Design, Synthesis, In Vitro Biological Activity Evaluation and Stabilized Nanostructured Lipid Carrier Formulation of Newly Synthesized Schiff Bases-Based TMP Moieties

A series of novel Schiff bases-based TMP moieties have been designed and synthesized as potential anticancer agents. The target Schiff bases were screened for their cytotoxic activity against the MDA-MB-231 breast cancer cell line. Most of the tested molecules revealed good cytotoxic activity, especially compounds 4h, 4j and 5d. Being the most potent, compound 4h showed good tubulin polymerization inhibition activity as revealed by immunofluorescence analysis and ELISA assay. Additionally, compound 4h was screened for cell cycle disturbance and apoptosis induction. Pre-G1 apoptosis and cell growth halt at the G2/M phase were discovered to be caused by it. Moreover, compound 4h induced apoptosis via p53 and Bax activation, as well as reduced the level of Bcl-2. Additionally, the most potent compound 4h was lodged on nanostructured lipid carriers (NLCs). 23 full factorial design was involved to govern the influence of the fabrication variables on the in vitro characters of the casted NLCs. F3 was picked as the optimum formula exhibiting dominant desirability value 0.805, EE% 95.6 ± 2.4, PS 222.4 ±18.7, PDI 0.23 ± 0.05 and ZP −39.2 ± 3.9 Mv. Furthermore, F3 affirmed improved solubility and release over the drug suspension. In the comparative cytotoxic activity, F3 was capable of diminishing the IC50 by around 2.15 times for pure 4h, while nearly close to the IC50 of the reference drug. Thus, NLCs could be a potential platform for boosted antitumor activity.


Introduction
Microtubules are among the most important molecular targets for cancer chemotherapeutic treatment [1][2][3]. The formation of microtubules is a dynamic process that involves the assembly and disassembly of α and β-tubulin subunits [4][5][6]. A chemotherapeutic agent binds to tubulin and interferes with this dynamic stability, and thus induces cell cycle disturbance, resulting in apoptosis induction and cell death [7][8][9]. Compounds containing trimethoxyphenyl (TMP) ring have been reported to have excellent potent cytotoxic activity against a broad range of cancer cell lines through inhibition of α and β-tubulin dynamic equilibrium [10][11][12][13]. The creation of tubulin polymerization inhibitors has been the subject of several investigations in recent years. As a result, anticancer medicines with tubulin polymerization inhibitory activity have been designed and developed using TMP-containing compounds. [14][15][16]. Colchicine (Col) I, combretastatin A-4 (CA-4) II and phenstatin III are natural compounds that bind to the colchicine binding site and prevent the polymerization of tubulins to form microtubules [17][18][19]. In addition, PIB-SA IV efficiently inhibited tumor proliferation similar to that of CA-4 [20]. Moreover, compound V showed strong antiproliferative activity against four human cancer cell lines with IC 50 values of 3-9 nm, as well as high selectivity to normal cells [21]. Additionally, the bis-hydrazide molecule VI displayed 83.1% in vitro tubulin polymerization inhibition activity at its IC 50 dose level [22] ( Figure 1).
A literature survey revealed that Schiff base derivatives are known for their various biological activities [23][24][25]. In addition, many Schiff bases exhibited promising antitumor potency against different cancer cell lines [26][27][28]. The Schiff base possessing TMP ring VII showed antiproliferative activity against the MCF-7 breast cancer cell line with an IC 50 value of 0.87 µM [26]. Furthermore, Schiff base VIII possessing Nfurylmethylidene moiety was identified as a potent antiproliferative agent against many cancer cell lines [28].
On the basis of the aforementioned structural analysis, as well as in keeping with our general enthusiasm for the design and development of novel anticancer agents, [29][30][31][32], as potential anticancer medicines, the current work focused on the design and synthesis of novel Schiff bases-based TMP moieties. The basic structural skeleton of the lead compound is formed from two TMP rings connected through amideacrylic acid hydrazide groups. The target molecules were intended to have different aliphatic and aromatic side chains at the hydrazide group to study the impact of these structural variations on antiproliferative activity. The hydrazide group in the lead compound was subjected to the replacement of the amino (NH 2 ) group to give N-ethylidene hydrazinyl derivative 2. In addition, further structure extension of hydrazide group with furan or substituted phenyl moieties afforded N-furylmethylidene hydrazinyl derivative 3 or N-arylidene hydrazinyl molecules 4a-5g, respectively ( Figure 2). The MDA-MB-231 cell line was used to test the cytotoxic activity of the target compounds. Additional studies were conducted on the most potent compound, 4h, including DNA flow cytometry and tubulin polymerization inhibition, as well as apoptotic-related tests. In addition, the most potent and promising drug was incorporated in nanostructured lipid carriers (NLCs) as an attempt to improve its solubility and other kinetic parameters, thereby boosting its cytotoxicity. This was attained by the formulation of 8 NLCs formulae of different compositions obtained from 2 3 full factorial design and the impact of the formulation variables on the EE%, PS and ZP was analyzed by Stat-Ease ® V.13 software (Design-Expert TM; Minneapolis, MN, USA). Then, the optimum formula was selected and involved in further characterization especially the comparative cytotoxicity study relative to the pure drug to demonstrate the influence of the NLCs of IC 50 on the compound.    S c hiff b ases ph armaco ph ore

