Introduction

In current years, the use of nanoparticles (NPs) has been noted with confined diameter size ranging from 10-100 nm (1) and has also created great promise with their use in biomedical photo-imaging, recovering medication, scaffolds studies, drug distribution, therapeutics, and tissue manufacturing (2-14). The last decade has seen a growing trend towards using nanoparticles for imaging (15,16). A considerable amount of literature is being focused on the theme of biogenic nanoparticles such as aragonite calcium carbonate nanoparticles (17) and gold nanoparticles AuNPs (18). Aragonite is a unique of the polymorphs of CaCO₃ that naturally and richly exists (95 - 98 %) in (Anadara granosa), a mollusc’s cockle shell that commonly originates in Malaysia (19). The effects of using calcium carbonate nanoparticles for imaging have been described with the Eu³⁺-doped calcium carbonate cubic nanoparticles produced by the carbonation synthetic route and the photoluminescence properties characterized by scanning electron microscope and x-ray diffractometer (20-22). In other related studies, it has been demonstrated that the technique used to develop fabricated oxygen-sensitive polymer nanocapsules using layer-by-layer (LBL) tactic using vaterite calcium carbonate nanoparticles as templates. Additionally, the buffer used was decisive in preserving their reliability and ensuring the nanoparticles were stable in alkaline sodium hydrogen carbonate (23). In addition, it also found that the nanoparticles revealed thermal stability and a notable adsorption capacity caused by vigorous spots like the amino and carbonyl groups (24-26). Regarding the AuNPs, there is a relatively small body of literature concerned with the development of AuNPs bioconjugates and their potential use in imaging or other biomedical applications (27,28). Also, due to their low or non-significant toxicity, they are increasingly used in diagnosis, therapeutics, disease treatment, and targeted drug delivery systems (29-34). In addition, AuNPs bioconjugates maintain high stability upon interaction with biomolecules such as proteins and antibodies (35, 36). Major attention is on the AuNP surface plasmon resonance property, which focuses on designing diagnostic biomaterials, drug-targeting agents, therapeutics, and contrast agents (37-43). Furthermore, existing works on AuNPs and their conjugates have extensively been employed in agriculture to enhance the visual detection of pesticides, the food industry to detect contaminants, and the extension of food shelf life (44,45). However, recent developments in biomedical imaging have caused limitations with the imaging probes meant for clinical usage. These include no biological disintegration or gentle elimination and great poisonousness, perplexing the manufacture of a robust imaging indicator, compromising their further evolution into clinical use (15). In addition, research investigations suggest that there are also several challenges associated with targeted tumor nanoparticles administered by intravenous route due to interaction with an intricate atmosphere (46). These include clearance of targeted nanoparticles by the phagocytes, either by effectively removing nanoparticles from circulation, leaving a trivial portion at the tumor sites, or by long retention of the nanoparticles, potentially developing into complications like toxicity (47). Secondly, tumor physiological properties like antigen expression and tumor permeability stop the buildup of nanoparticles or drug delivery in the region (48-50). It has also been elaborated in a prior study that nanoparticles in blood circulation habitually bind to plasma proteins (opsonization) that are phagocyted within the blood, spleen, bone marrow, and liver (51). Similar studies have shown techniques that alleviate these limitations by embracing the stabilization of particle dispersions using coatings and understanding the outcome of the nanoparticles in the bloodstream and their physiochemical properties; thus, there is a substantial need to produce biocompatible nanoparticles with ideal features (52-54). Herewith, Au-CsCaCO3NPs are prepared and assessed in vitro using Lactate Dehydrogenase LDH and Reactive Oxygen Species ROS assays. Cellular uptake of the Au-CsCaCO3NPs was evaluated using fluorescence and confocal microscopy. Primarily, the Au-CsCaCO₃NPs development is prompted by the need for cost-efficient and biocompatible nanomaterial for imaging. The preparation utilizes method-friendly approaches such as the classic Turkevich method (55) and dodecyl dimethyl betaine (BS - 12). The Au-CsCaCO₃NP's potential use for fluorescent imaging is also elaborately discussed.

