{"id":58716,"date":"2024-06-25T11:08:27","date_gmt":"2024-06-25T11:08:27","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=58716"},"modified":"2024-07-03T17:18:44","modified_gmt":"2024-07-03T17:18:44","slug":"green-synthesis-of-silver-nanoparticles-using-cardiospermum-halicacabum-leaf-extract-and-its-effect-on-human-colon-carcinoma-cells","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/green-synthesis-of-silver-nanoparticles-using-cardiospermum-halicacabum-leaf-extract-and-its-effect-on-human-colon-carcinoma-cells\/","title":{"rendered":"Green Synthesis of Silver Nanoparticles using Cardiospermum Halicacabum Leaf Extract and its Effect on Human Colon Carcinoma Cells"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Nanotechnology is a multidisciplinary field of\nresearch having applications in electronics, nanomedicine, aeronautics,\nbiomaterials and energy, cosmetics, and food. Metal nanoparticles such as\nplatinum, silver, and gold are broadly applied in diagnostic sensors,\nantimicrobials, and as agents for delivery of drug and gene (5, 38)<\/sup>. There is a dire need for\ndeveloping eco-friendly nanomaterial synthesis techniques that prevent harmful\nby-products associated with present physicochemical procedures. The relevance of green synthesis\nin today\u2019s times is immense and it must be the only way driving major\nscientific processes linked to nanotechnology in the future. Green synthesis\nallows for the reduction in generating any waste that is harmful to the\nenvironment, and prevents it by reducing the nanoparticles with the help of\nbiodegradable material like plant extracts, etc. Hazardous by-products that are\nunfriendly in nature could be reduced with the help of this method, and is\ntherefore a highly useful procedure to adhere to. <\/p>\n\n\n\n

Nanoparticles have greater surface to volume ratio\nas their size decreases. Specific surface area is important for catalytic reactivity\nand other features including antibacterial activity. The biological efficiency\nof nanoparticles can improve when their specific surface area increases due to\nthe rise in surface energy. Many bacterial strains and pathogens typically\nfound in industrial and medicinal operations have been known to be inhibited by\nsilver for a long time. Most of the therapeutic applications commonly utilize\nsilver and silver nanoparticles (AgNPs) due to their medicinal properties.\nAgNPs of bigger size (100 nm) generate these consequences more efficiently than\nthose of smaller size, although AgNPs of smaller size (10 nm) cause cellular\ntoxicity at a higher degree because they easily enter cells and get localised\ninside nuclei. AgNPs have unique features, such as conductivity, catalytic\nproperties, nonlinear optical behaviour, chemical stability, and antibacterial\nactivity, for which silver nanoparticles have been extensively researched (2)<\/sup>.<\/p>\n\n\n\n

Metal nanoparticles can be\nsynthesized and characterized effortlessly and involve easier modification of\nits surface. Furthermore, it has distinct physiochemical properties like\nhydrophilicity, optoelectronic property and non-toxicity, which make them a highly\ndemanding nanoscale delivery system. They are widely used as therapeutic agents\nfor several life-threatening ailments including cancer, sexually transmitted diseases,\nneurological diseases, cardiovascular diseases etc. Also, recent studies have shown that plant-mediated\nAgNPs demonstrate their anticancer activity by decreasing the rate of malignant\ncell proliferation through cell-cycle arrest (14)<\/sup>.\n<\/p>\n\n\n\n

Silver nanoparticles are fabricated using several\ntechniques, but chemical techniques are the most prevalent way of producing\nnanoparticles (AgNPs) among all other technologies. However, there is an\ninability to prevent the usage of harmful substances in the NP synthesis. Though\nAgNPs are commonly used in human contact regions, there is an increasing need\nto create environmentally acceptable nanoparticle syntheses that do not employ\nharmful chemicals.  <\/p>\n\n\n\n

In the biological method, plant extracts, microorganisms,\nand enzymes are recognized as environmentally safer replacement than physical\nand chemical nanoparticle synthesis. The capacity to manage and manipulate the\nconcentration of nanoparticles inside cell upto the sustained duration can\nresult in prolonged and effective therapeutic effect and diagnostic sensitivity\nin biological and clinical applications. Therefore, usage of expensive drugs\nfor cancer therapy would be eliminated because of this unique characteristic of\nAgNPs. The green synthesis of AgNPs from extracts of various types of plants having\nunique phytochemical composition contributes to rapid production, enhanced\nstability, and is very much cost\u2013effective (11)<\/sup>. Treatment using green\nsynthesised AgNPs is sustainable as the nanoparticles are not affected by any\nphysiological factors like enzymes, etc. from within and thus it results in\nincreased bioavailability of therapeutic agent.<\/p>\n\n\n\n