Tubulin Polymerization Inhibition Assays
Several investigations have shown that TMP-containing compounds such as Col and CA-4 interact with tubulin at the colchicine binding site, inhibiting microtubule assembly [33]. The effects of representative Schiff bases on the inhibition of tubulin assembly were evaluated. Compound 4h which demonstrated potent cytotoxic activity was assessed at its IC 50 concentration. Tubulin polymerization inhibitory activity against the MDA-MB-231 cell line was investigated by immunofluorescence analysis using a confocal microscope. The results demonstrated that compound 4h exhibited good tubulin polymerization inhibition activity compared with the untreated cells ( Figure 3A). methyl group attached to imine carbon. 13 C-NMR spectra of 5a-g confirmed the ca skeleton due to the presence of two signals at δ 145.73-149.84 ppm related to imine (C carbon and at δ 14.82-15.33 ppm corresponds to the methyl group attached to i carbon (CH3C=N). Furthermore, 13 C-NMR of 5a-g revealed a set of signals that appe in the aromatic region related to aromatic carbons.   Scheme 1. Synthesis of the target compounds 2-5g. Reagent and reaction condition: (i) acetaldehyde, furfural or respective aryl aldehyde, EtOH, AcOH, reflux 6-8 h; (ii) respective aryl ketone, n-butanol, reflux 6-8 h.  In addition, compound 4h was subjected to in vitro tubulin polymerization inhibitory activity assay using ELISA assay for β-tubulin. As anticipated, compound 4h inhibited the polymerization of tubulin with a percent inhibition of 78.14% compared with no treatment control ( Figure 3B). The results in this study suggested that the molecular target of the prepared Schiff base 4h is indeed tubulin.  All values are presented as mean value ± SD and statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (* p < 0.05, *** p < 0.001).

Apoptosis Inducing Effect of Compound 4h
A double staining examination of annexin V FITC/propidium iodide (PI) was used to evaluate compound 4h's influence on apoptosis and estimate the percentage of the total, early, and late apoptosis in MDA-MB-231 cells. Compound 4h treatment resulted in a higher proportion of overall apoptosis in the MDA-MB-231 cell line (31.27%) as compared to no treatment controls (1.63%). Furthermore, the proportion of early and late apoptotic cells rose by 23.12-and 63.67-fold, respectively, as compared to the untreated control cells. As supported by cell cycle effects and apoptosis assays of Schiff base 4h revealed cell growth arrest at the G2/M phase as well as apoptosis-inducing activity ( Figure 5).

