines. Differences in cellular uptake of NPs 8k, 10k, and 12k for the three tested cell lines in the presence of curcumin-NPs, curc-BTZ model-NPs, free curcumin, or the free curcumin-bortezomib model are depicted in Fig. 10(B). Let us first discuss the curcumin loaded NPs and their uptake in different cell lines (Fig. 10(B)). The most conspicuous feature is the very low uptake of curc-NPs-8k in cells from all three considered cell lines. This may be ascribed to the high negative values of the zeta potential at low sample concentrations for the curc-NPs-8k complexes (cf. Fig. 7), which can lead to repulsive electrostatic forces between the negatively charged cell membranes and the complexes and thereby to reduction of the cell uptake. It should be noted that the complexes with 10k and 12k NPs show lower negative values of the zeta-potential at low concentration than that for the complex with 8k. If we compare the three cell lines, it is obvious that the cell uptake is significantly higher for the HeLa cells than for the cells from the two breast cancer cell lines. The results clearly show that the cell uptake for MCF-7 cells is strikingly larger than for MDA-MB231 cells. A possible explanation for this can be the differences in organization of cell membrane that may influence the processes of endocytosis and/or the pore sizes of the cell membranes. It is

29

known that MDA-MB-231 cancer cells are more aggressive and less responsive to chemotherapy and one reason for this may be that the uptake of drug carriers is very poor. In general, cell characteristics reflect lipid composition of the cell membrane; changes of lipid components can influence the function of membrane proteins and cellular functions. He et al. studied membrane lipid phenotype of breast cancer cells, and they showed correlation of breast cell lines with changes in membrane lipids levels [48]. The flow cytometry results correspond to the images of HeLa, MCF-7, and MDA-MB-231 cells captured by spinning disc confocal microscope (Fig. 10 (C)) after identical treatment. The cellular uptakes of 10k and 12k NPs were clearly pronounced in the perinuclear area of HeLa cells (b,c) and MCF-7 cells (f,g). However, the MDA-MB-231 cells (j,k) revealed green fluorescence intensity of the apical part of the cells, without clear perinuclear localization. This is a further indication of that internalization of drug carriers into MDA-MB-231 cells is hindered. In a recent study, it was shown that native curcumin can be degraded within two hours, whereas curcumin encapsulated in glycerol monolete based NP was stabilized and protected against premature degradation [49]. The overall results in this work indicate that the carrier systems of curc-BTZ-loaded NPs10k and curc-BTZ-loaded NPs-12k seem to be the more efficient systems for cell uptake for the three cell lines considered in this study. This suggests that it is not favorable to have a PLA block that is too short. This may be due to both charge density of the nanoparticles and hydrophobicity. It is interesting to note that the cell uptake of free curcumin is low in all studied cell lines. This can probably be associated with the very low solubility of curcumin in aqueous media.

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cur c

cur c8 k

10k

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a

b

c

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e

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h

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Fig. 10. The cellular uptake of curc-NPs, curc-BTZ model-NPs, free curcumin and free curc-

BTZ model after 3 hours of incubation for a concentration of 40 µg/mL. (A-B) Flow cytometry, (A) Representative histograms of the cellular uptake of curc-BTZ model-loaded NPs in HeLa, 31

MCF-7, and MDA-MB-231 cells (control – grey, NPs 8k – blue, NPs 10k – purple). (B) Representative comparison of the efficiency of cellular uptake of curc-NPs, curc-BTZ modelNPs, free curcumin and curcumin-BTZ model for the three tested cell lines. (C) Confocal microscopy images of cells incubated with curc-BTZ model-loaded NPs or free curcumin. (a-d) HeLa cells, (e-h) MCF-7 cells, (i-l) MDA-MB 231 cells incubated with curc-BTZ NPs 8k (a, e, i), curc-BTZ NPs 10k (b, f, j), curc-BTZ NPs 12k (c, g, k) and free curcumin (d, h, l).

