Selective MW-assisted surface chemical tailoring of hydrotalcites for fluorescent and biocompatible nanocomposites

Abstract

ZnAl based hydrotalcite nanoparticles (ZnAl-HTlc NPs) were covalently modified by an organic oligothiophene fluorescent compound (T4Si) by using direct microwave (MW)-assisted silylation. Morphological and optical characterization proved that the MW-assisted method enables efficient grafting of the target fluorescent dye on the nanoparticles (NPs) surface in a few minutes with a predefined loading ratio only depending by the MW irradiation time. Moreover, the presented approach preserved the HTlc interlayer region, allowing further functionalization. Filmability, fluorescent properties, and biocompatibility of the silylated compound was also demonstrated highlighting the potential of the so-obtained lamellar NPs in applications broadening from diagnostic biomedical tools to photonics and sensing.

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1. Introduction

Layered solids are materials with interesting chemical and physical properties due to their structural anisotropy, and to the possibility of functionalization by intercalation of species having specific properties.¹ In particular, hydrotalcite-like compounds (HTlcs) attract particular interest due to their ready availability via inexpensive methodologies and peculiar structural and compositional features. HTlcs are the only clay with a positively charged surface balanced by interlayer anions. They can be presented by the formula [M(II)_(1−x)M(III)_x(OH)₂]^x+(A_x/nⁿ⁻·mH₂O), where M(II) and M(III) can be divalent and trivalent metal ions able to accommodate into octahedral cavities, Aⁿ⁻ can be any type of anion and x is defined as the M(III)/(M(II) + M(III)) molar ratio. Structurally, HTlc consists of octahedral brucite-like layers [M(II)_(1−x)M(III)_x(OH)₂]^x+ in which M(II) is partially substituted by M(III) to yield a net positive layer charge. Anionic species as well as the water molecules (A_x/nⁿ⁻·mH₂O), are located between the brucite-like layers, balancing the positive layer charge (Fig. 1a).^2–4 The possibility of anionic insertion into the interlayer region² makes HTlc versatile materials suitable for different industrial applications such as adsorbents, ion exchangers, pharmaceutics, catalysts, flame retardants^5–11 and nanostructured materials for photonic and opto-electronic devices.^11–14

Fig. 1 (a) Schematic representation of a sequence of layers of ZnAl-HTlc. (b) Molecular structure of T4Si.

However, major drawback of this approach is that the intercalation of anionic moieties in the interlayer region of HTlc limits the availability of ionic sites for further functionalization of the inorganic matrix. In order to overcome such issues, during the last decade, great efforts have been addressed toward the development of novel methods to chemically modify HTlc surface effectively and efficiently. Silylation, also known as silane grafting, is one of the most versatile approaches to modify many hydroxyls-rich organic and inorganic materials including glass, graphene oxide, zeolite-like porous materials, silicon chips, metals, and clays.¹⁵ Indeed, the abundant surface-OH groups can condense with silanes to form Si–O–Si networks. State-of-the-art on HTlc-silylation is based on a multistep, time consuming approach, that combines intercalation with anionic species and further reaction with excess of 3-aminopropyltriethoxysilane (APTS) as silylation agent.¹⁶ In this context, Park et al.^16a reported a two step approach to obtain silylated HTlc, firstly preparing dodecylsulfate intercalated HTlc and then adding in the reaction system 3-aminopropyltriethoxysilane (APTS). In situ coprecipitation of HTlc and APTS was then reported by Tao et al.^16b to realize a surface silylated hybrid. The effect of the surfactant amount on the chemo-physical surface properties and morphology of the resulting APTS-modified HTlc was also investigated by the same group.^16c,d The APTS based silylation approach was then exploited by Oh et al.^16e to post-graft luminescent fluorescein 5′-isothiocyanate (FITC) to HTlc through the formation of the thiourea bond, i.e. by reaction between the pendant NH₂-moieties of APTS with the NCS group of FITC. The use of the so-obtained fluorescent HTlc for cell fluorescent imaging has been reported highlighting the potential of modified HTlc for biological and biomedical purposes.