A ) B )
A  All values are presented as mean value ± SD and statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (* p < 0.05, *** p < 0.001).

Apoptosis Inducing Effect of Compound 4h
A double staining examination of annexin V FITC/propidium iodide (PI) was used to evaluate compound 4h's influence on apoptosis and estimate the percentage of the total, early, and late apoptosis in MDA-MB-231 cells. Compound 4h treatment resulted in a higher proportion of overall apoptosis in the MDA-MB-231 cell line (31.27%) as compared to no treatment controls (1.63%). Furthermore, the proportion of early and late apoptotic cells rose by 23.12-and 63.67-fold, respectively, as compared to the untreated control cells. As supported by cell cycle effects and apoptosis assays of Schiff base 4h revealed cell growth arrest at the G2/M phase as well as apoptosis-inducing activity ( Figure 5). compared to no treatment controls (1.63%). Furthermore, the proportion of early and late apoptotic cells rose by 23.12-and 63.67-fold, respectively, as compared to the untreated control cells. As supported by cell cycle effects and apoptosis assays of Schiff base 4h revealed cell growth arrest at the G2/M phase as well as apoptosis-inducing activity ( Figure 5). All values are presented as mean value ± SD and statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (* p < 0.05, *** p < 0.001).

Mitochondrial Membrane Potential
Most of the known anticancer molecules induce apoptotic cascade via activation of the intrinsic pathway via release of cytochrome-c after depolarization of mitochondrial membrane [34]. The potential status of the mitochondrial membrane is determined by the electrochemical gradient (∆ψ). In order to investigate the intrinsic pathway of apoptosis, ∆ψ dissipation was measured by cytofluorometry by exposing the MDA-MB-231 cell line to compound for 4h at a concentration equal to its IC 50 for 24 h. The results in Figure 6  were carried out using one-way analysis of variance (ANOVA) followed by Tukey's m comparison test (* p < 0.05, *** p < 0.001).

Mitochondrial Membrane Potential.
Most of the known anticancer molecules induce apoptotic cascade via activat the intrinsic pathway via release of cytochrome-c after depolarization of mitochon membrane [34]. The potential status of the mitochondrial membrane is determined b electrochemical gradient (Δψ). In order to investigate the intrinsic pathway of apop

Effect of Compound 4h on the Expression of p53
p53, a tumor suppressor gene, stops the formation of tumors. The activation of such a gene is known to be important in the regulation of the apoptotic pathway induced by various stimuli [35]. In order to understand the effect of TMP-based Schiff base 4h on p53 dependent apoptotic pathway, the MDA-MB-231 cells were treated with compound 4h at its IC 50 concentration for 24 h and the level of p53 was determined using ELISA assay. Figure 7 shows a rise in p53 levels of −10.97 fold when compared to the untreated control. a gene is known to be important in the various stimuli [35]. In order to understa dependent apoptotic pathway, the MDA its IC50 concentration for 24 h and the l Figure 7 shows a rise in p53 levels of −10  All values are presented as mean value ± SD and statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (*** p < 0.001).

Effect of Compound 4h on the Expression of Bax and Bcl-2
To further corroborate apoptosis induction by Schiff base 4h, the level of anti-apoptotic protein (Bcl-2) and pro-apoptotic protein (Bax) was determined in vitro by ELISA in MDA-MB-231 cells treated with Schiff base 4h. MDA-MB-231 cells were treated with compound 4h at its IC50 concentration for twenty-four hours. Compound 4h decreased the amount of Bcl-2 in MDA-MB-231 cells by −7.97-fold compared to untreated cells ( Figure 8). Furthermore, compound 4h increased the level of Bax by −4.42-fold compared to untreated cells. As shown by apoptosis experiments, compound 4h induces the intrinsic route of apoptosis by downregulating the levels of Bcl-2 and upregulating the levels of Bax.

