Targeted delivery of bee venom to A549 lung cancer cells by PEGylate liposomal formulation: an apoptotic investigation | Scientific Reports

Scientific Reports volume 14, Article number: 17302 (2024) Cite this article

1242 Accesses

1 Citations

Metrics details

This study focused on developing an optimal formulation of liposomes loaded with bee venom (BV) and coated with PEG (BV-Lipo-PEG). The liposomes were characterized using dynamic light scattering, transmission electron microscopy, and Fourier transform infrared spectroscopy. Among the liposomal formulations, F3 exhibited the narrowest size distribution with a low PDI value of 193.72 ± 7.35, indicating minimal agglomeration-related issues and a more uniform size distribution. BV-Lipo-PEG demonstrated remarkable stability over 3 months when stored at 4 °C. Furthermore, the release of the drug from the liposomal formulations was found to be pH-dependent. Moreover, BV-Lipo-PEG exhibited favorable entrapment efficiencies, with values reaching 96.74 ± 1.49. The anticancer potential of the liposomal nanocarriers was evaluated through MTT assay, flow cytometry, cell cycle analysis, and real-time experiments. The functionalization of the liposomal system enhanced endocytosis. The IC50 value of BV-Lipo-PEG showed a notable decrease compared to both the free drug and BV-Lipo alone, signifying that BV-Lipo-PEG is more effective in inducing cell death in A549 cell lines. BV-Lipo-PEG exhibited a higher apoptotic rate in A549 cell lines compared to other samples. In A549 cell lines treated with BV-Lipo-PEG, the expression levels of MMP-2, MMP-9, and Cyclin E genes decreased, whereas the expression levels of Caspase3 and Caspase9 increased. These findings suggest that delivering BV via PEGylated liposomes holds significant promise for the treatment of lung cancer.

Lung cancer is globally ranked as the second most common type of cancer, which makes this disease a notable healthcare issue1,2. Moreover, according to the Global Cancer Observatory, the World Health Organization has estimated that lung cancer mortality among both sexes and all ages will rise by 64.4% and 67.5%, respectively between 2020 and 20403. Since lung cancer therapy is challenging especially in advanced stages, finding a treatment method for improving existing therapies is necessary. The traditional strategy of treatment is surgical resection and other conventional treatment methods consist of chemotherapy therapy, radiation therapy, targeted therapies, laser therapy, immunotherapy, and photodynamic or4. Although single anticancer drugs such as paclitaxel, cisplatin, and etoposide or drug combinations in chemotherapy are used for the treatment of advanced cancers, severe toxic side effects are mostly a major problem of this therapy5,6.

Apitoxin also called bee venom (BV) is released from poison glands in the abdomen of the worker bee and has a main action of defense against the bee colony7,8. Also, Apitoxin has been used to manage painful disorders and treat various chronic inflammatory disorders including, skin conditions, rheumatoid arthritis, bursitis, tendinitis, and even neurologic disorders because of its non-steroidal anti-inflammatory properties9,10. Apitoxin consists primarily of melittin, which is the main component of bee venom (BV), along with other compounds such as ado-lapin, apamin, mast cell degranulating peptide, and two enzymes: phospholipase A2 and hyaluronidase. Additionally, it contains non-peptide components like histamine, dopamine, and norepinephrine7,11. The main compound of apitoxin is melittin, which acts as a cytotoxic compound against cancer cells through the induction of both caspase-dependent and caspase-independent apoptosis and PLA2 pathways7,12. As a result, recent studies have reported the growth inhibitory potential of melittin in multiple types of cancers namely, renal, lung, breast, liver, prostate, bladder, leukemia, and gastric10,13. Mohamed et al.14. reported the effects of Egyptian and Georgian bee venoms on the MCF-7 breast cancer cell line and A549 lung cancer cell line. They observed that the Egyptian bee venom showed greater toxicity on the two cell lines compared to the Georgian bee venom. Studies show that BV has various effects on apoptosis, necrosis, cytotoxicity, and inhibition of proliferation and it can manage a variety of cancer types, including prostate, breast, lung, liver, and bladder15.

The studies show that BV can inhibit cyclooxygenase-2 expression in human lung cancer cells. Moreover, it can induce apoptosis in synovial fibroblasts with rheumatoid arthritis by caspase-3 activation12,16. Also, BV prevented the proliferation of vascular smooth muscle cells because it can induce apoptosis through suppression of NF-kB and Akt and promotion of anti- apoptotic pathways17. In one study, Holle et al. assessed the cytotoxic effect of melittin-avidin conjugate on prostate malignant cells. The results showed that the size of tumors injected in melittin-avidin conjugate was less than that of non-injected tumors18. Studies have reported that melittin can disrupt erythrocyte membranes, which results in hemolysis. Moreover, it prevented tumor growth and induced the activation of matrix metalloproteinases (MMP) and caspase, which causes a cytotoxic effect on malignant cells by apoptosis and necrosis19,20.

However, some disadvantages of anti-cancer drugs, such as poor selectivity, the issue of multidrug resistance, and several side effects such as anemia, nausea, vomiting, nephrotoxicity, and, neurotoxicity, have made them restricted21,22. So, the use of nanoparticle carriers is a way to combat some limitations of apitoxin including the general cytotoxicity or resistance to apitoxin because incorporation of a therapeutic compound into an inert delivery system leads to carrying the compound to targeted cell or tissue, where it is released and can perform its purpose23,24,25.

Liposomes are small artificial vesicles with spherical structures, as well as the main compounds of liposomes, are cholesterol and natural non-toxic phospholipids26. Due to their biocompatibility, biodegradability, capacity to reduce drug toxicity, enhance therapeutic efficacy, and capacity to encapsulate both hydrophilic and hydrophobic drugs, liposomes have been widely used as drug delivery systems. Liposomes' unique properties make them a major breakthrough in the field of drug delivery systems27,28,29. Enhancing the bioavailability and stability of medicinal substances is liposomes' main benefit30,31. They can prolong systemic circulation, improve absorption, and shield medications from breaking down in the body, all of which increase the treatment's efficacy. Moreover, liposomes can be designed to target particular bodily tissues or cells, minimizing systemic side effects and raising the medication's therapeutic index. Liposomes have the ability to encapsulate these molecules, shielding them from enzymatic destruction and facilitating their transport to the intended region of action30,32. Liposome self-assembly is essential for medication delivery systems33. When phospholipids are dissolved in water, liposomes—self-assembling structures—spontaneously form spherical vesicles. This special quality is essential for encasing medications and other medicinal substances. The hydrophilic core and hydrophobic bilayer that are produced during the self-assembly process enable liposomes to transport both fat- and water-soluble medications. Because of their adaptability, liposomes can be used to administer a wider variety of medications34,35.

Additionally, by engineering the self-assembled liposomes to have varying sizes, charges, and surface characteristics, drug delivery systems can be tailored to the target site's requirements and the particular requirements of the therapeutic agent. For example, liposomes can be engineered to evade immune system recognition, extending their half-life in the body and boosting the likelihood of the medication reaching the intended location36,37.

A pH-responsive drug delivery system is a smart way to deliver a drug that reacts to pH changes around it. These systems are designed to release their therapeutic agents at specific pH levels, typically where an infection or disease is located. This is particularly useful for targeting areas like the stomach and tumor sites, which have different pH levels compared to the rest of the body. pH-responsive nanocarriers are essential for delivering certain drugs, such as bee venom, to cancerous cells. These carriers respond to stimuli, which enables them to adjust drug release in response to the acidity of the tumor microenvironment36,37.

Liposomes are vesicles made of lipids that consist of an aqueous core surrounded by a lipid bilayer membrane. Phospholipids and other amphiphilic compounds make up the lipid bilayer. The hydrophilic head groups and hydrophobic tails of these phospholipids enable liposomes to self-assemble into spherical vesicles. pH-responsive liposomes are made to adapt to changes in pH by changing either their structure or their characteristics. The primary factor accountable for pH responsiveness is frequently a liposomal membrane-integrated pH-sensitive lipid. Acidic environments, such as those found in tumor microenvironments, can cause pH-responsive liposomes to collapse. pH-sensitive lipids' protonation of functional groups modifies the liposome's surface charge and membrane characteristics. Drug release behavior, permeability, and liposome stability all alter as a result. The capacity of pH-responsive liposomes to take advantage of the acidic milieu seen at tumor locations is what makes them effective. This acidity is caused by an excess of metabolic byproducts, such as carbon dioxide and lactic acid, as well as an increased activity of proton pumps, or vacuolar-type (V-type) H + -ATPases. Because of these circumstances, the extracellular pH around the tumor cells is lowered, a feature that can be used to deliver drugs precisely where they are needed38,39,40,41. Lecithins, which are frequently present in liposome structures, have a major role in the pH-responsiveness of these vesicles. Both hydrophilic (which attracts water) and hydrophobic (which repels water) regions are present in these phospholipids. Lecithins preserve the fluidity of the membrane when liposomes are assembled into bilayers, which enables them to adjust to different pH levels. Lecithin-based liposomes inflate at low pH (like the tumor microenvironment), resulting in targeted medication release. Because of their sensitivity to pH, lecithins are crucial ingredients in liposomal formulations because they improve targeted drug delivery. Lecithin-based liposomes react to pH variations in their surroundings41,42,43,44.

