Sintesis Nanopartikel Emas Menggunakan Ekstrak Mimosa tenuiflora, Penilaian Sitotoksisitas, Serapan Seluler, dan Katalisis

Hasil dan Diskusi

Sintesis

Dengan inspeksi visual, terdeteksi bahwa sintesis NP sangat cepat di kedua sistem. Warna paling intens dari sistem AuMt1 yang ditunjukkan pada sisipan File tambahan 1:Gambar S1 menunjukkan kandungan NP yang lebih tinggi dari sintesis ini. Ini karena AuMt1 memiliki konsentrasi prekursor logam ganda dibandingkan dengan AuMt2. Dalam file tambahan 1:Tabel S1, reagen yang digunakan dalam sintesis nanopartikel memiliki pH asam. File tambahan 1:Gambar S1 menunjukkan perubahan pH reaksi saat sintesis AuMtNPs dilakukan. Reaksi dimulai dalam lingkungan asam (pH < 2,65), dan seiring berkembangnya sintesis NP, keasaman meningkat. Ini karena deprotonasi gugus hidroksil yang ada dalam molekul polifenol ekstrak Mt. Sebenarnya, ini adalah langkah pertama dari proses reduksi oksida yang menghasilkan transfer elektron dari gugus hidroksil yang terdeprotonasi ke Au ³⁺ ion. Sebagai produk reaksi reduksi oksida, Au ³⁺ ion direduksi menjadi atom logam Au ⁰ dan cincin polifenol yang menyumbang 2 elektron teroksidasi. Prosesnya dijelaskan di sisipan File tambahan 1:Gambar S1.

Uji Spektrum UV-Vis, DPPH, dan Uji Polifenol Total

Spektrum UV-Vis ekstrak kulit gunung ditunjukkan pada Gambar 1a, di mana sinyal terdiri dari pita yang terdefinisi dengan baik dengan maksimum 280 nm dan lebar 50 nm. Spektrum ini sangat mirip dengan yang dilaporkan untuk Rumex hymenosepalus ekstrak akar, yang memiliki kandungan senyawa polifenol yang tinggi [49]. Menentukan kandungan polifenol dalam ekstrak kulit Mt adalah penting karena molekul ini dapat berkontribusi secara signifikan sebagai agen pereduksi dalam sintesis AuNPs, menyediakan elektron yang diperlukan untuk reduksi Au ³⁺ ion menjadi emas metalik (Au ⁰ ). Setelah NP terbentuk, senyawa polifenol diserap pada permukaannya memberikan stabilitas pada bahan nano.

Karakterisasi Ekstrak Mt. a Ekstrak Mt spektrum UV-Vis dan b Penghambatan DPPH dengan analisis ANOVA satu arah (*p < 0,05)

Untuk uji DPPH, diamati bahwa untuk 12,5 mg/L ekstrak Mt, kami memperoleh penghambatan 50% (L50), sama seperti nilai yang dilaporkan untuk Vitamin C dan katekin (masing-masing 46 dan 58%). Hal ini menunjukkan bahwa ekstrak Mt memiliki kapasitas antioksidan yang sangat mirip dengan senyawa murni yang digunakan sebagai kontrol, Gambar 1b dengan perbedaan yang signifikan (*p < 0,05) dari nilai kontrol ditandai dengan tanda bintang. Nilai 425 mg/g yang diperoleh dari uji polifenol total menunjukkan bahwa hampir setengah dari massa yang diekstraksi setara dengan asam galat. Kapasitas antioksidan yang tinggi dan kandungan polifenol yang tinggi dalam ekstrak Mt menunjukkan bahwa itu dapat digunakan sebagai sintesis bahan nano bahan pereduksi dan penstabil yang baik dalam kerangka kimia berkelanjutan [50, 51].

