Biogenic Nanocopper: Eco-Friendly Synthesis,Characterization,and Their Anti- Trichomonas vaginalis,Anticancer,and Antimicrobial Effects

Abstract

Biogenic nanocopper (BNC) agents exhibit strong anticancer, antimicrobial, and antiparasitic effects. Their fewer side effects to normal cells cause them to be preferred to treat various diseases. Metal nanoparticles, particularly copper nanoparticles, are attracting more significant interest as therapeutic agents with the improvement of green synthesis methods. Studies to reduce the side effects of copper nanoparticles to exhibit strong pharmacological properties are progressing intensively. Here, BNCs with reduced side effects were synthesized using L-ascorbic acid as the reducing agent and various concentrations of copper (II) chloride. BNCs exhibited significant pharmacological activity on cancer, bacteria, and Trichomonas vaginalis cells. The newly synthesized BNCs were characterized by scanning electron microscopy, UV-Vis spectrophotometry, Fourier transform infrared spectroscope, and Differential/Thermal Gravimetric Analysis. The pharmacological activity of BNCs was evaluated by obtaining their inhibitor concentration and minimum inhibitory concentrations against some cancer, bacteria, and T. vaginalis cells. Newly synthesized BNCs have various shapes such as cubic, spherical, or rod and particle size distribution between 70 and 100 nm. According to experiment results, the newly synthesized BNCs were a significantly antiproliferative, antibacterial, and anti-T. vaginalis effect on cells compared to the control drugs. These findings confirm newly synthesized BNCs and their in vitro pharmacological potential. Further research should be targeted on the preclinical study of absorption, distribution, metabolism, excretion/toxicity (ADME/Tox) and in vivo effects on cancer and microbial pathogens.

INTRODUCTION

Nanotechnological products, which are increasing in use in today's medical science and technology, are materials with a three-dimensional structure ranging from 1 to 100 nm, and due to their unique properties, they have led to remarkable advances especially in advanced medical applications. Metal nanoparticles, one of the important nanotechnological products, potentially attract more attention especially in the treatment of cancer. The unique physicochemical and pharmacological properties of Biogenic Nano Copper (BNC) from metal nanoparticles have been previously shown.^1,2 Although nanocopper can be synthesized by various physical and chemical means, syntheses using biological reducers have gained importance. Plant extracts used for this purpose include various types of various agents such as phenolic compounds.

In recent years, the use of plant extracts for the production of BNCs nanoparticle has been largely defined and attracts the attention of many researchers, especially in the preferred biocompatible nanoparticle production.^3

–7 In addition, the pharmacological properties of BNCs obtained from plant extracts such as Eryngium caucasicum depend on the size, stability, and concentration of these BNCs.^8,9 The advantages of this method are, briefly, nontoxic, stable, low-cost, high purity, and homogeneous BNCs nanoparticle production.^10

–16 However, this biosynthesis method makes it difficult to bring the shape and size of BNCs into a more suitable form for use in chemotherapy or various biomaterials. Therefore, the use of plant-derived but pure reducers has gained importance. The natural and pure green synthesis method, such as L-ascorbic acid, is increasingly used to obtain ideal BNCs to be used in pharmacological and biomedical applications.^17

–21

With the current study, it was aimed to synthesize, characterize, and do pharmacological tests of L-ascorbic acid with smaller size and effective BNCs. Using different concentrations of L-ascorbic acid to reduce copper ions in the production of BNCs allows us to adjust the size and shape of the nanoparticles. To show this, morphological analyses of the BNCs were investigated by using scanning electron microscopy (SEM). The chemical analyses, including stability, of the BNCs were conducted by using Fourier transform infrared spectroscopy (FTIR), Thermo gravimetric Analysis (TGA), Differential Thermo gravimetric Analysis (DTG), and UV-visible spectroscopy. The pharmacological activities of the BNCs were evaluated to be used in anticancer, antibacterial and antiparasitic tests.

