Next Article in Journal
Aging Characteristics of Plant Hot-Mix Recycled Asphalt and Its Induced Aging on New Asphalt
Next Article in Special Issue
Relationship between Some Colloidal Properties of Non-Ionic-Anionic Surfactant Mixtures
Previous Article in Journal
Darcy–Forchheimer Relation Influence on MHD Dissipative Third-Grade Fluid Flow and Heat Transfer in Porous Medium with Joule Heating Effects: A Numerical Approach
Previous Article in Special Issue
Coating of Tomatoes (Solanum lycopersicum L.) Employing Nanoemulsions Containing the Bioactive Compounds of Cactus Acid Fruits: Quality and Shelf Life
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Processes
Volume 10
Issue 5
10.3390/pr10050907
processes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Paclitaxel and Myrrh oil Combination Therapy for Enhancement of Cytotoxicity against Breast Cancer; QbD Approach

by
Tamer M. Shehata
* and
Heba S. Elsewedy
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Alhofuf 36362, Saudi Arabia
*
Author to whom correspondence should be addressed.
Processes 2022, 10(5), 907; https://doi.org/10.3390/pr10050907
Submission received: 12 April 2022 / Revised: 30 April 2022 / Accepted: 2 May 2022 / Published: 5 May 2022
(This article belongs to the Special Issue Nanoemulsion Processes Design and Applications)
Browse Figures
Versions Notes

Abstract

:
Paclitaxel (PX), plant alkaloid, is a chemotherapeutic agent intended for treating a wide variety of cancers. The objective of the present study was to formulate and evaluate the anticancer activity of PX loaded into a nanocarrier, mainly PEGylated nanoemulsion (NE) fabricated with myrrh essential oil. Myrrh essential oil has been estimated previously to show respectable anticancer activity. Surface modification of the formulation with PEG-DSPE would help in avoiding phagocytosis and prolong the residence time in blood circulation. Various NE formulations were developed after operating (22) factorial design, characterized for their particle size, in vitro release, and hemolytic activity. The optimized formula was selected and compared to its naked counterpart in respect to several characterizations. Quantitative amount of protein absorbed on the formulation surfaces and in vitro release with and without serum incubation were evaluated. Ultimately, MTT assay was conducted to distinguish the anti-proliferative activity. PEGylated PX-NE showed particle size 170 nm, viscosity 2.91 cP, in vitro release 57.5%, and hemolysis 3.44%, which were suitable for intravenous administration. A lower amount of serum protein adsorbed on PEGylated PX-NE surface (16.57 µg/µmol) compared to naked counterpart (45.73 µg/µmol). In vitro release from PEGylated NE following serum incubation was not greatly affected (63.3%), in contrast to the naked counterpart (78.8%). Eventually, anti-proliferative effect was obtained for PEGylated PX-NE achieving IC50 38.66 µg/mL. The results obtained recommend PEGylated NE of myrrh essential oil as a candidate nanocarrier for passive targeting of PX.

1. Introduction

Breast cancer is a prevailing disease that gave rise to most cancer deaths between women in the world [1]. Although treatment of cancer has been an issue for many decades, it has gone through various strategies, one of them being chemotherapy. However, cancer chemotherapy has faced a lot of hurdles due to poor availability and difficult accessibility of drug to the cancer cells [2]. For that purpose, an innovative approach known as targeted drug delivery (TDD) has been evolved to facilitate delivering drugs to the specific tumor sites without affecting normal cells [3]. Two kinds of drug targeting are available; active and passive targeting. Active drug targeting is based on fitting certain ligands with their specific receptors, whereas passive targeting depends mainly on a well-known phenomenon in most tumor cells named enhanced permeability and retention (EPR) effect [4,5]. The EPR effect developed as a result of the physiological structure of the cancer cells that had abnormal architecture, leaky vasculatures, and wide gaps between cancer cells unlike normal ones. Exploiting these abnormalities, nanocarriers entrapping anticancer drugs could pass through these tissues improving the chance to reach cancer cells, which could not happen in normal tissues [6]. The problem with the nanocarriers following intravenous administration is the rapid removal by phagocytosis from blood circulation [7]. Therefore, prolonging the blood residence time of these nanocarriers is required, which could be achieved via surface modification using hydrophilic polymers such as polyethylene glycol (PEG) and polyvinyl alcohol (PVA) [8]. TDD consists of three chief constituents, namely the active substance, the targeting site, and the drug carrier. Drug carriers are systems in which the active ingredients are encapsulated in order to boost their selectivity and efficacy [9]. These carriers are mostly synthesized in a nanoscale range, thus being termed as nanocarriers. A wide variety of nanocarriers has been developed as a tool for drug targeting such as niosomes, liposomes, nanoparticles, nanoemulsions, and many others.
Nanoemulsion (NE) is one of the contemporary approaches of TDD systems that attracted the attention of researchers as a result of its great stability, biocompatibility, and efficient delivery of the active ingredients [10]. Additionally, it improved drug solubility, bioavailability, and drug loading capacity [11]. NE comprises two immiscible systems; oily phase that disperses into an aqueous phase or vice versa. It requires the presence of a surfactant and co-surfactant that play an essential role in preventing particle coalescence and consequently keeping the formulation stable [12]. PEGylated NE (PEG-NE) is a surface lipid-modified system that emerged and was applied extensively in the field of drug delivery [2]. PEG-NE was used in various routes of delivery including oral [13], ophthalmic [14], and intravenous adminstration [15].
The application of natural products in various treatment protocols became a novel trend that contributes to controlling several diseases, since they demonstrate the value of being safe and efficient [16]. Paclitaxel (PX) is a well-recognized natural anticancer drug obtained from the taxane family that proved to be efficient against a wide variety of cancers including breast cancer [17]. It mainly acts by interfering with the standard function of microtubule growth that represents an obstacle in front of the cell ability to use its cytoskeleton. The paclitaxel-microtubule complex poorly affects cell function as a result of microtubule shortening and lengthening. Moreover, PX might bind to an apoptosis stopping protein named B-cell leukemia 2, which would initiate an apoptosis in cancer cells [18,19].
However, the poor water solubility of PX is regarded as the main hurdle in its development. The approved PX formula, which was dissolved in Cremophor EL and dehydrated ethanol mixture, showed certain undesirable feedbacks mainly hypersensitivity reaction [20]. Therefore, it was important to search for an alternative dosage form that would improve PX solubility and diminish its associated problems.
Another class of natural products that proved to be therapeutically effective and safe is essential oils extracted from plants [21]. Myrrh essential oil is one of the extensively identified oils that demonstrated anti-inflammatory, anti-hyperlipidemic, analgesic, and anti-cancer activity [22,23,24,25]. The anti-cancer effect of myrrh oil has been widely investigated; it showed cytotoxic effect against human gastric cancer, hepatocellular carcinoma, and human prostate cancer cells. Unfortunately, myrrh oil was not extensively included in formulations intended for cancer therapy. Shi et al., 2012, reported the formulation and characterization of solid lipid nanoparticles loaded with frankincense and myrrh oil, which showed anti-tumor efficacy in H22-bearing Kunming mice [18,26,27,28,29]. Basically, myrrh consists of several constituents, including water-soluble gum, alcohol-soluble resins, and volatile oil. The gummy material contains polysaccharides and proteins, while the essential oils of myrrh were chemically identified to include α-pinene, dipentene, limonene, cuminaldehyde, cinnamic aldehyde, eugenol, m-cresol, sesquiterpene, a bicyclic sesquiterpene, a tricyclic sesquiterpene, formic acid, acetic acid, myrrholic acid, and palmitic acid [25,30]. Combining the EOs with active pharmaceutical agents would be considered an alternative strategy to boost net pharmacological influence [31].
At present, integrating natural bioactive compounds with several nanocarriers, especially NE, would provide great enhancement for their activities [32,33]. To find out, develop more efficient formulation, and gain the highest quality, an innovative methodology termed as quality by design (QbD) could be applied. Central composite design (CCD) is one of QbD approaches that help to implement a design depending on certain independent factors, mathematical equations, and statistical analysis to provide a desired, optimized formula [34].
The current investigation is an attempt to overcome the side effects of PX by incorporating it into PEG-NE as a drug delivery vehicle to be administered intravenously and to achieve a passive targeting. Various NE formulations were developed, characterized, and optimized by applying a QbD approach to get the best selected formula. The optimized NE formulation was subjected to number of evaluations including the quantitative estimation of serum protein associated on its surface and in vitro release study with and without serum incubation. Ultimately, cell viability of PX loaded into NE formulation was studied in MDA-MB-231 cancer cell line using MTT assay protocol.

