Carbonyl releasing Schiff base complex of Fe (III): synthesis, physicochemical characterization, antimicrobial and anticancer studies

REGULAR ARTICLE

Carbonyl releasing Schiff base complex of Fe (III): synthesis, physicochemical characterization, antimicrobial and anticancer studies

R V KUPWADE and V J SAWANT*

Department of Chemistry, Smt. Kasturbai Walchand College, Sangli, Maharashtra 416 416, India E-mail: v11131@rediffmail.com

MS received 20 August 2019; revised 14 October 2019; accepted 15 October 2019

Abstract. The carbonyl releasing Schiff base chelate derived Fe(III) complex has been synthesized fol- lowing the simple wet chemical method. The rhombohedral packing of ligands with the metal ion in the complex was confirmed by its XRD pattern and FTIR spectrum. Compounds were characterized by UV-Vis., PL, FTIR, XRD and TEM techniques. Better biocompatibility of the complex than that of the pure Schiff base had been elaborated on gram-positive and gram-negative bacteria by Agar well disc-diffusion antibacterial screening. The complex also displayed pH-responsive in vitro anticancer therapy on MCF-7 breast cancer cells as revealed by MTT assay. The release of CO and Schiff base as well as the interaction of Fe(III) with the mitochondria inside the cell are the key factors of cell biocompatibility. This CORM complex had exhibited higher anticancer activity on MCF-7 cells than the Schiff base and thus potential candidate for the future biomedical applications.

Keywords. Carbonyl releasing; Schiff base; chelate; anticancer.

1. Introduction

Carbonyl releasing complexes and molecules (CORM) are gaining more interest due to their ver- satile properties in biomedical fields, such as antimi- crobial, anticancer, antioxidant and anti-proliferative agents.

Alike the NO, carbonyl (CO) ligand is also cell signalling species which exhibits vasodilatory, anti-inflammatory and anti-proliferative activities.

Carbonyl releasing species are generally supramolecular complex agents which pass through the cell barriers. When their uptake takes place by lysosomes in cells at acidic pH, the release of CO molecule takes place and ROS (Reactive Oxygen Species) get generated by cell proton efflux. Hence, such complexes act as an antimicrobial and anticancer agent with better biocompatibility. Schiff bases are generally chelating ligands containing carbimide bonds formed after refluxing aldehydes with amines in synthetic protocols. These ligands are the best antimicrobial agents and form better complexes with a variety of transition and inner transition metals with good biocompatibility.

When such ligands combine together in a chelate of bioactive metals like Fe(III), these complexes play significant roles in bio signalling inside cells and give effects of biocompatibility or cytotoxicity. Hence, Schiff base complexes along with carbonyl ligands result in fast endocytosis and release of ligands inside cells to effect into ROS production at pH gradient of cells. This ROS produced into cells leads to antimi- crobial, anticancer or cytotoxic and biocompatible effects on cells. Parallel effects of biocompatibility have been shown by antibiotic ligand complexes with special biocompatible metals.

ROS-based antimicro- bial activity of Pd(II) complex has been reported by W Guerra et al.

Recent work on heterocyclic and Schiff base ligand complexes have been not only reported the antimicrobial properties but also DNA cleavage activities in cells leading to the production of ROS in cells and biomedical interface mechanisms.

Similar research of heterocyclic and carbimide containing Schiff base type ligands and their metal complexes have been reported for their biomedical applica- tions.

According to recent work on such hetero- cyclic species and Schiff bases along with their complexes with transition metals, these complexes have exhibited the supramolecular nature to pass from

*For correspondence

https://doi.org/10.1007/s12039-020-1746-ySadhana(0123456789().,-volV)FT3](0123456789().,-volV)

cell membranes easily and produce effects of bio- compatibility and DNA interactions to realize poten- tial effects on cell divisions and proliferations.

In continuation of these ideas, we had synthesized new carbonyl releasing Schiff base complex of Fe(III).

