Toxicological investigations of “naked” and polymer-entrapped AOT-based gold nanotriangles

Accepted Manuscript Title: Toxicological Investigations of “Naked” and Polymer-entrapped AOT-Based Gold Nanotriangles Authors: Ferenc Liebig, Silvia Moreno, Andreas F. Thunemann, ¨ Achim Temme, Dietmar Appelhans, Joachim Koetz PII: DOI: Reference:

S0927-7765(18)30269-8 https://doi.org/10.1016/j.colsurfb.2018.04.059 COLSUB 9312

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

19-1-2018 11-4-2018 28-4-2018

Please cite this article as: Ferenc Liebig, Silvia Moreno, Andreas F.Thunemann, ¨ Achim Temme, Dietmar Appelhans, Joachim Koetz, Toxicological Investigations of “Naked” and Polymer-entrapped AOT-Based Gold Nanotriangles, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.04.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Toxicological Investigations of “Naked” and Polymer-entrapped AOTBased Gold Nanotriangles Ferenc Liebig,a,§ Silvia Moreno,b,§ Andreas F. Thünemann,c Achim Temme,d,e Dietmar Appelhans,b Joachim Koetza,*

bLeibniz

for Chemistry, University of Potsdam, 14476 Potsdam, Germany

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aInstitute

Institute of Polymer Research Dresden, 01069 Dresden, Germany

cBundesanstalt

für Materialforschung und -prüfung, 12205 Berlin, (BAM), Unter den

dUniversity

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Eichen 87, Germany

Hospital Carl Gustav Carus, Department of Neurosurgery, Section

Experimental Neurosurgery/Tumor Immunology, TU Dresden, 01307 Dresden, eGerman

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Germany

Cancer Consortium (DKTK), partner site Dresden; German Cancer Research

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Center (DKFZ), Heidelberg, Germany, and National Center for Tumor Diseases (NCT),

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01307 Dresden, Germany

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§ Both authors contributed equally to this study and are considered as first authors.

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*Corresponding author at: University of Potsdam, Karl-Liebknecht-Straße 24-25, D14476, Potsdam, Germany. Tel.: +493319775220; Fax: +493319775054. E-mail address: [email protected] (J.Koetz)

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Statistical summary of the article: Total number of words: 5.668; Number of Figures: 6

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Graphical abstract

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Highlights

Toxicological behavior of AOT-stabilized gold nanotriangles (AuNTs)

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AuNTs coated with poly(ethyleneimine) (PEI), a maltose-modified PEI and

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heparin

Embryonic kidney carcinoma cell line HEK293T and leukemia cell line YTS

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Heparin-coated gold AuNTs with an improved biocompatibility by a factor of 50

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Heparin-coated gold nanotriangles with an enhanced cellular uptake of 70%

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ABSTRACT

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Negatively charged ultrathin gold nanotriangles (AuNTs) were synthesized in a vesicular

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dioctyl sodium sulfosuccinate (AOT)/phospholipid-based template phase. These “naked” AuNTs with localized surface plasmon resonances in the NIR region at about

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1300 nm and special photothermal properties are of particular interest for imaging and hyperthermia of cancerous tissues. For these kinds of applications the toxicity and the cellular uptake of the AuNTs is of outstanding importance. Therefore, this study focuses on the toxicity of “naked” AOT-stabilized AuNTs compared to polymer-coated AuNTs. Polymeric coating consisted of non-modified hyperbranched poly(ethyleneimine) (PEI), maltose-modified poly(ethyleneimine) (PEI-Mal) and heparin. The toxicological experiments were carried out with two different cell lines (embryonic kidney carcinoma

cell line HEK293T and NK-cell leukemia cell line YTS). This study revealed that the heparin-coating of AuNTs improved biocompatibility by a factor of 50 when compared to naked AuNTs. Of note, the highest nontoxic concentration of the AuNTs coated with PEI and PEI-Mal is drastically decreased. Overall, this is mainly triggered by the different surface charges of polymeric coatings. Therefore, AuNTs coated with heparin were selected to carry out uptake studies. Their promising high biocompatibility and cellular

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uptake may open future studies in the field of biomedical applications. Keywords: Gold nanotriangles, polymer-coating, toxicity, heparin, cellular uptake

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1. Introduction

The localized surface plasmon resonances (LSPR) of gold nanoparticles (AuNPs) are of particular interest for many applications, including photonics [1-3], drug delivery [4,5]

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and optical imaging [6,7]. In spite of wide variety of possibilities, a major challenge is to

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achieve different gold nanoparticle structures with well-defined shape, because the position of the LSPR band can be tuned by changing shape, size and surroundings [8-12].

