Nanoarchitectonics of an acetogenin-enriched nanosystem mediated by an aqueous extract of Annona cherimola Mill with anti-inflammatory and proapoptotic activity against HepG2 cell line

For the first time, this study shows the nanoarchitectonic process to obtain an acetogenin-enriched nanosystem (AuNPs-Ac) using an aqueous extract from Annona cherimola Mill (ACM) composed of gold nanoparticles embedded in an organic matrix that acts as stabilizing agent and presents anti-inflammatory activity and cytotoxical effect against HepG2 cell line, promoting apoptosis. The synthesis of AuNPs-Ac was confirmed by x-ray diffraction analysis, showing metallic gold as the only phase, and the scanning transmission microscope showed an organic cap covering the AuNPs-Ac. Fourier-transformed infrared suggests that the organic cap comprises a combination of different annonaceous acetogenins, alkaloids, and phenols by the presence of bands corresponding to aromatic rings and hydroxyl groups. High-Performance Liquid Chromatography has demonstrated the presence of annonacin, a potent acetogenin, in the extract of ACM. An in vitro anti-inflammatory activity of the extract of ACM and the AuNPs-Ac was performed using the albumin denaturation method, showing a nonlinear response, which is better than sodium diclofenac salt in a wide range of concentrations that goes from 200 to 400 μg ml−1 with both samples. The viability assay was studied using trypan blue, treating IMR90 and HepG2 at different concentrations of AuNPs-Ac. The results defined a median lethal dose of 800 μg ml−1 against HepG2 through apoptosis according to the ratio of caspase-cleaved 9/alpha-tubulin evaluated. It was also demonstrated that the nanosystem presents a higher cytotoxic effect on the HepG2 cell line than in IMR90, suggesting a targeted mechanism. In addition, the nanosystem performs better than using only the extract of ACM in the anti-inflammatory or antiproliferative test, attributed to their higher surface area.


Introduction
Hepatocellular carcinoma (HCC) is one of the most pernicious malignancies worldwide.Unfortunately, the incidence of HCC has significantly increased over the past few decades [1,2].The standard treatment of HCC includes chemotherapy, ablation by alcohol injection to the tumor, radiofrequency ablation, or surgical resection followed by chemotherapy [3,4].However, most liver cancers are diagnosed during advanced stages, where an invasive approach is the only treatment with a survival rate between 10% and 30%.Fortunately, nanotechnology has become a new area of medicine used to diagnose and treat cancer [5] with many clinically approved nanoparticles made of organic, inorganic, or hybrid materials [6].Recently, a postnanotechnology concept fuses nanotechnology with other research fields, including organic chemistry, supramolecular chemistry, micro/nano-fabrication, materials science, and biorelated sciences.This concept is named nanoarchitectonics.In this regard, it is known that gold nanoparticles represent one of the most studied classes of nanomaterials.Gold nanoparticles are highly sensitive, stable, reliable, and less toxic than other metallic nanoparticles.However, an important property of gold nanoparticles is their biocompatibility, making them widely used in biomedical applications, bio-imaging, drug delivery, and diagnosis.Several chemicals and mechanical methods could produce gold nanoparticles.Among them, green synthesis uses natural sources, such as plant extracts containing specific bioactive components such as alkaloids, phenols, flavonoids, proteins, and vitamins, responsible for reducing and capping the metallic nanoparticles to ensure their stability and add applicability [7][8][9].Considering the relevance of this method, some refer to it as nanoarchitectonic, such as You Jeong Lee and Youmie Park [10], which present green synthetic nanoarchitectonics of gold and silver nanoparticles prepared using Quercetin and applied as nanocarriers for drug delivery.
Here is presented the green synthesis of gold nanoparticles using Annona cherimola Mill (ACM) as a reductive agent.The ACM was selected due to its multiple beneficial molecules like alkaloids, flavonoids, and annonaceous acetogenins used to treat nervous disorders, diabetes, and cancer [11][12][13].Among the different biological compounds present in ACM, annonaceous acetogenins are known to be very potent cytotoxic compounds.Several members of the Annonaceae family, such as A. muricata, A. reticulata, A. squamosa, and ACM, contain acetogenins like annonacin [14,15].Annonacin is known for its potent cytotoxic activity against multidrug-resistant tumors, showing cytotoxicity against A-549 lung carcinoma, MCF-7 breast carcinoma, and HT-29 colon adenocarcinoma cell lines [14].This polyketide is an inhibitor of Complex I in the respiratory chain of tumor cells, a process that is closely related to tumor metabolism, cell death, apoptosis, and autophagy.The functional group related to their antineoplastic activity is suggested to be the mono tetrahydrofuran ring accompanied by two flanking hydroxy groups.In addition, exists numerous reports of the use of annonaceous to synthesize metallic nanoparticles to evaluate their cytotoxic effect against tumoral cell lines [ [16][17][18]; nevertheless, for the first time, an aqueous extract from ACM was used for the nanoarchitectonic process of an acetogeninenriched nanosystem (AuNPs-Ac) composed of gold nanoparticles and different acetogenins such as annonacin to evaluate their anti-inflammatory and cytotoxic effect against HepG2 cell lines.The use of gold nanoparticles as acetogenin-enriched nanosystems was chosen because they have been proven to be a control agent in the drug delivery process to reach desired targeted sites, protect against rapid degradation, and enhance drug concentration in target tissues.The size of the nanoarchitectonic system and its surface potential suggest that this smart drug delivery system can be achieved through accumulation and entrapment imposed in cancerous cells because of improper lymphatic flow and angiogenic vessels (enhanced permeability and retention effect or EPR).Therefore, the nanoparticles should accumulate more or selectively inside cancerous cells than normal cells, acting synergistically against tumors.
The AuNPs-Ac were characterized by UV-vis spectroscopy, scanning transmission electron microscopy (STEM), dynamic light scattering (DLS), zeta potential, x-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) describing semispherical NPs coated with biological compounds that present aromatic rings and hydroxyl groups responsible for their biological properties.The egg albumin model evaluated the anti-inflammatory effect of the AuNPs-Ac, quantifying the inhibited heat-induced protein denaturation, showing high performance, even better than the reference drug used (diclofenac sodium).The potentially toxic effects of the AuNPs-Ac were assessed through a cell viability assay with trypan blue, suggesting greater susceptibility in hepatocellular carcinoma (HepG2 cells) than nontumoral cell IMR90 with the median lethal dose in HepG2 cells at 800 μg ml −1 .The proapoptotic activity mechanism was elucidated by evaluating the cleaved caspase-9, showing that the AuNPs-Ac induce three times more apoptosis than the same amount of extract, indicating that the nanosystem is more efficient at inducing cell death than the pure extract.

Extract of ACM (ACM-extract)
Leaves of ACM were washed with distilled water to remove dust and dirt.Then, they were dried at 40 °C in a convection oven for eight hours, ground to powder using a Nutribullet Pro-1000 W, and homogenized with a sieve of 100 mesh.The extraction was made through ultrasound-assisted extraction (UAE) employing a Hielscher UP200Ht portable ultrasonic homogenizer for 5 min at 100% amplitude, one gram of powder leaves, and 15 ml of distilled water.Finally, it was filtered using filter paper, its pH measured (pH 5), and its annonacin concentration quantified through HPLC (0.44 mg ml −1 ).Finally, the extract was refrigerated at 4 °C in amber flasks until use.

Total phenolic content (TPC) analysis
The TPC of the extract was obtained through colorimetry techniques using ultraviolet-visible spectroscopy (UV-vis) based on ISO 14502-1-2005E.Briefly, 1 ml of diluted extract (1:100 dilution) is mixed with 5 ml of a 10% v/v solution of the Folin-Ciocalteu phenolic reagent and 4 ml of 7.5% Na 2 CO 3 solution.After one hour of resting in an amber bottle, its absorbance is measured at a wavelength of 765 nm.The results are expressed as equivalent gallic acid units with a previous calibration curve by scanning concentrations from 10 to 100 μg ml −1 .

Nanoarchitechtonic of acetogenin-enriched gold nanoparticles (AuNPs-Ac)
Several protocols have been developed to regulate the size, shape, and surface properties of AuNPs by altering physicochemical parameters such as pH, temperature, concentrations, and reactant ratios that affect the reaction kinetics, molecular mechanics, and thermodynamics, thereby modifying the characteristics of the reaction mixture [20][21][22][23][24].
For the green synthesis of the gold nanoparticles (AuNPs-Ac), a solution of 5 mM of HAuCl4 (pH = 4) was prepared as a gold precursor.Herein, our first effort to optimize the green synthesis of AuNPs was taken by varying the ratio between the precursor salt and the reducing agent (the ACM extract), because it is known that the concentration plays a significant role in maintaining the availability of reactants and in the electronegative balance of the reacting mixture, achieving changes in the size and size distribution of the synthesized nanoparticles [21] Three different concentrations were used 2:1, 1:1, and 1:2 volume ratio between HAuCL 4 :ACM extract.Then, the extract was added at the desired volume ratio, mixed with magnetic stirring for two hours, and left to rest for 24 h under refrigeration at 4 °C to allow a complete reaction.These conditions have been reported for other noble metal nanoparticles using similar acetogenic-rich extracts [16] The resultant particles were centrifugated at 8000 rpm for 20 min.The supernatant was removed while the sediment was redispersed in deionized water for 30 min using an ultrasonic bath.This process was repeated two times to remove unreacted precursors and extract.Finally, the samples were dried in a vacuum oven at 60 •C and re-dispersed in distilled water at 9.5 mg ml −1 .
Then, the influence of the concentration in the growth process of AuNPs was monitored by UV-vis absorption spectra.From the resultant curves, it was noticed that as the concentration of HAuCL 4 increases, the curves show a higher absorbance, indicating a higher amount of AuNPs.In addition, as the ratio increased, a red-shifted absorption spectrum was noticed, attributed to larger sizes in the AuNPs, as reported in the bibliography.Considering that the wavelength shift was small but the change in intensity was big, the highest ratio of 2:1 was chosen between the HAuCl 4 solution and the ACM extract (1:3448 molar ratio of gold to acetogenin).
Once the concentration was defined, three different pH values were evaluated (pH 4, 6, and 8).Different pH levels alter the concentration of H + ions in the solution, affecting the electronegativity of the reactants and their ability to react [21] From the UV-vis spectra, AuNPs tend to be smaller at lower pH values, leading to faster reaction kinetics and altered charge distributions that influence collision rates and reaction efficiency.AuNPs are preferred in the size order between 10 and 100 nm because it has been demonstrated that the EPR effect in tumors impacts the accumulation of antitumor drugs when the tumors are passively targeted with nanoparticles in this size range [25] Therefore, the use of pH 6 in the synthesis was proposed.
The chemical reaction rate also drastically depends on temperature, where the absorption spectra tend to blue-shift when the temperature increases [21] However, the temperature was kept at 25 °C to avoid annonacin degradation (<60 °C).Once the resultant AuNPs-Ac were redispersed at the desired concentration, the amount of organic compound capping the AunNps was evaluated through TGA [25] resulting in around 15% of the total mass.

Characterization
The UV-vis spectroscopy was recorded through Perkin Elmer-Lambda 365 equipment.Litesizer 500 from Anton Paar was used to analyze the size of the particles and their zeta potential.For the IR spectroscopy with Fourier transformation (FT-IR) and XRD analysis, the samples were dried at 50 °C to remove the solvent.A Perkin Elmer-Spectrum Two spectrometer recorded the FT-IR spectra from 4000 to 450 cm −1 , and a Rigaku MiniFlex diffractometer recorded the XRD patterns using radiation at 1.54 nm and a 2 °C min −1 scanning rate from 5 to 80°.The chromatographic analysis was performed in an LC 300 HPLC system (Perkin Elmer Inc., Waltham, MA, USA) equipped with an autosampler and a UV/Vis detector using an RP-C18 reversed-phase column (250 mm × 4.6 mm, 5 μm).The wavelength was set at 250 nm for annonacin detection, and modification of the protocol reported by Yang et al was performed [26].The first step was to reconstitute 1 mg of the dried ACM-extract in 1 ml of a methanol: water (85:15 v/v) solution and then filtered with a 0.45 μm nylon syringe Acrodisc.Then, an isocratic elution of 85% methanol (A) and 15% water (%) was used for 20 min with a 1 ml min −1 flow rate.This procedure was also followed for a standard annonacin (>98%, Chemfaces, Wuhan, China) solution, and results were compared for compound identification.An SEM/STEM Hitachi SU8230 cold-field emission microscope was used to obtain the morphology of the AuNPs-Ac.A sample drop was placed onto copper grids (mesh size 300 covered with an ultrathin carbon film) and let dry at room temperature before the analysis.

In vitro anti-inflammatory activity test
The described procedure is used to quantify the in vitro antiinflammatory activity of the nanoarchitectonic system of AuNPs-Ac using a reported standard approach of protein denaturation [27,28].Albumin was selected since it has one of the highest concentrations in humans, mainly found in blood plasma (also in other sources such as milk, egg white, and other plant resources); what is more, it experiences heat denaturation [29].
First, a solution using 0.2 ml of fresh hen organic albumin eggs was homogenized with 2.8 ml of a phosphate buffer saline solution (PBS, pH = 6.4).Then, 2 ml of different concentrations (100-500 μg ml −1 ) of the tested sample (ACM-extract or AuNPs-Ac) are added.The sample-buffered albumin mixture, now composed of 5 ml, was incubated for 15 min using a convection oven (DKN302, Yamato Scientific Co., Ltd; Tokyo, Japan), and after the time had passed, the chamber temperature was elevated to 70 °C and the mixture was left for five additional minutes to promote protein denaturation.Finally, samples are left to cool at room temperature outside the oven before turbidimetric analysis using UV-vis spectroscopy.Samples were placed on standard 3 ml cuvettes, and the absorbance was measured at a selected wavelength of 660 nm.Deionized water was used as a negative control, and sodium diclofenac salt (DFS) was used as a positive control for the test.The percentage of inhibition as proportional protein denaturation is calculated according to the following equation: where A C is the absorbance of the negative control and A S is the sample absorbance.All measurements are performed in triplicate, and data is presented as mean with standard deviation.

Viability with trypan blue
The dye exclusion test described by Strober [30] determined the number of viable cells in a suspension.Briefly, 1 × 10 6 cells were grown for 24 h in a 12-well plate in the incubator with 5% CO 2 and 37 °C.When the cell density was 70%, the cells were treated for another 24 h with DMEM, Triton 0.005%, 200, 400, 600, 800, and 1000 μg ml −1 AuNPs-Ac.After treatments, the cells were washed with PBS and trypsin added, then centrifuged and resuspended in DMEM.The cell suspension was mixed with trypan blue solution and counted in a Neubauer chamber, and the % of viability was calculated.

Cell death measurement by flow cytometry
HepG2 and fibroblast cells (3T3-L1) were seeded at a density of 10 000 cells per well in a 96-well plate and incubated at 37 °C in a 5% CO 2 atmosphere.Then, different concentrations of AuNPs-Ac from 100 to 500 μg ml −1 were added in a final volume of 100 μl of DMEM per well.After 24 h of incubation, treatments were discarded, and cells were rinsed thrice with PBS.Afterward, cells were stained with 700 μM of rezasurin and incubated for four h at 37 °C and 5% CO 2 atmosphere.After this, 2 μl of propidium iodide (500 ng ml −1 ) were added to each well and incubated for 15 min at 37 °C in a 5% CO 2 atmosphere.Then, cells were rinsed thrice and harvested.Cell viability and membrane permeability after AuNPs-Ac treatment were measured by flow cytometry.An Attune NxT flow cytometer monitored cell viability with the VL-3 channel using a 405 nm excitation and emission at 603 nm source.In contrast, dead cells by membrane permeability were measured with the BL-3 channel, using an excitation of 488 nm and an emission wavelength of 695 nm.Data acquisition consists of at least 10 000 events (cells) per sample.Experiments were performed in a triplicate manner.Data was analyzed using the Attune NxT software version 3.2.1 (Thermo Fisher Scientific, Waltham, MA, USA).

Characterization of ACM-extract
The leaves of ACM are a potential source of phenolic compounds related to anti-aging, anti-inflammatory, antioxidant, and antiproliferative properties.Our previous report [31] also established that the ultrasonic-assisted extraction (UAE) technique has an excellent extraction yield to obtain antibacterial and antioxidant aqueous extracts of ACM leaves.Then, the total soluble phenolic compound content (TPC) was quantified for the extract obtained using a modified Folin and Ciocalteu method [32].This method involves the reduction of the Folin-Ciocalteu reagent with phenolic compounds in an alkaline medium, forming a blue-colored complex with a maximum absorbance of 765 nm and reported against gallic acid equivalent (GAE).By this method, the extract presented a value of 6.90 mg of GAE per milliliter of extract (mg GAE ml −1 ), similar to our previous work [31].The results were derived from the calibration curve defined by y = 0.0239 + 0.01044x, with a coefficient of determination (R 2 ) of 0.9989, suggesting excellent linearity in the studied range of concentrations.
As was said earlier, the main constituents of ACM are annonaceous acetogenins, alkaloids, and phenols.All these compounds present at least one aromatics ring with one or more hydroxyl groups.These kinds of compounds contained in the leaves of the ACM can be detected through FT-IR analysis by observing the presence of alkanes, alkenes, aromatic rings, and hydroxyl groups.Besides, it has been reported that using UAE could result in the selective extraction of acetogenins in suitable conditions [31].Figure 1(a) shows the spectrum obtained from the ACM-extract with a broad and intense peak between 3700 and 2750 cm −1 , composed of superpose signals as presented by the first-order derivative spectra (figure 1(b)) [33].The first region between 3700 and 3200 cm −1 confirms the existence of hydroxyl compounds (−OH) [34][35][36].
The second region between 3200 and 3000 cm −1 replies to aromatic rings [36].Then the peaks in the derivative below 3000 cm −1 correspond to aliphatic compounds (sp 3 , sp 2 , and sp carbon hybridization) [34][35][36] OH in-plane bend causes signals between 1350 and 1260 cm −1 and phenol OH bend between 1410 and 1310 cm −1 [36].The presence of aromatic compounds is confirmed in the original spectrum by a strong band at 1614 cm −1 and a small signal at 1510 cm −1 , both attributed to the C=C-C aromatic ring stretch [36].In addition, aromatic C-H in-plane bend causes several bands between 1225 and 950 cm −1 , while aromatic C-H out-ofplane bend produces several bands between 900 and 670 cm −1 [36].Therefore, FTIR spectra suggests the presence of different annonaceous acetogenins in the extract.Since FTIR is not entirely conclusive, HPLC analysis was used to evaluate the ACM-extract and verify the presence of these compounds (figure 1(c)).As observed in the HPLC elution profiles, a fraction peak (1) at approximately 1.93 min suggests the selective extraction of the acetogenin annonacin since it elutes at the same time as the standard (CAS # 111035-65-5, ChemFaces, Wuhan, China).Additional fractions peaks (from 2 to 5) can be observed from approximately 4 to 6 min of elution; these peaks are not clearly defined/ separated, and the resolution between them is too close (less than 1 min retention time).However, it could be attributed to other similar acetogenins such as molvizarin or annonisin (2) [37], terpenoids such as isoboldine/isocorydine or anonaine (3 and 4) [38] or even flavonoids (6) such as rutin [39].Finally, we analyze the area under the curve of the highest peak of the chromatogram, which corresponds to the standard annonacin, and compare it with the same peak area of the extract to quantify the amount of annonacin, resulting in 0.44 mg ml −1 .From the results, the molar ratio between the HAuCl 4 salt used and the amount of annonancin in the ACM extract was calculated, obtaining 1:3448 HAuCl 4 :annonacin.

Characterization of acetogenin-enriched gold nanoparticles (AuNPs-Ac)
The antioxidant properties derived from the phenolic compound in the ACM-extract could be used as a reducing agent during the green synthesis of the AuNPs-Ac.After the synthesis process, the resultant purple solution was analyzed by UV-vis spectroscopy to corroborate the reduction of the Au ion into AuNPs-Ac.As is known, colloidal metallic nanoparticles can support a localized surface plasmon resonance (LSPR) caused by a displacement and coherent oscillation of the conduction electrons on the nanoparticle surface at a specific wavelength of incident light [40].LSPR depends strongly on several factors, including nanoparticle size, morphology, and interparticle distance, resulting in colloidal solutions of different colors [41].In gold, the LSPR is displayed between 500 and 600 nm.The position of this signal is inversely proportional to the size of the nanoparticles, while the intensity of the absorption band is directly proportional to the concentration [42].Hence, UV-vis spectroscopy could be a helpful technique that allows the estimation of the particle size by analyzing the extinction spectra of the AuNPs-Ac using the MIE theory.Figure 2(a) shows the UV-vis spectra obtained from the AuNPs-Ac with an LSPR centered at 552 nm.According to Haiss et al [43], assuming spherical nanoparticles, a surface plasmon resonance at around 550 nm responds to particles with diameters around 80 nm.
The size of the prepared nanoparticles was also analyzed by DLS, resulting in the size distribution pattern presented in figure 2(b).As can be seen, the sample shows two size distributions; the first one belongs to small nanoparticles with an average size of 75.88 nm, and the second one to larger nanoparticles with an average particle size of 240 nm.The standard deviation from the first size distribution is 14.19 nm and 57.10 nm from the second one.The polydispersity index (PDI = 18.96%) describes polydisperse particle sizes confirmed by the two peaks in the histogram.The sample was also analyzed by zeta potential, which provides information about surface functionality, stability, and the interaction of dissolved compounds with the solid surface.The sample presented a zeta potential value of −34.97 mV, indicating good physical colloidal stability caused by the organic coating, adding electrostatic repulsion to the individual particles.In addition, it is necessary to know the behavior of the AuNPs-Ac in the biological medium used for the in vitro cytotoxic test, in this case, DMEM.Then, the sample was analyzed by DLS and zeta potential but using DMEM as the new solvent (figure 2(b)).As can be seen, the size distribution is similar to the sample in water.It presents two peaks at 86.11 and 208.92 nm and a PDI of 19.86%.On the other hand, the zeta potential in this media was −10.6 mV, which is considerably lower than the reported in the water.It means that in DMEM, the AuNPs-Ac are less stable and will tend to agglomerate.
Figure 2(c) shows the AuNPs-Ac x-ray diffractograms.Both patterns are very similar, showing five reflections.These signals were indexed with the JCPDS 04-0784 card, corresponding to gold with a face-centered cubic structure.The reflections were displayed at 38°, 44°, 64°, 77°, and 82°, corresponding to (111), ( 200), ( 220), (311), and (222) crystallographic planes, respectively.The crystal size was evaluated by the Williamson-Hall method and was estimated to be 45 nm.These results confirm the crystalline nature of the samples and the presence of gold as a unique phase.
Then, the morphology of the sample was analyzed by STEM. Figure 3(a) shows the image obtained by bright-field scanning transmission electron microscopy (BF-STEM), exhibiting particles with mainly quasi-spherical morphologies and different sizes.Annular dark-field (ADF)-STEM images (figure 3(b)) exhibit Z-contrast; consequently, only the contribution of AuNPs-Ac can be appreciated.Thus, after applying the color look-up table (CLUT) (figure 3(c)), it can be seen clearly that an organic compound covered the AuNPs-Ac.The formation of an organic coating does not allow the NPs to agglomerate.Finally, the size distribution of the sample was determined (figure 3(d)), showing an average size of 68.69 nm.As can be seen, the AuNPs-Ac diameter measured by DLS and UV-vis are similar but more significant than STEM reported.The reason behind this is the way that the three techniques work.STEM is a punctual observation that does not provide information about acetogenin-enriched aggregation.On the other hand, DLS and UV-vis look at the entire particle suspension, including protective surfactants and stabilizers.In this case, the presence of the organic coating increases the diameter measured by UV-vis and DLS compared to STEM.
The resultant morphology of quasi-spherical structures, with average sizes lower than 100 nm, and their negative surface potential is essential in biological applications like drug delivery because it suggests a targeted mechanism.This mechanism is named the enhanced permeability and retention effect (EPR).It consists of targeting the drug delivery to cancer cells, taking advantage of the leaky vasculatures caused by tumor growth, reaching the interstitium nanocarriers with sizes ranging from 10 to 100 nm, and remaining there because the kidneys cannot filter positively charged particles nor particles smaller than 10 nm.In this way, it improves the circulation half-life and spares normal cells to reduce side effects [25].
A schematic of the possible reaction process to form the AuNPs-Ac is given in figure 4(a), with the functional groups determined by FTIR analysis (figure 4(b)).Providing an exact mechanism for the green synthesis of the nanoparticles is an extremely hard task since many organic compounds are present in the system.Also, more than one compound may intervene in the reduction and stabilization of the nanoparticles simultaneously.Regardless, an attempt  is to observe that the main moieties that intervene in the reduction reaction are −OH, in which an intensity change is clearly observed in the vibration band of 3250 cm −1 .Also, the carbonyl band (C=O), initially at 1614 cm −1 , has a bathochromic shift to 1712 cm −1, indicating a molecular bonding of the organic compound.With these two being the main functional groups that interact for the reduction and ligand in the gold salt into nanoparticles, the mechanism for phenolic compounds is described as the ortho and meta hydroxyl groups from the phenolic compound chelates Au 3+ ions to form a five-membered chelate ring that has the high oxidation-reduction potential of Au 3+ ions that oxidized into quinones with a concomitant reduction of the Au 3+ ion to Au°.Then, other Au°reduce atoms collide to form the gold nanoparticles.These can be stabilized by other compounds in the medium by negatively charged carbonyl groups forming part of the ligand.Meanwhile, the mechanism for acetogenins compounds could be attributed to the equilibrium reaction of chloroauric acid and gold chloride (III) with hydrochloric acid (HAuCl 4 → AuCl 3 + HCl) in which the latest acts as a catalyst to protonation [44] of the lactone to form and hydroxyl moiety which with the 5-(2-hydroxy) moiety form a chelate with the gold chloride (III) ion which also has a concomitant reduction into lactone/carbonyl and Au°, then the gold nanoparticle are further form as previously described.
In the spectrum, similar to the spectrum of the ACMextract, both samples show a broad band between 3000 and 3600 cm −1 , composed of the superposition of signals generated by hydroxyl compounds (an absorption band between 3650 and 3250 cm −1 ) and aromatic rings that caused peaks between 3200 and 3000 cm −1 .The presence of aliphatic compounds caused the narrow bands at 2935 cm −1 and 2860 cm −1 , which are attributed to C-H asymmetric and symmetric stretch vibrations, respectively.In the frequency region between 3000 and 3600 cm −1 , the wide band is less intense in the AuNPs-Ac than in the ACM-extract, while for the CH vibrations, the intensity increases.On the other hand, at lower frequencies, the spectrum of AuNPs-Ac shows the same peaks that the ACM-extract, confirming the presence of the aromatic rings and hydroxyl groups; nevertheless, those bands also present evident intensity variations, but in general, the spectrum suggests that the AuNPs-Ac inherit different surface chemistries from the leaves of ACM, which should provide anticancer activity.

Anti-inflammatory activity test
The denaturation of proteins can produce inflammation.Inflammation is a complex biological response to harmful stimuli such as an injury, infection, or disease.To prevent the inflammatory process, diverse drugs such as indomethacin, ketorolac, ibuprofen, or sodium diclofenac have been used but have several side effects that limit their use in chronic diseases.Fortunately, diverse green synthesized nanoparticles have been used to induce the anti-inflammatory mechanism, help in tissue regeneration, and effectively treat inflammation by natural pathways [45].The egg albumin denaturation inhibition assay is based on the increment of absorbance in the sample against a control, indicating a higher protein stabilization, as suggested in figure 5.
From the results, it is observed that the organic compounds of the ACM-extract and the AuNPs-Ac nano architectonic system could inhibit the heat-induced albumin denaturation process since, as observed, as the sample concentration increases, the inhibition percentage (traduce as protein stabilization) also increases.This is the first report on the anti-inflammatory activity of the ACM-extract.Also, it is quantified and observed more straightforwardly comparing the animal test directed to probe Annona Muricata L. antiinflammatory activity [46], considering the difficulty of using animals for experimental research since there are ethical concerns about their use when other suitable methods can be available for analysis.
At the highest concentration tested, more than 90% of inhibition is achieved using the AuNPs-Ac nanosystem (93.97%).Higher than the values obtained from the DFS model drug and the ACM-extract (86.21% and 79.74%, respectively).It is worth noting that the ACM-extract and AuNPs-Ac nanosystem display a nonlinear trend regarding their anti-inflammatory activity.Specifically, there is a significant increase in inhibition percentage between 100 and 200 μg ml −1 but no significant increase in values at higher concentrations.This nonlinear effect allowed the ACMextract to exceed the inhibition performance of DFS in the range between 200 and 400 μg ml −1 , while the AuNPs-Ac surpassed it in all the concentrations tested.The described effect can be explained by the compounds extracted from the leaves of the ACM.At first, the acetogenins extracted help in a rapid initial anti-inflammatory effect, and then the traces of other compounds, principally phenolic compounds, help to maintain the level of the process instead of increasing it.The increasing impact of the AuNPs-Ac nanosystem compared to the ACM-extract is due to this stabilization and organic capping of the annonaceous acetogenins and phenolic compounds that now have a higher surface area to promote the anti-inflammatory effect.

Viability with trypan blue
Cell viability assay was utilized to assess the potentially toxic effects of the AuNPs-Ac.The viability test chosen is based on the principle that a live cell possesses intact cell membranes that exclude certain dyes, such as trypan blue, whereas dead cells do not [47].The viability results from non-cancer IMR90 and HepG2 hepatocellular carcinoma cells treated for 24 h with different doses of the AuNPs-Ac were evaluated through the trypan blue technique, as shown in figure 6.As can be seen, cells without treatment and treated with 0.005% triton (as a toxicity inducer) are used as controls.Triton was used as a control due to its well-known cytotoxic effect caused by the disruptive action of its polar head group that destroys the compactness and integrity of the lipid membrane, driving 50% of mortality in HepG2 cells.The data suggest a dose-dependent decrease in cell viability observed in both cell lines.However, it is essential to note that in HepG2 cells at 800 μg ml −1 , 50% of the cells are dying.In contrast, in cells of non-tumoral origin IMR90, its viability remains above 75%, which indicates that AuNPs-Ac has a death-inducing effect with greater susceptibility in HepG2 cells.The less toxic results on normal cells suggest that this nanosystem could be used as an alternative to evaluate its antitumor potential further.

Cell death measurement by flow cytometry
An additional in-vitro assay analysis for cell viability was performed by measuring cellular metabolism through resazurin reduction in flow-cytometry, to corroborate the results obtained through trypan blue staining.Cell permeability was measured using propidium iodide to confirm if the treatment with AuNPs-Ac caused cell death due to damage to the plasma membrane.
As seen in figure 7(a), the viability of HepG2 cells depends on the concentration of AuNPs-Ac, as previously observed, with a concentration of 500 μg ml −1 being the most toxic, showing the highest number of cell deaths (approximately 70%) with a median lethal dose calculated as 377.61 μg ml −1 .The results shown are really interesting since it was previously reported that a rich ethanolic-dichloromethane fraction of acetogenin/alcaloid extract from Annona crassiflora has a toxic concentration of  250 μg ml −1 [38] which even though is lower than the reported in the present manuscript, is observed that in our system nontoxic solvents are needed and no further tedious steps of purification process are required to obtain the desired effect and also serve as evidence that the AuNPs are an effective vehicle to enhance antitumoral activity due to its surface area.Meanwhile, figure 7(b), depicts that the treatment of fibroblasts with the same concentrations of AuNPs-Ac showed slight toxicity (<10%) at concentrations from 100 to 300 μg ml −1 .In contrast, from 400 and 500 μg ml −1 AuNPs-Ac concentrations, cytotoxicity increased to more than 20% and 39%, respectively.However, in none of the cases did cell death in fibroblasts exceed the death induced in hepatocarcinoma cells, demonstrating that the toxicity caused by the different AuNPs-Ac treatments is much higher in neoplastic cells than in non-neoplastic cells.The overall results are in concordance with other reports [48] where mono-tetrahydrofuran ring from acetogenins have a higher activity toward hepatocarcinoma cell line compared to other tested cell lines.

Western-blot
To elucidate the mechanism through which the AuNPs-Ac could exert its antitumor activity in HepG2 cells, the dose of 800 μg ml −1 was selected since, in cell viability experiments at this concentration, the median lethal dose (LD 50 ) for this cell line was obtained.The cells were treated for 24 h with the controls: cells without treatment (DMEM), treated with 0.005% triton (inducer of cell death), and with the proportion of pure extract corresponding to 800 μg ml −1 of AuNPs-Ac (figure 8(a)).Western blot technique analyzed the apoptosis induction through the intrinsic pathway by evaluating the cleaved caspase 9 with a molecular weight of 35 kDa.No band appears in the control cells, while cell death is induced in triton, ACM-extract, and AuNPs-Ac treatments.To have a numeric value, alpha-tubulin was determined in the same membrane, and densitometric analysis was performed to calculate the ratio of caspase-cleaved 9/alpha-tubulin through the differences in band intensity (figure 8(b)).The data shows that treating cells with AuNPs-Ac induces three times more apoptosis than using the same concentration of extracts, indicating that the nanosystem is more efficient at inducing cell death than the pure extract.The significant increase in apoptosis by AuNPs-Ac compared to the same concentration and type of natural extracts places the AuNPs-Ac as a promising therapeutic tool.Even more so, it has been proposed that decreased cell death is also responsible for cell proliferation [49,50] establishing an essential role of apoptosis in preventing cancer.Therefore, further studies in other cancer cell types and even in vivo models are needed to better understand the possible therapeutic effects of this nanoarchitectonic system.

Conclusions
Here is demonstrated the ability of the aqueous extract of Annona cherimola Mill (ACM) to reduce and subsequently stabilize gold nanoparticles, obtaining an acetogenin-enriched nanosystem (AuNPs-Ac).According to the characterization, quasi-spheric-shaped NPs with a preferred size of around 80 nm and negative zeta potential were achieved.When the system is dispersed in water, it shows excellent stability, which is reduced in DMEM.Nevertheless, the negative surface charge and size suggest it can be used as a nanocarrier to deliver acetogenins with the enhanced permeability and retention effect (EPR) as a targeted delivery mechanism.Additionally, the system presents high anti-inflammatory activity, evaluated in vitro using albumin.The systems overtake the inhabitation values of the sodium diclofenac salt, used as control.Besides, in vitro biological assays confirmed that the AuNPs-Ac and the extract induce cell death by apoptosis to hepatocellular carcinoma HepG2 cell lines.The cell viability assay showed that the AuNPs-Ac present a median lethal dose in HepG2 cells at 800 μg ml −1 while nontumoral IMR90 cell line viability remains above 75%, suggesting greater sensitivity in liver tumor cells.It is important to add that, to our knowledge, ACM has not been reported to synthesize metallic NPs, neither the effect of the extract nor the NPs as anti-inflammatory agents or on the cellular viability and cytotoxic activity on tumoral and non-tumoral cell lines.Therefore, the presented AuNPs-Ac could be considered a safer and novel model for developing cancer therapy.

Figure 1 .
Figure 1.(a)) FTIR spectrum of the ACM-extract, (b) first derivative spectra of the FTIR spectrum in the region between 4000 and 2500 cm −1 , and (c) HPLC chromatogram of ACM-extract using a UAE method (green line) and grey dashed line annonacin standard.

Figure 2 .
Figure 2. (a) UV-vis analysis of the AuNPs-Ac, (b) DLS histogram of AuNPs-Ac synthesized using ACM-extract and dispersed in different solvents, and (c) x-ray diffraction patterns of the AuNPs-Ac.

Figure 4 .
Figure 4. Suggested scheme of a reaction mechanism for the synthesis of AuNPs-Ac and (b) comparison between the FTIR spectrum of the AuNPs-Ac and the ACM-extract.

Figure 5 .
Figure 5.Comparison of the anti-inflammatory activity from ACMextract and the AuNPs-Ac nano architectonic system against DFS.

Figure 6 .
Figure 6.Evaluation of cell viability with trypan blue.The graph represents the average of three independent experiments with triplicates per experiment.Cell lines HepG2, derived from human hepatocellular carcinoma (blue bars), and IMR-90 fibroblasts isolated from normal lung tissue (red bars) were analyzed.On the X axis are the concentrations of AuNPs-Ac administered and the percentage of cell viability on the Y axis.Statistical analysis was performed using a one-way ANOVA with a post hoc using the Dunnett method; * p < 0.33; ** p < 0.002; *** p < 0.001.

Figure 8 .
Figure 8. Analysis of cleave caspase 9. Western blot of HepG2 cells with different treatments: (a) shows a band at 35 KDa corresponding to cleaved-caspase 9 and alfa-tubulin, used as a constitutive protein with a molecular weight of 56 kDa.(b) The graphic represents the densitometric analysis for the chemiluminescence of each protein.The values are relative units of the normalization of cleave-caspase 9/alpha-tubulin.