2. Materials and Methods

2.1. Material

PX, Cremophore El, absolute ethyl alcohol, glycerol, egg phosphatidylcholine (EPC), corn oil, and acetonitril (HPLC grade) were procured from Sigma Aldrich (St. Louis, MO, USA). Distearoyl phosphatidylethanolamine-N-[methoxy poly (ethylene glycol)-2000] (PEG-DSPE) was purchased from Lipoid LLC., (Newark, NJ, USA). Myrrh essential oil was acquired from NOW® Essential Oils (NOW Foods, Bloomingdale, IL, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were supplied from Sigma Aldrich. Total protein and total lipid colorimetric kits were from United Diagnostics Industry, Dammam, Saudi Arabia. Tetrazolium dye (MTT reagent) was bought from Loba Chemie (Mumbai, India). All other chemicals were of the finest grade.

2.2. Central Composite Experimental Design

CCD methodology was implemented by running 11 formulations using two factor, two level (22) factorial design for selecting the optimized NE formulation using Design-Expert version 12.0 software (Stat-Ease, Minneapolis, MN, USA). Oil and surfactant concentration were selected as independent variables and are referred to as A and B, respectively. The impact of these variables on certain responses such as particle size (R1), in vitro release (R2), and hemolysis (R3) was studied. Table 1 demonstrated the selected factors along with their dependent responses at 2 levels (−1, 1). Statistical analysis was implemented using the analysis of variance test (ANOVA), in addition to some illustrative graphs that were generated, namely 2D-Contour plot, 3D-response surface plot, and linear correlation plot. All the data were supported by some mathematical equations as follow:
R = bo + b1A + b2B + b12AB + b11A2 + b22B2
Since R represents the dependent variable, while b0 denotes the intercept, b1, b2, b12, b11, and b22 refers to regression coefficients; A and B are main factors; AB indicates interactions between main factors, and A2 and B2 are polynomial terms.

2.3. Development of PEGylated PX-NEs

Various NE preparations containing PX were developed according to the method described earlier by Kan et al., with certain modifications [35]. Briefly, 3 mg of PX was dissolved in 250 µg cremophor EL, 250 µg absolute ethanol, and mixed with 100 mg of myrrh, different concentrations of corn oil, and 50 mg PEG-DSPE. Next, the oily phase was mixed with aqueous phase composed of 2.25% w/v glycerol solution. The mixture was homogenized using high shear homogenizer (T 25 digital Ultra-Turrax, IKA, Staufen, Germany) at 10,000 rpm for 10 min. The developed NE was sonicated using a probe sonicator (XL-2000, Qsonica, Newtown, CT, USA) for 20 s. Table 2 displays the matrix of 11 experimental NE formulations assembled by CCD along with their independent variables and the corresponding dependent responses.

2.4. Measurement of Size and Size Distribution (PDI)

First, 10 µL of PEGylated PX-NEs were diluted with about 3 mL distilled water in a disposable cuvette and checked at 25 °C for their particle size and PDI. This was analyzed by evaluating the dynamic light scattering at a scattering angle 90°, through Zetasizer apparatus (Malvern Instruments Ltd., Worcestershire, UK) [36].

2.5. In Vitro Release

A Franz diffusion cell apparatus (Logan Instruments Corp., FDC-6, New Jersey, USA) was adopted to estimate the profile of PX release from the NE formulations as previously reported by Paolino et al. [37]. In brief, 500 µL of each formulation constituting the donor compartment was held over a cellophane membrane (MWCO 2000–15,000). The receptor compartment containing up to 8 mL of the release media (PBS pH 7.4 and 0.1% Tween 80) adjusted at 37 °C and allowed to rotate at 100 rpm using a magnetic anchor of about 1 mm length. The study was continued up to 48 h where a sample of 300 µL was withdrawn at predefined time and substituted with the equivalent volume of fresh vehicle. The withdrawn samples were analyzed for PX using high-performance liquid chromatography (HPLC). The Shimadzu HPLC system consisted of a degasser (DGU-20A5), a liquid chromatograph (LC-20 AT), an auto sampler (SIL-20A), an ultraviolet–visible (UV/Vis) detector (SPD-20A), and a column oven (CTO-20AC) (SHIMADZU CORPORATION, Kyoto, Japan). A C18 reversed-phase column (100 mm × 4.6 mm, particle size 5-µm) was used. The mobile phase was acetonitrile: distilled water 50:50 (v/v), the flow rate was 1 mL/with retention time 10 min. UV detection was performed at 227 nm. Each experiment was executed in triplicate.

2.6. Animals

Male Wistar rats of average weight 200–220 g were supplied from the animal breeding center, King Saud University, Riyadh. The animals were kept at optimum temperature with free access to water and food using a 12 h light/dark cycle. All the animal experiments were performed in agreement with the Guidelines for the Ethical Conduct for Use of Animals in Research, and approved by the Animal Research Ethics Committee at King Faisal University, approval number (KFU-REC-2021-DEC-EA000306).