To increase the bioavailability of liposomes, the best way is to modify the surface of liposomes with inert polymeric molecules. PEG, as a modifier, has high solubility, biocompatibility, nontoxic, non-immunogenic, and non-antigenic potential. Several literatures have shown that the modification of liposomes through PEGylation, which is considered an agent to coat the exterior surface of liposomes with its long chains of polyethylene glycol (PEG), leads to a further increase in circulation times, greater tumor accumulation, and tumor growth repression. Moreover, this process permits liposomal nanocarriers to search their target sites by providing an added time in vivo, before being removed by macrophages45,46,47,48.

In pH-responsive liposomes, PEG is an essential component. Hydrophilic and biocompatible PEG polymers are used to adorn the liposome surface in order to inhibit the absorption of serum proteins. These PEG chains considerably lengthen liposomes' blood circulation duration. Nevertheless, full wrapping during endocytosis can be impeded and delayed because of the grafted PEG polymers' mobility on the liposome's surface. Tumor cell internalization is less likely when PEG polymers aggregate in the membrane-contact area, forming a ligand-free zone. PEGylated liposome internalization may be improved and aggregation may be limited by increasing repulsive interactions between grafted PEG polymers49,50. PEG does not react to pH, but its presence does change how liposomes behave50.

In conclusion, the main goal of this research is to develop a novel medication delivery method for bee venom to maximize its therapeutic potential in the treatment of lung cancer. Promising cytotoxic effects against different cancer cell types have been demonstrated by bee venom. Unfortunately, because of its poor stability and systemic toxicity, its utilization has been limited. We have used a liposomal medication delivery technology for bee venom to get over these obstacles. In addition, we have PEGylated the liposomes through engineering. PEGylation is the process by which polyethylene glycol (PEG) is attached to liposomes, increasing their stability and lengthening their duration in circulation within the body while reducing their size and release. This increases the liposomes' transport efficiency by giving them more time to reach the tumor location and decreasing the immune system's ability to recognize them. By doing this study, we hope to show that bee venom can be efficiently delivered to lung cancer cells by our PEGylated liposomal formulation, increasing its therapeutic efficacy while reducing its systemic toxicity.

For this purpose, the BV-loaded liposome was synthesized then it was modified with PEG (BV-Lipo-PEG). The pH-dependent release of BV-Lipo-PEG was studied. The cytotoxic effect and cell cycle of functionalized and non-functionalized BV-Lipo-PEG toward A549 cancer cells and HFF healthy cell lines were investigated to evaluate the therapeutic efficiency of the prepared liposomal nanocarrier. Also, the antiproliferative activity and mitochondrial dynamics of nanocarriers were evaluated through Real-time PCR and flow cytometry. The investigation of the expression levels of pro-apoptotic and anti-apoptotic genes in cancer cell lines revealed potential anticancer properties of bee venom. This was determined by observing the up-or down-regulation of these genes, indicating their role in promoting cell death or inhibiting apoptosis, respectively.

For detailed materials see SI.

Lipo-BV was prepared using a thin-layer hydration method according to a previous study with slight changes51. At first, the drug (apitoxin), lecithin, and cholesterol were dissolved in 10 mL chloroform then the organic phase was removed by a rotary evaporator (150 rpm, 60 °C, 30 min). After which, hydration of the dried thin films was carried out by 10 mL of PBS (1X) at 60 °C (120 rpm 30 min). To achieve uniform size distribution of the apitoxin-encapsulated liposomes, the suspension was sonicated for 1 min using a bath sonicator (UP50H compact laboratory homogenizer, Hielscher Ultrasonics, Germany). The tests of release, stability, and biological activity were carried out after keeping samples in a refrigerator (4 °C). PEG solution was prepared by dissolving the PEG grind in a cell culture medium (DMEM) under stirred conditions at room temperature for 15 min left overnight. Homogenization of the sonicated solution was done for 10 min at 12,000 rpm (Scheme 1). Based on the prepared samples, the constitution of the measured liposomal formulation is presented in Table 1. Three experiments were run; the BV loaded in liposomes were from F1 to F3 formulation then the influence of molar ratios (Lecithin: Cholesterol), while sonication time, and the content of drug: lipid was constant, on liposomal particle size (nm), Polydispersity index (PDI), and entrapment efficacy percentage (EE %) as dependent variables was devalued. The formulation with minimum particle size, as well as PDI, and maximum entrapment efficiency, had the best formulation. Finally, F3 was selected as the best formulation then F3-B (blank liposomal formulation) and F3-PEG3000 (PEGylate Lipo-BV) were synthesized based on F3 formulation.

Fabrication and PEGylation of the liposomal containing BV using thin-layer hydration method.

For detailed Optimization of PEG-Lipo-BV see SI.

For detailed Polydispersity Index, Size, Entrapment Efficiency, and Morphology see SI.

For detailed Fourier-Transform Infrared Spectroscopy (FTIR) see SI.

A comparison of in vitro drug release between free BV, Lipo-BV, and PEG-Lipo-BV was carried out in a dialysis bag (MWCO = 12 KDa). 2 mL of each sample was placed in a dialysis membrane and placed in 50 mL of PBS containing 0.1% (w/v) Tween 80 solution at 37 °C with stirring at pH = 5.4 and 7.4. (100 rpm) for 72 h. At specific time intervals (1, 2, 4, 8, 24, 48, and 72 h), one milliliter of the release medium was withdrawn and replaced with fresh medium. The amount of drug released at each time point was measured using ultraviolet (UV) spectrophotometry at a wavelength of 570 nm. Mathematical models were then used to analyze the drug release patterns52.

Stability was evaluated by storage of the optimum formulation containing the drug and the PEGylated formulation at 4 ± 1 °C and 25 ± 1 °C for three months. After storage, the stability of the formulations was evaluated by measurement of the particle size, PDI, and percentage of entrapment efficiency of samples at 4 °C and 25 °C53.

The cancer (A549, MCF-7, and HepG2) and healthy (HFF) cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine. All cells were maintained under standard cell culture conditions for 24 h. After which, 1.56, 3.12, 6.25, 12.50, 25, and 50 μg/mL concentrations of samples (Concentration of drug) were added to a medium then the incubation of cells (A549) was performed for 48 and 72 h. For control, different concentrations of Lipo, free BV, Lipo-BV, and BV-Lipo-PEG were added to HFF cell lines and incubated for 48 h. For comparison, different concentrations of Lipo, free BV, Lipo-BV, and BV-Lipo-PEG were added to HFF, MCF-7, and HepG2 cell lines and incubated for 48 h. Cell proliferation was estimated by cell viability assay. For this purpose, four types of cell lines with 0.5 mg/mL of MTT were incubated for 4 h at 37 °C. Then precipitated formazan was dissolved in 100 μL of DMSO. The absorbance of the resulting solution was measured at 570 nm using an ELISA reader (Organon Teknika, Oss, Netherlands). Finally, cell viability percentages were calculated based on the absorbance values54:

The percentage of cell viability was also calculated according to the above formula, and IC50 was measured afterward (With GraphPad prism software).

The level of apoptosis in A549 cells was assessed using the Annexin V-FITC/PI double staining method. Apoptosis was estimated by the treatment of cancer cells with empty liposome (Lipo), free BV, and PEG-Lipo-BV after incubation for 48 h. Initially, the cells were washed twice with PBS and suspended in 1X binding buffer (5 × 105 cells/well). Then, differentiation of apoptosis and normal cells was carried out using an annexin V-FITC (green fluorescence) and propidium iodide (red fluorescence). The samples were analyzed in triplicate using a benchtop flow cytometer (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). Finally, the IC50 values (concentration of the treatment inducing 50% cell death) were calculated for all samples55.

The effect of the samples on cell proliferation and cell cycle distribution was evaluated using PI staining56. Firstly, cells were seeded in the whole medium with 6-well plates at a density of 1 × 106 cells/well, then for fixation of cells, incubation of cells was performed overnight. Then both adhered and floated cells were gathered, then washed three times with PBS(1X). Cells were treated with non-coated drug-loaded liposomes and coated drug-loaded liposomes for 48 h. Staining of cells was done with 70% cold ethanol overnight at 4 °C after incubation of cells.

Fixed cells were then centrifuged, washed again, and resuspended in 500 μL of PI staining solution containing 50 μg/mL RNase A and 40 μg/mL PI in cold PBS. This staining step was performed in the dark for 20 min at room temperature. All treatments and staining procedures were performed in triplicate. At the end, a FACS Aria (BD Bioscience, USA) flow cytometer was employed to analyze DNA content.