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For example, anisotropic gold nanoparticles, like nanorods (AuNRs) or nanotriangles

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(AuNTs), adsorb in the near infrared (NIR) region [13-16]. This is essential for in vivo imaging of biological tissues [17,18]. In addition, the toxicological properties of AuNPs

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are of outstanding importance in this field of research [19]. Gold nanorods can be synthesized by a well-established seed-mediated, surfactantassisted, two-step procedure in presence of the cationic surfactant cetyltrimethylammonium chloride (CTAC), while their cytotoxic properties have been

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also investigated in more detail [13,14,16]. Molecular dynamics (MD) simulations have shown, that distorted cylindrical CTAB micelles are attached on positively charged gold

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nanorods [20]. However, the surface charge of Au NPs is of special relevance with regard to the cell toxicity [19]. The negative cell membrane potential interacts more efficiently

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with cationic particles according to electrostatic interactions [21]. Generally, cytotoxic assays revealed a higher cytotoxicity for positively charged nanoparticles. It is associated with an enhanced cellular uptake, exemplary shown for AuNPs with oppositely charged mercaptoundecanoic acid and the corresponding cationic ammonium salt [22]. Goodman et al. demonstrated that cationic AuNPs are moderately toxic, whereas anionic ones are quite nontoxic [23]. Therefore, in case of biomedical applications for AuNRs biosafe ligands such as peptides and phospholipids have to be

used to replace the more toxic cationic CTAB surfactant [24,25]. Kah et al. have shown that amphiphilic ligands (nonionic surfactants, cationic phospholipids or anionic lipids) can take over the role as capping agent [26]. Furthermore, the shape of the AuNPs also affects the toxicology [27,28]. Wang et al. have investigated the cytotoxicity of nanohexapods in comparison to nanorods and nanocages [27]. The nanohexapods show a negligible toxicity at a concentration of 200 µg/ml in contrast to the high toxicity of nanorods. Xie et al. have shown that different anisotropic gold nanoparticles, i.e., stars,

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rods and triangles, coated with methyl polyethylene glycol (mPEG) were nontoxic over

the concentration range from 2.5 to 40 µg/mL, but the most efficient cellular uptake was

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observed for nanotriangles [29].

Recently, we have shown that ultrathin gold nanotriangles (AuNTs) can be synthesized in a mixed vesicular template phase containing dioctyl sodium sulfosuccinate (AOT) and phospholipid PL90G [30]. The resulting AuNTs can be separated from spherical gold

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nanoparticles by a polyelectrolyte/micelle based depletion flocculation. After several

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purification steps a green colored AuNT dispersion is obtained. The ultrathin (7-8 nm)

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gold platelets with a mean edge length of 175 nm are stabilized by the anionic surfactant AOT and the phospholipid PL90G. The corresponding negative zeta potential of -25 mV

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indicates the presence of AOT molecules on the AuNT surface. This is in contrast to cationic CTAB-stabilized AuNTs synthesized by Scarabelli et al. [15], and the above

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discussed CTAB-based nanorods equipped with the more toxic CTAB molecules on the particle surface [31].

Recent studies have shown that our AuNTs can act as a nano-heat source [32]. At the

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plasmon resonance wavelength, they convert light into heat. In dependence on the laser influence a temperature jump of up to 150 °C can be calculated based on the shift of the

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symmetric (111) Bragg peak. These properties are most suitable for photothermal therapy and the high light absorption coefficient in the NIR region of the second optical window for in vivo imaging in biological tissues18 at 1300 nm. In addition, our AuNTs

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have an excellent photothermal stability up to a laser influence of 2.9 mJ/cm2 [33]. Moreover, photoacoustic imaging takes advantage of such features by using plasmonic nanoparticles as a contrast agent [34,35]. Additionally, hyperthermia can be induced by selective heating of the nanoparticles targeted regions of the tissues. This causes the desired cell death, which is vital for therapeutic applications [36,37]. Before using our AuNTs in tissue imaging and hyperthermia of cancer cells the goal of the study was to check the toxic properties of our negatively charged AuNTs as well as

their polymer-coated AuNTs. To the best of our knowledge cytotoxic properties as well as the cellular uptake of negatively charged flat nanotriangles have not been investigated so far. To validate the biomedical study of “naked” AOT/phospholipidstabilized AuNTs two kinds of cell lines have been selected. On the other hand, the embryonic kidney carcinoma cell line HEK293T is a good model to study the toxicity, several cell lines are derived from it, in addition, it is a cell line that can be easily transfected and modified for future experiments related to hyperthermia cancer

