A Facile Approach for Doxorubicine Delivery in Cancer Cells by

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A Facile Approach for the Doxorubicine Delivery in Cancer Cells by Responsive and Fluorescent Core/shell Quantum Dots. Enaam Jamal Al Dine, Sophie Marchal, Raphaël Schneider, Batoul Hamie, Jaafar Ghanbaja, Thibault Roques-Carmes, Tayssir Hamieh, Joumana Toufaily, Eric Gaffet, and Halima Alem Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00253 • Publication Date (Web): 15 Jun 2018 Downloaded from http://pubs.acs.org on June 16, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

A Facile Approach for Doxorubicine Delivery in Cancer Cells by Responsive and Fluorescent Core/shell Quantum Dots.

Enaam Jamal Al Dine1,2, Sophie Marchal3, Raphaël Schneider4, Batoul Hamie1,2, Jaafar Ghanbaja1, Thibault Roques-Carmes4, Tayssir Hamieh3, Joumana Toufaily3, Eric Gaffet1 and Halima Alem1* 1

Université de Lorraine, CNRS, IJL, BP 70239, 54506 Vandoeuvre-lès-Nancy Cedex, France

2

Laboratory of Materials, Catalysis, Environment and Analytical Methods, Faculty of Sciences I,

Lebanese University, Campus Rafic Hariri, Beirut, Lebanon 3

Institut de Cancérologie de Lorraine, 6 avenue de Bourgogne CS 30519 54519 Vandoeuvre-lès-

Nancy Cedex, France 4

Laboratoire Réactions et Génie des Procédés, Université de Lorraine, CNRS, LRGP, F-54000

Nancy, France

*Halima Alem mail: [email protected]

ACS Paragon Plus Environment

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Bioconjugate Chemistry 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 Biocompatible thermo-responsive copolymers based on 2-(2-methoxyethoxy) ethyl methacrylate (MEO2MA) and oligo (ethylene glycol) methacrylate (OEGMA) were grown from the surface of ZnO Quantum Dots (QDs) by Surface Initiated Atom Transfer Radical Polymerization with Activators Regenerated by electron Transfer (SI-ARGET ATRP) in order to design smart and fluorescent core/shell nanosystems to be used towards cancer cells. Tunable lower critical solution temperature (LCST) values were obtained and studied in water and in culture medium. The complete efficiency of the process was demonstrated by the combination of spectroscopic and microscopic studies. The colloidal behavior of the ZnO/copolymer core/shell QDs in water and in physiological media with temperature was assessed. Finally, the cytotoxicity towards human colon cancer HT29 cells of the core/shell QDs was tested. The results showed that the polymer-capped QDs exhibited almost no toxicity at concentrations up to 12.5 µg.mL-1 while when loaded with Doxorubicin hydrochloride (DOX), a higher cytotoxicity and a decreased HT29 cancer cell viability in a short time were observed.

KEYWORDS: core/shell QDs, responsive nanomaterials, drug delivery, HT29 cells.

BRIEFS. Fluorescent and aqueous dispersible core/shell nanoparticles and their further cytotoxicity towards cancer cells.

INTRODUCTION


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In the past decade, significant advances in the field of nanotechnology have been achieved, which include material science, nanophotonic, supramolecular assembly, and drug delivery. One of the great challenges for this revolution is medicinal application, which led to the so-called nanomedecine development.1 Different nanomaterials, such as 2D materials, can be considered for drug delivery purpose as they allow a high drug loading content (up to 700%) and release almost 100% of the encapsulated drug.2–4 Core/shell nanoparticles based on an inorganic core and a polymeric shell are also very attractive. As the core can display electric, optical or paramagnetic properties, which are crucial for imaging, and the polymeric shell can encapsulate a drug and release it in a targeted zone.5–7 These special properties of the polymeric shell are directly related to the flexibility of the macromolecular systems and to the fact that it exists an infinite variety of (co)polymer compositions that can easily be modulated.5,7 Inorganic/organic core/shell nanoparticles (NPs) based on ZnO quantum dots (QDs) are very promising in biomedical fields owing to their unique features which allow different bioapplications.5 Their interesting fluorescence properties and biocompatibility make these NPs ideal candidates to be used as fluorescent probes.8 Indeed ZnO QDs were found to be a good alternative to cadmium-based semiconductor QDs in terms of toxicity and cost and many efforts are currently developed to enhance their optical properties to improve their performances as biological labels.8 ZnO QDs have been found to exhibit unusual chemical and optical properties due to the quantum confinement effect and are promising tools for biological imaging applications.8–10 However, these QDs have been found to suffer from low stability in aqueous media because water molecules are able to exchange the organic protecting groups on the ZnO surface causing their aggregation and therefore the quenching of their luminescence.11,12 This low