The expression levels of key genes involved in apoptosis, proliferation, invasion, and metastasis were investigated using real-time PCR. These genes included Caspase 3, Caspase 9, MMP-2 (matrix metalloproteinase-2), MMP-9, and Cyclin E. Firstly, an RNA extraction kit based on instructions (Qiagen, Valencia, CA, USA) was used to extract total RNA from both treated and untreated cancer cell lines. Next, the concentration of extracted total RNA was estimated using a photonanometer (IMPLEN GmbH, München, Germany). Next, cDNA (complementary DNA) was synthesized from the extracted RNA using a Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). The reaction mixture for cDNA synthesis contained 5 µL of 5X reaction buffer, 1 µg of RNA template, 0.5 µL of random hexamer primer, 0.5 µL of oligo dT primer, 2 µL of 10 mM deoxynucleotide triphosphate (dNTP) mix, 1 µL of RNase inhibitor (20 U/µL), 1 µL of reverse transcriptase enzyme, and nuclease-free water to reach a final volume of 20 µL53. The temperature program was arranged as follows: 25 °C (5 min, for primer annealing); 42 °C (60 min); 70 °C (5 min); 4 °C (5 min). Eventually, the real-time PCR reaction was performed via a Light Cycler (Bioneer, Daejeon, Korea) by the following temperature program: 95 °C (1 min); 95 °C for 15 s; 60 °C (1 min). By the DDCt method, the relative gene expression could be calculated by considering 100% PCR efficiency57.

For detailed Statistical Analysis see SI.

In the current investigation, all the formulations of liposomes loaded with BV contained equal quantities of lipids (300 µmol), drug content (1 mg/ml), and sonication time (7 min). By changing the molar ratio of Lecithin: Cholesterol, the composition for the optimized formulation was measured. As can be seen in Table 1, among three BV-loaded liposomes (F1 to F3), the F3 formulation was selected as the best sample for further examination as it had the smallest size (193.72 ± 7.35 nm), lowest PDI (0.185 ± 0.010) and highest percentage of entrapment efficiency (93.90 ± 1.25). The results show that the particle size and PDI of Lipo-BV decrease with increasing the molar ratio of Lecithin: Cholesterol. When Lecithin: Cholesterol increased from 1:2 to 2:1, the particle size reduced to 274.3 ± 7.51 nm and 193.72 ± 7.35 nm, respectively. This trend could be due to the complex composition of lecithin. Moreover, when the concentration of Lecithin increases, more surfactants are available to stabilize and favor the formation of smaller vesicles58,59. Additionally, the results of optimization revealed that the ratio of cholesterol had a dramatic effect on the average size of the liposomal vesicles. Particle size rises with an increase in the amount of cholesterol. The hydrophobicity of the bilayer membrane increases after adding cholesterol to the solution, which may impart disturbance in the vesicular membrane. So, the increase of vesicle radius and formation of a more thermodynamic stable structure result from this disturbance60. According to Table 1, all the liposomal formulations (F1 to F3) showed a Polydispersity index value lesser than 0.5, and (F3) with the lowest PDI value (193.72 ± 7.35) showed a narrower size distribution and did not show agglomeration-related problems. The results indicated that Polydispersity index values lessened after increasing cholesterol concentration. The PDI of the F1 formulation with a Lecithin: Cholesterol molar ratio of 1:2 was 0.298 ± 0.015, while the PDI of the F3 formulation with a Lecithin: Cholesterol molar ratio of 2:1 reached 0.185 ± 0.010. A possible explanation is that the mobility and flexibility of phospholipids increase with enhancing the amount of Lecithin, which leads to higher fluidity of the bilayer. As a result formulations with smaller and more uniform vesicles can be produced59,61.

After F3 (Lipo-BV) was selected as the best formulation for further study, blank liposome (F3-B), and PEGylate-Lipo-BV formulations were performed. The results of particle size, PDI, and EE% of blank liposome, Lipo-BV, and PEG-Lipo-BV were summarized in Fig. 1. According to Fig. 1A, loading BV in liposomes leads to an increase in particle size. The particle size increased from 142.51 ± 5.74 to 193.72 ± 7.35 after adding BV. The reason why particle size increases with adding BV is that the volume of the hydration medium decreases with loading anti-cancer drug so additional material and low space result in the formation of an incomplete or distorted bilayer. As a result, the surface of prepared vesicles decreases and the particle size of nanocarriers increases for accommodation of the scarcity of water62. Also, coated liposomal formulations (PEG-Lipo-BV) showed smaller particle sizes (171.28 ± 5.25) compared to non-coated liposomes (193.72 ± 7.35). A decrease in the size of liposomes coated with PEG can be due to the affinity between PEG and the bilayer of liposomes. Physical bonding between PEG and Liposomes, such as hydrogen bonding, increases the cohesion of liposomes, so particle size can decrease63. According to Fig. 1B, the PDI of liposomal nanocarriers exhibited that the blank liposome has the smallest value of 0.160 ± 0.009. While the PDI value of Lipo-BV was 0.185 ± 0.010, it reached 0.173 ± 0.013 after coating Lipo-BV with PEG. The reason for the decrease in the PDI value of Lipo-BV after PEG modification is attributed to the change in the size of vesicles during the coating process.

Size, PDI, and EE% of free drug, coated and uncoated liposomes, (A) free drug, (B) Lipo-BV (C) BV-Lipo-PEG, and DSL of (D) free BV, (C) Lipo-BV, and (E) BV-Lipo-PEG.

The results of the effect of concentration of the molar ratio of Lecithin: Cholesterol on the EE% are indicated in Table 1. As can be seen, EE% increased with increasing the molar ratio of Lecithin: Cholesterol. The entrapment efficiency of BV liposomes was in the range of 79.64 ± 1.36 with Lecithin: Cholesterol equal 2:1 for formulation F1 to 93.90 ± 1.25% for formulation F3 with Lecithin: Cholesterol equal 1:2. The percentage of EE is affected by the hydrophilic and lipophilic nature of bioactive ingredients and the intensity of their interaction with the phospholipids. Also, the amount of the utilized water, membrane fluidity, and surface area of nanoparticles impact EE%64,65. EE% increases along with enhancing the amount of Lecithin, a possible reason is that EE is in line with the size of nanocarriers so larger nanoliposomes provide an appropriate volume for more entrapment of BV66. Results expressed that the entrapment efficiency escalated along with declining cholesterol concentration, which is consistent with previous studies. A possible reason why EE% increased with degreasing cholesterol concentration is that cholesterol enhances the hydrophobicity and stability of the bilayer and decreases the amount of permeability, which causes the hydrophobic drug to deliver into bilayers in an efficient way67,68.

The EE percentages of unmodified nanoliposomes loaded with BV reached 93.90 ± 1.25% whereas the EE percentages of BV-loaded into modified nanoliposomes boosted and reached 96.74 ± 1.49%, as shown in Table 1. According to Fig. 1C, the modification of nanocarriers leads to an increase in the amount of EE%. This better efficiency of PEGylated-Liposomes could be due to the long chain arms of PEG. PEG helps higher interaction and efficient immobilization of the drug with the surface of modified Liposomes69.

Figure 1D,E,F exhibits the comparison of the associated particle size distribution determined by using the DLS method between blank liposomes (Fig. 1D), Lipo-BV (Fig. 1E), and PEG-Lipo-BV (Fig. 1F). The result of the particle size distribution supported the PDI result. The obtained DLS indicated a single peak with a small Polydispersity index which signifies the insignificant aggregation of PEG-Lipo-BV in aqueous solution.

Field emission–SEM images of the optimized formulations for blank liposomes and liposome-loaded bee venom are shown in Fig. 2A,B. According to the SEM imaging results, the vesicle size of the blank liposome (127 nm) was smaller than that of the Lipo-BV vesicle (171 nm). Moreover, the SEM photograph of the PEG-BV-loaded liposome (Fig. 2C) demonstrated a more homogeneous distribution, a smooth surface, and a spherical shape without any aggregation than other samples. The size of PEG-Lipo-BV was estimated at 151 nm. The size distribution of nanocarriers obtained by DLS showed that the size of the functionalized liposomes containing BV was ∼175 nm (Fig. 1F). Results indicated that the size of liposomes achieved by DLS data was larger than that achieved by SEM. The size of nanoparticles was assessed using the Dynamic Light Scattering (DLS) method, which involved analyzing hydrated vesicles. This measurement was conducted in the presence of water, resulting in the samples exhibiting larger sizes compared to the dry vesicles observed under Scanning Electron Microscopy (SEM) or Transmission Electron Microscopy (TEM)70. TEM was applied to assess the morphology of optimum liposomes. Figure 2D–F illustrates the internal structure of blank liposome, Lipo-BV, and PEG-Lipo-BV, respectively. The size of the blank liposome is about 130 nm (Fig. 2D). Also, Lipo-BV (Fig. 2E) and PEG-Lipo-BV (Fig. 2F) are approximately 170 nm and 150 nm, respectively. The image shows a spherical shape of optimum liposomes. Furthermore, the rigid boundaries of the liposomes’ structure can be seen. After coating Liposomes with PEG, the size of formulations decreased. Its reason can be due to the affinity between PEG and the bilayer of Liposomes. Physical bonding between PEG and Liposomes, such as hydrogen bonding, can increase the cohesion of liposomes, so particle size can decrease by63,70.