In addition to physicochemical characterization of the simple Schiff base and its carbonyl complex with Fe(III), the antimicrobial biocompatibility and anti- cancer potentials have been tested by in vitro assay.

The higher antimicrobial and anticancer potential of the complex as compared to Schiff base has been elaborated on special MCF-7 human breast cancer cells by in vitro MTT assay. Complex exhibited increased endocytosis inside negatively charged cell membranes of bacteria and cancer cells and produced higher amount of ROS at acidic pH than Schiff base, CO, implying the effect of Fe(III) species towards intrinsic apoptosis of cancer cells.

Thus, carbonyl releasing Schiff base complex of Fe(III) can be con- sidered as a potential candidate for future biomedical applications.

2. Experimental

2.1 Materials and cell cultures

All the chemicals used for the synthesis of Schiff base and its carbonyl complex with Fe(III) and their in vitro bio- logical screening such as Ferric nitrate, P-nitro aniline, Benzaldehyde, Zinc powder, Calcium carbonate, Conc.

HCl, Ethanol were of A. R. grade. These chemicals were purchased from S. D. Fine Chem. Ltd. and Merck Ltd. and were used without further purification. The cell culture medium such as agar growth broth and bacterial culture, fetal bovine serum, trypsin buffer were obtained from Himedia Ltd. and NCCS cell repository center, Pune, India.

The human breast cancer cells MCF-7 were purchased from this cell repository. The double-distilled water was obtained from Millipore system and used throughout the synthesis andin vitrobiological screening tests.

2.2 Synthesis of Schiff base ligand

The simple Schiff base ligand was synthesized by refluxing P-nitroanilline and Benzaldehyde at 250 °C on the constant flame in R.B. flask with condenser for 3 h. The yellow product formed water-washed with ethanol to remove unreacted organic precursors and dried in the oven below 80°C. The Schiff base powder then subjected to physico- chemical the complex using UV-Vis., PL, FITR spectrom- etry. The formation of the product was confirmed on the basis of physical constant and TLC (see protocol in Scheme1).

2.3 Wet chemical synthesis of CORM, carbonyl - Schiff base complex of Fe(III)

The carbonyl-containing Schiff base metal complex of Fe(III) was synthesized by bubbling CO into the flask containing Schiff base and Fe(III) nitrate precursor using a wet chemical method (Scheme1). In detail, the Schiff base with Fe(III) in 2:1 mM proportion in the ethanol-water solvent system were refluxed in R. B. flask for 3 h and the precursor was then added in the flask with double distilled water. The gaseous CO ligand was produced in separate flask by the reaction of conc. HCl with calcium carbonate and zinc powder mixture. Then the CO ligands produced separately in another flask was passed and bubbled in ligand and metal precursor mixture to get carbonyl releasing CORM complex of Fe(III) containing CO ligands and Schiff base. The complex formed thus was washed with ethanol-water solvent system and dried in the oven.

2.4 Structural and morphological

characterization of CORM-Fe(III) complex

The structure, morphology, particle diameter range and types of bonding of functionalities in the CORM complex of Fe(III) was determined based on physicochemical char- acterization using UV-Vis., PL, FTIR, XRD spectrometry

OH

CHO

+ _{H2N NO2}

3h reflux in ethanol - H₂O

CH

N NO2

OH

CO bubbling Fe(NO₃)₃ stirred with ligand

N

NO₂ N O

NO₂

O Fe CO

CO

(NO3)3

Salicyladehyde P-nitro aniline

Schiff base ligand

CORM Fe (III) Schiff base complex

Scheme 1. Synthesis protocol of Schiff base ligand and carbonyl-schiff base Fe(III) complex.

techniques and TEM microscopic analysis. The spectronics double beam UV-Vis. spectrometer with water as blank was used to determine the absorption spectrum of complex to compare with Schiff base. To confirm functionalities pre- sent in complex and a with Schiff base, Perkin Elmer series FTIR spectrometer was used with KBr pellet technique. The PL emission spectrum of complex and Schiff base were determined using Jasco type spectrofluorometer with exci- tation identity of complex and Schiff base in ethanol solvent with the same ppm. Concentrations. The X-ray diffraction pattern of the complex was determined using X-ray spec- trometer by powder diffraction technique to elaborate the packing of ligands with metal in complex, hybridization and crystal system of complex. The particle range of complex to pass cell through membranes was determined using TEM microscopic analysis and SAED patterns were matched with XRD data to confirm the powder crystal system of complex.