MATERIAL AND METHODS

Green synthesis and characterization of BNCs

Copper (II) chloride dihydrate (CuCl₂.H₂O, 99.99% Pure) and L-ascorbic acid (C₆H₈O₆, ≥ 99.0% pure) from Sigma-Aldrich were used as received. The preparation of the BNCs was performed using the general procedure, the so-called green synthesis.²² To synthesize BNCs, 0.2 M of 10 mL CuCl₂.H₂O was put into each airtight lids flask and was kept at 90°C. Then, 0.5 M of 10 mL (CuNP-1), 1 M of 10 mL (CuNP-2), 1.5 M of 10 mL (CuNP-3), and 2 M of 10 mL (CuNP-4) of L-ascorbic acid solution were added to each flask by drop wise, and vigorously shaken in a shaker incubator at same temperature for 24 h. At the end of the whole process, the samples were divided into two groups, one of which was sonicated (which called -x). An ultrasonic processor at 20 kHz and 90 μm levels of amplitude for 15 min at 15-s intervals was used to obtain a different shape BNCs.

After, BNCs powder was obtained by filtering through the Whatman No 1 filter paper. IR spectra were measured by a Jasco FT-IR 430 spectrophotometer using KBr pellets in the range of 4000–400 cm⁻¹. Thermogravimetric analysis (TGA) studies were carried out on Perkin-Elmer Diamond TG/DTA Thermal Analysis instrument under an inert nitrogen atmosphere, with a heating rate of 10°C min⁻¹, at 35–900°C. To SEM, 20 mg BNCs powder was solved into 1 mL dimethyl sulfoxide (DMSO) and 10 μL solved BNCs added on the surface of a glass microscope slide. Samples were dried in the atmosphere and mounted on metal stub using double-sided adhesive carbon tapes and sputtercoated with gold. The shape and surface characteristics of BNCs were recorded using SEM at 20 kV (Zeiss LEO 440, Cambridge, United Kingdom).

Stability study

According to International Conference on Harmonisation Q2(R1) validation of analytical procedures: text and methodology, a spectrophotometric method was used for the stability features of these BNCs in physiological buffer. For the stability studies with these BNCs, a stock solution of each of the BNCs was prepared in Tris–HCl buffer. The working solution was prepared by adding 0.1 M phosphate buffer to obtain a volume range from 1.95 to 250 μg/mL. The BNCs in physiological buffer were analyzed over the 3-day study period.

Cell proliferation assay

HT29 (Human colorectal adenocarcinoma, ATCC^®HTB-38™), HeLa (Human cervix adenocarcinoma, ATCCCCL-2™), MCF7 (Human breast adenocarcinoma, ATCCHTB-22™), A549 (Human lung carcinoma, ATCCCCL-185™), C6 (Rattus norvegicus brain glioma, ATCCCCL-107™), and Hep3B (Human hepatocellular carcinoma, ATCCHB-8064™) cancer cell lines and FL (Human amnion cells, ATCCCCL-62™) normal cell line were maintained in a suitable medium containing fetal bovine serum and antibiotic solution. A Cell suspension was adjusted 1 × 10⁶ cells in 10 mL and transferred 100 μL into each well of culture plates. The BNCs were dissolved in sterile DMSO at final concentrations of 10–200 μg/mL and transferred the cells at 37°C with 5% carbon dioxide for overnight. The antitumor activities of the BNCs were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. In MTT assay, the percent inhibitions of test and control molecules were determined.

The percent inhibition was equal % inhabitations with following formula; % inhibition = (A sample – A control)/(A control) × 100. The half maximal inhibitory concentration (IC50) values of the BNCs were obtained by using Excel software and noted in microgram per milliliter at 95% confidence intervals. Three dose-response parameters (GI50, total growth inhibition [TGI], LC50) were calculated according to the following formulas using the absorbance measurements of time zero (Tz), control growth (C), and test growth in the presence of drug (Ti). Growth inhibition of 50% (GI50) was calculated from [(Ti–Tz)/(C–Tz)] × 100 = 50, which is the drug concentration resulting in a 50% reduction in the net growth increase in control cells during the drug incubation. The drug concentration resulting in TGI was calculated from Ti = Tz. The LC50 indicating a net loss of cells following treatment was calculated from [(Ti–Tz)/Tz] × 100 = −50.

