Spectroscopic Analysis of Copper Minocycline Novel Complex and Evaluation of its Potent Antibacterial, Antioxidant and Anti-breast Cancer (MCF-7) Properties

Minocycline is a second-generation, semisynthetic analogue of tetracycline (Arezoo et al., 2020), which inhibits Gram-positive and Gram-negative bacteria and can cross the blood-brain barrier (Kim and Suh, 2009). Its main mechanism of action is to stop protein synthesis. Minocycline has a longer half-life and more tissue absorption into the central nervous system and cerebrospinal fluid (Sapadin and Fleischmajer., 2006). Its higher efficacy is attributed to modification in ring D through 7-9 carbons (Nelson, 1998).
       
The FDA has approved 2^nd-generation tetracycline (Mino) for treating certain infectious diseases (Blum et al., 2004; Good and Hussey, 2003), with its rapid tissue penetration, longer half-life and strong bioavailability making it a promising treatment for breast cancer. Mino has shown anticancer effects through various pathways in different types of cancer (Barza et al., 1975; Kramer et al., 1978; Macdonald et al., 1973; Garrido-Mesa et al., 2013).
       
A recent study by Zihan et al., (2024) indicated that (Mino) may suppress chemotherapy-induced neuroinflammation,  but its effects in cancer survivors are still need more study. They evaluated the effects of (Mino) on affective behaviors and inflammations in women with breast cancer undergoing chemotherapy with proven attenuation of IL-8, but Mino did not alter self-effective symptoms or congestions in breast cancer survivors.
       
Breast cancer now accounts for 1 in 8 cancer diagnoses and 2.3 million new cases in both sexes combined, surpassing lung cancer as the most frequent disease diagnosed worldwide (Sung et al., 2021). It was by far the most frequent cancer in women in 2020, accounting for a quarter of all female cancer cases and its prevalence has been rising globally, especially in transitioning nations (Arnold et al., 2022). According to estimates, breast cancer claimed the lives of 685,000 women in 2020, accounting for 16% or one out of every six female cancer fatalities. The World Health Organization (WHO) has launched the Global Breast Cancer Initiative in response to the public health community’s inadequate response to this development (Anderson et al., 2021).
      
Copper metabolism is affected by cancer, infection and inflammation, with increased concentrations of ceruloplasmin and Cu. Interleukins (IL-1) and IL-6 stimulate hepatocytes’ production and secretion of plasma ceruloplasmin (Tapieroa et al., 2003). Copper supports cellular and humoral immunity (Percival.,1998; Huang and Failla., 2000) and is crucial for immune response, including the generation of IL-2 by activated lymphocytic cells (Hopkins and Failla.,1999).
       
Generally, neither humans nor rodents have any problems with the typical levels of Cu that are consumed, nor even with intakes that are significantly higher. For children ages 4 to 8, daily Cu intakes of up to 3 mg/d and for adults, 8 to 10 mg/d are deemed tolerable. These intakes are seven to eleven times higher than the daily allowance (Tanner.,1999).
       
A trace element, Cu is necessary for the operation of numerous cellular enzymes. Because Cu ions can assume 2 different redox states either oxidized Cu(II) or reduced Cu(I)-the metal can be an essential component of Fe absorption, mitochondrial respiration, elastin cross-linking, free radical scavenging and enzyme redox chemistry, as oxidative stress occurs when there is an imbalance between free radicals and antioxidants (Jain and Shakkarpude., 2024), such as lipid peroxidation that plays an essential role in many disorders (Gurgoze  et al., 2024). Copper (Cu), trace element likely to be of most practical significance (Tej et al., 2024). The amount of Cu in cells is determined by a delicate balance between the uptake and efflux of Cu ions. If present in excess, free Cu ions can damage cellular components. Molecular characterizations of Cu homeostasis in biological systems have been made.
       