2.5 Antimicrobial screening on gram-positive and gram-negative bacteria by agar well disc diffusion method

The cell-particle interactions of materials and complexes demonstrating their reactivity and biocompatibility can be elaborated using simple in vitro antibacterial screening in buffer solutions to maintain physiological mimicking pH at material cell interactions. As cell pH affects the biocom- patibility of molecules. Here in this work 15, 20 and 25 ppm concentrations of Schiff base and CORM complex were dosed on bacterial cell cultures grown in agar broth on discs, inside the wells bored on plates. The gram-positive bacteriaStaph. Aurues, and gram-negative bacteriaE. Coli, Kleb. were grown on culture plates and inhibited by dosing of Schiff base and complex solutions in buffer dispersions with physiological pH = 7.4 by use of phosphate buffer. The culture plates were incubated and zones of inhibition were measured, and biocompatibility/antimicrobial property of Schiff base and complex were compared.

2.6 In vitro MTT cell line anticancer assay on MCF-7 human breast cancer cells

The anticancer potential of Schiff base and complex were estimated and compared on the basis on in vitro MTT, MCF-7cellline spectrometric assay. Cytotoxicity of Schiff base and CORM complex were elaborated into MCF-7 (Human Breast cancer cells) with MTT assays. Cells (19 10³/mL) were seeded in 96-well plates and then incubated for 24 h at 37°C and 5% CO2atmosphere in an incubator with 100 lL of DMEM medium supplemented with 10%

fetal bovine serum (FBS) as a growth medium and 1%

penicillin-streptomycin solution per well. This medium was then replaced with 200 lL of the PBS, phosphate buffer saline (pH=7.4) medium containing either Schiff bases or complex doses (concentration 5, 10, 15, 20 and 25lg/mL).

Cells without treatment were considered as a control as well to evaluate its cytotoxicity. Cells were further incubated for 48 hours, and relative anticancer activity was assessed with MTT assays. After 48 h, MTT assay was carried out. In brief, MTT solutions (20lL of 5 mg/mL) were added after treatment and cell culture were again incubated for an additional 4 h. Dimethyl sulphoxide (150lL solution) was added to each well to solubilize the blue formazan crystals, and optical density at 492 nm was recorded for culture medium. Cell viability (%) for exposure of Schiff base and complex doses were calculated as follows,

%Cell viability¼ Optical density at492nm in test cells Optical density at492nm in control cells 100

The anticancer activities of Schiff base and complex doses were elaborated in terms of cell viability and plotted as a function of concentrations to compare the effects.

Finally, these cultures were subjected to DAPI staining to elaborate internalization of the complex into cancer cells, and determine the mechanism of cell deaths or apoptosis by interactions from the complex by the production of inter- anions/inter-cations or release of ligands and metal ion inside cell fluids.

2.7 DAPI staining and imaging of cancer cells after MTT assay

After the incubation with Schiff base/complex doses and further MTT interactions, the cells were washed with PBS (phosphate buffer saline) solution and fixed with 4%

paraformaldehyde for 30 min, then cells were added with 20lL of DAPI and were incubated for 20 min; finally, after spreading on slides, the cell were examined under a fluo- rescent microscope. The internalization of complex higher than Schiff base was examined by the staining and mech- anism of anticancer effects and biocompatibility/ cytotoxi- city was demonstrated.

3. Results and Discussion

3.1 Morphological and structural characterization

of Schiff base ligand and the CORM Fe(III) complex

3.1a UV-Vis. absorption and PL emission

spectrum: UV-Vis. and PL spectrum of

Schiff/complex were determined to study the

structural characteristics, metal to ligand charge

transfer transitions, bonding within the molecule and

absorption/emission property. Here UV-Vis, the

absorption spectrum of the complex (Figure

explains the charge transfer transition of Fe(III)

metal ion with Schiff base and carbonyl ligand, to

elaborate the bonding interactions in the complex. The

spectrum exhibits the diffused broad absorption peaks at 235 nm and 355 nm for the MLCT transitions from Fe(III) d electrons to CO and carbimide bond of Schiff base ligand respectively (metal to ligand charge transfer take place probably due to non bonded and Pi electrons of Schiff base and CO ligands and d electrons of Fe(III) in the complex). The Schiff base shows higher O.D. for a single peak near at same absorption wavelength of the complex at 365 nm. (Not shown), but dampening and peak shifting take place for absorption maxima of Schiff base after bonding interaction of carbimide bond with Fe(III) ions in the complex. Hence, UV-Vis spectrum of complex elaborates the bonding of ligands with Fe(III) and gives the idea of probable chelate type structure of the complex. The PL spectrum of Schiff base and complex determined separately explain the quenching of peak signal of Schiff base after bonding of Fe(III) with carbimide bonds in the complex. In Figures

the PL emission peaks of Schiff base and CORM complex respectively show the quenching of signal.

PL spectra of Schiff base and complex support for the bonding, structure and nature of complex con- taining Schiff base and CO ligand simultaneously linked with Fe(III) ions to give more stability for the chelate. According to PL spectra, the PL intensity of Schiff base at 815 nm. due to non bonded and Pi electrons was quenched by Fe(III) bonding in complex proving MLCT transition and coordinate/covalent linkages of carbimide bonds with a metal ion. Overall these spectra elaborate chelate nature of complex containing CO ligand liked with planer Schiff base

ligand with Fe(III) ion in the complex. So the spectra support for CO releasing nature of the complex, as Schiff base is firmly linked by coordinate bonds and CO electrons are representing different absorption maxima in UV-Vis spectrum of the complex at 235 nm representing more non-bonding free electrons over it. This elaborated the free site of CO ligands for easy release from complex to behave as CORM complex for better cell biocompatibility. Table

supports for MLCT transition identities in Schiff base and CORM complex determined from UV-Vis and PL spectrum.

The composition of the complex for its elements and ions C, H, O, N, Fe (III) along with functionalities of carbimide, carbonyl bonds to metal are confirmed by using UV-Vis and PL electron transitions and peaks in respective spectra. These evidence are supported by FTIR signals as per spectral studies in works of B. Sinha et al.,

hence these analyses of Schiff base and CORM complex not only prove the composition but also probable structure and functionalities in the complex.

3.1b FTIR spectrum of Schiff base and CORM complex: FTIR spectrum of Schiff base and complex were determined to estimate the functionalities present inside the molecules and to confirm the formation of CORM complex on the basis of signals represented by functional groups in fingerprint and functional regions of spectra.

Figure

base, in this spectrum presence of carbimide bond – C=N- was elaborated due to signal at 1918 and

2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0

0 1 2 3 4 5

d⁵ F e ( II I) M L C T w ith C O a n d c a r b im id e

Absorbance

W a v e le n g th n m

U V - V IS a b s o r p t io n s p e c tr a o f s c h if f b a s e c o m p le x

Figure 1. UV-Visible absorption spectrum of CORM Schiff base complex of Fe(III).

Figure 2. (A) PL emmission spectrum of schiff base. (B) PL emmission spectrum of complex.

Table 1. MLCT transitions of non bonded/Pi electrons of ligands and d electrons of Fe(III).

Absorption or emission peak shown in UV-Vis. Or

PL spectrum Schiff base transition Metal CORM complex charge transfer transition UV-Vis. Absorption at 355

nm.

n to Pi absorption maxima A_1gto T_2gand A_1gto A_2g from d⁵state of Fe(III) for splitting from bonding of carbimide and CO species

PL quenching at 815 nm. n to Pi relaxation- no quenching

Pi to Pi relaxation – quenching due to Fe(III) and carbimide linkage

1971 cm

. The signals at 531, 665 and 805 cm

represent the fingerprint identity of aromatic groups from benzaldehyde and nitroaniline. Signals at 2555 and 2673 cm

are attributed towards the aromatic nitro and nitrile entity of Schiff base. Signals from 3034 to 3379 cm

are raised in the spectrum due to the presence of aromatic –OH and carbimide hydroxyl side interactions in Schiff base. In Figure

spectrum proves functionalities present in CORM Fe(III) complex elaborates the presence of carbimide metal bond and side CO ligands over the metal ion.

Signals at 1760 and 1822 cm

elaborates the presence CO groups over Fe(III) metal ion in the complex. The signals at 1936 and 1969 cm

are attributed to Fe(III) bonded with carbimide linkage in the complex. The broadening of signals in complex FTIR spectrum at 2585 and 2884 cm

is attributed to carbimide and CO linkages at Fe(III) ion and covalent aromatic-O linkage to Fe(III) from Schiff base near to

carbimide metal bond. Overall all these observations lead to prove the chelate nature of the complex and octahedral site of Schiff base with metal ion and free carbonyl groups over it. Table

throws light on specific signals of the FTIR spectrum for the functionalities in the Schiff base and the complex.

3.1c XRD (X-ray diffraction) pattern of complex: As per XRD pattern of the complex in Figure

crystalline packing for metal ion and ligands, it elaborates the structure and morphology for CORM complex. The diffraction pattern gives signals for metal ion planes in an octahedral environment of Schiff base and carbonyl ligands, Fe(III) of the complex are in octahedral sites of packing with ligands in complex, hence phase purity of complex crystal have been proved by XRD patterns. The XRD data is determined using miller indices of pattern,

FTIR spectra of Schiff base

4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400

88.90 89.2 89.4 89.6 89.8 90.0 90.2 90.4 90.6 90.8 91.0 91.2 91.4 91.6 91.8 92.00

Frequency cm^-1

%T

FTIR spectra of CORM complex

4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400

33.0 34 36 38 40 42 44 46 48 50.0

Frequency cm^-1

%T

A

B

Figure 3. (A) FTIR spectrum of Schiff base ligand. (B) FTIR spectrum of CORM complex.

Scherer’s formula and packing of planes and it was matched with octahedral Fe(III) data in JCPDS card no. 85-3854. As per Table

rhombohedral packing of complex clearly represented the octahedral packing of the metal ion with ligands producing rhombohedral crystal. Hence, XRD data proved the phase purity, crystal packing, geometry, chelate octahedral nature of CORM complex.

3.1d TEM image and SAED pattern

of Complex: The TEM image of the complex in Figure

throws light on spherical elongated morphology and some aggregation of complex molecules due to supramolecular neighbouring bonding interactions. The particle sizes of complex crystals ranged from 60 nm to 75 nm. But most of the particles of complex molecules exhibited average size

0 8 0

6 0

4 0

2 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 1 2 0 0 0

[F e (III) in rh o m b o h e d ra ]

[1 1 1 ] [0 0 2 ]

[1 2 1 ] [0 0 1 ]

[0 2 0 ]

F e (III) io n in c a rb im id e a n d C O lig a n d e n v iro n m e n t O c ta h e d ra l rh o m b o h e d ra l c rys ta l s ym m e try

Intensity

2 T h e ta

X R D s p e c tra o f c o m p le x

Figure 4. XRD pattern of the Complex.

Table 2. Matching of FTIR signals elaborating the functionalities of Schiff base and CORM complex.

Signal in FTIR spectrum Schiff base functionality CORM complex functionality

1969 cm^-1 Carbimide bond -C=N- Carbimide linked to Fe(III)

1760 cm^-1 Absence of CO Presence of linked CO ligands over Fe(III)

3034 cm^-1 Aromatic -OH Ether aromatic-O- linkage with Fe(III)

1790 and 1824 cm^-1 Presence of an aromatic nitro group Out of plane nitro groups 2555 and 2585 cm^-1 Presence of nitrile entity Presence of linked nitrile entity

Table 3. Crystal parameters of CORM Fe(III) complex matched with standard Fe(III) octahedral JCPDS card.

Crystallite planes (Miller Indices) (h,k,l)

Rhombohedra of

octahedral chelate phase

d Calculated A⁰ d = a/H(h²?k²?l²)

or 2dSinh = nk

d Standard A⁰ JCPDS card no.- 89-3854

Fe(III)

Lattice Constant

a and b A⁰from main XRD peak at theta = 37.5 of CORM complex crystal

from rhombohedra

001 5.088 5.089 a standard = 3.249

b standard = 5.266

121 4.032 4.045

020 7.039 7.043

002 3.089 3.083 a calculated = 3.251 and

b calculated = 5.272

111 6.334 6.329

range 60 nm which can pass through cell membranes by supramolecular interactions easily. The TEM results were matched with XRD data and proved the chelate octahedral nature of complex with the crystalline state for good water solubility and further biocompatibility. The SAED patterns greatly matched with XRD data with crystalline dot patterns as per Figure

the complex had proved the morphology of the

complex for further water-loving nature and biomedical applications.

3.2 Antimicrobial properties Schiff base and complex for better biocompatibility

As per the images of bacterial cultures and zones of inhibition exhibited by Schiff base and complex on

Figure 6. (A) to (C) Anti microbial effects of schiff base ligand at 25 ppm. on (A) E-Coli.-Gram negative bacteria, (B)Kleb.-Gram negative bacteria,(C)Staph. aureus- Gram positive bacteria.

gram-positive and gram-negative bacteria in Fig- ures

the Schiff base and complex show better antimicrobial activities. The CORM complex shows higher activity than Schiff base and as per Table

gram-positive bacteria, higher activity was estimated than gram-negative bacteria. So the CORM complex exhibit higher biocompatibility than Schiff base, due to release of CO, Schiff base ligand and Fe(III) metal ion inside bacterial cells causing interactions with DNA. So, the complex show higher cell compatibility and internalization due to its chelate supramolecular nature proving its applicability in biomedical fields.

3.3 Anticancer potential of Schiff base

and complex by in vitro MTT assay on MCF-7 cells As per Figure

activity had been shown by complex than Schiff base compared with free control cells on the basis of in vitro MTT assay on MCF-7 breast cancer cells. The cell viabilities determined are related to cytotoxicity and anticancer effects on these cancer cells at 5, 10, 15, 20, 25 ppm. Concentrations of doses of Schiff base and CORM complex. Higher cell line apoptosis was observed at 25 ppm. Giving dose-dependent and pH triggered cytotoxicity of CORM complex. About 77%

Table 4. Anti-microbial activities of Schiff base and complex compared for gram-positive and gram-negative bacteria.

Type/name of bacterial culture in Agar broth [as per Figures 6A to 6C and7A to 7C]

Zones of inhibition for gram-positive bacteria/gram-negative bacteria as zone diameter in mm. for concentrations of drug/dose of complex or Schiff

base 25lg/mL (ppm.)in vitro on bacterial cells

For Schiff base For CORM complex

E. Coli (gram-ve) 10 mm. 35 mm.

Kleb.(gram-ve) 5 mm. 15 mm.

Staph aureus(gram?ve) 8 mm. 48 mm.

Figure 7. (A) to (C) Anti microbial effects of CORM complex at 25 ppm. on (A) E-Coli.-Gram negative bacteria, (B)Kleb.-Gram negative bacteria, (C)Staph. aureus- Gram positive bacteria.

apoptosis was given by CORM complex at 25 ppm dose, hence, the complex show good cytotoxicity on cancer cells and proved its potential application in the biomedical area. Supramolecular chelate nature to pass from cell barriers, presence key signaling CO ligand and Schiff base with electron providing Fe(III)

ion and pH triggered action on cancer cell endocytosis and apoptosis are the main entities which play role in anticancer effects of this new CORM complex. These all observations are supported and proved by DAPI staining image of MCF-7 cells after the action of CORM complex by MTT assay. In Figure

observed that the complex after endocytosis shrinks cancer cells fastly after incubation and show DAPI fluorescence at center of cells under a microscope.

This observation indicates the action of released spe- cies with DNA producing pH-sensitive ROS flux inside cancer cells from CORM complex resulting in intrinsic apoptosis of MCF-7 cells.

3.4 Mechanism for antimicrobial activity and anticancer potentials of the CORM complex As per Scheme

bacterial or cancer cells with production of ROS (re- active oxygen species) like O

, OH, OOH and H

O

after endocytosis and lysosomal digestion at acidic pH, it causes prominent biocompatible effects on these cells. CORM complex release CO ligand, Schiff base and dissociate Fe(III) inside acidic pH of cells, so by pH trigger it releases proton flux, CO, intercations inside cell organelles. The ROS produced inside bac- terial cells by these species on the basis of cell molecule interaction enters into rough organelles and mitochondria of cells disturbing membrane potentials of cell organelles and damage DNA or mutated DNA.

The free electrons from Fe(III) ions, CO ligand, Schiff base activity, inter cation and proton flux are key factors disturbing the cancer cell mitochondria and mutated DNA. Overall these intrinsic pathways of apoptosis by CORM complex cause higher anticancer effects than Schiff base on MCF-7 cells with probable ROS-lipid peroxidation mechanism which are pH-re- sponsive and CO signaling affecting biomedical activities.

Hence, this new CORM complex

5 10 15 20 25

0 10 20 30 40 50 60 70

% cell viability for MCF-7 cells after MTT assay

Concentrations of dosing for com plex and schiff base in ppm . action of CORM com plex action of Schiff base

Figure 8. Anticancer activities of schiff base and CORM complex by MTT assay on MCF-7 cells.

Figure 9. DAPI staining image of internalization of complex for apoptosis of MCF-7 cells.

pH trigger CORM Complex (Excitation at 385 nm.) emission at 815 nm.

[Release of H⁺ / Fe³⁺/ CO and Schiff base at acidic pH in lysosome]

Fe(III) to Schiff base CT transition VB⁺ + e^- to CB in Fe(III) - (free electrons) Dissociation-mitochondrial disruption

[CO] and Schiff base releasing at lysosomes pH change --- ROS in cells- O2., OH^., ^.OOH Mitochondrial cyt.-c destruction and caspase activation Apoptosis of cancer cell

Scheme 2. Mechanism for antimicrobial and anticancer activities of complex.

clearly indicates potential biomedical applications for antimicrobial and anticancer activities.

4. Conclusions

The new carbonyl-Schiff base CORM complex of Fe(III) exhibited rhombohedral packing and chelate nature with an octahedral site of ligands. The complex had exhibited 60 nm mean particle diameter and crystalline oval morphology as confirmed by XRD pattern and TEM analysis. This chelate with CO releasing ability has the potential to pass cell barrier with supramolecular nature and hence show better biocompatibility than Schiff base. The quenching of signal in PL spectrum elaborated the bonding of Fe(III) ion with Schiff base and CO ligand. Further- more, the complex exhibits ROS in bacterial and cancer cells after the release of the Schiff base, Fe(III) and CO by pH trigger. The CORM complex shows better biocompatibility by in vitro than Schiff base.

The synergic effects of the release of CO, Schiff base and interaction of Fe(III) with mitochondria inside the cells are the key factors for cell biocompatibility.

Hence, this CORM complex of Fe(III) exhibits an intrinsic pathway for apoptosis of cancer cells. Overall this CORM complex has potential application in biomedical fields for cytotoxicity and biocompatibility.

Acknowledgements

The author is thankful to analytical instrumentation labo- ratory, Jaysingpur College, Jaysingpur, India for providing some spectroscopic characterizations of samples, and to the Biotechnology laboratory of Smt. K. W. College, Sangli for providing cell cultures and in vitrotesting facilities.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

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