A Preliminary in-vitro Evaluation of Serum Capped Colloidal Silver Nanoparticles on Liver Cancer towards Development of Safe Personalized Nanomedicine

Abstract

Background

Serum-capped silver nanoparticles (AgNPs), synthesized using naturally occurring serum proteins and reducing agents, represent a green nanotechnology platform with potential for safer and more selective therapy. This study compared the safety and anticancer efficacy of serum-capped AgNPs with polyvinyl alcohol (PVA)-capped AgNPs.

Methods

Serum-capped AgNPs (~18 nm, zeta potential −8.9 mV) were synthesized from human serum and administered intravenously in mice (0.1 mL/dose). PVA-capped AgNPs of equivalent silver concentration served as controls. Effects were assessed in a mouse hepatoma model, WRL-68, and Hep-G2, two different liver-derived cell lines using histopathology, biochemical analysis, MTT assay, estimation of proliferative potential by cell cluster formation assay, scratch assay, and hexosamine assays.

Results

Serum-capped AgNPs showed a wider safety margin and lower toxicity in mice and normal WRL-68 cells compared with PVA-capped AgNPs, while exhibiting stronger cytotoxicity against hepatoma and Hep-G2 cancer cells. Treated mice showed improved survival, and favorable histopathological and biochemical profiles indicated reduced systemic toxicity. In the hepatoma model, serum-capped AgNPs, cisplatin, and their combination demonstrated significant anticancer effects, with combination therapy showing the strongest response.

Discussion

Serum-capped AgNPs provide a safer and more selective nanotherapeutic approach by enhancing compatibility with normal host cells while retaining potent anticancer activity. Serum-derived capping improves biological precision and reduces systemic toxicity compared with conventional AgNP formulations. These findings support further investigation of serum-capped AgNPs as potential broad-spectrum anticancer (and antimicrobial) agents and for future personalized nanomedicine applications.

Introduction

Therapeutically usable haemo-compatible small wide cluster-range, low anionic colloidal silver nanoparticles (AgNPs) have greater affinity towards high anionic microbes and some host cells, by charge shifting towards positivity at a narrow contact point of the tuneable outer diffuse-layer.^1,2 Smaller particles, after charge-affinity based attachment, can invade through disrupted membrane of microbial and mammalian cells, mainly by surface charge neutralization of phospho-lipid membrane.³ While larger particles can invade eukaryotic and mammalian cells by endocytosis following receptor-ligand attachment⁴ to recognisable foreign signature molecules. Carbon based or liposomal nanostructures for selective targeted delivery of anticancer drug, are also internalized into mammalian cells only by endocytosis.⁵ As their mechanism of anticancer action is indirect and according to the tagged drug, those will not be discussed here, while colloidal AgNPs carrying a cluster of cytotoxic silver atoms (Ag0) will be discussed, considering potential pan-cytocidal nature after access into the cell.^6–8 The greater surface–volume ratio of nanoparticle (NP), ie, smaller sized or triangular shaped NPs has greater probability of finding a larger contact area in respect of payload volume.⁹ This attributes stronger affinity and lethality. The efficacy of colloidal NPs also suitably alters at the charge-affinity attachment level by influence of local pH, temperature,¹⁰ and the presence of oxidative radicles. Exploiting all these properties of NPs, those can be made selectively toxic for rapidly multiplying, distinguished charge sign and molecular sign bearing cancer cells, sparing host cells.

After testing on 22 type cancer cells of different organs, it has been demonstrated by Le W et al that the stronger affinity of NPs towards cancer cells than originator normal cells [ZP ~ −15 mV, matching with suspending cells of blood] is due to greater differences of respective charge-signs.^11,12 The higher negative surface charge of cancer cells [ZP ~ −55 mV] is related to their high rate of glycolysis and excess production reactive oxygen species (ROS) by AgNPs inside hepatoma cells.¹³ In a study of positive electrostatic charge neutralization on human volunteers with late-stage solid tumour, synergistic drug response reiterates importance of charge signs.¹⁴

After adhesion of positive-ion rich AgNPs at narrow contact points of encountered cells, inclusion into depolarised membrane allows interactions of payload toxic contents to destabilise cell physiology at multiple levels. Other methods of relatively larger particle internalization can happen in eukaryotic cells only by receptor-ligand based endocytosis. In both cases subsequent events are the same. Sequentially, ion leaching of released silver (Ag⁰) atoms into cytosol^15,16 and production of excess reactive oxygen species (ROS) lead to further membrane damage by lipid-peroxidation,¹⁷ DNA damage, protein denaturation, enzyme inactivation and induction of programmed cell-death (PCD) at mitochondrial level through the caspase-3 pathway. Thus, AgNPs, according to their size, shape,^9,18,19 zeta potential,²⁰ and molecular structure of surface capping materials, display varying degrees of non-specific cytotoxic effects on in-vitro tested microbes²¹ and different types of cancer cells.²² But their systemic uses for controlling blood-stream infections or malignancies are prohibited for their potential cytotoxic effects on some host cells and environmental microbes.^23,24

It has been observed by in-vitro studies that AgNPs are gradually altered on exposure to well-buffered alkaline serum, by acquiring many higher affinity serum component proteins as corona cap and loose bio-functional efficacy at the expense of increased size and optimised low surface charge. Inspired by this phenomenon, a similar primary corona-protein capped AgNPs have been prepared with the aim of reasonably lower toxic nano-material formulation. Recently it has been observed by in-vitro studies on different microbes and by ex-vivo studies on the animal model that the host’s toxicity has been significantly eliminated by applying AgNPs capped with pooled human serum obtained from anonymized leftover clinical samples from healthy adult donors attending routine health check-up at SSKM Hospital, retaining potent anti-microbial actions.²⁵ As the chance of spillage or abuse for such personalised nanoproducts is remote with a safe disposal protocol,^26,27 no added risk on environmental microbes remains from proposed therapeutic use.

