1. Introduction
Cancer remains one of the most incident diseases globally with high mortality rates, having been estimated to affect almost 2 billion people just in the United States of America in the year of 2023, with more than 600 thousand related deaths, and having similar incidence rates all over the world [
1,
2,
3]. Although the fast-paced advances in medicine are today able to prolong the life of these patients, and even cure them in some cases, most common cancer therapies, such as chemotherapy, radiotherapy, immunotherapy, and surgery, are linked with poor prognosis and/or severely debilitating side-effects, such as nausea and vomiting, pain, fatigue, depression, hair loss, and immune system debilitation, which can lead to opportunistic infections due to the body’s higher vulnerability and low defenses [
4,
5,
6]. Hence, there is an urgent need for new, more effective, and safer therapies.
Bee venom, also known as apitoxin, is a substance produced in the venom gland under the abdominal cavity of female worker bees, and it is a colorless and odorless liquid, with an acid pH between 4.5 and 5.5. It is used by these insects to defend the hive against external threats [
7]. In its composition, we can find proteins like melittin, apamin, adolapin, and scapin, and enzymes such as phospholipase A2, hyaluronidase, and lysophospholipase, as well as other substances, including amino acids, carbohydrates, pheromones and minerals. Among all these compounds, melittin is the main active ingredient in bee venom, comprising 40% to 60% of its dry weight, followed by the enzyme phospholipase A2, present in 10-12% [
8]. The complex chemical composition of bee venom, and the potential synergy surrounding the interaction between its compounds, assures a vast range of biological activities, capable of targeting different diseases. These biological properties include anti-inflammatory, antioxidant, antiviral, antimicrobial and antitumor properties (
Figure 1) [
9]
. All these bioactivities could be potentially beneficial for a series of different diseases, from skin diseases, such as acne, wounds, psoriasis, or atopic dermatitis, to neurodegenerative diseases, such as Parkinson's disease, due to proven neuroprotective effects, and, of course, cancer [
10] [
11]. Nevertheless, while bee venom can be tolerated by the human body, it can cause some allergic reactions, as well as systemic and local inflammatory responses, other immune reactions, and anticoagulant effects, are the main drawbacks for its use as a potential therapeutic agent [
8]. New strategies are needed in order to increase the safety of bee venom administration and its incorporation into nanotechnological platforms to enhance its properties, avoid degradation, and reduce the potential side effects can be the answer [
12].
The use of nanotechnology for drug delivery can not only protect the drug from chemical and metabolic degradation, but also allow increased permeation through biological barriers, and enable a controlled, sustained and targeted drug delivery, hence leading to more localized therapeutic effects, and thus reduced systemic toxicity [
13,
14,
15]. Several types of nanosystems have been developed over the years, namely nanoemulsions, micelles, polymeric nanoparticles, and lipid nanoparticles, such as liposomes, and their more recent and innovative alternatives, ethosomes, transfersomes and niosomes [
16,
17,
18]. Niosomes are vesicular nanosystems capable of self-assembly, composed of amphiphilic molecules, such as non-ionic surfactants, which form an outer bilayered membrane, and an aqueous core [
19,
20]. This structure makes them highly versatile nanosystems for therapeutic application since they can deliver both hydrophobic molecules, within the bilayered membrane, and hydrophilic molecules, encapsulated within their aqueous core. Other molecules, such as cholesterol or similar lipids, can also be part of the niosomal membrane, giving them better properties, such as higher stability and deformability [
21,
22]. Thanks to their composition, niosomes are biodegradable and biocompatible, on account of the similarity between the lipids used for niosome production and the ones found in the human body, especially on the cellular membranes [
23,
24]. Additionally, when compared to their ancestors, liposomes, niosomes possess higher stability and longer shelf-life, a better capacity for controlled drug release, and lead to higher drug bioavailability [
25].