Molecular Docking Study
A molecular docking study with tubulin was performed for N-3,5-dibromo-4-hydroxybenzylidene hydrazinyl molecule 4h into the active site of tubulin crystal structure (PDB: 1SA0) in order to reveal insight into its binding mode. A molecular docking simulation of compound 4h into the tubulin protein binding site was performed using molecular operating environment (MOE) software, 2015.10. The docking results revealed that N-3,5-dibromo-4-hydroxybenzylidene hydrazinyl molecule 4h displayed good binding affinities (−25.49 kcal/mol). In addition, N-3,5-dibromo-4-hydroxybenzylidene hydrazinyl molecule 4h interacts with the active site of 1SA0 by three hydrogen bonds; Asn 101 with the oxygen of trimethoxyphenyl ring, Ser 140 with the oxygen atom of trimethoxy benzamide ring and Tyr 224 with the carbonyl oxygen of the amide group. Additionally, 3,5-dibromo-4hydroxyphenyl ring formed π-π interaction with Tyr 224 residue. These results reflect the higher activity and justify the experimental findings of compound 4h (Figure 9).

Effect of Compound 4h on the Expression of Bax and Bcl-2
To further corroborate apoptosis induction by Schiff base 4h, the lev anti-apoptotic protein (Bcl-2) and pro-apoptotic protein (Bax) was determined in vit ELISA in MDA-MB-231 cells treated with Schiff base 4h. MDA-MB-231 cells were tr with compound 4h at its IC50 concentration for twenty-four hours. Compoun decreased the amount of Bcl-2 in MDA-MB-231 cells by −7.97-fold compared to untr cells ( Figure 8). Furthermore, compound 4h increased the level of Bax by −4.42 compared to untreated cells. As shown by apoptosis experiments, compound 4h in the intrinsic route of apoptosis by downregulating the levels of Bcl-2 and upregu the levels of Bax. All valu presented as mean value ± SD and statistical analyses were carried out using one-way analy variance (ANOVA) followed by Tukey's multiple comparison test (*** p < 0.001).

Molecular Docking Study
A molecular docking study with tubulin was performed N-3,5-dibromo-4-hydroxybenzylidene hydrazinyl molecule 4h into the active s tubulin crystal structure (PDB: 1SA0) in order to reveal insight into its binding mo molecular docking simulation of compound 4h into the tubulin protein binding sit performed using molecular operating environment (MOE) software, 2015.10. docking results revealed that N-3,5-dibromo-4-hydroxybenzylidene hydra molecule 4h displayed good binding affinities (−25.49 kcal/mol). In add N-3,5-dibromo-4-hydroxybenzylidene hydrazinyl molecule 4h interacts with the a site of 1SA0 by three hydrogen bonds; Asn 101 with the oxygen of trimethoxyp ring, Ser 140 with the oxygen atom of trimethoxy benzamide ring and Tyr 224 wit carbonyl oxygen of the amide group. Additionally, 3,5-dibromo-4-hydroxypheny formed π-π interaction with Tyr 224 residue. These results reflect the higher ac and justify the experimental findings of compound 4h (Figure 9).

Nanostructured Lipid Carrier Fabrication Studies
Aiming to enhance the pharmacokinetic characteristics as very poor aqueous solubility estimated utilizing free web-based tool Swiss ADME (http://www.swissadme.ch/ accessed on 10 April 2022). Remarkably, the anticipated poor water-solubility of 4h was (LogS_SILICOS-IT = 6.7 × 10 −7 mg/mL; 9.27 × 10 −10 mol/L) which will impede its cytotoxic activity and possible clinical outcomes of compound 4h