So, time has come to evaluate the role of proposed safe systemic usable specific novel nanoparticles (NNP) in cancer therapy, either alone or in synergistic combination with other anticancer drugs. Tests with nonspecific AgNPs on the HCT116 cell line of colorectal carcinoma²⁸ also indicate a similar view. Greater uptake and anti-tumour effects have been observed after challenging with murine serum albumin capped AgNPs on murine breast carcinoma.²⁹ Though the globulin part of the murine serum has not been included there for capping purpose, the generated AgNPs have proved least toxic for healthy cells of experimental mice up to 2–8 mg/kg b.w. application. A higher margin of safety can be expected for the same NPs enriched with self and foreign molecular signature distinguishing the globulin part. So, a personalised colloidal silver nanoproduct may be a high precision anti-cancer drug in the future.

Methods

Preparation and Characterization of Systemic Usable AgNPs

The synthesis and characterization of AgNPs was performed as described in our previous publication in connection with anti-microbial studies of the same AgNPs.²⁵ Briefly, 5 mL of heat inactivated (at 56°C for 30 minutes) pooled human serum obtained from anonymized leftover clinical samples from 15 healthy adult donors was taken and 5 mL of 4 mM silver nitrate (AgNO₃) solution was added to this solution under continuous stirring to obtain an Ag⁺/serum complex solution. Then 10 mL of 2 mM dextrose solution was added to the Ag⁺/serum complex solution and the temperature of the solution increased to 75°C. Nano transformation is indicated by the colour change. Thus, prepared AgNPs contained an effective concentration of 1 mM silver (106.8 µg/mL silver or 168 µg/mL AgNO₃). Generated AgNPs were characterized by UV-vis absorption spectra, DLS, Zeta potential distribution, and TEM images. Thus, wide cluster range round form AgNPs was obtained by DLS measurement with an average size of 18 nm. The wide cluster range indicated participation of different serum proteins in different concentrations as capping molecules and size agglomeration of AgNPs also depended upon concentrations of different minor capping agents.³⁰ As the chemical reduction process of AgNO₃ was conducted in buffered alkaline pH of serum (~7.4) by multiple reducing contents of serum, uniform – 8. 9 mV zeta potential bearing average core size 18 nm, was obtained for the product termed as AgNP-Serum-18.

This was used for testing on cell lines, whereas for toxicity studies on small animal like mouse, 10-fold higher silver containing nanoparticles were used for assessing extended safety-level.²⁵ These strong AgNPs also showed somewhat similar size and zeta potential but narrow cluster-range, probably because serum contained enough capping and reducing substances.

In-vitro Study

Reagents

Penicillin-streptomycin, trypsin-EDTA, Dulbecco′s Modified Eagle′s Medium (DMEM) High Glucose (12100046, Gibco), sodium pyruvate, 1% antibiotic-antifungal agent, and Fetal bovine serum (FBS) (16140063, Gibco) were obtained from Gibco (Grand Island, NY, USA). Silver nitrate, cisplatin, and all other chemicals were purchased from Sigma-Aldrich.

Cell Line Details

The cell lines used in the study were human caucasian hepatocyte carcinoma cell (Hep-G2) and HeLa derivative normal human liver cell line (WRL-68). HepG2 was cultivated in Dulbecco′s Modified Eagle′s Medium (DMEM) High Glucose (12100046, Gibco), sodium pyruvate, supplemented with 10% US origin FBS (16140063, Gibco) 1% antibiotics (penicillin/100 U/mL and streptomycin/100 μg/mL).

WRL-68, similar to the human hepatocyte cell line, were procured from NCCS (National Centre for Cell Science, Pune, India, a government repository). This was cultivated in the same tissue culture medium. Cells were cultured in complete medium at 37°C in a 5% carbon dioxide incubator. After three to four days the cells grew to approximately 70% confluence. Afterwards, they were detached with trypsin and cultured in a new cell culture flask.

For cryopreservation, complete growth medium supplemented with 5% DMSO was used and the vials were placed at –20°C for 2–4 h then placed at −80°C for 24 h and then shifted to –196°C for long-term storage.

Cultivation with Nanoparticle Treatment

Depending on the doubling times, the cells were seeded in a 96-well plate with a specific number of cells and incubated for 3 days. On day 4, the medium was removed from each well and 200 µL of the various nanoparticle suspensions in varying concentrations in cell culture medium was added to the wells. Cells were treated with AgNPs or cisplatin at a specified concentration for exactly 48 hours before viability assessment. For the 24-hour time point, cells were treated for 24 hours followed immediately by 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazoliumbromide-assay and hexosaminidase assay.

MTT Assay

HeLa derivative normal human liver cell line representative WRL-68 and hepatocarcinoma representative Hep-G2 cell line were taken for cytotoxicity study. The MTT assay was used to determine the viability of the cells (Sigma-Aldrich, Steinheim, Germany). The reaction of the tetrazolium ring by mitochondrial dehydrogenase enzymes results in an insoluble blue reaction product in this assay. First, the medium from each well was removed, then 100 µL of fresh medium and 10 µL of sterile MTT solution (3 mg/mL PBS) were added to each well and incubated for 4 hours at 37°C in 5% CO₂. After incubation of the dye, 100 μL of DMSO were added and the plates were placed on a shaker for 15 min for complete crystal solubilization and the optical density of each well was then calculated. The quantity of the formazan product was measured by 595 nm using the scanning multi-well spectrophotometer (Bio-Rad, Model 680, Japan) with DMSO as blank. Absorption OD values proportionately indicated the number of living cells in culture. To calculate the standard error, each measurement was repeated three times with repetition of test in quintuple. The Student’s t-test was used for statistical analysis. Parallel wells containing AgNPs at all tested concentrations without cells were incubated with MTT reagent.

The IC50 value was calculated by using nonlinear regression (curve fit) followed by log (inhibition) vs response equation in graph pad prism software.