Hence, given the great therapeutic potential of bee venom, and the promising properties of niosomes for increased therapeutic efficacy and safety, the purpose of the present study was to develop and characterize bee venom-loaded niosomes, for potential anticancer therapy. Their physicochemical, therapeutic and safety profiles were assessed, including particle size and polydispersity index determination, in vitro cytotoxicity and anti-inflammatory potential assessment, and in vitro and ex vivo safety evaluation.
2. Materials and Methods
2.1. Materials and Reagents
Tween® 20, cholesterol, cetyl alcohol, chloroform, sulforhodamine B, lipopolysaccharide, trypan blue, dexamethasone, trichloroacetic acid, tris(hydroxymethyl)aminomethane buffer, cytochrome C from equine heart (purity ≥ 95%), melittin (purity ≥ 85%, HPLC grade) and phospholipase A2 (activity: 1775 units/mg solid) were purchased from Sigma-Aldrich (Saint Louis, Missouri, United States of America). Apamin (purity 98.3%) was purchased from CalBiochem (San Diego, California, United States of America). Fetal bovine serum, penicillin, streptomycin, trypsin, L-glutamine, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco Invitrogen Life Technologies (California, United States of America). Formic acid (HPLC grade) and acetonitrile (HPLC grade) were obtained from Fisher Scientific (Loughborough, Leicestershire, United Kingdom). The Griess reagent system kit was bought from Promega (Madison, Wisconsin, United States of America). Ultrapure water was obtained from adequate purification systems (Ellix Essential Millipore®, Darmstadt, Germany, and TGI Pure Water Systems, Brea, California, United States of America).
2.2. Bee Venom Production and Harvesting
The bee venom used as the active compound to be encapsulated within the developed niosomes was collected between August and November of 2018 from
Apis mellifera intermissa hives located in the northeast region of Morocco. To collect the bee venom, a double-face bee venom collector was used, especially developed for the purpose by the research team. The device was positioned in the hive at one of the outermost, diametrically opposed ends of the beehive, and produced mild electrical impulse shock waves on the beehive, which made the worker bees sting the glass, as a defense mechanism, leaving the bee venom deposited on it. Following the collection process, the venom was meticulously removed from the glass using a scraper and subsequently stored in pharmaceutical-grade vials. Samples were then freeze-dried, in a freeze dryer (Labconco FreeZone 4.5, Labconco Corporation, Kansas City, Missouri, USA), and kept at −20 ºC until further analysis [
26].
2.3. Ultra-High-Performance Liquid Chromatography Analysis
The ultra-high-performance liquid chromatography (UHPLC) analysis was executed utilizing a Dionex Ultimate 3000 UHPLC instrument (Thermo Scientific, Waltham, Massachusetts, United States of America), featuring a diode-array detector. The chromatographic system comprised a quaternary pump, an autosampler maintained at 5 °C, a degasser, a photodiode array detector, and an automatic thermostatic column compartment. Chromatographic separation was conducted on an XSelect CSH130 C18, 100 mm × 2.1 mm id, 2.5 µm XP column (Waters, Milford, MA, USA), with a constant temperature of 30 ºC. The mobile phase consisted of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile, previously degassed and filtered. The used conditions were in accordance with previous studies [
27]. Spectral data for all peaks were gathered within the range of 190–500 nm. Control and data acquisition were conducted using the Xcalibur
® data system (Thermo Scientific). Cytochrome C, employed as the internal standard, was prepared in deionized water at a concentration of 25 µg/mL. For analysis, the lyophilized bee venom (3 mg) was dissolved in 10 mL of internal standard. Each sample was filtered through a 0.2 µm polytetrafluoroethylene membrane. Bee venom peptide quantification was achieved using calibration curves for apamin (at a range of 2 - 60 µg/mL, y = 0.040x + 0.055,
R2 = 0.999), phospholipase A2 (at a range of 8 - 120 µg/mL, y = 0.049x − 0.356,
R2 = 0.999), and melittin (at a range of 15 - 250 µg/mL, y = 0.062x + 0.029,
R2 = 0.997).