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stability in aqueous media hindered the development of ZnO QDs for biomedical applications12,13 until the development of the NP surface functionalization processes.5 Thereby, a proper surface coating of ZnO QDs has been introduced and different strategies have been employed for this purpose, among them is the grafting of a polymer layer.9,12–14 A proper polymer coating has been demonstrated to enhance the stability of ZnO QDs, prevent their aggregation and render them water dispersible. Importantly, such a coating has been found to be a suitable way to prevent the release of Zn2+ ions which could be toxic to the cells.13–15 Xiong et al. prepared ZnO QDs with a copolymer shell composed of an internal hydrophobic polymethacrylate layer and an external hydrophilic poly(ethylene glycol) methyl ether groups allowing to disperse ZnO QDs in water without any alteration of their optical properties. The final core/shell NPs exhibited a very stable photoluminescence in water and were almost nontoxic when their concentration was kept below 0.2 mg.mL-1.16 Recently, we demonstrated that a layer of poly(N-isopropylacrylamide) with controlled thickness could also stabilize ZnO QDs optical properties in water.15 Stimuli-responsive polymers (SRPs) are known as smart materials, exhibiting a great potential in the biomedical field due to their behavior in response to an external stimuli, e.g. temperature, pH, light or magnetic field,17–19 particularly in drug delivery applications. SRPs are capable of encapsulating drugs and releasing them to the desired/targeted spots which could improve their therapeutic efficiency and minimize the undesired side effects of drugs.20–23 The combination of SRPs with fluorescent ZnO QDs resulting in core/shell responsive NPs appeared to be a highly interesting route to build an efficient drug delivery platform as the final nanomaterial could combine the physical properties of the core and the responsive particularity of the shell. Hence, temperature and pH are predominantly studied systems in drug delivery as tumor cells display higher temperature and lower pH due to fast cell metabolism.24 Polymers which respond to changes in temperature are called thermo-responsive polymers.17 The most studied family of


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these polymers are characterized by a lower critical solution temperature (LCST)17. The observed phase transition originates from a coil-to-globule transition governed by cooperative dehydration of hydrophilic chains.25–27 Their application in drug delivery systems are essentially performed by varying the interactions between the drug and the polymer chains (hydrophobicity, hydrophilicity,…). When the polymer chains are expanded in the solvent, some drugs with appropriate chemical structure may be embedded in the chains.5,22 Once heated above the LCST, the polymer chains show a conformation transition from expanded to collapse state inducing the drug release in the medium. As well-known biocompatible materials, copolymers of MEO2MA and OEGMA are very attractive in drug delivery due to their LCST that can be tuned by changing the monomer ratio in the co-polymer chains thus achieving a LCST close to the tumor temperature.28,29 It is also known that the thermo-responsive behavior and the LCST in aqueous solution can be affected by the presence of salts which are known to disrupt the hydration structure surrounding the polymer chains.30 This effect is ion dependent and be described by the Hofmeister series which originates from the ability of ions to precipitate proteins, for both anions and cations.30 The study of the core/shell NPs behavior both in aqueous media and in physiological media is therefore of high interest. In this work, thermo-responsive fluorescent core/shell NPs were designed by grafting a copolymer shell of xMEO2MA and (100-x)OEGMA (where x and (100-x) represent the amount of the monomer) from the surface of ZnO QDs via surface initiated Atom Transfer Radical Polymerization with Activators Regenerated by electron Transfer (SI-ARGET ATRP) as ecofriendly and effective polymerization technique.31 LCST values around the physiological temperature could be obtained by varying the monomers ratio and by considering the presence of salts in the solvent.32 Different characterizations revealed the successful grafting of the copolymer and the cytotoxicity of the core/shell nanoparticles loaded with a model cancer drug, i.e. the


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Doxorubicin hydrochloride (DOX) tests toward cancer cells indicate the high potential of these core/shell NPs in drug delivery. ZnO-based NPs were demonstrated to be of high efficiency on T98G cells, with moderate activity on KB cells and the less toxic on normal HEK cells.11 The high drug resistance properties of HT29 make them important cells to kill. To the best of our knowledge, only one report describes the effect of ZnO QDs on the HT29 cancer cells.33 In this work, Kakhroueian et al. showed that ZnO NPs exhibited a low toxicity in normal cells (MDBK) but a high toxicity against HT29 cancer cells. Indeed combining the both effects was thought to increase the both efficiencies, i.e. the ones from ZnO core and from DOX.33 This effect was clearly demonstrated in this work, where the most efficient against HT29 cancer was the system based on the core/shell nanostructures combined with the drug, for which the drug efficiency was at least 50% higher compared to the free DOX.