2.7. Hemolytic Activity

In this study, blood was obtained from rats using a syringe containing heparin traces, which was then centrifuged at 20 °C for 10 min at 1500× g. Subsequently, plasma was removed and exchanged with an identical amount of PBS pH 7.4, centrifuged over again, and repeated three times. Then, 1 mL of 2% w/v erythrocyte suspension in PBS pH 7.4 was incubated at 37 °C with equivalent volume of NE formulation for 30 min. Centrifugation at 3000× g for 10 min resulted in releasing hemoglobin and the supernatant was diluted with PBS and analyzed spectrophotometrically at λmax 550 nm. For control, to attain 100% hemoglobin release, erythrocytes were incubated with PBS or mixed with 1 mL of 1% Triton-X 100 [34].

2.8. Development of Naked PX-NE

According to the data obtained from the experimental study, one formula was optimized. Its naked counterpart (PX-NE without PEG-DSPE) was developed following same method of developing PEGylated PX-NE.

2.9. Characterization of Optimized PEGylated and Naked PX-NE

Particle size and PDI of PEGylated PX-NE and its naked counterpart were detected as previously mentioned in Section 2.4 using a Zetasizer apparatus (Malvern Instruments Ltd., Worcestershire, UK). Afterward, the viscosity of both naked and PEGylated formulations were evaluated via a Brookfield viscometer (DV-II+ Pro, Middleboro, MA, USA) at room temperature using spindle No. 63 [34].

2.10. Hemolytic Activity for Naked PX-NE

The same procedure performed in Section 2.7 was followed to figure out the hemolytic activity of the naked formulation in comparison with PEGylated one.

2.11. Quantitative Determination of Serum Protein Associated on PX-NE Surface

In order to estimate the amount of serum protein (opsonin) that could be absorbed on surface of the optimized formulation, naked and PEGylated PX-NE were incubated at 37 °C with equivalent amount of FBS for 30 min. The bulk serum protein was separated from the NE formulations by loading the preparation over Sepharose CL-4B gel column. NEs were received from the bottom of the column and quantitatively checked for protein associated on its surface via total protein and total lipid colorimetric kits [38].

2.12. In Vitro Release of PX from Optimized NE before and after Incubation with Serum

In vitro release study, as previously mentioned in Section 2.5, was followed in order to identify the percentage of PX released from marketed PX, optimized PEGylated PX-NE, and its naked counterpart. This experiment was performed before and after incubating the formulations with 10% serum [2].

2.13. Cell Line

In order to investigate the in vitro cytotoxicity study, MDA-MB-231cancer cells were selected and obtained from the American Type Culture Collection (ATCC; VA, New York, NY, USA) through college of science, King Faisal University, KSA. The cells were cultured in DMEM, with the addition of 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 μg/mL gentamicin. Here, 10% heat-inactivated FBS is required at 37 °C under 5% CO2/95% air.

2.14. In Vitro Cytotoxicity

The cytotoxicity of PX in different formulations including PX solution, blank NE, PEGylated PX-NE, and naked PX-NE was screened against MDA-MB-231cancer cells conducting MTT assay. The study was performed using 96-well plate where a volume of 5000 cells were seeded into the plate followed by adding a definite amount of the investigated formulations to the specified well. The plates were incubated 48 h at 37 °C, then subjected to further incubation for 4 h after treating with MTT reagent. Afterward, the media was removed and each well was treated with DMSO for 10 min and kept in a humidified incubator with 5% CO2. The absorbance was estimated at 570 nm [39].

2.15. Statistics

Data were statistically analyzed using one-way analysis of variance (ANOVA) via Design-Expert version 12.0 software (Stat-Ease, Minneapolis, MN, USA). Other analyses were executed by using SPSS statistics software, version 9 (IBM Corporation, Armonk, NY, USA). Data are considered statistically significant at p < 0.05.

3. Results

3.1. Statistical Analysis for the Experimental Design

CCD generated a matrix of 11 NE formulations as shown in Table 2, which offered four factorial and four axial points in addition to three central points. To demonstrate the results, statistical analysis using ANOVA was highly recommended. As per data in Table 3, the F-value for all responses suggests a significant model since it was 506.52, 568.05, and 282.72 for R1, R2, and R3, respectively. Moreover, the p-value was less than 0.05 in case of variables A and B for all responses, which indicates significant model terms. When values are greater than 0.1, the model terms are regarded as non-significant. Another term is lack of Fit F-value, which is advised to be non-significant in order to attain a fit model. In our study, lack of fit was 2.11, 0.5505, and 1.69 with relative p-value 0.3559, 0.7583, and 0.4179 for R1, R2, and R3, respectively.

3.2. Effect of Independent Variables on Particle Size Determination

Particle size is one of the responses that have an extensive effect on the fate of the in vivo investigation following intravenous administration. As shown in Table 2, the particle size of the developed PX-NEs ranged between 132 ± 2.5 and 238 ± 2.1 nm. It was apparent that increasing oil concentration (A) would result in subsequent increase in NE particle size. However, reduced particle size was noticed upon using higher concentration of EPC (B) while keeping (A) constant. Our result was parallel to Sarheed et al., whose findings proved that lower oil concentration resulted in decrease in lidocaine nanoemulsion droplet sizes with an increase in surfactant concentration [40]. This finding actually could be related to the increase in the dispersed phase. The instructed design matrix could illustrate the influence of R1 on the resulted response by the following equation:
R1 = 81.9119 + 149.953 A − 0.402665 B
As obvious from the equation, the positive influence of (A) on particle size was emphasized, since it carried a positive sign that indicates a synergistic effect; however, the negative sign of (B) signifies an antagonistic effect. Additionally, the data were further illustrated via certain graphs created from the design. Figure 1a represents a 3D response surface plot that clarified the effect of variables A and B on R1 response. Furthermore, Figure 1b and Table 4 demonstrate the fit statistics and the association between the adjusted and predicted R2 value for R1 response. Whereas the variation between adjusted R2 (0.9902) and predicted R2 (0.9841) was less than 0.2, the R2 value was recorded to be 0.9922 which could recommend the model and adequate precision was 59.587, which indicates adequate signal and proved that the model can be used to navigate the design space.

3.3. Effect of Independent Variables on In Vitro Release Study

Release of PX from the formulated NE preparations was estimated as displayed in Table 2 over a period of 48 h where it ranged from 47 ± 3.0 to 64.7 ± 2.6%. The percentage of PX released was decreased while using higher concentrations of oil, which is certainly ascribed to greater particle size that resulted in small surface area and consequently lower drug release [41]. On the other hand, higher EPC concentration while using same oil concentration would enhance drug release from the formulation which could be due to improving the drug solubility [42]. This could be explained in Figure 2a,b showing 3D response surface plot and linearity plot, respectively. Furthermore, as presented in Table 4, there was a reasonable agreement between predicted R2 (0.9870) and adjusted (0.9913) in addition to adequate precision value (62.5540), which is desirable and indicates a suitable signal. Moreover, the following mathematical equation would provide a description for the interaction between independent and dependent variables:
R2 = 71.5178 − 24.6658 A + 0.0783547 B
The negative sign of variable A denotes its antagonistic action; however, the positive one related to variable B confirms its synergistic effect.

