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Synthesis of Hydrophilic CuInS2/ZnS Quantum Dots with Different Polymeric Shells and Study of Their Cytotoxicity and Hemocompatibility Elena S. Speranskaya,*,† Chantal Sevrin,‡ Sarah De Saeger,§ Zeger Hens,∥,⊥ Irina Yu. Goryacheva,†,# and Christian Grandfils‡ †

Department of General and Inorganic Chemistry, Chemistry Institute, Saratov State University, Astrakhanskaya 83, 410012 Saratov, Russia ‡ Centre Interfacultaire des Biomatériaux (CEIB), University of Liège (ULg), Chemistry Institute, B6c, Allée du 6 aout, 11, B-4000 Liège (Sart-Tilman), Belgium § Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Gent, Belgium ∥ Department of Inorganic and Physical Chemistry Ghent University, Krijgslaan 281 S3, 9000 Gent, Belgium ⊥ Center for Nano- and Biophotonics, Ghent University, 9000 Gent, Belgium # Chemistry Institute, St. Petersburg State University, Universitetsky pr. 198504, St. Petersburg, Russia S Supporting Information *

ABSTRACT: In this work, there is a detailed description of the whole process of biocompatible CIS/ZnS QDs production. Special attention was paid to the stability of QDs against photooxidation. It was shown that Cu/In ratio greatly affected not only nanocrystals PLQYs but photostability as well. CIS/ ZnS QDs with Cu/In = 1:4 ratio showed high photostability under UV illumination both in toluene and aqueous solutions. Meanwhile, photoluminescence of CIS/ZnS QDs with Cu/In = 1:1 ratio was completely quenched after several hours under UV illumination, though their initial QY was as high as 40% with peak maximum at 740 nm. QDs were transferred to water by polymer encapsulation and were subsequently modified with polyethers Jeffamines, cheap analogues of PEG-derivatives. Three types of hydrophilic QDs differing in size, PEG content, and surface charge were obtained for further investigation and comparison of their cytotoxicity and hemocompatibility. It was shown that both leucocytes size distribution and coagulation activation change after introduction of polyethers into QDs polymeric shell, while red blood cells and platelets size distribution as well as hemolysis rate did not show any different results among different QDs and the polymer itself. All three types of QDs showed only slight cytotoxicity. Confocal microscopy proves penetration of hydrophilic CIS/ZnS QDs inside cells, so the low QDs cytotoxocity cannot be explained by low cellular uptake of the QDs and indicated low QDs toxicity in general. KEYWORDS: quantum dots, cadmium-free, amphiphilic polymers, Jeffamines, hemocompatibility, cytotoxicity

1. INTRODUCTION Quantum dots (QDs) are fluorescent colloidal nanoparticles possessing unique optical properties such as a tunable photoluminescence (PL), broad excitation spectra, and high photo- and chemical stability. Due to such properties, QDs are considered as prospective materials for biomedical diagnostics, light-emitting diodes, solar cells, and sensors.1,2 For imaging/ diagnostics applications in medicine, QDs should preferably have absorption and emission within the near-infrared (NIR) spectral window of 650−900 nm in order to minimize interferences of the light absorption/scattering and autofluorescence originating from the biological molecules.3−5 Cd-based NIR QDs such as CdTe, CdTeS, and CdTe/CdS were demonstrated as efficient fluorescent labels for different bioapplications such as in vivo imaging of tumor cells and targeted drug delivery.6−8 However, the well-known toxicity of © 2016 American Chemical Society

their components have remained a major obstacle for potential clinical use.9 Currently, much effort is devoted to the development of heavy-metal-free NIR nanocrystals such as silver chalcogenides5,10 and various I−III−VI type ternary compounds (CuInSe2, CuInS2, AgInS2).11−13 CuInS2-based QDs are the most studied I−III−VI2 semiconductor QDs so far. In comparison to CdSe-based QDs, CuInS2-based QDs have longer photoluminescence lifetime and can emit in the NIR region that is the most important for in vivo imaging. Typically, CuInS2 nanocrystals exhibit relatively broad emission spectra with full width half maximum (fwhm) on the order of 100−125 nm due to defect-related mechanism of photoReceived: November 20, 2015 Accepted: March 10, 2016 Published: March 10, 2016 7613