Culture of microorganisms and minimal inhibitory concentration

According to The European Committee on Antimicrobial Susceptibility Testing (EUCAST), the antibacterial activity of chemicals was assessed against seven gram (+) bacteria species; E. faecalis VRE ATCC 19433, E. faecalis ATCC 29212, S. aureus ATCC 25923, S. aureus MSSA ATCC 29213, S. aureus MRSA ATCC 46300, S. mutans ATCC 35668, S. gordonii NCTC 7870, four gram (–) bacteria species: E. coli ATCC 25922, E. coli ESBL ATCC 35218, P. aeruginosa AGME ATCC 27853, and A. actinomycetemcomitans ATCC 33384. As for bacterial cultures, a total of 11 bacterial cultures were grown in solid Mueller Hinton agar supplemented with 5% defibrinated sheep blood strains.

Bacterial inocula were prepared using Luria-Bertani broth (for E. coli), trypticase soy broth (for S. aureus, S. gordonii, P. aeruginosa, and A. actinomycetemcomitans), and brain heart infusion broth (for S. mutans, E. faecalis) cultures and suspensions were adjusted to 0.5 McFarland standard turbidity (1.5 × 10⁸ CFU/mL). The minimal inhibitory concentration (MIC) values of BNCs toward some human bacterial strains were examined with the help of a microwell dilution method. Each BNCs was dissolved in DMSO (20 mg/mL). A concentration gradient range from 7.81 to 1000 μg/mL in uncovered microplate wells containing nutrient broth was made by using serial two-fold dilutions of these BNCs. This plate was inoculated with bacteria and incubated at 35°C for 24 h. At the end of this period, the growth of microorganisms was determined visually and the point where no visible growth was accepted as the MIC.

Antiparasitic assay

Trichomonas vaginalis ATCC 50148 strain, which is susceptible to metronidazole (MTZ), was used. After the Cysteine Pepton Liver Maltose (CPLM) medium was prepared, it was autoclaved at 121°C for 15 min. Then, 10% inactive horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin were added to the medium that cooled to 50°C. T. vaginalis strain was planted in the prepared CPLM medium and incubated at 37°C under anaerobic conditions. In the logarithmic growth phase, T. vaginalis strain was counted in the Thoma slide and distributed in equal amounts (100 μL) to 96-well plates. The BNCs prepared in different concentrations (1.96, 3.91, 7.81, 15.625, 31.25, 62.5, 125, and 250 μg/mL) was added to the wells, and then CPLM medium was added to them with a final volume of 200 μL. The plate was incubated at 37°C under anaerobic conditions.

Protozoa at each dose were counted microscopically on the Thoma slide at the 3rd, 12th, and 24th h, and the presence of a possible inhibition was determined by comparison with the control group. Viability status of the protozoa was performed using a 1% eosin solution. The lowest dose with no viable parasites was detected microscopically and evaluated as the Minimum Lethal Dose. These validated parasites were cultured again and incubated for 2 days and microscopically confirmed that there were no live parasites.

RESULT AND DISCUSSION

Synthesis and characterization of BNCs

Confirmation of synthesis of BNCs was done by using UV–visible spectroscopy, FTIR, and SEM techniques. According to these measurements, CuNP-3 and CuNP-4 were successfully synthesized, and therefore, the sonication process applied to these, only. TGA data of the synthesized nanoparticles (CuNP-3 and CuNP-4) are given in the Supplementary Table S1. In addition, TGA and DTG curves of the BNCs (CuNP-3 and CuNP-4) are given in the Supplementary Figures S1 and S2, respectively. It can be seen in the Supplementary Table S1 that the CuNP-3 degrades in four steps, while the CuNP-4 degrades in five steps. Since the nanoparticles were prepared in deionized water, it is highly likely that water molecules are removed in the first step in the both nanoparticles (CuNP-3 and CuNP-4). The peak of -OH vibrations at 3500 cm⁻¹ in the FT-IR spectrum (Supplementary Fig. S3) confirms this (Supplementary Table S2).

As shown in Supplementary Table S1, CuNP-3 begins to decompose at higher temperature than the CuNP-4 (453, 118°C, respectively). It is known that thermal stability increases as decomposition temperature increases. Therefore, the CuNP-3 is thermally more stable than the CuNP-4. During nanoparticle synthesis using plant extracts, the reduction of Cu²⁺ ions to Cu⁰ can be followed using spectrophotometric techniques. Supplementary Figure S5 (B, line of a) exhibit that the absorption spectra of the BNCs synthesized in the reaction medium have absorbance peaks at 430 and 520 nm in Tris-HCl buffer. The absorbance peak at about 430 and 520 nm of the BNCs synthesized in this study is consistent with the previous reports, and it is thought that the small shifts in absorbance values are due to the shape and size of the particles.²³

The medium intensity bands observed at 3500–3000 cm⁻¹ in the FTIR spectrum of L-ascorbic acid (Supplementary Fig. S3) show intermolecular H-linked -OH stretching vibration frequencies in the free acid. In addition, the weak peaks in the region of 3000–2800 cm⁻¹ belong to aliphatic C-H stretching vibrations. The difference observed in O-H stretching vibrations in the FTIR spectrum of CuNP-3 and CuNP-4 is attributed into acid ionization and metal-ascorbate formation. It shows the presence of H₂O in the structure the peak observed at 3500 cm⁻¹ in the metal-ascorbate salts. Thermal analysis data also confirm this (Supplementary Fig. S1). While C = O stretching vibrations of the free ascorbic acid are observed at medium intensity at 1754 cm⁻¹, it is seen that the same peak shifts to lower frequencies in the metal salts and overlaps with C = C peaks.

The bands of the C-O and C-C stretching vibrations observed at 1120 cm⁻¹ and 821 cm⁻¹, respectively (Supplementary Table S2), in the spectrum of the free acid largely lost their density in the metal-ascorbate salts (CuNP-3 and CuNP-4). While the ring-C-O-C and C-C-C deformation modes of the free acid are seen as several bands of medium intensity in the region of 900–500 cm⁻¹, change of intensity of same peaks in the metal-ascorbate spectrum proves acid ionization and metal-ion salt formation. The spectral modifications observed for these skeletal vibrations are attributed into reorganization of H-bond systems and acid ionization on metal interactions.

Scanning electron microscopy data analysis of BNCs

The morphology and size of the BNCs were investigated using SEM. Cu nanocrystals have been easily synthesized by using ascorbic acid as organic-based reducing agent and copper chloride as precursor. In addition to this approach, sonication process may serve as a resizing agent to form convenient size of BNCs. While the BNCs nanostructured particles introduced sonication apparently show a cubical (∼20 nm) or amorphous (∼10 nm), nonsonicated BNCs (CuNP-3 and CuNP-4) display spherical (∼60 nm) or amorphous (∼5–20 nm) in shape morphology with dimensions generally less than 40 nm (Supplementary Fig. S4). However, the size of the nanoparticles was more than 60 nm because of cluster forming to get more close to each other. In addition, sonicated BNCs (CuNP-3x and CuNP-4x) nanoparticles were placed in a homogenous way, nonsonicated BNCs exhibited heterogeneous settlement.

Stability features of the BNCs in phosphate buffer

The BNCs exhibited good stability in phosphate buffer (Supplementary Table S3). Intraday measurements indicated that CuNP-4 remained more stable compared to CuNP-3. The spectra of the CuNP-3 exhibited a slight decrease in the peak from the second and the third day to the finish of the study (Supplementary Fig. S5 and Supplementary Table S3). The linearity, precision, and accuracy of the BNCs are within acceptable limits for the UV-Vis spectrophotometric method (Supplementary Fig. S5 and Supplementary Table S3). These results showed that the CuNP-4 in solution was found to be more stable up to 72 h at room temperature. Eight-point calibration curves were obtained in the concentration range from 1.95 to 250 μg/mL for these BNCs. The plots in measuring of the BNCs spectra were found to be linear in the scanning concentration range and the linearity values of CuNP-3 and CuNP-4 were 0.9866 and 0.9799 (Supplementary Table S3).

The UV-Vis spectrophotometric method results demonstrated that the % relative standard deviation (RSD) values of CuNP-3 and CuNP-4 for the repeatability for precision studies were within acceptable limits and were 0.7199 and 0.5461, respectively (Supplementary Table S3). The limit of quantification (LOQ) values of CuNP-3 and CuNP-4 were 48.515 and 59.601 μg/mL, respectively (Supplementary Table S3). It is recommended that drug stability be evaluated as one of the critical attributes and performance characteristics during clinical trials. Stability studies help determine the shelf life of the drug product by providing evidence of how the quality of active pharmaceutical ingredient (APIs) varies overtime under certain conditions. Good measured stability values of BNCs ensure their entry into further pharmacological studies.

Evaluation of anticancer properties of the BNCs

The burden of cancer, which still causes more deaths than similar incurable diseases, on people is increasing. Also, most of the cancer cases have difficulty in reaching effective chemotherapy services. Today, many studies are still carried out to find effective and cheap anticancer agents. For this purpose, BNCs were synthesized by using green synthesis method, and the anticancer effects of those four BNCs were evaluated according to the MTT protocol. Cisplatin and 5-FU are frequently used as chemotherapeutics in the clinic, and therefore, used as a positive control in this study. When TGI and inhibitor concentration (IC50) values of the BNCs were examined, it was concluded except some exceptions that all of those BNCs have strong anticancer effect on the cell line (Supplementary Tables S4 and S5).

When the anticancer effects of the BNCs were perused in C6 cells, all of those BNCs were detected to be effective (TGI: 2.32–7.92 μg/mL; IC50: 2.66–10.57 μg/mL) (Supplementary Table S4). CuNP-3x among BNCs was found to be more effective (TGI: 3.94 μg/mL), whereas CuNP-3 displayed a mild anticancer effect on the HeLa cell line (TGI: 7.79 μg/mL) (Supplementary Table S4). While only CuNP-3x (TGI: 188.88 μg/mL) among the BNCs reached sufficient antiproliferative activity on A549 cells, cisplatin and 5-FU caused strong anticancer effect at the desired level (TGI: 54.23 and 63.25 μg/mL, respectively) (Supplementary Table S4). On Hep3B, HT29, and MCF7 cells, CuNP-3x and CuNP-4x exhibited a potent effect (TGI: 4.41–165.12 and 6.90–7.07 μg/mL, respectively) (Supplementary Table S5).

Interestingly, sonicated BNCs (CuNP-3x and CuNP-4x) even at low concentrations provide enough antiproliferative effects when compared to the normal (CuNP-3 and CuNP-4) and control group (Cisplatin and 5-FU) (Supplementary Table S4 and S5) on the cancer cell lines. According to National Cancer Institute's (NCI) screening method, the low GI50 values (1–10 μg/mL) of the BNCs emphasized their pharmacological significance (Supplementary Table S4 and S5). However, the high values of lethal concentration (LC50) (>500 μg/mL) exhibited by the BNCs examined by NCI's screening method displayed their usability in clinic. When the antiproliferative effects of the BNCs on FL normal cells were examined, it was seen that CuNP-3x among BNCs showed strong toxic property (LC50: 25.72 μg/mL) (Supplementary Table S6). However, CuNP-4x exhibited low toxic feature against FL normal cells (LC50: > 500 μg/mL), and therefore, only CuNP-4x have reasonable LC50 values for pharmacological studies (Supplementary Table S6).

Evaluation of antibacterial effects of the BNCs

Bacterial infections, especially hospital-acquired ones, have become resistant to even strong antibiotics. This resistance generally develops as multidrug-resistance and decreasing the efficacy of the existing antibacterial drugs. In fact, for over 40 years, a new group of antibiotics has not yet managed to enter the clinic. In the guiding light of this literature of knowledge, to find a new agent for cure is still very significant. Therefore, the effects of BNCs on some pathogenic bacteria causing disease in human body have been revealed by using the MIC method. When the MIC values of sulbactam/cefoperazone (SCF) were used as positive control, the BNCs were considered to be antibacterial at 250 μg/mL and below the MIC values.

When the MIC values of BNCs displayed on gram (+) bacteria were examined, it was found that antibacterial effects of CuNP-3 against E. faecalis VRE ATCC 19433 (250 μg/mL) and S. mutans ATCC 35668 (125 μg/mL) and CuNP-4 against only S. mutans ATCC 35668 (250 μg/mL) were more or similar to the SCF antibiotic used as a positive control (Supplementary Table S7). Our test of BNCs failed the strong antimicrobial effect against pathogenic gram (–) bacteria. It could be concluded that BNCs prove to be suitable for advanced pharmacological investigations for only E. faecalis and S. mutans.

Evaluation of antiparasitic effects of the BNCs

T. vaginalis is one of the most common sexually transmitted protozoans that causes infection (trichomoniasis) in the urogenital system. Although trichomoniosis is usually asymptomatic, it is associated with serious complications such as vaginitis in women and urethritis in men. MTZ has been used as the only treatment option for many years. To increase the strength of MTZ and shorten the treatment period, there is a need to find more successful agents healing trichomoniosis without side effect. Herein, we report the in vitro analysis of BNCs against T. vaginalis. The CuNP-3 and CuNP-3x, at 4 μg/mL, induced complete parasite death after 12 h exposure time (Supplementary Table S9).

Analysis of data obtained from the microscopic count displayed that 3 h and 12 h exposure time of CuNP-3x reduced the parasite viability up to 100% at 8 μg/mL, and 4 μg/mL, respectively (Supplementary Tables S8 and S9). The other two BNCs, namely CuNP-4 and CuNP-4x, mildly reduced the parasite viability by 100% at 128 μg/mL and 16 μg/mL after 24 h exposure time, respectively (Supplementary Table S10). Our results showed that CuNP-3x is a more promising agent that can be entered the further pharmacological study for T. vaginalis treatment.

CONCLUSIONS

We have successfully synthesized stable copper nanoparticles using L-ascorbic acid. BNCs shapes may depend on the L-ascorbic acid rate and sonication process, which act as both reducing and resizing agents. FT-IR spectra of these BNCs pointed out that reducing process formed in the metal-ascorbate formation. The thermal decomposition behaviors of BNCs were understood to follow the usual pattern, in which the first water molecule is liberated. This observation implies that BNCs are sufficiently pure. The synthesized BNCs displayed remarkable antiproliferative activity against cancer cells when compared to positive controls. When it comes to the antibacterial effect of the BNCs, they displayed notable antibacterial properties against E. faecalis and S. mutans. The BNCs, especially CuNP-4x, exhibited high antiparasitic activity against T. vaginalis.

In fact, significant advantages of the BNCs synthesis were the low cost, ease, and safety of the synthesize method with the help of “green” approach. The positive controls used here are both very expensive and require many toxic chemicals to be synthesized. Therefore, it is essential that this method be developed enough to generate new drug molecules. Thus, the size and distribution control affecting the activity of the formed BNCs can be adjusted as desired. Taken together, the results described herein provide a good basis for further pharmacological and chemical studies about successful anticancer, antimicrobial, and antiparasitic administration of these nanoparticles.

Footnotes

AUTHORs' CONTRIBUTIONS

B.D., E.P., A.A., A.D., H.A., N.O., and A.K. participated in the study design and conducted experimental studies and prepared the article. All authors have read and approved the last article.

ACKNOWLEDGMENT

The authors are thankful to Dr. Şaban Tekin for generous assistance.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

FUNDING INFORMATION

This study was supported by the TÜBİTAK (Grant No. 2209A).

SUPPLEMENTARY MATERIAL

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

Supplementary Table S6

Supplementary Table S7

Supplementary Table S8

Supplementary Table S9

Supplementary Table S10

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