Here, we report the synthesis of functional hydrotalcite–quaterthiophene hybrid material through one-step MW-assisted silylation reaction. In our approach the silylated dye is conveniently prepared by reaction of a dye bearing an activated carboxylic end with stoichiometric amount of APTS.^15a,b,17 The silylated dye is then covalently grafted to the –OH surface groups of HTlc exploiting MW acceleration.^15a,b,17,18 We demonstrate that this procedure enables surface modification while preserves HTlc interlayer region and that the dye loading ratio is regulated only by the MW irradiation time. The so obtained hybrid material was fully characterized, and showed good solution processability and fluorescent properties. Moreover, the biocompatibility and cell internalization capability of the fluorescent HTlc composite was explored.

2. Experimental section

2.1 Preparation of ZnAl-HTlc nanoparticles and T4/ZnAl-HTlc hybrid composites

Zinc aluminum hydrotalcites (ZnAl-HTlc) nanoparticles with formula [Zn_0.72Al_0.28(OH)₂]Br_0.28·0.69H₂O were synthesized using the double microemulsion method reported in literature.¹⁹ The functionalization of ZnAl-HTlc, previously dried under vacuum at 80 °C, with T4Si dye was achieved by microwave assisted direct silanization,^15b,17 i.e. by irradiating a solution of ZnAl-HTlc (19 mg in 3.5 ml of DMF) and T4Si (C ∼ 10⁻⁴ M) with microwaves (MW) at 80 °C for 20 and 40 min. After cooling to room temperature, the crude powder was filtered and the T4/ZnAl-HTlc washed with DMF, acetone and dried under vacuum. T4Si was prepared according to previously described procedures.^17a

2.2 XRD

XRD patterns were taken with a Philips X'PERT PRO MPD diffractometer operating at 40 kV and 40 mA, with a step size 0.0170 2θ degrees, and step scan 20 s, using Cu Kα radiation and an X'Celerator detector.

2.3 FT-IR

Fourier-transform infrared spectra (FTIR) of different samples, dispersed in KBr pellets, were recorded using Bruker FT-IR IFS-133v spectrometer. Typically each spectrum was obtained averaging over 1064 scans at a resolution of 2 cm⁻¹.

2.4 TG/DTA

Thermogravimetric (TG) analyses were performed with a Netzsch STA 449C apparatus, in air flow and with a heating rate of 10 °C min⁻¹.

2.5 ICP-OES

Metal analyses were performed by Varian 700-ES series Inductively Coupled Plasma-Optical Emission Spectrometers (ICP-OES) using solutions prepared by dissolving the samples in some drops of concentrated HF and of concentrated HNO₃ solution and properly diluted.

2.6 Contact angle measurements

Water contact angles were measured by the static sessile drop method using a Digidrop GBX Model DS. For each film at least five drops were measured. The water droplets used for measurements had a volume of 1 μl.

2.7 AFM

Atomic Force Microscope (AFM) topographical images were collected using an NT-MDT Solver scanning probe microscope in tapping mode.

2.8 SEM

T4/ZnAl-HTlc silylated films were imaged with a Scanning Electron Microscope (SEM), ZEISS LEO 1530 FEG, after metallization with gold.

2.9 Photoluminescence spectroscopy and confocal laser scanning microscopy

UV-visible absorption spectra were recorded with a JASCO V-550 spectrophotometer. Photoluminescence was excited using a CW He–Cd laser at 325 nm with 10 mW power. No color filter was used for truncating the laser excitation. PL emission was collected with a calibrated optical multichannel analyzer (PMA-11, Hamamatsu). Confocal laser scanning microscopy (CLSM) images were performed with a Nikon Eclipse 2000-E laser scanning confocal microscope. The PL images were obtained exciting with a Ar⁺ laser with emission at 488 nm. The images were collected through a oil 60× objective with 1.4 numerical aperture and through a dichroic mirror that prevents PL modulation.

2.10 Cell culturing, treatment and imaging

Liver hepatocellular carcinoma HepG2 cells has been cultured as previously described.²⁰ Briefly cells were maintained in Dulbecco's Modified Eagle Media (DMEM)/HAM's-F12 (1 : 1) supplemented with 10% v/v of fetal bovine serum, 1% L-glutamine, 1% insulin and antibiotics (penicillin/streptomycin, 100 U ml⁻¹ and 100 mg ml⁻¹, respectively). All products were purchased by Gibco, Invitrogen, Life Technologies, Milan, Italy. Culture flasks were maintained in a humidified incubator with 5% CO₂ at 37 °C and cells has been split every third day. The day before experiment cells has been re-plated on 19 mm coverslips coated with poly-D-lysine, placed into 12-multiwell plate at a concentration of 1 × 10⁵ per sample. For cell viability assay and imaging, the pristine ZnAl-HTlc and T4/ZnAl-HTlc composite (sample c) were collected after repetitive washing with sterile water and immediately dispersed in cell culture media to avoid the formation of aggregates. The so obtained dispersions were sonicated for 10 minutes and used to treat cell culture at a final concentration of 50 μg ml⁻¹ and 100 μg ml⁻¹. After 24 h of incubation, cells were washed three times with phosphate buffer saline and fluorescein diacetate (FDA, Sigma Aldrich) cell viability assay, cell imaging and cell counting were next performed as previously described.²¹ Confocal imaging of FDA positive (viable) cells was performed by using Nikon TE 2000 inverted confocal microscope equipped with a 20× or 60× oil-objective and 488 nm Ar+ and 543 nm He–Ne lasers as excitation sources. Images reported are representative of 2 different experiments performed in triplicate. A series of confocal images (10 to 15 different fields of 0.6 × 0.6 mm for each sample) was taken for each sample in triplicate. Living cells were counted and the number of viable cells was calculated as percentage of viable cell averaged counting in untreated cells. Results were statistically analyzed using one-way analysis of variance (ANOVA) or independent t Student test. A statistically significant difference was reported if p < 0.05 or less. Data are reported as the mean ± standard error (SE) from separate experiments performed in triplicate. To verify internalization of nanocomposite images have been collected exciting samples with 488 nm Ar+ lasers as excitation sources and collecting fluorescent emission with the detection centred at >600 nm.

3. Results and discussion

3.1 Synthesis of T4/ZnAl-HTlc composites

Zinc aluminum hydrotalcites (ZnAl-HTlc) nanoparticles were synthesized using the double microemulsion method.¹⁹ Subsequent functionalization of ZnAl-HTlc, previously dried under vacuum at 80 °C, with N-(3-(triethoxysilyl)propyl)-[2,2′:5′,2′′:5′′,2′′′-quaterthiophene]-5-carboxamide (T4Si, Fig. 1b) was then performed via microwave (MW) assisted direct silanization,^15b,17 i.e. by irradiating a DMF solution of OH–ZnAl-HTlc and T4Si at 80 °C for 20 minutes (Scheme 1a and details in the Experimental section) rather than several hours required by the previously reported approaches. The effect of irradiation time on the final HTlc composition was also investigated by performing the same experiment using a doubled reaction time. Notably, no surfactants were needed as co-additive for promoting the grafting.^16a,e Scheme 1 shows the synthetic route and the type of samples herein analyzed (i.e. 1a pristine ZnAl-HTlc, silylated T4/ZnAl-HTlc composites after 20 and 40 minutes, both in form of powder, 1b, 1c and film 2b, 2c, respectively). Then, the crudes so obtained were treated by several washing–filtration cycles by using warm DMF and acetone as solvents to remove possible polymeric organic side-products. It is worthnoting that by casting the obtained dispersion we obtained HTlc nanocomposites films, enriching the potential applications of the silylated products. Scheme 1b shows the as obtained samples deposited on glass under UV-light illumination. The yellow emission is a first clear evidence of the presence of the T4 moieties.

Scheme 1 (a) Synthetic route to T4/ZnAl-HTlc composites. (b) Image of 1c deposited on glass illuminated by a Hg lamp, λ exc. range 330–380 nm.

3.2 X-Ray diffraction measurement

X-Ray diffraction patterns of pristine ZnAl-HTlc nanoparticles (sample 1a and 2a) and its silylated products T4/ZnAl-HTlc, both in form of powder and film (samples 1b, 1c and 2b, 2c respectively, Scheme 1a), are shown in Fig. 2. The ZnAl-HTlc basal spacing (d(003)) of 7.92 Å is compatible with the presence of bromide anions in the interlayer region.¹⁹ The T4/ZnAl-HTlc composites exhibit a basal of 7.75 Å, very close to those of pristine ZnAl-HTlc confirming the presence of bromide anions into the interlayer region. The slight spacing decreases, particularly evident for the (006) reflection (Fig. 2, 1b and 1c), can be attributed to the lower hydration water of silylated HTlc samples respect to the pristine ZnAl-HTlc, in agreement with thermogravimetric analysis and literature data.²² These findings indicate that the crystal structure of HTlc is not influenced by silylation. Additional reflections at lower 2θ angles were not detected in the silylated products, indicating that T4Si molecules are only located on the external surface of HTlc. Furthermore, in the casted ZnAl-HTlc and T4/ZnAl-HTlc films, the absence of any in-plane reflections (h, k ≠ 0) at high angle are evidence of a well c-oriented assembly of HTlc nanoplatelets, in agreement with already reported HTlc films.²³