3.4. Effect of Independent Variables on Hemolytic Activity

Hemolytic assay is very important in order to assure the safety of the NE formulation and avoid any unsafe consequences on biological membranes [43]. As displayed in Table 2, the percentage of hemolysis in all PX-NE formulations after being incubated with serum was within 2.8 ± 0.2 and 5.6 ± 0.2%. Hence, the erythrocytes were precipitated at the bottom whereas the above supernatant was transparent. The results indicate that all developed PX-NE formulations are safe and harmless and could be administered via intravenous route. Our results were in accordance with Wang et al., who confirmed the safety of paclitaxel micro-emulsion following intravenous administration since it did not show any hemolysis or erythrocyte agglutination [44]. Figure 3a related to the 3D response surface plot showed the substantial effect of independent variables A and B on the % of hemolytic activity, where there is a direct relation between them. Further, Figure 3b indicates the linear correlation between the predicted and actual values, which is clearly demonstrated in the close values of predicted (0.9719) and adjusted R2 (0.9826).
Moreover, and as shown in the following obtained mathematical equation, both variables A and B exhibited a significant influence on the hemolysis activity response (R3):
R3 = −1.05872 + 4.0799 A + 0.00868718 B

3.5. Optimizing the Independent Variables

The optimization step is very crucial in selecting the most appropriate formulation possessing the best characteristics and providing the most adequate response. Optimizing the formulation in the design software accomplished using numerical optimization depending mostly on the desirability value and the generated graphs. Basically, the achieved responses were oriented toward certain required objectives that were to keep both independent variables in range while minimizing all the responses R1, R2, and R3. Based on that consideration, and as shown in Figure 4, values of A and B were proposed to be 0.849 g and 100 mg, respectively. According to software point prediction and higher desirability obtained, the values of the optimized formulation were anticipated and compared to that of the developed NE as mentioned in Table 5. It was noted that the expected and the observed values were parallel to each other.

3.6. Characterization of Optimized PEGylated and Naked PX-NE

Based upon the former studies, an optimized PEGylated PX-NE formulation was developed. Comparable formulation without PEG-DSPE was fabricated to be the naked counterpart of PEGylated PX-NE and together underwent several investigations to assess the role of PEGylation. Primarily, both formulations were evaluated for their particle size and PDI. Figure 5A shows the particle size of the optimized PEGylated PX-NE formulation that was 170 nm with PDI 0.280, which was considered ideal for intravenous administration. Conversely, Figure 5B shows higher particle size of naked PX-NE that was 321.9 nm with corresponding PDI 0.287. Definitely, small particle size (as stated for PEGylated NE) is more preferable for intravenous administration in order to avoid emboli formation [45]. These records highlighted the significance of PEG and supported its role in reducing the particle size and maintaining a good distribution of particles within the NE formulation. With regard to the viscosity of NE formulation, it appeared to be of great importance, especially when intended for parenteral administration since it should be within an appropriate range. According to the data obtained, viscosity of PEGylated PX-NE was 2.91 ± 0.21 cP; however, for the comparable naked formulation, it was 3.32 ± 0.18 cP, which is considered in the satisfactory range. It was stated by Araújo et al. that viscosity of parenteral formulation should not exceed 3.9 cP [46].

3.7. Hemolytic Activity

Intravenous injection safety was assessed for the developed naked PX-NE using hemolytic test, and compared with that obtained for PEGylated NE formulation. It was noted that the erythrocytes did not survive since the solution turned into red. As observed in Figure 6, a significant difference was detected between hemolysis of PEGylated PX-NE (3.44 ± 0.2%) and its naked counterpart (6.8 ± 0.4%) p < 0.05. This could point toward the preferences of using the PEGylated formulation via intravenous administration.

3.8. Quantitative Determination of Serum Protein Associated on PX-NE Surface

Quantitative estimation of serum protein that might be adsorbed on the surface of the formulations was performed and the result is depicted in Figure 7. The amount of protein was found to be 16.57 ± 0.91 µg/µmol and 45.73 ± 1.55 µg/µmol for PEGylated PX-NE and its naked counterpart, respectively. It is highly obvious that total amount of protein adsorbed on surface of naked formulation is significantly greater than that associated on the PEGylated NE surface (p < 0.05). This could be accredited to PEGylation that resulted in forming fixed aqueous layer thickness, which is known as the FALT phenomenon. In that phenomenon, the layer of PEG could prevent the recognition of the developed NE by the serum protein and consequently inhibit their interaction [47]. The results encountered agree with Nandhakmar et al., who demonstrated lower amount of protein on the modified surface of paclitaxel nanoparticles and established that long circulating nanoparticles (with surface modification) could counterattack phagocytosis by preventing opsonin adsorption on its surface [38].

3.9. In Vitro Release of PX from Optimized NE before and after Incubation with Serum

A study of the in vitro release profile of PX from both formulations, PEGylated and naked NE, was carried out and the result is shown in Figure 8A. It was revealed that 93.3% of PX was released from PX solution within 12 h. Further, following 48 h, the percentage of PX released from PEGylated formulation (57.5 ± 2.7%) was noticeably lower than that released from its naked counterpart (63.3 ± 3.2%). This finding could be attributed to higher stability of PEGylated NE formulation, in addition to the rigid layer formed on the surface of NE by PEG that lowered the percentage of drug released from the formulation. On the other side, Figure 8B shows the percentage of PX released from each formulation after being incubated with equivalent amount of 10% serum. The figure revealed that serum speeds up the release of PX from naked NE, where it reached 78.8 ± 3.7% after 48 h. Nevertheless, it did not markedly affect the release from PEGylated NE (60.8 ± 2.3%) when compared with the release without serum incubation. This could be illustrated according to the MAC (membrane attack complex), which allows serum protein to attack naked NE, disrupting the formulation and facilitating the release of the drug [17]. The achieved results were confirmed by Gagliardi et al.’s study, where they investigated the influence of serum incubation with paclitaxel nanoparticle on different parameters such as particle size, entrapment, and in vitro study [48].

