Targeted Therapies for Breast and Lung Cancers by Using Propolis Loaded Albumin Protein Nanoparticles

Targeted Therapies for Breast and Lung Cancers by Using Propolis Loaded Albumin Protein Nanoparticles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeted Therapies for Breast and Lung Cancers by Using Propolis Loaded Albumin Protein Nanoparticles Mohamed G.M. Ghazy, Nemany A.N. Hanafy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2505179/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cancer is a popular disease among many others that can threaten defiantly human life. This is not only because of its invasiveness but also because of its resistance and the highly effective cost of its treatment. Propolis is rich in natural sources of bioactive and polyphenolic compounds that have proven their strong effect on cancer cells such as MCF-7 and A549 cell lines. Propolis extract was inserted into the Albumin protein, Bovine Serum (BSA) conjugated to folic acid (FA) to increase control of its delivery and to increase their cellular uptake. The growth of MCF-7 and A549 was significantly decreased by propolis extract and BSA-propolis NPs after their incubation for 72h by (54 ± 0.01% and 45 ± 0.005%, P ≤ 0.001) and (20 ± 0.01% and 10 ± 0.005%, P ≤ 0.0001) respectively. Similarly, there is a significant inhibition in the growth of A549 obtained after their incubation (propolis extract and albumin-propolis NPs) for 72 h (15 ± 0.03% and 5 ± 0.01%, P ≤ 0.00001). Propolis extract and BSA-propolis NPs exhibited a greater effect on protein expression of MCF-7 and A549 showing significant modulation of caspase-3, cyclin D1, and LC3II. The result was supported by the presence of nuclear fragmentations and activation of acidic/neutral autophagosomes in AO/EB and DAPI stains. In the recent investigation, the expression of phospho-GSK3β (Ser9) (p < 0.001) increased significantly in MCF-7 and A549 cells after their exposure to propolis extract and BSA-propolis NPs. Results support the potency application of propolis and its encapsulation as an alternative therapeutic agent for cancer treatments instead of chemotherapies because of its action on multi-signaling pathways. Biological sciences/Cancer Biological sciences/Cell biology Physical sciences/Nanoscience and technology Propolis Polyphenolic compounds Autophagosomes Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Cancer is still a serious threat to human life among many other health issues. Since 14.1 million new cases and 8.2 million cancer-related deaths had been reported [ 1 ]. The impact could reach 23.6 million by 2030 [ 2 ]. Many hard efforts had been employed to overcome cancer invasiveness by using radiotherapy, surgery, cytotoxic chemotherapy, targeted therapy, and immunotherapy [ 3 ]. However, the resistance of malignant cells to anticancer therapies caused a major issue in the performance of effective treatment of cancer patients [ 4 ]. The resistance leads to a lack of response to drug-induced tumor growth inhibition [ 5 ]. This often causes failure in treatment. At moment, many efforts were made to treat cancer using various natural polyphenolic compounds due to overcoming the undesirable side effects of chemotherapeutic agents, and their drug resistance [ 6 ]. Propolis is a diversified lipophilic material including resin collected from leaf, branch, and bark of certain tree species by the enzymatic metabolism in honey bees, which is then transported to the hive and mixed with wax glands by working bees, resulting in forming adhesive materials known as propolis [ 7 ]. Propolis consists of wax (30%), resin (50%), pollen (5%), essential oils (10%), and other substances (5%) [ 8 ]. The significant effect of propolis on the inhibition of cancer cell growth and increased cancer cell cycle arrest has been reported in many cancer cell lines such as MCF7 (breast cancer), HGC27 (human gastric carcinoma), A549 (non-small lung cancer), and also cell lines of stomach, cervical, esophageal, brain, laryngeal and skin [ 9 – 11 ]. Such these properties are due to its ability to induce inhibition of NF-κB and JNK signaling pathways; proline dehydrogenase/proline hydrogenase- and DNA fragmentation-initiated apoptosis; upregulation of p53, Bax, cleaved-caspase-3 and 9, and MAPK-associated proteins; regulation of glutathione and glutathione S-transferase; and modulation of the mitotic cell division [ 12 ]. Despite many publications in carcinogenesis, the possible therapeutic application of encapsulated propolis extract derived from the native species of Egyptian source is largely unknown and sparingly investigated. Encapsulation is a convenient method for minimizing the strong odor and taste of propolis (bitterness and pungent), which are odious for many users [ 13 – 15 ]. This technique inhibits the drug from electrostatic interactions with chemoreceptors by delaying its release into the oral cavity [ 16 ], which has the potential to hide bitter qualities and improve palatability might help with the incorporation of these chemicals into food items [ 17 – 18 ]. Albumin Protein Bovine Serum (BSA) is used widely as potential targeted delivery for cancer treatment because of its ability to interact with albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC) and glycoprotein 60 (gp60) providing a mechanism to increase its cellular mucoadhesion and penetration. Additionally, to raise its potential efficacy, folic acid was integrated into its moieties as a main targeted molecule used previously for folate receptors [ 19 ]. 2. Material And Methods 2.1. Chemicals Phosphate Buffer saline (PBS) tablets pH 7.4 was obtained from Oxoid Limited Basingstoke, Hampshire, England; Ethanol from Baker Analyzed, Fisher Scientific, Landsmeer The Netherlands. Bovine serum albumin (BSA); Folic acid (FA); Paraformaldehyde, Dimethyl sulfoxide (DMSO) from Sigma-Aldrich, St. Louis, MO, USA. 2.2. Isolation of propolis extract derived from an Egyptian source Propolis was collected manually from a beehive located in kafrelsheikh, Egypt. Propolis was then cut into the small part and soaked in 500 mL 70% ethanol at 37°C for 4 days, in a hermetically closed glass vessel under occasional shaking. The ethanolic extract of Egyptian propolis was then filtered through a Whatman filter paper no. 4 and the amount of ethanol was evaporated and then 100mL distilled water was added. Propolis extract was kept at -20°C for lyophilization [ 20 ]. 2.3. Identification and quantification of polyphenolic compounds High-Performance Liquid Chromatography (HPLC) analysis was obtained by using an Agilent 1260 series. The separation was performed by using the Eclipse C18 column (4.6 mm x 250 mm i.d., 5 µm). The fluid phase contained water (A) and trifluoroacetic acid (0.05% ) in acetonitrile (B) at a measurement rate of 1 ml/min. The fluid phase was assessed one by one in a linear gradient as follows: 0 min (82% A); 0–5 min (80% A); 5–8 min (60% A); 8–12 min (60% A); 12–15 min (82% A); 15–16 min (82% A) and 16–20 (82%A). The multi-wavelength detector was detected at 280 nm. The injection volume was 5 µL for each of the sample solutions. The column temperature was maintained at 40°C[ 21 ]. 2.4. Preparation of propolis albumin nanoparticles Albumin NPs loaded by propolis were obtained by the desolvation method. Briefly, 10 mL of ethanolic extract propolis was added to 50 mg BSA conjugated folic acid (dissolved in 100mL, 10 mM NaCl, pH 9) and allowed to interact using magnetic stirring for 30 minutes. In this condition, ethanol in the extract was also used as a desolvation agent which gradually increases the crosslink of protein moieties in the solution leading to agglomeration. Since the covalent bonds contributed to forming crosslinking network structures between albumin moieties and polyphenolic compounds of propolis extract. The unreacted materials were removed by dialysis bag against distilled water. Albumin-propolis NPs were kept at -20°C and then were lyophilized [ 22 ]. 2.5. Characterization 2.5.1. Transmission Electron Microscopy (SEM) Samples for TEM analysis were prepared by drop-casting a few microliters of solutions onto standard TEM carbon-coated Cu grids, allowing the solvent to evaporate completely. Prepared samples were imaged utilizing a 2100 TEM-JEOL JEM microscope operating at 200 kV [ 23 ]. 2.5.2. Zeta Potential and DLS measurements The electrophoretic mobility of samples was determined by photon correlation spectroscopy (Brookhaven) by using a Zeta Nano Sizer. All measurements were performed at 25°C. Five following measurements were taken for analysis. 2.5.3. UV-Vis spectrophotometer The absorbance of samples was measured by using (Jasco V-770) UV Visible Absorbance Spectrophotometer. 500µl of the fabricated assembly was diluted into 4 ml by using distilled water and then scanned at the range of 200–800 nm. The results were analyzed by Origin 8. 2.5.4. Fourier Transform Infrared Spectroscopy (FTIR) FTIR experiments were carried out by using JASCO Fourier Transform Infrared Spectrometer (JASCO, JAPAN, model no. AUP1200343) used to detect the surface molecular structures in the scale bar of 400–4000 cm − 1 by using the KBr pellet method. For all of the tests, at least three scans were recorded on different regions of the samples and representative spectra were analyzed [ 24 ]. 2.5.5. Estimation of polyphenolic compounds The phenolic compounds as a total (TPC) of samples were measured using the method described by Jansen-Alves et al. (2018). At first, 0.2 M Folin-Ciocalteu reagent was mixed with the samples in a ratio of 5:1. Then, 2 ml of freshly sodium carbonate saturated solution was added to each sample. After that, all samples were vortexed for around 15 s, followed by incubation for 2 h in a dark place at room temperature. Finally, the absorbance of each sample was measured using a spectrophotometer (Beckman) at 725 nm. Before the experiment, a calibration curve using gallic acid at the concertation of 10–600 µg/mL had plotted. The experiment was run out in triplicate and all the results are reported as mg gallic acid equivalent per gram of dried samples (mg GAE/g dried sample) [ 25 ]. 2.5.6. Percentage yield and entrapment efficiency The percentage of yield was calculated by dividing the weight of obtained NPs by the total weight of the amount of propolis and BSA. In this case, the yield (%) was calculated from the following equation The difference between total polyphenolic compounds found inside BSA NPs (TPC) and total polyphenolic compounds found on the surface (TPCS) was used to calculate % of entrapment efficiency. Briefly, 25 mg of propolis-loaded BSA NPs were suspended with 5 ml of 96% ethanol and centrifuged at 15000 rpm for 15 min at 25°C to wash unreacted materials. After phase separation, 500µL of the supernatant was used to calculate the total polyphenolic compounds found on the surface (TPCS). The other 4.5 mL was homogenized for 10 min at 14,000 rpm and then kept in a water bath for 24 h at 25°C to decompose NPs and to calculate the total polyphenolic compounds found inside BSA NPs (TPC). The % of entrapment efficiency was measured by using the following equation [ 26 ]. 