Enhanced apoptotic activity of Pluronic F127 polymer-encapsulated chlorogenic acid nanoparticles through the PI3K/Akt/mTOR signaling pathway in liver cancer cells and in vivo toxicity studies in zebra ﬁ sh

: In this study, chlorogenic acid nanoparticles encapsulated in Pluronic F127 polymer were synthesized and characterized to determine if they could treat human liver cancer. The nanoparticles were synthesized using standard procedures and characterized using physical and biological techniques such as X-ray di ﬀ raction, Fourier transform infrared spectroscopy, UV-Vis, dynamic light scattering, Photoluminescence, scanning electron microscopy, and transmission electron microscopy. The anticancer e ﬀ ects were assessed using MTT analysis, acridine orange/ethidium bromide, reactive oxygen species (ROS), COMET assay, annexin-V/FITC, cell cycle analysis, and expression of marker genes against HepG2 cell lines. The results showed signi ﬁ cant cytotoxicity, apoptosis induction, and increased ROS production in treated cells compared to control cells. The nanoparticles also activated the apoptotic cascade and regulated the PI3K/AKT/mTOR pathways. The nanocomposites exhibited unique characteristics such as anticancer e ﬃ cacy in vitro . Further research was conducted using zebra ﬁ sh to model hematological parameters, liver enzymes, and histopathology to study e ﬀ ectiveness. Green-synthesized Pluronic F127 –


Introduction
Liver cancer, categorized into primary and secondary forms, is the third major cause of cancer-associated mortalities globally.The primary forms include hepatocellular carcinoma (HCC), cholangiocarcinoma, and mixed liver cancer, while secondary forms spread through blood or other ways (1).Liver cancer risk factors include hepatitis-B, -C, and -D virus (HBV, HCV, HDV), autoimmune hepatitis, alcoholic hepatic cirrhosis, non-alcoholic steatohepatitis (NASH), diabetes, and aflatoxin-B1 exposure (2).Geographic region, gender, and age may also play a role in the development of liver cancer (3).While various treatment options are available for liver cancer, surgical intervention remains the primary method.
Postoperative liver cancer treatment can lead to metastasis, high recurrence rates, and poor prognosis, affecting patients' quality of life and survival (4).Therefore, a new avenue in cancer treatment is the pursuit of a therapeutic approach that can effectively cure liver cancer while minimizing side effects.Thus, exploring the therapeutic mechanisms of bioactive compounds in liver cancer is crucial for the advancement of treating cancer, as they can enhance chemotherapy drug effectiveness in cancer cells and prevent adverse reactions in healthy cells through various pathways (5).
Phytocompounds or their derivatives are being recognized progressively as efficient alternatives for treating cancer (6).Chlorogenic acid (CA, 3-CQA), the common isomer of caffeoylquinic acid, is at present identified as 5-CQA.This polyphenolic antioxidant occurs in various foods, like coffee, tea, apples, berries, and spinach, and has exhibited antibacterial, anti-inflammatory, anticancer, anti-ulcerogenic, and anti-diabetic properties (7,8).However, the advancement of optimal delivery systems is crucial to maximizing their effectiveness.Several studies have previously demonstrated the potential of CA in combating breast cancer, inducing apoptosis via the mitochondrial-dependent pathway, which decreases mitochondrial membrane potential and stimulates the production of reactive oxygen species (ROS) in multiple cancer cell types (9,10).In addition, numerous studies have shown that CA can work in interaction with other plantderived and natural compounds to enhance their anticancer effects (11).Numerous studies have investigated the cytotoxic effects of CA and consistently observed that CA displayed minimal or no cytotoxicity on normal cells in comparison to cancer cells (12)(13)(14).
At present, polymeric micelles have been recognized as a potential vehicle for delivering anticancer drugs due to their formation from amphiphilic block copolymers consisting of hydrophilic and hydrophobic chains that selfassemble in water to create nanosized structures (15).Recent development of nanomedicine-based therapy has revealed that the use of targeted nanoparticles along with stimuli-reactive polymers can increase the efficacy of various cancer treatments via augmentation of the clinical indices of the bioactive compounds engineered within the metal-based nanocomposites (16,17).The numerous biomedical applications of nanoparticles have made a great influence on the diagnosis of several diseases/disorders, such as cancer and other infectious diseases.Furthermore, the combined use of biomedicine and nanomedicine has formed a platform called nano-theranostics (therapeutics and diagnostics).Yet, the synthesis method of natural polymeric nanoparticles involves complicated steps compared to synthetic ones, and this may propose an efficient alternative for the preparation of numerous nanocarriers (18).Nanocarriers have demonstrated greater build-up in solid tumors through the improved penetrability and retention (EPR) effect when compared to free drugs; specifically, Pluronic F127, a biocompatible drug delivery vehicle that is a commonly used type of polymeric micelle (19).The incorporation of chlorpromazine (CPZ) into Pluronic nano micelles has increased drug selectivity and cytotoxicity toward chronic myeloid leukemia cells, indicating its possible efficacy for cancer treatment, while solasodine, a steroidal alkaloid with antifungal, antiviral, and anti-tumor properties, has also been encapsulated in Pluronic F127 nanocarriers to enhance its anticancer efficacy in A549 and Hela cells, and doxorubicin hydrochloride-encapsulated Pluronic F127 nanocapsules exhibited delayed drug release using a similar approach (20)(21)(22).
This research aims to create and examine nanoparticles made of Pluronic F127 and CA.It also examines their potential anticancer properties in vitro and in vivo against HCC.The authors are also interested to determine if the nanoparticles can induce apoptosis and generate ROS in HepG2 cancer cells.The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling is used to accomplish this.This pathway is essential for controlling cellular proliferation and promoting apoptosis.

Materials and methods
2.1 Synthesis of Pluronic F127 chlorogenic acid nanoparticles (PF127 CA NPs) About 50 mg of CA was mixed with 500 mg of Pluronic F127 in 20 mL of distilled water and left at room temperature for 6 h.The unreactive constituents were then discarded by dialyzing in water for 1 day, resulting in PF127 CA NPs.

Characterization analysis
X-ray diffraction (XRD) was employed to study PF127 CA NPs.The diffraction patterns were recorded using a monochromatic Cu Kα diffraction beam of wavelength 1.5406 Å in the 2θ range of 25-80°.To examine the Pluronic F127, a CA nanoparticle, an FE-SEM field emission-scanning electron microscope (FE-SEM) with energy-dispersive X-ray (EDAX) spectroscopy was employed (Carl Zeiss Ultra-55 FE-SEM) with EDX (model: INCA).The transmission electron microscopy (TEM) instrument (Tecnai-F20), which operated at an increasing voltage of 200 kV, was utilized to investigate the morphologies of the PF127 CA NPs.The Perkin-Elmer spectroscope was used to record the Fourier transform infrared (FTIR) spectrum in the wavenumber of 400-4,000 cm −1 .The absorption spectrum of PF127 CA NPs was recorded using a Lambda 35 spectrometer in the range of 200-1,100 nm, while the spectrometer was used to capture the photoluminescence (PL) spectra (LAMBDA 465, Perkin Elmer).

Cell culture and maintenance
HCC (HepG2) cell lines were obtained from ATCC, USA.HepG2 cell lines were grown in MEM with 10% FBS and 1% antimycotic cocktail.The cells were incubated at 37°C in an incubator with 5% CO 2 .

Assessment of the cytotoxic effect of PF127 CA NPs by MTT analysis
To analyze the cellular cytotoxicity of nanoparticles and to study the growth of cells, MTT analysis was performed.HepG2 cells were loaded into 96-well plates.The cells were incubated for 24 h at 37°C and 5% CO 2 .About 100 mL of freshly prepared media was mixed with 2, 4, 16, 32, 64, 128, and 256 µg‧mL −1 PF127 CA NPs.The plates were sustained for 24, 48, and 72 h at 37°C in an incubator containing 5% CO 2 .MTT (100 µL) was mixed up into cells and incubated for 4-5 h.Following incubation, the DMSO solution was added to dissolve the crystals formed at the bottom.Then, the optical density was measured at a wavelength of 650 nm, and IC 50 was calculated (23).

Evaluation of apoptotic efficacy of PF127 CA NPs by AO/EtBr staining
To understand the apoptotic effect of PF127 CA NPs on HepG2 cells, AO/EtBr staining protocols were performed.About 100 μg‧mL −1 of double stain (AO/EtBr) were mixed with HepG2 cells and treated with nanocomposites for 24 h.A coverslip was positioned over the cells to spread the dye.The slide was then held at 37°C for 5 min.The apoptotic cells were visualized under a fluorescence microscope (magnification 20×).The images were obtained using three individual experiments (24).

Analysis of cell apoptosis by DAPI staining
The DAPI staining was employed to study the apoptosis in the control and treated HepG2 cells.PF127 CA NPs at IC 50 were administered to the cells after seeding them into the 12-well plate.After incubation, the cells were rinsed thrice with PBS and then fixed using 1 mL of methanol at 37°C for 10 min.After rinsing with PBS, the cells were stained with DAPI at 37°C in the dark and then investigated using a fluorescence microscope (25).

Evaluation of increased ROS generation using P127 CA NPs
To examine the efficacy of PF127 CA NPs on ROS production, HepG2 (1 × 10 5 cells‧well −1 ) cells were placed in 6-well plates and exposed to IC 50 concentration of PF127 CA NPs for 24 h.After treatment, the cells were cleaned with sterile PBS and resuspended with DCFH-DA (10 µM) in serum-free culture media.The cells were subjected to hydrogen peroxide (H 2 O 2 ), a positive control for 10 min.The release of intracellular ROS was identified after incubation at 37°C using a detection reagent following the instruction of the ROS assay kit.The image thus obtained was visualized and captured using fluorescence microscopy (25).

Comet assay
Fully grown HepG2 cells were treated with IC 50 concentration of PF127 CA NPs in Petri dishes for 3 h.Cells were then trypsinized and the cell suspension was homogenized in 1 mL of media.The cells were centrifuged at 500g for 5 min and an equal proportion of cell suspension was suspended with 1% of low melting agarose.The solution was then placed on slides containing a melting agarose base.Following that, the cells were immersed in 0.045 M tris-borate-EDTA (TBE) solution comprising 2.5% sodium dodecyl sulfate (SDS) (pH 8.4) for 10 min.Then, the slides were again exposed to TBE buffer in a comet assay tank without SDS for 10 min.Finally, the cells were stained with 20 g‧mL −1 ethidium bromide stain and washed properly.The slides were then placed under coverslips and maintained in humified atmosphere.From each group, about 100 cells were examined under an upright fluorescence microscope.The fluorescence microscope would be fitted with a digital camera to measure the tail length of nuclear DNA.Using image analysis software, the tail length was measured and photographed (26).

Cell cycle analysis by flow cytometry
Flow cytometry is used to analyze cell cycle phase distribution.HepG2 cells were incubated with IC 50 concentration of PF127 CA NPs for 24 h.They were removed, cleaned twice, fixed, treated with RNase A (50 µg‧mL −1 ), and stained with PI (100 µg‧mL −1 ).Fluorescence was read using a flow cytometer (Becton Dickinson, USA), and the proportion of cells in each stage was calculated (27).

Evaluation of apoptotic cell death by flow cytometry
HepG2 cells were developed for 24 h in 24-well plates.The solution of IC 50 concentration of NPs was added and kept for 24 h at 37°C.Cells were then removed after 24 h and cleaned with ice-cold PBS thrice.Following centrifugation, the supernatant solution was removed, and the pellets formed were suspended again in annexin-V-FITC/PI buffer and placed for 15 min in dark conditions at 37°C.The cells that underwent apoptosis were then analyzed using an annexin V-FITC kit using manufacturer instructions.The cells were studied by flow cytometer (Becton Dickinson, USA) using Cell Quest Software (27).
2.12 Effect of PF127 CA NPs on the PI3K/AKT/ mTOR pathway in HepG2 cells by RT-qPCR HepG2 cells exposed to PF127 CA NPs were evaluated for mRNA expression of PI3K/Akt/mTOR signaling proteins.About 1 g of total RNA was obtained utilizing the TRIzol reagent, and cDNA synthesis was performed following the instructions of the manufacturer.The concentration and quality of RNA obtained were assessed using a spectrophotometer.RT-qPCR analysis was performed using the SYBR green PCR kit.The real-time PCR was performed on a StepOnePlus (Life Technologies, USA).The intensity of the target genes obtained was analyzed.The primer sequences of the target genes were obtained from PUBMED using evidence from the earlier literature.mRNA expression of target genes was standardized using housekeeping genes and their relative expression was represented using the 2 −ΔΔCT formula (28).The primers are as follows: PI3Kupstream-5′-AACACAGAAGACCAATACTC-3′, downstream-5′-TTC GCCATCTACCACTAC-3′, AKT upstream-5′-AGAAGCAGGAGGAG GAGGAG-3′, downstream-5′-CCCAGCAGCTTCAGGTACTC-3′ and mTOR upstream-5′-AGGCCGCATTGTCTCTATCAA-3′, downstream-5′-GCAGTAAATGCAGGTAGTCATCCA-3′ (Phoenix Flow Systems, USA).

Animal acclimatization
Adult zebrafishes in good condition were procured from the local vendor.The zebrafishes were acclimatized in the standard laboratory conditions as per OECD guidelines (203).The guidelines to maintain zebra fishes, the static system of water, the ideal temperature, pH, salinity, and dissolved oxygen levels were observed on a regular basis.The zebrafishes were sustained at an optimum temperature and pH range of 27 ± 2°C and 6.8-7.4 , respectively.The healthy zebrafishes were fed with fish pellets twice per, which were available from commercial sources from pet shops.The healthy adult zebrafishes without any deformities or other infections were preferred for the present experiments.Adult zebrafishes were separated into different groups and maintained in several tanks for toxicity validations of the nanocomposite.

LC 50 determination for PF127 CA NPs
PF127 CA NPs were checked for their toxicity toward the zebrafishes.Various concentrations of PF127 CA NPs such as 25, 50, and 100 mg‧L −1 were added into the tank, and their toxicities were determined.The acute toxicity was assessed for 7 days in static water treatment.

Experimental model
The zebrafishes of similar weights of both sexes were selected for the study.The fishes were divided into four groups that contained six fishes each.The procedure was duplicated to ensure precise results.Group 1: Control fish/untreated Group 2: 25 mg‧L −1 of PF127 CA NPs Group 3: 50 mg‧L −1 of PF127 CA NPs Group 4: 100 mg‧L −1 of PF127 CA NPs Throughout the investigation, the fishes were fed with a standard fish pellet diet.

Hemotoxic and hepatotoxic effects of PF127 CA NPs on zebrafish
The fishes were euthanized on day 8.The blood samples were collected from the entire group and hematological parameters such as red blood cells (RBC), white blood cells (WBC), and differential count were analyzed.The liver enzymes such as aspartate aminotransferase (AST), alanine transaminase (ALT), and alkaline phosphatases (ALP) were estimated using biochemistry kits (Biosystems Diagnostic, Cat.Nos 11830, 11832, and 11592, respectively).The protocol was followed as per the manufacturer's instructions.The liver tissue was excised and stored in formalin for histopathology study using hematoxylin and eosin (H&E) staining method (29,30).

Statistical analysis
The results of the study were shown as mean ± SD.The variation among the groups was assessed using a one-way ANOVA and Tukey postdoc assay using SPSS software.

Characterization studies
PF127 CA NPs were dispersed in the water using the UVvisible (UV-Vis) spectrum by ultrasonication.PF127 CA NPs were found to have an absorbance edge at 285 and 335 nm.
Various functional groups of PF127 CA NPs were observed from the FTIR spectrum at 3,432, 2,901, 1,637, 1463, 1,348, 1,103, 951, 843, and 568 cm −1 .The O-H and C-OH bending of phenol groups was at 1,348 and 1,103 cm −1 for the CA group.The PF127 signals associated with the functional groups were present in this polymer peak at 3,432 cm −1 for hydroxyl stretching, 2,901 cm −1 for asymmetric stretching, and CH 2 rocking (951, 843, and 568 cm −1 ) on the oleic acid.The FTIR spectrum results confirmed that NPs from CA NPs have successfully interacted with PF127 of the PF127 CA NPs surface matrix.These interactions are due to the electrostatic interaction between the chlorogenic acid with PF127 molecules.The PL spectra of PF-127/CA NPs with an excitation wavelength of 325 nm are shown in Figure 1.
The deconvolution of the PL spectra of PF-127 CA NPs revealed seven peaks, classified as UV at 366, 388, and 398 nm, violet at 424 nm, blue at 446, 447, 461, and 480 nm, and green at 501 and 508 nm.Figures 2 and 3 show the surface morphologies of PF127 CA NPs investigated using FE-SEM/TEM and SEAD at several magnifications (10 μm to 5 nm).The prepared PF127 CA NPs have a PF-127 nanoplatelike structure with CA-decorated small particles, as shown by FE-SEM/TEM images.The mean particle size is 50-60 nm.PF-127 NPs are conjugated to CA surfaces.Figure 3 depicts the chemical composition of the synthesized PF127 CA NPs.Carbon and oxygen were found to have the highest atomic percentages in PF127 CA NPs. Figure 4 depicts the XRD patterns of the synthesized PF127 CA NPs.The amorphous nature of PF127 CA NPs is confirmed by the broad peaks in the XRD spectrum (Figure 4a).PF127 CA NPs have decreased crystallinity properties.Albumin CA NPs have a mean crystallite size of 52 nm.The hydrodynamic diameter of PF127 CA NPs was determined using dynamic light scattering and found to be 117 nm, as shown in Figure 4b.

Anti-cancer activity in vitro screening of
PF127 CA NPs

Cytotoxicity analysis
Figure 5 depicts the influence of PF127 CA NPs on the growth of HepG2 cells.After 24, 48, and 72 h of incubation with NPs (2-256 µg‧mL −1 ), it was observed that the exposure to NPs remarkably reduced the cell growth when compared with the control.As for cytotoxicity, the IC 50 concentration of PF127 CA NPs was observed after 24 h at 12 µg‧mL −1 , and these concentrations were chosen for further analysis.

Effect of PF127 CA NPs on the O/EtBr and DAPI nuclear staining, and ROS-induced apoptotic cell death in the HepG2 cells for 24 h
As shown in Figure 6, HepG2 cells were treated with PF127 CA NPs, stained using AO/EtBr, and examined under a fluorescence microscope.The control cells revealed green fluorescence demonstrating live cells (Figure 6a), while Anti-cancer potential of PF127 with chlorogenic acid-encapsulated NPs  5     6h), whereas control cells had significantly lower ROS production (Figure 6g).

Assessment of Comet assay DNA damage in HepG2 cells exposed to PF127 CA NPs
HepG2 cells exposed to PF127 CA NPs resulted in Comet assay DNA damage, as evidenced by an increase in the length of the tail compared to control cells (Figure 7b).This increase in tail length was examined only in cells exposed to NPs, suggesting that the damage was caused by the treatment.As shown in Figure 7c, cisplatin (10 µM) is a positive control drug and may have caused DNA damage as a result of the production of free radicals by NPs.Our findings support the idea that exposure to nanoparticles can lead to DNA damage by the generation of ROS, which can trigger apoptosis.Similar findings have been reported in previous studies on the DNA damage induced by silver nanoparticles.

Cell cycle arrest in HepG2 cells induced by PF127 CA NPs
Flow cytometry analysis was conducted to investigate the effect of PF127 CA NPs on the HepG2 cancer cell line.The analysis revealed sub-G1 cell cycle arrest, indicating significant inhibition of cell proliferation after 24 h of treatment (Figure 8b and c).Moreover, the cellular proportion in the sub-G1 stage was high in the NP-treated group than control, suggesting the stimulation of cell cycle arrest.In comparison, cisplatin (10 µM) and PF127 CA NPs caused a high proportion of cells arrested at the G2/M phase.These results indicate that PF127 CA NPs possess anti-proliferative properties, which could be ascribed to their ability to induce cell cycle arrest.

Annexin-V/FITC/PI flow cytometry analysis of HepG2 cells exposed to PF127 CA NPs
To assess the impact of PF127 CA NPs on inducing apoptosis, annexin-FITC/PI dyes were used to stain HepG2 cells and analyzed using flow cytometry (Figure 9).The control cells exhibited a higher proportion of viable cells (Figure 9a) and fewer early, late, and necrotic cells.In contrast, HepG2 cells treated with PF127 CA NPs showed a decrease in viable cells and an increase in apoptotic and necrotic cells (Figure 9b).The apoptotic rate was significantly higher in HepG2 cells treated with PF127 CA NPs than control.These outcomes demonstrate that PF127 CA NPs induce apoptosis in HepG2 cells, which inhibits the proliferation of cancer cells, consistent with the results of the MTT assay and earlier studies.

Effect of PF127 CA NPs on the PI3K/AKT/mTOR pathway in HepG2 cells
RT-PCR was employed to evaluate the expression of PI3K, AKT, and mTOR cell signaling molecules in HepG2 cells (Figure 10).Compared to control cells, the cells treated    In Figure 11, compared with control, the sample showed considerable reduction in AST, ALT, and ALP levels (P < 0.0001) on exposure to PF127 CA NPs at different dosages such as 25, 50, and 100 mg‧L −1 for 7 days indicating the non-toxic character of the nanoparticle.

Hemotoxic properties of PF127 CA NPs in zebrafish
Hematological studies showed (Figure 12) non-significance in RBC and WBC cell counts on treatment when compared to control.In the case of the differential count of WBC, there was a significant (P < 0.0001) decrease in the proportion count of lymphocytes, monocytes, neutrophils, eosinophils, and basophils confirming the absence of hemotoxicity.

Histopathological evaluation of PF127 CA NPs in zebrafish
Representative H&E-stained micrographs of the zebrafish liver tissue following exposure to sample 1 for 7 days are shown in Figure 13 (black arrows indicate the hepatic cells).

Discussion
The need to create effective drug agents for the treatment of both human and plant diseases is gaining more attention (31).The use of nanotechnology to deliver drugs for cancer treatment is a targeted approach that facilitates the attachment of drugs to the surface of cancer cells.Nanoparticles are well-known for their potent anticancer properties.These materials possess several advantageous characteristics, including the ability to disrupt hormones, interfere with the normal cell cycle, interact with nucleic acids,  and promote the synthesis of proteins, all of which can inhibit the growth of cancer cells (32).Additionally, nanocomposite materials are capable of interacting with arteries, veins, and stroma tissues that surround tumor cells, resulting in minimal side effects while hindering cancer cell growth (33).Despite the development of potent drugs for cancer and other infectious diseases, resistance to drugs has increased, resulting in many failures and posing a challenge for pharmaceutical companies and research groups (34).As a result, the search for natural plant-based agents for treating cancer has become increasingly popular, and there is now a greater focus on developing multi-composite nanomaterials that utilize phytochemicals from plant-based products (35,36).It has been observed that the combination of phytochemicals derived from plants together with a  potent drug carrier, PF127, in the form of NPs results in a higher level of precision in cancer treatment and other clinical applications (37).As a result, in our current study, we have produced a drug nanoparticle using PF127 and CA, a naturally occurring plant phytocomponent.After the synthesis, we evaluated the nanoparticles to validate their physical properties using XRD, FE-SEM, TEM, EDAX spectra, UV, and DLS that demonstrated the nanocomposites had properties similar to those of previously reported nanoparticles utilizing phytocompounds from plants.A combination of polymeric drug carriers along with CA was examined to understand the ability of these compounds as an anticancer agent against HepG2 in vitro.
Additionally, we investigated the potential anticancer activity of PF127 CA NPs using HepG2 cells.The considerable cytotoxicity noted in HepG2 cells treated with the nanoparticles suggests that the cytotoxic effect was induced by the accumulation of ROS and pro-oxidants, which stimulated oxidative stress and cellular internalization (38).Cells treated with higher concentrations of nanoparticles exhibited a decrease in the percentage of viable cells, whereas those exposed to lower concentrations of nanoparticles displayed higher concentrations of viable cells.These findings confirm that PF127 CA NPs possess excellent cytotoxic capabilities and can stimulate ROS production, which is consistent with previous studies (39).
Cellular uptake of nanoparticles can induce oxidative stress and interfere with DNA function (40,41).Green fluorescence observed in PF127 CA NP-treated cells suggests that these nanoparticles can stimulate oxidative stress and ROS production in HepG2 cells by NADPH oxidation.This may result in oxidative damage to cells and macromolecules, which is consistent with previous research (42)(43)(44).Thus, our findings support the ability of PF127 CA NPs to induce oxidative stress and trigger ROS production in HepG2 cells.
These outcomes reveal that the nanocomposites could induce significant anticancer effects by modulating the PI3K/AKT/mTOR axis, which performs a vital role in pathological and physiological processes.Activation of this pathway has been reported in numerous tumors.The findings of this study are consistent with earlier evidence (21) (45,46).In our studies, no significant changes in biochemical and H&E-stained micrographs of zebrafish liver tissue were observed, indicating that the synthesized nanoparticles are non-toxic.

Conclusions
From the research findings, we have concluded that PF127-encapsulated CA NPs show better anticancer properties against liver cancer cells.These anti-cancer activities of nanocomposites in liver cancer cells are by modulating the PI3K/AKT and mTOR pathways.As a result, from in vivo studies, PF127 CA NPs are promising for use in nanoparticles.These results indicate the non-toxic character of liver function tests and histopathological evaluation of treated nanoparticle groups with no notable changes.In conclusion, the current work highlights that PF127 CA NPs are effective candidates for the treatment of hepatic cancer.Additionally, preclinical works are still highly recommended in the future to understand the exact mechanisms and promote the clinical use of PF127 CA NPs.

Figure 2 :
Figure 2: TEM micrographs of PF127 CA NPs: lower and higher magnification TEM images (a-c).

Figure 3 :
Figure 3: FE-SEM images of PF127 CA NPs: lower (a) and higher (b) magnification.Elements, weight%, and atomic% of the composition obtained by EDX (c).

Figure 5 :
Figure 5: PF127 CA NPs cause cytotoxicity in HepG2 cells.HepG2 cells were treated with different concentrations (2-256 µg‧mL −1 ) of PF127 CA NPs for 24, 48, and 72 h.The cells were subjected to MTT assay, and the values were depicted as ±SD of three individual experiments.

Figure 6 :
Figure 6: Effect of PF127 CA NPs on the AO/EtBr DNA damage, DAPI nuclear staining, and apoptotic cell death, and ROS induction in liver cancer HepG2 cells for 24 h.The control cells (a), respectively, with condensed or fragmented nuclei and necrotic cells (b).Cisplatin (10 µM) (c) positive control drug.A DAPI nuclear condensation test followed by 24 h treatment with PF127 CA NPs (e), treatment with cisplatin (10 µM) as a positive control (f), and untreated control (d).ROS stained with DCF-DA.Cells treated with PF127 CA NPs (h) and cisplatin (10 µM) (i) as positive controls, untreated control (g).This is a representative image of the experiment performed in triplicate with magnification at 20×.

Figure 7 :
Figure 7: DNA damage was measured by comet assay after treatment of HepG2 cells with PF127 CA NPs.The (a) control and (b) nanoparticle-treated cells (IC 50 concentration); (c) cisplatin (10 µM) positive control drug.The extent of DNA damage was expressed in terms of comet% tail length.

Figure 8 :
Figure 8: Cell cycle analysis using flow cytometry after staining with PI.HepG2 cells were treated with IC 50 concentration of Pluronic F127 chlorogenic PF127 CA NPs for 24 h and standard drug cisplatin (10 µM) at a concentration of 5 µM‧mL −1 compared to the control.(a, b) PF127 CA NP-treated cells (IC 50 concentration), (c) cisplatin (10 µM) positive control drug, and (d) the percentage of cell cycle distribution.

3. 3
In vivo toxicology of PF127 CA NPs 3.3.1 Hepatotoxic effects of PF127 CA NPs in zebrafish

Figure 9 :
Figure 9: HepG2 cells were treated with IC 50 concentration of PF127 CA NPs for 24 h under annexin-V/-FITC/PI flow cytometry.(a) Control (untreated cells), (b) PF127 CA NP-treated cells (IC 50 concentration), and (c) cisplatin (10 µM) positive control drug administration in HepG2 cells.The percentage of apoptotic cell (d) data were mean ± SD of two independent experiments; *p < 0.05 compared with control, **p < 0.001 compared with control.

Figure 10 :
Figure 10: Effect of PF127 CA NPs on the PI3K/AKT/mTOR pathway in HepG2 cells.Values were presented as mean ± SD of triplicates.Data are analyzed by one-way ANOVA and Tukey postdoc assay using SPSS software; *p < 0.01 and **p < 0.05 compared with control.
and demonstrate the efficacy of PF127 CA NPS in blocking the growth of tumor cells and promoting apoptosis in HepG2 cells.The toxicity of metal oxide nanoparticles was assessed using a zebrafish model, including TiO 2 , ZnO, Fe 3 O 4 , Al 2 O 3 , and CrO 3 .Jeng and Swanson noted apoptotic effects in ZnO NPs in cells

Figure 13 :
Figure 13: Histopathological analysis of the livers of adult zebrafish treated with PF127 CA NPs.