Hexosaminidase Assay

The proliferation of hepatocarcinoma cells and normal liver cells were determined by using endogenous enzyme hexosaminidase. Cells were grown in 96 well plates and treated with Serum-AgNP-18 for 24 hours as per the designed experiments. 75 µL of substrate solution (53.3 mg of p-nitrophenol-N-acetyl-beta-D-glucosaminide, 384 mg of sodium citrate, and 10 mL of 0.5% Triton X 100 dissolved in 20 mL of water, pH 5) was added to each well and were incubated for 20 minutes at 37°C under 5% CO₂ atmosphere. After incubation, 112.5 µL of developer solution (112 mg glycine, 55.8 mg EDTA in 30 mL of water, pH 10.4) was added and the absorbance was recorded at 400 nm in a microtiter plate reader.

Estimation of Proliferative Potential by Cell Cluster Formation Assay

WRL-68 and Hep-G2 cell lines were taken for this study. Estimation of proliferative potential by cell cluster formation assay was performed for cytotoxicity assessment of newly developed AgNP-serum-18.²⁵ Briefly, 2 mL of MEM and DMEM high glucose medium was added subsequently into each well (flat bottom 6 wells tissue culture plate), followed by addition of after 24 h incubation at 37°C in a CO₂ incubator, plates were checked for cell adherence which was continued for another 3 days (72 hours) incubation to observe colony formation. After confirming colony formation, wells were washed with appropriate incomplete medium (without FBS). Then, up to 4 mL of fresh complete medium was added with the addition of normal AgNP-serum-18 suspensions in varying concentrations. Thus, approximate effective concentrations of silver were achieved to 0, 0.5, 1, 5, 10, and 15 µg/mL at wells in chronological order. After another 24 hours of incubation at 37°C in a CO₂ incubator, wells were washed with PBS and fixed for 15 minutes in 50 μL of methanol. Wells were washed twice with PBS before being stained with Hematoxylin for 2 minutes and then washed in tap water. Images were taken from an inverted microscope at 10x magnification. The experiments were performed independently in triplicate, and the percentage inhibition of proliferative potential, as determined by the cell cluster formation assay, was calculated relative to the negative control.²⁷ The student’s t test was used for statistical analysis.

Wound Closure Assay

The scratch assay was performed on cell migration of in-vitro wound’s approximating cells, by a modified method using an aqueous extract from the leaves of Chromolaena odorta (Eupolin) in place of Achillea eriophora (spider) extract.^31,32 Human hepatocarcinoma equivalent Hep- G2 cells were seeded in 6-well plates at densities of 5×10⁴ cells/well in the growth medium. The dishes were properly incubated for approximately 6 h at 37°C, allowing cells to adhere and spread on the substrate completely. The required number of cells for a confluent monolayer depends on both the particular cell type and the size of dishes was adjusted appropriately. For the WRL-68 cell line with doubling time 24 h in MEM + 1% sodium pyruvate + 10% FBS, per well 8,000 cells were inoculated. For Hep-G2 cell line with doubling time 50–60 h in DMEM + 10% FBS, per well 10,000 cells were inoculated. After reaching near 100% confluency, the media of the cells were changed. Then, on the grown monolayer cells, scratches were made using a sterile micro-pipetting tip. The tip was drawn firmly across the diameter of the dish. Cells were washed by PBS in order to remove the loosened debris. A range of concentrations (5–15 µg/mL) of Serum-AgNP-18 were added. The cells were treated with an equivalent amount of DMSO, as control.

For the migration assay, cultures were rinsed twice in PBS, fixed in absolute methanol, and examined at a magnification of 40x using a light microscope with ocular calibration. Images were taken instantly after the wound was created, followed by imaging at 0, 4, 8, 16, 20, and 24 hours. Using Image J software, cell migration rates were calculated by measuring the change in wound area (pixels).

In-vivo Study

Male Swiss albino mice weighing 25–30 g were used in this study (n = 120). The mice were maintained at thermoneutral temperature conditions before starting the experiments. The mice were kept at 20°C and 26°C and fed with pellets. All animal experiments and sample collection were performed at the Institute of Post-Graduate Medical Education and Research. The protocols were approved by the Institutional Animal Ethics Committee (IAEC) at IPGME &R (Ethical No.-Inst/IEC-2018/488).

Development of Hepatocarcinoma

Male Swiss albino mice (n = 78) were first injected with DENA (a single intraperitoneal injection of 1 mg/kg at 14 days of age). Second, CCl₄ (0.2 mL/kg, 2 times intraperitoneally injected per week, beginning at 8 weeks of age) was given for up to 14 weeks.³³ At the same time, the mice in the control group (n = 42) were injected with an equal volume of physiological saline. Throughout the trial, all animals were kept in a temperature-controlled facility with a 12-hour light–dark cycle and had free access to food and drink. To track tumour progression, the mice were sacrificed at various points throughout, with or without DENA therapy to track tumour progression. Mice were euthanized by a licensed veterinarian using an overdose of sodium pentobarbital in accordance with AVMA and ARRIVE guidelines. Death was confirmed by the absence of respiration, heartbeat, and reflexes, and carcasses were disposed of following institutional biosafety protocols.

Several biochemical liver function tests were performed on blood samples. For histological analysis, liver tissues were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

Grouping of Experimental Animals

Group-I (Normal Control) mice (n = 50) were included for studying the margin of safety assessment between non-serum capped AgNP-PVA-13 and AgNP-Serum-18. Those were used for viability, histopathology (n = 25), blood biochemistry test for five sets (n = 5 each) after injection of saline, silver equivalent AgNO₃ solution, AgNP-PVA-13, AgNP-Serum-18 and ten-fold higher concentration AgNP-Serum-18 for determining the respective margin of safety on heathy mice. Of the 120 mice used, 50 were assigned to safety assessments (Group I) and 70 were designated for HCC induction. We initiated induction in 78 mice to account for an anticipated 10–15% attrition; 60 mice successfully developed HCC and were randomized (n = 10/group) into treatment arms (Groups II–V). For survival monitoring, we used n = 6 per group, while the remaining animals were used for biochemical and histopathological endpoints. To ensure objectivity, we used computer-generated randomization, and both the pathologist and biochemist were blinded to the group assignments. Mice were housed at a stable 24 ± 2°C.