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treatment. On the other hand, NK-cell leukemia cell line YTS is used in clinic

experiments. This offers us a model which is closer to our immune system. Furthermore,

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YTS cells represents a candidate transporter system for selectively delivering gold

nanotriangles into tumors since they can be genetically modified to express chemokine receptors which enables their infiltration into tumors [38]. Yet, both cell lines allow an unspecific uptake of gold nanotriangles. Furthermore both cell lines are attributed by

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different size and shape, while one is adherent and the other is in suspension. This

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thoroughly enables us to study how the kind of the cell line can affect the

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biocompatibility and cellular uptake of the NPs. For a more comprehensive discussion of the role of surface charge triggered by polymer shell surrounding particles, the “naked”

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AuNTs were additionally coated with cationic and anionic polyelectrolytes. Small-angle X-ray scattering (SAXS) experiments were performed for determination of the AuNTs

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concentration, a method which was proved recently for its accuracy in an interlaboratory comparison [39]. Firstly, we studied what kind of functionalization leads to a better biocompatibility of the NPs. The second step of the study was to validate a

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satisfied cellular uptake in both cell lines. First experiments have shown that negatively charged AuNTs can be reloaded by adding the cationic polyelectrolyte) polyelectrolyte

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(PEI) and maltose-modified poly(ethyleneimine) (PEI-Mal) due to electrostatic interactions. Also, an adsorption process of the anionic polyelectrolyte heparin on “naked” AuNTs has been shown, while this adsorption process can be explained by H-

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bonds between phospholipid molecules at the particle surface and heparin. The combination of AuNTs and (biological) macromolecules such as PEI, PEI-Mal or heparin is of intense interest because of the possibility of synergistic properties. Several functionalized nanomaterials have recently been investigated for its beneficial chemical and biological properties and its capacity to improve the biocompatibility of their resulting biohybrid structures and have been used to increase their performance in various biological and diagnostic applications [40-45].

2. Experimental 2.1. Materials Water was purified using a Milli-Q system (Millipore). Dimethyl sulfoxide was obtained from Sigma Aldrich. Heparin sodium salt was obtained from PanReac AppliChem. AlamarBlue, cell viability reagent was obtained from Thermo Fisher Scientific. The fluorescence labeled heparin-FITC was obtained from Nanocs. The branched

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poly(ethyleneimine) (PEI) with a molar mass of 25,000 g/mol was obtained from BASF. The maltose-modified poly(ethyleneimine) (PEI-Mal) with open shell (structure B) was

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synthesized by an earlier described reductive amination [46]. 2.2. Cell culture

The adherent HEK293T cell lines were grown in Dubelcco's modified Eagle medium

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(DMEM; Gibco, Germany) and the suspension YTS cell lines were grown in Roswell Park

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Memorial Institute medium (RPMI-1640; Gibco, Germany) [47,48]. Both media were

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supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES, and an antibiotic cocktail (100 IU/mL Penicillin and 0.1 mg/mL Streptomycin). RPMI-1640

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was additionally supplemented with 2 mM L-glutamine. All eukaryotic cell lines were cultured at 37°C, 5% CO2 and 80% relative humidity, in appropriate cell culture medium.

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Exhausted medium was replaced at least once a week and cells were passaged when reaching high confluence or on demand. All experiments with cell cultures were performed in laminar flow work bench Laminair HB2472 (Heraeus Instruments GmbH).

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Cells were kept in the BBD 6220 CO2 incubator (Thermo Scientific Heraeus) under

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constant conditions at 37 °C in 5% CO2 atmosphere. 2.3. Methods

UV-vis-NIR spectra were recorded at a Shimadzu UV–2600 spectrophotometer. TEM

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images were collected with a JEM-1011 (JEOL) at an acceleration voltage of 80 kV to specify the shape and size of the AuNTs. To determine the polyelectrolyte adsorption process, zeta potential measurements were performed with the Malvern Nano Zetasizer 3600. SAXS measurements were performed in a flow through capillary with a Kratky-type instrument (SAXSess from Anton Paar, Austria) at 21 ± 1°C. The SAXSess has a low sample-to-detector distance of 0.309 m, which is appropriate for investigation of

dispersions with low scattering intensities. The measured intensity was converted to absolute scale according to Orthaber et al [49]. The scattering vector is defined in terms of the scattering angle q and the wavelength of the radiation (λ = 0.154 nm): thus q = 4π n / λ sinθ. Deconvolution (slit length desmearing) of the SAXS curves was performed with the SAXS-Quant software. Samples analyzed with SAXS were used as prepared. Curve fitting was conducted with software SASfit and McSAS for determination of particles’ size parameters and their volume fraction [50]. The