RESULTS AND DISCUSSION

Synthesis and characterization of ZnO@P(MEO2MAX-OEGMA100-X) NPs. P(MOE2MAxOEGMA100-x) copolymers were grown from silane-functionalized ZnO QDs as described in previous reports22,23,37 and illustrated in Figure 1. The efficiency of the grafting process is confirmed by FT-IR as described and shown in the Supporting Information (see also Figure S1). The NPs microstructure was studied by the combination of High resolution Transmission Electron Microscopy (HR-TEM) and X-ray diffraction (XRD) experiments. The XRD patterns of ZnO NPs exhibit diffraction peaks located at 2θ = 31.7, 34.3, 36.3, 47.4, 56.5, 62.8 and 67.8° corresponding to the typical diffractions of (100), (002), (101), (102), (110), (103) and (112) planes of wurtzite ZnO (see Figure S2).


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Figure 1. Schematic illustration of the synthetic process for ZnO@P(MEO2MAX-OEGMA100-X) NPs by SI-ARGET ATRP. For all the investigated ZnO@P(MEO2MAX-OEGMA100-X) NPs, the TEM images show high crystallinity and size uniformity (Figure 2a and the inset). The average particle size was measured to be of 5-6 nm (inset of figure 2a). In Figure 2, bright and dark field TEM micrographs have been selected to show the homogeneous dispersion of ZnO@P(MEO2MA60-OEGMA40) NPs. At high magnification (inset Figure 2c), the NPs were found to exhibit a spherical crystalline shape and an amorphous phase around the crystalline core could be observed (Figure 2c). This amorphous phase confirmed the presence of a polymer layer. The interplanar spacing was found to be 0.25 nm, which is in good agreement with the (101) plane of ZnO wurtzite structure.


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a

b

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c

Figure 2. a) Low magnification TEM micrographs of ZnO@P(MEO2MA80-OEGMA20) and the corresponding Gaussian size distribution (inset of Figure 2a), b) Dark field HR-TEM images of ZnO@P(MEO2MA60-OEGMA40) NPs and c) Dark field image of ZnO@P(MEO2MA60-OEGMA40) NPs with a high magnification image showing the interplanar spacing.

The UV-visible absorption spectra of ZnO@oleate, ZnO@Ph-Cl and ZnO@P(MEO2MA60OEGMA40) NPs exhibit a blue-shift of their excitonic peak (λ = 345 nm for ZnO@oleate, ZnO@Ph-Cl and λ = 340 nm for ZnO@P(MEO2MA60-OEGMA40)) compared to bulk ZnO (375 nm) due to their small size (Figure 3a-c). The relationship between the absorption coefficient α and the bandgap Eg of a direct bandgap semiconductor is given by the Tauc plot (αhν)2 = A(hν – Eg) where hν is the photon energy and A a constant. Eg values, determined by plotting (αhν)2 vs hν and considering the tangent of the linear section of the absorption band edge with the x axis, are 3.41, 3.27 and 3.28 eV for ZnO@oleate, ZnO@Ph-Cl and ZnO@P(MEO2MA60-OEGMA40), respectively (Figure S3). These values are higher than Eg of bulk ZnO (3.3 eV) due to the confinement of photogenerated charge carriers in ZnO QDs. The photoluminescence (PL) emission spectra of these QDs have been found to show a single broad band emission centered at 532 nm (2.33 eV) (Figure 3a-c). The large Stokes shift of the PL emission band relative to Eg