Nanostructured Lipid Carrier Fabrication Studies
Aiming to enhance the pharmacokinetic characteristics as very poor aqueous solubility estimated utilizing free web-based tool Swiss ADME (http://www.swissadme.ch/ accessed on 10 April 2022). Remarkably, the anticipated poor water-solubility of 4h was (LogS_SILICOS-IT = 6.7 × 10 −7 mg/mL; 9.27 × 10 −10 mol/L) which will impede its cytotoxic activity and possible clinical outcomes of compound 4h (Figure 10). Thus, Nanostructured lipid carriers (NLCs) fabrication was proposed as a simple and economic tactic for boosting the kinetic properties of compound 4h. NLCs aspired to promote the absorption of the compound besides improving its therapeutic response and reduce its predicted side effects. The composition of the eNLC matrix encompasses a blend of lipid molecules of various configurations, originally a gathering of both solid and liquid lipid, which is predisposed to the creation of more crystalline imperfections which in turn grant a greater gap for drug molecules accommodation [36]. Moreover, those crystalline imperfections allow the promotion of drug charging capability, and the suppression of the expulsion of the drug on storage, besides the modulation of the drug release pattern which is correlated to the variation in the composition of the lipid matrix [37]. There are colloidal dispersions of solid lipid and hydrophilic surfactant in the SLN form. Owing to the exclusive characteristics of NLCs, such as minute size, huge surface area, and improved drug loading potentiality [37], thus, loading compound 4h on NLCs was assumed to promote its cytotoxic activity via densifying its biological manifestations at the tumor site.

Compound 4h Loaded NLCs Design, Preparation and Optimization
Emulsification-ultrasonication technique was adopted for the fabricati drug-loaded NKCs formulations. The design of the formulations was cons 2 3 -factorial design using Stat-Ease ®-V.13 software (Design-Expert TM; Minne USA) ( Table 2). The picked independent variables were the type of lipid (A concentration (B; X2), along with the concentration of surfactant (C; X3). The formulae were formulated, assessed for the pre-determined dependent encapsulation efficiency (Y1: EE%), particle size (Y2: PS) and zeta potential (Y analyzed using Stat-Ease ®-V.13 software. The compositions and the res

Compound 4h Loaded NLCs Design, Preparation and Optimization
Emulsification-ultrasonication technique was adopted for the fabrication of eight drug-loaded NKCs formulations. The design of the formulations was constructed via 2 3 -factorial design using Stat-Ease ® -V.13 software (Design-Expert TM; Minneapolis, MN, USA) ( Table 2 sulation efficiency (Y1: EE%), particle size (Y2: PS) and zeta potential (Y4: ZP) and analyzed using Stat-Ease ® -V.13 software. The compositions and the results of the dependent responses of eight drug-NLCs formulations were displayed in Table 2. Drug estimation at different concentrations was performed using HPLC at λmax 254 nm [38].