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reference material RM 8012 from NIST containing gold nanoparticles with a nominal

diameter of 30 nm and a gold concentration of 48.17 ± 0.33 µg g-1 was used as quality

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control material [51].

The SAXS measurements of the “naked" nanotriangles, the triangles functionalized with heparin and heparin-FITC were performed in situ in aqueous solution and in HEPES buffer. The scattering curve of the nanotriangles functionalized with heparin and

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heparin-FITC is shown as an example in Figure 1. Therein the scattering vector range of

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0.06 nm-1 ≤ 𝑞 ≤ 7.0 nm-1 corresponds to a length scales of 52 nm to 0.5 nm. The

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scattering intensity scales with q-2 at low q-values which is a characteristic of plate-like nanoparticles (indicated by a straight line in Figure 1). To facilitate curve fitting, a disk

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of the same height and radius of gyration was used instead of a triangle as used earlier (compare reference [52]). The relation between the side length of a nanotriangle a and

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the radius of a corresponding cylinder is 𝑟𝑐 =

𝑎 √6

. We calculated this relation from

comparing the radius of gyration of a nanotriangle (regular triangular prism) 𝑅g, NT = [(𝑎2 + ℎ2 )/12]1/2 and the radius of gyration of a cylinder of 𝑅g,c = (𝑟𝑐2 /2 + ℎ2 /12)1/2 . In

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the present case for a = 170 nm we calculate rc = 70 nm. This value was held constant for SAXS data interpretation since it is larger than the dmax in our SAXS data. The resultant

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curve fit of the data and the corresponding size distribution of the heights are shown in Figure 1 (red solid line and red bars in the inset, respectively). The height distribution

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can be fitted with a Gaussian function with a maximum at 7 nm and a width of 2 nm (inset: black solid line). The concentration of the nanotriangles is 𝑐 = 𝑛 𝑣NT 𝜚 where n is the particle number concentration as derived using SASfit, the 𝑣NT =

√3 4

𝑎2 ℎ is the volume of a nanotriangle

and 𝜚 is the density of the nanotriangles. Here we assume that the nanotriangles have the same density as gold in bulk form of 19.3 g/cm3. Since a is known form TEM and h from SAXS we were able to derive the concentration of the nanotriangles without the

presence of a Guiner region in the SAXS data. The concentration of the “naked” nanotriangles as measured in their stock solution was 0.16 mg/mL with an estimated uncertainty of ca. 5% (see Reference [39]). 2.4. Synthesis of gold nanotriangles and their polymer coating By using a vesicular template phase containing the phospholipid PL90G, AOT and the strongly alternating polyampholyte poly(N,N-diallyl-N,N-dimethylammonium-alt-3,5-

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bis-carboxyphenyl-maleamic carboxylate) (PalPhBisCarb), the AuNTs were synthesized according to the protocol described earlier by Liebig et al. [30]. For a separation of the

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triangles from spherical particles a higher amount of AOT (above the critical

micellization concentration) was added to the dispersion to initiate depletion flocculation [30].

In the next step, the obtained negatively charged AuNTs were coated with different

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nanotriangle dispersion at a volume ratio 0.3:1.

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polymers. Therefore, 2 wt% polymer solutions in buffer were added to the gold

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In the case of PEI and PEI-Mal, the purification step leads to instability of the particles. Therefore, 2% wt PEI or PEI-Mal solutions in buffer were added to the gold

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nanotriangles dispersion at a volume ratio 0.3:1 and incubated for 30 min. It is important to note that these dispersions could feature an excess of polymer and this

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could affect the following biomedical studies. To prove, if there is an excess of PEI in solution, we have performed experiments by adding a gold chloride solution at 100 °C. In absence of AuNTs, PEI in solution reduces the gold chloride solution and spherical

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gold nanoparticles are formed, indicated by a localized surface plasmon resonance (LSPR) peak at about 520 nm [53]. In presence of AuNTs at a molar ratio of PEI : AuNT =