Group-II (Cancer Induced) HCC served as the tumour control group (n = 30) for viability, histopathology, and blood biochemistry tests, sacrificing at different stages of tumour development.

Group-III (Cancer Standard drug) HCC tumour administered with Cisplatin (2 mg/kg, i.p.) for continuous 14 days served as the treatment control (n = 10).

Group-IV (Cancer + Serum-AgNP-18) treated with Serum-AgNP-18 (2 mg/kg) (intravenous, i.v) for 14 days weekly served as the test group (n = 10).

Group-V (Cancer + Serum-AgNP-18 + Cisplatin) HCC treated with Serum-AgNP-18 (intravenous, i.v) for 14 days weekly with Cisplatin (2 mg/kg) daily for 14 days served as the combination treatment test group (n = 10). The standard drug, Cisplatin 2 mg/kg, and the strong Serum-AgNP-18 were administered (intravenous, i.v) after confirming the HCC by blood biochemistry and Histopathology (n = 10). All the drugs were administered for 14 days weekly for Serum-AgNP-18 (intravenous, i.v) and 14 days continuously for Cisplatin. The biochemical parameters, histopathology, and immunohistochemistry were evaluated using three mice for each test with three repetitions from common nine mice, leaving one.

Sample Collection and Biochemical Testing

The blood samples from each mouse were collected by sino-orbital puncture using micro-capillary tubes. Blood samples were withdrawn in clean and dry test tubes and then centrifuged at 3,000 rpm for 15 min. The supernatant was used for biochemical tests of liver function. The alteration of biochemical test was determined after treatment with a single and combined dose of Serum-AgNP-18 and cisplatin. An auto-analyser system was used to perform routine liver function tests from sera, including total bilirubin (TBIL), alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), and alkaline phosphatase (ALP).

Histopathological Confirmation

Tissue specimens from liver were collected then fixed in 10% neutral buffered formalin and processed by the conventional methods to obtain paraffin sections. The embedded paraffin sections were cut at 4 μm thickness, then stained by H&E stain for histopathological examination.

Microscopic grading and scoring of the liver were carried out to determine the degree of severity of the observed histopathological lesions. Grading of hepatocellular damage was performed according to the following parameters: necrosis, vascular congestion, and hemorrhage.

Immunohistochemistry Studies

Tissue sections with a thickness of 3 µm were mounted on poly-L-lysine-coated slides. The slides were deparaffinized in xylene for 10 minutes after being maintained in a hot air oven for 1 hour at 56°C (three changes). The slides were treated with 0.3% hydrogen peroxide in methanol for endogenous peroxidase blocking after antigen retrieval with 0.01 m citrate buffer (pH 6.0). The rest of the procedure was carried out using an immunohistochemistry (IHC) kit according to a standard manufacturer’s protocol (DAB150, Millipore). Caspase 3, BAX, and BCL2 antibodies were purchased from Santa Cruz Biotechnologies. All antibodies were used at dilutions of 1:200.

Results

In-vitro Study: Comparative Efficacy of AgNPs on Normal and Tumour Cell Lines

MTT Assay and Hexosaminidase Assay

Our study results of MTT assays after 24 h exposure to Serum-AgNP-18 showed a dose-dependent decrease in viability percentage of tumour cell line Hep-G2, whereas it showed almost no toxic effects on the normal liver cell line, WRL-68 (Figure 1a and b).

Figure 1.

MTT Assay (a) Hep-G2 cells treated with Serum-AgNP-18 (0–25 μg/mL) (b) WRL-68 cells treated with same AgNPs (0–25 μg/mL). (c) Obtained IC ₅₀ values of Hep-G2 and (d) WRL-68 cells. (e) Hexosaminidase Assay: Hep-G2 cells treated for 24 h with Serum-AgNP-18 (0–25 μg/mL). (f) WRL-68 cells treated for 24 h with same AgNPs (0–25 μg/mL).

Viability percentage obtained by MTT assay of Serum-AgNP-18 for 24 h on the Hep-G2 cell line exposed to 0, 1, 5, 10, 15, 20, 25 μg/mL represented a dose–response pattern, as shown in Figure 1a. Data were reported as mean ± SD of three independent repetitions of experiment. Using the dose-response curves, IC50 was calculated as 5.5 μg/mL (Figure 1c; p < 0.001).

While on WRL-68 cell line, viability percentage obtained by MTT assay exposed to 0, 1, 5, 10, 15, 20, and 25 μg/mL of Serum-AgNP-18 for 24 h represented a different dose–response pattern, as shown in Figure 1b. Data were reported as mean ± SD of three independent experiments. Using the dose–response curves from our previous related study for assessing the margin of safety,²⁵ IC50 was obtained, 282 μg/mL for Serum-AgNP-18 on WRL-68 cell line (Figure 1d; p < 0.0001).

The results of Hexosaminidase assays after 24 h exposure to Serum-AgNP-18 showed a dose-dependent decrease in viability percentage of the Hep-G2 cell line, whereas it reflected almost no toxic effects on the WRL-68 cell line (Figure 1e and f).

In MTT assay, no significant formazan formation was detected containing AgNPs at all tested concentrations without cells (absorbance <0.05 at 570 nm). No significant substrate conversion was observed in cell-free AgNP-containing wells in Hexosaminidase assays.

Estimation of Proliferative Potential by Cell Cluster Formation Assay

The estimation of proliferative potential by the cell cluster formation assay at graded concentrations of AgNP-serum-18 in both malignant (HepG2) and non-malignant (WRL-68) liver cell lines (Figure 2) showed a dose-dependent decline in the viability and survival of HepG2 cells with increasing concentrations of AgNP-serum-18 from 0.5 to 15 μg/mL. In contrast, the WRL-68 cell line remained largely unaffected, maintaining a considerable percentage of viable cells. These findings indicate that AgNP-serum-18 exhibited greater cytotoxicity toward HepG2 cells than WRL-68 cells under identical treatment conditions.

Figure 2.

Hep-G2 and WRL-68 cells were treated with different doses AgNPs (0–15 µg/mL) for 24 hours. (a) Serum-AgNP-18 significantly inhibits colony formation of Hep-G2 cells. (b) The percentage of colonies of Hep-G2 cells. (c) No cytotoxic effect was found in WRL-68 cells. (d) The percentage of colonies of WRL-68 cells.

Wound Closure Assay

In the cell migration study, the cessation of cellular migration indicated the effects of AgNP-serum (Figure 3). The carcinomatous cell line was treated with three graded concentrations of AgNPs and observed at 8 h intervals up to 24h. The control was also set without challenge of AgNP-serum.

Figure 3.

Representative scratch assay images (40x magnification) over a 24 h period. (A–D) Cellular migration of control cells; (E–H)Cellular migration of cells treated with 5 µg/mL Serum-AgNP-18; (I–L) Cellular migration of cells treated with 10 µg/mL Serum-AgNP-18; (M–P) Cellular migration of cells treated with 15 µg/mL Serum-AgNP-18; (Q) The statistical analysis of the wound healing area in Hep-G2 cells treated with various concentrations of Serum capped AgNPs. Data shown are the mean ± standard deviation of three independent experiments. * p < 0.05 vs the control (0 µg/mL).

With increasing doses of AgNP-serum the approximating edges of cancer cells stopped migrating, whereas the control tissue culture showed no such migration arrest resulting in complete closure of the growing edge in 4 hours.

For statistical analysis, raw images were analyzed using an automated software (eg, in MATLAB), which initially measured wound width, then calculated percent closure and finally area under the curve (AUC). Error bars represented standard error of the mean. The 5 µg/mL, 10 µg/mL, and 15 µg/mL Serum-AgNP-18 treatment group statistically slowed cellular migration of the hepatocarcinoma cell line (Hep-G2), showing a statistically lower AUC value compared to all other treatment groups by using a one-way ANOVA with a Tukey post hoc adjustment (p < 0.05). The wound healing assay measures net wound closure, reflecting both migration and proliferation. Without mitotic inhibition, reduced closure in AgNP treated groups indicates reduced “wound closure potential” rather than pure migration inhibition. Future studies using transwell assay with mitomycin C are needed to isolate the migration mechanism.

Following treatment for 24 hours, the percentage of wound healing area was 89.59±2.75% for the negative control cells, 59.32±3.32% for the cells treated with 5 μg/mL serum capped AgNPs, 32.56±4.17% for the cells treated with 10 μg/mL serum capped AgNPs, and 11.67±1.46% for the cells treated with 15 μg/mL serum capped AgNPs.

In-vivo Study: Comparative Efficacy of AgNPs and Cisplatin on Hepatoma Induced Mice

Alteration of the Biochemical Profiles of Serum During the Development of HCC

We induced liver tumours in Swiss albino mice by injecting DENA as a single intraperitoneal injection of 1 mg/kg at 14 days of age. Second, CCl₄ (0.2 mL/kg, 2 times per week intraperitoneal injections beginning from at 8 weeks of age) was given for up to 14 weeks. Starting 6 weeks after initiation of DENA treatment, the body weight (BW) of DENA-injected mice became significantly reduced (p < 0.05) than that of control mice (Figure 4a). Administration of DENA + CCl₄ also led to an increase in serum TBIL, AST, ALT, and ALP, which indicated liver damage (Figure 4b–e). To monitor tumour development, the mice were sacrificed at different times during the DENA+ CCl₄ administration phase. Safety level of serum and non-serum capped AgNPs and ionic silver on healthy mice tested earlier was as follows (Table 1).

Figure 4.

After several weeks application of hepatoma inducer DENA into mice keeping untreated control, the changes of (a) body weight with time of study decreased in carcinogen treated group; (b) liver-function indicator total bilirubin (TBIL) in mg/dL serum significantly increased after 12 W; (c) serum alanine aminotransferase (ALT IU/L) significantly increased after 12 W; (d) serum aspartate aminotransferase (AST, IU/L) significantly increased after 8 W; (e) serum alkaline phosphatase (ALP, IU/L) significantly increased after 8 W, expressed as mean ± SD on the vertical axis.

Table 1.

Serum Biochemical Values of Swiss Albino Male Mice After 4 Weeks of AgNP Administration (n = 4)

Dose (µg/kg)	Control	Silver 3570 (AgNP-Serum-18)	Silver 357 (AgNP-PVA-13)
BUN1 (mg/dL)	109.75±7.5	139±12	280±17
CRE2(mg/dL)	0.45± 0.15	0.5±0.1	1.675±0.2
TBIL3 (mg/dL)	0.225±0.15	0.2±0.1	1.025±0.35
CBIL4(mg/dL)	0.02±0.015	0.0325±0.1	0.37±0.36
UNCBIL5 (mg/dL)	0.015±0.005	0.035±0.005	0.08±0.01
ALP6 (IU/L)	144.75±14	148.5±13.5	255.5±26
AST7 (IU/L)	164.75±44	144.25±16.5	378±12.5
ALT8(IU/L)	63±3.5	62.75±6.5	73.5±12

Note: 1, blood urea nitrogen; 2, creatinine; 3, total bilirubin; 4, conjugated bilirubin; 5, unconjugated bilirubin; 6, alkaline phosphatase; 7, aspartate amino transaminase; 8, alanine amino transaminase. All data were presented as the mean ± SEM. Data were subjected to statistical analysis using one-way ANOVA with GraphPad Prism 8.0.2(263)). Mean values were considered to be statistically significant at p < 0.05.²⁵

Histological examination indicated the evolution of liver disease caused by hepatotoxicity of DENA+ CCl₄. Mice in the DENA+ CCl₄ group developed mild liver fibrosis during week 4 and severe fibrosis and cirrhosis during week 8. HCC nodules arose from cirrhotic livers after 12 weeks of DENA+ CCl₄ administration. By week 16, all mice in the DENA group had developed diffuse HCC in cirrhotic livers (Figure 4a).

Alteration of the Liver-Function Profiles of Serum After Treatment

On the day of sacrifice, loss of mean body weight of the mouse treated with cisplatin and AgNP was least in comparison to the control mouse. But those treated with AgNPs and cisplatin showed minimum loss of mean body weight. Mouse with HCC left untreated served as control and were affected the most. The mean body weight of this control sample was reduced by around 50% in comparison to healthy control sample (Figure 5a).

Figure 5.

Body weight and biochemical tests after treatment with AgNPs and cisplatin of HCC mice. (a) Body weight, (b) serum total bilirubin (TBIL, mg/dL), (c) serum alanine aminotransferase (ALT, IU/L), (d) serum aspartate aminotransferase (AST, IU/L), and (e) serum alkaline phosphatase (ALP, IU/L). All the values were expressed as mean ± SD on the vertical axis. (f) Mouse survival. The median survival in the Control, HCC, cisplatin, AgNPs, and cisplatin+ AgNPs groups was > 2, 4, 6, 8, 10, and 12 weeks, respectively. The longest median survival time was noted in the Cisplatin+ AgNPs group.

Mean total bilirubin of the HCC affected mice was tested and the group treated with cisplatin and AgNP showed the lowest concentration of total bilirubin in comparison to thev HCC affected untreated group serving as positive control (Figure 5b). This, however, was lowest in the unaffected group serving as a healthy control.

Mean ALT level was lowest in the healthy control group. Among the HCC affected group those treated with a combination of cisplatin and AgNP showed the lowest ALT level (< 100 IU/mL) signifying the least affected hepatic physiology (Figure 5c). Untreated diseased control showed the highest ALT level (>200 IU/mL).

The mean AST value of the mice group (Figure 5d) reflected a similar scenario, having the highest value in untreated disease-control (> 900 IU/mL). In the group treated with cisplatin and AgNP, the mean AST was lowest among the diseased group (< 200 IU/mL).

Mean Alkaline phosphatase value was lowest in the healthy control group (< 100 IU/mL). As the graph suggests, among the HCC affected group, the parameter was least in those treated with cisplatin and AgNPs (Figure 5e). The untreated group showed an Alkaline phosphatase level > 200 IU/mL.

Kaplan–Meier Curve and Survival Rate of Swiss Albino Mice

The Kaplan-Meier curve illustrated the survival rate of control Swiss albino mice over time in different groups (Figure 5f). Six mice in each group were observed for 12 weeks after the completion of the treatment with cisplatin, AgNP alone, and combination of AgNP-cisplatin. One group of mice served as healthy control, whereas another group did not receive any treatment and served as diseased control. The untreated mice died within 5 weeks. No mortality was observed in the mice group treated with cisplatin-AgNP combination during the period of 11 weeks, while all others died within this period.

Histopathological and Immunohistochemical Examination

Hepatic tissues of normal control mice showed hepatic lobules with intact lobular architecture and portal tracts are of normal shape and thickness (Figure 6A). Loss of hepatic lobular architecture was observed in DEN+CCl₄-injected mice and liver tissue was composed of solid sheets of necrotic cells with mild-to-moderate nuclear atypia, nuclear hyperchromasia, and increased nucleo/cytoplasmic ratio. Some portal tracts showed dilated bile ducts. There was focal necrosis with pericentral and periportal mononuclear inflammatory cell infiltration (Figure 6B). Mice injected with DEN+CCl₄ and treated with a Cisplatin were improved with restoration of normal lobular architecture and disappearance of inflammation and cholestasis (Figure 6C). Hepatic tissues of mice injected with DEN+CCl₄ and treated with AgNPs showed restoration of normal lobular hepatic architecture and a marked reduction in the extent of cholestasis and inflammation (Figure 6D). Mice injected with DEN+CCl₄ and then treated with AgNPs+ Cisplatin showed restoration of the normal lobular architecture, an improvement in the cytological features, and disappearance of cholestasis and inflammation (Figure 6E).

Figure 6.

Histopathological examination stained with H&E and Immunohistochemical staining for Caspase-3, Bax, and BCL2 in liver tissues sections. (A) Normal mice; (B) Hepatocellular carcinoma, grade 2: disturbed architecture; massive centrilobular hepatocellular necrosis associated with haemorrhage and mononuclear inflammatory cells infiltration; dilated of hepatic radicles and bile canaliculi (H&E 200×); (C) Mice treated with Cisplatin showing normal hepatocytes; (D) Mice treated with AgNPs show preserved lobular architecture; (E) Mice treated with AgNPs+ Cisplatin shows restoration of the normal lobular architecture; (F) Normal control group showing few numbers of positive hepatocytes for caspase-3; (G) HCC group also showing few numbers of positive hepatocytes for caspase-3; (H) Mice treated with Cisplatin and (I) Mice treated with AgNPs, showing brownish cytoplasmic stain; (J) Mice treated with AgNPs+ Cisplatin showing many positive hepatocytes with moderate density for caspase-3; (K) Normal control group showing few numbers of positive hepatocytes for Bax; (L) HCC group also showing few numbers of positive hepatocytes for BAX; (M) Mice treated with Cisplatin showing mild density for BAX; (N) Mice treated with AgNPs; (O) Mice treated with AgNPs+ Cisplatin showing many positive hepatocytes with moderate density for BAX; (P) Normal mice showing few numbers of positive hepatocytes for BCL2; (Q) HCC group showing many positive hepatocytes with moderate density for BCL2; (R) Mice treated with Cisplatin showing mild density for BCL2; (S) Mice treated with AgNPs showing mild density for BCL2; (T) Mice treated with AgNPs+ Cisplatin showing few numbers of positive hepatocytes for BCL2.

Immunohistochemical analysis shows that no cleaved caspase-3 immune-reactive hepatocytes in liver sections of normal control mice (Figure 6F). Few numbers of positive hepatocytes were noticed in HCC mice group for caspase-3, proapoptotic changes (Figure 6G). When mice treated with Cisplatin and AgNPs showed brownish cytoplasmic stain (Figure 6H and 6I). On the other hand, strong positive cleaved caspase-3 immune expression was noticed in examined sections from groups treated with AgNPs+ Cisplatin (Figure 6J).

Immunohistochemical analysis showed that no cleaved BAX immune-reactive hepatocytes in liver sections of normal control mice (Figure 6K). Few numbers of positive hepatocytes were noticed in the HCC mice group (Figure 6L). Mice treated with Cisplatin and AgNPs showed a brownish cytoplasmic stain (Figure 6M and N). On the other hand, strong positive cleaved BAX immune expression was noticed in examined sections from groups treated with AgNPs+ Cisplatin (Figure 6O).

Immunohistochemical analysis showed no cleaved BCL2 immune-reactive hepatocytes in liver sections of normal control mice (Figure 6P). Strong positive cleaved caspase-3 immune expression was noticed in examined sections from groups treated with HCC (Figure 6Q). When mice treated with Cisplatin and AgNPs showed mild expression (Figure 6R and S), few positive hepatocytes were noticed in the AgNPs+ Cisplatin mice group (Figure 6T).

BAX and Caspase-3 being pro-apoptotic, induce in their expressions in malignant cells. BCL-2 being anti-apoptotic, did not express themselves in malignancy. On treatment with AgNP-cisplatin the expression of BAX and Caspase-3 was moderately higher than BCL-2. When treated with AgNP and cisplatin independently, BAX and Caspase-3 showed mild expression, whereas BCL-2 showed higher expression.

Discussion

For a long time, scientists have been trying to develop effective anticancer drugs applying nanotechnology. For higher affinity to graphene, fullerene, or other carbon-based nano-structures to cancer cells without much deleterious effects on normal cells, are tested for targeted drug delivery of anticancer drugs, which are selectively tagged with such polymer coated nanoparticles^5,34 for controlled diffusion from polymeric membrane. This approach has limited the range of action with resistance possibility like the original drug. On the other hand, most of the colloidal heavy-metal based nanoparticles (NPs) having property of charge-sign guided target delivery of large quantity cytotoxic heavy-metal atoms into accessible cells resulting in oxidative stress mediated programmed cell death (PCD) or apoptosis.^28,35,36 So, earlier version AgNPs carried alarming risk for some rapidly multiplying host cells apart from any rapidly multiplying microbes in blood stream or cancer cells. But such NPs can be made selective for some different immune-marker³⁷ bearing cells sparing normal host cells, by incorporating self-serum proteins capped structures. Apart from low charge-sign differences in normal cells, the molecular signature of this novel class of NPs may block receptor-ligand mediated endocytosis into such cells.

It has been shown that high anionic nanoparticles in circulation adsorb diverse group of proteins to attain haemo-compatible colloidal state by forming corona like additional cap on nanoparticles.^38–40 Such NP-protein complex may elicit reduced cellular uptake and toxicity with enhanced circulation time. Most other AgNPs when mixed with human blood, though acquire secondary corona-protein cap within 2–3 h to attain blood-matched surface property, but during the window-period of experiment, sufficient haemolysis indicates instant targeting high oxygen carrying red-blood-cells. Thereby released ROS in circulation may be a reason for toxicity to many other host cells.⁴¹ However, secondary corona protein capped AgNPs when added to blood in-vitro, are found to guard haemolysis totally.⁴²

Recently a matched homologous serum capped AgNPs have been shown as safe systemic usable anti-microbial agent, where corona proteins are sole capping agents. Those may have nonspecific anticancer effects for similar mechanisms of action sparing toxicity to host cells. Results of our study on the hepatoma cell line and mice model support this. For practical limitation on small animal experiment, human serum capped AgNPs have been tested in our study on the representative murine model. Use of host-specific serum for capping purposes is expected to increase the margin of safety in such experiment.

So, further experiments in larger animals either suffering from natural or induced cancer in any organ, should be carried out by treating with such colloidal AgNPs capped with that animal’s serum. The reason for using matched serum as capping agent from the same host or similar cancer patient is to include any antibody against cancer biomarkers on surface that may increase target selectiveness, while eliminating any risk of targeting heathy cells due to mismatched isoantibodies or alloantibodies. Receptor-specific internalization of nano size viral particles can be compared with the situation except high zeta potential based affinity towards oppositely charged cells of nanoparticles. On the contrary, AgNPs can destabilize viral lipid envelop or receptor protein to reduce viral infectivity,⁴³ even against COVID-19.⁴⁴

The differences of cytotoxic effects of human serum capped AgNPs on normal and liver cancer cell lines clearly indicate potent anticancer effects by the tested safe systemic usable nano preparation, which is reiterated by mice animal model studies. So, it is expected that AgNPs capped with serum of the same cancer patient may be more target-specific at sub-toxic dose for the host. However, the possibility of complication cannot be ruled out from excess tumour lyses, as sometimes occur following conventional anticancer therapy in the form of “tumour lyses syndrome”.

Cancer stem cells are more drug resistant, slow growing cells, which are often responsible for recurrence of the disease.⁴⁵ They have a slight difference in surface marker HLA pattern. It is expected that self-serum capped AgNPs will also target cancer stem cells and will be a better option for cancer therapy. A similar experience is noted for AgNPs to act on the high resistance dormant persisted sub-population of microbes within biofilms.⁴⁶

According to in-vitro and in-vivo experimental results, the maximum single dose AgNP-Serum-18 required for killing or inducing apoptosis of cancer cell population is much lower than the dose required for killing normal cells or inducing adverse physiological functions in mice. The margin of safety indicated by IC-50 is about 50 times higher in the normal cell line (282 μg/mL) than that of the cancer cell line (5.5 μg/mL). Estimation of proliferative potential by cell cluster formation assay and Hexosaminidase assay results also reiterate this observation. Wound closure assay study and liver function test results also point towards potent anticancer effect of the test nanoparticles. Immunohistochemistry results point towards a possible pathway of programmed cell death in NP-exposed cancer cells.

Interpolating dose requirement data, infusion of 100 mL AgNP-Serum-18 to an adult individual (about 100 kg body weight with 5L of blood in circulation) will be sufficient (derived from 25 mL serum) to achieve higher blood concentration of around 21.6 µg/mL silver, which is higher than obtained lethal dose assay results for the cancer cell line while a safe lower dose for the normal cell line. Paradoxically, pharmacokinetics of target specific drugs is not simply plasma concentration dependent but is proportionate to higher MLD for target cell mass. Theoretically for 1 kg tumour mass of same person, 1 mL test nanoproduct derived from 0.25 mL serum should be sufficient in lieu of 100 mL. However, according to our combination treatment experiment on mice model, priming by single dose nano-therapy followed by conventional anticancer therapy has been proved more rewarding. Such a combination of AgNPs and cisplatin action with the nano-medicine may be due to effective killing on “Programmed Cell Death” initiated cancer cells. In such case, even infusion of < 1 mL AgNP-Serum-18 with other anti-cancer measure may be sufficient for effective management. Anticipating competitive consumption of NPs by microbes in blood as a co-infective agent or excretion through the kidney or gallbladder before target attachment, a tolerable higher dose may be considered based on subsequent phase trials. This is particularly applicable for localised solid tumours due to target attachment of small amount NPs in small volume blood passing through the tumour in each blood-cycle, while affinity based instant attachment to blood-cancer cells ensure better bioavailability of nanoproducts.

Such drugs should be infused slowly to avoid disproportionate consumption by early encountered cells. Even much lower volume of AgNPs may be required for cancer management in lower body weight individuals and in children. Intravenous use of a minimum amount of safe colloidal nanoparticles may not significantly alter gut or surface commensals⁴⁷ and may be reasonably safe for the ecosystem. Such personalized nanomedicine has no chance for spillage into the environment with a minimum safe disposal policy. The multi-target mechanism of Serum-AgNP-18 observed in our HCC model suggests that resistance development may be less likely than with single target drugs. Unlike heavy-metal resistance possibility in bacteria at plasmid level mutation followed by dissemination by conjugation not applicable for eukaryotic cell with rare probability by transposition without risk for another patient. On the other hand, nanoparticles (NPs) may help overcome or delay resistance to combined chemotherapeutic agents.⁴⁸ Better cancer management by early empirical use of AgNPs are expected as additional benefits, along with management of microbial co-infections, if any.

Histological and immunohistochemical findings were primarily evaluated qualitatively to support the proof-of-concept nature of this study. Future studies should incorporate validated histopathological scoring systems and quantitative image analysis to provide a more objective evaluation of tissue changes and biomarker expression. Though the limitations of this preliminary study are testing on a hepatic carcinoma model only with small repetitions of animal-experiments; the expected outcome for other cancers may be similar for common non-specific actions. Therefore, the successful hepatoma induction experiments conducted in 60 mice may still be considered significant, despite only three repetitions being performed for each anticancer treatment group. These findings should be validated in future studies using authenticated HCC cell lines (eg, Huh-7 and SNU-449) and primary human hepatocytes. This will open-up subsequent phases of trials in higher animals with self-serum-capped AgNPs, the best green synthesis product that do not contain any unused intolerable components for intravenous use and obviously eco-friendly with zero chance of spillage.

Abbreviations

AgNPs, Silver Nanoparticles; CCL4, Carbon Tetrachloride; DENA, N-nitrosodiethylamine; DMEM, Dulbecco′s Modified Eagle′s Medium; DMSO, Dimethyl sulfoxide; EDTA, Ethylenediamine tetra acetic acid; FBS, Foetal bovine serum; HE, Hematoxylin and eosin; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCCS, National Centre for Cell Science; NNP, Novel Nano Particles; OD, Optical density; PBS, Phosphate buffered saline; PVA, polyvinyl alcohol; TBIL, total bilirubin; AST, Aspartate amino-transferase; ALT, Alanine amino-transferase; ALP, Alkaline phosphatase; HCC, Hepato-cellular carcinoma; nm, nano-meter; ZP, Zeta potential; mV, milli-volt.

Ethics Statement

Discarded human serum samples obtained after testing in the Serology Department were collected for nanoparticle preparation with permission from the Head of the Institution and approval from the Institutional Ethics Committee of the Institute of Post Graduate Medical Education and Research (IPGME&R), Kolkata-20, India (Ethics No. Inst/IEC/2018/488). All animal experiments and sample collections were performed at IPGME&R. The study protocols were approved by the Institutional Animal Ethics Committee (IAEC) of IPGME&R (Ethics No. IAEC/PKM/2020/1). All experiments were conducted in compliance with the ARRIVE guidelines and the CPCSEA guidelines for the care and welfare of laboratory animals.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

P. K. Maiti applied for a patent in India related to this work (Application No. 201831002809, filed on 24/01/2018). The patent has been granted with IPO No. 572744.