Karakterisasi

Kinetika Formasi dan Spektrum UV-Vis AuMt

Gambar 2a menunjukkan evolusi temporal absorbansi pada puncak SPR AuNPs (masing-masing 550 dan 560 nm untuk AuMt1 dan AuMt2) saat reaksi sintesis nanomaterial berlangsung. Data eksperimen sesuai dengan fungsi sigmoidal Boltzmann [52], di mana setidaknya tiga tahap pertumbuhan diamati. Yang pertama, absorbansi tumbuh perlahan pada awal reaksi sintesis ketika Au ³⁺ ion direduksi menjadi Au ⁰ dan membentuk agregat dari beberapa atom yang bergabung untuk membentuk NP kecil. Pada tahap kedua, NP kecil meningkatkan ukurannya dengan pertumbuhan autokatalitik dan absorbansi tumbuh dengan cepat. Pada tahap terakhir, dalam rekristalisasi NP, absorbansi mencapai fase diamnya. Seperti dapat dilihat pada Gambar. 2, absorbansi maksimum dicapai dalam 60 detik untuk AuMt1 dan 120 detik untuk AuMt2. Menariknya, tahap pertama pertumbuhan adalah 20 detik untuk AuMt1, sementara itu hampir nol (kurang dari 1 detik) untuk AuMt2, yang dijelaskan karena proporsi yang lebih tinggi dari molekul pereduksi (ekstrak Mt) sehubungan dengan prekursor logam. Ini mendukung pembentukan nukleus yang cepat dalam NP AuMt2 sehubungan dengan AuMt1; namun demikian, tahap berikutnya dari pertumbuhan NP rendah untuk AuMt2, dan NP dengan ukuran yang lebih besar diperoleh. Telah dilaporkan bahwa sintesis AuNPs, menggunakan maltosa dan tween80 sebagai penstabil, menunjukkan kinetika pertumbuhan dengan waktu reaksi yang sangat mirip dengan yang dilaporkan dalam penelitian ini [53]. Dalam laporan sintesis hijau lainnya [54], ditunjukkan bahwa proporsi terendah dari agen pereduksi/prekursor menghasilkan NP ukuran yang lebih kecil.

Karakterisasi UV-Vis AuMt1 dan AuMt2. a Formasi kinetik dan b Spektrum UV-Vis

Gambar 2b menunjukkan spektrum serapan karakteristik AuMt di wilayah yang terdiri dari 250–875 nm. SPR untuk AuMt1 menunjukkan pita simetris dengan serapan maksimum pada 550 nm dan lebar 200 nm. Puncak plasmon AuMt2 mengalami sedikit pergeseran merah yang terlokalisasi sekarang di 560 nm dengan pita asimetris dan lebar yang lebih besar dari 300 nm, yang disebabkan oleh perbedaan ukuran antara kedua nanomaterial (d_AuMt1 AuMt2 )_{1 2} . Perilaku serupa telah dilaporkan dalam sintesis AuNPs dengan natrium citratum sebagai agen pereduksi, di mana pergeseran merah dan pelebaran plasmon dikaitkan dengan mode osilasi yang lebih tinggi yang mempengaruhi penampang kepunahan dengan meningkatkan ukuran NP [55]. Selain itu, kedua spektrum menunjukkan pita penyerapan pada 280 nm yang sesuai dengan molekul polifenol ekstrak, yang menunjukkan bahwa ekstrak Mt bertindak sebagai penstabil AuMtNP.