2.6. In vitro studies 2.6.1. Cytotoxicity evaluation To assess the effect of BSA NPs, propolis extract, and propolis-loaded BSA NPs on tumor cell growth. The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium-bromide assay was used to evaluate cell bio-viability. Briefly, A549 and MCF-7 cells were exposed to serial concentrations (1, 10, 50, and 100 µg/mL) for 48h and 72h. Then, 5 mg/mL MTT reagent was added to each well and the reaction was allowed to proceed for 4 h at 37°C. The culture medium was then removed and precipitated formazan crystals were dissolved by adding DMSO (200 µL). The absorbance of each well was read at 570 nm and directly correlated to several remaining viable cells. Absorbance data were normalized to the percentage of control, then graphed [ 27 ]. 2.6.2. Evaluation of protein expression with flow cytometry For protein expression analysis, A549 and MCF-7 cells were seeded separately as 5×105 cells per well. After 24h, cells were then treated separately by using 20µg/mL propolis extract and BSA-propolis NPs. After treatment, detached cells were washed once and suspended in Permeafix (Ortho Diagnostic Systems, Roissy, France) and incubated for 40 min at 20°C. The material was then centrifuged, and the pellet was purified twice with washing buffer (0.2 mM EDTA, 5% FCS in phosphate-buffered saline [PBS]). Labeling was performed by adding to the cells 100 ml of washing buffer containing 5 ml of polyclonal phycoerythrin (PE)–attached anti–active caspase 3 antibodies (PharMingen–Becton Dickinson, Le Pont de Claix, France) [ 28 ] cyclin D1-Alexa Fluor 647 antibodies and Anti-LC3A/B antibody attached to Alexa Fluor 488, Cell Signaling, cat. no. 13082] [ 29 ]. Cells were then analyzed by flow cytometry. 2.6.3. Western blotting analysis Whole-cell lysates were prepared as previously described [ 28 ]. Briefly, cells were lysed with cell RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF) after two washes with ice-cold PBS. Bradford protein assay kit was used to evaluate the protein concentration. 20–30 µg of protein was electrophoresed by 10–12% sodium dodecyl sulfate-PAGE and transferred onto polyvinylidene fluoride membranes. After blocking with 5% non-fat dry milk at room temperature for 1 h, the sheet was incubated at 4°C overnight with the primary antibodies. Then sheets were purified three times with PBST to remove any non-bound primary antibodies and incubated with horseradish-peroxidase-conjugated secondary antibodies at concentrations of 1:2000–10,000 dilutions for 1 h at room temperature. Membranes were cleaned three times with PBST and incubated with ECL substrate. The immunoreactive signals were detected under an Amersham Image600 (USA) [ 30 ]. 2.7. Cellular bio-image 2.7.1. Fluorescent days (AO/EB) Staining Assay Acridine orange (AO), is a cationic day, that can penetrate the cell membrane of viable and dead cells emitting green fluorescence after its interaction with double DNA strands. Ethidium bromide is a nucleic acid stain that cannot penetrate a live cell. The dual staining can be used to distinguish between live cells, early-stage apoptosis, late-stage apoptosis, necrosis, and autophagosomes. Since AO can diffuse through the cell membrane making the nuclei and autophagosomes appear green. Ethidium bromide (EB) can be adsorbed only when loss of cellular integration causes the nucleus to be orange. Thus, live cells present a homogeneously green nucleus; early apoptotic cells have a bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; cells that have died from direct necrosis show a structurally normal orange nucleus (19). In the current study, MCF-7 and A549 were treated with propolis extract and BSA-propolis NPs for 24, 48, and 72h, and then, they were washed with phosphate buffer saline pH 7.2 and were fixed by 4% paraformaldehyde for 30 min. Cells were washed with PBS and then Image was captured by fluorescence microscopy (Nikon 500, 515 nm) with 400x amplification [ 31 ]. 2.7.2. DAPI Staining Assay DAPI (4’,6- diamidine- 2’- phenindole dihydrochloride) is a blue emission color that can be detected under fluorescence microscopy. it can bind specifically to DNA double strands. For analysis of chromosomal DNA changes, MCF-7 and A549 tumor cells were treated with propolis extract and BSA-propolis NPs at the concentration of 0.7 µM for 48 and 72h. Then, cells were washed with phosphate buffer saline (PBS) and fixed in methanol (70%) for 20 minutes. Cells were incubated for 30 minutes with 0.4 µg/mL of DAPI (Sigma Chemical Co., USA). DNA alterations were observed by fluorescence microscopy (Nikon, USA, 385–410 nm) [ 32 ]. 2.8. Biostatistics analysis The results were expressed as mean ± standard division of mean (SEM). Data were analyzed by SPSS version 20 using one-way analysis of variance (ANOVA), followed by Duncan's test for comparison between different treatment groups. The data are shown as *P < 0.05 **P < 0.01 and ***P < 0.001. Three independent experiments were used to present DATA. 3. Results 3.1. Flavonoids composition of ethanolic propolis extraction The individual Flavonoids in propolis extract and encapsulated propolis were identified and quantified by using HPLC to provide a better understanding of the concentration of each flavonoid separately. In the recent study, the constituents of flavonoids in free propolis and encapsulated one were summarized in Table 1 and Fig. 1 . Since quercetin showed good concentration in encapsulation (7.01 ± 0.1 µg/mL). While chlorogenic acid observed abundant value (7.01 ± 0.1 µg/mL) in free propolis. The other contents of encapsulated flavonoids were arranged as follows; Querectin > Gallic acid > Chlorogenic acid > Kaempferol > Ellagic acid > Syringic acid > Coffeic acid > Rutin > Coumaric acid > Cinnamic acid > Naringenin > Methyl gallate > Ferulic acid. The concentration of encapsulated each flavonoid depends mainly on the ability of this molecule to react with moieties of capsules. Besides that, solubility, conjugation with other phenolic compounds, degree of polymerization, and chemical structure of each flavonoid are additional factors that can affect encapsulation [ 21 ]. Meanwhile, all polyphenolic compounds were estimated as (400 mg) according to the standard curve obtained by using gallic acid. Table 1 Quantification and identification of flavonoids in ethanolic propolis extract and their encapsulation. Polyphenol components Ethanolic propolis extract Encapsulated Propolis Area Conc. (µg/mL) Area Conc. (µg/mL) Gallic acid 215.77 1.5 33.68 6.90 Chlorogenic acid 5201.61 830 27.22 4.30 Methyl gallate 10.2 1.2 10.58 0.27 Caffeic acid 502.51 39 18.86 1.47 Syringic acid 150.10 14 27.06 2.68 Rutin acid 6.40 1.57 5.10 1.25 Ellagic acid 34.3 3.6 9.65 2.79 Coumaric acid 120.37 2.6 21.93 0.73 Ferulic acid 27.85 1.7 1.90 0.15 Naringenin 225.7693 21 2.69 0.26 Quercetin 12.8 15 60.19 7.01 Cinnamic acid 81.58 1.51 12.24 0.23 Kaempferol 4.86 0.46 25.72 3.07 3.1. Formulation and Characterization of albumin-propolis NPs The morphology of the fabricated albumin-propolis NPS was shown by using SEM, as illustrated in Fig. 2 . The nanoparticles demonstrated spherical shape and agglomerations with an average size of about ~ 200 nm, with a smooth structure. Since, albumin is cross-linked by propolis polyphenolic content in presence of ethanol, showed assembled 3D structure. The result indicates that polyphenolic compounds can be used as natural crosslinkers due to their possible interaction between amino-carboxyl groups of albumin. The result also highlights the advantage of using natural cross-linkers that make them possible to overcome toxic crosslinkers or initiators. The mean zeta potential values of the albumin-propolis NPs (Fig. 3 ), as estimated by the DLS technique, were found to be 31 mV. The zeta potential of 30 mV was within the recommended range (− 30 mV to + 30 mV), indicating that encapsulated propolis would not quickly settle and would be suspended and distributed in the physiological media [ 33 ]. The encapsulation efficiency was calculated to identify the loading capacity of albumin NPS as an effective carrier for propolis encapsulation. The percentage of encapsulation efficiency and yield depends mainly on the amount of encapsulated propolis. In the recent work, the percentage of loading capacity and yield was estimated as 85% and 80% respectively. Folic acid conjugated BSA has exhibited abroad bands at 3432 cm − 1 that were assigned to stretching vibration of O-H and N-H. The amide band I appeared at ~ 1663 cm − 1 . Many small bands located at 1394 cm − 1 were assigned to the presence of aromatic ring stretches of the pyridine and p-amino benzoic acid moieties (Fig. 4 ; A) [ 34 ]. A spectrum of propolis extract showed a significant band at 3391 cm − 1 that was attributed to the stretching vibration of OH in phenolic compounds. While symmetric and asymmetric C–H stretching was located at 2937 cm − 1 . Meanwhile, the carbonyl group (C = O) exhibited stretching vibration at 1689 cm − 1 . while, C = C and C-O were obtained starching vibration at 1623 cm − 1 and 1273 cm − 1 respectively [ 35 ].. A spectrum of albumin-propolis NPs showed an intrinsic abroad peak at 3392 that was attributed to the stretching vibration of O-H and N-H2. Likewise, a significant characteristic peak of symmetric and asymmetric C–H was shown at 2937 cm − 1 . While the carboxylic group was detected at 1743 cm − 1 and the carbonyl group was presented at 1689 cm − 1 . A spectrum of propolis extract exhibits an extensive peak with two crests at 295 nm and 323 nm. While this peak was shifted towards 277nm in the spectrum of albumin-propolis NPs. Phenolic compounds generally exhibit an absorption peak in the ultraviolet light range of 250 and 350 nm for spectrophotometric analysis (Fig. 4 ; B). . 