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(more than 1.0 eV) and its high full-width at half-maximum (128 nm, 0.54 eV) are typical of electron-hole radiative recombination involving charge traps at the surface of ZnO QDs (oxygen vacancies V0* and other defects) with energy levels located within the bandgap with holes in the valence band.12,16,33 The PL quantum yield (QY) of ZnO QDs is linked to the concentration of these defects but also to the ligand anchored at their periphery and to the solvent. The PL QY of the native ZnO@oleate QDs dispersed in toluene is of 24%. The surface functionalization with the CMPETMS chlorosilane didn’t significantly affect the PL QY but a decrease to ca. 15% was observed after the growth of the P(MEO2MAX-OEGMA100-X) polymer at their surface and the subsequent transfer in aqueous phase. This decrease of PL QY is systematically observed after the transfer of ZnO nanocrystals in water, regardless of the ligand used.8 Figure 3a-d shows that aqueous dispersions of the ZnO@P(MEO2MAX-OEGMA100-X) NPs exhibit a bright yellow luminescence under UV light irradiation. The stable PL peak maximum at 532 nm and the fullwidth at half-maximum of the PL emission peak indicate that the surface and the defects involved in the visible emission of ZnO QDs were not affected by the copolymer composition and the grafting process.


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ZnO

b

ZnO@silane

Absorbance

a

c ZnO@P(MEO MA -OEGMA ) 2 60 40

300

400

500

PL Intensity (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

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600

Wavelength (nm)

d

e

f

g

Figure 3. Absorption and PL emission spectra of (a) ZnO@oleate QDs, (b) ZnO@Ph-Cl and OEGMA40) NPs. The bottom of the figure shows the core/shell NPs dispersed in PBS solution under UV light (d) ZnO@P(MEO2MA80-OEGMA20), (e) ZnO@P(MEO2MA75-OEGMA25), (f) ZnO@P(MEO2MA65-OEGMA35), (g) ZnO@P(MEO2MA60-OEGMA40)


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Thermo-responsive behavior of ZnO@P(MEO2MAX-OEGMA100-X) NPs. In water, the aqueous dispersed ZnO@P(MEO2MAX-OEGMA100-X) NPs behavior with temperature was monitored by DLS at a concentration of 0.1 mg.mL-1. For each sample, the temperature was increased from 20 to 70 °C (Figure 4). The ZnO@P(MEO2MA75-OEGMA25) NPs hydrodynamic diameter evolution was constant upon heating until 36 °C and then it started to increase gradually from 38 °C to reach a steady state from 45 to 70 °C, which is related to the aggregation of the NPs as discussed in previous works.15,37 The same behavior was witnessed for all the samples (Figure 4). The LCST could be tuned by changing the content of MOE2MA and OEGMA units in the copolymers as shown in previous works.22,23,37 As reported by Lutz et al., the increase of the OEGMA amount leads to the shift of the LCST through higher temperature.27

500 400

HD (nm)

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


ZnO@P(MEO2MA 60-OEGMA40) ZnO@P(MEO2MA 65-OEGMA35) ZnO@P(MEO2MA 75-OEGMA25) ZnO@P(MEO2MA 80-OEGMA20)

300

ZnO

ZnO

200

ZnO

ZnO

100 ZnO

ZnO

0 ZnO

ZnO

20

30

40

50

60

Temperature (°C)

Figure 4. Evolution of ZnO@P(MEO2MA75-OEGMA25) diameter with temperature; the drawing on the left illustrates the NPs in their dispersed state and the one on the right illustrates the aggregation of the NPs.


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Heating and cooling cycles demonstrate the complete reversibility of the responsive behaviour of the core/shell NPs (see Figure S4). The same experiments were conducted in culture media (RPMI). As expected, due to the RPMI composition (mixture of salts and proteins), a decrease of 4-6 °C of the LCST was observed for all the samples (Figure 5, Table 1). Nevertheless, in RPMI, the reversible behavior of the core/shell NPs is preserved when performing heating and cooling cycles as shown in Figure S5.

Figure 5. Evolution of ZnO@P(MEO2MA65-OEGMA35) NPs diameter showing a shift in the LCST in RPMI (1) and increase of the aggregate sizes (2).