Fig. 2 XRD patterns of pristine ZnAl-HTlc nanoparticles in form of powder (1a) and film (2a), silylated products T4/ZnAl-HTlc, both in form of powder and film (samples 1b, 1c and 2b, 2c respectively, Scheme 1a).

3.3 Infrared spectroscopy

Infrared spectroscopy was used to verify the covalent grafting of T4Si molecules onto HTlc surface. Fig. 3a shows the IR absorption spectra of the pristine ZnAl-HTlc 1a, T4Si molecules and of the T4Si modified ZnAl-HTlc 1b and 1c.

Fig. 3 (a) FT-IR spectra of T4Si molecules, pristine ZnAl-HTlc nanoparticles 1a, T4/ZnAl-HTlc 1b, T4/ZnAl-HTlc 1c recorded in the 400–4000 cm⁻¹ region. (b) FT-IR spectra of 1a and 1c recorded in the 400–1200 cm⁻¹ region.

The broad band at around 3500 cm⁻¹ is attributed to the vibration modes of hydroxyl groups, both those in the brucite-like layers (Zn/Al–OH) and from the interlayer water molecules. The bands below 1000 cm⁻¹ are due to the M–O stretching vibrations (M = Zn²⁺ and Al³⁺).²⁴

After silanization, a series of bands arising from T4Si pendants^15a are detected in the T4/ZnAl-HTlc silylated products (see Fig. 3b). The intensity of these new features depends on the reaction time: longer the MW exposure, stronger are these peaks. The new band between 1000 and 1100 cm⁻¹ and the one centered at 795 cm⁻¹ can be assigned, respectively, to Si–O–C and Si–C stretching vibrations of silanes, clearly showing the presence of siloxane bridge bonds in silylated samples. The M–O stretching vibration of HTlc at 657 cm⁻¹ decreases significantly in the silylated sample transferring the oscillator strength to a new band at 671 cm⁻¹ which can be ascribed to Si–O–M deformation vibrations. Similar spectral features were observed for other types of silylated HTlc.^16d

In addition, in the T4/ZnAl-HTlc sample, the intensity of the band at around 830 cm⁻¹ decreases and appears a new peak at 880 cm⁻¹. The IR bands in this spectral region have been ascribed to the out of plane CO₃²⁻ bend or to the M–OH vibration modes.^24b Considering that it has been shown that carbonate ions are not present in HTlc compounds obtained following the double microemulsion method,^19a we can exclude that, in our case, the 830 cm⁻¹ mode is related to CO₃²⁻ and we can assign this band to O–H modes. In silylated compound this mode shifts at 880 cm⁻¹ due to the interaction of T4Si molecules with the hydroxyl groups of the brucite layers. The intensity reduction of the mode at 830 cm⁻¹ is not compensated by the appearance of the new band at 880 cm⁻¹ (see Table 1), this is probably due to the reduction of the number of OH groups because of a partial substitution with T4Si species. This is consistent with the observed intensity decrease of the metal–OH stretching mode at about 3470 cm⁻¹ in the silylated compounds (see Fig. 3a).

Table 1 Energies E (cm⁻¹), bandwidths FWHM (cm⁻¹), Lorentzian/Gaussian percentages L/G (%) and normalized intensities I of the IR bands between 400 and 1100 cm⁻¹ for samples 1a and 1c

	E	FWHM	L/G	I1a	I1c
1	427.4	60	100	1.00	1.00
2	553.3	62	47	0.51	0.35
3	599.2	61	100	0.71	0.65
4	657.4	161.5	3	0.84	0.42
5	670.8	98.5	100		0.23
6	795	45.8	0		0.06
7	830.4	257.9	100	0.86	0.58
8	880.6	51.7	0		0.08
9	1081.7	175.3	65		0.23

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Finally, another evidence of the occurred silylation is the weak appearance of C–H stretching vibrations of the thiophene rings (region 2970–2880 cm⁻¹) in the T4/ZnAl-HTlc composites (Fig. 3a).

3.4 Thermogravimetric and ICP analyses

The chemical composition of the T4/ZnAl-HTlc products, reported in Table 2, was determined by TG/DTA and ICP-OES analyses. The TG/DTA curves of the pristine ZnAl-HTlc 1a nanocrystals and of the silylated products are showed in Fig. 4. Three stages of endothermic weight loss are evident in all samples. For sample 1a (Fig. 4a), the first step, ranging from 80 °C to 150 °C, with a total mass loss of 8.0% can be assigned to the loss of adsorbed water and interlayer water. The second region, up to 350 °C, with a mass loss of 14.5% corresponds to the weight loss due to the dehydroxylation of the brucite layers. The last step with a mass loss of 18.5% is related to the loss of interlayer bromide anions. The total mass loss observed is 41.0%.^19a The water loss stage (80–150 °C) of the T4/ZnAl-HTlc composites 1b and 1c is quite different to that of the pristine 1a: the decrease in mass loss for the silylated samples (7.0% for sample 1b and 5.6% for sample 1c, Fig. 4b and c, respectively) indicated less adsorbed water molecules on the surface of HTlc. According to previous studies,^15b this is likely due to the change of the HTlc surface properties from hydrophilic to hydrophobic following the modification with T4Si molecules. The weight loss of the last step (400–800 °C), related to the loss of interlayer bromide anions, allows to obtain the mol of bromide anions per 100 g of 1b and 1c composites and then the weight percentage of [Zn_0.72Al_0.28(OH)₂]Br_0.28. The weight percentage of HTlc in the composites was 53.9% and 39.2% for sample 1b and sample 1c, respectively. Considering the hydration water the weight percentage of T4Si molecules was 39.1% in the sample 1b and 55.2% in the sample 1c. Noteworthy, the presence of broad exothermic peaks confirms the presence of the organic moieties.

Table 2 Elemental content of samples 1a–c

Sample	Elemental amount (mmol g^−1)
	Zn	Al	Si
1a	4.76	1.80	0
1b	3.57	1.39	0.84
1c	2.80	1.04	1.06

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Fig. 4 TG/DTA curves of (a) pristine ZnAl-HTlc 1a, (b) T4/ZnAl-HTlc 1b and (c) T4/ZnAl-HTlc 1c. (Operative conditions: heating rate: 5 °C min⁻¹, air flow.)

The effect of irradiation time was investigated by elemental analysis, indicating that the loading efficiency increases on increasing the MW-reaction time (Table 2).

According to the TG results the content of silicon (mmol g⁻¹) increased of nearly 20% in case of longer MW exposition (composite 1c). These results demonstrate that complete –OH functionalization with T4Si dye is achieved upon only 40 minutes of MW irradiation. Moreover, in good accordance with TG and ICP results, also the surface wettability determined by water contact angle measurements, reveals an higher content of the organic (i.e. hydrophobic) component for sample 1b and 1c with respect to pristine sample 1a. Indeed, the water contact angle value changes from 70.2° (±2.7) to 77.0 (±2.6) and 82.5 (±1.6) on going from pristine 1a to 1b and 1c respectively (see ESI, Fig. S1†). This evidence can be ascribed to the strong hydrophobic character of the grafted T4Si having an extended π-conjugated framework and to the increase of the T4Si moieties on increasing the functionalization time (i.e. the T4 loading).

3.5 Optical characterization and confocal microscopy

Applications for HTlc nanoplatelets films in sensing, catalysis and photonics have been envisioned and demonstrated.^11–14 In this context, we analyze optical and fluorescent properties of obtained sylilated films. The absorption and emission spectra of T4Si in solution, cast film and of T4/ZnAl-HTlc (1c) are shown in Fig. 5a. The absorbance spectrum of isolated T4Si molecules is unstructured and broad with maximum absorption wavelength peaked at 405 nm. On the other hand, the emission spectrum is well-structured with at least three features clearly visible (471, 500 and 550 nm) as expected in the case of a planar and fully-conjugated molecular structure of the emitting state.

Fig. 5 (a) Absorption and emission spectra of T4Si diluted solution (black line), emission spectrum of a film of T4Si obtained by drop-cast of a saturated solution (green line) and emission spectrum of a cast film of T4/ZnAl-HTlc 1c (red line). (b and c) Confocal Laser scanning microscopy images at different magnification of drop cast film of 1c onto glass.

The emission spectrum of a T4Si drop-casted thin-film, is largely red-shifted (∼90 nm) with respect to the diluted solution pointing at possible aggregate formation in solid-state.²⁵ Indeed, the PL spectrum of T4/ZnAl-HTlc nanoparticle film, sample 1c (red line in Fig. 5a), shows an emission maximum located in-between the emission of the diluted solution and of the thin-film of T4Si molecules. Moreover, in the case of T4/ZnAl-HTlc nanoparticle film, the emission is broad (FWHM ∼ 180 nm) and partially structured, extended even at higher wavelength with respect to the solution. Collectively these evidences suggest that the T4 emitting moieties in films of sample 1c experienced different environments and aggregate states with respect to both T4Si diluted solution and thin-film cases. It is likely that the interaction among doped nanoparticles plays a role in determining the broad emission profile which is collected from the drop-casted film.

Given that from XRD data, Fig. 2, we can exclude the intercalation of T4 molecule in the interlayer region of the ZnAl-HTlc NPs, we can suppose that the broad emission spectrally extending at higher-energy region with respect to the diluted solution is possibly due to the steric hindrance and arrangement disorder among T4 moieties onto the NPs surfaces.

The spectroscopic findings are well-corroborated by the CLSM investigation. As it can be seen in Fig. 5b and c, the lamellar nanoparticles are fluorescent after the functionalization with the thiophene-derivative dye, with emission ranging from green (spectral range 515 ± 20 nm) to yellow (spectral range >600 nm). This feature is in accordance with the broad photoluminescence spectrum collected from the film.

Moreover, the crystals display a wide range of diameter dimension, from 6 μm (largest dimension) (Fig. 5b) to 1 μm (smallest dimension) (Fig. 5c) while maintaining the typical hexagonal-like morphological features. Interestingly, the fluorescence emission shifts from green to yellow and the emission intensity increases on increasing the crystal size. This suggest that the larger crystals result from aggregation of smaller particles, that suitably arrange without fluorescence quenching.

3.6 Morphological characterization of T4/ZnAl-HTlc composites

The morphology and organization of the ZnAl-HTlc nanoparticles before and after silanization with the T4Si dye were investigated by SEM and AFM analyses. Fig. 6 shows the SEM images of the pristine ZnAl-HTlc, 1a (Fig. 6a) and of the T4/ZnAl-HTlc 1c (Fig. 6b–d). The typical hexagonal shape,¹⁹ particularly evident for the larger particles (yellow arrows in Fig. 6a), and the dimension (diameter of about 150–200 nm) of the HTlc nanoplatelets is retained after silylation (see Fig. 6a and b and inset of 6b). However, the formation of large aggregates (around 20 μm) can be observed for the silylated product (Fig. 6c). As indicated in Fig. 6d these aggregates are constituted by the stacking of the nanoplatelets rather than by a single crystal; these features are in agreement with the confocal images shown in Fig. 5b and c where different emission can be observed depending on the aggregation state of HTlc nanoparticles. It is likely that cross-linking by the Si–O–Si network between different particles or by T4–T4 π–π interactions are underpinning the nanoplatelets aggregation observed in the films. Indeed, the prediction of the mechanism of reaction and type of bonding in heterogeneous silylation processes is still a matter of debate and is difficult to precisely define due to the multiple possibility of hydrolysis and Si–O bond formation.^16c Finally, AFM investigation of T4/ZnAl-HTlc, 1c substrate shows a grainy surface with a roughness comparable to that of the pristine ZnAl-HTlc (RMS roughness of about 130 nm) (see Fig. S2†).

Fig. 6 (a) SEM images at different magnifications of pristine ZnAl-HTlc 1a (a) and of T4/ZnAl-HTlc composite 1c (b–d). The yellow arrows indicate the particles with more evident hexagonal shape.