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3.2 : 1 the gold reduction process is preferentially performed in the PEI shell surrounding the AuNTs, and the LSPR peak at 520 nm can be minimized, experimentally shown in the supplementary part (Figure S1) [54]. Therefore, we can conclude that in all

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experiments shown here at a significant lower PEI concentration (PEI:AuNT = 1.9 : 1) we have no excess of PEI in solution. In the case of heparin, another purification step was carried out. For that, 300 µL of heparin or rather heparin-FITC were added to 1 mL of the gold nanotriangle stock solution. The samples were vortexed for 10 s. Subsequently, the samples were centrifuged for 8 min at 13000 rpm. Then, the supernatant was removed and the precipitation was filled up to 1ml with Milli-Q-water. To that, 300 µl of the polymer was

added again and the process was repeated. The whole procedure was done six times for each sample. Subsequently, SAXS measurements of diluted dispersions were performed to determine the concentration of the AuNTs. Thus, a stock solution of 0.16 mg/mL was used to fabricate polymer-coated AuNTs. The resulting polymer-coated solution was used for all biological experiments after additional dilution of naked and polymer-coated AuNTs. Further details of stock solution concentration for polymer-coated AuNTs are

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mentioned in the figure captions.

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2.5. Biocompatibility

The biocompatibility of the structures was proved with the viability assay using AlamarBlue. HEK293T and YTS cells were seeded in a 96-well plate at an initial density of 2×104 cells per well in 150 µL of complete medium. All compounds were individually

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evaluated by treating YTS and HEK293 cells with different concentrations of the

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corresponding samples for 24 h which were considered toxic when the survival rate was

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<80%. AlamarBlue reagent (10% v/v) was added to each well and cells were further incubated for 4 h at 37 °C. Finally, the fluorescence intensities were recorded by a

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microplate reader. Dimethyl sulfoxide 20% (DMSO) was used as positive control of cellular death. Cells only with medium were used as the untreated control (NT). Each

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experiment was performed by triplicate and the data are shown as the mean value plus standard deviation (SD). Fluorescence intensities in the cytotoxicity experiments were recorded on Synergy 2 Multi Mode Reader (BioTek Instruments) at room temperature

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(25 °C).

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2.6. Cellular uptake

In order to study the cellular uptake in different cell lines, the nanotriangles have been quantitatively evaluated using flow cytometry analysis. Both cell lines, were treated with

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25 µg/mL of heparin-FITC systems (highest nontoxic concentration) for different incubation times (0, 8, and 24 h). Cells were seeded at a density of 2×105 cells per well for HEK293T and 2.5×105 cells per well for in 1.5 mL of complete medium in a 12-well plate and incubated for 24 h. Cells were then incubated with 54 µL of stock solution (CF = 0.7 mg/mL) at 37 °C in a humidified 5% CO2 incubator for different incubation times. After desired incubation time, cells were washed with PBS two times followed by centrifugation at 1100 rpm for

5 min at 4°C. The resulting pellets were resuspended in 200 μL PBS and analyzed by the flow cytometer. Flow cytometry experiments were performed on MACSQuant® Analyzer 10 (Miltenyi Biotec) which is equipped with three lasers (violet, blue and red with excitation wavelengths 405 nm, 488 nm and 635 nm, respectively) and 10 optical channels. 2.7. Statistical analysis

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The statistical analysis, including the calculation of the mean, standard deviation, and Pvalues, was performed using the Mann-Whitney U nonparametric test. The significance

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level was set as P ≤ 0.05. GraphPad Prism 5.0 software was used. All data were

generated from duplicate or triplicate wells in at least three independent experiments. 3. Results and discussion

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The “naked” AuNTs were synthesized in the vesicular template phase and separated by a

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polyelectrolyte/micelle depletion flocculation in analogy to our former experiments.29

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TEM micrographs show sharp edged nanotriangles with a mean edge length of 175 nm ± 15 nm (Fig. 2B). AFM measurements in combination with TEM micrographs after tilting

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the stage and ultrafast X-ray diffraction have already shown that the thickness of the ultrathin platelets is 7 nm ± 1 nm [30,32,33]. HRTEM micrographs reveal that the top

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and bottom surface of the triangles are composed by (111) facets [30]. The characterization by UV-vis-NIR spectroscopy show a shoulder at about 850 nm and an absorption maximum (LSPR band) at 1300 nm (compare Figure 2A). The UV shoulder is

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in the first optical NIR window for biological tissues (between 650 and 950 nm, pink shaded area in Figure 2) because of minimal light absorption by hemoglobin and water