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Table 1. LSCT of the investigated ZnO@P(MEO2MAX-OEGMA100-X) NPs. LCST in water

LCST in CM

(°C)

(°C)

ZnO@P(MEO2MA80-OEGMA20)

36

32


40

34


42

38


46

40

Core/shell NPs


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Cell viability. The results obtained from the cell viability MTT assay showed that ZnO@P(MEOxMA-OEGMA100-x) NPs, regardless of the copolymer composition, exhibit low toxicity up to 12.5 µg.mL-1 Zn concentration with a cell viability above 90 %. At higher ZnO concentration, the best biocompatibility was obtained with 20 and 25 % of OEGMA groups (90100 % and 70-80 % cell viability at 25 µg.mL-1 and 50 µg.mL-1, respectively) (Figure 6), suggesting that the number of OEGMA units in the copolymer shell has an unexpected influence on the cytotoxicity of the NPs (Figure 7). In addition, increasing the exposure time from 24 to 72 h did not induce cell viability loss in case of low OEGMA content whereas it decreased to some extent for the NPs composed of 35 and 40 % OEGMA groups (Figure 7). At 50 µg.mL-1, the viability of HT29 cells was 81.0 ± 10.0% for ZnO@P(MEO2MA80-OEGMA20) NPs whatever the incubation time but only 23.1 ± 5.3% and 8.8 ± 7.3% for ZnO@P(MEO2MA60-OEGMA40) NPs at 24 h and 72 h, respectively (Figure 6).

Figure 6. Viability of HT29 cells after 24 and 72 h of exposure to ZnO@P(MEO2MAXOEGMA100-X) NPs.


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Figure 7. Viability of HT29 cells after 24 and 72 h of exposure to ZnO@P(MEO2MAXOEGMA100-X) NPs at 50 µg.mL-1 Zn2+.

Cytotoxicity resulting from ZnO dissolution in culture medium and endosomes is mediated in cells by the generation of reactive oxygen species.38 Cai et al. showed that ZnO QDs incubated with human carcinoma A549 cells for 48 h exhibited about 70% cell viability at 10 µg.mL-1 Zn2+ concentration.39 A careful coverage of the ZnO surface should decrease the release of Zn2+ ions, especially in aqueous media. We recently show that increasing the thickness of the shell could ensure the stability in aqueous media of the ZnO QDs optical properties.15 However, the decrease of the MEO2MA/OEGMA ratio lead to smaller amount of copolymer grafted at the surface of the NPs due to the OEGMA encumbered structure.37 It is thus not surprizing that the release of Zn2+ from the surface of the NPs would be easier from the core/shell NPs where the shell is composed of the smaller amount of OEGMA.


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ZnO@P(MEO2MAX-OEGMA100-X) NPs loaded with DOX. The results showed that ZnO@P(MEO2MAX-OEGMA100-X)-DOX NPs exhibited an enhanced toxicity in HT29 cells (Figure 8). At Zn concentration of 25 µg.mL-1 for ZnO@P(MEO2MA65-OEGMA35) and 12.5 µg.mL-1 for ZnO@P(MEO2MA60-OEGMA40) NPs, the viability of HT29 cells could be assumed to be higher than 80 %, while it significantly dropped after 5 h of incubation at 37 °C with the corresponding DOX-NPs. As clearly seen, high the DOX concentration led to high toxicity of both ZnO@P(MEO2MA65-OEGMA35)-DOX and ZnO@P(MEO2MA60-OEGMA40)-DOX NPs.

Figure 8. Viability of HT29 cells after 24 or 5 h of exposure to a) ZnO@P(MEO2MA65OEGMA35) and ZnO@P(MEO2MA65-OEGMA35)-DOX NPs, and b) ZnO@P(MEO2MA60OEGMA40) and ZnO@P(MEO2MA60-OEGMA40)-DOX NPs, respectively.


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In agreement with the results of Cai et al.

39

with ZnO-PEG-DOX NPs, a DOX concentration-

dependent cytotoxicity has been demonstrated, nevertheless to a lesser extent than free DOX.

140

DOX ZnO@P(MEO2MA65-OEGMA35) ZnO@P(MEO2MA60-OEGMA40) ZnO@P(MEO2MA65-OEGMA35)-DOX ZnO@P(MEO2MA60-OEGMA40)-DOX

120

100

Cell viability (%)

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


80

60

40

20

0 0

1,3

2,5

5,6

10

20

DOX concentration (mg/ml)

Figure 9. Viability of HT29 cells after 5 h of exposure to free DOX, ZnO@P(MEO2MA65OEGMA35), ZnO@P(MEO2MA60-OEGMA40 ZnO@P(MEO2MA65-OEGMA35)-DOX and ZnO@P(MEO2MA60-OEGMA40)-DOX NPs.