3.10. In Vitro Cytotoxicity

In order to figure out the anti-tumor proliferation capability of PEGylated PX-NE, MTT assay investigation was executed using MDA-MB-231 culture cells at various concentrations. As depicted in Figure 9, cell viability was extensively and significantly high in control group that recorded 96.85 ± 2.49% when compared to other groups under examination (p < 0.05). The cytotoxicity in all other groups was seemingly a concentration-dependent cytotoxicity. Cell viability in blank NE could reach 53.52 ± 4.4%, which appeared to be significantly higher than cell viability in PX-solution, naked, and PEGylated PX-NE which showed 24.53 ± 1.76, 37.16 ± 3.01, and 31.59 ± 2.29%, respectively. However, it seemed to possess a certain cytotoxic effect toward MDA cells that point toward the presence of definite anti-cancer activity for myrrh essential oil [49]. Apparently, upon increasing the concentration, a lower significant difference was distinguished between PX solution and PX formulations whether naked or PEGylated PX-NE. This is presumed to be due to the higher release of PX from the solution than from the NE formulations that could result in greater availability of PX for the cellular uptake providing a quick cytotoxic effect in the media [17]. In addition, it is a well-known fact that there is no endocytosis of NE in cancer cell lines and the effect depends on the drug released from the NE formulation. Nandhakumar et al.’s results confirmed the lower % of cell viability of free paclitaxel when compared to paclitaxel nanoparticle formulation [38]. Additionally, the obtained results were further supported by Najlah et al., who certified the higher cytotoxic influence of paclitaxel alone compared with paclitaxel in clinoleic and intralipid nanoemulsions [50]. Moreover, lower IC50 was exhibited by PX solution (23.82 ± 5.96 µg/mL), which was in a significant difference (p < 0.05) with that of naked (47.68 ± 7.64 µg/mL) and PEGylated PX-NE (38.66 ± 5.51 µg/mL).

4. Conclusions

In the present study, paclitaxel was incorporated within an efficient nanocarrier formulation, namely, nanoemulsion formulated using myrrh essential oil and PEG-DSPE. Different formulations were optimized based on certain observed responses with respect to particle size, in vitro release, and hemolytic test. The optimized formula was compared to its naked counterpart in order to investigate the role of PEGylation in protecting the intravenous formulation from being recognized by serum protein. The PEGylated formulation provided acceptable particle size, proper viscosity, lower amount of protein, and lower percentage of hemolysis to be suitable for intravenous administration. Additionally, cytotoxicity was emphasized for the formulation using MTT assay and proved the influence of myrrh oil and its expected synergism with paclitaxel. In conclusion, a nanoemulsion developed using myrrh essential oil could be a prospective nanocarrier for paclitaxel delivery.