3.5.

In vitro cytotoxicity

In the light of sufficient internalization of curcumin and curcumin-bortezomib modelloaded NPs in the tested cell lines, cytotoxicity tests were employed to study the efficiency of NPs drug delivery carriers to treat cancer in comparison to free drugs. Fig. 11 shows MTT results depicted as cell viability after 24 h of incubation in dependence on sample concentration of bare NPs (blank), curcumin-loaded NPs, curc-BTZ-loaded NPs, free curcumin, and free curc-BTZ for all three cell lines (HeLa, MDA-MB-231 and MCF-7). The concentrations of the polymer systems range from 6.25 to 800 µg/mL, and the concentration of curcumin in curcumin-loaded NPs and free curcumin varied in the range from 0.625 to 80 µg/mL. According to the drug content loading experiments, curc-NPs contain about 10% of the polymer concentration. It should be noted that it was necessary to dilute the original curc-BTZ model by a factor of 1000 because of the high toxicity of BTZ. Therefore, the concentration domain utilized for the curcBTZ complex ranged from 1 to 128 nM. Cell viability of HeLa cells are presented in Fig. 11a. Since there are only minor differences among the copolymer types (8k, 10k, or 12k), the data are only displayed for 8k. The

32

horizontal line in the panels indicates the level, above which the effect of cytotoxicity is ignored. The lowest concentration (ca. 11 µg/mL) for a progressive increase of cytotoxicity is observed for curcumin loaded nanoparticles (curc-NPs-8k); in contrast, the transition concentration for free curcumin is much higher (ca. 40 µg/mL). This difference can probably be attributed to the higher amount of soluble curcumin in the curcumin-loaded micelles compared to free curcumin. The addition of BTZ to the curcumin-loaded nanoparticles (curc-BTZ-NPs-8k), curcumin-BTZ complexes have a higher transition concentration (ca. 100 µg/mL) more than twice in contrast to complex–loaded NPs. In the case of the bare NPs, the cell viability is rather high (ca. 60%) even at the highest concentration (800 µg/mL) so the cytotoxicity of the polymer particles is low. The effect of particles and drug complexes on the cell viability of MDA-MB-231 cells is quite different from the results for the HeLa cells. The impact of free curcumin on the cell viability is tricky. At concentrations up to 50 µg/mL, no cytotoxicity is detected, whereas at concentrations above 100 µg/mL the cytotoxicity is prominent. Curcumin-loaded 8k NPs virtually lack toxicity over the studied concentration region, whereas a progressively stronger concentration-induced cytotoxicity effect is observed for the other loaded NPs (10k and 12k). It seems that the concentration-dependent cytotoxicity becomes more dominant for the carrier with the longest PLA block. This is not unexpected because NPs with higher hydrophobicity can probably accumulate more curcumin that can be released. In the case of the MCF-7 cell line, cytotoxicity of curcumin-loaded NPs was more pronounced with a similar pattern (data not shown). Fig. 11c illustrates the higher cytotoxic effect of curcumin-BTZ complex loaded NPs for the MCF-7 cell line compared to the free curcumin-BTZ complex. Complex loaded 8k NPs revealed significantly higher toxicity than the corresponding 10k and 12k NPs. A similar trend was observed for MDA-MB-231 cells, where 8k NPs were significantly more toxic compared to

33

free complexes and for 10k and 12k NPs. As discussed above, the nanoparticles with the shortest lactide chain are the least stable ones, and this may lead to higher cytotoxicity. b) MDA-MB 231 cells

C

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Bare NPs 8k Free Curcumin Curc-NPs 8k Free CurcBTZ CurcBTZ-NPs 8k

on t

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Cell Viability (%)

a) HeLa cells

Concentration (µg/mL)

Fig. 11. Cell viability determined from MTT assay for HeLa, MCF-7 and MDA-MB-231 cells

exposed for 24 hours in the presence of the systems indicated as a function of concentration. (a) HeLa cells exposed to the indicated systems. (b) MDA-MB-231 cells in combination with the indicated systems. (c) MCF-7 cells in the presence of indicated systems. The horizontal lines in the panels indicate a cell viability level where data above is considered non-cytotoxic and below cytotoxic.

4.

Conclusions Biodegradable mPEG-PLAy diblock copolymers were synthesized by employing ROP. The

mPEG block was kept constant, whereas the length of the hydrophobic PLA group was altered (y= 35, 78, and 115). By utilizing nanoprecipitation, nanoparticles were prepared from these copolymers (8k, 10k, and 12k) and they were loaded with curcumin or curc-bortezomib (BTZ) complex. To test cell uptake and cytotoxicity of these systems, HeLa, MCF-7, and MDA-MB231 cell lines were engaged in this study. It was found that after internalization into the cells, nanoparticles (NPs) of different PLA lengths and loading material were all located in the 34

cytoplasm but not in the nucleus. The result advocated that particles from the copolymer with intermediate PLA length are the most efficiently internalized. The DLS results revealed that the size of the particles rose markedly as the NPs were loaded with curcumin and an additional size-increase was detected upon further loading with BTZ. The DLS results, as well as the cryo-TEM measurements on systems with particles together with additives indicate that bare and loaded particles coexist. The zeta-potential experiments disclosed that the lowest particle concentration and particles prepared from the copolymer with the shortest PLA-block favor the highest value (-27 mV) of the zeta-potential and at this condition, the particles can be considered as electrostatically stabilized. The results show that the cell uptake is significantly higher for the HeLa cells than for the breast cancer cells. When it comes to a comparison between MCF-7 and MDA-MB-211 cells, the cell uptake for MCF-7 is significantly larger than for MDA-MB-231 cells. Furthermore, flow cytometry experiments revealed that internalization of drug carriers into MDA-MB-231 cells constitutes a difficulty and may signalize a problem in the treatment of this type of cancer. The cytotoxicity results demonstrate that bare nanoparticles are in general not cytotoxic at the concentrations studied here. For curcumin loaded particles, the cell viability with MDAMB-231 cells is highest for NPs prepared from the copolymer with the shortest PLA block. As the concentration increases, free curcumin turns out to be quite cytotoxic. It can be concluded that the data from the MTT assay show that the efficiency of curcumin-bortezomib is enhanced by using NPs as carrier systems. Our results therefore suggest that the presented mPEG-b-PLA nano-formulations can be useful as carriers of drug for breast cancer treatment. Therefore for successful use of nanoparticles as drug carrier the origin of the cancer cells has to be considered.

35

Acknowledgements

Financial support from Norwegian Financial Mechanism 2009-2014 under Project Contract MSMT-28477/2014 (project 7F14009) is gratefully acknowledged. This work was also supported by grants from the Czech Science Foundation (P302/12/G157), from Charles University (UNCE 204022, Prvouk/1LF/1) and by the grant CZ.2.16/3.1.00/24010 (OPPK program) of the European Regional Development Fund. The authors thank Mgr. Viktor Cerny for his kind help with flow cytometric analysis. We are also thankful to Dr. Jitka Forstova for putting to our disposal instrumentation necessary for biological experiments.

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Graphical Abstract

Curcumin-Bortezomib Loaded Polymeric Nanoparticles for Synergistic Cancer Therapy

Sandra Medel, Zdenka Syrova, Lubomir Kovacik, Jiri Hrdy, Matus Hornacek , Eliezer Jager , Martin Hruby , Reidar Lund , Dusan Cmarko , Petr Stepanek , Ivan Raska , Bo Nyström

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Highlights

• Synthesized methoxy-poly(ethylene glycol)-block-polylactic acid (mPEG-b-PLA) copolymers. • Nanoparticles for drug delivery of bortezomib and curcumin. • Cell uptake and cytotoxicity have been tested on different breast cancer cell lines.

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