Influence of the surface modification of BaTiO3 nanoparticles by hydrolyzed chitosan obtained from shrimp exoskeletons on the optical response intensity of the second harmonic

Ferroelectric nanoparticles, due to their high non-linear optical response, are of considerable practical interest for their use as nanoprobes for the study of biological materials by multiphoton microscopy methods. To prevent toxic effects, it is necessary to modify the surface using biocompatible polymers. Here we report the results of the study on the effect of surface modification of BaTiO3 nanoparticles with hydrolyzed chitosan obtained from shrimp exoskeletons (Penaeus vannamei). It was found that increasing of the hydrolyzed chitosan concentration in the solution during surface modification from 1 to 3% leads to a significant reduction in nanoparticle agglomeration, while the intensity of the second harmonic generation changes insignificantly.


Introduction
Ferroelectric BaTiO 3 nanoparticles, due to their non-centrosymmetric structure, have been considered potential candidates in the manufacture of nanoprobes for biomedical research at the cellular level by means of two-photon microscopy or second harmonic generation (SGH) under continuous wave or femtosecond laser excitation, which can provide high contrast in bioimaging applications [1,2]. Physically, SGH is a non-linear optical process that converts two photons into one photon with half the wavelength of the excitation laser [3,4]. In addition, they are promising candidates for tissue engineering, cell stimulation, drug delivery, as well as for cancer therapy [5]. For reducing the cytotoxic effect of the BaTiO 3 nanoparticles, which is caused by to Ba 2+ leaching occurrence from the surface in aqueous solutions at pH < 12 [6], the surface is modified using various organic compounds, for example hydroxyl groups or amine ring groups [5]. The most widely investigated ways of modifying BaTiO 3 nanoparticles are by silane coupling agents [7]. The influence of different solvents and coating methods on the structure and properties of the obtained nanocomposites was investigated for them [8].
However, the use of biopolymers as modifying agents has been studied to a lesser extent. Chitosan is one of the most promising materials, because of the previously demonstrated high biocompatibility of BaTiO 3 -chitosan nanocomposites in a wide range of concentrations in aqueous solutions (0-100 μg/mL) [9,10]. One of the most promising sources of chitosan is shrimp waste, which not only has high commercial potential, but also makes a significant contribution to recycling [11]. Earlier it was shown that nanostructured chitosan obtained by this method has high purity and advanced optical characteristics [12].
Structurally, the nanoprobes used for diagnosis and imaging are core-layer nanocomposites in which the ferroelectric core is encapsulated in a biocompatible layer. It should be noted that the size of barium titanate nanoparticles must be larger than the critical size in order to retain their ferroelectric properties [13]. Previously, we showed a significant decrease in the non-linear optical and piezoelectric response of barium titanate nanoparticles when their surface is modified with chitosan [14,15]. This fact can affect the intensity of the SGH generation, affecting the brightness and stability of the probe radiation. This is primarily caused by the thickness of the modifying coating capable of absorbing and scattering the response generated by the probe. Therefore, the aim of the paper is to investigate the effect of the surface modifying hydrolyzed chitosan obtained from shrimp exoskeletons on the intensity of the optical response of the second harmonic.

Materials and methods
BaTiO 3 ferroelectric nanoparticles were obtained by thermal treatment (900 °C, 6,5 h) of commercially available nanoparticles (Sigma Aldrich, average size 50 nm) with a spherical shape initially in a paraelectric cubic phase in air [16]. Then, 1 g of BaTiO 3 nanoparticles was refluxed in a 400 mL 30% aqueous solution of H 2 O 2 (J.T. Baker) to enrich the surface by hydroxyl groups. The surface modification was carried out with chitosan hydrolyzed (CH) in sulfuric acid at concentrations of 1, 2 and 3 (%v/v) (CH1, CH2 and CH3). Chitosan hydrolyzed 0.125 g was added and diluted in 50 mL of acetic acid (CH 3 COOH) and then was being mixed for 24 h at 40 °C. The pH of the solution was regulated to 4.7 with 10% aqueous solution NaOH and then the solution was filtered through a 0.45 μm membrane. Then 0.1 g of BaTiO 3 treated with H 2 O 2 was added and mixed for 4 h at temperature of 40 °C. To that solution, 50 mL of sodium tripolyphosphate (STPP)-0.4 mg/mL dissolved in milli-Q water were added dropwise under magnetic stirring, for reducing BaTiO 3 nanoparticles agglomeration. Finally, they were washed, filtered and dried at 90 °C during a day; posteriorly, were dispersed in aqua by ultrasonic treatment (60 W, 5 min.).
The modified nanoparticles crystal structure and morphology were evaluated by XRD Bruker D8 Eco Advance and STEM Vega 3 LMU-Tescan, respectively. For that study, 4 μL of each sample were deposited on copper grids covered with a carbon film; the images were obtained through a bright transmitted electron detector (TE bright) applying voltage of 30 kV. The dynamic light scattering analysis (Nicomp Nano Z3000) was used to evaluate the dispersibility of BaTiO 3 nanoparticles modified with hydrolyzed chitosan in aqueous solutions and for the measurement of the zeta potentials of the obtained solutions. The surface chemistry of each sample was characterized by FTIR-ATR (Thermo Fisher Nicolet iS-50) in absorbance mode; the spectra were taken in the region of 500 to 4000 cm −1 , with 6 scans for each sample, at a resolution of 1 cm −1 . To demonstrate the generation of the second harmonic, the samples (nanoparticles powders) were irradiated with pulsed infrared laser radiation from a Nd: YAG laser. (1064 nm, 34.78 mJ and 42.72 mJ, 10 ns). The laser radiation was directed to the surface of the samples by a mirror 100% reflective of infrared radiation. To collect the light emitted by the samples, a suitable optical system was installed, consisting of an optical fiber and an HR4000 spectrometer from Ocean Insight.

Results and discussion
The observed splitting of the reflexes (001/100), (002/200), (012/120) demonstrates the formation of the ferroelectric tetragonal phase with non-linear optic response in nanoparticles during their annealing (Fig. 1). At the same time, the estimation of the particle size from the broadening of the diffraction reflex according to the Scherrer equation, demonstrates an increase in the average particle size to 77 nm. While amorphous halo around 17-18 degrees is caused by the chitosan [17].
The scanning transmission electron microscopy images (Fig. 2) demonstrate that the chitosan concentration increase in the solution with the used modification technique from 1 to 3 (%v/v) significantly reduces particle agglomeration.
The aqueous dispersions stability of the modified with chitosan BaTiO 3 nanoparticles in the solutions was investigated by DLS method. The obtained data shows no significant change in the average particle size after 24 and 48 h since the suspensions preparations (Fig. 1, Supporting  materials). Figure 3 represents the FTIR spectra of the BaTiO 3 nanoparticles enriched with hydroxyl groups (BaTiO 3 -OH) and of the subsequent modifications with chitosan hydrolyzed at 1, 2 and 3% in sulfuric acid (BaTiO 3 -OH@CH1, BaTiO 3 -OH@CH1 and BaTiO 3 -OH@CH1). The intense band at 560 cm −1 shown in all spectra represents the vibration of the Ti-O bond of BaTiO 3   attributed to the -NH groups in the chitosan and the broadening are possibly due to physical interaction with STPP [11]. The bands at 1640 cm −1 , 1550 cm −1 and 1416 cm −1 , are referred to the presence of C=C stretch, to stretching vibrations of C-O group and aromatic C-C stretch, respectively; all associated with chitosan. Similar absorption peaks were obtained in the commercial chitosan samples [17]. The absorption band shown at 890 cm −1 may be attributed to the shift of the P-O stretching that comes from the ionotropic gelation process with STPP [18]. Therefore, these results demonstrated the surface modification of the BaTiO 3 nanoparticles with the hydrolyzed chitosan.
The results of the experiments on the SGH investigation in the samples under the study (Fig. 4) show that even the particle surfaces modification in a solution containing 1 (%v/v) hydrolyzed chitosan significantly (almost tenfold for pulses with energy of 34.78 mJ and sixfold for pulses with energy of 42.72 mJ) reduces the signal intensity as compared to the unmodified nanoparticle sample. Apparently, this can be related both to the fact that the modifying biopolymer layer can absorb the excitatory radiation as it propagates toward the ferroelectric particle core and the second harmonic signal. However, the results demonstrate that the chitosan concentration changing in the solution has almost no effect on the intensity of the second harmonic generation in the case of laser pulses 34.78 mJ and has little effect in the case of pulses 42.72 mJ. Thus, it should be concluded that the chitosan concentration changing in the solution during the modification in the studied interval does not lead to a significant increase in its layer thickness.

Conclusions
The obtained results demonstrate the significant decrease in the agglomeration of BaTiO 3 ferroelectric nanoparticles when they are modified in the hydrolyzed chitosan solution obtained from the shrimp exoskeletons as its concentration in the solution increases from 1 to 3 (%v/v). At the same time, the surface modification leads to the significant decrease in the intensity of the second harmonic generation. However, the chitosan concentration changing from 1 to 3 (%v/v) practically does not change the intensity of SGH by the nanoparticles. Thus, it should be concluded that the increase in the concentration of the chitosan in the solution in the range under study does not lead to an increase in the thickness of the biopolymer layer on the surface of BaTiO 3 nanoparticles, but promotes the formation of a more uniform structure of this layer, which can prevent more effectively their agglomeration.