2.4. Niosome Formulation Development and Characterization
For formulation preparation, the thin-film hydration method was applied (
Figure 2), which is the most commonly used method for niosome production [
20,
28]. Firstly, the non-ionic surfactant and lipid fraction (3.5:2 molar ratio) were weighed into a round bottom flask and then dissolved in 6 mL of chloroform. The organic solvent was subsequently evaporated, in a rotary evaporator (Rotavapor R-210/215, BÜCHI, Switzerland), combined with a vacuum pump (V-700/710, BÜCHI, Switzerland), and a vacuum controller (V-850/855, BÜCHI, Switzerland), in a heating water bath (40 ºC to 60 ºC), with a rotation speed of 8 rpm, and under reduced pressure (120 mbar), for 60 minutes. After full organic solvent evaporation, a thin layer was formed on the interior of the flask, and this thin layer was then hydrated with either deionized water (6 mL) for the formulation vehicle (empty niosomes), or an aqueous compound solution (2 µg/mL) for the bee venom-loaded niosomes, under mild magnetic stirring, for 60 minutes. Although after thin-film hydration vesicles were already formed, to attain a nanometric and homogeneous particle size, the mixture was extruded (Avanti Mini-Extruder, Avanti Polar Lipids, USA) through a synthetic polycarbonate membrane with a 200 nm pore size (Avanti Polar Lipids, USA). Various extrusion cycles were performed (11, 21, 31, 41, and 51), and the particle size and polydispersity index (PDI) for every performed cycle series were subsequently measured. Particle mean hydrodynamic size and PDI of the developed formulations were measured by dynamic light scattering, using a Zetasizer apparatus (ZetaSizer Nano ZS, Malvern Instruments, Malvern, United Kingdom). Samples were diluted 40-fold in deionized water, and measured in disposable polymethyl methacrylate 12 mm square diameter cuvettes, at 25°C. All the samples were measured in triplicate and the mean and standard deviation are reported. Formulations were stored in a refrigerator at 4 °C until further characterization.
2.5. In Vitro Therapeutic Potential
2.5.1. Cytotoxic Activity
The developed formulations’ cytotoxic activity was tested in several different human cancer cell lines, namely: AGS (gastric adenocarcinoma), Caco-2 (colorectal adenocarcinoma), MCF-7 (breast adenocarcinoma), NCI-H460 (lung carcinoma), and HeLa (cervical carcinoma). For assessment of the potential toxicity of the developed formulations on non-cancerous tissues, non-tumor cell lines Vero (African green monkey kidney) and PLP2 (primary pig liver culture) were also tested. All cell lines were maintained in RPMI-1640 medium, supplemented with 10% fetal bovine serum, glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 mg/mL), except Vero cells, which were maintained in DMEM medium. The culture flasks were incubated at 37 ºC, under a 5% CO2 and high humidity atmosphere. Cells were used only upon reaching a 70 to 80% confluence.
Formulations were dissolved in water:dimethyl sulfoxide (DMSO) (1 mL; 1:1) to obtain stock solutions with a concentration of 1 μg/mL, from which successive dilutions were made, obtaining the samples at the concentrations to be tested (0.05 – 0.0008 μg/mL). Each sample (10 μL) was incubated with the cell suspension (190 μL) in 96-well microplates, for 72 hours. The microplates were incubated at 37 ºC, under a 5% CO2 and high humidity atmosphere. All cell lines were tested at a concentration of 10,000 cells/well, except for Vero cells, in which a density of 19,000 cells/well was used. Subsequently to this incubation period, plates were incubated again, for 1 hour, at 4 ºC, after the addition of previously cooled trichloroacetic acid (10% w/v; 100 μL). The plates were then washed with water and, after drying, a sulforhodamine B solution (0.057% w/v, 100 μL) was added, and then left to stand at room temperature for 30 minutes. To remove non-adhered sulforhodamine B, the plates were washed three times with an acetic acid solution (1% v/v) and left to dry. Finally, the adhered sulforhodamine B was solubilized with Tris(hydroxymethyl)aminomethane (Tris) buffer (10 mM, 200 μL), and sample absorbance was measured at a wavelength of 540 nm, in a microplate reader (ELX800 Biotek microplate reader, Bio-Tek Instruments, Inc., Winooski, VT, United States of America). The results are expressed in terms of the sample concentration with the ability to inhibit cell growth by 50% (GI50).