Ukuran, Potensi Zeta, dan Stabilitas AuMtNPs

Ukuran AuMtNP oleh DLS dan Z-potensial diuji pada kondisi yang berbeda untuk satu konsentrasi (50 μg/mL) seperti yang ditunjukkan pada Tabel 1. AuMt1 dan AuMt2 menunjukkan nilai negatif yang tinggi (≤ 30 mV) dalam air, yang mendukung stabilitas elektrostatik kedua nanopartikel sistem. Menurut Qu et al. [56], ζ nilai secara bertahap meningkat dengan ukuran NP; dalam kasus kami, AuMt1 memiliki ukuran lebih kecil dari AuMt2 dalam air, ukuran yang dikendalikan oleh sintesis NP. Nilai ukuran ini cocok dengan NP ζ nilai, di mana semakin tinggi ζ sesuai dengan NP ukuran yang lebih tinggi. Potensi Zeta (ζ ) AuMtNP yang tersebar di s-DMEM menunjukkan nilai negatif yang lebih sedikit dibandingkan dengan yang diperoleh dalam air ultra murni (Tabel 1). Pengurangan ini dapat dikaitkan dengan kation yang ada DMEM dan protein yang ada di FBS yang menutupi permukaan AuMtNP yang menyebabkan penurunan interaksi elektrostatik. Meskipun demikian ζ reduksi, nilainya tetap mendekati 25 mV untuk kedua sistem yang menunjukkan bahwa nanopartikel mempertahankan stabilitas elektrostatiknya setelah inkubasi s-DMEM [57]. Selain itu, Tabel 1 menunjukkan hasil yang diperoleh DLS untuk diameter hidrodinamik AuMtNP (2R_H ) diukur pada 37 °C dalam air ultra murni dan media kultur. Dalam s-DMEM, ukuran kedua sistem meningkat karena adsorpsi protein pada permukaan nanopartikel [58]. Untuk AuMt1, pertumbuhan 2R_H karena korona protein adalah 33,8 nm dan untuk AuMt2 adalah 42,9 nm. Diharapkan semakin besar ukuran nanopartikel maka akan semakin besar pula permukaan untuk penyerapan protein [59]. Ini bisa menjelaskan nilai yang sedikit lebih kecil pada ζ untuk AuMt2 dibandingkan dengan AuMt1 di s-DMEM. Untuk AuMt1 dan AuMt2, interaksi dengan protein s-DMEM disebabkan oleh molekul ekstrak yang menempel pada permukaan nanopartikel. Molekul-molekul ini sedikit berbeda antara AuMt1 dan AuMt2, seperti yang ditunjukkan pada hasil XPS. Kami juga mengukur pH larutan dalam rentang konsentrasi yang sama. Ditemukan bahwa tidak ada perubahan pH dan yang nilai rata-ratanya sekitar 7,5 untuk AuMt dalam air ultra murni dan 7,2 dalam s-DMEM (Tabel 1).

File tambahan 1:Gambar S2 menunjukkan diameter hidrodinamik AuMtNP saat terdispersi dalam air ultra murni dan s-DMEM pada 37 °C, dalam kisaran konsentrasi antara 25 dan 200 μg/mL. Untuk setiap sistem yang dipelajari, diameter hidrodinamika tidak berubah dengan konsentrasi nanopartikel, dan hanya untuk, AuMt2 s-DMEM pada 100 μg/mL, ukuran partikel meningkat sehubungan dengan konsentrasi terendah yang dievaluasi, yang mungkin mengindikasikan proses agregasi NP pada konsentrasi ini [32].

Spektroskopi Inframerah Transformasi Empat (FTIR)

Spektrum FTIR, ditunjukkan pada Gambar. 3, sesuai dengan ekstrak Mt, AuMt1, dan AuMt2. Pita lebar khas yang berpusat di sekitar 3250 cm ⁻¹ terkait dengan OH fenolik dari tanin dan flavonoid terutama. Puncak pada 1594 cm ⁻¹ sesuai dengan getaran lentur N-H, pada 1705 cm ⁻¹ untuk peregangan asiklik keton dan daerah antara 1000 dan 1300 cm ⁻¹ untuk meregangkan C–O. Puncak berkisar antara 1600 hingga 500 cm ⁻¹ diidentifikasi dengan polifenol, sinyal pada 1235 dan 1160 cm ⁻¹ terkait dengan regangan ikatan C–O aromatik, dan pada 1020 cm ⁻¹ terhadap regangan pita C–O alifatik dan pada 1235 cm ⁻¹ secara khusus terkait dengan karakteristik sifat siklik eter. Sinyal ini dapat dikaitkan dengan senyawa yang paling melimpah dalam ekstrak Mt seperti tanin Mimosa, flavon sakuranetin, saponin triterpenoid, kalkon, dan N ,T -dimethyltryptamine alkaloid (File tambahan 1:Gambar S3). Sampel AuMt1 dan AuMt2 menunjukkan puncak karakteristik yang sama di wilayah polifenol mengkonfirmasikan bahwa NP distabilkan oleh molekul ekstrak Mt [60]. Kami mengamati perubahan 1331 cm ⁻¹ di pita lebar dan penurunan intensitas puncak untuk AuMt1 dan AuMt2 sesuai dengan ikatan antara AuNPs dan kelompok C-H polifenol; 1723 cm ⁻¹ digeser oleh oksidasi polifenol menjadi senyawa karboksilat selama reduksi Au ³⁺ ke Au ⁰ [51, 61, 62].