3.7 Biocompatibility of T4/ZnAl-HTlc composites on HepG2 cells

The crucial role of functionalized HTlc nanoparticle size for successful cellular uptake and viability has been clearly demonstrated.^16e

In this context, to explore the potential of T4/ZnAl-HTlc products for biomedical applications, the biocompatibility of the T4/ZnAl-HTlc (sample c) nanocomposite was estimated on liver hepatocellular carcinoma HepG2 cell line, after exposure to the dispersed nanoparticles for 24 h at 37 °C in cell culture media (Fig. 7). Single plane confocal images of FDA assay positive cells revealed that the viable (fluorescent) cells were visible in not-treated HepG2 cells, as well as in cells incubated for 24 h with pristine HTlc (100 μg ml⁻¹, Fig. 7a and b). Notably, cells treated with the same amount of silylated-HTlc (Fig. 7c, sample c, 100 μg ml⁻¹) displayed comparable viability. Histogram plot reporting averaged percentage of living cells (Fig. 7d) confirmed that sylilation of ZnAl-HTlc nanoparticles is not altering their well-known biocompatibility, proven for different cell types.²⁶

Fig. 7 (a–c) Single plane confocal images representative of fluorescein diacetate emission collected by HepG2 FDA positive cells (viable cells) in not-treated (a) pristine HTlc treated (100 μg ml⁻¹, b) and after 24 h of incubation with sample c (100 μg ml⁻¹, c). (d) Histogram plot reporting mean and SE of % of viable cells counted in different conditions. (e) High magnified (60×) confocal image representative of fluorescent emission collected at wavelength >600 nm, from HepG2 cells after 24 h of incubation with 100 μg per ml of sample c, and by not-treated cells (inset).

Moreover, to explore the potential of sample 1c as a fluorescent diagnostic tool, we collected a series of confocal images of cells loaded with dispersion of sample c for 24 h and then washed several times with PBS. In some cells, we observed a compartmentalized, spotty or patched fluorescence, surrounding cell nucleus or in the proximity of the inner cell membrane compartments (Fig. 7e, arrows), suggesting that the nanocomposite was internalized into living cells. FITC–sylilated-HTlc, with a diameter lower than 200 nm, were shown to be uptaken by secondary cell culture and to co-localize with subcellular caveolar structures located in the proximity of the internal plasma membrane.^16e

In agreement T4–sylilated-HTlc with an average size of 150–200 nm (Fig. 6b) are capable to internalize into living cells and displayed the patchy distribution pattern resembling the one reported by Oh et al.^16e Future studies will clarify, possible involvement of lipid raft component in the internalization mechanisms or rather in the subcellular compartmentalization of HTlc.

4. Conclusions

In summary, we have here described a novel and efficient surfactant-free method to chemically tailor the HTlc NPs surface by a synthetic procedure requiring only few minutes rather than several hours or days. A new class of fluorescent, filmable and biocompatible composites with high potential for photonics sensing and biological purposes can be achieved by exploiting this method, and by varying the type of HTlc and/or the dye counterpart. X-Ray diffraction study demonstrates that surface modification of the HTlc NPs exclusively occurs, enabling further functionalization of the interlayer region with other organic or inorganic moieties. Elemental analysis according to TG data shows that the dye loading can be tuned by changing the MW irradiation time, this suggesting the possibility of grafting different dyes in predefined ratio.

Sequential surface MW-assisted silylation and intercalation experiments are currently under way to simultaneously tailor the NPs surface and their interlayer region.

Acknowledgements

This work was supported by FP7-PEOPLE-2012-ITN 316832-OLIMPIA by Italian MIUR project FUTURO in RICERCA – RBFR12SJA8, by Fabbrica del Futuro: Silk-IT and by the FESR Operational Programme 2007–2013 of the Emilia-Romagna Region – Activities I.1.1. The authors wish to thank Dr Franco Corticelli (IMM-CNR) for the precious assistance for SEM analysis.

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures, synthesis, DSC analyses, hot stage polarized optical microscopy. Supplementary figures S1 and S2. Fig. S1: shapes of a water droplet (1 μl) on the surface of pristine ZnAl-HTlc film and T4/ZnAl-HTlc composites. Fig. S2: AFM images of pristine ZnAl-HTlc and T4/ZnAl-HTlc films. See DOI: 10.1039/c3ra46669h

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