Fabrication Variables Influence on the Selected Responses
The characterization of the pre-determined responses for each drug-loaded formulae of NLCs: Entrapment Efficiency (Y1: EE%), particle size (Y2: PS) and zeta potential (Y3: ZP). From Table 2, it can be depicted that there was a discrepancy in the outcomes of the measured responses which denoted the great impact of the fabrication variables on the preselected NLCs characters.
The entrapment efficiency of the fabricated NLCs formula ranged from 37.4 ± 1.8 for F6 to 95.6 ± 4.1 for F3 and the impact of the variable's type of lipid (A; X1), lipid concentration (B; X2), along with the concentration of surfactant (C; X3) on EE% can be obviously displayed by the cubic plot and predicted versus actual values plots ( Figure 11). Concerning the type of lipid (A), Precirol ATO-5 originated NLCs formulae exhibited significantly (p = 0.0065) greater EE% values than those prepared utilizing Geleol. This may be attributed to the nature of the incorporated lipids which greatly affect the extent of creation of a well-organized crystalline lattice, where lipids enclosing fatty acid acyl glycerol of various chain length grants a higher space for the drug accommodation based on that they were able to configure a less organized particle [39]. Thus, Precirol ATO-5 constituted to form a mixture of various fatty acids of different chains (palmitic C16 and stearic C18) predisposes to a higher possibility to configure less ordered particles than that of Geleol (mono acid acyl glycerol), hence a higher gap for drug loading. Moreover, lipids that possess a blend of diverse chain fatty acids have a greater capability of drug solubilization, and hence, higher drug loading efficiency [40]. Moreover, increasing the lipid concentration (B; X2) from 2% to 6% results in a significant (p = 0.0019) increase in EE%. This is plausible as a higher concentration of lipid means a greater amount of lipid was feasible to endorse drug molecules [41]. Meanwhile, increasing surfactant concentration (C; X3) resulted in subsequent significant (p = 0.0119) reduction in EE%. The existence of surfactant results in a diminished interfacial tension between the aqueous phase and lipid phase predisposing to the production of particles of diminished size which in turn reduces the NLCs capacity to endorse higher amount of the drug [36,39]. was feasible to endorse drug molecules [41]. Meanwhile, increasing surfactant concentration (C; X3) resulted in subsequent significant (p = 0.0119) reduction in EE%. The existence of surfactant results in a diminished interfacial tension between the aqueous phase and lipid phase predisposing to the production of particles of diminished size which in turn reduces the NLCs capacity to endorse higher amount of the drug [36,39].  solid lipid possessing a higher melting point predisposed to gradual crystallization, and thus a higher opportunity to conform more organized crystals with larger particle size [42]. According to Das et al. who found that investigated particle size of formulated clotrimazole NLCs was highly affected by the alteration of type of lipids and following this descending order Compritol < Precirol < Geleol [36]. Moreover, increasing the lipid concentration (B; X2) from 2% to 6% results in significant (p = 0.028) enlargement in PS, this may be attributed to the availability of a higher amount of lipids to enclose more drugs, thus increasing the PS. Moreover, increasing the amount of lipids will elevate the system viscosity which in turn will increase the tendency of particle aggregation [43]. On another hand, increasing the surfactant concentration significantly (p = 0.0326) reduces the NLCs formulation PS. This can be justified owing to the fact that the increase in the concentration of surfactant will diminish lipid droplet surface tension, which will be able to be subdivided into a finer size. In addition, sufficient surfactant concentration will be able to wrap the minute lipid droplets and hinder their tendency to coalescence, thereby elevating the system stability [44]. Turning to ZP (Y4), its values ranged from −12.5 ± 1.8 to −39.2 ± 3.4 Mv denoting the higher stability of produced NLCs as the increase in ZP will subsequently positively affect the system stability. Figure 13   ANOVA results revealed that changing the type of lipid (A; X1) from Precirol ATO-5 to Geleol predisposed to a significant (p = 0.0014) elevation in PS, owing to the fact that solid lipid possessing a higher melting point predisposed to gradual crystallization, and thus a higher opportunity to conform more organized crystals with larger particle size [42]. According to Das et al. who found that investigated particle size of formulated clotrimazole NLCs was highly affected by the alteration of type of lipids and following this descending order Compritol < Precirol < Geleol [36]. Moreover, increasing the lipid concentration (B; X2) from 2% to 6% results in significant (p = 0.028) enlargement in PS, this may be attributed to the availability of a higher amount of lipids to enclose more drugs, thus increasing the PS. Moreover, increasing the amount of lipids will elevate the system viscosity which in turn will increase the tendency of particle aggregation [43]. On another hand, increasing the surfactant concentration significantly (p = 0.0326) reduces the NLCs formulation PS. This can be justified owing to the fact that the increase in the concentration of surfactant will diminish lipid droplet surface tension, which will be able to be subdivided into a finer size. In addition, sufficient surfactant concentration will be able to wrap the minute lipid droplets and hinder their tendency to coalescence, thereby elevating the system stability [44].
Turning to ZP (Y4), its values ranged from −12.5 ± 1.8 to −39.2 ± 3.4 Mv denoting the higher stability of produced NLCs as the increase in ZP will subsequently positively affect the system stability. Figure 13 revealed the 3D surface plots variables type of lipid (A; X1), lipid concentration (B; X2), along with the concentration of surfactant (C; X3) and predicted versus actual values plots of ZP. From ANOVA results, it can be depicted that changing the type of lipid (A; X1) from Precirol ATO-5 to Geleol predisposed to a significant (p = 0.0021) suppression in ZP and this came in accordance with Das et al., who revealed that the ZP values of the prepared SLN will increase in the following order: Geleol < Precirol < Compritol (1). This may be due to the higher negative charged groups associated with the blend of fatty acid incorporated in Precirol relative to the mono acid acyl in case of Geleol. However, increasing the lipid concentration led to a significant (p = 0.0004) elevation in ZP values owing to the higher entrapment of drug-bearing anionic group within the particles, in addition to the increase in anionic groups of the fatty acids on increasing the lipid concentration [39]. Meanwhile, increasing the surfactant concentration significantly (p = 0.0048) reduces the ZP values. the virtue of utilizing nonionic surfactant (Cremophore RH 40), which will not impart any additional charge besides the surfactant bulkiness, will aid in disguising the particles charge on surrounding it [39]. besides the surfactant bulkiness, will aid in disguising the particles charge on surrounding it [39].