2.5.2. Anti-Inflammatory Activity
The anti-inflammatory activity of the developed formulations was also assessed. First, the formulations were diluted in a water:DMSO solution to obtain a final concentration of 1 μg/mL, from which successive dilutions were carried out. Final concentrations ranged from 0.05 to 0.0008 mg/mL. A RAW 264.7 mouse macrophage cell line was used (Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH), and grown in DMEM medium, supplemented with heat-inactivated fetal serum (10%), glutamine and antibiotics, and kept in an incubator at 37 ºC, with a 5% CO2 and highly humid atmosphere. Cells were detached with a cell scraper, and an aliquot of macrophages cell suspension (300 μL), with a cell density of 5 x 105 cells/mL, and with a proportion of dead cells below 5% (according to the trypan blue exclusion test), was placed in each well. The microplate was incubated for 24 hours, in an incubator, at the previously indicated conditions, to allow for adequate cell adherence and multiplication. After that period, the cells were treated with different concentrations of the developed formulations (15 μL) and incubated for one hour, with the range of tested concentrations being between 0.05 and 0.0008 μg/mL. Stimulation was performed with the addition of 30 μL of a liposaccharide (LPS) solution (1 mL/mL) and incubation for an additional 24 hours. Dexamethasone (50 mM) was used as a positive control, and samples in the absence of LPS were used as a negative control. Quantification of nitric oxide was performed using a Griess reagent system kit (nitrophenamide, ethylenediamine, and nitrite solutions). The produced nitric oxide was determined by reading absorbances at 540 nm (ELX800 Biotek microplate reader, Bio-Tek Instruments, Inc., Winooski, VT, United States of America), on a 96-well microplate, and by comparison with a standard calibration curve. Results are depicted by a graphical representation of the percentage of inhibition of nitric oxide production versus sample concentration, and expressed in relation to the formulation concentration that causes the 50% inhibition of nitric oxide production (IC50).
2.6. Safety Assessment
2.6.1. In Vitro Safety Assessment – Cytotoxicity Evaluation on HaCaT and HFF-1 Cell Lines
To assess the safety of the developed formulations in vitro, the colorimetric sulforhodamine B assay was conducted on two human cell lines: HFF-1 (human skin fibroblasts) and HaCaT (human immortalized keratinocytes). This assay measured cell survival after treatment with the developed formulations. The cell lines were cultured in DMEM medium, supplemented with fetal bovine serum (10%), glutamine, and antibiotics (penicillin and streptomycin 1%), in an incubator at 37 °C, with a 5% CO2 atmosphere, and with saturated controlled humidity. Trypsin was used to disperse the cells from the inside of the flask where they were cultured, since these cells are adherent. The cell density for this assay was 10,000 cells per well. X-triton (1%), a detergent, served as a positive control due to its capacity to disrupt and destroy all cells. For the negative control group, only cells and medium were added (no formulation or other compound).
After the cells were dispersed by trypsin in the culture medium, centrifuged at 3000 rpm for 5 minutes, and resuspended in the medium, 10,000 cells per well were plated in a 96-well optical-bottom plate to adhere overnight. Afterwards, the bee venom-loaded niosomes and empty niosomes (formulation vehicle) samples were prepared by diluting them in water, for final concentrations equal to 0.1, 0.05, 0.025, and 0.0125 mg/mL, which were added to the plate. Samples at the exact same concentrations were also prepared for the bee venom compound solution, also by dilution with water. Each concentration level was tested in triplicate. The plates were then incubated for 48 hours at 37 °C and in a 5% CO
2 atmosphere. After 48 hours, the cells were fixated with trichloroacetic acid, for 1 hour, at 4 °C. Afterwards, the liquid inside the plate was discarded, and the plate was washed 3 times with water, and left to dry. Once dried, 100 µL of sulforhodamine B was added to each well and left there for 30 minutes at room temperature. Acetic acid at 1% was used to remove the unbounded dye from the cells, and the bounded dye was dissolved with a 10 mM Tris buffer. The IC
50 values were expressed as the concentration (µg/mL) of each formulation that caused 50% inhibition of cell growth. Samples were quantified by using UV-visible spectrophotometry in a microplate reader (ELX800 microplate reader, Bio-Tek Instruments, Winooski, Vermont, United States of America) at 540 nm, based on previously described methods [
27,
29].
2.6.2. Ex Vivo Safety Assessment – HET-CAM Test
The hen's egg-chorioallantoic membrane (HET-CAM) test method was used to assess the irritation-inducing potential of the developed formulations, upon contact with a highly sensitive biological membrane, focusing on its ability to induce toxicity in the chorioallantoic membrane of a chicken egg. This type of membrane is known to resemble the human eye, and even if the product is not intended for ocular use, the test can still be quite useful since a formulation that is reasonably safe for eye application is probably also safe for contact with most other human organs [
30].
The assay was done following the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommended test method [
31]. The experimental protocol consisted on the incubation of forty Leghorn fertilized chicken eggs, for 10 days, in an automatic rotating incubator, at 37 °C and 65% humidity. To confirm the presence of an embryo, a flashlight was used against each egg. If no embryo was detected that egg would be excluded from the experiment, and not used. For each tested formulation, a total of three eggs were used. After the 10-day incubation period, the top part of the eggshells was cut open to expose the chorioallantoic membrane, so that the formulation could then be applied. Nevertheless, before formulation application, all the membranes were hydrated using a 0.9% NaCl solution, for 30 minutes. Then, three formulation drops (approximately 0.1 mL per drop, for a total of 0.3 mL of formulation) were applied to the chorioallantoic membrane of each egg [
31]. The negative control group were three eggs where a 0.9% NaCl solution was applied (no reaction intended), while for the positive control group a 10% NaOH solution was applied on the membrane of three eggs (inflammatory reaction intended). The irritancy degree of each formulation was observed and monitored by the appearance of the following events: hemorrhage (bleeding of the vessels), lysis (disintegration of the vessels), and coagulation (intra and/or extra-vascular protein denaturation). The occurrence or non-occurrence of these events was observed at specific time points, namely 0.5, 2, and 5 minutes. A total score was then attributed, from 0 to 21, being the sum of the values attributed to each event and corresponding to a level of irritability (
Table 1). Hence, after the 5-minute time interval, formulations were given an irritation score (IS), with the following corresponding irritation levels: an IS score between 0 and 4.9 being slightly/non-irritative; an IS score between 5 and 8.9 being moderately irritative; and an IS score between 9 and 21 being strongly irritative [
31,
32].
2.7. Data Analysis
For a better understanding and interpretation of the results, whenever possible statistical analysis was performed, using GraphPad Prism® (GraphPad, San Diego, CA, USA), version 8.0 software. More specifically, either a one-way ANOVA or a two-way ANOVA test was applied, with a Tukey's multiple comparisons post-test. A 95% confidence level was considered in all analysis, with a p value < 0.05 being considered statistically significant.
Author Contributions
Conceptualization, F.V., A.C.P.-S. and P.M.; methodology, P.C.P., A.C.P.-S., R.C.C., A.R.S., M.J.S. and S.I.F.; validation, P.C.P., F.V., A.C.P.-S., M.V.-B. and S.I.F.; formal analysis, M.B.P., P.C.P., F.V., A.C.P.-S., R.C.C., A.R.S. and M.J.S.; investigation, M.B.P., P.C.P. and P.M.; resources, F.V., A.C.P.-S., P.M. and S.I.F.; data curation, M.B.P, P.C.P., R.C.C., A.R.S., M.J.S. and S.I.F.; writing—original draft preparation, M.B.P., P.C.P. and A.C.P.-S.; writing—review and editing, P.C.P., F.V., A.C.P.-S., P.M., M.V.-B. and S.I.F.; supervision, P.C.P., F.V., A.C.P.-S. and S.I.F.; project administration, F.V., A.C.P.-S. and P.M.; funding acquisition, F.V. and A.C.P.-S.. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Schematic representation of beehive derived compounds, including a photograph of the beehive at Polytechnic Institute of Bragança’s apiary, Bragança, Portugal (produced with Biorender).
Figure 1.
Schematic representation of beehive derived compounds, including a photograph of the beehive at Polytechnic Institute of Bragança’s apiary, Bragança, Portugal (produced with Biorender).
Figure 2.
Schematic illustration of niosome composition and production, using the thin-film hydration method, followed by extrusion through a nanometric pore membrane (produced with Biorender).
Figure 2.
Schematic illustration of niosome composition and production, using the thin-film hydration method, followed by extrusion through a nanometric pore membrane (produced with Biorender).
Figure 3.
Example chromatogram after UHPLC analysis of the bee venom sample, at 220 nm. IS – internal standard (cytochrome C, 25 µg/mL); UHPLC - ultra-high-performance liquid chromatography.
Figure 3.
Example chromatogram after UHPLC analysis of the bee venom sample, at 220 nm. IS – internal standard (cytochrome C, 25 µg/mL); UHPLC - ultra-high-performance liquid chromatography.
Figure 6.
Anti-inflammatory potential of the developed bee venom-loaded niosomes (BVN), compared to the free compound (bee venom solution, BVS), and the formulation vehicle (empty niosomes, EN), evaluated in a mouse macrophage cell line (RAW 264.7); IC50 values are depicted, and correspond to formulation concentrations providing 50% of inhibition of nitric oxide production; data is represented as mean ± standard deviation; **** p < 0.0001 (schematic representation produced with Biorender).
Figure 6.
Anti-inflammatory potential of the developed bee venom-loaded niosomes (BVN), compared to the free compound (bee venom solution, BVS), and the formulation vehicle (empty niosomes, EN), evaluated in a mouse macrophage cell line (RAW 264.7); IC50 values are depicted, and correspond to formulation concentrations providing 50% of inhibition of nitric oxide production; data is represented as mean ± standard deviation; **** p < 0.0001 (schematic representation produced with Biorender).
Figure 8.
HET-CAM assay schematic representation, and photographs of the hen's eggs-chorioallantoic membranes after formulation application, either the developed bee venom-encapsulated niosomes [a)], or the formulation vehicle [empty niosomes, b)].
Figure 8.
HET-CAM assay schematic representation, and photographs of the hen's eggs-chorioallantoic membranes after formulation application, either the developed bee venom-encapsulated niosomes [a)], or the formulation vehicle [empty niosomes, b)].
Table 1.
Numerical time-dependent scoring scheme for the HET-CAM test method.
Table 1.
Numerical time-dependent scoring scheme for the HET-CAM test method.
Effect |
Score |
0.5 min |
2 min |
5 min |
Lysis |
5 |
3 |
1 |
Hemorrhage |
7 |
5 |
3 |
Coagulation |
9 |
7 |
5 |
Table 2.
Chemical characterization of the bee venom encapsulated within the developed niosomes, or solubilized within the produced compound solution, performed by UHPLC analysis. Major compounds are presented, with values corresponding to the amount present in each analyzed sample (µg/mL).
Table 2.
Chemical characterization of the bee venom encapsulated within the developed niosomes, or solubilized within the produced compound solution, performed by UHPLC analysis. Major compounds are presented, with values corresponding to the amount present in each analyzed sample (µg/mL).
Compounds |
Bee venom solution (300 µg/mL) |
Bee venom-loaded niosomes (2 µg/mL) |
Correspondent percentage (%) |
Melittin |
193.43 |
1.29 |
63 |
Phospholipase A2 |
37.52 |
0.25 |
12 |
Apamin |
3.61 |
0.024 |
1.2 |
Table 4.
Significance matrix of the HaCaT and HFF-1 cell viability assay results (depicted in
Figure 7), comparing the viability percentage variation with concentration variation within each studied formulation, for each cell type. The presented values correspond to
p values, after the application of one-way ANOVA with Tukey’s multiple comparisons’ test.
Table 4.
Significance matrix of the HaCaT and HFF-1 cell viability assay results (depicted in
Figure 7), comparing the viability percentage variation with concentration variation within each studied formulation, for each cell type. The presented values correspond to
p values, after the application of one-way ANOVA with Tukey’s multiple comparisons’ test.
HFF-1 cells |
HaCaT cells |
Bee venom-loaded niosomes |
Bee venom-loaded niosomes |
Concentration (µg/mL) |
0 |
25 |
50 |
100 |
200 |
Concentration (µg/mL) |
0 |
25 |
50 |
100 |
200 |
Overall |
< 0.0001 |
Overall |
< 0.0001 |
0 |
- |
NS |
NS |
0.0352 |
< 0.0001 |
0 |
- |
0.0025 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
25 |
- |
- |
NS |
NS |
< 0.0001 |
25 |
- |
- |
NS |
< 0.0001 |
< 0.0001 |
50 |
- |
- |
- |
NS |
< 0.0001 |
50 |
- |
- |
- |
0.0002 |
< 0.0001 |
100 |
- |
- |
- |
- |
< 0.0001 |
100 |
- |
- |
- |
- |
< 0.0001 |
200 |
- |
- |
- |
- |
- |
200 |
- |
- |
- |
- |
- |
Empty niosomes |
Empty niosomes |
Concentration (µg/mL) |
0 |
25 |
50 |
100 |
200 |
Concentration (µg/mL) |
0 |
25 |
50 |
100 |
200 |
Overall |
< 0.0001 |
Overall |
< 0.0001 |
0 |
- |
NS |
< 0.0001 |
< 0.0001 |
< 0.0001 |
0 |
- |
NS |
NS |
< 0.0001 |
< 0.0001 |
25 |
- |
- |
< 0.0001 |
< 0.0001 |
< 0.0001 |
25 |
- |
- |
NS |
< 0.0001 |
< 0.0001 |
50 |
- |
- |
- |
< 0.0001 |
< 0.0001 |
50 |
- |
- |
- |
< 0.0001 |
< 0.0001 |
100 |
- |
- |
- |
- |
0.0392 |
100 |
- |
- |
- |
- |
< 0.0001 |
200 |
- |
- |
- |
- |
- |
200 |
- |
- |
- |
- |
- |
Bee venom solution |
Bee venom solution |
Concentration (µg/mL) |
0 |
25 |
50 |
100 |
200 |
Concentration (µg/mL) |
0 |
25 |
50 |
100 |
200 |
Overall |
< 0.0001 |
Overall |
< 0.0001 |
0 |
- |
NS |
NS |
0.0003 |
< 0.0001 |
0 |
- |
NS |
NS |
< 0.0001 |
< 0.0001 |
25 |
- |
- |
NS |
0.0002 |
< 0.0001 |
25 |
- |
- |
NS |
< 0.0001 |
< 0.0001 |
50 |
- |
- |
- |
0.0021 |
< 0.0001 |
50 |
- |
- |
- |
< 0.0001 |
< 0.0001 |
100 |
- |
- |
- |
- |
0.0087 |
100 |
- |
- |
- |
- |
< 0.0001 |
200 |
- |
- |
- |
- |
- |
200 |
- |
- |
- |
- |
- |
Table 5.
Significance matrix of the HaCaT and HFF-1 cell viability assay results (depicted in
Figure 7), comparing the viability percentage variation between studied formulations, for the same concentrations, for each cell type. The presented values correspond to
p values, after application of two-way ANOVA with Tukey’s multiple comparisons’ test.
Table 5.
Significance matrix of the HaCaT and HFF-1 cell viability assay results (depicted in
Figure 7), comparing the viability percentage variation between studied formulations, for the same concentrations, for each cell type. The presented values correspond to
p values, after application of two-way ANOVA with Tukey’s multiple comparisons’ test.
HFF-1 cells |
HaCaT cells |
Bee venom-loaded niosomes vs empty niosomes |
Bee venom-loaded niosomes vs empty niosomes |
Overall |
0 µg/mL |
25 µg/mL |
50 µg/mL |
100 µg/mL |
200 µg/mL |
Overall |
0 µg/mL |
25 µg/mL |
50 µg/mL |
100 µg/mL |
200 µg/mL |
< 0.0001 |
0.0037 |
0.0104 |
0.0025 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
0.0058 |
< 0.0001 |
0.0015 |
NS |
< 0.0001 |
Bee venom-loaded niosomes vs bee venom solution |
Bee venom-loaded niosomes vs bee venom solution |
Overall |
0 µg/mL |
25 µg/mL |
50 µg/mL |
100 µg/mL |
200 µg/mL |
Overall |
0 µg/mL |
25 µg/mL |
50 µg/mL |
100 µg/mL |
200 µg/mL |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
Empty niosomes vs bee venom solution |
Empty niosomes vs bee venom solution |
Overall |
0 µg/mL |
25 µg/mL |
50 µg/mL |
100 µg/mL |
200 g/mL |
Overall |
0 µg/mL |
25 µg/mL |
50 µg/mL |
100 µg/mL |
200 µg/mL |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
0.0004 |
0.0001 |
< 0.0001 |
< 0.0001 |
< 0.0001 |
Table 6.
Cell viability of the developed bee venom-loaded niosomes, formulation vehicle (empty niosomes), and compound solution (bee venom solution), evaluated in HaCaT and HFF-1 cell lines, and represented by the calculated IC50 value.
Table 6.
Cell viability of the developed bee venom-loaded niosomes, formulation vehicle (empty niosomes), and compound solution (bee venom solution), evaluated in HaCaT and HFF-1 cell lines, and represented by the calculated IC50 value.
|
Cell viability (IC50, µg/mL) |
|
HFF-1 cells |
HaCaT cells |
Bee venom-loaded niosomes |
165.7 ± 1.0 |
161.0 ± 1.0 |
Empty niosomes |
138.2 ± 1.0 |
69.8 ± 1.00 |
Bee venom solution |
> 200.0 |
> 200.0 |
Table 7.
HET-CAM results after application of the developed bee venom-loaded niosomes or formulation vehicle (empty niosomes), with respective positive (0.1 N NaOH) and negative (0.9% NaCl) controls, on hen's eggs-chorioallantoic membranes, and represented by the calculated average irritation score (IS) ± standard deviation, and visible vascular reactions and overall irritation degree.
Table 7.
HET-CAM results after application of the developed bee venom-loaded niosomes or formulation vehicle (empty niosomes), with respective positive (0.1 N NaOH) and negative (0.9% NaCl) controls, on hen's eggs-chorioallantoic membranes, and represented by the calculated average irritation score (IS) ± standard deviation, and visible vascular reactions and overall irritation degree.
Formulation |
Average IS |
Standard deviation |
Vascular reactions following treatment |
Irritation degree |
Bee venom-loaded niosomes |
7.7 |
0.47 |
No lysis |
Slightly irritative |
Empty niosomes |
14 |
0 |
Hemorrhage |
Strongly irritative |
0.9% NaCl |
0.00 |
- |
None |
Non-irritant |
0.1 N NaOH |
19.00 |
- |
Lysis and hemorrhage |
Strongly irritative |