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[17]. On the other hand the peak maximum is in the desired second NIR window

(between 1000 and 1350 nm, grey shaded area in Figure 2), where the absorption and scattering from oxygenated and deoxygenated blood, skin and fatty tissue is at the

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lowest level, ideally suited for in vivo imaging [18]. Previously, we have shown that the growth mechanism of gold nanotriangles in the vesicular template phase (self-assembled by mixing the anionic surfactant AOT, phospholipid PL90G and the polyampholyte PalPhBisCarb at pH 9) can be described by an Ostwald ripening process [52]. Thus, the negative zeta potential of the “naked” AuNTs with -25 mV (shown in Table 1) indicates that the triangles are surrounded by an AOT

double layer. After adding the polycation poly(ethyleneimine) (PEI) the zeta potential shifts to a positive value of +13 mV indicating the reloading of the AuNT surface due to polymer adsorption. This phenomenon can be well understood by electrostatic interactions between the negatively charged AOT double layer and the positively charged PEI. Similar results of reloading to +12 mV were observed using maltosemodified PEI (PEI-Mal). Additional experiments with heparin show an opposite effect. This smoothly indicates

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that the absolute value of the negative zeta potential is increased from -25 mV to -53 mV. This change can be explained only by the adsorption of the strong negatively

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charged heparin. The extraordinary effects of heparin on adhesion and vesiculation of

phospholipid membranes is a well-known phenomenon, which is of special relevance for using heparin as efficient treatment of acute thrombosis [43]. Due to our synthetic approach in presence of the phospholipid PL90G, it can be assumed that in addition to

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the AOT molecules phospholipids are also adsorbed on the nanotriangle surface.

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Moreover supposedly, heparin macromolecules are attached to the surface of AuNTs due

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to heparin phospholipid interactions [55].

Following, the biocompatibility study was carried out, for that, two cell lines have been

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selected, the embryonic kidney carcinoma cell line and NK-cell leukemia cell line YTS. The aim is to study the cellular uptake of functionalized NPs up to 24 h as maximum

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time treatment, therefore, in the biocompatibility assay the cells were treated for 24 h with different concentrations of gold nanotriangles. The concentration of negatively charged “naked” gold nanotriangles was determined by SAXS experiments (CStock = 0.16

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mg/mL). With this, the concentration-dependent biocompatibility of negatively charged triangles was studied first, obtaining in both cell lines a highest non-toxic concentration

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of 1 μg/mL (Figure 3). At 5 µg/mL the viability of the YTS cell line is drastically decreased in contrast to the HEK293T cell line. This also exhibits the more toxic

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properties of negatively charged triangles for leukemia cells (Figure 3). The next step was to study the nanotriangles loaded with PEI, PEI-Mal and heparin. When the “naked” AuNTs are entrapped by a PEI shell the viability is decreased in the order of 3 magnitudes. This denotes that the toxicity increases drastically. These results are in full agreement with the cytotoxic properties of hyperbranched PEI in vitro and in vivo [39,40]. The highest nontoxic concentration is shifted to 0.001 µg/mL (Figure 4A). At higher concentrations (≥ 0.01 µg/mL) the positively charged AuNTs are more toxic

for the embryonic kidney carcinoma cells than for the leukemia cells, quite opposite to the “naked” AuNTs discussed above. On the other hand one has to consider that excess PEI was not separated due to colloidal instability of PEI-coated AuNTs. This may also contribute to the low highest nontoxic concentration of PEI-coated AuNTs. However, when the sugar modified PEI (PEI-Mal) is used as capping component, the highest nontoxic concentration is shifted to 0.01 µg/mL. This means that the toxicity for

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both cell lines is decreased by one order of magnitude as found for naked AuNTs (Figure 3). Also, PEI-Mal is less toxic for the HEK293T cells in comparison to the YTS cells

(Figure 4B). Compared to PEI-coated AuNTs (Figure 4A), the presence of maltose groups

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in PEI-Mal is responsible for the increased highest nontoxic concentration. Due to

colloidal instability of PEI-Mal-coated AuNTs, excess PEI-Mal could not be separated as given in the case of PEI-coated AuNTs. This fact is truly responsible for the lower non-

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toxic concentration as found for naked AuNTs and also confirms the higher toxicity of positively charged Au nanoparticles [23].