SEM, TEM of coated and uncoated liposomes, (A) SEM of blank Lipo, (B) Lipo-BV, and (C) Lipo-BV-PEG and TEM of (D) blank Lipo, (E) Lipo-BV, and (F) Lipo-BV-PEG.

Lipid content (µmol), and Lecithin: Cholesterol: PEG (mol ratio) were evaluated as individual variables (Table S1). In contrast, particle size, and entrapment efficiency (EE) were dependent factors in the optimization studies. Table S2 shows the results of the CCD experiments. The size of BV-Lipo-PEG ranged between 165.3 and 210.4 nm and EE of BV-Lipo-PEG ranged between 80.49 and 95.73%. The analysis of variance for particle size and EE is shown in Table S3. The polynomial response was modified in the quadratic model. It shows that the individual parameters A, and B could all have a significant impact on particle size and EE. As presented in Table S4, the particle size regression model shows the increased influence of individual variable A and the possible decreased effect of B variables on particle size, and the increased influence of individual variable A and B on EE. Table S5 offers the necessary criteria for responses. The ideal preparation was successfully formulated (Table S5). The particle size, and EE for optimum preparations were investigated and found to be 177.4, and 95.73, respectively, for BV-Lipo-PEG formulation. A possible explanation is that increase in the amount of lipid from 200 to 300 µmoL results in increase of the area occupied by lipid molecules as an increase in the membrane fluidity and the size of the liposomes are observed71,72.

Figure 3 presents the chemical structure of lecithin, cholesterol, naked liposome, free BV, liposome-BV, and PEGylate-liposome-BV. The FTIR pattern for lecithin (Fig. 3a) shows the wave numbers 2923–2810 cm-1, 1740 cm-1, 1210–1140 cm-1, and 1465 cm-1 attributed to Alkanes groups stretching, Aliphatic ester stretching, PO2 groups stretching, and CH2 stretching, respectively. Cholesterol (Fig. 3b) displayed peaks at 3400 cm-1 and 3000–2850 cm-1 due to 1 O–H stretching and C–H stretching, respectively. Also, the peaks at 1463 cm-1 and 1054 cm-1 were attributed to C–H bending and C–O stretching, respectively. C–H stretching from 3124 to 2863 cm-1 belongs to cholesterol. Naked liposomes (Fig. 3c) showed the characteristic band peak at 1749 cm-1 due to CH2 bending vibration. Also peaks at 3478 and 1645 cm-1 due to O–H stretching and bending vibration. The wave number 1749 cm-1 is attributed to C=O stretching. Bee venom as a pure drug (Fig. 3d) showed the characteristic band peak at 3060 and 3392 cm-1 due to N–H stretching vibration and, 1386 and 1454 cm-1 due to COO-. Vibration on a broad band 1240–1290 cm-1 due to Amid III, 1541 cm-1 due to Amid II, and 1653 cm-1 due to Amid I. Also, a peak at 617 cm-1 was exhibited due to primarily amid O=C–N bending. The formulation of the drug encapsulated in liposomes (Lipo-BV) (Fig. 3e) shows the peaks at the wave number 804 cm-1 C–N–C stretching, 1533cm-1 Amid II stretching, and 1116cm-1 amid III stretching vibrations. Compared to free BV, all peaks for Lipo-BV were observed at lower wavelengths, which confirms loading BV in liposomes. In the FTIR spectra of BV-Lipo-PEG (Fig. 3f), C-O bonding related to PEG appeared at 1299 cm-1, which confirms the presence of PEG in liposomes.

FTIR spectra of (a) lecithin, (b) cholesterol, (c) liposome, (d) free bee venom, (e) Lipo-BV, (f) BV-Lipo-PEG, and (g) PEG.

The release of bee venom from uncoated and coated liposomes was studied at various pH values (physiological pH (~ 7.4), tumoral microenvironment (~ 5.4). As shown in Fig. 4A, the burst-primary release within 10 h is ascribed to diffusion of BV from the outer layer of the liposomes, then all formulations displayed a steady release from 10 to 72 h.

In vitro release of (A) BV from the free drug, Lipo-BV, and BV- Lipo-PEG at pH 5.4 and 7.4, and (B, C, D) the physical stability of Lipo-BV and (E, F, D) BV -Lipo-PEG at 4 and 25 °C after 90 days (Mean ± SD, n = 3).

The release rate of the free bee venom was 88% in the first 10 h, which remained almost steady up to 72 h, when the whole of the drug was released. Free and adsorbed BV on the liposome is the main reason for primary burst. After the immediate release of drugs into the resolved medium, a primary burst occurs. While the drug release rate of the optimum Lipo-BV was 55% at pH 7.4, that of optimum PEG-Lipo-BV reached 40% after72h. It seems that the entrapment of bee venom in liposomes could prevent burst release. In addition, after coating BV-loaded Lipo with PEG, a significant reduction was observed in burst release at both pH (7.4, and 5.4). Its reason is that the barrier layer is provided for drug diffusion after coating liposomes73. Also, the release rates of BV from liposomal nanoparticles coated with PEG at physiological pH were significantly lower than that of formulations at pathological (cancerous) pH, 65% and 50% after 24 h, respectively. Also, the same result was observed for Lipo-BV (the release rates of BV from liposomal nanoparticles at physiological pH were significantly lower than that of cancerous pH). The reason for this is that liposomes show different behavior under different conditions. Liposome nanoparticles swell and break in acidic conditions. However, liposomes could efficiently hold drugs under physiological conditions. Moreover, drug release at physiological pH was lower than tumoral microenvironment as the drug and the surfactant interact with each other by electrostatic interaction74.

The release kinetics of free BV, Lipo-BV, and BV-Lipo-PEG formulation at various pH were studied using different kinetic models (Table 2). The value of R2 determines the best model for each formulation. The sample with the highest value of R2 was chosen as a suitable model for the release mechanism. As can be seen in Table 2, the first-order model is considered as the best-fitting model for free BV whereas Higuchi and Korsmeyer-Peppas model was defined as the best model at pH = 7.4 and pH = 5.4, respectively. Also, the best model for Lipo-BV-PEG at pH = 7.4 was Higuchi. In the Korsmeyer-Peppas kinetic model, the amount of observed n for all samples was 0.43 < n < 0.85, which indicates the Anomalous transport mechanism for determining the release of BV molecules from prepared formulations.

The physical stability of coated and non-coated Lipo-BV formulations was evaluated by characterization of vesicle size, Polydispersity index (PDI), and entrapment efficiency after storage at 4 °C and 25 °C for 90 days. As seen (Fig. 4), an increase in the percentage of size and PDI, and a decrease in entrapment efficiency were shown during storage time because the leakage of the drug occurred for the liposomal formulations. Mean vesicle size, Polydispersity index, and entrapment efficiency of non-coated liposomes (Fig. 4B–D) showed more changes after 60 days compared to coated liposomes (Fig. 4E–G). So, the stability of the coated samples stored at 4 °C and 25 °C is greater than the non-coted samples. Furthermore, the stability of the samples stored at 4 °C is higher compared to the non-coated samples stored at 25 °C. This trend is because of the higher stability of hydrophobic liposomes stored at low temperatures since liposomal formulations swell and break at high temperatures, leading to a significant boost in the size and PDI of liposomes and a reduction in entrapment efficiency. According to Fig. 4A,B,C after 30, 60, and 90 days, the size of Lipo-BV was significant (p < 0.001). However, the EE% of Lipo-BV was significant just after 90 days (p < 0.001). Also, the results showed that the size of Lipo-BV-PEG, PDI, and the EE% of Lipo-BV-PEG was significant just after 90 days (p < 0.001). In total, the vesicle size of the decorated drug-loaded liposomes (389.43 nm) was found to be larger than undecorated drug-loaded liposomes (329.92 nm) at 4 °C after 90 days. Also, the PDI of non-coated liposomes and liposomes coated with PEG were observed to be 0.319 and 0.287, respectively. The rise in particle size following the coating of drug-loaded liposomes with PEG is attributed to an increase in viscosity, leading to an increase in particle size compared to drug-loaded liposomes lacking PEG. Additionally, the Polydispersity Index (PDI) of non-coated liposomes is lower than that of coated liposomes. This outcome could arise from alterations in vesicle size during the coupling process.