The\nuse of medicinal plants has expanded the prospects and treatment options for\ncancer. They aid in the development of cutting-edge cancer treatment strategies\nlike the green production of AgNPs and serve as a source of new chemicals\nthrough phytochemical screening. The Cardiospermum\nhalicacabum <\/em>belonging to Sapindaceae family is a widely known medicinal\nplant. This plant is cheap and widely available; it is rife with phytochemicals.\nIt contains alkaloids, tannins, flavonoids, glycosides and terpenoids, and is\nconsidered highly pertinent in the treatment of cancer. According to reports, C. halicacabum’s<\/em> main chemical\ncomponents include quercetin3-O-\u03b2-D-glucoside, quercetin3-O-\u03b1-L-rhamnoside, quercetin,\nkaempferol3-O-\u03b1-L-rhamnoside, kaempferol, apigenin7-O-\u03b2-D-glucuronidebutylester,\napigenin7-O-\u03b2-D-glucuronidemethylester, apigenin, and luteolin. These phytochemicals\nmake Cardiospermum halicacabum<\/em> a rich\nsource of antioxidants (24)<\/sup>. In cancer treatment, there is\nan emerging concern towards therapeutic agents using naturally sourced leaves\nfor eco- friendly Ag nanoparticle synthesis. Therefore, it is hypothesized that\nCardiospermum halicacabum <\/em>is rich in phytochemicals\nthat may help in inhibition of cancer cells. <\/p>\n\n\n\n

Cancer\nis one of the world’s deadliest diseases, killing millions of people every\nyear. Colon cancer ranks\nthird in the world amongst the central reason for cancer related deaths. It\nemerges in the colon epithelium which leaps to poly-adenoma and from where\nfinally proliferates to carcinoma stage. Due to various environmental and\ngenetic factors, sedentary lifestyle, alcohol abuse and smoking, there is an\nincrement in Colon cancer. Colon cancer is very badly affecting people\nacross the world and needs proper attention. In this study, AgNPs have been\nproduced, utilizing the extract from the leaf of Cardiospermum halicacabum<\/em>.\nThey have been tested on cell lines for toxicity and anticancer\nactivity.(36)<\/sup><\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Preparation\nof plant extract<\/strong><\/p>\n\n\n\n

Cardiospermum halicacabum <\/em>(Modakathan) leaves were collected\nfrom Guduvanchery, Tamil Nadu. The leaves were separated from the collected\nplant, rinsed with ordinary water and then washed thoroughly several times with\nMillipore water, to remove debris and other unwanted materials present in it.\nThe leaves were then dried under Sun for few days and kept ready for obtaining\nthe extract. 5g of dried leaves were weighed, followed by finely cutting them\ninto small pieces using sterile scalpel blade. The cut leaves were then added\nin a sterile beaker containing 100 ml Millipore water. The plant extract was\ncarefully boiled at 60o<\/sup>C for 10 minutes to ensure prevention of phytochemical degradation due to increased\ntemperature. The filtrate of plant extract obtained was then collected\nby using Whatman filter paper and stored at 4\u00baC until further use. <\/p>\n\n\n\n

Silver\nnanoparticle synthesis<\/strong><\/p>\n\n\n\n

For synthesis of nanoparticles, the\nconcentration of 1mM of silver nitrate was utilized. 1mL of the extract of leaf\nwas dissolved in 9 mL of silver nitrate and the resultant volume was incubated in\ndark room for 24 hours at room temperature. It was then spun to observe colour\nchange and then subsequently centrifuged (Thermo Scientific, ST 16R, USA) for\n20 min. The supernatant was discarded and pellets of silver nanoparticles were\ncollected and dried in the oven for further analysis.<\/p>\n\n\n\n

UV-Vis\nspectroscopy analysis<\/strong><\/p>\n\n\n\n

The silver nitrate reduction to\nAgNP was analysed using UV-Visible Spectrophotometer (Agilent, Cary 60, USA).\nIt is a fast, simple, easy, and sensitive procedure which analyses the Surface\nPlasmon Resonance to confirm the presence of silver nanoparticles (18)<\/sup>. Strong\nelectromagnetic wave absorption is generally shown by Silver Nanoparticles in\nthe visible range that helps in analysis of Surface Plasmon Resonance. In a quartz cuvette, briefly 2 ml of the\nreaction mixture was taken and absorption spectrum was recorded in the range\nfrom 300 to 800nm. The spectroscopic readings were taken after 24 hours of\nincubation. <\/p>\n\n\n\n

Zeta\nAnalyser<\/strong><\/p>\n\n\n\n

Size distribution of bio-reduced\nAgNPs was measured using Zetasizer (Horiba, SZ-100, Japan). This measures the\nparticle size in dispersed systems from sub-nanometres to several micrometres\nin diameter. It determines the average size of the particles using photon correlation\nspectroscopy using Zetasizer which utilises the Brownian motion of the particle\nto analyse the changes in scattering of light. Furthermore, the net surface\ncharge of the nanoparticles called as Zeta Potential, was also studied to\ndetermine the colloidal stability of the nanoparticles (19)<\/sup>. It involves centrifuging the\nreaction mixture for 20 minutes at 10,000 rpm followed by discarding the\nsupernatant and taking the pellet. Then, the pellets are resuspended in Millipore\nwater and were analysed for Zeta potential. The average size of the particles in\nterms of the most appropriate one was reported. <\/p>\n\n\n\n

FTIR\nanalysis<\/strong><\/p>\n\n\n\n

FTIR (Shimadzu, Irtracer 100,\nJapan) analysis was employed for investigating the superior functional groups\nthat are responsible for bio reduction of Ag+ ions and stabilization and capping\nof the AgNPs. This also indicates on how well the functional groups are bonded\nwith each other. The peaks that are obtained in FTIR will provide information\non the presence or absence of expected functional groups. Briefly, the\nsynthesized nanoparticles were coated onto KBr, made into thin pellet film and\ndried before measurements. Potassium bromide is\nused as a carrier for the sample in IR spectrum because it is transparent in the IR range 500-4000 cm-1<\/sup>.\nTherefore, it doesn\u2019t exhibit absorption in this range and no interference\nwould occur in this region. Then, the pellet was subject to FT-IR\nspectroscopy analysis in the range of 500\u20134000 cm-1<\/sup>.<\/p>\n\n\n\n

XRD\nanalysis<\/strong><\/p>\n\n\n\n

XRD (Bruker, D8 Advance, USA)\nanalyses the nature (crystal or amorphous structure) of silver nanoparticles\nand the protocol was adapted from (20)<\/sup>. The sample was exposed to\nEnergy Dispersion, which involves projecting an X-ray beam onto the sample and\ndisrupting the incident beam scattered by the atoms, resulting in the\ndevelopment of patterns. XRD spectrum checks the quality and formation of\ncompounds. The pattern of XRD was measured using an X-ray diffraction. The\npowdered sample was obtained as pellet by drying it, which was then loaded onto\na glass sample holder and analysed.<\/p>\n\n\n\n

Scanning\nElectron Microscope analysis (SEM)<\/strong><\/p>\n\n\n\n

SEM (Thermo Scientific, Apreo 2\nSEM, USA) was performed to study the morphological nature of silver\nnanoparticles according to (30)<\/sup>. HRSEM uses a high-energy\nelectron beam to scan the silver nanoparticles surface, and then the electrons\nbackscattered were seen to reveal the sample’s characteristics. AgNPs were\nplaced on specimen stubs double sided adhesive tape and was subsequently\nobserved under HR-SEM. Finally, the presence of the elements in the nanoparticle\nwas determined by EDX analysis along with SEM.<\/p>\n\n\n\n

Anticancer\nactivity<\/strong><\/p>\n\n\n\n

COLO 205 is a colon cancer cell\nline, acquired from NCCS, Pune. It was cultured in Roswell Park Memorial Institute\nMedium (RPMI-1640) using 5% FBS and\n1% antibiotic solution incubated in CO2<\/sub> incubator at 37 \u00b0C.<\/p>\n\n\n\n

Cell\nproliferation assay<\/strong><\/p>\n\n\n\n

The samples to be studied for cell\nproliferation were analysed by MTT colorimetric assay using Colo 205 and the methodology\nwas adapted from (41)<\/sup>.\nHere, the MTT assay is used as an investigative tool to analyse the metabolic\nactivity of the cells and to measure their cell proliferation, which could be\ncorrelated indirectly with the anti-cancer activity. Briefly, the cultured cells were plated\nat a density of 1\u00d7105<\/sup> cells\/well in 96-well plate and increasing\nconcentrations of the\nsilver nanoparticles were added to it, followed by incubation for 24 hours. After that, each well\nplate was treated with 50 \u00b5L MTT (5mg\/mL), followed by 4 hours incubation at 37o<\/sup>c.\nFinally, the MTT along with the medium were aspirated. Furthermore, 100 \u00b5L DMSO\nwas affixed as a solvent for Formazan crystals to dissolve, that were obtained\nduring the experiment. The absorbance was measured using a\nmicro plate reader at 570 nm (Thermo Fisher Scientific, USA).<\/p>\n\n\n\n

Cell\nviability assay<\/strong><\/p>\n\n\n\n

Trypan blue assay was\ndone to investigate the viability of cells using Colo 205 cells upon\nadministration of drug according to(41)<\/sup>.\nThis dye-based test differentiates the dead and live cells depending on the intactness of the cell\nmembranes. The live cells have intact cell membrane whereas it is ruptured in\ndead cells.  Trypan blue is an azo dye\nwhich cannot permeate cell membrane, while it can through a ruptured one. Upon\nseeding of Colo 205 cells, various concentrations of the silver nanoparticles were added to\neach well for 24 hours. After 24 hours, they were trypsinized, followed by 5\nminutes of centrifugation at 1,000 rpm. The pellet obtained was suspended in PBS. Cell suspension was taken and trypan\nblue was added. Haemocytometer was used to count the number of viable cells. The cells were then viewed under light\nmicroscopy.  (Leica, Germany)<\/p>\n\n\n\n

Cell viability (%) = (Number of cells which are unstained \/Total number of cells) X 100<\/strong><\/p>\n\n\n\n

Cell toxicity assay<\/strong><\/p>\n\n\n\n

The cell toxicity of the sample\nwas measured by the release of Lactate Dehydrogenase (LDH) into the culture\nmedia which provides information about the cell membrane disruption. Hence, the\nLDH Assay was carried out using Colo 205 cells (22)<\/sup> by plating the cultured cells on the\n96 well plate and treating it with varying concentrations of silver\nnanoparticles 24 hours. After 24 hours, the medium was centrifuged at 1500 rpm\nfor 5 minutes and 4 mL of Tris EDTA buffer was mixed with 100\u00b5L of supernatant\nand was incubated for 15 minutes. After incubation, 400 \u00b5L of sodium pyruvate\nsolution (14 mM) was added to the solution and the absorbance was measured\ncontinuously for 3 minutes at 340 nm. <\/p>\n\n\n\n

LDH activity (\u03bcM\/Min\/L) = (OD\nchange x total volume\/ min) \/ (Sample volume \u00d7 6.3 \u00d7 10\u22123<\/sup>)<\/strong><\/p>\n\n\n\n

Apoptotic assay<\/strong><\/p>\n\n\n\n

The cell apoptosis study was done by dual fluorescent staining using\nHoechst 33342\/propidium iodide(32)<\/sup>.\nBriefly, the Colo 205\ncells were plated into 24-well plate and were treated with increasing\nconcentration of silver\nnanoparticles for 24 hours. After the incubation, the media was aspirated and\nPBS was used to wash the cells. The wells were stained (50 \u00b5L HOE, 50 \u00b5L P1 in\n5mL PBS). Following it, the cells underwent 15 minutes incubation in dark at 37o<\/sup>C. The cells were then viewed under\nfluorescence microscopy (Olympus. CKX-41-TR, Japan).<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

Cancer remains a\ndreaded disease despite technological advancements, capable of tilting the\nmortality index if not properly attended to (25)<\/sup>. In the year 2020 alone, the\nnewly registered cases of cancer throughout the world, inclusive of all its\ntypes, amounts to 10.1 million for men, with about 5.5 million deaths, and 9.2\nmillion for women with about4.4 million deaths, and most of these deaths had\noccurred in the Asian countries(10)<\/sup>.Even amongst these cancers,\ncolon cancer is known to be second in mortality relating to cancer, and is\nknown for its trend of occurring mostly in high HDI countries (37)<\/sup>. In order to efficiently treat\ncancer, newly developed technologies play a major role by effectively\novercoming the limitations of conventional treatment. Nanotechnology can play a\nkey role in treating cancer, and its success is evident from the voluminous\nresearch papers on that topic published in the current century (15)<\/sup>. It addresses many concerns that\nconventional therapy fails to address, such as increased solubility of the\ntherapeutic agent, preventing degradation of the drug from various metabolic\nprocesses, enhanced specificity of drug action, etc (36)<\/sup>. In this context, Ag\nnanoparticles were used to encapsulate Cardiospermum\nhalicacabum<\/em> extract in order to efficiently treat cancer. <\/p>\n\n\n\n

Silver\nnanoparticle synthesis<\/strong><\/p>\n\n\n\n

When the silver nitrate solution\nwas added to the aqueous extract of Cardiospermum halicacabum <\/em>and\nincubated, the colour of the solution changed to dark brown from yellow as\nshown in figure 1, which confirmed the synthesis of silver nanoparticles. The\ncolour change occurred as a result of reduction of Ag+ <\/sup>into Ago\n<\/sup>by the reducing agent, which are the phytochemicals present in the\naqueous extract. These phytochemicals and the related colour change are the\nindicators of silver nanoparticle synthesis. The\ncolour shift from golden yellow to dark brown (shown in fig. 1a and 1b) was\nobserved as a result of the reduction reaction mediated by the phytochemicals\nof plant extract.This is the first observable change that explains the\ninteraction of cationic silver ions with bioactive components of leaf. The\nobtained change of colour was due to the presence of compounds like alkaloids,\nphenols, tannins, saponins, triterpenoids, etc., specifically the important\ncarbonyl groups contained therein, that are essential for the bio-reduction of\nAgNO3<\/sub> to Ag nanoparticles,(27,33,34)<\/sup>and also the excitation of\nthe surface plasmon vibrations present in Ag nanoparticles as is confirmed with\nthe UV-vis spectroscopy (26)<\/sup>.\nThe absorption spectra measured in nm for Ag is directly a function of its size\nand the lesser the diameter of the metal, the more is the scattering of the\nconducting electrons, which could be one plausible reason for the colour change\nof nano Ag particles (7)<\/sup>.\nThe oxidation of the hydroxyl groups to carbonyl groups in the solution by Ag\nions and themselves getting reduced to elemental Ag is the initial process for\nthe bio-reduction. This process is followed by the oxidation of the hydroxyl\ngroups to potent reducing carbonyl groups like aldehyde by dissolved air, which\nare responsible for additional reduction of the Ag ions(3)<\/sup>. One useful inference from\nthis is that, the concentration of plant extract in which the Ag ions are\ndissolved is directly proportional to the bio-reduction of Ag ions. This study\nis similar with two of the studies that\nuse Parthenium hysterophorus<\/em>\nleaf extract and Nocardiopsis dassonvillei\n<\/em>extract wherein similar pattern of colour change was reported suggesting\nthe occurrence of the aforementioned reduction reaction (1,21<\/sup>)<\/sup><\/p>\n\n\n\n\n\n
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Figure 1: Silver nanoparticles synthesis (A) aqueous leaf extract of Cardiospermum halicacabum<\/em> (B) Silver nanoparticles.<\/strong><\/p>\n

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UV-Vis\nspectroscopy analysis<\/strong><\/p>\n\n\n\n

The reduction of the\nsilver nitrate to AgNPs was analysed using UV-Visible spectrophotometer. The\nspectrum has been frequently used to characterize the metal nanoparticles with\nan absorbance range 300-700 nm. The sample exhibited maximum absorbance value\nat 430nm after 24 hours, indicating appropriate silver nitrate reduction to\nAgNPs as shown in figure 2. Strong electromagnetic wave absorption is\ngenerally shown by Silver Nanoparticles in the visible range that indicates the\npresence of Surface Plasmon Resonance of AgNPs. UV spectral analysis was performed in order to\nestimate at what absorbance range do the electrons of Ag absorb maximum light,\nand that is indicative of whether they have reached nano-size, based on the\npoints mentioned from (29)<\/sup>.\nUV spectral analysis (shown in fig. 2) showed an absorbance peak at around 430\nnm which falls within the range of 400-450nm; the characteristic range of Ag\nparticles in nano-range (31)<\/sup>.These values are in line with the values obtained\nfrom studies that synthesized Ag nanoparticles from soda lime borate glasses,\nwherein the peaks correspond to 420 nm that comes under the above-mentioned\nrange. They contain similar organic bioactive compounds that mediate the\nreduction reaction, and the peak obtained in the same range only corroborates\nour result (9)<\/sup>.<\/p>\n\n\n\n\n\n
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Figure 2: Analysis of silver nanoparticles using UV-Vis spectrum<\/strong><\/p>\n

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Size\nand charge analysis<\/strong><\/p>\n\n\n\n

The Zetasizer was used to measure\nthe size of the bio-reduced nanoparticles based upon Brownian motion of the\nparticle upon the illuminated scattered light on the nanoparticle. The principle of Dynamic light\nscattering is used to scatter light in all directions as the particle is in\nmotion, and thereby measure the intensity of fluctuation of the patches created\non a nearby screen as the scattering occurs. The nanoparticle size analysis\nconfirmed the synthesized AgNPs were extensively dispersed in solution. The\nmean size of the synthesized nanoparticles was 150.1 nm as shown in figure 3. PI or Polydispersity Index is a measure of\nhow broad the molecular weight distribution is, and falls within the range of\n0-1 to indicate if the sample is monodisperse or polydisperse. The PI\nwas also noted to be 0.450 (shown in figure 3). Since the value falls below\n0.5, the nanoparticles are found to be monodisperse. Nanoparticles obtained had a zeta average\nof 150.1nm (shown in fig. 3) as is concordant with the study that used Rhus\nChinensis <\/em>wherein similar size range was reported for the nanoparticles,\nthereby confirming its applicability on cells (28)<\/sup>.Our PI indicates that the\nnanoparticles synthesised are of uniform size or are monodisperse, and this\nuniformity implies that the therapeutic agent, is able to reach the targeted\nsite in optimum amounts, as nanoparticles that are too big in size (i.e. >\n150 nm) would not properly penetrate the cell, and those that are too small\nwould lead to low intracellular bioavailability (23)<\/sup>.\n<\/p>\n\n\n\n\n\n
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Figure 3: Zeta analysis of silver nanoparticles.<\/strong><\/p>\n

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FTIR\nanalysis<\/strong><\/p>\n\n\n\n

Fourier Transform Infrared\nspectroscopy was performed to find the possible functional groups present as\nshown in figure 4. The peaks obtained in the FTIR report conveys the essential\ninformation that the functional groups of the phytochemicals in leaf extract\nand the nanoparticle have formed a stable nano-complex by showing peaks which\ncorrespond to different functional groups, thereby confirming their presence. Specific\nband at 3422.04 cm\u22121<\/sup> correlates to OH stretching and 2354.16 cm\u22121<\/sup>\nof C\u2013H stretching corresponds to aromatic compounds. However, the peak at\n1771.65 cm\u22121<\/sup> corresponding to carbonyl group stretching may be\nrepresentative of phenyl ester. The band at 1274.97 is indicative of C-N\nstretching, corresponding to an aromatic amine, 1580.69 indicates N-H bending,\nrepresentative of primary amines, and 1752.36 cm\u22121 <\/sup>represents C=O\nstretching, indicating esters. Peak representing 1063.76 cm\u22121 <\/sup>is of\nC-O stretching that corresponds to presence of primary alcohols and 1037.72 cm\u22121<\/sup>\ncorresponds S=O stretching, indicative of sulfoxides. These values correspond\nto functional groups present containing the elements C, H, O, N, and S, which\nare abundantly present in the organic components of the plant extract, thereby\nconfirming their role in capping and biological reduction of nanoparticles.\nStudies relating to the synthesis of Ag nanoparticles show FTIR results wherein\npeaks corresponding to the same functional groups are observed, especially\naround 3477 cm-1<\/sup>, 2360.1 cm-1<\/sup>, 2342.93 cm-1<\/sup>,\n1762.21 cm-1<\/sup>, 1657.7 cm-1<\/sup>, 1398.21 cm-1<\/sup>, 1038\ncm-1<\/sup>, and 1024 cm-1<\/sup>thereby corroborating the activity of\nthe functional groups like O-H stretching, C=O stretching, C=C stretching, C-O\nstretching, C-N stretching, and S=O stretching (31, 8)<\/sup>.<\/p>\n\n\n\n\n\n
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 Figure 4: Characterization of silver nanoparticles using FTIR<\/strong><\/p>\n

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XRD\nanalysis<\/strong><\/p>\n\n\n\n

XRD analysis is carried out to\nprimarily analyse the structural nature of the nanoparticles formed. The characteristic\npeaks of silver nanoparticles were further investigated using XRD as shown in\nthe Figure 5. In the pattern of XRD, specific peaks of diffraction were\nidentified at 27.72o<\/sup>, 32.29o<\/sup>, 38.76o<\/sup>, 43.17o<\/sup>,\n54.47o<\/sup>, 64.04o<\/sup> and 77.75\u25e6, all of which correspond to\n(111), (333), (111), (200), (311), (220) and (111) face-centred cubic (fcc)\nstructure, respectively, which was calculated using Debye\u2013Scherer equation. The\nresults obtained had revealed the crystalline nature of the nanoparticles. The\nnano-dimensional condition of the synthesized system is inferred from the crisp\nand wide diffraction pattern such that the many peaks on the particles\nrepresent their multi-faceted growth direction. The broadness in the peak is\nalso thought to be caused by local crystal defects in the nanocrystals. The 2\u03b8\nvalues shown in fig. 5 are in agreement with the reported XRD analysis of 27o<\/sup>,\n32o<\/sup>, 46o<\/sup>, 54o<\/sup>, 68o<\/sup> and 77o<\/sup>\nin silver nanoparticles stabilized by Eugenia\nroxburghii<\/em>(13)<\/sup>.\n<\/p>\n\n\n\n\n\n
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Figure 5: XRD pattern of the silver nanoparticles.<\/strong><\/p>\n

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Scanning\nElectron Microscopy (SEM)<\/strong><\/p>\n\n\n\n

The SEM analysis is carried out to\nobserve the shape, size and structural studies of the synthesized\nnanoparticles. Figure 6 has shown clearly developed Ag nanostructures with\nlarge distribution of size, and rod-shaped structures are formed. The silver\nnanoparticles synthesized showed a large distribution of size. The visible\nagglomeration of nanoparticles was observed. The bioactive components from\naqueous leaf extract of Cardiospermum halicacabum <\/em>were capped with the AgNPs are well exhibited by\nthe visible\nagglomeration of the nanoparticles in the images obtained. The\nclustering was found to be similar to studies wherein synthesis of silver\nnanoparticle was achieved through aqueous leaf extracts of Alhagi graecorum<\/em> in which the obtained silver nanoparticles were\nclustered and spherical in shape indicating successful stabilisation by\nphytochemicals (16)<\/sup>. \nFigure 7 shows the\nEDS pattern of silver nanoparticles. The peak around 3 KeV confirms the\nposition of elemental silver which is caused as a result of Surface Plasmon\nresonance in silver nanoparticles. Elemental silver as the major constituent in\nthe nanoparticle was confirmed. The negligible presence of oxygen as\nevident from the elemental analysis graph might be from the phytochemicals\npresent in the therapeutic agent and is not of any significant consequence to\nour analysis. The value of obtained peak is in accordance with the analysis of\nsilver nanoparticles using Olive and Green tea extract (4)<\/sup>.<\/p>\n\n\n\n\n\n
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Figure 6: SEM micrograph of the silver nanoparticles<\/strong><\/p>\n

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Figure 7: EDS pattern of synthesized silver nanoparticles.<\/strong><\/p>\n

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Anticancer assays<\/strong><\/p>\n\n\n\n

Cell\nproliferation assay<\/strong><\/p>\n\n\n\n

The cell\nproliferation assay is performed primarily to check whether there is a decrease\nin the proliferation of cells when treated with the synthesized nanoparticles,\nas uncontrolled proliferation is one undeniable hallmark indicative of cancer.\nThis MTT assay essentially checks for the mitochondrial function, and\ndetermines whether the cell is proliferating or not. The in-vitro proliferation effect\nof AgNPs against colon cancer cell lines (Colo 205) & cell viability (%)\nwas carried out by MTT assay. In this analysis, various concentrations (50-300\n\u03bcg\/mL) of silver nanoparticles were used to treat the cell-line for 24 hours.\nWe observed that the proliferation rate of colon cancer cells reduced with\nincrease in the amount of silver nanoparticles. From about 75% to 20%, the drop\nin the rates is evident with the increasing concentration. The cell inhibition\nwhich was seen after 24 hours exposure was dose dependent as shown in figure 8.\nThe reduction in the cells percentage with\nenhancing concentration suggests inhibition of cell proliferation. This\ninhibition of cell proliferation is indicative of the cell\u2019s growth arrest as a\nresponse to treatment and thus implies the success of the synthesized\nnanoparticles. The antiproliferative effect was reported in a similar fashion\neven for another study wherein AgNPs were synthesized with Cucumis prophetarum<\/em>(17)<\/sup>. Here, a similar dose-dependent\ndecrease in cell proliferation was observed, and upon administering more\namounts of synthesized nanoparticles, the cell proliferation decreased. This\nstudy utilized various cell lines such as A549, MDA-MB-231, HepG2, and MCF-7,\nwhich were checked for its antiproliferative activity,  whereby they had different IC50<\/sub>\nvalues in descending order: 105.8, 81.1, 94.2, and 65.6 \u00b5g\/mL. Another study\nthat used the aqueous extract of Acer\noblongifolium<\/em>, showed anti-proliferative activity on a comparitive basis\nbetween MCF-7 and HeLa cells, with the highest activity in MCF-7, and this too\nwas in a dose-dependent manner, i.e. decreased cell proliferation with\nincreased dosage (6)<\/sup>.\nThis proves that our results, being similar to theirs, is thus appropriate. <\/p>\n\n\n\n\n\n
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Figure 8: <\/strong>Cell proliferation of colo 205 after treatment with silver nanoparticles after 24 hours<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Cell viability\nassay<\/strong><\/p>\n\n\n\n

Cell viability\ntest is performed to evaluate whether the cells are alive or not. After treating the cells with\nvarious concentrations (50- 300 \u03bcg\/mL) of silver nanoparticles, they were\ntested for 24 hours to check the number of viable cells, using trypan blue\nstain. It is quite obvious from the results that the viability of colon cancer cell\nline decreased with increased concentration from 60 % to about 20%. The drop in\ncell viability seen after 24 h exposure is shown in the Figure 9. Here, a drop in the cell viability was noted after\nincubation for 24 hrs post treatment with Ag nanoparticles (fig. 9). The drop\nwas dose-dependent, linear, and gradual, as can be seen from figure 9. Similar\ndecreasing trends in the viability was observed in MCF-7 and A549 cells on\ntreatment with NPs synthesized from Syzygium\naromaticum <\/em>extract (35)<\/sup>. Here, the cell viability was\ntested for treatment with just the plant extract and alongside synthesized Ag\nnanoparticles, to prove that the latter was much efficient in achieving low\ncell viability as is similar with our results. In fact, their study even\nproclaimed that lesser quantity of Ag nanoparticles is required to induce this\neffect. <\/p>\n\n\n\n\n\n
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Figure 9: <\/strong>Viability of Colo 205 after 24 hours of treatment with silver nanoparticles<\/strong><\/p>\n

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Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Cell toxicity assay<\/strong><\/p>\n\n\n\n

Cytotoxicity assay through LDH\nactivity evaluation is a good measure of whether post treatment with Ag\nnanoparticles, there is still cancerous activity. Lactate Dehydrogenase enzyme\nreleases during cell death into the medium. The lactate to pyruvate conversion\nis catalysed by this enzyme in the presence of NAD+\/NADH. High LDH levels\nindicate high cytotoxicity and this is a good indicator of cancerous\nconditions. Thus, estimation of LDH activity confirms whether necrotic cell\ndeath or apoptotic cell death has occurred after treatment with silver\nnanoparticles for 24 h with increasing concentrations (50-300\u03bcg\/mL). The LDH\nreleased was quantified after 24 hours of treatment. There is a gradual\ndecrease in the LDH activity from around 27 \u00b5M\/min\/L to about 23 \u00b5M\/min\/L as\nthe concentration increased from 50 \u00b5g\/ml to 300 \u00b5g\/ml. There was no\nsignificant increase in LDH activity, as shown in figure 10 which indicated\nthat cell cycle arrest has taken place due to the treatment. The LDH activity\nshowed an increment from 50 to 100\u00b5g\/mL wherein the activity increased from 27\nto 29\u00b5M\/min\/L, which was then followed by an overall decreasing trend, from\nabout 29 to 24 \u00b5M\/min\/L and then getting evened in and around the same range\n(fig. 10). This is indicative of cell growth arrest, which was facilitated by\nthe NPs. This study has results that can be related to another study that uses Albizia adianthifolia <\/em>leaf to synthesize\nAg nanoparticles and test it on A549 cell line (12)<\/sup>. Here, the LDH activity is\nreduced to 36% from 101% following treatment with initially 10 \u00b5g\/mL and then\n50 \u00b5g\/mL Ag nanoparticles, thereby proving the arrest of cell growth.  <\/p>\n\n\n\n\n\n
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Figure 10: <\/strong>LDH release after 24 hours of treatment silver nanoparticles at various concentrations.<\/strong><\/p>\n

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Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Apoptosis assay<\/strong><\/p>\n\n\n\n

Apoptosis assay was performed to\ncheck which of the cells had died as a result of apoptosis, and is measured\nagainst treatment with increasing concentration of Ag nanoparticles. The\ninduction of apoptosis was tested using HOE\/PI fluorescent staining with\ntreatment of varied concentration of50-300\u03bcg\/mL of silver nanoparticles for 24\nhr. Pale blue spots were observed as live cells or early apoptotic cells, as\nstained by HOE. The distinction could be identified based on the intensity of\nthe colour; higher intensity signifies apoptotic cells as evidenced by the\nability of the stain to permeate the cell, while lesser intensity signifies\nearly apoptotic or live cells. The red spots are indication of the stain PI,\nand they stain late apoptotic cells. The higher the intensity more advanced the\napoptotic stage. It can be observed that apoptosis rate of colon cancer cells\nincreased as there is increment in silver nanoparticles concentration. At 150\n\u00b5g\/ml, there are maximum red spots visible as an indication of apoptosis\noccurring in those many numbers of cells as shown in figure 11. Similarly at\n150 \u00b5g\/ml and 200 \u00b5g\/ml, the intensity of pale blue spots does increase in a\ndose-dependent manner, indicating that some cells pass through the early\napoptotic phase, an indication of the effect of prepared silver nanoparticles. These\nresults suggest that the AgNPs induce apoptosis on the cells as a reason of\nmorphology change and DNA condensation, indicating that these biologically\nreduced nanoparticles have apoptotic potential as reported in (39)<\/sup>. In another study, wherein Clinacanthus nutans <\/em>leaves were used to\nsynthesize Ag nanoparticles and apoptosis was induced, even for low\nconcentrations of Ag nanoparticles such as 4 \u00b5g\/mL, late apoptosis was induced (40)<\/sup>.\nThe differences in phytochemical composition and amount, also followed by\nnumber of nanoparticles formed due to the phytochemicals, could have determined\nhow much of the therapeutic agent acted on the cells to induce the apoptotic\npathway. <\/p>\n\n\n\n\n\n
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Figure 11: <\/strong>Cell death detection using HOE\/PI fluorescent staining after 24 hours of treatment with silver nanoparticles<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

To\nsummarise the study, the main objective of designing a suitable delivery system\nfor intracellular transportation of the therapeutic agent was successful for\nthe following reasons: the presence of intended functional groups as shown by\nFTIR data was corroborated by SEM EDX analysis wherein the presence of silver is clearly noted.\nThe intended morphology, size, and charge were achieved through SEM, XRD and\nZetasizer, and though negligible deviations existed, it amounts to no big change concerned with the\nfunctioning of our system. The\nXRD values are in line with previous studies and show diffraction peaks\nindicative of silver, while the Zetasizer highlights the proper size range of\nthe synthesized nanoparticles and also its proper Polydispersity index. Lastly,\nthe most important part that concerns the effect of the drug on cancer cell line\nColo 205 was evaluated by several cell-based assays that revealed the\nefficiency of the drug nanoparticle system on the cancer cell line. A huge drop in the proliferative\nactivity was observed in the cells after the treatment with nanoparticles,\nfollowed by a reduction in viability to from 60% to 20%, and also minimal LDH\nactivity indicative of anti-cancerous activity, and lastly the increased\nintensity of the red fluorescence signifies apoptotic activity, all of which\ncumulatively representing the success of the nanoparticles in eliciting\nanti-cancer activity. Therefore, in conclusion, a suitable drug delivery\nsystem is designed and it shows considerable anti-cancer activity on Colo 205\ncell line. <\/p>\n\n\n\n

Acknowledgments<\/strong><\/p>\n\n\n\n

The authors would like to\nacknowledge deep regards and gratitude to Dean, School of bioengineering, and\nHoD, Department of Biotechnology, SRM Institute of Science and Technology. We\nalso extend our gratitude to the Management of SRM Institute of Science and\nTechnology for providing the facility to complete our project. <\/p>\n\n\n\n

Conflict\nof Interest<\/strong><\/p>\n\n\n\n

There is no conflict of interest\namongst the authors<\/p>\n\n\n\n

Funding\nSource<\/strong><\/p>\n\n\n\n

There is no source of financial\nsupport.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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