Compared to free DOX, we observed that both NPs were more cytotoxic from 5.5 µg.mL-1 DOX with the most significant effect for ZnO@P(MEO2MA60-OEGMA40)-DOX NPs. This result was unexpected because the NPs, particularly those with the MEO2MA60-OEGMA40 copolymer, should retain DOX at 37 °C. In fact, it could be assumed that cell internalization of ZnO@P(MEO2MA60-OEGMA40) NPs was more efficient than for their counterparts and free DOX and thus higher quantity of DOX could be delivered over the incubation time. In addition, the cytotoxic effect of NPs alone cannot be ruled out (without DOX) at this concentration range (5.5-20 µg.mL-1 DOX corresponding to 25-100 µg.mL-1 Zn2+). One explanation for these results could be in part given by Xiong et al. who showed with ZnO@polymer-DOX NPs, an enhanced


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toxicity of DOX loaded in ZnO@polymer NPs with respect to free DOX in brain cancer cells.40 These authors suggested that polymeric ZnO NPs are engulfed continuously in cells by the endosomal pathway that finally decomposes the NPs in lysosomes to release high concentrations of DOX molecules (Figure 9).35 ZnO@P(MEO2MA65-OEGMA35) and ZnO@P(MEO2MA60-OEGMA40) NPs are both materials with LCST at 38 and 40 °C, respectively, higher than the temperature required for living cells in normal conditions. By subjecting the cells over a short period of time (5 h in our conditions) at 41 °C, it was expected that DOX release was increased to obtain higher cytotoxicity. Figure 10 represents the cell viability obtained when the cells were exposed for 5 h at 37 and 41°C with ZnO@P(MEO2MA65-OEGMA35)-DOX, ZnO@P(MEO2MA60-OEGMA40)-DOX NPs or free DOX at 2.6 µg.mL-1 DOX concentration (12.5 µg.mL-1 Zn2+). At this concentration, about 80% cell viability was maintained at 37 °C with the NPs alone or the free DOX whereas the cytotoxicity of DOX-NPs was moderate with respectively 69.9 ± 20.7 % and 67.4 ± 3.3 % cell viability.


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100 Free DOX ZnO@P(MEO2MA65-OEGMA35)-DOX ZnO@P(MEO2MA60-OEGMA40)-DOX

80

Cell viability (%)

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


60

40

20

0 41

37

Temperature (°C)

Figure 10. HT29 cell viability in presence of 2.6 µg.mL-1 free DOX, ZnO@P(MEO2MA65OEGMA35)-DOX and ZnO@P(MEO2MA60-OEGMA40)-DOX NPs at 37 or 41 °C for 5 h.

When increasing the temperature from 37 to 41 °C for 5 h, the loss of cell viability for the both types of DOX-NPs significantly increased and dropped to 36.4 ± 15.2% and 28.8 ± 19.7%. In the same conditions, DOX produced only 10% more cytotoxicity (from 78.6 ± 9.9% cell viability at 37 °C to 72.2± 2.4 % at 41°C). Figure 11 shows the impact of the temperature on the DOX cytotoxicity. In all cases (free DOX or DOX-NPs), increasing the temperature at 41 °C enhanced the DOX effect. Above 2.5 µg.mL-1 DOX, the cytotoxicity increased dramatically with both NPs whereas the free DOX remained by far less toxic with the viability of cells maintained above 40 %. From 5 µg.mL-1, DOX ZnO@P(MEO2MA60-OEGMA40)-DOX NPs at 41 °C for 5 h produced the almost complete death of HT29 cells (Figure 11). The decrease in cell viability from 37 to 41 °C could be attributed to the release of more DOX molecules due to the phase transition of the copolymer grafted on the surface of ZnO NPs.


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Cell viability (%)


100 75 50 25 0 100 75 50 25 0 100 75 50

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a

b

c

0

5

10

15

20 -1

DOX concentration (µg.mL )

Figure 11. HT29 cell viability after incubation with a) ZnO@P(MEO2MA65-OEGMA35)-DOX, b) ZnO@P(MEO2MA60-OEGMA40)-DOX NPs and c) free DOX at 37 and 41°C for 5 h.

CONCLUSIONS Core/shell ZnO@P(MEO2MAX-OEGMA100-X) NPs were synthesized via SI-ARGET ATRP to graft MEO2MAX-OEGMA100-X copolymers from the surface of ZnO QDs. These core/shell NPs were found to exhibit a thermo-responsive behavior that could be controlled by tuning the molar ratio of MEO2MA and OEGMA monomers. The thermo-responsive behavior denoted by a LCST showed lower values in CM. The presence of salts following the Hofmeister series caused the LCST of the NPs to decrease by 4 – 6 °C in CM. The cytotoxic tests towards human colon cancer HT29 cells of the core/shell QDs showed that those functional nanomaterials exhibited almost no


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toxicity at concentrations up to 12.5 µg.mL-1 while when loaded with DOX, a higher cytotoxicity and a decreased HT29 cancer cell viability in a short time were observed. This work paves a new way for the design of new tools towards cancer therapy and theragnostic. MATERIALS AND METHODS Materials All

the

chemical

reagents

were

purchased

from

Sigma-Aldrich,

except

((chloromethyl)phenylethyl)-trimethoxysilane (CMPETMS) (Gelest, ˃ 95%). All the reagents were used as received. Characterization techniques Dynamic light scattering (DLS) was performed at room temperature using a Malvern Zetasizer has instrument with a He-Ne laser (4 10-3 W) at a wavelength of 633 nm. The NPs aqueous solutions were filtered through Millipore membranes (0.2 µm pore size). The data were analyzed by the CONTIN method to obtain the hydrodynamic diameter and size distribution in each aqueous dispersion of NPs. Transmission Electron Microscopy (TEM) measurements were conducted by placing one drop of NPs solution on holey carbon grids34–36. Samples were studied using an ARM 200F instrument (JEOL). For the TGA measurements, a SETSYS Supersonic thermobalance (SETARAM) was used. The furnace is made up of a graphite element operating from room temperature up to 1600°C. The apparatus is controlled by software appointed Calisto. Dry samples of 30 mg were put in an alumina crucible with a volume of 30 µL. The samples were heated from room temperature to 600°C at a heating rate of 5°C/min under argon atmosphere.


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UV-visible absorption spectra were obtained with a Thermo Scientific Evolution 220 spectrophotometer, while PL spectra were measured on a Horiba Fluoromax-4 Jobin Yvon spectrofluorimeter. Photoluminescence (PL) spectra were spectrally corrected and PL quantum yields (QYs) were determined relative to Rhodamine 6G in ethanol (PL QY = 94%).

Synthetic Methods Synthesis of ZnO@P(MEO2MAX-OEGMA100-X) NPs Synthesis of hydrophobic ZnO@oleate QDs: Oleate-capped ZnO QDs were synthesized by using standard airless technique. In brief, anhydrous zinc acetate (220 mg, 1.2 mmol) was dissolved in 20 mL of ethanol at 50 °C. When the solution became clear, 70 µL (0.22 mmol) of oleic acid were added, the mixture was stirred for 5 min and the temperature was increased to reflux. In a separate flask, 360 mg (1.99 mmol) of tetramethyl ammonium hydroxide pentahydrate (TMAH) were dissolved in 5 mL of hot ethanol and injected quickly to the reaction flask. After 2 min, the temperature was decreased to 0 °C by using an ice bath and addition of 50 mL of ethanol (or 25 mL EtOH + 25 mL MeOH). Then the QDs were separated with centrifugation (4000 rpm for 30 min) and washed two times with ethanol.

Silanization of ZnO QDs:

a)

First step: ZnO@oleate QDs were redispersed in 10 mL of toluene and

((chloromethyl)phenylethyl)trimethoxysilane (CMPETMS) (0.2 mmol, V = 49.1 µL) was injected to the reaction mixture and stirred for 2 min. Next, 2 mL of TMAH solution (108.75 mg in 6 mL of ethanol) were added and the mixture stirred for 15 min in reflux. The mixture was cooled with a water bath and QDs were separated by centrifugation and washed two times with EtOH.


22

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b)

Second step: QDs were redispersed in 10 mL of toluene and 4 mL TMAH solution were

injected. The reaction was conducted for 30 min in reflux. After cooling, QDs were separated by centrifugation and washed two times with EtOH.

Synthesis of ZnO QDs coated with the P(MEO2MAX-OEGMA100-X): The growths of the P(MEO2MAX-OEGMA100-X) (X/100-X = 100/0, 80/20 or 60/40) were initiated in 10 mL of a mixture of N,N-dimethylformamide/dimethyl sulfoxide (DMF/DMSO, 10/90, v/v) using CuCl2/TPMA (tris(2-pyridylmethyl) amine) in the presence of hydrazine. In a 30 mL Schlenk reactor, 50 mg of silanized QDs were dispersed under stirring in a DMF/DMSO mixture (10/90, v/v). Next, 17.7 mmol of a mixture of MEO2MA/OEGMA at different ratio (four different ratios MEO2MAX-OEGMA100-X (%) were used: 80/20, 75/25, 65/35 and 60/40 which led to ZnO@P(MEO2MAX-OEGMA100-X) QDs and 200 µL of stock solution (in DMSO) of CuCl2/TPMA (0.884 µmol, 4.3 µmol) were added. The reaction mixture was degassed by three freeze-pump-thaw cycles. The reaction was conducted at room temperature. When the media was completely homogeneous, 250 μL of a solution of hydrazine in DMSO (7.1 mg.mL-1) was added. The reaction was conducted during 2 h for each sample. The polymerization mixtures were poured into hot Milli-Q water to precipitate the insoluble components. The solution was then centrifuged (4000 rpm for 30 min ) three times using hot Milli-Q water.

DOX Release measurements The DOX was encapsulated in ZnO@P(MEO2MAX-OEGMA100-X) NPs by mixing 1 mL of 2.5 mg.mL-1 of the NPs with 5 mL of 1 mg.mL-1 of DOX solutions. The suspension was left under stirring for 24 h at room temperature. Afterwards the solution was subjected to a 24 h dialysis at room temperature to separate the NPs/DOX system from the free DOX (until no DOX could be


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detected by UV-visible spectroscopy in the dialysis bath). The NPs/DOX were separated from the solution by ultracentrifugation and were re-dispersed in 5 mL of culture media (CM).

Biological Characterization Cytotoxicity tests : The colorimetric MTT test based on the cell metabolic reduction of the tetrazolium

dye

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide)

in

formazan crystals was used to analyze the cytotoxic effect of ZnO@P(MEO2MAX-OEGMA100-X) NPs on human colon adenocarcinoma HT29 cells. The procedure was as follows:

Cell culture : Three days before adding the NPs, HT29 cells were trypsinized and recovered in complete RPMI containing 9 % fetal calf serum and 5 % glutamine 200 mM. After counting, 200 µL of either 105 cells.mL-1 or 2.5 x 104 cells.mL-1 were seeded in 96-well plate and received increasing doses of NPs for respectively 24 or 72 h at 37 °C in 95 % humidified air with 5 % CO2. Control cells did not receive NPs. MTT tests for cell viability test : After incubation, cells were washed twice with RPMI and 150 µL complete medium were added before 50 µL of MTT solution (2.5 mg.mL-1 phosphatebuffered solution (PBS)). The plates were then incubated for 2 h. After removing the contents of the wells, 100 µL of pure DMSO was added to solubilize the formazan crystals. The absorbance of each well was then read at 540 nm with the MSC multi scan plate reader.

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ACKNOWLEDGMENT: The financial support was given by the Centre National de Recherche Scientifique (CNRS) and the Lorraine University.

Supporting Information Available: [Description of the information]. This material is available free of charge via the Internet at http://pubs.acs.org.


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Dox

37°C 41°C 120

100

Free DOX ZnO@P(MEO2MA65-OEGMA35)-DOX ZnO@P(MEO2MA60-OEGMA40)-DOX

Cell viability (%)

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

Page 28 of 30

80

60

40

20

0 41

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Temperature (°C)

Fluorescent and aqueous dispersible core/shell nanoparticles and their cytotoxicity towards cancer cells.


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SYNOPSIS TOC A Facile Approach for the Doxorubicine Delivery in Cancer Cells by Responsive and Fluorescent Core/shell Quantum Dots. Enaam Jamal Al Dine, Sophie Marchal4, Raphaël Schneider, Jaafar Ghanbaja, Thibault Roques-Carmes, Tayssir Hamieh, Joumana Toufaily, Eric Gaffet1 and Halima Alem* *Halima Alem mail: [email protected]

120

100

Free DOX ZnO@P(MEO2MA65-OEGMA35)-DOX ZnO@P(MEO2MA60-OEGMA40)-DOX

Cell viability (%)

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


80

60

40

20

0 41

37

Temperature (°C)

Fluorescent and aqueous dispersible core/shell nanoparticles and their cytotoxicity towards cancer cells.




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A Facile Approach for Doxorubicine Delivery in Cancer Cells by

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