Cu is the body’s 3rd most common trace element, after Fe and Zn and it is an essential catalyst for hematopoiesis and iron absorption (Kui et al., 2024). Because of the risk of cellular damage from both copper overload and deficiency, copper transport and levels within cells are carefully controlled. Overly high Cu concentrations have the ability to trigger the production of toxic reactive oxygen species (ROS), which are highly cytotoxic and can cause damage to cells (Scheiber et al., 2013). Numerous studies have shown that clinical signs of anemia, leukopenia, neutropenia and allogeneic cytopenia are correlated with Cu deprivation (Halfdanarson et al., 2008).
       
Clinical signs of Cu deficiency can also resemble the neurological symptoms of vitamin B12 deficiency-related spinal cord neuropathy (Jaiser and Winston., 2010). Notably, growth, angiogenesis and metastasis are three aspects of tumor progression where Cu may be a limiting factor (Denoyer et al., 2015).
       
Cu compounds exhibit strong antitumor and anti-metastatic properties when applied to solid tumors (Santini et al., 2014; Balsa et al., 2023; Leon et al., 2017). They are potential therapeutic agents for cancer treatment due to their biocompatibility, decreased toxicity, improved biological availability and higher levels in cancer tissue (Ron et al., 1988). Cu complexes offer a substitute for breast cancer treatment, producing stable copper complexes with distinct functionalities. However, issues with solubility in physiological buffers and complex, unpredictable mechanisms of action hinder their use in clinical settings (Fisher et al., 2007).
       
At present as appeared, the majority of reports have concentrated on minocycline’s non-antibiotic characteristics. We provide experimental research regarding the synthesized novel complex (Mino/Cu) with the obtained data regarding the antibacterial activity against three strains (Salmonella enteritidis, Klebsiella pneumonia and Bacillus subtilis), anti-breast activity (MCF-7) and antioxidant activity regarding the novel complex capability of free radicals scavenging activities via (ORAC test) for evolving of potent and novel effectiveness of Mino.

Experimental work
 
Chemicals
 
All chemicals used: minocycline hydrochloride (C₂₃H₂₈ClN₃O₇), copper chloride (CuCl₂), Ethanol are procured from The Sigma-Aldrich Chemical Company which provides pure grade Chemicals.
 
Synthesis of copper complex
 
Copper minocycline complex was prepared by mixing minocycline hydrochloride (2 mmol of 30 mL ethanolic solution) with copper chloride (1 mmol of 20 mL of distilled water). After that, reflux for six hours. The green solid product was filtered, cleaned with a water-ethanol mixture and vacuum-dried over calcium chloride (CaCl₂), all the analyses were performed in both labs of Taif University (Synthesis) and Zagazig University (Spectroscopic analysis) between January and August 2024; as shown in Fig (1).


 
Instruments
 
The infrared spectra of minocycline hydrochloride and its copper complex were captured as KBr disks using an IR spectrophotometer set to 400-4000 cm^-1. The Spectrophotometer UV2 Unicam UV/Vis was used to record UV spectra between 800-200 nm and the CHN 2400 Perkin Elmer was used to measure the C, H and N spectra. The HACH conductivity meter model was used to determine the conductivity data for the compound manganese minocycline compound using a DMSO solution at a concentration of 10^-3M. Using a Shimadzu thermo-gravimetric analyzer and Joel JSM-6390 equipment, thermogravimetric analysis (TG/DTG-50H) was carried out. SEM images were measured. The X-ray diffraction was recorded. JEOL 100s microscopy was used to measure TEM images.
       
Inoculum preparation (colony suspension method)
 
100 milliliters of broth medium were mixed with strains of Salmonella enteritidis (ATCC 13076), Klebsiella pneumoniae (ATCC 13883) and Bacillus subtilis (ATCC 6633). The mixture was then incubated for 24 hours at 35°C. After streaking each broth onto an Agar medium with a loopful, the aforementioned conditions were allowed to develop, with the exception of B. subtilis, which was cultured on a fresh agar plate for 24 hours at 30°C ± 1.0. To make a direct sterile 0.1 N NaCl solution, three to four colonies were inoculated. The Densi CHEK© optical device was then used to adjust the suspension in order to achieve turbidity, resulting in a suspension with 1-2 x 10⁸ CFU/ml (Al-Thubaiti  et al., 2022).
 
Broth Macro-dilution Method
 
The study involved inoculating samples with Muller Hinton Broth (MHB) into 24- or 12-well plates. Each well was inoculated with two milliliters, then transferred to the next well. This procedure was repeated eight times for each sample. The plates were then incubated and set on a dark surface to track bacterial growth (Al-Thubaiti  et al., 2022).
 
Antibacterial Activity against Salmonella enteritidis (ATCC13076), Klebsiella pneumoniae (ATCC13883) and Bacillus Subtilis (ATCC 6633)
 
Bacillus subtilis, Salmonella enteritidis and Klebsiella pneumoniae were injected into broth medium and incubated at 30°C for 24 hours. A loop from each broth was streaked into the agar medium and the mixture was incubated at 30°C. Control wells were given to each plate and each well was incubated at 30°C for one day. After incubation, the well plates were placed on a dark surface to evaluate bacterial growth (Al-Thubaiti  et al., 2022). The test’s validity was confirmed by the presence of turbid growth in all growth wells and clear, turbidity-free (El-Megharbel  et al., 2024) control wells.
 
Cell culture
 
MCF-7 breast cancer cells were procured from Cairo, Egypt. The cells were cultured at 37°C in 5% CO₂ atmosphere using DMEM medium supplemented with streptomycin, penicillin and bovine serum (Promega Co., USA) (Skehan et al., 1990).
 
Cytotoxicity assay
 
The study tested MCF-7 breast cancer cells and their supplements in DMEM medium using the SRB assay. After three days of exposure to the Mino/Cu complex, the cells were fixed with 10% TCA and washed three times with acetic acid. The absorbance at 540 nm was measured using a German microplate reader called “BMGLABTECH®-FLUOstar (Allam et al., 2018). The concentration of the complex inhibited or killed 50% of the cells.
 
Antioxidant activity
 
ORAC assay
 
The antioxidant activity of Mino/Cu novel metal complex was measured using Liang et al.’s (2014) methodology. Samples were incubated with fluoresceine for 10 minutes at 37°C, followed by 3 cycles of fluorescension measurements and 70µL of newly prepared 2,2-Azobis dihydrochloride, taking approximately 60 minutes with 40 cycles.
 
Statistical analysis
 
The treated groups were compared using a One-Way Analysis of Variance with post-hoc testing and the data were displayed as mean ± S.E. P£0.05 indicates statistical significance Dean et al., 2013.

Microanalytical and molar conductance value
 
Copper minocycline chelate is solid, stable and insoluble in water. Molar conductance value measured in 1.0 × 10^-3 mol/cm³ DMSO for copper compound was Λm = 21 (Ω-1 mol^-1 cm^-1), confirming the non-electrolytic character (El-Megharbel and Hamza., 2022) due to absence of Cl-1 ions inside and outside chelation sphere. For [Cu (Mino)2] · H₂O ratio is 1:2 for Cu (II): minocycline according to values of elemental analysis C, H , N and molar conductance. The sites of donation for minocycline with Cu (II) were confirmed IR, UV, XRD.
 
Infrared spectra (FT-IR)
 
Fig 2 shows the infrared data for minocycline and its Cu (II) chelate. The stretching vibrations of (O-H), (N-H) and (C-H) aromatic molecules are responsible for the vibrational bands in the 3400-2800 cm^-1 range observed in the infrared spectrum of the free ligand minocycline (Nakamoto., 1986). Chelation with the Cu (II) metal ion causes some shifts in wave numbers because of the change in the distribution of electronic density for the aromatic rings and the major attached functional groups. The band at 3350 cm^-1 that was assigned to v (OH) for the minocycline free ligand was absent from the Cu (II) complex. The (C=O) amide group was not involved in the complexation process, as evidenced by the appearance of the Cu (II) complex of free minocycline and its v band at 1649 cm^-1. Two bands at 1754 and 1727 cm^-1 on minocycline are indicative of (C=O) ketonic rings; these two bands vanish on Cu (II) chelate. These data indicate that the compound minocycline chelates with the Cu (II) metal ion via the oxygen atoms of the hydroxyl and (C=O) ketonic groups. For  C-C, C=C and C-H variation of modes of vibration may change due to  complex formation (Masoud et al., 2005). There are new bands at 605 and 550 cm^-1 appeared for Cu(II) complex, corresponding to ν(M-O) (Nakamoto.,1970; Bellamy, 1975). According to the information above, as illustrated in Fig 2, minocycline functions as a bidentate ligand and Cu(II) metal ion is chelated through the oxygen atoms of ketonic and hydroxyl groups.


 
UVVis spectra
 
The electronic spectra of minocycline and its Cu (II) chelate in DMSO solvent are shown in Fig 3. Minocycline has absorption bands at 215,230 and 250 nm that are attributed to n → π∗  transitions due to its organic moiety. Additionally, a band that appears at 355 nm is attributed to n → π∗ transitions. The Cu (II) complexity exhibits weak bands at 220, 230, 240, 250, 275 and 315 nm as transitions between π → π∗ and n → π∗.



 
XRD, SEM and TEM investigations
 
X-ray diffraction was carried out in Fig 4. XRD patterns explain the crystallinity arrangement. Using Cu Ká radiation and X-ray diffraction, Mino and its Cu (II) chelate were examined at room temperature. From 10° to 80°, the diffractograms were analyzed. The Scherrer formula (Cullity,1972), D=k/Cosq  is used to determine the crystalline size, where k is a constant equal to 0.94, λ is the X-ray wavelength (0.154 nm) and β is the full width at half maxima peak of the XRD pattern. 2.91 nm was determined to be the computed crystalline size for manganese complexity.


       
The TEM images for Mino and its Cu (II) complexity are shown in Fig 5 (A,B). For Mino and its Cu (II) chelate, the orderly matrix of the pictograph was revealed. This proves the existence of the Cu (II) /Mino complex’s homogenous phase material. The spherical black spot-like shape of Cu (II)/Mino chelate is accompanied by particle sizes that range from 2.91 to 9.95 nm.


       
Fig 5 (C.D) shows SEM pictures of Mino and its Cu (II) /Mino complex. The pictures demonstrate how tiny and within the nanoscale the particles are. Because of their uniform sizes, all of the particles had a greater capacity for agglomerate formation, according to the surface morphology of minocycline and Cu (II)/Mino.
       
Mino showed up as tiny rectangular protrusions.
 
Thermogravimetric analyses
 
As shown in Fig 6, thermogravimetric and differential thermogravimetric analysis (TGA-DTG) for Cu (II) chelate was performed up to 1000 °C. The presence of an external water molecule is confirmed by a weight loss of up to 154 °C caused by the loss of uncoordinated water. Cu (II) chelate’s thermal analysis curve reveals a weight loss of up to 819 °C as a result of the breakdown of two minocycline ligands and the loss of coordinated water molecules. There are two phases involved in the breakdown of Cu (II) chelate. CuO that has been tainted with a few carbon atoms is the most stable final product.

@figurue6
 
Antioxidant activity
 
The antioxidant capacity of Mino/Cu metal complex obtained by measuring the oxygen radical absorbance (ORAC) is shown in Table 1. Activity of Mino itself was lower by 80.30 % than that of Mino/Cu, this result is very promising result and confirm the greatest free radical absorbance activity of Mino/Cu than Mino itself. 

@table 1
 
Anti-breast cancer activity (MCF-7)
 
The cellular inhibitory concentration (IC50) of the viability of the cells was determined using MasterPlex 2010 software. For the novel complex (Mino/Cu), the percentage of viable cells was 94.91 µg/mL at concentrations of 10 ug/mL and 57.54 µg/mL at concentrations of 100 ug/mL. The faint color in the column of the cellular viability test on the well plate at concentrations of 100 ug/mL, which clearly shows the death of the majority of breast cancer cells, demonstrated the novel potent record of the newly synthesized complex of Mino/Cu in inhibiting the growth of breast cancer cells (MCF-7) to very low cellular viability percentages (Fig 7).


 
Antibacterial activity evaluation
 
The target complexes were assessed biologically using bacterial strains (Bacillus subtilis, Klebsiella pneumonia and Salmonella enteritidis). The results of the antimicrobial activities of the Mino/Cu metal complex are shown in (Fig 8). It was discovered that the Mino/Cu complex exhibited high antimicrobial activities against a variety of bacterial strains and was sufficient. It was discovered that the complex had high levels of inhibition against Gram-positive and Gram-negative bacteria (K. pneumonia, S.enteritidis and B. subtilis) at incredibly low concentrations of 0.625 mg/ml for B. subtilis and 1.25 mg/ml for Mino/Cu against both S.enteritidis and K. pneumonia (Fig 8).


       
An efficient antibiotic for treating a variety of skin, respiratory and genital infections is minocycline “Mino”. In patients who are allergic to penicillin treatment, it is also used as a 2nd-line medication for infections. Due to the adverse reactions that occur after systemic administration, Mino’s application for these infections has been restricted. The intriguing anti-inflammatory and neuroprotective efficacy of Mino has been the subject of numerous studies recently; this subject was initially brought up in case reports in 2007 (Arezoo et al., 2020).
       
After zinc (Zn) and iron (Fe), copper (Cu) is the 3^rd most common trace element in the body and it is an essential catalyst for hematopoiesis and Fe- absorption (Kui et al., 2024). Because of the risk of cellular damage from Cu- overload and deficiency, Cu transport and levels within cells are strictly controlled. Overabundance of Cu has the ability to trigger the production of toxic reactive oxygen species (ROS), which are highly cytotoxic and can cause damage to the cells , which supports the current findings by anti-cancer potency of the novel synthesized Mino/Cu complex against MCF-7 cellular growth (Scheiber et al., 2013; Denoyer et al., 2015).
       
Interestingly, growth, angiogenesis and metastasis are three aspects of tumor progression that Cu may limit (Denoyer et al., 2015) . Against a variety of solid tumors, several Cu complexes have shown strong anti-tumor and anti-metastatic effects (Santini et_al2014; Balsa et al., 2023; Leon et_al2017). Moreover, copper’s lower toxicity and greater biological availability, combined with its elevated levels in cancer tissue, make it a viable therapeutic agent for the treatment of cancer (Ron et al., 1988).
       
Numerous studies have shown that clinical signs of anemia, leukopenia and allogeneic cytopenia are correlated with Cu deficiency (Halfdanarson et al., 2008). Additionally, low birth weight babies and young children with Cu deficiency frequently have abnormalities in their bones, such as osteoporosis, fractures and skeletal deformities (Sutton et al., 1985). Clinical signs to the neurological symptoms of spinal cord neuropathy linked to vitamin B12 deficiency can also result from Cu deficiency (Jaiser and Winston., 2010).
       
Chemotherapy, radiotherapy, immunotherapy and surgery are all used to treat breast cancer. However, only a small number of patients can receive immunotherapy and the quality of life of patients receiving chemotherapy and radiation therapy is significantly impacted. As a result, copper complexes represent a novel therapeutic option for breast cancer. A wide variety of stable copper complexes with distinct functionalities are produced when copper ions combine with metal nuclei in suitable organic ligands to form these complexes. However, issues remain with their solubility in physiological buffers and their complex and unpredictable mechanisms of action, which hinder their use in clinical settings (Fisher et al., 2007) . With continued research efforts, complexes seem to have limitless potential for treating cancer.
       
The current study revealed that the novel complex Mino/Cu potent antibacterial efficacy against risky bacterial strains such as: Salmonella Enteritidis, Previous study (Doorduyn et al., 2006) confirms known risk factors for salmonellosis. Mino/Cu complex inhibited it’s growth at concentration 1.25 µg/ml, which is a bacterial disease of poultry and can cause foodborne illness in humans such as gastroenteritis (commonly known as ‘gastro’) when contaminated food is consumed. Symptoms of a salmonella infection usually include diarrhea, fever, abdominal cramps, headache, nausea, or vomiting. The risk may involve infection of salmonella, it may enter the bloodstream, it can infect tissues throughout your body, including: The urinary system (urinary tract infection), or the tissues surrounding the brain and spinal cord (meningitis), thus the current results present potent data regarding inhibition of this risky bacterial growth.
       
Klebsiella species cause infections in various sites, including the lung, urinary tract, bloodstream, wound and brain. Preexisting health conditions increase the risk of these infections. Klebsiella pneumoniae is a major global concern due to hypervirulent and carbapenem-resistant strains (Chang et al., 2021). The prognosis is poor, especially in patients with diabetes or nosocomial infections. Mortality rates are above 50%. A novel complex, Mino/Cu, showed promising activity at a concentration of 1.25 µg/ml.
       
B.subtilis is a Gram (+ve) spore-forming bacteria. B. subtilis contains B. subtilis subsp.  It is commonly found in the environment and in the human gut (Brutscher et al., 2022).
       
The current study revealed activity against B.subtilis at very low conc. at 0.625 µg/ml, which can be beneficial results in case of resistant strains of B. subtilis.
       
Copper (Cu) can produce reactive oxygen species (ROS) that are crucial for various pathologies, including cancer, nervous system disorders and aging (Halliwell and Gutteridge., 1984). Cu can also exhibit antioxidant activity by complexing with other drug ligands. This investigation used the ORAC test to show the novel complex (Mino/Cu)’s antioxidant activity, which could be a strong regulatory mechanism to prevent toxic accumulation of Cu ions. The addition of an antibiotic increases its antibacterial activities and the novel complex could serve as a potent antioxidant mechanism (Halliwell and Gutteridge., 1990).
       
The study reveals the strong anticancer activity and genuine insight into the strong anticancer activity of the Mino/cu novel complex in inhibiting MCF-7 breast cancer cell proliferation at 57.54 µg/ml at conc. 100 ug/ml of the tested novel complex. Cu-complexes can have therapeutic effects on breast cancer cells through the non-Cu death pathway, which is more selective for the mode of action of recently developed Cu complexes (Chu et al., 2023). This study provides evidence that Cu complexes can inhibit breast cancer cells by targeting proteins associated with cellular death, which in turn causes oxidative stress, apoptosis, oxidative stress and autophagy. More synthesis of novel Cu-complexes with increased specificity may lead to effective treatments for breast cancer in the future.

Utilizing elemental analysis and spectroscopic methods like infrared, 1HNMR, ultraviolet, X-ray diffraction, magnetic susceptibility, SEM and TEM, the novel complex known as minocycline/Cu (Mino/Cu) was produced and investigated. For the novel Mino/Cu novel complex induced high potent anticancer activities by inhibition of MCF-7 proliferation growth by 57.54 µg/ml at concentration 100 ug/ml. Mino/Cu has potent antibacterial activities against the bacterial strains (Salmonella enteritidis, Klebsiella pneumonia and Bacillus subtilis), at very low concentration recording at 1.25 ,1.25 and 0.625 mg/ml respectively. The novel complex Mino/Cu exhibited a potent high antioxidant activity via ORAC activity at 1581 (µM Trolox eq/ml).

The authors extend their appreciation to Taif University, Saudi Arabia, for supporting this work through project number (TU-DSPP-2024-155).
 
Funding
 
This research was funded by Taif University, Saudi Arabia, Project No. (TU-DSPP-2024–155).

The authors would like to confirm that there are no known conflicts of interest associated with this publication and there has been no conflict with any financial support that could have influenced its outcome.