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By coating the “naked” AuNTs with heparin the picture is completely changed. The

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particles become much more biocompatible and the highest nontoxic concentration is

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increased up to 50 µg/mL (Figure 5A). This value is 10 µg/mL higher than the value obtained by Xie et al. [29]. Certainly, in comparison to the “naked” AuNTs the

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biocompatibility is improved by a factor of 50 and in comparison to the PEI-capped ones by a factor of 5 * 104. Due to these results, cellular uptake studies were performed with heparin-coated AuNTs. For this, they were coated with heparin-FITC, while the labelling of heparin only leads to a very weak additional toxic effect in the error range (Figure 5B).

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Considering the cellular uptake of the fluorescence-labelled heparin-entrapped AuNTs significant differences between the two cell lines can be seen (compare Figure 6). An

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important point for the biomedical applications is cellular uptake that depends on both particle and the cell mechanical state [29, 56,57]. When nanoparticles are next to a cell,

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the interactions between the nanoparticles and the cell membrane generate forces from different origins. One of the first steps is that the membrane of the cells wraps the nanoparticles, producing finally the process of cellular uptake. There are several studies about the kinetics, energetics, and forces for the uptake of nanoparticles. This final uptake for nanoparticles is strongly dependent on the size and shape of nanoparticles, the biomechanical properties of the cell membrane and also the local environment of the cells [58]. It was also shown that large cell size facilities cellular uptake, while high

membrane tension inhibits this process. High membrane tension requires high membrane deformation energy during the wrapping of NPs which also reduced the cellular uptake efficiency [58]. In our study two kinds of cell lines have been selected which have different shape, size, tension in the membrane and local environment as well. Although a higher uptake would be expected in HEK293 due to their largeness compared to YTS cells, the results show that the cellular uptake of human YTS cells after 24 hours is much higher by a

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factor of 4 in comparison to the embryonic kidney carcinoma cells (HEK293). Perhaps, the interaction between NPs and the membrane of HEK293 cell line could be more

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complex leading to a lower uptake. Furthermore, the YTS cells are in suspension. This can also influence the endocytosis process of the NPs which is more favoured than in

HEK293 cells. This thoroughly confirms that beside the adequate good biocompatibility much more AuNTs (70%) will be taken up from the leukemia cells indicating a high

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internalization and more specific interaction between the biocompatible AuNTs and the

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kidney cells. In general the high cellular uptake is in agreement with other investigations

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demonstrating specific shape effects of nanotriangles [29]. It is important to emphasize that these hybrid systems are able to show a variable selectivity to cells with different

4. Conclusions

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and environment as well.

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characteristics. This is caused by the fact that tumours can feature different sizes, shapes

Negatively charged “naked” gold nanotriangles, synthesized in a vesicular template

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phase and stabilized by the anionic surfactant AOT and phospholipids reveal a moderate cell toxicity in comparison to the well-established more toxic nanorods stabilized by the

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cationic surfactant CTAB. By coating the AuNTs with the polycation PEI via Coulombic interactions the toxicity is drastically increased but can be leveled off after incorporating maltose units into the branched poly(ethyleneimine). In contrast to the cationic

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modified AuNTs heparin can be used as a preferable coating for the “naked” triangles. The resulting heparin-entrapped AuNTs are much more biocompatible at a high cellular uptake of 70%. These hybrid systems offer a good biocompatibility and a selective uptake depending on the characteristics of the cell line used, while their optical properties may play a vital role in the future research of in vivo tissue imaging and hyperthermia of cancer cells.

Acknowledgement Silvia Moreno has been supported by a grant from Fundación Alfonso Martín Escudero. The financial support from the German Research Foundation (KO 1387/14-1; INST 336/64-1) is gratefully acknowledged.

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We thank Kerstin Brademann for help in SAXS measurements.

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103 102

0

10

2

4

6

8

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q-2

10 12

Height (nm)

1

100 0.05

0.1

1

7

-1

q (nm )

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Intensity (m-1)

104

Frequency

Figure captions

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Figure 1. SAXS curve of nanotriangles, functionalized with heparin-FITC, in HEPES

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buffer. The measured scattering intensity is given as a function of the scattering vector (symbols). The curve fit is given using a model of gold nano-plates with a mean height of

A

7 ± 1 nm and a radius of 70 nm (diameter of 140 nm, red solid line). The straight dashed

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line represents an intensity scaling proportional to q-2 which is typical for plate-like particles. The inset shows the volume-weighted distribution of the heights of the plates

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(red bars) and a curve fit with a maximum at 7 nm and a width of 2 nm (black solid line).