This work aims to evaluate the gold-near infrared dye conjugated cockle shell calcium carbonate nanoparticles through bio-cellular uptake, and confocal and fluorescence imaging.

Materials and Methods

Ethical approval

The work procedure was permitted via IACUC UPM; AUPR015/2015.

Materials and chemicals

The gold colloid solution was bought from Malaysia (Prima Nexus Sdn Bhd). The breast cancer cell line (JCRB: MCF-7) and the fibroblast cell line (JCRB: NIH-3T3) were commercially bought from the Japanese Collection Research Bioresource (JCRB). All other materials and chemicals used were purchased from (Sigma-Aldrich (in Steinheim, Germany, and (USA); Naclai tesque, Inc., Kyoto, Japan; and Cell Biolabs, Inc., San Diego, CA, USA). Incorporation of NIR dye and production of Au-CsCaCO3NPs, characterization of Au-CsCaCO3NPs through Transmission Electron Microscope (TEM), zeta potential and measurement size distribution, UV-VIS spectrophotometer and cell biocompatibility were discussed in (56).

Cells seeding and treatment

The cultured flasks' cells were detached using trypsin and seeded into ninety-six well sterile dishes at a concentration of 1×10⁵ cells each well. The ninety-six well dishes were then placed into the 5% carbon dioxide incubator at 37˚C for 24 hours. The media in the wells was aspirated, and the cells were treated and co-cultured in replicates with Au-CsCaCO₃NPs solution (concentration of one mg/ml in ten percent serum-free DMEM media), for twenty-four hours, forty-eight hours, and seventy-two hours. The following treatment experience was finished, and the media in the wells was removed and splashed with phosphate-buffered saline. Earlier, it was exchanged with additional media previous to additional new treatments, for example, LDH Assay (58) and ROS Assay (71).

Lactate dehydrogenase assay LDH

Published LDH studies were identified using a search strategy (57) and colleagues developed. Subsequently, cell seeding of MCF-7 cells and NIH3T3 cells in ninety-six-well dishes and treatment, the dishes were incubated for 72 hours after treatment with different concentrations in µg (100, 50, and Control) of the nanoparticle solutions. The 96-well plates were cleared of all previous media, and cell membrane integrity was assessed using a protocol adopted by (58) and colleagues. The protocol measures the amount of LDH out through the lysed cells directly related to the damaged cells.

Lactate dehydrogenase treatment protocol

The 96-well plates were removed from the incubator with the previous treatment. Sterile water and Triton X-100 provided were supplementary to individual wells in triplicates for the pre-seeded and treated plates. The dishes were incubated for ten minutes in a room environment. Approximately 90 µl of media was carefully transferred from each well to clean 96-well plates suitable for micro-plate readers. Around 10 µl of LDH assay reagent was supplementary to the wells and permitted to incubate for 1 hour to allow for LDH activity of the cells. After incubation, the dishes were positioned on a shaker for 10 minutes. The suspension's visual concentration was examined with a micro-plate reader at a wavelength of 450 nanometers; the values were recorded, and a graph was plotted with Excel.

Reactive oxygen species assay ROS

This part of the study was achieved using a protocol from the ROS assay kit. For the preparation of reagents, 1X DCF-DA (20X DCF-DA stock solution was diluted to 1x in serum-free DMEM media and mixed uniformly using a sonicator vortex), and Hydrogen Peroxide (H₂O₂) dilutions were prepared in DMEM serum-free media. For the preparation of a typical curve, about one to ten serial dilutions of DCF standards were prepared in a concentration variety of zero µM to ten µM by attenuating the one mM DCF stock in DMEM serum-free media. Approximately 75 µl of individual DCF standard was transferred to a ninety-six-well dish appropriate for fluorescence measurement, followed by 75 µl of the 2X cell lysis buffer. Fluorescence data readings were obtained using a fluorescence microplate reader, measured at 480 nanometres excitation and 530 nanometres secretion.