Statistical Optimization of Fabrication Variables for Election the Optimum Formula
The optimization aimed to pick the best formula that possesses the predetermined criteria as maximum EE%, ZP and diminished PS. This was attained via analysis of the influence of the independent variables on the determined dependent criteria using The

Statistical Optimization of Fabrication Variables for Election the Optimum Formula
The optimization aimed to pick the best formula that possesses the predetermined criteria as maximum EE%, ZP and diminished PS. This was attained via analysis of the influence of the independent variables on the determined dependent criteria using The Design-Expert V.13 software. Based on 2 3 factorial design and its subsequent analysis F3 constituted of Precirol ATO 5 as lipid used in concentration 6% w/v along with 1% w/v Cremophore RH40 was chosen to be the optimum formula possessing a desirability value of 0.805. Moreover, as shown in Table 3 the predicted and actual values of EE%, PS and ZP were not gapped by more than 10% assuring the appropriateness of the design and the liability of its outcomes. (Figure 14) arise the optimum criteria for 4h-loaded NLCs.      Figure 15) displayed the attained image of the optimum 4h-loaded NLC fo (F3) using the TEM technique. It can be depicted as the spherical soft-shaped nanoparticles devoid from any crystals for the drug.

In vitro drug release experiment
The release attitude of the drug from drug-loaded NLC relative to drug susp was conducted by adopting in vitro drug release experiment. The cumulative amo drug released over 24 h was computed and found to be 92.45% ± 3.37 compared to ± 1.2 for 4h suspension ( Figure 16). Thus, NLCs as a carrier for the drug will aid extended release of the drug and its densifying in the tumor location in a solubilized form [38].

In Vitro Drug Release Experiment
The release attitude of the drug from drug-loaded NLC relative to drug suspension was conducted by adopting in vitro drug release experiment. The cumulative amount of drug released over 24 h was computed and found to be 92.45% ± 3.37 compared to 20.8% ± 1.2 for 4h suspension ( Figure 16). Thus, NLCs as a carrier for the drug will aid in the extended release of the drug and its densifying in the tumor location in a more solubilized form [38].

Comparative Cytotoxicity Study of Optimized Formula Versus Pure 4h
In order to highlight the significance of the drug lodging on NLCs, the cyto study was conducted for optimized NLCs (F3) relative to the pure drug. (

Comparative Cytotoxicity Study of Optimized Formula versus Pure 4h
In order to highlight the significance of the drug lodging on NLCs, the cytotoxicity study was conducted for optimized NLCs (F3) relative to the pure drug. (Figure 17) revealed the % viability of MDA-MB-231 cancer cells post-exposure to both preparations and reference drugs. The IC 50 values of the results were displayed in F3, pure 4h and CA-4 were (0.59 ± 0.07, 1.27 ± 0.18 and 0.54 ± 0.04 µM), respectively. The magnitude of cytotoxicity was found to increase on the following trend: compound 4h < F3 < CA-4. F3 exhibited the chief cytotoxicity over 4h and this may be attributed to the large surface area of NLCs, promoted drug solubility and release along with the promotion of the drug cellular uptake, all of these lead to a boost in the anticancer activity.