Figure 2. UV-vis spectrum of purified gold nanotriangles indicating the first (pink shaded area) and second window (grey shaded area) for in vivo imaging (A) and the corresponding TEM micrograph (B).

YTS

120

HEK293T

80 60 40

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% Viability

100

20 NT DMSO 10

5

1

0.5

0.1 0.05 0.01 0.001

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0

Concentration (μg/mL)

Figure 3. Viability of “naked” gold nanotriangles in YTS and HEK293T cell lines by

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Alamar Blue assay. Cells were treated for 24 h in a range of concentrations 0.001-10

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µg/mL with gold nanotriangles. Dimethyl sulfoxide 20% was used as positive control of

A

cellular death. Each experiment was performed by triplicate. Stock solution of naked

A

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M

gold nanotriangles: 0.16 mg/mL.

A YTS

% Viability

120

HEK293T

100

80 60 40

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20 0 NT

DMSO

0.1 µg/mL

0.05 µg/mL

0.01 µg/mL 0.001 µg/mL AuNTs

120

YTS

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+ 24 µg/mL + 12 µg/mL + 2.4 µg/mL + 0.24 µg/mL PEI

B

HEK293T

U N

80

A

60 40

M

% Viability

100

0

NT

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20

DMSO

0.1 µg/mL

0.05 µg/mL 0.01 µg/mL 0.001 µg/mL AuNTs

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+ 24 µg/mL + 12 µg/mL + 2.4 µg/mL + 0.24 µg/mL PEI-Mal

Figure 4. Viability of PEI-adsorbed gold nanotriangles (A) and PEI-Mal-adsorbed gold

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nanotriangles (B) in YTS and HEK293T cell lines by Alamar Blue assay. Cells were treated for 24 h in a range of concentrations 0.001-1 µg/mL with modified gold nanotriangles. Dimethyl sulfoxide 20% was used as positive control of cellular death. Each experiment was

A

performed by triplicate. Both stock solutions used for dilution: 0.008 mg/mL of AuNTs + 19 mg/mL of PEI or PEI-Mal.

A

120

YTS

HEK293T

% Viability

100 80 60 40

0

50

NT DMSO 100

B

25

10

5

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20

0.1 0.01

1

YTS

HEK293T

U

100

80

N

60

A

40 20 0

25

10

5

1

0.1 0.01

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NT DMSO 50

M

% Viability

120

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Concentration (μg/mL)

Concentration (μg/mL)

Figure 5. Viability of heparin-adsorbed gold nanotriangles (A) and heparin-FITC-

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adsorbed gold nanotriangles (B) in YTS and HEK293T cell lines by Alamar Blue assay. Cells were treated for 24 h in a range of concentrations 0.01-100 µg/mL with gold

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nanotriangles. Dimethyl sulfoxide 20% was used as positive control of cellular death. Each experiment was performed by triplicate. Heparin-adsorbed gold nanotriangles CStock solution of AuNTs = 1.73 mg/mL; heparin-FITC-adsorbed gold nanotriangles CStock solution of

A

AuNTs =

0.7 mg/mL.

B

90 80 70 60 50 40 30 20 10 0

HEK293T YTS

0

**

8

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% uptake

A

24

Time (h)

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B1-A:AuNTs-FITC

Figure 6. Cellular uptake of modified gold nanotriangles with heparin-FITC was studied in YTS and HEK293T cell lines for different incubation times (0, 8 and 24 h) by flow

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cytometer (A) and an example of cellular uptake in YTS is shown after 24 h of incubation

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(B). Cells were treated with 25 µg.mL-1 of heparin-FITC systems. The data represent the

A

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M

A

mean ± SD of three independent experiments (**P ≤ 0.01; vs. NT).

Table 1. Zeta potential of AuNTs in absence and presence of positively charged polymers (PEI and PEI-Mal) and negatively charged polymers (Heparin and HeparinFITC) Zeta potential [mV]

+13 ± 2 +12 ± 2

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-53 ± 2 -51 ± 1

N A M TE D EP CC A

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-25 ± 1

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“Naked” AuNTs (stock solution: 0.16 mg/mL) in absence of polymers AuNTs (stock solution: 0.008 mg/mL) in presence of PEI AuNTs (stock solution: 0.008 mg/mL) in presence of PEI-Mal AuNTs (stock solution: 1.73 mg/mL) in presence of Heparin AuNTs (stock solution: 0.7 mg/mL) in presence of Heparin-FITC