The cytotoxicity of blank Lipo, free BV, Lipo-BV, and BV-Lipo-PEG was investigated by MTT assay against A549 cell lines. According to Fig. 5A,B, the cytotoxicity of free BV, Lipo-BV, and BV-Lipo-PEG was significant (p < 0.001) during 48 h (Fig. 5A) and 72 h (Fig. 5B) of treatment toward A549 cell lines. However, the cytotoxicity of blank liposomes was insignificant (p < 0.05). Also, the investigation of the sample’s effect on A549 cells indicated that the percentage of cell viability in concentrations of 1.56, 3.12, 6.25, 12.50, 25, and 50 μg/mL decreased significantly through 48 to 72h. Based on Fig. 5C, the half-maximal inhibitory concentration (IC50) amount of BV-Lipo-PEG (11.74 (48 h), 5.89 (72 h)), Lipo-BV (22.86(48 h), 10.86 (72 h)), and free BV (38.56 (48 h), 29.81 (72 h)) showed a statically significant decrease (p < 0.001) against A549 cell line compared to blank Lipo (456.65 (48 h), 421.36 (72 h)) (p < 0.05). Some literature shows that bee venom can increase the cytotoxicity against cancer cells as bee venom has compounds such as calmodulin inhibitors, which have the capability of enhancing the intracellular distribution of anti-cancer agents through inhabitation of the exterior transport. As a result, the cytotoxicity of samples increases directly against tumor cells12,75. Also, results indicate that BV-Lipo-PEG is more cytotoxic on both cancer cell lines after 48 and 72 h of treatment. BV-Lipo coated with PEG exhibited more cytotoxicity on cancer cells compared to combined non-coated BV-Lipo. The use of BV-containing PEGylated liposomes leads to the assembly of PEGylated-liposomes in the solid tumor so a high percentage of BV is released from PEGylated due to permeability and retention effect leading to an increase of cytotoxicity on tumor cells76.

In vitro cytotoxic impacts of (A) blank Lipo, free BV, Lipo-BV, and BV-Lipo-PEG in A549 cell line with different concentrations after 48h. (B) In vitro cytotoxic impacts blank Lipo, free BV, Lipo-BV, and BV-Lipo-PEG in A549 cell line with different concentrations in A549 cell line with different concentrations after 72h, and (C) Comparison between IC50 of all samples in A549 cell line in 48 and 72 h (Data are represented as mean ± SD and n = 3; p < 0.0001****, p < 0.001***, p < 0.01 **, p < 0.05 *.

As can be seen from Fig. 6A, the toxicity of BV-Lipo-PEG and BV-Lipo toward the HFF cell line enhanced with increasing the concentration of both samples, leading to a reduction of cell viability. According to Fig. 6A after 48h, the cytotoxicity of blank liposome, free BV, Lipo-BV, and BV-Lipo-PEG was significant (p < 0.001) towards HFF cell lines. Pristine liposome has the best viability comparison with other formulations. These data for HFF indicate that the high cytotoxicity of BV-Lipo is because of the release of BV inside the cells, and it is not related to the release of components from empty liposomal formulation77. In general, the possible anti-cancer function of Lipo-BV could be attributed to various factors, such as enhancing its delivery and release mechanisms, ensuring sustained release and efficacy over an extended period, its biological activity level, boosting immune response and cytokine production, and enhancing its bioactivity against cancerous cells78,79,80,81,82.

(A) In vitro cytotoxic impacts of blank Lipo, free BV, Lipo-BV, and BV-Lipo-PEG in HFF cell line with different concentrations after 48h. (B) In vitro cytotoxic impacts of blank Lipo, free BV, Lipo-BV, and BV-Lipo-PEG in MCF-7 cell line with different concentrations after 48h. (C) In vitro cytotoxic impacts of blank Lipo, free BV, Lipo-BV, and BV-Lipo-PEG in HepG2 cell line with different concentrations after 48h (Data are represented as mean ± SD and n = 3; p < 0.0001****, p < 0.001***, p < 0.01 **, p < 0.05 *).

The cytotoxicity results of the samples against the two cell lines (MCF-7 and HepG-2) compared to A2780 are given in Fig. 6B,C. These results indicated that BV-Lipo-PEG showed strong anticancer activity against the two tested cell lines although it was more effective against the MCF-7 cell line compared to HepG-2 and A2780. The viability was increased significantly by decreasing the drug concentration in both cancer cell lines and in all samples. The recorded IC50 of BV (for 48 h) was 17.52, 11.16, and 38.56 μg/ml for HepG-2, MCF-7, and A2780, respectively. However, the IC50 for Lipo-BV was 9.57, 5.83, and 22.86 μg/ml for HepG2, MCF-7, and A2780, respectively, and the IC50 for BV-Lipo-PEG recorded 5.58, 3.88, and 11.47 μg/ml for the HepG-2, MCF-7, and A2780 cell lines, respectively.

Since the examination of apoptotic activity is essential in the development of anticancer agents and is beneficial in the evacuation of drug delivery systems, flow cytometry was carried out to evaluate the early and late apoptosis or necrosis induced in tumor cell lines. Following the double staining of cells with annexin V-FTTC (fluorescein thiocyanate-labeled annexin V) and PI (propidium iodide), the quantitative measurement of apoptosis in lung cancer cells (A549 cells) was conducted using flow cytometry. Treatment of the A549 cells with Lipo, Free BV, Lipo-BV, and Lipo-BV@PEG (Fig. 7A) resulted in the induction of apoptosis in both types of breast cancer cells. The results of apoptotic studies for all samples are demonstrated in Fig. 7B. Biocompatibility of pristine liposomes was higher than in other samples as a result of the apoptosis rate being very low for lung cell lines treated with pristine liposomes. Totally, According to Fig. 7B, the apoptotic activity of free BV, Lipo-BV, and BV-Lipo-PEG was significantly higher (p < 0.001) than blank liposome and the control group (p < 0.05) towards A549 cell lines in. Moreover, free BV, Lipo-BV, and PEG-Lipo-BV resulted in a significantly greater percentage of cells in the late phase compared to blank Lipo and control group (p < 0.001). Meanwhile, results of the late phase indicate that the percentage of cells in PEG-Lipo-BV is greater than those of free BV, and Lipo-BV (p < 0.001). The Lipo-BV has led to 35.9% apoptosis (21.8% early apoptosis and 14.8% late apoptosis) and 3.59% necrosis in A549 cells. Nevertheless, PEG-Lipo-BV has led to 45.65% apoptosis (31.35% early apoptosis and 14.3% late apoptosis) and 2.9% necrosis in the treated A549 cells. In this study, we used BV involved in preventing tumor cell growth and metastasis and inducing cancer cell apoptosis83. Additionally, BV loaded in coated nanocarriers improves the apoptosis rate as this nanocarrier system causes drug molecules to be delivered into cancer cells with higher dosages84,85.

(A) The effects of control, and different formulations on vehicle Lipo, Free BV, Lipo-BV, and Lipo-BV@PEG of A549 cells that had undergone 48 h of treatment. Q1 (necrotic cells), Q2 (late apoptosis), Q3 (early apoptosis), and Q4 (alive cells). (B) Flow cytometric analysis of A549 cells after treatment with IC50 concentration of vehicle Lipo, Free BV, Lipo-BV, and Lipo-BV@PEG formulations; Means and standard deviations are represented by the data (n = 3). For all charts, ***: p < 0.001; **: p < 0.01; *: p < 0.05.

To estimate the effects of loading nanoliposomes with BV and coating BV-loaded nanoliposomes with PEG on the cell cycle, A549 cell lines were treated with empty Lipo, free BV, BV-Lipo, and BV-Lipo-PEG at their IC50 concentrations and examined by flow cytometry (Fig. 8A). During the cell cycle, all cells enter the following stages from G/S to G2/ M. Treatment with free BV, BV-Lipo, and BV-Lipo-PEG increased the proportion of sub-G1 phase cells (Fig. 8B). However, sub-G1 phase cells increased significantly after treatment with BV-Lipo and BV-Lipo-PEG compared to free BV, which indicates apitoxin-loaded-PEG-liposome enhances the anticancer potential of the chemotherapeutic agent. In contrast, treatment with the empty Lipo did not cause any significant changes in the cell cycle distribution under similar conditions (5.16% for empty Lipo, 18.98% for free BV, 33.13% for BV-Lipo, and 39.11% for BV-Lipo-PEG). Moreover, the number of cells in G2 was less after treatment with PEG-liposomal BV (2.65%) compared to the control group (p < 0.001). According to a previous study, BV inhibits cancer growth at the cell cycle level. This inhibition occurs in the sub-G1 phase at G0/G1 phase86,87. Furthermore, the results show that entrapment of BV in nanoliposomes and coating BV-loaded liposomes with PEG helps multiple targets during different cell cycle phases and shows lower doses of the drug are used in these liposomal structures88.

(A) Histograms showing DNA content of cell cycle progression of all samples in A549 cell lines; (B) Cell cycle distribution for A549 cells after treatment with IC50 concentration of vehicle Lipo, Free BV, Lipo-BV, and Lipo-BV@PEG formulations. Means and standard deviations are represented by the data (n = 3). For all charts, ***: p < 0.001; **: p < 0.01; *: p < 0.05.

The principal branches of gene expression are proapoptotic and antiapoptotic. To investigate the effectiveness of different liposomal formulations (Lipo, BV, BV-Lipo, and PEG-Lipo-BV), Gene expression levels in the A549 cell lines were estimated by real-time PCR The selected five genes were Caspase 3, Caspase 9, MMP-2, MMP-9, and Cyclin E in A549 (Fig. 9A–E). Free BV, BV-Lipo, and PEG-Lipo-BV resulted in significant upregulation in the expression levels of Caspase 3, Caspase 9 (Fig. 9A,B) compared to blank liposomes and the control group (p < 0.001) in A549 cells. Caspase 3 and Caspase 9 genes showed higher activity in the presence of the PEG-Lipo BV formulation compared to Free BV and BV-Lipo. Also, the expression levels of Caspase 3 and Caspase 9 genes indicated that the increase of these genes in A549 cell lines treated with BV-Lipo was more than those in cells exposed to the free BV (p < 0.001).