3.2. In vitro studies 3.2.1. Cytotoxicity MTT assay is a colorimetric assay, used to evaluate the activity of mitochondrial metabolism as an indicator of a healthy state or cytotoxicity. In this assay, the viable cells can form formazan purple crystals by reduction of yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) in the presence mitochondrial succinate dehydrogenase. The formazan is further dissolved by DMSO and the intensity of the color is proportional to the percentage of live cells at absorbance 570 nm [ 36 ]. Here, the MTT assay was applied to explore the potential cytotoxicity of albumin NPs, free propolis, and albumin-propolis NPs at serial concentrations (1, 10, 50, 100 µg/mL) in lung and breast cancer cells lines using A549 and MCF-7 cells. Based on cellular cytotoxicity, MCF-7 cells were significantly inhibited after 48h and 72h incubation with 100µg/mL (propolis extract and albumin-propolis NPS) by(54 ± 0.01% and 45 ± 0.005%, P ≤ 0.001) and (20 ± 0.01% and 10 ± 0.005%, P ≤ 0.0001) respectively (Fig. 5 ) [ 37 ]. Similarly, there is a significant decrease in the growth of A549 was obtained after their incubation (propolis extract and albumin-propolis NPs) for 72 h (15 ± 0.03% and 5 ± 0.01%, P ≤ 0.00001) [ 38 ]. While empty albumin NPs were obtained no significant reduction in the growth of A549 and MCF-7 cells indicating their potential use as a safety vector for the drug delivery system. 3.2.2. Evaluation of Protein Expression with Flow Cytometry 3.2.2.1. Caspase − 3 Caspase-3 is an executioner caspase, found mainly as inactive pro-caspase in viable cells, which is further activated by initiator caspases such as (caspase 8 and caspase 9) during apoptosis. Active caspase-3 can cleave and fragment DNA and has the ability also to denature proteins. Thereby leading to cell death [ 38 ]. In Fig. 6 & 7 , the caspase-3 level was significantly overexpressed in A549 and MCF-7 cells treated for 48h by propolis extract and BSA-propolis NPs. Compare to the control (untreated cells), the regulation of caspase-3 with propolis extract and BSA-propolis NPs treated A549 and MCF-7 cells were significantly increased by (64.5 ± 1.2% and 80.7 ± 1.09%, p ≤ 0.00001) respectively and (54.5 ± 2.5% p ≤ 0.0001 and 82.8 ± 2.3%, p ≤ 0.00001) respectively [ 38 – 41 ]. 3.2.2.2. Cyclin Cyclin D1 is a G1 cyclin that organizes the shifting from G1 to the S phase, thus its level increased during the G1 phase of the cell mitotic division. Whereas cyclin A is regarded as a regulator of the transition to mitosis since it reaches its maximum level during the S and G2 phases [ 42 ]. Cyclins regulate the cell cycle by binding to and activating cyclin-dependent kinases (Cdks). For this reason, cyclin–Cdk complexes can phosphorylate specific targets leading to activating the cell cycle at the right moment [ 43 ]. In the recent investigation, propolis extract and albumin propolis NPs can down-regulate Cyclin D1. Since cyclin D was significantly inhibited in MCF-7 and A549 cells after their exposure to propolis extract and albumin propolis NPS by (39.3 ± 2.9% p ≤ 0.0001 and 26.5 ± 5.5% p ≤ 0.0001) and (36.2 ± 3.3% p ≤ 0.00001 and 23 ± 2.6% p ≤ 0.00001) compared to MCF-7 cells that were untreated (77.8 ± 4% ) and untreated A549 cells (87.7 ± 2.5%) (Fig. 6 & 7 ) [ 44 ]. 3.2.2.3. LC3 Light chain 3 (LC3), Microtubule-associated protein 1A/1B distributed in cultured cells and mammalian tissues ubiquitously because it is highly soluble. Once autophagosome engulfs components of cytoplasm including organelles and cytosolic proteins. LC3 (cytosolic form) is conjugated to phosphatidylethanolamine forming LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. For this reason, LC3-II is a standard marker for accounting for the number of autophagosomes [ 45 – 46 ]. In the recent work, there was a significant regulation in the percentage of LC3 level in both MCF-7 and A549 treated by propolis extract and BSA-propolis NPs. Since, MCF-7 cells exhibited higher evaluation of LC3-II by (77 ± 1.2% p ≤ 0.00001 and 92.2 ± 0.3% p ≤ 0.0001) respectively. Similarly, A549 cells showed (77.8 ± 0.7% p ≤ 0.00001 and 89.8 ± 0.8% p ≤ 0.00001) respectively. LC3-II is a specific marker associated with the formulation of autophagosomes. The result indicates an increased number of autophagosomes in MCF-7 and A549 cells after their treatment with propolis extract and BSA-propolis NPs (Fig. 6 & 7 ) [ 47 – 48 ]. 3.2.3. Cellular bio-images 3.2.3.1. Analysis of Apoptosis by AO/EB Staining to explore the phenotype expression of MCF-7 and A549 cells during treatment, the dual fluorescent staining composed of the dual fluorescent days (AO/EB) was used to distinguish apoptotic cells, necrotic cells, and autophagosomes. Since, early-stage apoptotic cells, shown by crescent-like shape and nuclei were seen as yellowish-green color (Fig. 8 & 9 ,). While late-stage apoptotic cells are marked by orange nuclear EB staining. Meanwhile, cell volume was increased in necrotic cells showing orange-red emission at their periphery. Additionally, protonation of acridine orange occurs at acidic vesicles (autolysosomes) causing bright red emission for AO, while the nucleus becomes bright green. At neutral pH, AO becomes de-protonation and green color is emitted. By this mechanism, AO can track acidic vesicular organelles and distinguish autophagy [ 49 – 51 ]. 3.2.3.2. DNA staining assay To elucidate the changes in nuclear morphology, DAPI stain was used as a specific dye for nucleic acid. Nuclear fragmentation was observed after 72h incubation with propolis extract and BSA-propolis NPs Fig. 8 & 9 [ 52 ]. 3.2.4. Western blot analysis Glycogen synthase kinase 3 (GSK3B) is a proline-directed serine⁄threonine protein kinase involved in modulating numerous processes, including metabolism, proliferation, apoptosis, autophagy, development, and differentiation. The regulation of GSK3 depends on the phosphorylation at the specific sites of GSK3; phospho-GSK3α (Ser21) and phospho-GSK-3β (Ser9) both of them can inhibit the GSK3β activity [ 26 ]; whereas phospho-GSK3β (Tyr216) increases the activity of GSK3β [ 24 , 26 ]. In our DATA, the upregulation of phospho-GSK3β (Ser9) (p < 0.001) was increased significantly in MCF-7 and A549 cells after their exposure to propolis extract and BSA-propolis NPs. Such phosphorylation led to inhibiting the activity of GSK-3β (Fig. 10 ) [ 53 ]. 4. Discussion Propolis is rich in phenolic compounds, which may act as natural antioxidants [ 54 ]. In fact, because of the present different groups of phenolic acids, the ethanolic extracts of propolis affect the inhibition of free radicals formation, reducing or complexing transition metal ions, capturing the superoxide amino radicals and peroxy radicals, and breaking the free radical reactions preventing lipid peroxidation. All of these properties, make propolis has strong antibacterial, antifungal, antiviral and anti-inflammatory properties [ 55 ]. Recently the potent activity of propolis as an antioxidant has been considered significantly suppress tumor growth by directly inhibiting tumor cell growth and inducing apoptosis or indirectly by scavenging ROS and increasing antioxidant enzyme activities [ 56 ]. In the recent work, ethanolic propolis extract was inserted into BSA-FA moieties with strong binding affinity because of its abundant chemical interactions. FTIR illustrated the strong interaction between propolis extract and the amino acid structure of BSA. Likewise, zeta potential revealed net charge of albumin propolis NPs was estimated as 31mV ± 0.8. Meanwhile, albumin-propolis NPs showed strong mono dispersion with 3D assemble structure having a diameter of ̴330nm. The potential cytotoxicity of propolis was estimated after 48h and 72h by using MCF-7 and A459. Therefore, there is a significant inhibition in the growth of A549 and MCf-7 after 72h incubation. Since, 100µg/mL of albumin propolis NPs can inhibit significant growth of MCF-7 and A549 by (10 ± 0.005%, P ≤ 0.0001 and 5 ± 0.01%, P ≤ 0.00001). To induce a better understanding of the mechanism of propolis, protein expression was evaluated by using flow cytometry for Caspase-3, Cyclin D1, and LC3 in MCF-7 and A549. The expression of caspase-3 with propolis extract and BSA-propolis NPs treated MCF-7 and A549 cells were significantly increased by (64.5 ± 1.2% and 80.7 ± 1.09%, p ≤ 0.00001) respectively and (54.5 ± 2.5% p ≤ 0.0001 and 82.8 ± 2.3%, p ≤ 0.00001) respectively. The result confirms the strong ability of propolis to activation of caspase-3 and induction cell death in both MCF-7 and A549. Another confirmation is that Cyclin D1 was also significantly inhibited when cells were exposed to propolis extract and albumin propolis NPs. Cyclin D1 is a nuclear regulator, responsible for the transition cells from the G1 phase to the S phase. Its inhibition could block cell division and induce cell cycle arrest. Increasingly, LC3-II, as a standard marker for autophagosomes showed a higher expression in both MCF-7 and A549 after their exposure to propolis extract and albumin propolis NPs. Since, MCF-7 cells exhibited higher evaluation of LC3-II by (77 ± 1.2% p ≤ 0.00001 and 92.2 ± 0.3% p ≤ 0.0001) respectively. Similarly, A549 cells showed (77.8 ± 0.7% p ≤ 0.00001 and 89.8 ± 0.8% p ≤ 0.00001) respectively. Such a result indicates an increased number of autophagosomes meaning that propolis can activate protein expression of apoptotic and autophagy pathways. To support these data, AO/EB and DAPI stains were used to investigate apoptotic stages, DNA fragmentation, and autophagosomes. In the recent study, live cells showed uniformly green color compared to early apoptotic cells that observed condensed or in some cases, fragmented nuclei with bright green color. While late apoptotic cells exhibited condensed and fragmented orange chromatin. In autophagy, neutral autophagosomes, characterized by green vesicle color, were located close to the cell center. While acidic autophagosomes characterized by red vesicle color were observed close to the cell periphery [ 57 – 58 ]. The result was supported by DAPI stain as a very specific nuclear stain can distinguish nuclear fragmentations. Therefore, MCF-7 and A549 cells that were exposed to propolis extract and albumin propolis NPs for 72h showed higher nuclear fragmentation and rapture of the cell membrane. To induce a better understanding of how propolis can modulate LC3II and Cyclin D1. The GSK3 beta was also estimated in the recent work by using western blot in both MCF-7 and A549. Therefore, GSK3 beta was significantly inhabited by an increase in the phosphorylation of ser-9. Since the phosphorylation of serine-9 inactivates GSK3b [ 59 ]. Since, GSK3β can positively control the cell viability and apoptotic state of tumor cells by its controlling of cyclin D1, beta-catenin, c-Jun, c-Myc, LC3, and cAMP-response element binding protein. With this result, the reduction of GSK3β minimizes survival and proliferation and induces apoptosis in various types of cancers, such as colorectal cancer, pancreatic cancer, and bladder cancer [ 60 ] 5. Conclusion Cancer is still a serious threat now with more health issues generated continuously during its treatment. Neither surgery, chemotherapies nor radiotherapies, or immunotherapies are obtaining great recovery for cancer treatment. Owing to cancer resistance, their non-selection, and their undesirable side effects. The efforts have turned recently into using a natural product such as propolis because it is rich in many different polyphenolic compounds that may potentiate antioxidant activity and can modulate multi-signaling pathways in cancer cells. In the recent investigation, propolis extract was loaded with albumin-folic acid to minimize the strong odor and taste of propolis and to control its delivery. Propolis extract and albumin propolis NPs have proven their strong ability to activate caspase-3 and LC3II and to negatively regulate Cyclin D1. Increasingly, propolis extract can inactivate GSK3 beta by its ability to phosphorylate serin-9. The result was also confirmed by AO/EB and DAPI stains to observe the real nuclear fragmentation after 72h incubation of MCF-7 and A549. Propolis and its derivatives as natural products are b recommended to use as therapeutic agents to fight exactly the invasiveness of cancer. Declarations Ethics approval and consent to participate: All experiments were conducted following the h US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and cell line experiments Guide for the Care and Use of Laboratory Animals after being approved by the relevant Ethical Committee and authorized by the Italian and German Ministry of Health. This study was also approved by the Research Ethics Committee of Kafrelsheikh University. Consent for publication: All authors have provided their agreement to publish the submitted manuscript. Availability of data and materials: Data is available in a publicly accessible repository (https://scholar.google.com/citations?user=_nvSCFgAAAAJ&hl=en). Competing interests: The authors declare no conflict of interest. Funding: This is no fund was received. Authors' contributions : NANH: Methodology, data curation, investigation, writing original draft, preparation, design of the manuscript, format analysis, and supervision. MGMG: Methodology, data curation, investigation, format analysis, revised original draft. All authors have read and agreed to the published version of the manuscript. Acknowledgments: NANH and MGMG would like to thank Dr. Moataz M.M. Rashad and Amal for their great support. Authors' information : Data of authors will be available in the proofreading manuscript after being accepted. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2505179","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":174237055,"identity":"6047052f-27e5-46b4-92e5-bdac88e3e38f","order_by":0,"name":"Mohamed G.M. Ghazy","email":"","orcid":"","institution":"Agriculture Research Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"G.M.","lastName":"Ghazy","suffix":""},{"id":174237056,"identity":"22b01cf0-2332-4f97-ac10-3aa581196470","order_by":1,"name":"Nemany A.N. Hanafy","email":"data:image/png;base64,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","orcid":"","institution":"Kafrelsheikh University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Nemany","middleName":"A.N.","lastName":"Hanafy","suffix":""}],"badges":[],"createdAt":"2023-01-22 23:59:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2505179/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2505179/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32709504,"identity":"b246f35d-bfc2-4bdc-bb6f-0543f2c2f7a9","added_by":"auto","created_at":"2023-02-09 15:13:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":107402,"visible":true,"origin":"","legend":"\u003cp\u003eChromatography result showed peaks of \u003ca href=\"https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/flavonoid\" title=\"Learn more about flavonoids from ScienceDirect's AI-generated Topic Pages\"\u003eflavonoids\u003c/a\u003e in ethanolic propolis extract and their peaks after encapsulation.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/e1a77bc794fc274b195985d5.jpeg"},{"id":32711670,"identity":"a4be2605-1b9e-478d-be6e-266601202d14","added_by":"auto","created_at":"2023-02-09 15:21:39","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":142520,"visible":true,"origin":"","legend":"\u003cp\u003eSEM image showed the morphology of BSA-propolis NPs. A) Low magnification. B) High magnification. C) Quantification analysis of BSA-propolis NPs diameter by using the image j program.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/3a495daa6ece257fb108eee0.jpeg"},{"id":32709506,"identity":"e1418db1-58fb-4ea8-b45d-60401c7b8b8c","added_by":"auto","created_at":"2023-02-09 15:13:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112628,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential measurement. A) Net charge of BSA-propolis NPs. B) Mono distribution and hydrodynamic diameter of BSA-propolis NPs.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/37f816392e98da14055827ed.jpeg"},{"id":32709511,"identity":"780cb4da-c1c6-4c3e-acc4-d38e78ec98ee","added_by":"auto","created_at":"2023-02-09 15:13:39","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":197439,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR and UV-visible spectrophotometer. A) Modification of chemical bands. B) Absorbance of BSA-propolis NPs.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/877a97b0348106c6a37d228b.jpeg"},{"id":32711669,"identity":"c71b3e9f-5a58-4ff5-91a7-a81a0238f278","added_by":"auto","created_at":"2023-02-09 15:21:39","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":281185,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability assay after 48h and 72h for MCF-7 and A549 incubated with propolis extract and BSA-Propolis NPs.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/68c4320d72e41a04a917917e.jpeg"},{"id":32711668,"identity":"7f004da9-9f62-48f0-98ae-db8441e4313c","added_by":"auto","created_at":"2023-02-09 15:21:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":235627,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry of protein expression for MCF-7. Capsase-3 (a-c). Cyclin D1(d-f). LC3II (g-i).\u003c/p\u003e\n\u003cp\u003eQuantification analysis of protein expression. A) Capsase-3. B) Cyclin D1. C) LC3II\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/0a2343a6f464b7a0a2c7774a.png"},{"id":32709508,"identity":"f5f54f9e-2358-4ccc-b525-e813d076948d","added_by":"auto","created_at":"2023-02-09 15:13:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":233430,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry of protein expression for A549. Capsase-3 (a-c). Cyclin D1(d-f). LC3II (g-i).\u003c/p\u003e\n\u003cp\u003eQuantification analysis of protein expression. A) caspase-3. B) Cyclin D1. C) LC3II\u003c/p\u003e","description":"","filename":"F7.png","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/a180ec36a30ed1dced73667e.png"},{"id":32711672,"identity":"6f2da1ca-3c11-4160-a815-d1de3c370707","added_by":"auto","created_at":"2023-02-09 15:21:39","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":316672,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopy showed AO/EB and DAPI stain in breast cancer after 24, 48, and 72h incubation with propolis extract and BSA-propolis NPs.\u003c/p\u003e","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/149546d21104012eada46d1b.jpeg"},{"id":32709513,"identity":"fa541159-da67-4345-8c62-3892d88569df","added_by":"auto","created_at":"2023-02-09 15:13:39","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":215601,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopy showed AO/EB and DAPI stain in lung cancer after 24, 48, and 72h incubation with propolis extract and BSA-propolis NPs.\u003c/p\u003e","description":"","filename":"floatimage13.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/8d79b0cd17340e16075bf15b.jpeg"},{"id":32709514,"identity":"f32ed801-04cb-4eee-bd6f-196c44f46444","added_by":"auto","created_at":"2023-02-09 15:13:40","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":215069,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of serine 9 phosphorylation of GSK-3β in MCF-7 and A549 cells. (\u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003e B\u003c/strong\u003e) Western blot band of p-GSK-3β(S9)). Beta-actin was analyzed as a loading control. Densitometric analysis of p-GSK-3β(S9)) levels after their exposure to propolis extract and BSA-propolis NPs for 48h.\u003c/p\u003e","description":"","filename":"F10.png","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/095a5f15051c5003dd379bf7.png"},{"id":42396748,"identity":"1838bf3f-4c9c-402d-b063-d2517765e809","added_by":"auto","created_at":"2023-08-31 02:37:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1822405,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/94b64479-4d4f-4e31-9af8-77a97355e3e3.pdf"},{"id":32712075,"identity":"12c20257-bdab-46ed-abc8-453989b4d859","added_by":"auto","created_at":"2023-02-09 15:29:39","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":212492,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblotanalysis.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2505179/v1/d02d1bac3bff78cc3f22a3dc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeted Therapies for Breast and Lung Cancers by Using Propolis Loaded Albumin Protein Nanoparticles","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer is still a serious threat to human life among many other health issues. Since 14.1\u0026nbsp;million new cases and 8.2\u0026nbsp;million cancer-related deaths had been reported [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The impact could reach 23.6\u0026nbsp;million by 2030 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Many hard efforts had been employed to overcome cancer invasiveness by using radiotherapy, surgery, cytotoxic chemotherapy, targeted therapy, and immunotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the resistance of malignant cells to anticancer therapies caused a major issue in the performance of effective treatment of cancer patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The resistance leads to a lack of response to drug-induced tumor growth inhibition [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This often causes failure in treatment. At moment, many efforts were made to treat cancer using various natural polyphenolic compounds due to overcoming the undesirable side effects of chemotherapeutic agents, and their drug resistance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Propolis is a diversified lipophilic material including resin collected from leaf, branch, and bark of certain tree species by the enzymatic metabolism in honey bees, which is then transported to the hive and mixed with wax glands by working bees, resulting in forming adhesive materials known as propolis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Propolis consists of wax (30%), resin (50%), pollen (5%), essential oils (10%), and other substances (5%) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The significant effect of propolis on the inhibition of cancer cell growth and increased cancer cell cycle arrest has been reported in many cancer cell lines such as MCF7 (breast cancer), HGC27 (human gastric carcinoma), A549 (non-small lung cancer), and also cell lines of stomach, cervical, esophageal, brain, laryngeal and skin [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Such these properties are due to its ability to induce inhibition of NF-κB and JNK signaling pathways; proline dehydrogenase/proline hydrogenase- and DNA fragmentation-initiated apoptosis; upregulation of p53, Bax, cleaved-caspase-3 and 9, and MAPK-associated proteins; regulation of glutathione and glutathione S-transferase; and modulation of the mitotic cell division [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite many publications in carcinogenesis, the possible therapeutic application of encapsulated propolis extract derived from the native species of Egyptian source is largely unknown and sparingly investigated.\u003c/p\u003e \u003cp\u003eEncapsulation is a convenient method for minimizing the strong odor and taste of propolis (bitterness and pungent), which are odious for many users [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This technique inhibits the drug from electrostatic interactions with chemoreceptors by delaying its release into the oral cavity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which has the potential to hide bitter qualities and improve palatability might help with the incorporation of these chemicals into food items [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Albumin Protein Bovine Serum (BSA) is used widely as potential targeted delivery for cancer treatment because of its ability to interact with albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC) and glycoprotein 60 (gp60) providing a mechanism to increase its cellular mucoadhesion and penetration. Additionally, to raise its potential efficacy, folic acid was integrated into its moieties as a main targeted molecule used previously for folate receptors [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Material And Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e\n \u003cp\u003ePhosphate Buffer saline (PBS) tablets pH 7.4 was obtained from Oxoid Limited Basingstoke, Hampshire, England; Ethanol from Baker Analyzed, Fisher Scientific, Landsmeer The Netherlands. Bovine serum albumin (BSA); Folic acid (FA); Paraformaldehyde, Dimethyl sulfoxide (DMSO) from Sigma-Aldrich, St. Louis, MO, USA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Isolation of propolis extract derived from an Egyptian source\u003c/h2\u003e\n \u003cp\u003ePropolis was collected manually from a beehive located in kafrelsheikh, Egypt. Propolis was then cut into the small part and soaked in 500 mL 70% ethanol at 37°C for 4 days, in a hermetically closed glass vessel under occasional shaking. The ethanolic extract of Egyptian propolis was then filtered through a Whatman filter paper no. 4 and the amount of ethanol was evaporated and then 100mL distilled water was added. Propolis extract was kept at -20°C for lyophilization [\u003cspan\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. Identification and quantification of polyphenolic compounds\u003c/h2\u003e\n \u003cp\u003eHigh-Performance Liquid Chromatography (HPLC) analysis was obtained by using an Agilent 1260 series. The separation was performed by using the Eclipse C18 column (4.6 mm x 250 mm i.d., 5 µm). The fluid phase contained water (A) and trifluoroacetic acid (0.05% ) in acetonitrile (B) at a measurement rate of 1 ml/min. The fluid phase was assessed one by one in a linear gradient as follows: 0 min (82% A); 0–5 min (80% A); 5–8 min (60% A); 8–12 min (60% A); 12–15 min (82% A); 15–16 min (82% A) and 16–20 (82%A). The multi-wavelength detector was detected at 280 nm. The injection volume was 5 µL for each of the sample solutions. The column temperature was maintained at 40°C[\u003cspan\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. Preparation of propolis albumin nanoparticles\u003c/h2\u003e\n \u003cp\u003eAlbumin NPs loaded by propolis were obtained by the desolvation method. Briefly, 10 mL of ethanolic extract propolis was added to 50 mg BSA conjugated folic acid (dissolved in 100mL, 10 mM NaCl, pH 9) and allowed to interact using magnetic stirring for 30 minutes. In this condition, ethanol in the extract was also used as a desolvation agent which gradually increases the crosslink of protein moieties in the solution leading to agglomeration. Since the covalent bonds contributed to forming crosslinking network structures between albumin moieties and polyphenolic compounds of propolis extract. The unreacted materials were removed by dialysis bag against distilled water. Albumin-propolis NPs were kept at -20°C and then were lyophilized [\u003cspan\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. Characterization\u003c/h2\u003e\n \u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e2.5.1. Transmission Electron Microscopy (SEM)\u003c/h2\u003e\n \u003cp\u003eSamples for TEM analysis were prepared by drop-casting a few microliters of solutions onto standard TEM carbon-coated Cu grids, allowing the solvent to evaporate completely. Prepared samples were imaged utilizing a 2100 TEM-JEOL JEM microscope operating at 200 kV [\u003cspan\u003e23\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e2.5.2. Zeta Potential and DLS measurements\u003c/h2\u003e\n \u003cp\u003eThe electrophoretic mobility of samples was determined by photon correlation spectroscopy (Brookhaven) by using a Zeta Nano Sizer. All measurements were performed at 25°C. Five following measurements were taken for analysis.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e2.5.3. UV-Vis spectrophotometer\u003c/h2\u003e\n \u003cp\u003eThe absorbance of samples was measured by using (Jasco V-770) UV Visible Absorbance Spectrophotometer. 500µl of the fabricated assembly was diluted into 4 ml by using distilled water and then scanned at the range of 200–800 nm. The results were analyzed by Origin 8.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e2.5.4. Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e\n \u003cp\u003eFTIR experiments were carried out by using JASCO Fourier Transform Infrared Spectrometer (JASCO, JAPAN, model no. AUP1200343) used to detect the surface molecular structures in the scale bar of 400–4000 cm\u003csup\u003e− 1\u003c/sup\u003e by using the KBr pellet method. For all of the tests, at least three scans were recorded on different regions of the samples and representative spectra were analyzed [\u003cspan\u003e24\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e2.5.5. Estimation of polyphenolic compounds\u003c/h2\u003e\n \u003cp\u003eThe phenolic compounds as a total (TPC) of samples were measured using the method described by Jansen-Alves et al. (2018). At first, 0.2 M Folin-Ciocalteu reagent was mixed with the samples in a ratio of 5:1. Then, 2 ml of freshly sodium carbonate saturated solution was added to each sample. After that, all samples were vortexed for around 15 s, followed by incubation for 2 h in a dark place at room temperature. Finally, the absorbance of each sample was measured using a spectrophotometer (Beckman) at 725 nm. Before the experiment, a calibration curve using gallic acid at the concertation of 10–600 µg/mL had plotted. The experiment was run out in triplicate and all the results are reported as mg gallic acid equivalent per gram of dried samples (mg GAE/g dried sample) [\u003cspan\u003e25\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e2.5.6. Percentage yield and entrapment efficiency\u003c/h2\u003e\n \u003cp\u003eThe percentage of yield was calculated by dividing the weight of obtained NPs by the total weight of the amount of propolis and BSA. In this case, the yield (%) was calculated from the following equation\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003cp\u003eThe difference between total polyphenolic compounds found inside BSA NPs (TPC) and total polyphenolic compounds found on the surface (TPCS) was used to calculate % of entrapment efficiency. Briefly, 25 mg of propolis-loaded BSA NPs were suspended with 5 ml of 96% ethanol and centrifuged at 15000 rpm for 15 min at 25°C to wash unreacted materials. After phase separation, 500µL of the supernatant was used to calculate the total polyphenolic compounds found on the surface (TPCS). The other 4.5 mL was homogenized for 10 min at 14,000 rpm and then kept in a water bath for 24 h at 25°C to decompose NPs and to calculate the total polyphenolic compounds found inside BSA NPs (TPC). The % of entrapment efficiency was measured by using the following equation [\u003cspan\u003e26\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e2.6. In vitro studies\u003c/h2\u003e\n \u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e2.6.1. Cytotoxicity evaluation\u003c/h2\u003e\n \u003cp\u003eTo assess the effect of BSA NPs, propolis extract, and propolis-loaded BSA NPs on tumor cell growth. The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium-bromide assay was used to evaluate cell bio-viability. Briefly, A549 and MCF-7 cells were exposed to serial concentrations (1, 10, 50, and 100 µg/mL) for 48h and 72h. Then, 5 mg/mL MTT reagent was added to each well and the reaction was allowed to proceed for 4 h at 37°C. The culture medium was then removed and precipitated formazan crystals were dissolved by adding DMSO (200 µL). The absorbance of each well was read at 570 nm and directly correlated to several remaining viable cells. Absorbance data were normalized to the percentage of control, then graphed [\u003cspan\u003e27\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e2.6.2. Evaluation of protein expression with flow cytometry\u003c/h2\u003e\n \u003cp\u003eFor protein expression analysis, A549 and MCF-7 cells were seeded separately as 5×105 cells per well. After 24h, cells were then treated separately by using 20µg/mL propolis extract and BSA-propolis NPs. After treatment, detached cells were washed once and suspended in Permeafix (Ortho Diagnostic Systems, Roissy, France) and incubated for 40 min at 20°C. The material was then centrifuged, and the pellet was purified twice with washing buffer (0.2 mM EDTA, 5% FCS in phosphate-buffered saline [PBS]). Labeling was performed by adding to the cells 100 ml of washing buffer containing 5 ml of polyclonal phycoerythrin (PE)–attached anti–active caspase 3 antibodies (PharMingen–Becton Dickinson, Le Pont de Claix, France) [\u003cspan\u003e28\u003c/span\u003e] cyclin D1-Alexa Fluor 647 antibodies and Anti-LC3A/B antibody attached to Alexa Fluor 488, Cell Signaling, cat. no. 13082] [\u003cspan\u003e29\u003c/span\u003e]. Cells were then analyzed by flow cytometry.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e2.6.3. Western blotting analysis\u003c/h2\u003e\n \u003cp\u003eWhole-cell lysates were prepared as previously described [\u003cspan\u003e28\u003c/span\u003e]. Briefly, cells were lysed with cell RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF) after two washes with ice-cold PBS. Bradford protein assay kit was used to evaluate the protein concentration. 20–30 µg of protein was electrophoresed by 10–12% sodium dodecyl sulfate-PAGE and transferred onto polyvinylidene fluoride membranes. After blocking with 5% non-fat dry milk at room temperature for 1 h, the sheet was incubated at 4°C overnight with the primary antibodies. Then sheets were purified three times with PBST to remove any non-bound primary antibodies and incubated with horseradish-peroxidase-conjugated secondary antibodies at concentrations of 1:2000–10,000 dilutions for 1 h at room temperature. Membranes were cleaned three times with PBST and incubated with ECL substrate. The immunoreactive signals were detected under an Amersham Image600 (USA) [\u003cspan\u003e30\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e2.7. Cellular bio-image\u003c/h2\u003e\n \u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e2.7.1. Fluorescent days (AO/EB) Staining Assay\u003c/h2\u003e\n \u003cp\u003eAcridine orange (AO), is a cationic day, that can penetrate the cell membrane of viable and dead cells emitting green fluorescence after its interaction with double DNA strands. Ethidium bromide is a nucleic acid stain that cannot penetrate a live cell. The dual staining can be used to distinguish between live cells, early-stage apoptosis, late-stage apoptosis, necrosis, and autophagosomes. Since AO can diffuse through the cell membrane making the nuclei and autophagosomes appear green. Ethidium bromide (EB) can be adsorbed only when loss of cellular integration causes the nucleus to be orange. Thus, live cells present a homogeneously green nucleus; early apoptotic cells have a bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; cells that have died from direct necrosis show a structurally normal orange nucleus (19). In the current study, MCF-7 and A549 were treated with propolis extract and BSA-propolis NPs for 24, 48, and 72h, and then, they were washed with phosphate buffer saline pH 7.2 and were fixed by 4% paraformaldehyde for 30 min. Cells were washed with PBS and then Image was captured by fluorescence microscopy (Nikon 500, 515 nm) with 400x amplification [\u003cspan\u003e31\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e2.7.2. DAPI Staining Assay\u003c/h2\u003e\n \u003cp\u003eDAPI (4’,6- diamidine- 2’- phenindole dihydrochloride) is a blue emission color that can be detected under fluorescence microscopy. it can bind specifically to DNA double strands. For analysis of chromosomal DNA changes, MCF-7 and A549 tumor cells were treated with propolis extract and BSA-propolis NPs at the concentration of 0.7 µM for 48 and 72h. Then, cells were washed with phosphate buffer saline (PBS) and fixed in methanol (70%) for 20 minutes. Cells were incubated for 30 minutes with 0.4 µg/mL of DAPI (Sigma Chemical Co., USA). DNA alterations were observed by fluorescence microscopy (Nikon, USA, 385–410 nm) [\u003cspan\u003e32\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e2.8. Biostatistics analysis\u003c/h2\u003e\n \u003cp\u003eThe results were expressed as mean ± standard division of mean (SEM). Data were analyzed by SPSS version 20 using one-way analysis of variance (ANOVA), followed by Duncan's test for comparison between different treatment groups. The data are shown as *P \u0026lt; 0.05 **P \u0026lt; 0.01 and ***P \u0026lt; 0.001. Three independent experiments were used to present DATA.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Flavonoids composition of ethanolic propolis extraction\u003c/h2\u003e \u003cp\u003eThe individual Flavonoids in propolis extract and encapsulated propolis were identified and quantified by using HPLC to provide a better understanding of the concentration of each flavonoid separately.\u003c/p\u003e \u003cp\u003eIn the recent study, the constituents of flavonoids in free propolis and encapsulated one were summarized in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Since quercetin showed good concentration in encapsulation (7.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;g/mL). While chlorogenic acid observed abundant value (7.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;g/mL) in free propolis. The other contents of encapsulated flavonoids were arranged as follows; Querectin\u0026thinsp;\u0026gt;\u0026thinsp;Gallic acid\u0026thinsp;\u0026gt;\u0026thinsp;Chlorogenic acid\u0026thinsp;\u0026gt;\u0026thinsp;Kaempferol\u0026thinsp;\u0026gt;\u0026thinsp;Ellagic acid\u0026thinsp;\u0026gt;\u0026thinsp;Syringic acid\u0026thinsp;\u0026gt;\u0026thinsp;Coffeic acid\u0026thinsp;\u0026gt;\u0026thinsp;Rutin\u0026thinsp;\u0026gt;\u0026thinsp;Coumaric acid\u0026thinsp;\u0026gt;\u0026thinsp;Cinnamic acid\u0026thinsp;\u0026gt;\u0026thinsp;Naringenin\u0026thinsp;\u0026gt;\u0026thinsp;Methyl gallate\u0026thinsp;\u0026gt;\u0026thinsp;Ferulic acid.\u003c/p\u003e \u003cp\u003eThe concentration of encapsulated each flavonoid depends mainly on the ability of this molecule to react with moieties of capsules. Besides that, solubility, conjugation with other phenolic compounds, degree of polymerization, and chemical structure of each flavonoid are additional factors that can affect encapsulation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMeanwhile, all polyphenolic compounds were estimated as (400 mg) according to the standard curve obtained by using gallic acid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantification and identification of flavonoids in ethanolic propolis extract and their encapsulation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolyphenol components\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eEthanolic propolis extract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eEncapsulated Propolis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConc. (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eConc. (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGallic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e215.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5201.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl gallate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e502.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyringic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRutin acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEllagic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoumaric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFerulic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaringenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e225.7693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCinnamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKaempferol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Formulation and Characterization of albumin-propolis NPs\u003c/h2\u003e \u003cp\u003eThe morphology of the fabricated albumin-propolis NPS was shown by using SEM, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The nanoparticles demonstrated spherical shape and agglomerations with an average size of about\u0026thinsp;~\u0026thinsp;200 nm, with a smooth structure. Since, albumin is cross-linked by propolis polyphenolic content in presence of ethanol, showed assembled 3D structure. The result indicates that polyphenolic compounds can be used as natural crosslinkers due to their possible interaction between amino-carboxyl groups of albumin. The result also highlights the advantage of using natural cross-linkers that make them possible to overcome toxic crosslinkers or initiators.\u003c/p\u003e \u003cp\u003eThe mean zeta potential values of the albumin-propolis NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), as estimated by the DLS technique, were found to be 31 mV. The zeta potential of 30 mV was within the recommended range (\u0026minus;\u0026thinsp;30 mV to +\u0026thinsp;30 mV), indicating that encapsulated propolis would not quickly settle and would be suspended and distributed in the physiological media [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The encapsulation efficiency was calculated to identify the loading capacity of albumin NPS as an effective carrier for propolis encapsulation. The percentage of encapsulation efficiency and yield depends mainly on the amount of encapsulated propolis. In the recent work, the percentage of loading capacity and yield was estimated as 85% and 80% respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFolic acid conjugated BSA has exhibited abroad bands at 3432 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that were assigned to stretching vibration of O-H and N-H. The amide band I appeared at ~\u0026thinsp;1663 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Many small bands located at 1394 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to the presence of aromatic ring stretches of the pyridine and p-amino benzoic acid moieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e;\u003cb\u003eA)\u003c/b\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA spectrum of propolis extract showed a significant band at 3391 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that was attributed to the stretching vibration of OH in phenolic compounds. While symmetric and asymmetric C\u0026ndash;H stretching was located at 2937 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Meanwhile, the carbonyl group (C\u0026thinsp;=\u0026thinsp;O) exhibited stretching vibration at 1689 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. while, C\u0026thinsp;=\u0026thinsp;C and C-O were obtained starching vibration at 1623 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1273 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]..\u003c/p\u003e \u003cp\u003eA spectrum of albumin-propolis NPs showed an intrinsic abroad peak at 3392 that was attributed to the stretching vibration of O-H and N-H2. Likewise, a significant characteristic peak of symmetric and asymmetric C\u0026ndash;H was shown at 2937 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. While the carboxylic group was detected at 1743 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the carbonyl group was presented at 1689 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A spectrum of propolis extract exhibits an extensive peak with two crests at 295 nm and 323 nm. While this peak was shifted towards 277nm in the spectrum of albumin-propolis NPs. Phenolic compounds generally exhibit an absorption peak in the ultraviolet light range of 250 and 350 nm for spectrophotometric analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e;\u003cb\u003eB).\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.2. In vitro studies\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Cytotoxicity\u003c/h2\u003e \u003cp\u003eMTT assay is a colorimetric assay, used to evaluate the activity of mitochondrial metabolism as an indicator of a healthy state or cytotoxicity. In this assay, the viable cells can form formazan purple crystals by reduction of yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) in the presence mitochondrial succinate dehydrogenase. The formazan is further dissolved by DMSO and the intensity of the color is proportional to the percentage of live cells at absorbance 570 nm [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here, the MTT assay was applied to explore the potential cytotoxicity of albumin NPs, free propolis, and albumin-propolis NPs at serial concentrations (1, 10, 50, 100 \u0026micro;g/mL) in lung and breast cancer cells lines using A549 and MCF-7 cells.\u003c/p\u003e \u003cp\u003eBased on cellular cytotoxicity, MCF-7 cells were significantly inhibited after 48h and 72h incubation with 100\u0026micro;g/mL (propolis extract and albumin-propolis NPS) by(54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% and 45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005%, P\u0026thinsp;\u0026le;\u0026thinsp;0.001) and (20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% and 10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005%, P\u0026thinsp;\u0026le;\u0026thinsp;0.0001) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Similarly, there is a significant decrease in the growth of A549 was obtained after their incubation (propolis extract and albumin-propolis NPs) for 72 h (15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% and 5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%, P\u0026thinsp;\u0026le;\u0026thinsp;0.00001) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. While empty albumin NPs were obtained no significant reduction in the growth of A549 and MCF-7 cells indicating their potential use as a safety vector for the drug delivery system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Evaluation of Protein Expression with Flow Cytometry\u003c/h2\u003e \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.1. Caspase \u0026minus;\u0026thinsp;3\u003c/h2\u003e \u003cp\u003eCaspase-3 is an executioner caspase, found mainly as inactive pro-caspase in viable cells, which is further activated by initiator caspases such as (caspase 8 and caspase 9) during apoptosis. Active caspase-3 can cleave and fragment DNA and has the ability also to denature proteins. Thereby leading to cell death [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the caspase-3 level was significantly overexpressed in A549 and MCF-7 cells treated for 48h by propolis extract and BSA-propolis NPs. Compare to the control (untreated cells), the regulation of caspase-3 with propolis extract and BSA-propolis NPs treated A549 and MCF-7 cells were significantly increased by (64.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% and 80.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09%, p\u0026thinsp;\u0026le;\u0026thinsp;0.00001) respectively and (54.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5% p\u0026thinsp;\u0026le;\u0026thinsp;0.0001 and 82.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%, p\u0026thinsp;\u0026le;\u0026thinsp;0.00001) respectively [\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.2. Cyclin\u003c/h2\u003e \u003cp\u003eCyclin D1 is a G1 cyclin that organizes the shifting from G1 to the S phase, thus its level increased during the G1 phase of the cell mitotic division. Whereas cyclin A is regarded as a regulator of the transition to mitosis since it reaches its maximum level during the S and G2 phases [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Cyclins regulate the cell cycle by binding to and activating cyclin-dependent kinases (Cdks). For this reason, cyclin\u0026ndash;Cdk complexes can phosphorylate specific targets leading to activating the cell cycle at the right moment [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the recent investigation, propolis extract and albumin propolis NPs can down-regulate Cyclin D1. Since cyclin D was significantly inhibited in MCF-7 and A549 cells after their exposure to propolis extract and albumin propolis NPS by (39.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9% p\u0026thinsp;\u0026le;\u0026thinsp;0.0001 and 26.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5% p\u0026thinsp;\u0026le;\u0026thinsp;0.0001) and (36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3% p\u0026thinsp;\u0026le;\u0026thinsp;0.00001 and 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6% p\u0026thinsp;\u0026le;\u0026thinsp;0.00001) compared to MCF-7 cells that were untreated (77.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4% ) and untreated A549 cells (87.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.3. LC3\u003c/h2\u003e \u003cp\u003eLight chain 3 (LC3), Microtubule-associated protein 1A/1B distributed in cultured cells and mammalian tissues ubiquitously because it is highly soluble. Once autophagosome engulfs components of cytoplasm including organelles and cytosolic proteins. LC3 (cytosolic form) is conjugated to phosphatidylethanolamine forming LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. For this reason, LC3-II is a standard marker for accounting for the number of autophagosomes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the recent work, there was a significant regulation in the percentage of LC3 level in both MCF-7 and A549 treated by propolis extract and BSA-propolis NPs. Since, MCF-7 cells exhibited higher evaluation of LC3-II by (77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% p\u0026thinsp;\u0026le;\u0026thinsp;0.00001 and 92.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% p\u0026thinsp;\u0026le;\u0026thinsp;0.0001) respectively. Similarly, A549 cells showed (77.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7% p\u0026thinsp;\u0026le;\u0026thinsp;0.00001 and 89.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8% p\u0026thinsp;\u0026le;\u0026thinsp;0.00001) respectively. LC3-II is a specific marker associated with the formulation of autophagosomes. The result indicates an increased number of autophagosomes in MCF-7 and A549 cells after their treatment with propolis extract and BSA-propolis NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. Cellular bio-images\u003c/h2\u003e \u003cdiv id=\"Sec32\" class=\"Section4\"\u003e \u003ch2\u003e3.2.3.1. Analysis of Apoptosis by AO/EB Staining\u003c/h2\u003e \u003cp\u003eto explore the phenotype expression of MCF-7 and A549 cells during treatment, the dual fluorescent staining composed of the dual fluorescent days (AO/EB) was used to distinguish apoptotic cells, necrotic cells, and autophagosomes. Since, early-stage apoptotic cells, shown by crescent-like shape and nuclei were seen as yellowish-green color (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e,). While late-stage apoptotic cells are marked by orange nuclear EB staining. Meanwhile, cell volume was increased in necrotic cells showing orange-red emission at their periphery. Additionally, protonation of acridine orange occurs at acidic vesicles (autolysosomes) causing bright red emission for AO, while the nucleus becomes bright green. At neutral pH, AO becomes de-protonation and green color is emitted. By this mechanism, AO can track acidic vesicular organelles and distinguish autophagy [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section4\"\u003e \u003ch2\u003e3.2.3.2. DNA staining assay\u003c/h2\u003e \u003cp\u003eTo elucidate the changes in nuclear morphology, DAPI stain was used as a specific dye for nucleic acid. Nuclear fragmentation was observed after 72h incubation with propolis extract and BSA-propolis NPs Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4. Western blot analysis\u003c/h2\u003e \u003cp\u003eGlycogen synthase kinase 3 (GSK3B) is a proline-directed serine\u0026frasl;threonine protein kinase involved in modulating numerous processes, including metabolism, proliferation, apoptosis, autophagy, development, and differentiation. The regulation of GSK3 depends on the phosphorylation at the specific sites of GSK3; phospho-GSK3α (Ser21) and phospho-GSK-3β (Ser9) both of them can inhibit the GSK3β activity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]; whereas phospho-GSK3β (Tyr216) increases the activity of GSK3β [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our DATA, the upregulation of phospho-GSK3β (Ser9) (p \u0026lt; 0.001) was increased significantly in MCF-7 and A549 cells after their exposure to propolis extract and BSA-propolis NPs. Such phosphorylation led to inhibiting the activity of GSK-3β (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePropolis is rich in phenolic compounds, which may act as natural antioxidants [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In fact, because of the present different groups of phenolic acids, the ethanolic extracts of propolis affect the inhibition of free radicals formation, reducing or complexing transition metal ions, capturing the superoxide amino radicals and peroxy radicals, and breaking the free radical reactions preventing lipid peroxidation. All of these properties, make propolis has strong antibacterial, antifungal, antiviral and anti-inflammatory properties [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Recently the potent activity of propolis as an antioxidant has been considered significantly suppress tumor growth by directly inhibiting tumor cell growth and inducing apoptosis or indirectly by scavenging ROS and increasing antioxidant enzyme activities [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In the recent work, ethanolic propolis extract was inserted into BSA-FA moieties with strong binding affinity because of its abundant chemical interactions. FTIR illustrated the strong interaction between propolis extract and the amino acid structure of BSA. Likewise, zeta potential revealed net charge of albumin propolis NPs was estimated as 31mV\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8. Meanwhile, albumin-propolis NPs showed strong mono dispersion with 3D assemble structure having a diameter of ̴330nm.\u003c/p\u003e \u003cp\u003eThe potential cytotoxicity of propolis was estimated after 48h and 72h by using MCF-7 and A459. Therefore, there is a significant inhibition in the growth of A549 and MCf-7 after 72h incubation. Since, 100\u0026micro;g/mL of albumin propolis NPs can inhibit significant growth of MCF-7 and A549 by (10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005%, P\u0026thinsp;\u0026le;\u0026thinsp;0.0001 and 5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%, P\u0026thinsp;\u0026le;\u0026thinsp;0.00001).\u003c/p\u003e \u003cp\u003eTo induce a better understanding of the mechanism of propolis, protein expression was evaluated by using flow cytometry for Caspase-3, Cyclin D1, and LC3 in MCF-7 and A549. The expression of caspase-3 with propolis extract and BSA-propolis NPs treated MCF-7 and A549 cells were significantly increased by (64.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% and 80.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09%, p\u0026thinsp;\u0026le;\u0026thinsp;0.00001) respectively and (54.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5% p\u0026thinsp;\u0026le;\u0026thinsp;0.0001 and 82.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%, p\u0026thinsp;\u0026le;\u0026thinsp;0.00001) respectively.\u003c/p\u003e \u003cp\u003eThe result confirms the strong ability of propolis to activation of caspase-3 and induction cell death in both MCF-7 and A549. Another confirmation is that Cyclin D1 was also significantly inhibited when cells were exposed to propolis extract and albumin propolis NPs. Cyclin D1 is a nuclear regulator, responsible for the transition cells from the G1 phase to the S phase. Its inhibition could block cell division and induce cell cycle arrest. Increasingly, LC3-II, as a standard marker for autophagosomes showed a higher expression in both MCF-7 and A549 after their exposure to propolis extract and albumin propolis NPs. Since, MCF-7 cells exhibited higher evaluation of LC3-II by (77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% p\u0026thinsp;\u0026le;\u0026thinsp;0.00001 and 92.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% p\u0026thinsp;\u0026le;\u0026thinsp;0.0001) respectively. Similarly, A549 cells showed (77.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7% p\u0026thinsp;\u0026le;\u0026thinsp;0.00001 and 89.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8% p\u0026thinsp;\u0026le;\u0026thinsp;0.00001) respectively. Such a result indicates an increased number of autophagosomes meaning that propolis can activate protein expression of apoptotic and autophagy pathways.\u003c/p\u003e \u003cp\u003eTo support these data, AO/EB and DAPI stains were used to investigate apoptotic stages, DNA fragmentation, and autophagosomes. In the recent study, live cells showed uniformly green color compared to early apoptotic cells that observed condensed or in some cases, fragmented nuclei with bright green color. While late apoptotic cells exhibited condensed and fragmented orange chromatin. In autophagy, neutral autophagosomes, characterized by green vesicle color, were located close to the cell center. While acidic autophagosomes characterized by red vesicle color were observed close to the cell periphery [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe result was supported by DAPI stain as a very specific nuclear stain can distinguish nuclear fragmentations. Therefore, MCF-7 and A549 cells that were exposed to propolis extract and albumin propolis NPs for 72h showed higher nuclear fragmentation and rapture of the cell membrane.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo induce a better understanding of how propolis can modulate LC3II and Cyclin D1. The GSK3 beta was also estimated in the recent work by using western blot in both MCF-7 and A549.\u003c/p\u003e \u003cp\u003eTherefore, GSK3 beta was significantly inhabited by an increase in the phosphorylation of ser-9. Since the phosphorylation of serine-9 inactivates GSK3b [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Since, GSK3β can positively control the cell viability and apoptotic state of tumor cells by its controlling of cyclin D1, beta-catenin, c-Jun, c-Myc, LC3, and cAMP-response element binding protein. With this result, the reduction of GSK3β minimizes survival and proliferation and induces apoptosis in various types of cancers, such as colorectal cancer, pancreatic cancer, and bladder cancer [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eCancer is still a serious threat now with more health issues generated continuously during its treatment. Neither surgery, chemotherapies nor radiotherapies, or immunotherapies are obtaining great recovery for cancer treatment. Owing to cancer resistance, their non-selection, and their undesirable side effects. The efforts have turned recently into using a natural product such as propolis because it is rich in many different polyphenolic compounds that may potentiate antioxidant activity and can modulate multi-signaling pathways in cancer cells. In the recent investigation, propolis extract was loaded with albumin-folic acid to minimize the strong odor and taste of propolis and to control its delivery. Propolis extract and albumin propolis NPs have proven their strong ability to activate caspase-3 and LC3II and to negatively regulate Cyclin D1. Increasingly, propolis extract can inactivate GSK3 beta by its ability to phosphorylate serin-9. The result was also confirmed by AO/EB and DAPI stains to observe the real nuclear fragmentation after 72h incubation of MCF-7 and A549. Propolis and its derivatives as natural products are b recommended to use as therapeutic agents to fight exactly the invasiveness of cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eAll experiments were conducted following the h US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and cell line experiments Guide for the Care and Use of Laboratory Animals after being approved by the relevant Ethical Committee and authorized by the Italian and German Ministry of Health. This study was also approved by the Research Ethics Committee of Kafrelsheikh University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eAll authors have provided their agreement to publish the submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e Data is available in a publicly accessible repository (https://scholar.google.com/citations?user=_nvSCFgAAAAJ\u0026amp;hl=en).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis is no fund was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e :\u0026nbsp;NANH: Methodology, data curation, investigation, writing original draft, preparation, design of the manuscript, format analysis, and supervision. \u0026nbsp;MGMG: Methodology, data curation, investigation, format analysis, revised original draft. \u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e NANH and MGMG would like to thank Dr. Moataz M.M. 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Glycogen synthase kinase 3 inhibition promotes lysosomal biogenesis and autophagic degradation of the amyloid-β precursor protein. Mol Cell Biol. 2012 Nov;32(21):4410\u0026ndash;8. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/MCB.00930-12\u003c/span\u003e\u003cspan address=\"10.1128/MCB.00930-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Propolis, Polyphenolic compounds, Autophagosomes, Cancer ","lastPublishedDoi":"10.21203/rs.3.rs-2505179/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2505179/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCancer is a popular disease among many others that can threaten defiantly human life. This is not only because of its invasiveness but also because of its resistance and the highly effective cost of its treatment. Propolis is rich in natural sources of bioactive and polyphenolic compounds that have proven their strong effect on cancer cells such as MCF-7 and A549 cell lines. Propolis extract was inserted into the Albumin protein, Bovine Serum (BSA) conjugated to folic acid (FA) to increase control of its delivery and to increase their cellular uptake. The growth of MCF-7 and A549 was significantly decreased by propolis extract and BSA-propolis NPs after their incubation for 72h by (54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% and 45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005%, P\u0026thinsp;\u0026le;\u0026thinsp;0.001) and (20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% and 10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005%, P\u0026thinsp;\u0026le;\u0026thinsp;0.0001) respectively. Similarly, there is a significant inhibition in the growth of A549 obtained after their incubation (propolis extract and albumin-propolis NPs) for 72 h (15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% and 5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%, P\u0026thinsp;\u0026le;\u0026thinsp;0.00001). Propolis extract and BSA-propolis NPs exhibited a greater effect on protein expression of MCF-7 and A549 showing significant modulation of caspase-3, cyclin D1, and LC3II. The result was supported by the presence of nuclear fragmentations and activation of acidic/neutral autophagosomes in AO/EB and DAPI stains. In the recent investigation, the expression of phospho-GSK3β (Ser9) (p \u0026lt; 0.001) increased significantly in MCF-7 and A549 cells after their exposure to propolis extract and BSA-propolis NPs. Results support the potency application of propolis and its encapsulation as an alternative therapeutic agent for cancer treatments instead of chemotherapies because of its action on multi-signaling pathways.\u003c/p\u003e","manuscriptTitle":"Targeted Therapies for Breast and Lung Cancers by Using Propolis Loaded Albumin Protein Nanoparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-09 15:13:34","doi":"10.21203/rs.3.rs-2505179/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1065cb61-b5d1-4f3d-a349-cb3816ade7ac","owner":[],"postedDate":"February 9th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":19033699,"name":"Biological sciences/Cancer"},{"id":19033700,"name":"Biological sciences/Cell biology"},{"id":19033701,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2023-08-31T02:29:20+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-09 15:13:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2505179","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2505179","identity":"rs-2505179","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}