Synthesis and Characterization of Nanocomplexes by Green Chemistry and Their Applications in Different Fields

In this study, a convenient synthesis of a series of La(III) complexes (in nanoscales) with different organic ligands was prepared by green chemical method. The physicochemical properties of the nanocomplexes were obtained. The SEM and TEM techniques were used to confirm the nanosize and morphology. The cytotoxicity of the newly synthesized compounds was demonstrated and tested for their anticancer activity against common pathogenic organisms and against human epithelial colorectal adenocarcinoma cells (Caco2), Vero, and MCF-7 cells. By examining its pharmacological activity, we were able to identify new potent and selective anticancer agents. Also, the structures succeeded to affect and stop the growth of Klebsiella pneumonia strain, and the results revealed promising activity. In addition, an electrospinning technique was used for the fabrication of novel bio-medicated nanofibers which are applied for wound dressing as local chemotherapy for skin injuries.


Introduction
The Environmental Protection Agency defined the green chemistry or sustainable chemistry as "the production of chemical products and processes which have the ability to reduce or eliminate the use or generation of hazardous substances." In recent years there is an expectation that chemists and chemical engineers should design greener and more sustainable chemical processes, and it was expected that this trend will continue to grow over the next few decades [1].
Studies on metal-based drugs offer promise in the fight against cancer, which is the main aim of the inorganic chemistry [2,3].
The metal-based anticancer drugs are driven by the necessity to fill the gap in tumor chemotherapy in order to minimize the undesirable side effects and enlarge the spectrum of activity for more tumor types and metastatic cancers. The Schiff base structures play a key role in studying the mechanism of the transformation and racemization reaction in biological systems due to the similarity of their structures to the natural biological system [4][5][6][7][8][9][10][11].
Among various metal complexes, La(III) complexes have been intensively investigated due to their more physiological activities and lower toxicities after coordination with ligand, so, in recent years people have paid great interest to synthesis, DNA interaction, and anticancer activity of La(III) complexes [12][13][14][15][16].
Dressings play multimodal roles in wound healing process, such as preventing infections, absorbing exudates, and maintaining moisture, and thus are indispensable [17]. Electrospinning is the method to fabricate ultrafine fibers from polymer solution(s) or melt(s) with diameters ranging from nanometers to micrometers [18].
The obtained results revealed the successful implementation of the nanoparticlenanofiber as a local wound dressing especially for skin injuries.

Materials and reagents
In this study all chemicals used were of highest purity. Organic solvents such as ethyl alcohol and dimethylformamide (DMF) were spectroscopically pure from the British Drug House (BDH). The other materials were purchased from Sinopharm Chemical Reagent Co., Ltd. and used without further purification. In addition to the 5-bromosalicylaldehyde (Sigma-Aldrich), La(NO 3 ) 3 Á6H 2 O (Merck) were also used.

Instrumentation
In open capillaries melting points (°C, uncorrected) were determined on a Gallenkamp melting point apparatus (Sanyo Gallenkamp, Southborough, UK). In the Microanalytical Center, Cairo University (C, H, N, and S) were carried out. 1 H NMR spectra (DMSO-d 6 ) were measured at Bruker FT-400MHZ spectrophotometer. The IR spectra were recorded on a Perkin Elmer 437 IR spectrophotometer (400-4000 cm À1 ) (KBr technique). Thermogravimetric analysis was performed using a Shimadzu TGA-50H with a flow rate of 20 ml min À1 . The UV-vis spectra were recorded on a Perkin Elmer Lambda 3B UV-vis spectrophotometer. X-ray diffraction were recorded at room temperature ($25°C) on Empyrean X-ray diffractometer equipped. The patterns were run with Cu target (CuKα radiation), and the tube operated at 45 kV and 30 mA. The size and morphology of the nanocomplexes were characterized with a scanning electron microscope (SEM) (Philips XL 30) with gold coating and TEM.

Cytotoxicity assay
Different concentrations of the nanometal complexes were tested for their cytotoxicity against Vero, Caco2, and MCF-7 cell lines using (MTT) Thiazolyl Blue Tetrazolium Bromide method according to [19,20]. In brief, Vero, Caco2, and MCF-7 cells (10 Â 10 3 ) were cultured in a 96-well plate overnight at 37°C, 5% CO 2 , and 88% humidity. The total volume of used DEMEM supplemented medium and the synthesized compound supernatant was 200 μL with final concentrations of 10, 20, 30, 40, 60, 80, and 100 mg/L. The plate was incubated at 37°C and 5% CO 2 for 3 days. After incubation, debris and dead cells were removed by washing three times with fresh medium. Twenty microliters of MTT solution (5 mg/mL of MTT in PBS buffer) was added to each well and shook for 5 min at 150 rpm to thoroughly mix the MTT into the media. The cells were incubated at 37°C and 5% CO 2 for 3-5 h to metabolize MTT by viable cells. Two hundred microliter dimethyl sulfoxide (DMSO) was added to each well and shook again for 5 min at 150 rpm, and then the viability of the cells was calculated by measuring the optical density at 630 nm subtracted from optical density at 570 nm [21]. The percentage of viability cells was calculated by comparison with control cells (without adding the synthesized compounds to the cells) using the equation (A) test/(A) control Â 100.

Flow cytometry
To discriminate living cells from dead cells or for cell cycle analysis, propidium iodide (PI) can be used according to Léonce et al. [22]. This analysis is based on the stoichiometric binding of PI to intracellular DNA. At the end, cells were washed with PBS and collected by trypsinization. Cells were then resuspended in warm PBS and fixed with 4 ml ice-cold ethanol. Finally, in darkness, cells were stained with 0.5 ml of warm PI solution; 7 ml of PI solution consists of 0.35 ml of PI stock solution (1 mg/ml), 0.7 ml RNase A stock solution (1 mg/ml), and 6 ml of PBS and incubated in darkness for 30 minutes. The samples were kept on ice until flow cytometric analysis.

Preparation of (chitosan-La(III) nanocomplex) composite nanofibers
Chitosan (CS) was dissolved in 50% acetic acids at room temperature under moderate stirring for 12 h to form a homogeneous solution, and then the solution of La complex nanoparticles was quietly adding. The total solution was fixed to 3 wt.%. The solution was filled in a 20 ml NORM-JECT Luer Lock tip plastic syringe having an 18-gauge stainless steel needle with 90°blunt end. The electrospinning setup included a high voltage power supply, purchased from the NanoNC, Inc. (S. Korea), and a nanofiber collector of aluminum foil that covered a laboratoryproduced roller with the diameter of 10 cm. The collector was placed at 10 cm tip-to-collector distance (TCD) [39].

Synthesis of complex nanoparticles by sonochemistry method (green chemistry)
Ten milliliter of 0.1 M solution of La(NO 3 ) 3 .6H 2 O in ethanol was positioned in a high-density ultrasonic probe, operating at 24 kHz with a maximum power [23] output of 400 W. Ten milliliter of 0 The obtained precipitates were allowed to evaporate at room temperature to obtain the complex nanoparticle in powder form.

Elemental analyses for nanometal complexes
In the present investigation, the analytical data of the prepared metal complexes suggest the structures as given in Figure 6. The physicochemical results of the newly synthesized compounds were presented in Table 1. The obtained results were in good agreement with those calculated for the suggested formulae. The melting points were sharp, indicating the purity of the prepared nanometal complexes. The proposed formula of the nanometal complexes is according to the following general equations:

1 H NMR spectra
The 1 HNMR spectra ( Figure 1, Table 2) of the La (III) nanocomplex 1 showed a singlet peak at 8.00 ppm assigned to azomethine proton, in addition to the multiplet signals due to aromatic protons. It was found that the spectra of complex 2 display singlet peaks due to SH and NH groups at δ3.5 and 5.4 ppm indicating the involvement of these groups in coordination with the metal atoms without deprotonation.
In addition to the 1 H NMR spectra of complex 3, showed singlet peaks due to the O▬H proton at 10.4 ppm and the S▬H proton peak at 3.47 ppm. This analysis showed that the O▬H and S▬H groups share in the complexation a loss of their protons. Moreover, the spectra of the complexes showed multiplet signals at 6.99-7.48 ppm attributed to the aromatic protons for all complexes [24,25].

IR spectra and mode of bonding
In order to ascertain the mode of bonding, the IR spectrum of the free ligands was compared with those of their La complexes. By careful comparison of the spectra of the complexes with those of the free ligands, it was found that the band at 3430 cm À1 is found to be disappeared by complexation suggesting the involvement of OH groups of the phenolic groups in complex formation with their deprotonation. The bands due to NH and SH are shifted to higher or lower frequencies indicating the participation of -N and -S donors in the coordination, while the bands due to C=O are unchanged. The strong bands at 1622 and 1298 cm À1 due to υ C=N and υ C▬O (azomethine and phenolic groups) are shifted to lower wave number 1609-1565 and 1280-1242 cm À1 , respectively, in metal complexes indicating the participation of nitrogen of azomethine and oxygen of phenolic group in complexation. The complexes showing also three bands at the range 1480-1400, 1380-1350, and 1170-1020 cm À1 indicate the unidentate coordination mode of the nitrate group. The curves also, showed that shift in the peaks of O▬H, C▬O, C▬S groups (complex 3) due to their sharing in the complexation and that recorded by the M▬O, and M▬S peaks. All these results are in good agreement with the conductance data. Another evidence for the coordination is the observance of new bands in the far IR region at 570-505, 520-440, and 432-524 cm À1 which may be due to υ M▬O υ M▬N and υ M▬S , respectively, that are not observed in the spectrum of the free ligands [26,27].

Molar conductivity measurements
The conductivity Λ m value of the La nanocomplexes 1-3 can be calculated by using the relation Λ m = K/C. The complexes were (10 À3 M) dissolved in DMF, and the molar conductivities of their solutions at 25AE2°C were measured ( Table 1). It is concluded from the results that complexes 1 and 3 are found to have molar conductance values of 20.51 and 3.39 ohm À1 mol À1 cm 2 , respectively, indicating that these complexes are nonelectrolytic and monomeric in nature. Also the values indicate the bonding of the nitrate ions to metal cations [28,29]. The molar conductivity value of complex 2 is seen to be 57.88 ohm À1 mol À1 cm 2 indicating the ionic nature of this complex, and the nitrate ion is outside the coordination sphere.

Electronic spectral data
The assignments of the significant electronic spectral absorption band of the nanometal complexes are given in Table 3. Two absorption peaks were observed in the spectra range of 210-227 and 279-337 nm due to п-п* and n-п* transitions, respectively, due to benzene and the azomethine (CH=N) function [26,29]. Moreover, the spectra of complexes indicate no significant absorption in the visible region due to the absence of f-f transition, since f-f transitions are Laporteforbidden and very weak in nature.

Magnetic susceptibility
The following equation was used to calculate the magnetic susceptibility of the La nanocomplexes: where C, calibration constant of standard; l, length of sample in filled tube; R 0 , reading of empty sample tube; R, reading with sample; χ g gram magnetic susceptibility.
The gram magnetic susceptibility of the complexes was found in the range À2.2098 Â 10 À6 -(-2.3013 Â 10 À6 ) cm 3 mol À1 , respectively. Negative values of χ g show that these complexes are diamagnetic in nature or there is no significant magnetic moment ( Table 3).

Thermal analysis
Thermogravimetric analyses (TGA) of the La nanocomplexes were used to determine the thermal stability of the complexes, decide whether the water molecules are lattice or coordinated, and finally suggest a scheme for thermal decomposition of these chelates. In the present investigation, heating rates were suitably controlled at 10 o C min À1 under nitrogen atmosphere, and the weight loss was measured from the ambient temperature up to $ 600 o C. The weight loss for each chelate was calculated within the corresponding temperature ranges.
Also, TGA curve of the La(III) complex of nanoscale [(La)(L 1 )(NO 3 )(H 2 O)] (1) showed three thermal stages. The first stage represents the loss of H 2 O molecule of coordination, HNO 3 and ½O 2 with a found weight loss of 11.72% (cacld. 11.54%) within the temperature range of 130À180 o C; the subsequent second step (180À300 o C) corresponds to the loss of C 5 Table 3.
Magnetic moment and electronic spectral data of La(III) nanocomplexes.