spektrum FTIR. Ekstrak gunung (hijau), AuMt1 (hitam) dan AuMt2 (merah)

Spektroskopi Fotoelektron Sinar-X (XPS)

Dalam analisis pemindaian survei XPS untuk AuMt1 dan AuMt2, sampel telah dengan jelas menunjukkan adanya oksigen (O 1s ), karbon (C 1s ) dan emas (Au 4f ), yang puncaknya berpusat di sekitar 532, 284, dan 85 eV, masing-masing, seperti yang ditunjukkan pada Gambar. 4a, b. Eksperimen XPS resolusi tinggi dibuat untuk menetapkan kelimpahan relatif berbagai kelompok fungsional molekul yang melapisi permukaan AuNP. Au4f spektrum XPS resolusi tinggi untuk AuMt1 dan AuMt2 terdiri dari dua puncak simetris yang dipisahkan oleh 3,7 eV (Gbr. 4b, e). Peaks associated with 4f _5/2 spin-orbital coupling are located on binding energy (BE) of 88.6 and 87.7 eV for AuMt1 and AuMt2, respectively. For 4f 7/2, spin-orbital coupling peaks are located on 84.9 and 84.0 eV. The link of intensities (I4f _7/2  > I4f _5/2 ) and location and separation (ΔBE = 3.7 eV) between peaks confirm that gold ions (Au ³⁺ ) are reduced completely to metallic gold Au ⁰ [63]. Au4f signals, for AuMt1, are slightly shifted (~ 0.9 eV) at higher energies with respect to sample AuMt2. This can be explained in terms of NP size differences between samples. AuMt1 has a half population of NPs with size less to 40 nm, while AuMt2 NPs have a mean diameter of 150 nm, determined by TEM. Peak shift for Au4f signals, due to the presence of small NPs, has been reported by other authors who relate the Au4f BE increase with decreasing NP size [64, 65]. Also, the shift effect could be due to the interaction of functional groups capped on surfaces of AuNPs [66]. In Fig. 4c, f, the high-resolution XPS spectra of C1s are shown for AuMt1 and AuMt2. Spectra were deconvoluted by 3 Gaussian bands associated with C=O, C–O, and C–C or C=C. For AuMt1, peaks are centered on 286.9, 286.1, and 284.5 eV, for AuMt2 on 287.0, 286.3, and 284.7 eV, respectively. Comparing the experimental XPS curves for C 1s , we see appreciable differences between AuMt1 and AuMt2. The main difference comes from a significant decrease in AuMt1 of the signal associated with C–O group. Comparing the percentage contributions of each group, obtained from the deconvolutions (Additional file 1:Table S2), we see that in AuMt2 contribution of C–O signal is 27.8% while in AuMt1 is 16.6%. This difference can be explained in terms of the oxide-reduction reaction that gives rise to the process of AuNP formation. The synthesis of AuMt1 is added twice the metal precursor (HAuCl₄ is 0.01 M) than in synthesis of AuMt2. In both cases, the same amount of extract is used as a reducing agent, so in AuMt1, more hydroxyl groups (−C–OH) are consumed to reduce a greater number of Au ³⁺ ion. Thus, a decrease of C–O signal in AuMt1 confirms that hydroxyl groups participate in the synthesis reaction. High-resolution XPS of O 1s revealed that carbonyl C=O is the most abundant group (Additional file 1:Figure S4 and Table S2). In addition, the content of the C=O group is higher in the AuMt1 sample, which confirms what was previously discussed.

XPS spectra of AuMt1 and AuMt2. a , d Survey spectra, b , e Au4f resolusi tinggi, dan c , f C1s high resolution

XPS and FTIR indicate that AuMtNPs interact mainly with carbonyl groups (ketones) in addition to hydroxyl groups of Mimosa tannins, saponins, and other molecules that participate in the reduction of Au ³⁺ to Au ⁰ and stabilization of AuMtNPs [63, 67, 68].

Transmission Electron Microscopy

AuMt1 TEM micrographs are shown in Fig. 5a, b and AuMt2 in Fig. 5d, e showing products’ shape distribution. AuMt1 has the biggest diversity in shapes. AuMt shape is determined by the relationship between the variation of metal precursor concentration and Mt extract at a fixed concentration. In this case, NPs were observed without cleaning the extract to observe the interaction that forms around the AuMt. As observed in the micrographs, an extract is placed on the surface; however, NPs are kept dispersed and no aggregation is shown. Figure 5c, f show size distribution for each sample, and AuMt1 have an average size dispersion of 40 nm and AuMt2 of 150 nm.

Size distributions by TEM. a , b , c AuMt1 and d , e , f AuMt2

In Fig. 6a, AuMt TEM micrographs were also analyzed by EDS (Fig. 6b), which showed Au presence. Other chemical elements such as Cl, O, and Ca, on EDS spectrum, come from the extract that surrounds NPs. According to the crystallographic tab (JCPDS file:04-0784), the obtained distances between 2.35 and 2.03 Å (Fig. 6c) correspond to Au crystalline planes (111) and (200).

Nanostructural characterization of AuMt. a TEM, b EDS, c single HRTEM, and FFT and d XRD

X-ray Diffraction (XRD)

Figure 6d shows the characteristic AuMt XRD diffraction peak at 2Ɵ , which are in 38.17, 44.37, 64.81, and 77.66 ^o corresponding with the planes (111), (200), (220), and (311), respectively; these planes correspond with the face-centered cubic Au (space group Fm ³ m, JCPDS File No. 89-3722). High Score Plus and Origin software were used for the analysis [69].

Biological Tests

Cytotoxicity by MTT and Live/Dead Assay

To evaluate AuMt1 and AuMt2 toxicity, tests were performed on HUVEC cells using MTT. Four concentrations and two times for both materials were evaluated. In Fig. 7a, it is observed that at 24 h for AuMt1, cell viability decreases between 10 and 20%, only in concentrations higher than 25 μg/mL. For AuMt2, a similar effect is obtained in cell viability; however, the concentration of 100 μg/mL seems to have no effect on these tests. In Fig. 7b, MTT tests at 48 h for AuMt1 and AuMt2 are shown. For AuMt1, it is easy to notice that concentration with the greatest effect is 50 μg/mL, where the viability drops almost 30% compared to the control. The concentration of 50 μg/mL seems to be the concentration with the highest toxic effect; however, when the obtained data were analyzed, it is found that there is no significant difference between the obtained data on 24 and 48 h, a similar result obtained for 100 and 200 μg/mL, Fig. 7c. For AuMt2, a toxic effect between 20 and 30% is observed only on 100 and 200 μg/mL, while 25 and 50 μg/mL show no significant difference, compared to the observed effect at 24 h, Fig. 7d. This seems to correlate with AuMt2 size growth in s-DMEM (Additional file 1:Figure S2) where at a concentration of 100 μg/mL, they begin to aggregate. In the work published by Chandran et al. [70], they used gold nanoparticles coated with branched polyethyleneimine (BPEI), lipoic acid (LA), and polyethylene glycol (PEG), where they see an important toxicity in HUVEC cells by nanoparticles coated with BPEI, which have sizes of 40 and 80 nm, where viability is between 20 and 30%. When these particles are covered with human serum proteins, it is found that toxicity decreases; this is due to the corona effect. Recently, Zhaleh et al. [71] have reported the biogenic synthesis of 40-nm gold nanoparticles using leaf extracts from Gundelia tournefortii L. tanaman. Interestingly and in contrast to our results, the authors indicate that MTT cell viability tests for these particles in HUVEC, the cell viability was 95% at 1000 μg/mL; however, they do not establish if the low cytotoxicity is due to the fact that there is no material internalization or if the particles are harmless due to protein corona. In this sense, bioreductive compounds present in Gundelia tournefortii L extract are different from those reported for Mimosa tenuiflora extract (Additional file 1:Figure S3). Thus, the interactions of these two nanoparticle systems with proteins present in FBS are very different, which may explain the differences in cytotoxic responses.

Viability assay using MTT in HUVEC cell. a For 24 h and b 48 h. 0ne-way ANOVA analysis with (*p < 0,05). Comparison between 24 and 48 h for c AuMt1 and d AuMt2 with ANOVA analysis with Tukey tests (n.s. no significance and (*p  < 0.05))

As mentioned above, AuMt1at a 50-μg/mL concentration shows the highest toxicity and cellular uptake. We believe that toxicity may be due to the fact that the nanomaterial has a low affinity to s-DMEM proteins, since it has only 16.6% of hydroxyl groups on the surface to promote hydrogen bonding with S-DMEM proteins. The fact that the material toxicity decreases as AuMt1 concentration increases may be due to a cellular detoxification response, like an exocytosis caused by high intracellular content of gold [70]. For AuMt2, the toxicity effect at 100 and 200 μg/mL may be due to nanomaterial agglomeration, which could be attaching to the membrane causing adverse effects for the cells; however, more experiments are required to confirm this hypothesis.

Only one concentration (50 μg/mL) was chosen to be evaluated by live/dead fluorescent dye; this is due to the purpose of confirming the MTT results and later analyzing the metallic NP internalization in HUVEC cells, avoiding a field saturation by NPs. When cells were stained with live/dead fluorescent dye kit, it was found that a large part of the cell population favorably marked for calcein and just a few for ethidium homodimer, indicating that cell culture is viable, as shown in Fig. 8.

Live/dead assay in HUVEC cells. a , d , dan g with calcein; b , e , dan h with ethidium homodimer; dan c , f , dan i merge by confocal microscopy

Confocal Laser Scanning Microscopy:Fluorescence of AuMt

In Fig. 9a, d are shown micrographs of AuMt1 and AuMt2 in bright field and in Fig. 9b, e, their corresponding fluorescence, captured by confocal microscopy. Red fluorescence of AuMt (collected emission 650–700 nm) was excited employing 640 nm diode laser, and a mayor size of NPs can be appreciated in AuMt2 sample than AuMt1. In the merge images in Fig. 9c, f, it can be observed how the luminescence comes exclusively from the dark points associated with the NPs. This indicates that the cleaning process effectively removed the extract that is not complexed to the nanomaterial, so there is no background emission. It is interesting to observe that an intense fluorescence of the NPs captured by the confocal system is achieved at a very low excitation power of the laser (below 0.5 mW). So, this NPs system can be fluorescently traced efficiently in cellular systems with little risk of phototoxicity. Some authors have reported fluorescent emission about 610 nm, suggesting intrinsic Au fluorescence [72, 73]. AuNPs emission is related to the core size confinement effect that generates discreet electronic states [74]. However, in our case, the metal surface is covered with flavonoids, which show fluorescence, and when complexing with AuNPs, the fluorescence of both is enhanced. Different authors have reported that fluorescence is largely enhanced by charge transfer from the surface ligands to the metal core via S–Au bonds [75]. It has also been reported that ligands (thiol molecules, DNA oligonucleotides, dendrimers, polymers, peptides, and proteins) affect AuNPs optical and electronic properties since its fluorescent properties can be significantly affected by their surface chemistry [76].

AuMt1 and AuMt2 fluorescence. a , d Bright field. b , e Confocal. c , f Merge

Cellular Internalization

Cells were also analyzed, by confocal microscopy, after 24 h incubation, with AuMt1 and AuMt2 at a concentration of 50 μg/mL. The nucleus is shown in blue color using DAPI Fig. 10a, and the cytoskeleton structure was stained with anti-beta actin in green color Fig. 10b and merge Fig. 10c. The observed micrographs were obtained through 3 different channels on separate tracks, where the excitation wavelengths were 405, 488, and 640 nm for DAPI, anti-beta actin, and AuMt, respectively.

AuMt1 and AuMt2 cellular internalization. a , d , dan g with DAPI; b , e , dan h with beta-actin; dan c , f , dan i merge by confocal microscopy

As previously described, cells were also analyzed by confocal microscopy, for AuMt1 and AuMt2 internalization, at a concentration of 50 μg/mL. Confocal micrographs show that AuMt are internalized in HUVEC cells cytosolic space, and many of these particles are surrounding the nucleus, without being internalized in it. When not observing particles in the nucleus, a more meticulous analysis was carried out, by cell orthogonal projection and a 3-D reconstruction. Observing the micrographs of both reconstructions, it is possible to notice that AuMt is distributed differentially. In Fig. 11a, b for AuMt1, it can be observed that a material is dispersed in the cytoplasm, while in AuMt2, the material is concentrated in the nuclear periphery, as shown in Fig. 11c, d. We were not able to find NPs in the nucleus, and this suggests that the nanomaterial has little or no genotoxic potential, since it has no way of interacting with nuclear DNA, which is a quality for a nanocarrier. Efficient cellular uptake depends on NP size, shape, charge, and coating, the parameters that can affect their interactions with cell proteins. The fact that polyphenolic compounds are found on AuMt surface could facilitate the nanomaterial internalization, which would make it a candidate as a possible pharmacological nanocarrier [77,78,79].

Orthogonal projections and 3-D images. a , c AuMt1 and AuMt2 cellular internalization analysis through orthogonal projection and b , d 3-D reconstruction by confocal microscopy

The obtained results for an AuMtNP cellular uptake in HUVEC by a confocal microscopy at 50 μg/mL suggests that AuMt1 interacts with the cells in a greater quantity than AuMt2 in a 3:1 ratio, as seen in Additional file 1:Figures S5–S7. If we consider that protein corona in AuMt1 is 9.1 nm smaller than in AuMt2, we can suggest that AuMt1-efficient internalization by HUVEC cells is given by a combination of factors such as AuMt1 smaller size, the highest absolute value of z potential and the lower thickness of protein corona. This indicates a poor protein coverage that allows partial exposure of the nanoparticle surface, which is rich in extract molecules. Therefore, nanoparticles can interact by means of extract molecules with surface-specific membrane receptors that facilitate the internalization of AuMt1.

Catalytic Tests

Catalysis

Analysis of catalytic reaction was realized to calculate the degradation percentage ( %D) using the Eq. (5):

using the A ₀ absorbance at t  = 0 and A is the absorbance at time t . Langmuir-Hinshelwood equation was used to calculate the slope of the regression plot \( \ln \left(\frac{A}{A_0}\right) \) versus irradiation time [80], which is expressed in Eq. (6) and K is the first-order rate constant of the degradation ratio:

For the analysis of catalytic activity on MB degradation, the absorbance at 660 nm was monitored. Figure 12 shows the AuMt1 and AuMt2 catalytic activity, where a decrease on maximum absorption of MB is observed as time progresses Fig. 12a, d. MB degradation and its conversion to leucomethylene is confirmed by progressive decreases of the absorbance at 292, 614, and 660 nm correspond to MB and by the increase in time of the absorbance at 256 nm associated with leucomethylene. Homogeneous catalysis reaches a 50% MB degradation at 190 s Fig. 12b, while the degradation ratio K for the total process is 8.24 × 10 ⁻³ s to AuMt1 Fig. 11c. AuMt2 reaches a 50% of MB degradation in 400 s, Fig. 12e, and K takes the value of 3.54 × 10 ⁻³ /s, Fig. 12f.

AuMt1 and AuMt2 Catalysis. a , d UV-Vis spectra. b , e Percentage. c , f Ratio of MB degradation

On this way, AuMt1 have a more efficient response than AuMt2. We observed a size-dependent effect (AuMt) in degradation ratio [81], and a total surface area of NPs is inversely proportional to the NP size [37]. Table 2 shows a comparison between different green syntheses of AuNPs and their K obtained in size function.