Author Contributions

T.M.S., funding acquisition and data curation. T.M.S. and H.S.E., methodology, project administration, software, writing—original draft, software, writing—original draft, writing—review, editing, formal analysis, conceptualization, and interpretation. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by The Deanship of Scientific Research (DSR), Vice presidency for Graduate Studies and Scientific Research, King Faisal University (KFU), Annual funding track, grant number AN0008.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Animal Research Ethics Committee at King Faisal University, approval number (KFU-REC-2021-DEC-EA000306).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors thank the Deanship of Scientific Research, King Faisal University for funding and also College of Clinical Pharmacy for facilities support.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Redig, A.J.; McAllister, S.S. Breast cancer as a systemic disease: A view of metastasis. J. Intern. Med. 2013, 274, 113–126. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Elsewedy, H.S.; Aldhubiab, B.E.; Mahdy, M.A.; Elnahas, H.M. Brucine PEGylated nanoemulsion: In vitro and in vivo evaluation. Colloids Surf. A Physicochem. Eng. Asp. 2021, 608, 125618. [Google Scholar] [CrossRef]
  3. Vasir, J.K.; Labhasetwar, V. Targeted drug delivery in cancer therapy. Technol. Cancer Res. Treat. 2005, 4, 363–374. [Google Scholar] [CrossRef] [PubMed]
  4. Salahpour Anarjan, F. Active targeting drug delivery nanocarriers: Ligands. Nano-Struct. Nano-Objects 2019, 19, 100370. [Google Scholar] [CrossRef]
  5. Elsewedy, H.S.; Dhubiab, B.E.A.; Mahdy, M.A.; Elnahas, H.M. Development, optimization, and evaluation of PEGylated brucine-loaded PLGA nanoparticles. Drug Deliv. 2020, 27, 1134–1146. [Google Scholar] [CrossRef]
  6. Greish, K. Enhanced permeability and retention (EPR) effect for anticancer nanomedicine drug targeting. Methods Mol. Biol. 2010, 624, 25–37. [Google Scholar] [CrossRef]
  7. Blanco, E.; Shen, H.; Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 2015, 33, 941–951. [Google Scholar] [CrossRef]
  8. Suk, J.S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L.M. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv. Drug Deliv. Rev. 2016, 99, 28–51. [Google Scholar] [CrossRef] [Green Version]
  9. Trucillo, P. Drug Carriers: Classification, Administration, Release Profiles, and Industrial Approach. Processes 2021, 9, 470. [Google Scholar] [CrossRef]
  10. Jaiswal, M.; Dudhe, R.; Sharma, P.K. Nanoemulsion: An advanced mode of drug delivery system. 3 Biotech 2015, 5, 123–127. [Google Scholar] [CrossRef] [Green Version]
  11. Elsewedy, H.S.; Al-Dhubiab, B.E.; Mahdy, M.A.; Elnahas, H.M. Basic Concepts of Nanoemulsion and its Potential application in Pharmaceutical, Cosmeceutical and Nutraceutical fields. Res. J. Pharm. Technol. 2021, 14, 3938–3946. [Google Scholar] [CrossRef]
  12. Aswathanarayan, J.B.; Vittal, R.R. Nanoemulsions and Their Potential Applications in Food Industry. Front. Sustain. Food Syst. 2019, 3, 1–21. [Google Scholar] [CrossRef] [Green Version]
  13. Santander-Ortega, M.J.; Plaza-Oliver, M.; Rodríguez-Robledo, V.; Castro-Vázquez, L.; Villaseca-González, N.; González-Fuentes, J.; Cano, E.L.; Marcos, P.; Lozano, M.V.; Arroyo-Jiménez, M.M. PEGylated Nanoemulsions for Oral Delivery: Role of the Inner Core on the Final Fate of the Formulation. Langmuir 2017, 33, 4269–4279. [Google Scholar] [CrossRef] [PubMed]
  14. Balguri, S.P.; Adelli, G.R.; Janga, K.Y.; Bhagav, P.; Majumdar, S. Ocular disposition of ciprofloxacin from topical, PEGylated nanostructured lipid carriers: Effect of molecular weight and density of poly (ethylene) glycol. Int. J. Pharm. 2017, 529, 32–43. [Google Scholar] [CrossRef] [PubMed]
  15. Devalapally, H.; Zhou, F.; McDade, J.; Goloverda, G.; Owen, A.; Hidalgo, I.J.; Silchenko, S. Optimization of PEGylated nanoemulsions for improved pharmacokinetics of BCS class II compounds. Drug Deliv. 2015, 22, 467–474. [Google Scholar] [CrossRef] [Green Version]
  16. Atanasov, A.G.; Zotchev, S.B.; Dirsch, V.M.; Orhan, I.E.; Banach, M.; Rollinger, J.M.; Barreca, D.; Weckwerth, W.; Bauer, R.; Bayer, E.A.; et al. Natural products in drug discovery: Advances and opportunities. Nat. Rev. Drug Discov. 2021, 20, 200–216. [Google Scholar] [CrossRef]
  17. Yoshizawa, Y.; Kono, Y.; Ogawara, K.-i.; Kimura, T.; Higaki, K. PEG liposomalization of paclitaxel improved its in vivo disposition and anti-tumor efficacy. Int. J. Pharm. 2011, 412, 132–141. [Google Scholar] [CrossRef]
  18. Shi, F.; Zhao, J.-H.; Liu, Y.; Wang, Z.; Zhang, Y.-T.; Feng, N.-P. Preparation and characterization of solid lipid nanoparticles loaded with frankincense and myrrh oil. Int. J. Nanomed. 2012, 7, 2033. [Google Scholar]
  19. McCloskey, D.E.; Kaufmann, S.H.; Prestigiacomo, L.J.; Davidson, N.E. Paclitaxel induces programmed cell death in MDA-MB-468 human breast cancer cells. Clin. Cancer Res. 1996, 2, 847–854. [Google Scholar]
  20. Araki, T.; Kono, Y.; Ogawara, K.-i.; Watanabe, T.; Ono, T.; Kimura, T.; Higaki, K. Formulation and evaluation of paclitaxel-loaded polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer. Biol. Pharm. Bull. 2012, 35, 1306–1313. [Google Scholar] [CrossRef] [Green Version]
  21. Abdallah, M.H.; Elsewedy, H.S.; AbuLila, A.S.; Almansour, K.; Unissa, R.; Elghamry, H.A.; Soliman, M.S. Quality by Design for Optimizing a Novel Liposomal Jojoba Oil-Based Emulgel to Ameliorate the Anti-Inflammatory Effect of Brucine. Gels 2021, 7, 219. [Google Scholar] [CrossRef] [PubMed]
  22. Soliman, W.E.; Shehata, T.M.; Mohamed, M.E.; Younis, N.S.; Elsewedy, H.S. Enhancement of Curcumin Anti-Inflammatory Effect via Formulation into Myrrh Oil-Based Nanoemulgel. Polymers 2021, 13, 577. [Google Scholar] [CrossRef] [PubMed]
  23. Shehata, T.M.; Khalil, H.E.; Elsewedy, H.S.; Soliman, W.E. Myrrh essential oil-based nanolipid formulation for enhancement of the antihyperlipidemic effect of atorvastatin. J. Drug Deliv. Sci. Technol. 2021, 61, 102277. [Google Scholar] [CrossRef]
  24. Shalaby, M.A.; Hammouda, A.A.-E. Analgesic, anti-inflammatory and anti-hyperlipidemic activities of Commiphora molmol extract (Myrrh). J. Intercult. Ethnopharmacol. 2014, 3, 56–62. [Google Scholar] [CrossRef]
  25. Chen, Y.; Zhou, C.; Ge, Z.; Liu, Y.; Liu, Y.; Feng, W.; Li, S.; Chen, G.; Wei, T. Composition and potential anticancer activities of essential oils obtained from myrrh and frankincense. Oncol. Lett. 2013, 6, 1140–1146. [Google Scholar] [CrossRef] [Green Version]
  26. Sun, M.; Hua, J.; Liu, G.; Huang, P.; Liu, N.; He, X. Myrrh induces the apoptosis and inhibits the proliferation and migration of gastric cancer cells through down-regulating cyclooxygenase-2 expression. Biosci. Rep. 2020, 40, BSR20192372. [Google Scholar] [CrossRef]
  27. Zheng, P.; Huang, Z.; Tong, D.-C.; Zhou, Q.; Tian, S.; Chen, B.-W.; Ning, D.-M.; Guo, Y.-M.; Zhu, W.-H.; Long, Y. Frankincense myrrh attenuates hepatocellular carcinoma by regulating tumor blood vessel development through multiple epidermal growth factor receptor-mediated signaling pathways. World J. Gastrointest. Oncol. 2022, 14, 450. [Google Scholar] [CrossRef]
  28. Anwar, H.M.; Moghazy, A.M.; Osman, A.A.E.E.; Rahman, A.A.A. The Therapeutic Effect of Myrrh (Commiphora molmol) and Doxorubicin on Diethylnitrosamine Induced Hepatocarcinogenesis in Male Albino Rats. Asian Pac. J. Cancer Prev. APJCP 2021, 22, 2153. [Google Scholar] [CrossRef]
  29. Wang, X.-l.; Kong, F.; Shen, T.; Young, C.Y.; Lou, H.-x.; Yuan, H.-q. Sesquiterpenoids from myrrh inhibit androgen receptor expression and function in human prostate cancer cells. Acta Pharmacol. Sin. 2011, 32, 338–344. [Google Scholar] [CrossRef]
  30. Hanuš, L.O.; Řezanka, T.; Dembitsky, V.M.; Moussaieff, A. Myrrh-commiphora chemistry. Biomed. Pap. 2005, 149, 3–28. [Google Scholar] [CrossRef] [Green Version]
  31. Langeveld, W.T.; Veldhuizen, E.J.; Burt, S.A. Synergy between essential oil components and antibiotics: A review. Crit. Rev. Microbiol. 2014, 40, 76–94. [Google Scholar] [CrossRef] [PubMed]
  32. Ben Jemaa, M.; Falleh, H.; Riadh, K. Encapsulation of Natural Bioactive Compounds: Nanoemulsion Formulation to Enhance Essential Oils Activities. In Microencapsulation-Processes, Technologies and Industrial Applications; 2019; Available online: https://doi.org/10.5772/intechopen.84183 (accessed on 11 April 2022).
  33. Elsewedy, H.S.; Younis, N.S.; Shehata, T.M.; Mohamed, M.E.; Soliman, W.E. Enhancement of Anti-Inflammatory Activity of Optimized Niosomal Colchicine Loaded into Jojoba Oil-Based Emulgel Using Response Surface Methodology. Gels 2021, 8, 16. [Google Scholar] [CrossRef] [PubMed]
  34. Khalil, H.E.; Alqahtani, N.K.; Darrag, H.M.; Ibrahim, H.-I.M.; Emeka, P.M.; Badger-Emeka, L.I.; Matsunami, K.; Shehata, T.M.; Elsewedy, H.S. Date Palm Extract (Phoenix dactylifera) PEGylated Nanoemulsion: Development, Optimization and Cytotoxicity Evaluation. Plants 2021, 10, 735. [Google Scholar] [CrossRef] [PubMed]
  35. Kan, P.; Chen, Z.B.; Lee, C.J.; Chu, I.M. Development of nonionic surfactant/phospholipid o/w emulsion as a paclitaxel delivery system. J. Control. Release Off. J. Control. Release Soc. 1999, 58, 271–278. [Google Scholar] [CrossRef]
  36. Soliman, W.E.; Khan, S.; Rizvi, S.M.D.; Moin, A.; Elsewedy, H.S.; Abulila, A.S.; Shehata, T.M. Therapeutic Applications of Biostable Silver Nanoparticles Synthesized Using Peel Extract of Benincasa hispida: Antibacterial and Anticancer Activities. Nanomaterials 2020, 10, 1954. [Google Scholar] [CrossRef]
  37. Paolino, D.; Celia, C.; Trapasso, E.; Cilurzo, F.; Fresta, M. Paclitaxel-loaded ethosomes®: Potential treatment of squamous cell carcinoma, a malignant transformation of actinic keratoses. Eur. J. Pharm. Biopharm. 2012, 81, 102–112. [Google Scholar] [CrossRef]
  38. Nandhakumar, S.; Dhanaraju, M.D.; Sundar, V.D.; Heera, B. Influence of surface charge on the in vitro protein adsorption and cell cytotoxicity of paclitaxel loaded poly(ε-caprolactone) nanoparticles. Bull. Fac. Pharm. Cairo Univ. 2017, 55, 249–258. [Google Scholar] [CrossRef]
  39. Ismail, T.A.; Shehata, T.M.; Mohamed, D.I.; Elsewedy, H.S.; Soliman, W.E. Quality by Design for Development, Optimization and Characterization of Brucine Ethosomal Gel for Skin Cancer Delivery. Molecules 2021, 26, 3454. [Google Scholar] [CrossRef]
  40. Sarheed, O.; Dibi, M.; Ramesh, K.V.R.N.S. Studies on the Effect of Oil and Surfactant on the Formation of Alginate-Based O/W Lidocaine Nanocarriers Using Nanoemulsion Template. Pharmaceutics 2020, 12, 1223. [Google Scholar] [CrossRef]
  41. Laxmi, M.; Bhardwaj, A.; Mehta, S.; Mehta, A. Development and characterization of nanoemulsion as carrier for the enhancement of bioavailability of artemether. Artif. Cells Nanomed. Biotechnol. 2015, 43, 334–344. [Google Scholar] [CrossRef]
  42. Efentakis, M.; Al-Hmoud, H.; Buckton, G.; Rajan, Z. The influence of surfactants on drug release from a hydrophobic matrix. Int. J. Pharm. 1991, 70, 153–158. [Google Scholar] [CrossRef]
  43. Cabello-Alvarado, C.; Andrade-Guel, M.; Pérez-Alvarez, M.; Cadenas-Pliego, G.; Cortés-Hernández, D.A.; Bartolo-Pérez, P.; Ávila-Orta, C.; Cruz-Delgado, V.; Zepeda-Pedreguera, A. Graphene nanoplatelets modified with amino-groups by ultrasonic radiation of variable frequency for potential adsorption of uremic toxins. Nanomaterials 2019, 9, 1261. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Wang, Y.; Wu, K.-C.; Zhao, B.-X.; Zhao, X.; Wang, X.; Chen, S.; Nie, S.-F.; Pan, W.-S.; Zhang, X.; Zhang, Q. A novel paclitaxel microemulsion containing a reduced amount of Cremophor EL: Pharmacokinetics, biodistribution, and in vivo antitumor efficacy and safety. J. Biomed. Biotechnol. 2011, 2011, 854872. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Danaei, M.; Dehghankhold, M.; Ataei, S.; Hasanzadeh Davarani, F.; Javanmard, R.; Dokhani, A.; Khorasani, S.; Mozafari, M. Impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems. Pharmaceutics 2018, 10, 57. [Google Scholar] [CrossRef] [Green Version]
  46. Araújo, F.; Kelmann, R.; Araújo, B.; Finatto, R.; Teixeira, H.; Koester, L. Development and characterization of parenteral nanoemulsions containing thalidomide. Eur. J. Pharm. Sci. 2011, 42, 238–245. [Google Scholar] [CrossRef] [Green Version]
  47. Shehata, T.; Ogawara, K.-i.; Higaki, K.; Kimura, T. Prolongation of residence time of liposome by surface-modification with mixture of hydrophilic polymers. Int. J. Pharm. 2008, 359, 272–279. [Google Scholar] [CrossRef]
  48. Gagliardi, A.; Bonacci, S.; Paolino, D.; Celia, C.; Procopio, A.; Fresta, M.; Cosco, D. Paclitaxel-loaded sodium deoxycholate-stabilized zein nanoparticles: Characterization and in vitro cytotoxicity. Heliyon 2019, 5, e02422. [Google Scholar] [CrossRef] [Green Version]
  49. Cao, B.; Wei, X.C.; Xu, X.R.; Zhang, H.Z.; Luo, C.H.; Feng, B.; Xu, R.C.; Zhao, S.Y.; Du, X.J.; Han, L.; et al. Seeing the Unseen of the Combination of Two Natural Resins, Frankincense and Myrrh: Changes in Chemical Constituents and Pharmacological Activities. Molecules 2019, 24, 3076. [Google Scholar] [CrossRef] [Green Version]
  50. Najlah, M.; Kadam, A.; Wan, K.-W.; Ahmed, W.; Taylor, K.M.G.; Elhissi, A.M.A. Novel paclitaxel formulations solubilized by parenteral nutrition nanoemulsions for application against glioma cell lines. Int. J. Pharm. 2016, 506, 102–109. [Google Scholar] [CrossRef] [Green Version]
Processes 10 00907 g001 550
Figure 1. (a) 3D-response surface plot and (b) linear correlation plot between predicted versus actual values for illustrating the influence of variables A and B on particle size (R1).
Figure 1. (a) 3D-response surface plot and (b) linear correlation plot between predicted versus actual values for illustrating the influence of variables A and B on particle size (R1).
Processes 10 00907 g001
Processes 10 00907 g002 550
Figure 2. (a) 3D-response surface plot and (b) linear correlation plot between predicted versus actual values for illustrating the influence of variables A and B on in vitro release (R2).
Figure 2. (a) 3D-response surface plot and (b) linear correlation plot between predicted versus actual values for illustrating the influence of variables A and B on in vitro release (R2).
Processes 10 00907 g002
Processes 10 00907 g003 550
Figure 3. (a) 3D-response surface plot and (b) linear correlation plot between predicted versus actual values for illustrating the influence of variables A and B on hemolytic activity (R3).
Figure 3. (a) 3D-response surface plot and (b) linear correlation plot between predicted versus actual values for illustrating the influence of variables A and B on hemolytic activity (R3).
Processes 10 00907 g003
Processes 10 00907 g004 550
Figure 4. Optimization ramp for the investigated independent variables, (A) oil concentration; (B) EPC concentration, along with their expected values of responses; (C) particle size; (D) in vitro release; (E) hemolysis, in addition to the desirability value.
Figure 4. Optimization ramp for the investigated independent variables, (A) oil concentration; (B) EPC concentration, along with their expected values of responses; (C) particle size; (D) in vitro release; (E) hemolysis, in addition to the desirability value.
Processes 10 00907 g004
Processes 10 00907 g005 550
Figure 5. Particle size and PDI of (A) PEGylated PX-NE and (B) naked PX-NE.
Figure 5. Particle size and PDI of (A) PEGylated PX-NE and (B) naked PX-NE.
Processes 10 00907 g005
Processes 10 00907 g006 550
Figure 6. Hemolytic activity of PX-NE formulations after incubation with rat red blood cells (2% w/v in PBS) at 37 °C for 30 min. Results are expressed as the mean ± S.D. n = 3. * p < 0.05, compared to naked PX-NE.
Figure 6. Hemolytic activity of PX-NE formulations after incubation with rat red blood cells (2% w/v in PBS) at 37 °C for 30 min. Results are expressed as the mean ± S.D. n = 3. * p < 0.05, compared to naked PX-NE.
Processes 10 00907 g006
Processes 10 00907 g007 550
Figure 7. Total amount of serum proteins adsorbed on the surface of naked and PEGylated PX-NE. * p < 0.05, compared with naked PX-NE.
Figure 7. Total amount of serum proteins adsorbed on the surface of naked and PEGylated PX-NE. * p < 0.05, compared with naked PX-NE.
Processes 10 00907 g007
Processes 10 00907 g008 550
Figure 8. Profile of PX in vitro release from PX solution, PEGylated, and naked NEs (A) without serum incubation; (B) after serum incubation. Results are expressed as the mean ± SD. n = 3.
Figure 8. Profile of PX in vitro release from PX solution, PEGylated, and naked NEs (A) without serum incubation; (B) after serum incubation. Results are expressed as the mean ± SD. n = 3.
Processes 10 00907 g008
Processes 10 00907 g009 550
Figure 9. Cell viability of MDA-MB-231 cancer cells upon using naked and PEGylated PX-NE formulations compared with blank NE and PX solution. Data are represented as mean ± SD. * p < 0.05, compared to control, # p < 0.05, compared to blank NE, and • p < 0.05, compared to PX solution.
Figure 9. Cell viability of MDA-MB-231 cancer cells upon using naked and PEGylated PX-NE formulations compared with blank NE and PX solution. Data are represented as mean ± SD. * p < 0.05, compared to control, # p < 0.05, compared to blank NE, and • p < 0.05, compared to PX solution.
Processes 10 00907 g009
Table
Table 1. Selected independent variable with their level of variation and the responses.
Table 1. Selected independent variable with their level of variation and the responses.
Independent VariableSymbolLevel of Variation
−1+1
Corn oil concentration (g)A0.751.25
EPC concentration (mg)B100140
ResponsesSymbolConstraints
Particle size (nm)R1Minimize
In vitro drug release (%)R2Minimize
Hemolysis (%)R3Minimize
Table
Table 2. Experimental design for PEGylated PX-NE formulations along with their corresponding observed values of response.
Table 2. Experimental design for PEGylated PX-NE formulations along with their corresponding observed values of response.
FormulaIndependent VariablesResponse
A (mg)B (mg)R1 (nm)R2 (%)R3 (%)
F11120182 ± 3.856.5 ± 3.14.1 ± 0.4
F21148.2168 ± 2.659.0 ± 2.64.4 ± 0.5
F31120184 ± 2.555.7 ± 1.73.9 ± 0.3
F41.25140218 ± 3.051.5 ± 2.25.2 ± 0.2
F51.35120238 ± 2.147.0 ± 3.0 5.6 ± 0.2
F60.75140140 ± 1.863.6 ± 2.93.1 ± 0.3
F70.75100154 ± 2.861.2 ± 2.82.9 ± 0.1
F81.25100226 ± 3.249.0 ± 3.15.0 ± 0.3
F91120179 ± 2.0 57.0 ± 2.54.0 ± 0.4
F10191.71198 ± 2.153.6 ± 3.43.7 ± 0.3
F110.64120132± 2.564.7 ± 2.62.8 ± 0.2
A: Corn oil concentration; B: EPC concentration; R1: Particle size; R2: In vitro release; R3: Hemolysis.
Table
Table 3. Statistical analysis of all responses showing F-value, p-value, and lack of fit.
Table 3. Statistical analysis of all responses showing F-value, p-value, and lack of fit.
SourceR1R2R3
F-Valuep-ValueF-Valuep-ValueF-Valuep-Value
Model506.52<0.0001 *568.05<0.0001 *282.72<0.0001 *
A968.36<0.0001 *1067.18<0.0001 *549.49<0.0001 *
B44.690.0002 *68.92<0.0001 *15.940.0040 *
Lack of Fit2.110.35590.55050.75831.690.4179
A: Corn oil concentration; B: EPC concentration; R1: Particle size; R2: In vitro release; R3: Hemolysis; *, significant.
Table
Table 4. Fit statistics for the independent variables.
Table 4. Fit statistics for the independent variables.
Independent VariableR1R2R3
R20.99220.99300.9860
Adjusted R20.99020.99130.9826
Predicted R20.98410.98700.9719
Adeq Precision59.587362.554044.8864
Table
Table 5. Expected and observed values of the optimized PX-NE formulation.
Table 5. Expected and observed values of the optimized PX-NE formulation.
ResponseExpected ValuesObserved Values
R1 (nm)168.9 ± 3.4170.0 ± 2.6
R2 (%)58.4 ± 0.5357.5 ± 2.7
R3 (%)3.27 ± 0.123.44 ± 0.2
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Shehata, T.M.; Elsewedy, H.S. Paclitaxel and Myrrh oil Combination Therapy for Enhancement of Cytotoxicity against Breast Cancer; QbD Approach. Processes 2022, 10, 907. https://doi.org/10.3390/pr10050907

AMA Style

Shehata TM, Elsewedy HS. Paclitaxel and Myrrh oil Combination Therapy for Enhancement of Cytotoxicity against Breast Cancer; QbD Approach. Processes. 2022; 10(5):907. https://doi.org/10.3390/pr10050907

Chicago/Turabian Style

Shehata, Tamer M., and Heba S. Elsewedy. 2022. "Paclitaxel and Myrrh oil Combination Therapy for Enhancement of Cytotoxicity against Breast Cancer; QbD Approach" Processes 10, no. 5: 907. https://doi.org/10.3390/pr10050907

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop