2. Experimental

#### 2.1 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(NO3)36H2O (Merck) were also used.

#### 2.2 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-d6) were measured at Bruker FT-400MHZ spectrophotometer. The IR spectra were recorded on a Perkin Elmer 437 IR spectrophotometer (400–4000 cm<sup>1</sup> ) (KBr technique). Thermogravimetric analysis was performed using a Shimadzu TGA-50H with a flow rate of 20 ml min<sup>1</sup> . 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.

#### 2.3 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 <sup>10</sup><sup>3</sup> ) were cultured in a 96-well plate overnight at 37°C, 5% CO2, 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% CO2 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% CO2 for 3–5 h to

Synthesis and Characterization of Nanocomplexes by Green Chemistry and Their Applications… DOI: http://dx.doi.org/10.5772/intechopen.83558

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 100.

### 2.4 Flow cytometry

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 [12–16]. Dressings play multimodal roles in wound healing process, such as preventing

indispensable [17]. Electrospinning is the method to fabricate ultrafine fibers from polymer solution(s) or melt(s) with diameters ranging from nanometers to

The obtained results revealed the successful implementation of the nanoparticle-

In this study all chemicals used were of highest purity. Organic solvents such as ethyl alcohol and dimethylformamide (DMF) were spectroscopically pure from

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.

H NMR spectra (DMSO-d6) were measured at Bruker FT-400MHZ spectrophotometer. The IR spectra were recorded on a Perkin Elmer 437 IR spectrophotometer

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)

Different concentrations of the nanometal complexes were tested for their cyto-

) were cultured in a 96-well plate overnight at 37°C, 5% CO2,

toxicity against Vero, Caco2, and MCF-7 cell lines using (MTT) Thiazolyl Blue Tetrazolium Bromide method according to [19, 20]. In brief, Vero, Caco2, and

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% CO2 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% CO2 for 3–5 h to

using a Shimadzu TGA-50H with a flow rate of 20 ml min<sup>1</sup>

(Philips XL 30) with gold coating and TEM.

) (KBr technique). Thermogravimetric analysis was performed

. The UV-vis spectra

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(NO3)36H2O (Merck)

infections, absorbing exudates, and maintaining moisture, and thus are

nanofiber as a local wound dressing especially for skin injuries.

micrometers [18].

Green Chemistry Applications

2. Experimental

were also used.

1

20

2.2 Instrumentation

(400–4000 cm<sup>1</sup>

2.3 Cytotoxicity assay

MCF-7 cells (10 <sup>10</sup><sup>3</sup>

2.1 Materials and reagents

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.

#### 2.5 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].

### 2.6 Synthesis

### 2.6.1 Synthesis of complex nanoparticles by sonochemistry method (green chemistry)

Ten milliliter of 0.1 M solution of La(NO3)3.6H2O 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 0.1 M solution of N'-((E)-5-bromo-2 hydroxybenzylidene)-6-(((E)-5-bromo-2-hydroxybenzylidene)amino)-4-oxo-2 thioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine-7-carbohydrazide H2L<sup>1</sup> , N-(2 mercaptophenyl) benzamide H2L<sup>2</sup> , and 2-((2-mercapto- phenyl) imino)-1,2 diphenylethan-1-ol H2L<sup>3</sup> was added dropwise. The obtained precipitates were allowed to evaporate at room temperature to obtain the complex nanoparticle in powder form.

### 3. Results and discussion

#### 3.1 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:

3.3 IR spectra and mode of bonding

DOI: http://dx.doi.org/10.5772/intechopen.83558

H NMR data for La(III) nanocomplexes.

Table 2. 1

number 1609–1565 and 1280–1242 cm<sup>1</sup>

3.4 Molar conductivity measurements

3.5 Electronic spectral data

forbidden and very weak in nature.

23

ductance values of 20.51 and 3.39 ohm<sup>1</sup> mol<sup>1</sup> cm2

ligands [26, 27].

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<sup>1</sup> is found to be disappeared by complexation suggesting the involvement

Compd. no. δph δCH=N δSH δNH δOH 6.39–8.46 8.00 ––– 6.99–7.48 – 3.5 5.4 – 7.00–8.00 – 3.47 – 10.4

Synthesis and Characterization of Nanocomplexes by Green Chemistry and Their Applications…

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<sup>1</sup> due to υC=<sup>N</sup> and υC▬<sup>O</sup> (azomethine and phenolic groups) are shifted to lower wave

ing 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<sup>1</sup> 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<sup>1</sup> which may be due to υM▬<sup>O</sup> υM▬<sup>N</sup> and υM▬S, respectively, that are not observed in the spectrum of the free

The conductivity Λ<sup>m</sup> value of the La nanocomplexes 1–3 can be calculated by using the relation Λ<sup>m</sup> = K/C. The complexes were (10<sup>3</sup> M) dissolved in DMF, and the molar conductivities of their solutions at 252°C were measured (Table 1). It is concluded from the results that complexes 1 and 3 are found to have molar con-

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<sup>1</sup> mol<sup>1</sup> cm2 indicating the ionic nature of this complex, and the nitrate ion is outside the coordination sphere.

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 Laporte-

, respectively, in metal complexes indicat-

, respectively, indicating that

#### 3.2 <sup>1</sup> H NMR spectra

The <sup>1</sup> 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 <sup>1</sup> 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].


#### Table 1.

Elemental analysis and some physical measurements of La(III) nanocomplexes (1-3).

Figure 1.

1 H NMR data for La(III) diamagnetic complex (1).

Synthesis and Characterization of Nanocomplexes by Green Chemistry and Their Applications… DOI: http://dx.doi.org/10.5772/intechopen.83558


Table 2.

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

HNMR spectra (Figure 1, Table 2) of the La (III) nanocomplex 1 showed

H NMR spectra of complex 3, showed singlet peaks due to the

29.90 (30.01)

2.94 (3.06)

2.83 (2.90)

(%) found (calcd.)

1.32 (1.56)

27.75 (27.87)

36.54 (36.31)

C HN S Λm\*

9.92 (10.00)

> 5.40 (5.71)

4.55 (4.83)

7.58 (7.62)

9.80 (10.00)

> 8.33 (8.47)

20.51

57.88

3.39

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

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].

> Color (yield %)

<350 Faint orange (80.54)

110 Dark gray (85.49)

165 Page (79.80)

Elemental analysis and some physical measurements of La(III) nanocomplexes (1-3).

(°C)

following general equations:

Green Chemistry Applications

H NMR spectra

In addition to the <sup>1</sup>

1 [(La)(L1

3 [La(H2L<sup>3</sup>

2 [La(H2L<sup>2</sup>

(Empirical formula) M.P.

)(NO3) (H2O)]

). (NO3)2(H2O)2]NO3. H2O

> )(NO3)3. H2O]

H NMR data for La(III) diamagnetic complex (1).

3.2 <sup>1</sup>

The <sup>1</sup>

deprotonation.

Compd. code

\*ohm <sup>1</sup> cm2 mol<sup>1</sup>

Table 1.

Figure 1. 1

22

1 H NMR data for La(III) nanocomplexes.