Powder X-ray diffraction studies
The diffraction of X-rays from the planes of a crystal (diffraction analysis) is considered as one of the important methods of the analytical work. By using X-ray diffraction which depends on the crystal properties of solids, we can identify the crystal structure of various solid compounds and identify the actual compounds from its structure, and we can also determine the arrangement of molecules in crystal [30]. X-ray diffractometry is an important technique as it is a nondestructive, non-contrast, fast, and sensitive one. Obtained XRD data of H 2 L 1 ligand and its La(III) complex 1 of nanoparticle ( Figure 2) show that XRD pattern of the ligand is different in comparison with the XRD pattern of the nanocomplex. The obtained data indicate that the complex is more crystalline in nature than that of the ligand which has an amorphous phase. The average particle size of the complex (1) can be estimated from Debye-Scherrer equation (Eq. (2)): where β the breadth of the observed diffraction line at its half intensity maximum; K, the shape factor, 0.89; λ, the wavelength of the X-ray source used in XRD; θ, the corresponding incidence angle.
The average particle size of the nanoparticles is 15 nm. The sharp diffraction peaks of the samples indicate that the well-crystallized lanthanum complex nanocrystals can be prepared by our procedure [23].

EDX spectra
The amount of elements present in the percentage level of the metal complexes was identified by EDX data [31]. EDX spectra are used to calculate the percentage level of the elements present in the metal complexes like C, O, N, S, and La that are present in the La(IIl) nanocomplex 2, shown in Figure 3. The revealed data are in good agreement with that of the elemental analysis.

SEM analysis
SEM technique was used to study the morphology and size of nanoparticles ( Figure 4). It seems that the particles are semispherical with some agglomerations of the particles. The SEM images further revealed the stabilization of La(III) 1 nanoparticles due to interaction with the Schiff base ligand; this stabilization facilitates penetration of tumor cell membrane and causes the destruction of tumor cell [32].

TEM analysis
Transmission electron microscopy (TEM) can be used to directly image nanoparticles at scales approaching a single atom. TEM analysis is performed to examine the size and shape of the nanoparticles. The La(III) complex 2 nanoparticle was fairly uniform in size, spherical in shape, and with the average diameter  ranging from 3.2 to 44.9 nm ( Figure 5). Electron microscopy analysis allowed confirming visually the observed stability of the obtained nanocomplex.
Correlation of all results obtained for the complexes under study gives us information regarding the suggested structure of the complexes to be as in Figure 6. The fully optimized geometries of the ligand and its metal complexes were shown in Figure 7.

Cytotoxicity and anticancer activity
The cytotoxicity of the prepared nanocomplexes has been demonstrated and tested for their anticancer activity against Caco2, Vero, and MCF-7 cell lines. The obtained results revealed that complex 1 had the best recorded anticancer activity against Caco2 cells with inhibition percentage of 72.13 ( Figure 8B). The IC 50 (μg) (the value which corresponds to the concentration required for 50% inhibition cell viability) for the noncytotoxic doses against human primary dermal fibroblasts cells were recorded as 2.482 μg/ml. The IC 50 for these structures were recorded as 0.3772 μg/ml for compound 1 on Caco2 cells. In spite of the potency of the sample as anticancer agents, the superior selectivity index values against Caco2 cells than noncancerous cells were recorded with a value of 46.6.
On the other hand, the other tested compounds have shown moderate activities against all cells; the anticancer activity is less than that recorded for 1.