Comparative Cytotoxicity Study of Optimized Formula Vers
In order to highlight the significance of the drug lodging on study was conducted for optimized NLCs (F3) relative to the p revealed the % viability of MDA-MB-231 cancer cells post-exposur and reference drugs. The IC50 values of the results were display CA-4 were (0.59 ± 0.07, 1.27 ± 0.18 and 0.54 ± 0.04 μM), respectiv cytotoxicity was found to increase on the following trend: compou exhibited the chief cytotoxicity over 4h and this may be attribute area of NLCs, promoted drug solubility and release along with the cellular uptake, all of these lead to a boost in the anticancer activity

General
See Supplementary Materials-Appendix SA.

General
See Supplementary Materials-Appendix SA. Hydrazide 1 (0.46 g, 1 mmol) was added to a solution of an appropriate aldehyde; namely acetaldehyde, furfural or respective aryl aldehyde (1 mmol) in absolute ethanol (20 mL) containing a catalytic amount of acetic acid glacial (1 mL). The reaction mixture was refluxed for 6-8 h and then filtered. The residue obtained was dried, and crystallized from absolute ethanol to yield pure compounds 2, 3 and 4a-l.

Conclusions
Finally: new Schiff base-based TMP compounds were synthesized and evaluated for their cytotoxic activity against the breast MDA-MB-231 cell line and the normal breast MCF-10A to conclude this research. The tested Schiff bases revealed good activity over the MDA-MB-231 cell line, especially compounds 4h, 4j and 5d with IC 50 values 1.27 ± 0.18 µM, 2.84 ± 0.18 µM and 1.98 ± 0.19 µM, respectively, compared to CA-4 (IC 50 = 0.54 ± 0.04). The cytotoxic activity of Schiff base 4h is correlated to tubulin polymerization inhibitory activity as revealed by immunofluorescence analysis and β-tubulin polymerization inhibition percentage on MDA-MB-231 cells (78.14% polymerization inhibition at 1.27 µM). In addition, compound 4h caused cell cycle arrest at the G2/M phase (fivefold more than control MDA-MB-231 cells) and cellular apoptosis as ascertained by an increase in the percentage of the pre-G1 phase by almost 19-fold compared with negative control cells and Annexin V FITC/PI staining assay. Moreover, compound 4h was found to be an apoptotic inducer via a decrease in the level of MMP and Bcl-2 by 3-and 8-fold, respectively, compared to negative control cells and an increase in the level of p53 and Bax by 11-and 5-fold, respectively, compared to the negative control cells. Additionally, the most potent compound 4h was lodged on nanostructured lipid carriers (NLCs). Eight formulae resulted from 2 3 full factorial design which was conducted to govern the influence of the fabrication variables on the in vitro characters of the casted NLCs. Based on the outcomes of the experimental design and factorial analysis, F3 was picked as the optimum formula exhibiting a dominant desirability value of 0.805, EE% 95.6 ± 2.4, PS 222.4 ± 18.7, PDI 0.23 ± 0.05 and ZP −39.2 ± 3.9 Mv. Moreover, F3 exhibited an improved release and solubility profile over that of the drug suspension. In the comparative cytotoxic activity, F3 was capable of diminishing the IC 50 by around 2.15 times for pure 4h, while nearly close to the IC 50 of the reference drug. Thus, NLCs could be a potential platform for boosted antitumor activity of compound 4h against MDA-MB-231 cancer cells.