Introduction

Cancer is one of the most dangerous threats to human health and is considered the second leading cause of death for all human beings. The early stages of cancerous lesions are the most important steps for successful treatment. Choosing the right treatment for cancer patients faces many challenges, such as age, side effects, and patient compliance. Therefore, it is urgent to find targeted and personalized treatments. The possible uses of nanomaterials to improve human life and its environment are numerous [1]. One of the methods used to synthesize nanoparticles is environmentally friendly green chemistry, represented by plants, which has the advantage of overcoming the shortcomings of chemical and physical methods without the need for toxic chemicals, energy, pressure and temperature [2, 3]. Bioavailability is the amount and rate at which a drug is absorbed. The rate of absorption should be fast or slow depending on the treatment condition, which can be achieved by changing the physicochemical properties of the drug and the properties of the dosage form. Nano-formulations have many advantages, especially in botanical medicines, including improved bioavailability, reduced toxicity, enhanced pharmacological activity, etc. [4, 5]. Thus, nano-phytopharmaceuticals hold great future potential in enhancing activity and overcoming problems associated with herbal medicines. Chamomile, also known as Chamomilla recutita or Matricaria recutita, has a long history of use as an herbal remedy. The plant contains various biologically active compounds, including coumarins and sesquiterpenes. Flavonoids have also been identified in chamomile, with apigenin being the most abundant and contributing to the observed pharmacological properties of the plant's flowers, known as "Chamomillae flos" [6,7,8]. Developmental studies have been conducted to investigate the total apigenin content throughout the life cycle of both diploid and tetraploid forms of chamomile. The interest in chamomile research continues due to its reported anti-inflammatory, anti-viral, and particularly anticarcinogenic properties [9,10,11]. Apigenin has been extensively studied for its anti-inflammatory and anticarcinogenic properties. It has been shown to inhibit the growth of various cancer cell lines and to induce apoptosis (programmed cell death) in cancer cells, making it a promising compound for cancer prevention and treatment. Apigenin has also been reported to have neuroprotective effects, as well as to improve glucose metabolism and insulin sensitivity [8, 12]. Theranostics in nuclear medicine exploit the properties of certain isotopes, such as 131I, for imaging and therapy [13]. This approach helps identify specific molecular targets in patients, monitor treatment effects, and monitor potential toxicity. 131I is one of the most commonly used radioisotopes in nuclear medicine. 131I decays to extremely stable xenon-131 with a half-life of 8.03 days. Negative beta particles and gamma photons are released during the decay process. The average and maximum energies of primary beta emissions are 191.5 keV and 606.3 keV, respectively, while primary photons (gamma emissions) have an energy of 364.5 keV [14], and are therefore used for targeted radionuclide therapy (TRT) [15].

Experimental

Dry chamomile flowers were purchased from Haraz, Egypt. All other reagents were purchased from Sigma (St. Louis, MO) and were of analytical reagent grade or HPLC grade where applicable. Apigenin 7-O-glucoside (> 95% pure), were obtained from Sigma. HR TEM (Ted Pella, Redding, CA, USA), DLS (BIC, USA and PCS), Zetasizer (Beckman Coulter, Miami, FL, USA) were used for characterization of nanoplatforms. 131I was obtained from RPF, EAEA, Egypt. NaI (Tl) γ-counter (SPECTECH, ST450 SCA, USA) for radioactivity measuring.

Extraction of chamomile extract

Dry chamomile flowers were weighed and crushed to fine powder (mesh 50). The chamomile flower powder was extracted in heat reflux extractor under stirring for 18 h, (Pt CELL for pharmaceutical industries) by hydroalcoholic solvent (water: ethanol ratio 3:1). After that, the suspension was filtered. The filtered aqueous extract was concentrated and dried in vacuum oven (Pt CELL for pharmaceutical industries) at 50 °C, whereas organic extracts were dried at room temperature and stored at − 20 °C until use.

Determination the content of apigenin-7-glucoside in chamomile powder extract by HPLC

The Content of Apigenin-7-glucoside in chamomile flowers and chamomile powder extract was determined by Waters HPLC Acquity Arc with PDA Detector, Pt CELL for pharmaceutical industries.

  1. a-

    Chromatographic conditions:

    • Column:—Waters XSELECT C18, 5µ, (250 * 4.6) mm.

    • Flow Rate: 1.5 ml/min.

    • Detection Wave Length: 335 nm.

    • Injection volume: 20 µL.

    • Mobile phase:

      • (A) 5 mM KH2PO4 pH 2.5 by H3PO4.

      • (B) Acetonitrile: Methanol (65: 35) %

Time

(A)

(B)

0

75

25

2

75

25

15

15

85

18

75

25

25

75

25

  1. b-

    Standard preparation:

    • Accurately weigh 5 mg of Apigenin-7-glucoside standard.

    • Transfer it to 100 ml volumetric flask.

    • Add 80 ml methanol and sonicate for 10 min.

    • Cool to room temperature and add methanol to the volume.

    • Filter using syringe filter 0.22µ and discarding the first 5 ml of the filtrate.

  2. c-

    Sample preparation:

    • Accurately weigh 1 g of Chamomile powder extract.

    • Transfer into Suitable flask fitted with reflux condenser and stirrer.

    • Add 80 ml methanol then heat and reflux the mixture with stirring for 1 h.

    • Cool to room temperature and filter then collect the filtrate into 100 ml volumetric flask.

    • Complete to volume with methanol then take 30 ml into round flask fitted with reflux condenser and stirrer.

    • Add 5 ml of NaOH solution prepared by dissolving 0.4 g NaOH in 5 ml water.

    • Heat and reflux the mixture for 25 min then cool to room temperature. Adjust pH with HCl of 5 to 6.2.

    • Transfer the solution to 50 ml volumetric flask then complete to volume with methanol.

    • Filter using syringe filter 0.22 µ and discarding the first 5 ml of the filtrate.

  3. d-

    Sample testing.

    Accurately inject the standard solution for three times when the chromatographic system is stabilized under the condition. The RSD should be not more than 2.0%. Inject equal volumes of sample solution to the chromatograph, record the chromatograms and measure the peak responses of Apigenin-7-glucoside.

  4. e-

    Calculation.

    Calculate the percentage of Apigenin-7-glucoside taken by the formula:

    $$Assay = \frac{Average\;area\;of\;test}{{Average\;area\;of\;standard}} \times \frac{Conc.\;of\;standard}{{Conc.\;of\;test}} \times Potency\;of\;standard$$

Synthesis and characterization of Ch-AgNPs and Ap-AgNP

First; 0.7 ml of 1 mM silver nitrate was added dropwise to 3 ml of 2 mM sodium borohydride, then mixed for 10 min to form AgNPs. Second; 0.5 mL of chamomile extract (5 mg/mL) or 0.5 mL of apigenin 7-glucoside (1 mg/mL) was added to the AgNPs and mixed well, then left for a day to form Ch-AgNPs or Ap-AgNPs. Ch-AgNP and Ap-AgNP platforms were well characterized using HR-TEM to determine shape and core diameter, DLS to determine hydrodynamic diameter, and Zeta Sizer to determine the zeta potential of the formed nanoplatforms.

Antioxidant capacity determination, DPPH assay

The antioxidant capacity of chamomile extract, apigenin 7-glucoside, Ch-AgNP, and Ap-AgNP was tested using the DPPH assay. Free radical scavenging activity by 2,2-Diphenyl-1-trinitrophenylhydrazine hydrate (DPPH) assay [16] and comparison of chamomile extract, apigenin-7-glucoside, AgNPs, Ch-AgNPs, and Ap-AgNPs in terms of antioxidant potency: first add 100 μL of freshly prepared DPPH reagent (0.1% methanol), then add 100 μL of sample into the well plate, and incubate at room temperature for 30 min in the dark. Measure the density of the reaction color at 540 nm using a FluoStar Omega microplate reader, and the result is expressed by the following equation:

$$\% RSA = \frac{A0 - A1}{{A0}} \times 100$$

*RSA is the radical scavenging activity, A0 is the absorbance of the control and A1 is the absorbance of the test.

Cytotoxicity assay

Cell lines: HepG2 and MCF-7 cell lines were obtained from ATCC through its Biologics and Vaccines Holding Company (VACSERA) in Cairo, Egypt. Chemical reagents: reagents RPMI-1640 medium, MTT and DMSO (Sigma Co., St. Louis, USA), fetal bovine serum (GIBCO, UK). For comparison, doxorubicin was used as a standard cancer drug. MTT Assay: Using the various cell lines described above, the inhibitory effect of compounds on cell growth was determined by MTT assay. This colorimetric assay is based on the conversion of yellow tetrazolium bromide (MTT) to a purple formazan derivative by mitochondrial succinate dehydrogenase in living cells.

Radioiodination of Ch-AgNPs and Ap-AgNP

[131I]I-Ch-AgNPs and [131I]I-Ap-AgNPs platforms were prepared by optimized values of the substrate (Ch-AgNPs or Ap-AgNP), chloramine-T (CAT), pH and time. First; the substrate was placed in a dark, sealed reaction vessel, then CAT was dissolved in ethanol to a concentration of 8.8 mM and added, followed by Na13I. All factors were studied as the following amounts: CAT amount (25–125 µg) at different pH ranges (6–10) and substrate amount (0.25–1.25 mL) over 5–60 min. After the reaction was complete, the reaction was terminated with 5 μL of sodium thiosulfate (190 mM). The reaction was monitored by RTLC technique by using methanol 70% as a solvent system and radiochemical purity was determined in the same way, where free iodide migrated at Rf = 0.6 to 1.00, while the iodinated platform at Rf = 0.00 remained at the origin. Then purified from any free iodide before in-vivo studies.

$${\text{Radiolabeling}}\;{\text{yield = Total}}\;{\text{activity}}\;{\text{(Rf = 0 - 1) - activity}}\;{\text{of}}\;{\text{free}}\;{\text{131I}}\;{\text{(Rf = 0}}{\text{.6 - 1)}}$$

Biodistribution study of [131I]I- Ch-AgNPs and [131I]I- Ap-AgNPs

Animal biodistribution studies have been approved by the Ethics Committee of the Egyptian Atomic Energy Authority (EAEA). In this study, tumor-bearing Swiss albino mice weighing approximately 25–30 g were divided into five groups (5 mice per group). Each mouse was intravenously injected with [131I]I-Ch-AgNPs or [131I]I-Ap-AgNPs 10 μl at a concentration of 2 mCi, then the mice were anesthetized with isoflurane and weighed at different times (0.25, 0.5, 1.0, 2.0, 4.0 and 24 h)) after injection. Fresh blood, bone and muscle samples were collected in vials that were weighed first and counted, where the weight of bone, muscle and blood had to be 10, 40 and 7% of the total body weight, respectively [17,18,19], other organs and tissues were rinsed with saline and observed in the container, and counted with a NaI(TI) well-type γ counter to determine the radioactivity in each sample. All results were calculated using percent injected dose/gram organ (%ID/g organ).

Results and discussion

Extraction and purification of chamomile and Apigenin-7-glucoside

The retention time (Rt) for Apigenin-7-glucoside was quantitatively determined by injecting standard solutions of each into the UHPLC chromatographic column. The determined Rt for Apigenin-7-glucoside standards was 4.849 min (Figs. 1, 2, 3 and 4). The Chamomile extract was also subjected to HPLC separation (Fig. 3), which showed the presence of Apigenin-7-glucoside at the previously assigned Rt. Finally, the precipitated Apigenin-7-glucoside was identified similarly, the chromatogram showed 10.29% recovery of the total Apigenin-7-glucoside content in the original extract.

Fig. 1
figure 1

HPLC of Apginin-7-glucoside

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Fig. 2
figure 2

HPLC of chamomile herb

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Fig. 3
figure 3

HPLC of chamomile extract

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Fig. 4
figure 4

HPLC of chamomile herb after extract

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Synthesis and characterization of Ch-AgNPs and Ap-AgNP

Chamomile extract and apigenin 7-glucoside are well known antioxidants. AgNPs were formed from Ag ions using sodium borohydride as a reducing agent. Subsequently, chamomile extract and apigenin 7-glucoside were used to functionalize and stabilize the formed Ch-AgNP and Ap-AgNP, respectively. Figure 5, 6 show the average size of Ch-AgNPs and Ap-AgNPs determined using different techniques (TEM and DLS), respectively. TEM revealed spherical nanoparticles with mean diameters of 10 and 8 nm for Ch-AgNPs and Ap-AgNPs, respectively; while DLS showed mean hydrodynamic diameters of Ch-AgNPs and Ap-AgNPs were 357 and 308 nm, respectively, and their stability was determined by zeta potential which give values of 19.44 and 11.17 mV for Ch-AgNPs and Ap-AgNPs, respectively, indicating the stability of the colloidal nanoplatform. This stability may be due to the electrostatic repulsion created by the presence of charged surfaces.

Fig. 5
figure 5

Ch-AgNPs core diameter, hydrodynamic diameter and zeta potential

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Fig. 6
figure 6

Ap-AgNPs core diameter, hydrodynamic diameter and zeta potential

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Antioxidant capacity

One of the main causes of DNA damage is free radicals that represent the first stage of carcinoma, natural products consist of many of antioxidant source act as exogenous which stimulate function of the endogenous antioxidant system to prevent free radicals formation inside the body as its main responsibility [20, 21]. Chamomile was reported to induce apoptosis in cancer cells [22]. Ch-AgNPs showed the highest scavenging activity with IC50 of 5.88 ± 0.77 μL/mL followed by Ap-AgNPs with IC50 of 11.34 ± 0.61 μL/mL, while the value of IC50 for both apigenin 7-glucoside and chamomile extract > 1000.

Cytotoxicity of the newly synthesized nano-platform against HepG2 and MCF-7

Liver cancer and breast cancer are the two most common types of cancer in humans. Therefore, we chose HepG2 and MCF-7 human cancer cell line models for testing. The cytotoxic activity of the synthesized NPs was tested against HepG2 and MCF-7 and compared with DOX (doxorubicin) as a known reference. The results showed that both nanoplatforms (Ch-AgNPs and Ap-AgNPs) had high cytotoxic effects on both cell lines tested. The IC50 values of Ch-AgNPs against HepG2 and MCF-7 were 6.2 and 6.9 μg/mL, while the IC50 values of Ap-AgNPs against HepG2 and MCF-7 were 5.2 and 6.1 μg/mL, respectively.

Formation of [131I]I-Ch-AgNPs and [131I]I-Ap-AgNPs

The maximum RCP of 95.04 ± 2.85 and 97.36 ± 2.92% were obtained for both Ch-AgNPs and Ap-AgNPs, respectively. Briefly ([131I]I-Ch-AgNPs), 1 mL total reaction volume by the addition of 5 µL Na131I containing 2 mCi to a mixture of 750 µL of nano-platform and 75 µg CAT (2 CAT: 1 EtOH) at pH 8 at room temperature for 0.5 h; while in case of [131I]I-Ap-AgNPs the same conditions were used with only 50 µg CAT. RCP was determined by means of TLC for each experiment. NPs were radiolabeled with 131I by direct electrophilic substitution with CAT as the oxidant. The above-mentioned factors studied in this experiment are all affected by the reaction pathway. The highest RCP obtained depends mainly on the amount of CAT, which is responsible for the formation of iodonium ions from iodide ions, thereby enabling the electrophilic reaction. When the amount of CAT was reduced or increased above the optimal value, RCP was greatly reduced (Figs. 7a and 8a). This reduction may be due to the formation of unwanted by-products at high CAT levels and insufficient oxidation of radioiodine at low CAT levels [23, 24]. RCP showed a maximum at pH 8, and RCP decreased at pH values below or above 7 (Fig. 7b and 8b), probably due to the formation of (IO-) and (IO3-) ions, which do not participate in ( Formation of IO-) and (IO3-) Ion in the radioiodination process [25, 26]. At 750 µL and more of NPs values of RCP give us the highest yield, but before this value, RCP decreases (Figs. 7c and 8c) due to insufficient substrate which is required to capture all (I +) from the solution [27, 28]. Furthermore, timing is one of the key factors for optimal RCP (Figs. 7d and 8d). These data indicates that the time before 30 min is insufficient to complete the reaction. Finally, the radioiodinated nanoparticles formed were stable for more than 48 h.

Fig. 7
figure 7

Effect of reaction parameters on RCP of [131I]I-Ch-AgNPs: a- CAT amount. b- pH, c- Substrate amount, d- Reaction time

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Fig. 8
figure 8

Effect of reaction parameters on RCP of [131I]I-Ap-AgNPs: a- CAT amount. b- pH, c- Substrate amount, d- Reaction time

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Biodistribution study of [131I]I- Ch-AgNPs and [131I]I- Ap-AgNPs

Biodistribution studies of [131I]I-Ch-AgNPs and [131I]I-Ap-AgNPs are shown in Fig. 9a and b, respectively, at different times (0.25, 0.5, 1, 2, 4 and 24 h after injection, The elimination of both nanoplatforms was mainly through the hepatobiliary route, as the uptake in the liver was relatively high over time [29]. As shown in Fig. 9, the nanoplatforms rapidly spread to various sites due to rapid blood clearance. Regarding tumor uptake, 1 h Compared with normal muscle, [131I]I-Ch-AgNPs and [131I]I-Ap-AgNPs were collected at the same time in a high proportion. The tumor muscles to normal muscles ratio were shown in Fig. 10 which indicates that Ap-AgNPs accumulate in a ratio of 8.24 after two hours while the ratio in the case of Ch-AgNPs was 4.19 at the same time. These results indicates that radioiodinated NPs was able to efficiently target cancer tissue, and also indicates that both [131I]I-Ch-AgNPs and [131I]I-Ap-AgNPs may be used as the best opportunity for tumor theranostics.

Fig. 9
figure 9

Biodistribution of a- [131I]I- Ch-AgNPs, b- [131I]I- Ap-AgNPs in tumor bearing mice

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Fig. 10
figure 10

Tumor muscle/Normal muscles for [131I]I- Ch-AgNPs, and [131I]I- Ap-AgNPs

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Conclusions

Since anticancer drugs need to be developed further and more efficiently, this study aims to develop novel dual anticancer drugs that can be used in chemotherapy and radiotherapy. Ch-AgNPs and Ap-AgNPs were synthesized using a green method for in vitro chemotherapy and TRT testing. The two nanoplatforms showed high potency against the two cancer cells tested, HePG2 and HCF7, and also showed high accumulation at the cancer site, leading us to conclude that the two new formulations could be used as anticancer chemotherapeutics. These nanoplatforms can also be used for TRT by radioiodination of these platforms with 131I (used as a therapeutic radioisotope). In conclusion, after further preclinical studies, the synthesized nanoplatforms can be used for chemo/radioisotope therapy.