(A) Expression levels of Caspase 3 and (B) Caspase 9 genes in A549 cells, and (C) Expression levels of MMP-2 and (D) MMP-9 genes in A549 cells, and Expression levels of Cyclin E genes in A549 cells were exposed blank Lipo, Free BV, Lipo-BV, and Lipo-BV-PEG formulations. Data represent means ± standard deviations (n = 3). For all charts, ***: p < 0.001; **: p < 0.01; *: p < 0.05.

For all samples, the expression levels of MMP-2 and MMP-9 in the A549 cancer cells boosted significantly compared to the control group (p < 0.001; Fig. 9C,D). The reduction of the expression of Cyclin E (Fig. 9E) in the presence of BV-Lipo and free BV formulations was milder than that of the BV-Lipo-PEG formulation (p < 0.001). Moreover, the expression level of Cyclin E for cells treated with BV-Lipo was less than that of cells treated with Free BV (p < 0.001). Additionally, the expression levels of MMP-2 and MMP-9 in BV-Lipo-PEG were lower than those in the BV-Lipo (p < 0.001); MMP-2 and MMP-9 genes indicated more reduction after treatment with BV-Lipo compared to free BV (p < 0.001).

The release of mitochondrial cytochrome c can both affect and does not affect Caspase 3 activation. However, the process of Caspase 9 activation is reliant on mitochondrial-cytochrome c. The investigation of apoptosis is necessary for numerous processes because apoptotic chromatin condensation and DNA fragmentation can occur through Caspase 3 and Caspase 953. In this study, BV was encapsulated in nanocarriers to enhance delivery to cancer cells. Bee venom can prevent cancer growth by activation of Caspase 9 and Caspase 389. The inhabitation of MMPs is imperative because they can mediate extracellular matrix degradation and influence cancerous cell proliferation, inflammatory disorder, invasion, and angiogenesis90. The studies show that BV leads to the block of the NF-kB and PI3K/Akt/mTOR pathway, which helps to inhibit the EGF-induced MMP-9 expression91.

Liposomal nanocarriers coated with PEG were prepared using a thin film layer to deliver BV simultaneously for increasing the chemotherapeutic effect of lung cancer. The physical characterization of functionalized nanoscale liposomes indicated that BV-Lipo-PEG had higher stability and lower changes in entrapment efficiency at physiological pH compared to non-coated liposomes. The BV release from liposomal formulations in physiological pH (7.4) was lower than in acidic conditions (pH = 5.4). The cellular results also exhibited that the controlled release of the BV from functionalized Liposomes results in high toxicity on A549 cancer cells over 48 and 72 h. The results of the late phase indicate that the percentage of cells in PEG-Lipo-BV is greater than those of free BV, Lipo-BV In this study, BV was encapsulated in nanocarriers to enhance delivery to cancer cells. Bee venom can prevent cancer growth by activation of Caspase 9 and Caspase 3. Moreover, BV-Lipo-PEG demonstrated significant apoptosis of the tested cancer cells. Lipo-BV, and PEG-Lipo-BV showed a selective cytotoxic effect on cancer cells since they were more effective against A549 cell lines. Therefore, this liposomal drug delivery system could be used as a promising nanocarrier for lung cancer treatment.

The data generated and/or analyzed during the current study are not publicly available for legal/ethical reasons but are available from the corresponding author on reasonable request.

Yoder, L. H. Lung cancer epidemiology. Medsurg. Nurs. 15, 171 (2006).

PubMed Google Scholar

Rahib, L., Wehner, M. R., Matrisian, L. M. & Nead, K. T. Estimated projection of US cancer incidence and death to 2040. JAMA Netw. Open 4, e214708–e214708 (2021).

Article PubMed PubMed Central Google Scholar

Abdulbaqi, I. M. et al. Pulmonary delivery of anticancer drugs via lipid-based nanocarriers for the treatment of lung cancer: An update. Pharmaceuticals 14, 725 (2021).

Article CAS PubMed PubMed Central Google Scholar

Timmerman, R. et al. Stereotactic body radiation therapy for inoperable early stage lung cancer. Jama 303, 1070–1076 (2010).

Article CAS PubMed PubMed Central Google Scholar

Trüeb, R. M. in Seminars in cutaneous medicine and surgery. 11–14 (No longer published by Elsevier).

Zadeh, E. S. et al. Smart pH-responsive magnetic graphene quantum dots nanocarriers for anticancer drug delivery of curcumin. Mater. Chem. Phys. 297, 127336 (2023).

Article Google Scholar

Raghuraman, H. & Chattopadhyay, A. Melittin: A membrane-active peptide with diverse functions. Biosci. Rep. 27, 189–223 (2007).

Article CAS PubMed Google Scholar

Zhang, Z., Zhang, H., Peng, T., Li, D. & Xu, J. Melittin suppresses cathepsin S-induced invasion and angiogenesis via blocking of the VEGF-A/VEGFR-2/MEK1/ERK1/2 pathway in human hepatocellular carcinoma. Oncol. Lett. 11, 610–618 (2016).

Article CAS PubMed Google Scholar

Kwon, Y.-B. et al. Bee venom injection into an acupuncture point reduces arthritis associated edema and nociceptive responses. Pain 90, 271–280 (2001).

Article PubMed Google Scholar

Pascoal, A. et al. An overview of the bioactive compounds, therapeutic properties and toxic effects of apitoxin. Food Chem. Toxicol. 134, 1–11 (2019).

Article Google Scholar

Song, H. S., Ko, M. S., Jo, Y. S., Whang, W. K. & Sim, S. S. Inhibitory effect of acteoside on melittin-induced catecholamine exocytosis through inhibition of Ca2+-dependent phospholipase A2 and extracellular Ca2+ influx in PC12 cells. Arch. Pharm. Res. 38, 1913–1920 (2015).

Article CAS PubMed Google Scholar

Oršolić, N. Bee venom in cancer therapy. Cancer Metastasis Rev. 31, 173–194 (2012).

Article PubMed Google Scholar

Gurunathan, S., Qasim, M., Kang, M.-H. & Kim, J.-H. Role and therapeutic potential of melatonin in various type of cancers. OncoTargets Ther. 14, 2019 (2021).

Article Google Scholar

Mohamed, A. F., El-Fiky, A. A., Hassan, O. S. & El-Megharbel, N. A. Controlled study of application of bee products as new medicine against breast and lung cancer. Int. J. Adv. Res. 4(6), 392–402 (2016).

Article CAS Google Scholar

Heinen, T. E. & da Veiga, A. B. Arthropod venoms and cancer. Toxicon 57, 497–511 (2011).

Article CAS PubMed Google Scholar

Ip, S.-W. et al. Bee venom induced cell cycle arrest and apoptosis in human cervical epidermoid carcinoma Ca Ski cells. Anticancer Res. 28, 833–842 (2008).

CAS PubMed Google Scholar

Lee, W.-R. et al. Protective effects of melittin on transforming growth factor-β1 injury to hepatocytes via anti-apoptotic mechanism. Toxicol. Appl. Pharmacol. 256, 209–215 (2011).

Article CAS PubMed Google Scholar

Holle, L. et al. A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo. Int. J. Oncol. 22, 93–98 (2003).

CAS PubMed Google Scholar

Moreno, M. & Giralt, E. Three valuable peptides from bee and wasp venoms for therapeutic and biotechnological use: Melittin, apamin and mastoparan. Toxins 7, 1126–1150 (2015).

Article CAS PubMed PubMed Central Google Scholar

Kasravi, M. et al. MMP inhibition as a novel strategy for extracellular matrix preservation during whole liver decellularization. Biomater. Adv. 156, 213710 (2024).

Article CAS PubMed Google Scholar

Hussain, Z. et al. PEGylation: A promising strategy to overcome challenges to cancer-targeted nanomedicines: A review of challenges to clinical transition and promising resolution. Drug Deliv. Trans. Res. 9, 721–734 (2019).

Article CAS Google Scholar

Estanqueiro, M., Amaral, M. H., Conceição, J. & Lobo, J. M. Nanotechnological carriers for cancer chemotherapy: The state of the art. Colloids Surf. B Biointerfaces 126, 631–648 (2015).

Article CAS PubMed Google Scholar

Aufschnaiter, A. et al. Apitoxin and its components against cancer, neurodegeneration and rheumatoid arthritis: Limitations and possibilities. Toxins 12, 66 (2020).

Article CAS PubMed PubMed Central Google Scholar

Hojabri, M. et al. Wet-spinnability and crosslinked Fiber properties of alginate/hydroxyethyl cellulose with varied proportion for potential use in tendon tissue engineering. Int. J. Biol. Macromol. 240, 124492 (2023).

Article CAS PubMed Google Scholar

Tokmedash, M. A., Zadeh, E. S., Balouchi, E. N., Salehi, Z. & Ardestani, M. S. Synthesis of smart carriers based on tryptophan-functionalized magnetic nanoparticles and its application in 5-fluorouracil delivery. Biomed. Mater. 17, 045026 (2022).