DCF Dye filling

The 96-well plates were cleared of all previous media and washed well with PBS twice. About 100 µl of 1X DCF-DA prepared solution was supplementary to the cells and incubated for 45-60 min at 37°C. The 96-well dishes were cleared of all solutions and washed away well with PBS twice. The DCF-DA-loaded cells were treated with the prepared hydrogen peroxide in 100 µl media.

Quantitation of Fluorescence

After treatment with the oxidant, the 96-well plates were cleared of all previous media and washed well with PBS twice. About 100 µl of media was supplementary to individually well along with 100 µl of 2X cell lysis buffer, which was carefully miscellaneous and then incubated for 10 minutes. Around 150 µl of the combination was then moved into a hygienic ninety-six-well dish for fluorescent measurement. The fluorescence was documented at 480 nm excitation and 530 nm emission with a fluorescent micro-plate reader. The experiment was conducted in triplicates.

In-vitro imaging and cellular acceptance of Au-CsCaCO₃NPs

Breast cancer cells MCF-7 were seeded into six-well dishes and permitted to cultivate in 100% DMEM media. Upon accomplishment 80% cell confluence, the media was removed and then exchanged with 1ml of new culture media complemented with 25 mM HEPES comprising Au-CsCaCO₃NPs suspension and incubated at 37˚C for 6 hours. The cells were splashed in PBS solution (3) periods before fluorescent and confocal microscopy examination. Fluorescence images were documented to quantify conjugate nanomaterial uptake by the cells, and fluoresce emission was visualized using a fluorescent microscope (Immunofluorescence microscopy system, Tokyo, Japan).

Fluorescent preparation protocol

The cells were seeded onto four well-chambered sterilized slides (SPL Life sci, made in Korea) and incubated for twenty-four hours overnight. The medium in the wells was detached, and the cells were treated and co-cultured in replicates with Au-CsCaCO₃NPs solution for 72 hours. Once the treatment experience was accomplished, the medium in the wells was removed and splashed with PBS twice. The cells were re-suspended after trypsinization, and 0.5 ml of Devil’s stain (Acridine Orange (AO) and Propidium Iodide (PI) in the ratio of 1:1) was supplementary to individually well for 60 minutes in a room environment. Afterward, a drop was placed on a clean slide with the coverslip. The slides were examined using fluorescence microscopy (Immunofluorescence microscopy system, Tokyo, Japan).

Confocal preparation protocol

The cells were seeded onto 4-wells chambered sterile slides (SPL life sci, made in Korea) and incubated for twenty-four hours overnight. The medium in the wells was detached, and the cells were treated and co-cultured in replicates with Au-CsCaCO₃NPs for 72 hours. Once the treatment experience was accomplished, the medium in the wells was removed and splashed with PBS twice. The cells were stable in 3.7% pre-cooled paraformaldehyde in a room environment for fifteen minutes and afterward splashed twice with PBS. Around 500 µl of Devil's stain was supplementary to individually well for 60 minutes at room temperature and afterward splashed with PBS twice, followed by the counter stain of 50 µl prepared intermediate DAPI (4', 6-diamidino-2-phenylindole) for two mins at room environment. The wells were finally splashed thrice with PBS in the dark, and coverslips were mounted with Prolong gold antifade reagent (Molecular probe, USA). The slides were finally studied using a confocal laser scanning microscope (Zeiss, Germany).

Statistical examination

All data examination was achieved by SPSS software (Version 10, Chicago, USA) by student’s t-test and one-way ANOVA and stated as Mean ± Standard deviation. The level of statistical consequence was P<0.05.

Results

Lactate dehydrogenase assay LDH

This analysis was further intended to evaluate the cell membrane integrity of MCF-7 cell line and NIH3T3 cell line treated with the synthesized Au-CsCaCO3NPs. Analyzing LDH levels after exposure to the concentrations in µg (100, 50, and Control) designed from the assay dose-response for 72 hours. The findings displayed in (Figure 1) validate that (the LDH) percentage (%) release of MCF-7 cells treated with Au-CsCaCO₃NPs was slightly more significant than that of the Au-CsCaCO₃NPs treated NIH3T3 cells.

Figure 1: Comparative LDH released by Au-CsCaCO3NPs treated MCF-7 cells and Au-CsCaCO3NPs treated NIH3T3 cells, presenting higher LDH % release with MCF-7 as compared to NIH3T3 at p< 0.05.

Reactive oxygen species ROS

The objective of ROS experimental assay analysis was to further investigate the ROS of Au-CsCaCO3NPs on MCF-7 and NIH3T3 by measuring the relative fluorescence units (RFU) adapted from the standard as presented in (Figure 2).

Figure 2: DCF Standard curve.

The results show that Au-CsCaCO₃NPs treated MCF-7 cells also showed greater ROS release as matched to the Au-CsCaCO₃NPs treated NIH3T3 cells. They displayed minimal ROS release of less than 400 RFU at a concentration of 100 µg in matched to their MCF-7 counterparts, as indicated in (Figure 3).

Figure 3: Comparative ROS generation by Au-CsCaCO3NPs treated MCF-7 cells and Au-CsCaCO3NPs treated NIH3T3 cells, presenting higher ROS generation with MCF-7 as compared to NIH3T3 at p<0.05.

Fluorescent imaging and confocal imaging

The purpose of fluorescent and confocal imaging of MCF-7 and NIH3T3 was to morphologically visualize cell death and the possibility of cellular uptake of the nanoparticles using the AO and PI double staining method and DAPI. The results in the MCF-7 fluorescent micrographs display most live cells with the Control, unlike the Au-CsCaCO₃NP treated cells, which show more cell death, as revealed in (Figure 4). The NIH3T3 fluorescent micrographs show more live cells with the Control and Au-CsCaCO₃NPs, as revealed in (Figure 5).

Figure 4: Fluorescent images of MCF-7 cells subsequent treatment with (AO), (PI), and merged (PI and AO). Images (a) and (b) show live cells' subsequent treatment with (AO), with the Control having extra cells equal to Au-CsCaCO3NPs treated cells. Images (c) and (d) show dead cells in the next treatment with (PI), with the Control having fewer cells equal to Au-CsCaCO3NPs treated cells. Images (e) and (f) show live and dead cells together after merging. (Magnification ×10, scale bar 100 µm).

Figure 5: Fluorescent images of NIH3T3 cells next treatment with (AO), (PI), and merged (PI and AO). Images (a) and (b) show live cells next treatment with AO, Au-CsCaCO3NPs treated cells showing not much difference with the Control. Images (c) and (d) show fewer or no dead cells in the next treatment with PI. Images (e) and (f) show live and dead cells together after merging. (Magnification ×10, scale bar 100 µm).

However, the most interesting fact is that the MCF-7 treated cells observed more cell deaths than the NIH3T3 treated cell counterparts. The findings in the confocal micrographs are an overview of the mechanisms of intracellular uptake by MCF-7 and NIH3T3 which show the appearance of the nanoparticles inside the cellular compartment under four different fluorescent filters (blue, green, red, and merged filters) unlike the controls as indicated by arrows within their figures illustrated in (Figures 6-9).

Figure 6: Confocal micrographs of MCF-7 Control presentation cellular morphology under different fluorescent filters using DAPI (blue), AO (green), PI (red), and merged filter. ((A) ×63, scale bar 50 µm (B) ×20, scale bar 100 µm).

Figure 7: Confocal micrographs of Au-CsCaCO3NPs treated MCF-7 cells presentation cellular acceptance and morphology under different fluorescent filters using DAPI (blue), AO (green), PI (red) and merged filter (living cells (control cells) pointer by black arrows). ((A) ×63, scale bar 50 µm (B) ×20, scale bar 100 µm).

Figure 8: Confocal micrographs of NIH3T3 Control presentation cellular morphology under different fluorescent filters using DAPI (blue), AO (green), PI (red), and merged filter. ((A) ×100, scale bar 20 µm (B) ×20, scale bar 100 µm).

Figure 9: Confocal micrographs of Au-CsCaCO3NPs treated NIH3T3 cells presentation cellular acceptance and morphology under different fluorescent filters using DAPI (blue), AO (green), PI (red) and merged filter (living cells (control cells) pointer by black arrow). ((A) ×100, scale bar 20 µm (B) ×20, scale bar 100 µm).

Discussion

The results show a biocompatibility assessment of the nanoparticles on the cell lines by evaluating the cell membrane integrity. It was initially established that a normal cell membrane is impermeable to LDH release activity, and a common and standard biocompatibility test was used in in vitro cytotoxicity studies (57). A bioassay was used to quantify the number of dead cells through LDH leaked from the damaged cell plasma membrane correlating to cell death rate (59). This study surprisingly illustrates that the conjugated nanoparticles LDH % release of NIH3T3 was significantly lower compared to that of MCF-7. This result could be clarified because nanoparticle internalization could prompt intracellular responses along with the growing accumulation of LDH within the cytosol, facilitating the reverse conversion of lactate to pyruvate following a decrease of NAD⁺ to NADH within the cell. Furthermore, these results propose the possibility of conjugated particles for biomolecular and cellular bio-medical uses, such as imaging and drug distribution.

Several reports have proven LDH to oxidize lactate to pyruvate while consuming NADH (60). The cytosolic enzymes are released into the extracellular liquids only when the cell membrane integrity is absent, assessing the cell membrane integrity by calculating the quantity of LDH released from the lysed cells, which is directly proportional to the damaged cells (58). Recent literature on LDH findings with work done on cancer cell line HeLa and MCF-7 using nanoparticle delivery systems and surface functionalized nanoparticles have reported higher LDH leakages (61). In the same regard, high LDH leakages have been confirmed with MCF-7 as a result of metallic nanoparticles (62), but also known reports have argued that lower LDH release with slight comprises of the cell membrane integrity by fibroblast cells L929 (63). However, this outcome is contrary to that of Smith (58), Lanari (64), Abdullah (65), Koren (66) who found that LDH assay studies on HeLa, MCF-7, and NIH3T3 using gold nanoribbons revealed utmost LDH discharge in HeLa than MCF-7 and reported no significant difference with the LDH% discharge between MCF-7 and NIH3T3 (67). In addition, previous studies on nanomaterials have reported that they induce high LDH levels, attributed to nanoparticle morphology inducing oxidative stress (68). Therefore, the current LDH results revealed no toxicity, as demonstrated by the conjugated nanoparticles.

Collectively, the overall ROS assay findings of the nanoparticles on MCF-7 and NIH3T3 offer beneficial thought into O₂ metabolism. ROS refers to chemicals, radicals, or molecules that include reactive oxygen composites, for example, peroxides subsequent from typical O₂ metabolism with vigorous characters in homeostasis and cell signaling (69). This study was intended to determine the cell lines' relative fluorescence units (RFU). The results in this work indicate that the Au-CsCaCO₃NPs treated MCF-7 cells showed greater ROS release as paralleled to the Au-CsCaCO₃NPs treated NIH3T3 cells. ROS generation of the NIH3T3 was considerably lesser than on MCF-7 and showed reliable ROS generation, likewise in agreement with earlier biocompatibility outcomes highlighting the remarkability of Au-CsCaCO₃NPs.

Consequently, these outcomes could be explained by the statement that the internalization of the nanoparticles probably caused oxidative stress, which, in sequence, caused cell death, as displayed in the outcomes. This work supports evidence that the introduction of environmental stress significantly raises ROS levels, producing considerable cellular injury, furthermore, known as oxidative stress (70). Also, it corroborates the results of earlier studies in which cancer cells create significant levels of ROS. This is further explained by increased metabolic activities of oxidases and peroxisomes, mitochondria malfunctioning, or cellular dysfunction (71-73). Constant with prior works, this research found literature that reported nanoparticle cellular acceptance to make mitochondrial membrane penetrability and damaging the respiratory chain, producing apoptosis (74). Similarly, it has been argued for native cancer treatment that (Burkitt lymphoma B cells) and (epithelial breast cancer cells) produced probable injury using directed X-ray and AuNPs causing great ROS generation leading to cellular necrosis or apoptosis nonetheless, whereas supporting slight injury to neighboring, particle-free tissue (75).

Furthermore, it has been proven that carbon and metallic nanomaterials produce slight ROS. But, their morphology, size, positive surface charges, aggregation, cellular interface, and nano metallic ions guide oxidative stress discharge, producing physiological dysfunction of the cell, which in sequence stimulates DNA injury (76,77). This finding contradicts previous studies, which have suggested a significantly higher generation of ROS over time by metallic nanomaterial as described by Xue (78) with works on human liver HepG2 cells. Similarly, no significant effects were found with the work on NIH3T3 cells (79). Conclusively, these results offer competencies of the conjugated nanoparticles for cellular imaging applications.

These results provide interesting insights into fluorescence and confocal imaging of the Au-CsCaCO₃NPs treated cells of MCF-7 and NIH3T3 using AO, PI double staining, and DAPI. The current work was intended to define the cell death and cellular acceptance of the synthesized nanoparticles where the most prominent finding to arise from the fluorescence imaging is the fact that the MCF-7 treated cells observed more cell deaths in comparison to the NIH3T3 treated cells counterparts. Additionally, from the confocal imaging, it is abundantly clear that the cells could take up nanoparticles within their cellular compartments. These results reflect those of Feng (80) and Yuan (81), who also found that fabricated bio-conjugated nanoparticles confirmed cellular internalization into MCF-7 and MDA-MB-231 over NIH3T3. Furthermore, these results encourage agreement with further work. In this water, soluble fluorescent conjugated polyelectrolytes with self-assembly 3D nanostructures were established to use for bioimaging of cancer and normal cell lines, including MCF-7 and NIH3T3 which also showed comparable figures as well (82).

In addition, the present study also supports similar evidence from Zhang (83) that conjugated nanoparticles were booked up by the treated MCF-7 and NIH3T3 cells. More importantly, the results align with recent studies that confirmed cellular acceptance of the nanoparticles, cell death findings, and non-toxicity to the normal cell line over the cancer cell line (84-86). Although these results differ from some published studies Muehlmann (87) and Li (88) they are consistent with several recent works Li (89), Li (90) and Yang (91). It seems likely that these results could be due to a numeral of features affecting the efficiency of the nanoparticles and cellular uptake, for example, size, shape, charge, and surface modification of the nanoparticles, explained by pinocytosis, a type of endocytosis associated with internalization of the nanoparticles. Elaborately, smaller particles, such as nanoparticles, can easily be internalized by the cell (92-95). Regarding shape and charge, it has been confirmed that more positive charges and spherical particles are easily booked up by the cell caused by the highly negative charge present in the cell membrane (96-100). These are useful results; therefore, it is safe and possible that our conjugated nanoparticles could be used for bio-imaging.

Conclusions

This research aimed to assess fluorescent imaging and bio-cellular uptake of the Au-CsCaCO3NPs. Based on the results of this study, it is now possible to state that the easily synthesized conjugated nanoparticles are biocompatible, environmentally friendly, and could be used for bio-imaging. Moreover, they allowed for cellular uptake by cancer cells and standard cells and imparted cell death to the cancer cells in contrast to the standard cells. Altogether, these findings recommend the possible opportunity the synthesized conjugated nanoparticles could play in progressing cancer imaging. This research extends our knowledge of bio-imaging using Au-CsCaCO3NPs and its potential application in diseased-cellular diagnostics.

Conflict of interests

The authors announce that they have no competing interests in the publication of this research article.

Acknowledgments

We would like to thank all the staff of (Institute of Bioscience, Universiti Putra Malaysia), and (Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia) who assisted in obtaining the data. This work is financially supported by Fundamental Research Grant Scheme (FRGS) provided by Malaysian Government [Grant Project no. 5524842] and Islamic Development Bank M.Sc. Scholarship Programme.