DOI: 10.1021/acsami.5b11258 ACS Appl. Mater. Interfaces 2016, 8, 7613−7622

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ACS Applied Materials & Interfaces luminescence.14 Such broad spectra make them inappropriate for use as labels in multicolour labeling or assays due to spectra overlapping; on the other hand, with such broad spectra, QDs can be excited with wavelengths close to their emission maximum, and only the longer wavelength part of the spectrum can be detected, which can be useful for in vivo applications. Lately, hydrophobic CuInS2-based QDs have reached the photoluminescence quantum yields comparable to that of the Cd-based QDs, though the results on CIS-QDs photostability are still scarce. For the bioapplications, especially for long-term tracing, not only high initial PLQY matters, but sufficient photostability is required. On the basis of results published before, it can be concluded that both CIS structure and ZnS passivating shell significantly influence QDs photostability in organic solutions.13,15,16 To the best of our knowledge, the photostability in aqueous solutions was studied only in one work,17 where it was surprisingly shown that hydrophilic CIS/ ZnS QDs covered with CTAB showed better photostability than the initial hydrophobic QDs. For in vivo applications, QDs must be demonstrated to be safe for humans, which means that they should at least be noncytotoxic, hemocompatible, and stable in the biological medium. 3,18,19 The cytotoxicity of QDs is frequently determined; particularly, it was shown that heavy-metal-free QDs such as CuInS2 and Ag2S are much less cytotoxic.9,13 Meanwhile, the mechanism of interaction of nanoparticles with blood is not well understood, although it is clear that this biological medium is sensitive to many factors including size, shape, nature, and composition of nanoparticles.20 Upon dilution in the bloodstream, nanoparticles can activate toxicological reactions and trigger several biological cascades..5,20 Thus, establishing the hemocompatibility of nanoparticles is critical in determining the safe and successful outcome of any further clinical trials. Only a few studies of nanoparticles hemocompatibility have been published so far,5,21,22 but their growing number reflects the clinical relevance of such research. Concerning CuInS2-based QDs, to the best of our knowledge, only hemolytic capacity of CuInS2/ ZnS QDs encapsulated with lipid micelles was studied.23 Here, we demonstrate the whole process of biocompatible CIS/ZnS QDs production. Special attention was paid for the stability of QDs against photooxidation. Particularly, it was shown that the Cu/In ratio greatly affected not only nanocrystals PLQYs but on photostability as well. CIS/ZnS QDs with a Cu/In ratio of 1:4 showed high photostability under UV illumination both in toluene and aqueous solutions. Meanwhile, photoluminescence of CIS/ZnS QDs with a Cu/In ratio of 1:1 was completely quenched after several hours under UV illumination, though their initial QY was as high as 40% with peak maximum at 740 nm. The cytotoxicity and hemocompatibility of the synthesized CIS/ZnS QDs as well as of the amphiphilic polymer used for QDs hydrophilization were studied. It was shown that the observed slight cytotoxicity of the QDs and the polymer was not a result of their low cellular uptake due to data from confocal microscopy indicates that the hydrophilic CIS/ZnS QDs successfully entered cells. The hemocompatibility studies were done for CIS/ZnS QDs with three polymeric shells with different polyethylene glycol content and for the amphiphilic polymer itself to investigate which QDs parameters could be crucial for QDs hemocompatibility. One important conclusion was the absence of difference in hemocompatibility of QDs covered with the polymer and the polymer itself. It means that QDs did not lose their hydrophilic

shell in the used conditions and that the hemocompatibility is highly dependent on QDs surface. It was shown that both leucocytes size distribution and coagulation activation change after introduction of polyethers into QDs polymeric shell, while red blood cells and platelets size distribution, as well as hemolysis rate, did not show any different results among different QDs and the polymer itself. Those in vitro assays were made as prescreening tests to assess the hemocompatibility of the QDs. To the best of our knowledge this is the first work investigating the hemocompatibility of CuInS2-based QDs in such detail.

2. EXPERIMENTAL SECTION 2.1. Materials. 1-Dodecanethiol (DDT, ≥ 98%), octadecene (ODE, 90%), copper(I) iodide (CuI, ≥ 99.5%), zinc stearate (Zn(St)2, purum), poly(maleic anhydride-alt-1-octadecene) (PMAO, M ∼ 30 000−50 000), rhodamine 6G and dimethylformamide (DMF) were purchased from Sigma-Aldrich (Bornem, Belgium). Indium(III) acetate (In(Ac)3, 99.99%) and oleic acid (OA, 90%), ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDC) and phosphate buffered saline (PBS) tablets were purchased from Alfa Aesar (GmbH & Co. KG, Germany). Jeffamine M1000 (∼1000 g/mol), Jeffamine ED2003 (∼2000 g/mol) were kindly provided by Huntsman (Belgium). All organic solvents (ethanol, toluene, and chloroform) were purchased from Sigma-Aldrich (Bornem, Belgium) and were used without further purification. Trypan blue from Invitrogen, Life Technologies (Carlsbad, CA). MEM, 1X, FBS and Trypsin were purchased from Gibco (Grand Island, NY). The Pierce Protein Concentrators (9K, 20 mL) were purchased from Thermo Scientific (Rockford, IL). 2.2. CIS/ZnS QDs Synthesis. CIS cores (Cu/In = 1:4 and 1:1 molar ratio) and CIS/ZnS QDs were synthesized as it was described in our previous work.24 The detailed description can be found in the Supporting Information (sections S1 and S2). 2.3. Hydrophilization of QDs. Hydrophobic QDs were transferred to an aqueous solution by encapsulation with an amphiphilic polymer (Figure 1). The synthesis of the amphiphilic polymer

Figure 1. Scheme of hydrophobic QDs encapsulation with PMAO− Jeffamine M1000 polymer (QDs 1) and subsequent conjugation of the obtained QDs with Jeffamine M1000 (QDs 2) and Jeffamine ED-2003 (QDs 3) using EDC chemistry. (poly(maleic anhydride-alt-1-octadecene), Jeffamine M1000 (PMAO− Jeffamine M1000), and QDs water-solubilization process were performed as described in ref 24, and the detailed description is included in the Supporting Information (section S3). 2.4. Purification of Hydrophilic QDs. QDs were transferred to aqueous solutions using a large excess of the polymer. In this work, sucrose gradient ultracentrifugation was used to remove free polymeric micelles. Briefly, 3.2 mL of QDs solution was placed on the top of two layers of sucrose solutions composed of 1 mL of 50% sucrose solution and 0.8 mL of 10% sucrose solution carefully and sequentially placed in 5 mL ultracentrifuge tubes. Ultracentrifugation was conducted at 275 000g for 1.5 h at 15 °C using a Beckman L8-70 M Ultracentrifuge equipped with a SW 55 Ti rotor. After this fractionation step, the layer of QDs was taken out and purified from sucrose by centrifugation using Pierce Protein Concentrators (9K MWCO). To monitor the sedimentation of the PMAO−Jeffamine polymer during centrifugation, 7614


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Figure 2. Optical properties of CuInS2 QDs prepared using (A−C) Cu/In = 1:1 and (D−F) Cu/In = 1:4 molar ratios. Absorption (A, D) and photoluminescence spectra (B, E) of CuInS2 aliquots at 220 °C after the indicated reaction time. The intensities of the absorption and emission peaks were normalized. (C, F) Quantum yield of the aliquots is presented as a function of reaction time. we conduced its hydrophilization and ultracentrifugation in exactly the same conditions used for the QD. Thanks to the blue fluorescence signal given by the polymer and red fluorescence from QDs, the polymer and QDs were easily located in the ultracentrifuge tubes under UV radiation. After purification, the QDs were buffer exchanged with phosphate buffered saline (PBS) solution using Pierce Protein Concentrators (9K MWCO). 2.5. Binding Hydrophilic QDs with Jeffamine Polymers. PEGbased Jeffamine (Jeffamine M1000 or Jeffamine ED 2003) was dissolved in PBS solution of QDs covered with PMAO−Jeffamine M1000 polymer (Figure 1) with the concentration of QDs ∼ 0.8 μM (OD 500 nm (QDs PBS solution): 0.68); Jeffamine/QDs = 15000:1 molar ratio. After that EDC dissolved extemporaneously in PBS (0.39 M) was added to the QD−Jeffamine mixture; EDC/QDs adopting a 320 000:1 molar ratio, respectively. The solution was stirred for 2 h at 37 °C and then the excess of EDC and Jeffamine were removed by ultrafiltration with Pierce Protein Concentrators (9K MWCO). 2.6. Determination of Cytotoxicity. The in vitro cytotoxicity was measured using trypan blue staining. MA-104 (embryonic rhesus monkey kidney) cells were cultured in minimum essential medium (MEM, 1X, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY). These cells were seeded into 24-well cell-culture plate at a cell density of 6 × 104/well in a 95% (v/v) humidified atmosphere and 5% (v/v) CO2 at 37 °C (MCO-18AC, Sanyo, Japan). After their overnight cultivation, the cells were incubated with hydrophilic CuInS2/ZnS QDs or with PMAO− Jeffamine M1000 polymer dispersed in the culture medium in order to achieve final concentrations of 50, 100, 150, and 200 μg/mL of QDs or PMAO−Jeffamine M1000 polymer. The medium without addition of QDs or the polymer was added to the negative control group. The cells were incubated overnight at 37 °C under 5% CO2. Cells were observed under inverted microscopy Biolam P1 (LOMO, Saint Petersburg, Russia) to monitor cell morphology, adhesion and proliferative activity. Cell viability was quantified using an automated live cells counter Countess Automated (Invitrogen Life Technologies, USA). 2.7. Cell Culture and Observation of Intracellular Location of QDs in HEP2 Carcinoma Cells. Human HEP2 carcinoma cells were cultured in Minimum Essential Medium (MEM, 1X, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY). These cells were seeded into cell-culture plate at a cell density of 10 × 105 in a 95% (v/v) humidified atmosphere and 5% (v/v) CO2 at 37 °C (MCO-18AC, Sanyo, Japan).

After their overnight cultivation, the cells were incubated with hydrophilic CuInS2/ZnS QDs dispersed in the culture medium in order to achieve final concentrations 100 μg/mL of QDs. The cells were incubated overnight at 37 °C under 5% CO2. After incubation cells were washed three times with PBS and then fixed with 4% formaldehyde. 2.8. Hemocompatibility Studies. Hemocompatibility tests were performed with hydrophilic CuInS2/ZnS QDs with 3 different polymeric shell compositions (structures QDs 1, QDs 2, QDs 3 in Figure 1) and with the polymer PMAO−Jeffamine M1000 which was used for the initial QDs water−solubilization process (Figure 1). QDs and the polymer dispersed in PBS were diluted in whole blood in order to achieve a final nanoparticles concentrations of 100, 10, and 1 μg/mL. The hemocompatibility of QDs and the polymer was evaluated by studying hemolysis, the morphology and size distribution of blood cells, and coagulation activation, both through the extrinsic pathway (prothrombin time, PT assay) and the intrinsic pathway (activated partial thromboplastin time, APTT assay). The experiments were done as described in our previous work5 investigating Ag2S QDs hemocompatibility, and the detailed description is also included in the Supporting Information (S4 section). 2.9. Characterization of QDs. UV−visible absorption spectra of CIS core and CIS/ZnS core/shell QDs were recorded on a PerkinElmer Lambda 2 UV/vis Spectrometer. PL emission spectra were collected by a fluorescence spectrometer FS920 (Edinburg Instruments, UK). The PLQY of the QDs dispersed in toluene or aqueous solutions was measured comparing their integrated emissions with rhodamine 6G (QY of 94%, in ethanol) under excitation at 488 nm. All emission spectra were corrected for the detector sensitivity. The optical density of the samples was around 0.05. Photostability tests of the samples was conducted under light irradiation (Consort E2107 lamp: λ(excitation) = 365 nm, power 610 μW/cm2). Fourier transform infrared (FTIR) spectra were collected adopting film prepared by evaporating sample solutions and using a Nicolet 6700 spectrometer. Ultracentrifugation was performed on a Beckman L8− 70 M Ultracentrifuge equipped with a SW 55 Ti rotor. Gel electrophoresis analysis was performed on a Bio-Rad system. The samples were run on 1% agarose gel. The agarose gel was prepared by dissolving the agarose powder in Tris-borate-EDTA (TBE) buffer. The electrophoretic run was carried out for 60 min at 100 V. Afterward the samples were checked under UV illumination (365 nm). 7615


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Figure 3. Absorption, photoluminescence spectra and QY of CuInS2 and CuInS2/ZnS QDs after successive addition of the Zn precursor; (A−C) Cu/In = 1:1 and (D−F) Cu/In = 1:4 molar ratio.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Hydrophobic CuInS2/ZnS QDs. Hydrophobic CIS/ZnS QDs were prepared by “one-pot” noninjection synthesis in organic solvent.19,25 CIS cores were prepared using Cu/In molar ratios of 1:1 and 1:4. As shown in Figure 2, absorption and emission peaks only slightly shifted during synthesis at the given Cu/In ratio, although the QY significantly increased with time. Only the last aliquot (taken at 8 min) of QDs with Cu/In 1:1 molar ratio showed a much lower fluorescence and a considerably wider peak. Sediments were noticed rapidly after the onset of the reaction in spite of it running under continuous agitation. Aliquots containing the nonfluorescent red-colored precipitate of QDs were withdrawn 6 min after initiation of the reaction. In a former study,4 QDs aggregation has been attributed to either the complete decomposition of DDT or the presence of fatty acids. However, a supplementation of an extra amount of DDT during the synthesis or the elimination of OA during the CIS core synthesis did not improve the colloid stability of QD in this reaction medium. Whatever the conditions used after 5−6 min the reaction mixture contained aggregated QDs as well. In addition, fluorescence wavelengths were much shorter and the PLQY of the cores was lower compared to nanocrystals synthesized with the use of OA (Figure S1). So, OA seemed to be one of the essential factors to obtain high-quality CIS QDs by this method. The use of a Cu/In = 1:1 molar ratio resulted in a fluorescence at longer wavelengths compared to QDs prepared with a Cu/In = 1:4 ratio and a significantly lower PLQY. This result is in accordance to data published earlier where the Cu/ In ratio was demonstrated to be a key factor in adjusting fluorescence wavelength and PLQY. The decrease of Cu concentration leads to the increase of copper vacancies which are one of the possible acceptors in “donor−acceptor pair” recombination in CuInS2 nanocrystals.13,19,26 To the best of our knowledge, the reported QY for CIS (Cu/ In = 1:4) cores (up to 32%) in this manuscript is the highest published so far.13 For further coating with ZnS shell, the

synthesis of CIS QDs was stopped after 8 min (in case of Cu/ In = 1:4) and after 4 min (in case of Cu/In = 1:1). The obtained QDs had fluorescence maximum at 696 nm (QY ∼ 28%) and at 790 nm (QY ∼ 6%) using Cu/In = 1:4 and Cu/In = 1:1 molar ratios, respectively. As the most appropriate shell material ZnS shell was used to passivate CIS surface states.26,27 In this work Zn precursor was added gradually, in five cycles. In each injection of Zn precursor, the molar amount of Zn was equal to the initial amount of In. The excess of DDT in the solution acted as a source of sulfur. The fluorescence maximum considerably shifted to the blue wavelength region after the first addition of Zn precursor and showed only little further shifts after the additional Zn precursor injections (Figure 3). The absorption shoulder of QDs shifted in a lesser extent to shorter wavelengths compared to the fluorescence spectrum. In our previous study28 it was shown that in these reaction conditions, two processes apparently occur during Zn precursor addition: cation exchange resulting in the peaks blue shifts and growth of a ZnS shell, where the former mainly takes place during the first shell growth cycle. As highlighted in Figure 3(C,F) the PLQY steadily increases to reach a value as high as ∼40% for CIS/ZnS with Cu/In = 1:1 and ∼80% for CIS/ZnS with Cu/In = 1:4 molar ratio after 5 additions of Zn precursor. Subsequent additions of Zn precursor did not lead to a further increase of PLQY (Figure 3). A higher QY of the initial CIS cores resulted in a higher QY of the core−shell nanocrystals (Table 1), indicating that the synthesis of highly luminescent cores is a key factor to produce core−shell QDs with good QY. The uncovered cores were initially bright (their QY was measured 1 day after synthesis), but they dramatically lost their brightness (from 24 to 0.01% after 2 weeks) even after storage at 4 °C in the darkness. In contrast, core−shell QDs showed much better photostability during storage in a refrigerator and under constant UV illumination (Figure 4). However, a high difference in the photostability of CIS/ZnS prepared with 1:1 and1:4 Cu/In molar ratio was observed. Core−shell QDs prepared with a Cu/In = 1:1 molar ratio showed a very low photostability in time; even storing in darkness at 4 °C resulted 7616


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the initial hydrophobic ligands are not removed from the QDs surface. The most frequently used amphiphilic polymer for QDs hydrophilization is PMAO due to its commercially availability and low cost. QDs covered with unmodified PMAO are stabilized by electrostatic repulsion, so they are stable only at basic pH. In our previous work,28 it was shown that simple modification of PMAO with Jeffamines can sufficiently increase QDs stability in different buffers. Jeffamines are polyethers and can serve as cheap analogues of expensive PEG-derivatives. The PMAO polymer was modified by reaction between the maleic anhydride groups of PMAO and amine groups of Jeffamine M1000.24 It can be anticipated that the hydrocarbon chains of this copolymer should interact by hydrophobic interactions with the initial ligands covering the QDs, while its hydrophilic groups (carboxylic and PEG) should provide colloidal stability in aqueous solutions (Figure 1). The procedure consists of mixing the QDs and the polymer chloroform solution with an aqueous basic solution, followed by the subsequent slow evaporation of chloroform under rotor evaporator Figure 5A

Table 1. PLQY of CuInS2 or CIZ/ZnS QDs (Cu/In = 1:4 Molar Ratio) sample CuInS2 (3 min growth) CIS/ZnS CuInS2 (8 min growth) CIS/ZnS

ligands on QDs surface

solvent

quantum yield (%)

DDT

toluene

14

DDT, stearate DDT

toluene toluene

63 28

DDT, stearate

toluene

80

Figure 4. (A) Fluorescence spectra of CIS/ZnS QDs (Cu/In = 1:4) (a) 2 days after the synthesis; (b) after storage in a refrigerator for 6 months; and after (c) 1 and (d) 2 days under constant UV illumination (365 nm). (B) CuInS2/ZnS QDs (Cu/In = 1:1 molar ratio) (e) 2 days after the synthesis and (f) after storage in a refrigerator for 3 months.

in dramatic changes in PLQY and in blue-shifted emission spectrum (Figure 4B). Under UV illumination (365 nm), they were completely quenched after several hours. On the contrary, CIS/ZnS QDs prepared using Cu/In = 1:4 ratio did not show any changes in fluorescence spectra and in QY after 6 months in a refrigerator at 4 °C. They retained considerably good QY after constant illumination under UV lamp (365 nm) during 2 days (Figure 4A). These data about photostability showed that the estimation of stability against photo-oxidation is as important as the determination of initial PLQY in order to make a decision about QDs quality and suitability for further use. In our case, the initial PLQY of both CIS/ZnS QDs are among the highest ones published so far for such wavelengths. The low photostability can go unnoticed if the QDs are used for some applications just after their synthesis, but it is crucial for such applications as long-term visualization and for storage. Thereby, despite the fact that CIS/ZnS QDs (Cu/In 1:1) with fluorescence at 740 nm are more preferable as labels for bioapplication, their low photostability made their further use impossible. For this reason, subsequent evaluations have been conducted with CIS/ZnS QDs (Cu/In = 1:4) characterized by a fluorescence peak at 650 nm. The initial dramatic increase in QDs PLQYs after ZnS shelling for both CIS cores (with 1:1 and 1:4 Cu/In molar ratios) indicated the successful formation of ZnS layer passivating the surface defects. As we suggest, the low photostability indicates that the ZnS layer on CIS QDs with Cu/In = 1:1 ratio is not thick or uniform enough, so it cannot serve as a good isolating layer for CIS cores for substantial amount of time. 3.2. Hydrophilization of CuInS2/ZnS QDs. To transfer CIS-based QDs to aqueous solutions, we used two common approaches: encapsulation with amphiphilic polymers24,28,29 or micelles9,23 and ligand exchange.12,17 As a rule, the first approach leads to higher PLQY after hydrophilization due to

Figure 5. (A) Fluorescence and absorbance spectra of QDs in toluene and QDs transferred to aqueous solution. (B) Change of fluorescence and absorbance spectra of hydrophilic QDs after constant UV illumination (365 nm).

displays the absorbance and emission spectra of the initial toluene QDs solution and the aqueous solution of QDs covered with PMAO−Jeffamine M1000 polymer. Compared to hydrophobic QDs, the absorbance spectra of the hydrophilic QDs did not shift while a 25 nm red shift in the PL peak position was observed. The PLQY of the hydrophilic QDs was considerably lower than of the hydrophobic QDs but at 50%, it remained very high for water-soluble CuInS2/ZnS nanocrystals. The PL decay traces of the QDs before and after water-solubilization (Figure S2) show that hydrophilic QDs have a higher intensity of the fast component and shorter average PL lifetime compared to the initial hydrophobic QDs. According to literature, the short component (tens of nanoseconds) is due to nonradiative recombination induced by surface defects.14,30 It thus appears that hydrophilic QDs have an increased surface defect concentration despite the fact that the initial hydrophobic ligands still stayed on the QD surface. The hydrophilic QDs were purified from the excess of the polymer by sucrose gradient ultracentrifugation (5−20% mass of sucrose). Although demonstrated efficient in our previous study,28 this approach only yields a relatively small amount of QDs purified per tube (0.4 mL for a 5 mL tube). As shown in Figure 6, the adoption of a discontinuous sucrose gradient resulted in the accumulation of purified QDs in the 10% sucrose layer while the excess of polymer still stayed in the aqueous layer after ultracentrifugation and did not percolate to the 10% sucrose layer (Figure 6A). The 10% sucrose solution was optimal for 7617


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Figure 7. Gel migration of CuInS2/ZnS QDs: (A) − QDs modified with Jeffamine M1000 and Jeffamine ED 2003 using different EDC/ QDs ratios; QDs modified with Jeffamine M1000 using different Jeffamine/QDs ratios: EDC/QDs = 160 000 (160k) (B), EDC/QDs = 320 000 (320k) (C). The photos were taken under excitation by a UV lamp.

Figure 6. Distribution of fluorescence of QDs after their ultracentrifugation on a discontinuous sucrose gradient exposed under UV lamp: (A) PMAO−Jeffamine M1000 polymer and (B) QDs. The polymer (blue under UV) did not penetrate a 10% sucrose layer, while most of QDs (red under UV) were concentrated in a 10% sucrose layer.

groups. Figures 7, S3 show the crucial role of the EDC/QDs ratio in the degree of polymer binding with QDs. In addition, the influence of Jeffamine/QDs molar ration on conjugation degree was shown. From Figure 7B it is clear that using EDC/ QDs = 160 000 molar ratio even high Jeffamine excess could not increase the conjugation degree, while using EDC/QDs = 160 000 molar ratio an increased Jeffamine amount led to the higher conjugation degree as it was shown in Figure 7C. Figure S4 represents the absorbance and PL spectra of the initial QDs covered with PMAO−Jeffamine M1000 (QDs 1) and QDs obtained after binding with Jeffamine M1000 (QDs 2) and Jeffamine ED 2003 (QDs 3) (EDC/QDs = 350 k, Jeffamine/ QDs = 20 k). The PLQY decreased after the modification (from 55 to 38 and 32% after binding with Jeffamine M1000 and ED 2003 respectively) and there were small blue-shifts of the emission peaks, whereas the absorption spectra hardly changed. QDs modified with additional Jeffamine M1000 (QDs 2) stayed at the start line and QDs bound with Jeffamine ED 2003 (QDs 3) moved toward the negative electrode (Figure 7) which proved that the QDs 2 were almost uncharged and QDs 3 were positively charged in the used buffer. Meanwhile, IR spectra of the QDs still showed the presence of carboxylic groups on the QDs which were left unreacted presumably due to dense layer of PEG chains (Figure S5). TEM images of the initial hydrophobic QDs and QDs after water-solubilization and modification are shown in Figure S6. It is clear that the sample of QDs 1 contains both single and multiple nanocrystals covered with the polymer. After modification with Jeffamine polymers, the QD samples contain even more particles that consist of several quantum dots embedded in the polymers. So, three types of hydrophilic QDs differing in size, PEG content, and surface charge were obtained for further investigation and comparison of their cytotoxicity and hemocompatibility. 3.4. In Vitro Cytotoxity. Toxicity of three different hydrophilic CuInS2/ZnS QDs (QDs 1−3, Figure 1) to MA104 (embryonic rhesus monkey kidney) cells was evaluated using trypan blue staining. In the 24 h incubation, MA-104 cells were only slightly affected by the presence of QDs, but overall, the QDs 1 (covered with PMAO−Jeffamine M1000 polymer without modification) showed better compatibility (Figure 8). It is interesting to note that there was no evident decrease in cell viability with increase of QDs concentration in the studied range. As shown in Figure 8, incubation of cells with the PMAO−Jeffamine M1000 polymer resulted in a slight cytotoxity despite the absence of toxic groups in its structure. The cell viability after incubation with QDs covered with the

QDs concentration: lower concentrations of the layer led to penetration of polymer in this layer, while QDs did not penetrate the layers with higher sucrose concentrations. The bottom layer consists of 50% sucrose and is used to prevent any accidental QD precipitation on the tube’s bottom; QDs have a density that is 50% that of sucrose, and they cannot percolate to this layer. This approach can be used for purification of large amount of QDs. Besides, preparation of the gradient is easy. The purified hydrophilic QDs stored in a refrigerator for 6 months did not show any decrease in fluorescence brightness. Under constant UV illumination during 2 days, their PLQY reduced, but they still had relatively high brightness (Figure 5B), which is crucial for long-term storage and such applications as in vivo visualization and tracking. It is important to note that the hydrophilic QDs which were not purified from the excess of the polymer were much less photostable. They completely lost their brightness after 1 week of storage at room temperature unprotected from daylight, or after only several hours when exposed to UV irradiation. 3.3. Modification of QDs Polymeric Shell. The carboxylic groups available on the surface of the hydrophilic QDs make for an easy starting point to further modify the QD surface. The QDs with three different polymeric shells were obtained for further investigation of polymeric shell influence on QDs cytotoxicity and hemocompatibility. QDs covered with PMAO−Jeffamine M1000 polymer were conjugated either with Jeffamine M1000 or Jeffamine ED 2003 in order to increase PEG content and obtain QDs different in surface charge (Figure 1). Conjugation with Jeffamine ED-2003 allows us to introduce simultaneously amino groups of potential interest for further functionalization (Figure 1). Both reactions were made through EDC classical chemistry.31 The efficiency of the conjugation process was monitored by gel-electrophoresis. The QDs coated with the Jeffamine-modified polymers were retarded in gel electrophoresis compared to particles coated with the initial polymer (Figure 7, and section S3). The actual shift of the conjugates on the gel was caused by changes in size and charge. The more Jeffamine attached to the polymer, the bigger the polymer-coated QDs and the more retarded their bands during gel-electrophoresis. The charge of particles became less negative in case of conjugation with Jeffamine M1000 and became positive in case of binding with Jeffamine ED 2003 due to partial protonation of the amine 7618


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Table 2. Percentage of Hemolysis and Hemostasis Activation (Quick and TCA) after Incubation of Quantum Dots and PMAO−Jeffamine M1000 Polymer at 37 °C in Whole Blood sample

concentration

Ctrl − Ctrl + QDs 1

0.49 ± 0.01 48.8 ± 0.01 100 μg/mL 10 μg/mL 1 μg/mL 100 μg/mL 10 μg/mL 1 μg/mL 100 μg/mL 10 μg/mL 1 μg/mL 100 μg/mL 10 μg/mL 1 μg/mL

QDs 2

Figure 8. Change in the cell viability of embryonic rhesus monkey kidney cells after 24 h incubation with QDs 1−3 and PMAO− Jeffamine M1000 polymer as determined by trypan blue staining.

QDs 3

polymer was not lower than the cell viability after incubation with the polymer itself, so it was concluded that the additional cytotoxicity caused by inorganic part of QDs was not observed. The low level of the QDs toxicity is not a result of low cellular uptake of the QDs. As it was shown with confocal microscopy, the hydrophilic CIS/ZnS QDs successfully entered the cells (Figure S7 and video, Supporting Information). 3.5. Blood Compatibility. Due to the large surface expressed by nanoparticles and knowing the reactivity of blood when in contact with foreign materials, the exposure of the QDs to blood could induce significant reactions of the humoral and cellular blood components. Hemolysis, activation or inhibition of the coagulation cascades, activation of the complement are among the most typical reactions observed when blood is incubated in vitro with foreign surfaces.5,20 The hemocompatibility studies were done for CIS/ZnS QDs with three polymeric shells with different polyethylene glycol content and surface charge and for the amphiphilic polymer itself to investigate which QDs parameters could be crucial for QDs hemocompatibility. The experiments were done according to general guidelines of ISO 10993. In practice, blood cell reactivity was controlled following hemolysis and any change in their morphology and size distributions. Humoral reactions were checked analyzing the coagulation activation through the extrinsic and intrinsic pathways (Table 2). The results of the hemolytic test attest that in the concentration of QDs assessed (up to 100 μg/mL) the hemolysis percentage did not exceed 0.3%. Therefore, according to the ASTM Standard Practice for Assessment of Hemolytic Properties of Materials, our nanomaterials can be considered as non-hemolytic (the threshold is fixed to 2%). The microscopic analysis of the blood smears (Figure S8) also supports the resistance of erythrocytes after their incubation with the different QDs. Indeed all micrographs of the blood cells at a magnification of 500× do not highlight any morphological changes of the red blood cells, the most prominent blood cell population. The other cell types, in particular platelets, which are well-known to be the most reactive against foreign surfaces, remain with a physiological appearance, that is, nonaggregated and with irregular size and shape. The absence of any cell reaction is also confirmed by the counting and analysis of the size distribution of the two main blood cell types (i.e., erythrocytes and platelets). Indeed, the comparison of their relative size distributions (Figure 9) clearly attests of their stability, an observation also given by their global cell count which are not affected. If these results support that the QDs, no more than the polymer, do not interact or promote cell aggregation, then a

polymer

hemolysis (%)a 100.0 0.23 0.19 0.17 0.24 0.16 0.23 0.30 0.30 0.30 0.28 0.28 0.31

± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.03 0.03 0.02 0.02 0.03 0.04 0.04 0.04 0.02 0.04

APTT/TCA activation (%)b

PT1/quick activation (%)

100.0 112.6 83.0 98.5 100.3 101.9 100.9 100.6 97.6 99.4 99.1 89.2 97.6 98.8

90.6 96.7 99.1 100.9 100.9 100.9 98.3 100.0 100.0 89.9 93.5 95.1

a

Hemolysis was calculated as the percent of free hemoglobin (released after contact with the QDs or polymer) in relation to the total blood hemoglobin. Ctrl +: blood incubated with saponin. Ctrl −: blood incubated with PBS. bQuick and TCA (total coagulation activity) hemostasis assays are reported in percentage of the clotting ability of the sample compared to the clotting ability of a standard human plasma normalized to 100. Ctrl +: blood incubated with kaolin. Ctrl −: blood incubated with PBS.

significant change is noticed in the size distribution of WBCs. Indeed, at the highest concentration of 100 μg/mL all QDs and the free polymer induce a left shift of the subpopulation of the largest leucocytes. Moreover, for QDs 2 and 3 the same trends remained even for the smallest 1 μg/mL concentration, while for the initial polymer as well as for QDs covered with this polymer (QDs 1) the size distribution was different from the control sample only at the highest concentration (Figure S9). Such evolution in size distribution of this population of leucocytes which should correspond to the polymorphonuclear leukocytes, mostly neutrophils, cannot be readily explained on the basis of these size changes only. More investigations would be needed indeed in order to analyze in more depth the reaction of this subcell population. But leucocytes are wellknown for their unique ability for particle phagocytosis, a process known to be followed by changes in cell morphology.32 The effect on hemostasis control was determined by coagulation assays, both through the extrinsic pathway (PT assay) and the intrinsic pathway (APTT assay). Although distinguished in clinical laboratory, it is worth mentioning that these two coagulation pathways are intrinsically linked. Tissue factor−factor VIIa complex initiating the extrinsic pathway is also capable of activation of factor IX of the intrinsic pathway. In turn, the intrinsic tenase complex influences the tissue factordependent pathway.33 Clot formation was determined after blood incubation with QDs and the polymer. Longer clotting times correspond to lower clotting ability expressed in percent to the standard plasma normalized to 100%. As shown in Table 2, neither the extrinsic nor the intrinsic pathway was affected in the presence of QDs 2 and QDs 3 samples. However, and interestingly enough, both pathways were inhibited at the highest concentrations of the polymer and QDs covered with this polymer (QDs 1). As reported for other types of nanoparticles, the inhibition of the intrinsic and extrinsic pathways of coagulation can be 7619


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Figure 9. Blood cell distribution in control samples (1, blood not incubated with PBS; 2, blood incubated with PBS) and in the samples incubated with QDs 1, QDs 2, QDs 3, and the polymer (at concentration 100 μg/mL).

explained by the nonspecific adsorption of the proteins involved in the coagulation cascades onto the surface of materials.34 So, the fact that QDs covered with polymers containing more PEG (QDs 2 and 3) did not show any influence on the coagulation is well in correlation with the steric repulsion against opsonisation expected from this polyether sequence.35,36 One important observation was the absence of difference in hemocompatibility of QDs covered with the polymer and the polymer itself. It means that QDs did not lose their hydrophilic shell in the used conditions and that the hemocompatibility is highly dependent on QDs surface.

Hemocompatibility studies have shown that both leucocytes size distribution and coagulation activation change after the introduction of polyethylene glycol into QDs polymeric shell, while red blood cell and platelet size distribution, as well as hemolysis rate, did not show any different results among modified QDs. Also, the results in hemocompatibility experiments for QDs covered with the amphiphilic polymer and for the polymer itself were very similar. It demonstrates that QDs did not lose their hydrophilic shell in the used conditions and that the hemocompatibility is highly dependent on QDs surface. On the basis of the obtained results, the synthesized CuInS2based polymer-covered QDs are therefore very promising as fluorescent labels for in vivo applications due to their high quantum yield, photostability, and cyto- and hemocompatibility, though additional investigations are needed to fully evaluate QD hemocompatibility and applicability for real clinical tests.

4. CONCLUSIONS CuInS2-based QDs are considered to be very promising substitutes of heavy-metal-based QDs in different bioapplications due to their suitable optical properties and lower cytotoxicity. Recently, much progress has been made in CIS or CIS/ZnS synthesis, modification, and application, though there are still problems and questions that need to be investigated for successful use of such QDs in the future in real devices. Our work contains a detailed description of the whole process of biocompatible CIS/ZnS QD production. Particularly, photostability of hydrophobic and hydrophilic CIS-based QDs was investigated for bioapplications, especially for longterm tracing; not only does high initial PLQY matter, but sufficient photostability is required. It was shown that photostability of core−shell CIS/ZnS QDs under constant UV illumination dramatically depends on Cu/In ratio used for CIS cores synthesis. CIS/ZnS QDs with Cu/In = 1:4 ratio showed high photostability under UV illumination both in toluene and aqueous solutions. Meanwhile, photoluminescence of CIS/ZnS QDs with Cu/In = 1:1 ratio was completely quenched after several hours under UV illumination, though their initial QY was as high as 40% with peak maximum at 740 nm. So, CIS/ZnS (Cu/In = 1:4) with higher photostability was chosen for further bioapplication, despite the fact that other less photostable CIS/ZnS (Cu/In = 1:1) QDs had much more appealing fluorescence wavelengths. The synthesized QDs were transferred to aqueous solutions by amphiphilic polymer encapsulation and were further modified with polyethylene glycol-based polymers to obtain QDs with three polymeric shells differing in polyethylene glycol content and surface charge. All three types of QDs showed only slight cytotoxicity. Confocal microscopy proves penetration of hydrophilic CIS/ZnS QDs inside cells, so the low QD cytotoxocity cannot be explained by low cellular uptake of the QDs and indicated low QD toxicity in general.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b11258. Video of QD incorporation in cells by a confocal laser fluorescence microscope, 3D reconstruction. (ZIP) CuInS2 core synthesis. CuInS2/ZnS core/shell synthesis. Hydrophilization of QDs. Hemocompatibility studies. Fluorescence spectra of CuInS2 prepared using Cu/In = 1:4 molar ratio without OA addition. PL lifetime decays of QDs samples under excitation wavelengths of 380 nm. Gel migration of CuInS2/ZnS QDs modified with Jeffamine M1000 and Jeffamine ED 2003. Absorption and fluorescence spectra of CuInS2/ZnS QDs in toluene and in aqueous solutions. IR spectra of PMAO− Jeffamine M1000 polymer and QDs covered with different polymers. TEM images of CuInS2/ZnS hydrophobic and hydrophilic QDs. Confocal laser scanning fluorescence microscopy images showing fluorescence of human HEP2 carcinoma cells after incubation with CIS/ QDs covered with PMAO−Jeffamine M1000 polymer and a subsequent fixation with formaldehyde. Microphotographs of blood smears after incubation of blood samples with QDs. Comparison of the white blood cell distributions after incubation with PMAO−Jeffamine M1000 polymer and QDs of different concentrations. (PDF) 7620


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AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the Russian Science Foundation (project 14-13-00229).

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