Flow cytometry
Flow cytometric analyses of propidium iodide-stained nuclei cells were performed to investigate whether treatment could induce cell cycle perturbations in human epithelial colorectal adenocarcinoma. Cell cycle parameters were compared for Caco2 cells that had been incubated for 24 h with 1 ml of IC 50 concentrations of sample 1 with untreated (control) cells. As shown in Figure 9, following 24 h incubation with treatment, there was an increase in the proportion of cells in the G2/M phase (51.10%, for treatment 1).
Also, an additional peak was observed, consistent with aneuploid cells which contain more than 4n DNA. This peak may represent a population of cells that has escaped mitotic arrest and continued to replicate as multinuclear cells without dividing (i.e., endoreduplication), or it may be due to a small fraction of tetraploid

Chitosan-nanocomplex 2 composite nanofibers
The chitosan played an important role in the synthesis of the composite nanofiber. The amino group has been used to stabilize metal nanoparticles [33], so we suggested that the free NH 2 ligands of chitosan might bind to the nanoparticles and stabilized them.

FTIR
The formation of the CS-stabilized NPs was further investigated by FTIR spectroscopy. The FTIR spectra of the CS-NP nanofiber and CS nanofiber attributed a broad band at the range of 3350-3510 cm À1 corresponding to the amine and hydroxyl groups. For the nanocomposite, there is a change in the shape and frequencies of the bands in this region, indicating participation of the NH 2 group in the reaction. Although there is some difference in the two figures, both of them showed the basic characteristic peaks for CS at 3422 cm À1 (O▬H stretch) and 2960-2970 cm À1 (C▬H stretch), which indicated that the CS can adsorb on the formed nanoparticles in the preparation process [34,35].

TGA
Thermogravimetric analysis (TGA) is widely used to investigate the thermal decomposition of a polymer. The TGA thermograms of pristine CS nanofiber, CS-NP composite nanofiber, at a heating rate of 20°C/min under nitrogen atmosphere are investigated. Chitosan's nanofiber weight change between 28.27 and 100°C is associated with the loss of adsorbed and bound water. Chitosan polymer degradation started at 100°C and continued up to 333.80°C, with a 75.5% polymer weight loss [36,37]. The CS-NP composite nanofiber exhibited a three-step degradation pattern. The first step occurred between 24.68°C and 100.86°C, which is due to water evaporation. The second and the major mass loss were observed between 100°C and 261.34°C, with a 40.42% polymer weight loss. The third step occurred between 261.34 and 367.43°C, with a 19.95% polymer weight loss with total loss of 71.27% at 367.43°C.

TEM
The NP complex was encapsulated by CS, and their sizes were in the range of 15-50 nm as shown in the TEM image of the CS-NP composite nanofiber ( Figure 10). It could be observed that a considerable amount of non-spherical particle was observed. These non-spherical particles seemed to be fabricated by coalescence of two spherical particles during the nucleation process [38].

SEM
The morphology of electrospun (chitosan-nanoparticle) composite nanofiber is shown in Figure 11. It is clear from these figures that no phase separation has been observed and the homogeneity of the obtained nanofibers can be easily observed and the composite of the nanoparticles has been proven. It is also clear that the diameter of chitosan-nanoparticle composite nanofiber is smaller than chitosan nanofiber.

Antimicrobial activity
The ability of prepared La nanoparticle-coated nanofibers to cease the growth of microbes known for its human pathogenicity has been investigated. La nanoparticlecoated nanofibers succeeded to locally cease the growth of all tested microbes. Each disc succeeded to stop the growth of tested microbes at the boundaries between the disc and the plates' surfaces, forming clear zones with 7 mm diameter. It is worth mentioning that La nanoparticle-coated nanofibers showed the same pattern of antimicrobial activity against all tested microbes. The ability of prepared nanoparticlenanofiber hybrid to affect the microbes locally is definitely promising for biomedical applications. These results would support the coexistence of the microbes and their antimicrobial agent at the same location and hence improve the overall microbe drug interaction process. Moreover, this kind of antimicrobial agent localization would prevent the probability of its migration to other sites inside the body that would help to avoid the expected subsidiary complications. It would be recommended that these nanoparticle-coated nanofibers are used as wound dressing for skin injuries.

Conclusion
La(III) nanocomplexes with different organic ligands were synthesized and characterized by different techniques. In addition to the nanosize was proved by different techniques also. All prepared metal complexes were screened against different human cell lines. It was found that we were able to identify new potent and selective anticancer compounds. Also, one of these nanocomplexes was tested as wound dressing which is recommended to be used as local chemotherapy for skin injuries.