Article ADS CAS Google Scholar

Akbarzadeh, A. et al. Liposome: Classification, preparation, and applications. Nanoscale Res. Lett. 8, 1–9 (2013).

Article Google Scholar

Egbaria, K. & Weiner, N. Liposomes as a topical drug delivery system. Adv. Drug Deliv. Rev. 5, 287–300 (1990).

Article CAS Google Scholar

Zylberberg, C. & Matosevic, S. Pharmaceutical liposomal drug delivery: A review of new delivery systems and a look at the regulatory landscape. Drug Deliv. 23, 3319–3329 (2016).

Article CAS PubMed Google Scholar

Salimi, A. Liposomes as a novel drug delivery system: Fundamental and pharmaceutical application. Asian J. Pharm. https://doi.org/10.22377/ajp.v12i01.2037 (2018).

Article Google Scholar

Bhuimali, M. et al. Evaluation of liposomes for targeted drug delivery in lung cancer treatment. Int. J. Polym. Mater. Polym. Biomater. 73, 395–404 (2024).

Article CAS Google Scholar

Ali, M. S. et al. Advancements in 5-fluorouracil-loaded liposomal nanosystems: A comprehensive review on recent innovations in nanomedicine for cancer therapy. J. Drug Deliv. Sci. Technol. 96, 105730. https://doi.org/10.1016/j.jddst.2024.105730 (2024).

Article CAS Google Scholar

Sanati, M. et al. Advances in liposome-based delivery of RNA therapeutics for cancer treatment. Progress Mol. Biol. Trans. Sci. 204, 177–218 (2024).

Article Google Scholar

Zhang, Y. et al. Self‐assembled nanocarrier delivery systems for bioactive compounds. Small https://doi.org/10.1002/smll.202310838 (2024).

Article PubMed Google Scholar

Binaymotlagh, R., Hajareh Haghighi, F., Chronopoulou, L. & Palocci, C. J. G. Liposome-hydrogel composites for controlled drug delivery applications. Gels. 10, 284 (2024).

Article CAS PubMed PubMed Central Google Scholar

Tan, X., Liu, Y., Wu, X., Geng, M. & Teng, F. Layer-by-layer self-assembled liposomes prepared using sodium alginate and chitosan: Insights into vesicle characteristics and physicochemical stability. Food Hydrocoll. 149, 109606 (2024).

Article CAS Google Scholar

Wei, Y. et al. Enzyme-responsive liposomes for controlled drug release. Drug Discov. Today 29(7), 104014. https://doi.org/10.1016/j.drudis.2024.104014 (2024).

Article CAS PubMed Google Scholar

Tzror-Azankot, C., Anaki, A., Sadan, T., Motiei, M. & Popovtzer, R. in Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XXI. 41–49 (SPIE).

Shah, H. et al. pH-responsive liposomes of dioleoyl phosphatidylethanolamine and cholesteryl hemisuccinate for the enhanced anticancer efficacy of cisplatin. Pharmaceutics 14, 129 (2022).

Article CAS PubMed PubMed Central Google Scholar

Yang, Q. et al. Advances in the study of liposomes gel with stimulus responsiveness in disease treatment. J. Clust. Sci. 35, 701–714 (2024).

Article CAS Google Scholar

Karanth, H. & Murthy, R. pH-Sensitive liposomes-principle and application in cancer therapy. J. Pharm. Pharmacol. 59, 469–483 (2007).

Article CAS PubMed Google Scholar

Cuomo, F. et al. pH-responsive liposome-templated polyelectrolyte nanocapsules. Soft Matter 8, 4415–4420 (2012).

Article ADS CAS Google Scholar

Chen, S., Morrison, G., Liu, W., Kaur, A. & Chen, R. A pH-responsive, endosomolytic liposome functionalized with membrane-anchoring, comb-like pseudopeptides for enhanced intracellular delivery and cancer treatment. Biomater. Sci. 10, 6718–6730 (2022).

Article CAS PubMed Google Scholar

Shao, X.-R. et al. Effects of micro-environmental pH of liposome on chemical stability of loaded drug. Nanoscale Res. Lett. 12, 1–8 (2017).

Article ADS Google Scholar

Shinitzky, M. & Barenholz, Y. Dynamics of the hydrocarbon layer in liposomes of lecithin and sphingomyelin containing dicetylphosphate. J. Biol. Chem. 249, 2652–2657 (1974).

Article CAS PubMed Google Scholar

Awasthi, V., Garcia, D., Goins, B. & Phillips, W. Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits. Int. J. Pharm. 253, 121–132 (2003).

Article CAS PubMed Google Scholar

Caddeo, C. et al. Stability, biocompatibility and antioxidant activity of PEG-modified liposomes containing resveratrol. Int. J. Pharm. 538, 40–47 (2018).

Article CAS PubMed Google Scholar

Minato, S., Iwanaga, K., Kakemi, M., Yamashita, S. & Oku, N. Application of polyethyleneglycol (PEG)-modified liposomes for oral vaccine: Effect of lipid dose on systemic and mucosal immunity. J. Controll. Release 89, 189–197 (2003).

Article CAS Google Scholar

Trucillo, P. & Reverchon, E. Production of PEG-coated liposomes using a continuous supercritical assisted process. J. Supercrit. Fluids 167, 105048 (2021).

Article CAS Google Scholar

Rustad, E. A. et al. The pH-responsive liposomes—the effect of PEGylation on release kinetics and cellular uptake in glioblastoma cells. Pharmaceutics 14, 1125 (2022).

Article CAS PubMed PubMed Central Google Scholar

Shen, Z., Ye, H., Kröger, M. & Li, Y. J. N. Aggregation of polyethylene glycol polymers suppresses receptor-mediated endocytosis of PEGylated liposomes. Nanoscale. 10, 4545–4560 (2018).

Article CAS PubMed Google Scholar

Pourmoghadasiyan, B. et al. Nanosized paclitaxel-loaded niosomes: Formulation, in vitro cytotoxicity, and apoptosis gene expression in breast cancer cell lines. Mol. Biol. Rep. 49, 3597–3608 (2022).

Article CAS PubMed Google Scholar

Choi, K. Y. et al. Smart nanocarrier based on PEGylated hyaluronic acid for cancer therapy. ACS Nano. 5, 8591–8599 (2011).

Article CAS PubMed Google Scholar

Akbarzadeh, I. et al. Niosomal delivery of simvastatin to MDA-MB-231 cancer cells. Drug Dev. Ind. Pharm. 46, 1535–1549 (2020).

Article CAS PubMed Google Scholar

Ahmadi, S. et al. In vitro development of controlled-release nanoniosomes for improved delivery and anticancer activity of letrozole for breast cancer treatment. Int. J. Nanomed. 17, 6233–6255 (2022).

Article Google Scholar

Karimifard, S. et al. pH-responsive chitosan-adorned niosome nanocarriers for co-delivery of drugs for breast cancer therapy. ACS Appl. Nano Mater. 5, 8811–8825 (2022).

Article CAS Google Scholar

Lu, W. et al. Free iron catalyzes oxidative damage to hematopoietic cells/mesenchymal stem cells in vitro and suppresses hematopoiesis in iron overload patients. Eur. J. Haematol. 91, 249–261 (2013).

Article CAS PubMed Google Scholar

Ståhlberg, A., Zoric, N., Åman, P. & Kubista, M. Quantitative real-time PCR for cancer detection: The lymphoma case. Expert Rev. Mol. Diagn. 5, 221–230 (2005).

Article PubMed Google Scholar

Shahi, S., Sonwane, U., Zadbuke, N. & Tadwee, I. Design and development of diphenhydramine hydrochloride topical liposomal drug delivery system. Int. J. Pharm. Pharm. Sci. 5, 534–542 (2013).

CAS Google Scholar

Zhao, L., Temelli, F., Curtis, J. M. & Chen, L. J. F. R. I. Preparation of liposomes using supercritical carbon dioxide technology: Effects of phospholipids and sterols. Food Res. Int. 77, 63–72 (2015).

Article CAS Google Scholar

Moghddam, S. R. M. et al. Formulation and optimization of niosomes for topical diacerein delivery using 3-factor, 3-level Box-Behnken design for the management of psoriasis. Mater. Sci. Eng. C 69, 789–797 (2016).

Article CAS Google Scholar

Zook, J. M. & Vreeland, W. N. Effects of temperature, acyl chain length, and flow-rate ratio on liposome formation and size in a microfluidic hydrodynamic focusing device. Soft Matter. 6, 1352–1360 (2010).

Article ADS CAS Google Scholar

Sankhyan, A. & Pawar, P. K. Metformin loaded non-ionic surfactant vesicles: optimization of formulation, effect of process variables and characterization. DARU J. Pharm. Sci. 21, 1–8 (2013).

Article Google Scholar

Cheng, R., Xu, T., Wang, C. & Gan, C. The stabilization and antioxidant performances of coenzyme Q10-loaded niosomes coated by PEG and chitosan. J. Mol. Liq. 325, 115194 (2021).

Article CAS Google Scholar

Maherani, B., Arab-Tehrany, E., Kheirolomoom, A., Cleymand, F. & Linder, M. J. F. C. Influence of lipid composition on physicochemical properties of nanoliposomes encapsulating natural dipeptide antioxidant l-carnosine. Food Chem. 134, 632–640 (2012).

Article CAS PubMed Google Scholar

Maherani, B. et al. Optimization and characterization of liposome formulation by mixture design. Analyst. 137, 773–786 (2012).

Article ADS CAS PubMed Google Scholar

Nii, T. & Ishii, F. Encapsulation efficiency of water-soluble and insoluble drugs in liposomes prepared by the microencapsulation vesicle method. Int. J. Pharm. 298, 198–205 (2005).

Article CAS PubMed Google Scholar

Akbari, V., Abedi, D., Pardakhty, A. & Sadeghi-Aliabadi, H. Release studies on ciprofloxacin loaded non-ionic surfactant vesicles. Avicenna J. Med. Biotechnol. 7, 69 (2015).

PubMed PubMed Central Google Scholar

Deniz, A. et al. Celecoxib-loaded liposomes: Effect of cholesterol on encapsulation and in vitro release characteristics. Biosci. Rep. 30, 365–373 (2010).

Article CAS PubMed Google Scholar

Davarpanah, F., Khalili Yazdi, A., Barani, M., Mirzaei, M. & Torkzadeh-Mahani, M. Magnetic delivery of antitumor carboplatin by using PEGylated-Niosomes. DARU J. Pharm. Sci. 26, 57–64 (2018).

Article CAS Google Scholar

Dovlatabadi, S. Effects of kenaf filler reinforcement on mechanical properties of molded polypropylene composites: A particle size study. Polym. Renew. Resour. 11, 64–68 (2020).

Google Scholar

Moghddam, S. R. M., Ahad, A., Aqil, M., Imam, S. S. & Sultana, Y. Formulation and optimization of niosomes for topical diacerein delivery using 3-factor, 3-level Box-Behnken design for the management of psoriasis. Mater. Sci. Eng. C 69, 789–797 (2016).

Article CAS Google Scholar

Wang, Z. & He, X. Dynamics of vesicle formation from lipid droplets: Mechanism and controllability. J. Chem. Phys. 130, 094905 (2009).

Article ADS PubMed Google Scholar

Lim, E.-B., Haam, S. & Lee, S.-W. Sphingomyelin-based liposomes with different cholesterol contents and polydopamine coating as a controlled delivery system. Colloids Surf. A Physicochem. Eng. Asp. 618, 126447 (2021).

Article CAS Google Scholar

Nogueira, D. R. et al. In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles. Biomaterials. 34, 2758–2772 (2013).

Article CAS PubMed Google Scholar

Tsuruo, T., Iida, H., Tsukagoshi, S. & Sakurai, Y. Increased accumulation of vincristine and adriamycin in drug-resistant P388 tumor cells following incubation with calcium antagonists and calmodulin inhibitors. Cancer Res. 42, 4730–4733 (1982).

CAS PubMed Google Scholar

Lila, A. S. A., Doi, Y., Nakamura, K., Ishida, T. & Kiwada, H. Sequential administration with oxaliplatin-containing PEG-coated cationic liposomes promotes a significant delivery of subsequent dose into murine solid tumor. J. Controll. Release 142, 167–173 (2010).

Article Google Scholar

Wiranowska, M. et al. Preferential drug delivery to tumor cells than normal cells using a tunable niosome–chitosan double package nanodelivery system: a novel in vitro model. Canc. Nanotechnolgy 11, 1–20 (2020).

Google Scholar

Alalawy, A. I. et al. Effectual anticancer potentiality of loaded bee venom onto fungal chitosan nanoparticles. Int. J. Polym. Sci. 2020, 1–9. https://doi.org/10.1155/2020/2785304 (2020).

Article CAS Google Scholar

Park, J. H., Yim, B. K., Lee, J.-H., Lee, S. & Kim, T.-H. Risk associated with bee venom therapy: a systematic review and meta-analysis. PloS One 10, e0126971 (2015).

Article PubMed PubMed Central Google Scholar

Qiao, M. et al. Effect of bee venom peptide–copolymer interactions on thermosensitive hydrogel delivery systems. Int. J. Pharm. 345, 116–124 (2007).

Article CAS PubMed Google Scholar

Lee, J. et al. Nasal delivery of chitosan/alginate nanoparticle encapsulated bee (Apis mellifera) venom promotes antibody production and viral clearance during porcine reproductive and respiratory syndrome virus infection by modulating T cell related responses. Vet. Immunol. Immunopathol. 200, 40–51 (2018).

Article CAS PubMed Google Scholar

Khalil, A., Elesawy, B. H., Ali, T. M. & Ahmed, O. M. J. M. Bee venom: From venom to drug. Molecules 26, 4941 (2021).

Article CAS PubMed PubMed Central Google Scholar

Yusuf, N., Irby, C., Katiyar, S. K. & Elmets, C. A. J. P. Photoprotective effects of green tea polyphenols. Photodermatol. Photoimmunol. Photomed. 23, 48–56 (2007).

Article PubMed Google Scholar

Schmid, D. et al. Efficient drug delivery and induction of apoptosis in colorectal tumors using a death receptor 5-targeted nanomedicine. Mol. Ther. 22, 2083–2092 (2014).

Article CAS PubMed PubMed Central Google Scholar

Yang, C., Liu, H.-Z. & Fu, Z.-X. Effects of PEG-liposomal oxaliplatin on apoptosis, and expression of Cyclin A and Cyclin D1 in colorectal cancer cells. Oncol. Rep. 28, 1006–1012 (2012).

Article CAS PubMed Google Scholar

Swellam, T. et al. Antineoplastic activity of honey in an experimental bladder cancer implantation model: In vivo and in vitro studies. Int. J. Urol. 10, 213–219 (2003).

Article CAS PubMed Google Scholar

Aliyu, M. et al. Acacia honey modulates cell cycle progression, pro-inflammatory cytokines and calcium ions secretion in PC-3 cell line. Cancer Sci Ther 4, 401–407 (2012).

Google Scholar

Jiménez-López, J. et al. Paclitaxel antitumor effect improvement in lung cancer and prevention of the painful neuropathy using large pegylated cationic liposomes. Biomed. Pharmacother. 133, 111059 (2021).

Article PubMed Google Scholar

Rady, I., Siddiqui, I. A., Rady, M. & Mukhtar, H. Melittin, a major peptide component of bee venom, and its conjugates in cancer therapy. Can. Lett. 402, 16–31 (2017).

Article CAS Google Scholar

Zhang, L. et al. Cryptotanshinone inhibits the growth and invasion of colon cancer by suppressing inflammation and tumor angiogenesis through modulating MMP/TIMP system, PI3K/Akt/mTOR signaling and HIF-1α nuclear translocation. Int. Immunopharmacol. 65, 429–437 (2018).

Article CAS PubMed Google Scholar

Choi, K. E. et al. Cancer cell growth inhibitory effect of bee venom via increase of death receptor 3 expression and inactivation of NF-kappa B in NSCLC cells. Toxins 6, 2210–2228 (2014).

Article PubMed PubMed Central Google Scholar

Download references

These authors contributed equally: Samireh Badivi and Sara Kazemi.

Department of Physics, Science and Research Branch, Islamic Azad University, Tehran, Iran

Samireh Badivi

Bogomolets National Medical University, Kyiv, Ukraine

Sara Kazemi

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, 15260, USA

Mohammadmahdi Eskandarisani

School of Mechanical Engineering, College of Engineering, University of Tehran, P.O. Box 11155-4563, Tehran, Iran

Mohammadmahdi Eskandarisani

Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, Iran

Nastaran Asghari Moghaddam

School of Pharmacy, International Campus, Tehran University of Medical Sciences, Tehran, Iran

Ghazal Mesbahian

Stem Cells Research Center, Tissue Engineering and Regenerative Medicine Institute, Islamic Azad University, Central Tehran Branch, Tehran, Iran

Sara Karimifard

Department of Chemistry, Science and Research Branch, Islamic Azad University, Tehran, Iran

Elham Afzali

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

N.A.M. and E.A. developed the idea and designed the experiments. S.B., S.K., G.M., S.K., and E.A. conducted the experiments. N.A.M. analyzed the data. M.M.E., E.A., and N.A.M. wrote the manuscript. All authors confirmed the final manuscript before the submission. All authors read and approved the final manuscript.

Correspondence to Nastaran Asghari Moghaddam or Elham Afzali.

The authors declare no competing interests.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

Badivi, S., Kazemi, S., Eskandarisani, M. et al. Targeted delivery of bee venom to A549 lung cancer cells by PEGylate liposomal formulation: an apoptotic investigation. Sci Rep 14, 17302 (2024). https://doi.org/10.1038/s41598-024-68156-6

Download citation

Received: 22 March 2024

Accepted: 21 July 2024

Published: 27 July 2024

DOI: https://doi.org/10.1038/s41598-024-68156-6

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative