Research Article |
Corresponding author: Khalid Al-Kinani ( khalidalkinani@copharm.uobaghdad.edu.iq ) Academic editor: Milen Dimitrov
© 2024 Taif Abdullah, Khalid Al-Kinani.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Abdullah T, Al-Kinani K (2024) Propranolol nanoemulgel: Preparation, in-vitro and ex-vivo characterization for a potential local hemangioma therapy. Pharmacia 71: 1-12. https://doi.org/10.3897/pharmacia.71.e115330
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A lack of safe and effective topical alternatives to oral propranolol HCl (PHCl) hampers optimal management of infantile hemangioma (IH), particularly in complex cases with severe side effects or treatment failures. This study aimed to develop a nanoemulsion gel (NEG) for topical PHCl delivery. A meticulously formulated nanoemulsion (NE) encapsulated with clove oil, Tween 20, and polyethylene glycol 400 emerged as the standout candidate (NE3) due to its exceptional stability, resilience, and favorable drug loading. NE3 exhibited a remarkable globule size of 14.57 nm, a low polydispersity index (PDI) of 0.282, and a stabilizing zeta potential of −19.89 mV. The subsequent formulation of PHCl-NEG displayed desired rheological and spreadability properties for topical application. Ex-vivo skin retention and permeation studies revealed effective PHCl deposition within the dermal layer with minimal systemic exposure. This promising approach offers a potential alternative to oral PHCl, potentially mitigating severe side effects and improving outcomes in complex IH cases.
clove oil, ex-vivo permeation study, infantile hemangioma, nanoemulgel, propranolol-HCl
Propranolol HCl (PHCl), a pharmaceutical compound classified as a β-adrenergic receptor antagonist, was first developed for the purpose of managing arrhythmias. Previously, particularly in 2008, it was shown that the oral administration of PHCl to newborns produced significant efficacy in treating infantile hemangioma (IH). Consequently, PHCl has since been established as the primary and sole medicine approved by the FDA for the treatment of IH (
Administration of PHCl by the oral route has the potential to result in significant complications. Infants should be carefully monitored due to the possibility of systemic exposure to PHCl, which can lead to alterations in sleep patterns, acrocyanosis, and gastrointestinal symptoms. Furthermore, there is a risk of experiencing severe adverse reactions such as bronchospasm, symptomatic hypotension, hypoglycemia, and bradycardia (
When it comes to the treatment of superficial hemangiomas, dermal application of PHCl produces far less adverse effects in comparison to the oral administration method. This is due to the advantages of achieving a higher concentration of the medication locally and diminishing its systemic exposure (
In contemporary times, there has been a significant surge in the level of attention and fascination directed towards the field of nanotechnology (
Topical formulations of PHCl have been the subject of several investigations that have demonstrated their safety and effectiveness to some extent, but some cases of partial resolution have been reported in the context of newborns with IH. To name several of these pharmaceutical hydrophilic preparations, a gel containing 3% PHCl (
To overcome the challenges of oral PHCl in complex IH, including significant side effects and treatment resistance, this study explores a topical nanoemulsion gel (NEG) containing PHCl. This novel approach seeks to target affected skin regions directly, potentially minimizing systemic exposure and improving outcomes in cases where complete resolution remains elusive.
Propranolol-HCl, Tween 20, glycerin, and Carbopol (934) were procured from Awamedica Pharmaceuticals Factory (Erbil, Iraq). PEG400 was obtained from Sigma-Aldrich Co., (St Louis, MO, USA). Clove essential oil and triethanolamine were supplied by Alpha Chemika Co., Ltd. and Thomas Baker Pvt., Ltd. (Mumbai, India) respectively. Double distilled deionized water was produced by a Milli-Q purification instrument (Baghdad, Iraq).
All additional reagents utilized in the experiments were of analytical grade. The full-thickness skin samples of male “Wister Albino” rats, with an average weight of 140 ± 10 g, were acquired from the SLAC Laboratory Animal at the Pharmacy College in Baghdad, Iraq. Ex-vivo permeation investigations and drug deposition experiments involving rat skin were conducted with the consent of the Institutional Animal Care and Use Committee at the Pharmacy College, Baghdad University.
For the development of drug-loaded NEs, the evaluation and optimization of formulation components relied on how well the payload dissolved in the saturation solubility study and how the phases behaved between the formulation excipients. The saturated solubility of PHCl was determined in various oils, surfactants, and co-surfactants using the shake-flask method. In this approach, an excess amount of PHCl was added to 5 g of each individual component (
By using a vortex mixer and the optimal component ratio determined from pseudo-ternary phase diagrams, PHCl-loaded NEs were generated by the low-energy emulsification approach (
The study assessed the in-vitro diffusion rates of PHCl in NE structures using a Franz diffusion cell system. The receptor section of the cell has a volume of 12 mL and an effective diffusion area of (1.767 cm2). This section was separated from the donor section by a synthetic semipermeable membrane with a molecular weight cut off of 8,000–14,000 D, which served as a barrier for diffusion. A sample of the prepared PHCl-loaded NE was placed in the donor compartment of the Franz diffusion cell, which contained acetate buffer saline at a pH of 5.6. The stirring rate was fixed at 50 rpm, and the temperature was maintained at 37 +/- 0.5 °C (
For topical application, nanoemulgel containing the optimum NE structure were produced with Carbopol 934 at a concentration of 1% w/w. The measured quantities of the selected gelling polymer were dissolved in deionized distilled water, and the mixture was allowed to sit overnight for the purpose of achieving homogeneous swelling (
A rotational viscometer (Myer rotary viscometer VR 3000, spindle no.7; Vendrell Instruments Ltd., Barcelona, Spain) was used to examine the PHCl-NEGs’ rheological behaviors at 25 +/- 1.0 °C. In order to characterize the PHCl-NEGs’ shear-stress profiles and thixotropic behaviors, we subjected them to a series of simulated topical administrations at shear rates ranging from 10 to 200 s−1 over the course of eight stages, with 30 s of equilibration time between each step (Md et al. 2020).
The spreadability of the PHCl-NEG and a commercially available emulgel product (Voltaren emulgel) was assessed by inserting a precisely measured quantity (1.0 g) of the sample between two glass plates having a surface area of 20 × 20 cm2 and a width of 6 mm for a duration of 1 min. The upper plate was used as a normal-weight compressor of 240.0 g. The measurement of spreadability was stated as a diameter function relating the spreading area to the applied weight (
A total of 50 mL of DI DW was added to a known weight of the prepared nanoemulgel (5.0 g). Using a digital pH meter (Hanna Instruments HI 98107 Bucharest, Romania), the PHCl-NEG system was diluted to a concentration of 10% w/v, stirred thoroughly for 15 min, and its pH was measured (
A quantity of 1.0 g of the formulations was collected from random sections of the prepared PHCl-NEG. The samples were diluted using methanol and subjected to sonication for 15 min. The extracts underwent centrifugation at a speed of 3000 rpm for a duration of 15 min. Subsequently, the resulting supernatants were subjected to filtration using a syringe filter made of a 0.45 µm pore diameter membrane. The concentration of PHCl in each extract was quantified utilizing a UV spectrophotometer (
The evaluation of the ex-vivo skin permeation of PHCl from the NEG system was conducted utilizing the Franz diffusion cell. A specimen of shaved and dissected posterior skin from Wister Albino rats was affixed to the two compartments of the cell, as displayed in Appendix
The permeation profile was determined by plotting the cumulative amount (Q, in µg/cm2) of PHCl that penetrated per unit area across the skin as a function of time. By conducting a plot, various parameters can be derived. These parameters encompass:
The rate of permeation, denoted as Jss, is measured in µg/cm2/min. It was determined by calculating the slope of the linear section of the regression line, as shown in the equation below:
Jss = dQ/dt
The permeability coefficient, denoted as Kp and measured in cm/min, was determined using the following equation:
Kp = Jss/C0
The variable C0 represents the initial drug concentration in the donor compartment.
The enhancement ratio, denoted by Er, was calculated by dividing the flux of PHCl-NEG by the flux of PHCl-gel, as shown in the following equation:
Er = Jss of PHCl-NEG/Jss of PHCl gel
Simultaneously, investigations were conducted to analyze the skin deposition of both PHCl-NEG and PHCl gel (
The stability of the developed PHCl-NEG system was assessed by storing it at ambient temperature (25 +/- 2 °C) and in the refrigerator (4 +/- 2 °C) for a period of three months. The specimens were inserted into photoprotective glass tubes and retrieved afterwards at regular intervals (0, 1st, 2nd, and 3rd month) in order to examine the alterations in their physical characteristics, pH levels, rheological properties, and drug content percentage (
The experimental results are presented as the average of three samples along with their standard deviations. The data were analyzed using one-way ANOVA (Tukey’s post-hoc test). Differences were considered statistically significant when p < 0.05.
The solubility of PHCl, a solid substance with a white crystalline structure, was determined at room temperature. It was observed that PHCl had a significantly higher solubility in clove oil compared to that in other oils which is clearly shown in Fig.
Clove oil is a potent essential oil derived from the distillation process of the flower buds, stems, and foliage of clove trees (Eugenia aromatica or caryophyllata, Fam. Myrtaceae) (
PHCl had favorable solubility when combined with Tween 20 as a surfactant and PEG 400 as a co-surfactant (Table
Constituents | Solubility (mg/g) |
---|---|
Tween 20 | 13.12 +/- 1.05 |
Cremaphor EL | 9.82 +/- 0.98 |
PEG 400 | 46.06 +/- 1.93 |
Transcutol P | 27.71 +/- 1.15 |
The elevated solubility of PHCl in clove oil as an oil phase can be attributed to the demonstrated strong hydrogen bonding capacity of clove oil. This characteristic potentially elucidates the rationale behind its pronounced solubility for PHCl. It is possible that clove oil’s solubility is due, in part, to the presence of eugenol. The structure of PHCl is depicted in Fig.
The hydrophilic-lipophilic balance (HLB) value of the surfactant structure is critical for monitoring the emulsification of the aqueous and oil phases and developing a nanoemulsion. Non-ionic surfactants with high HLB values, such as Tween 20 at 16.7, improve globule stability, making them better for drug delivery systems (
A study of phase behavior was conducted to examine the effects of different component ratios in relation to the mixture of the surfactant and co-surfactant in the formulation on the development of NEs. Using a pseudo-ternary phase diagram, we can see how the mass proportion of surfactant to co-surfactant (Km) is related to the phase behavior of an NE (Md et al. 2020).
The structure experienced changes in its optical properties as it transitioned from a transparent state to a translucent state, and eventually to an opaque state. These transitions were the result of reorganization occurring within the constituents present in the non-equilibrium phase, leading to alterations in the system’s light-scattering characteristics. The phase behaviors of the preformulated NE comprising these components at different ratios are presented in Table
Behavior investigation in the preformulation phase for developing of PHCl-NEs.
Oil phase | Drug load | Km phase | Km ratio | Deduction |
---|---|---|---|---|
Clove Oil | Blank | Tween 20 and PEG 400 | 1:2 | Translucent emulsion >100 nm |
Clove Oil | Blank | Tween 20 and PEG 400 | 1:1 | Transparent nanoemulsion <100 nm |
Clove Oil | Blank | Tween 20 and PEG 400 | 2:1 | Transparent nanoemulsion <50 nm |
Clove Oil | Blank | Tween 20 and PEG 400 | 3:1 | Transparent nanoemulsion <20 nm |
The NE region was utilized for the assessment of Km, and a positive correlation was seen between the size of the NE region and the efficiency of nanoemulsification in the overall structure. Many formulae are feasible for the pseudo-ternary phase diagram’s NE region. A transparent dispersion NE system and globule size of less than 100 nm can be achieved with up to 10% oil phase w/w. Consequently, by aqueous titration, the generally recognized as safe (GRAS) grade Km phase (Tween 20 and PEG 400) and the oil phase (clove oil) were utilized to investigate phase diagrams. Clove oil is thought to be cytotoxic, yet the phenoxyl radical produced by eugenol is much more stable and therefore, far less reactive than reactive oxygen species (ROS), which provide protection (
Fig.
From the results obtained from the saturation solubility study, PHCl was dissolved in clove oil as the oil phase, and then the Km phase (Tween 20 and PEG 400) was blended using a vortex mixer. Transparent dispersion NE structures with a drug payload were generated as the aqueous phase was then titrated promptly, and the resulting solution was homogenized by ultrasonication.
The present experiment aimed to evaluate the impact of varying the surfactant-co-surfactant mass ratio (Km) on the globule size and polydispersity index (PDI) of PHCl-loaded nanoemulsions (NEs). This investigation was conducted by preparing a series of NEs with fixed composition and altering the Km ratios (1:1, 2:1, and 3:1) of the selected optimal components. The findings of the study revealed that an increase in surfactant concentration, in relation to the co-surfactant content, had a substantial impact and was inversely correlated with the average globule size of the resulting nanoemulsion. Furthermore, it was observed that this had also had an inverse impact on the PDI of the NEs structures. The PDI followed the order of NE1 > NE2 > NE3, as depicted in Fig.
These results indicated that the formulations characterized by a greater Km ratio exhibited better-performing PHCl-NE structures. The observed phenomenon can be attributed to the enhanced solubilization and improved hydrophilicity of PHCl, which are facilitated by greater amounts of Tween 20 in the Km ratio (
The prepared NE formulations utilized in the investigation of the phase diagram were stressed by thermodynamic stability assessment, encompassing procedures such as heating-cooling cycles, centrifugation tests, and freeze-thaw cycles.
All formulations (Table
Formulation | Nanoemulsion Composition (%w/w) | |||
---|---|---|---|---|
Oil | Tween20 | PEG 400 | DI DW | |
NE1 | 10.0 | 30.0 | 30.0 | 30.0 |
NE2 | 10.0 | 40.0 | 20.0 | 30.0 |
NE3 | 10.0 | 45.0 | 15.0 | 30.0 |
Furthermore, all the formulations successfully completed the stress tests. The percentage of light transmittance, %T, for the generated NE structures (NE1–NE3) was found to be greater than 98%, suggesting that the structures under investigation were in a finely dispersed state (Table
Formulation | Conductivity (σ) µS/cm | %T | % Drug content | Mean globule size (nm) | PDI | Dilutability |
---|---|---|---|---|---|---|
NE1 | 68.0 | 98.18 ± 0.93 | 98.43 ± 0.61 | 90.06 ± 1.16 | 0.531 ± 0.13 | Opaque ×× |
NE2 | 75.0 | 99.06 ± 0.52 | 98.79 ± 0.36 | 47.78 ± 0.61 | 0.385 ± 0.06 | Clear √√ |
NE3 | 81.0 | 99.33 ± 0.49 | 99.02 ± 0.42 | 14.57 ± 0.25 | 0.289 ± 0.008 | Very clear √√√ |
The electrical conductivity of the produced NEs was tested, and the observed pattern in conductivity, NE3 > NE2 > NE1, indicated that an increase in the surfactant ratio resulted in higher conductivity levels. The successful synthesis of oil-in-water (o/w) nanoemulsions (
In the context of topical applications, it is desirable for the globular size of NEs to be smaller than 50 nm, accompanied by a PDI value of less than 0.30, and effective stabilization with a zeta potential ζ that is as modest as −30 mV (
The observations of in-vitro diffusion of PHCl from the NEs (NE2 and NE3) indicated that there was a complete diffusion of HPCl from both NE2 with 96% and NE3 with 99% after a duration of 120 min (Fig.
The NE3 formulation structure exhibited the highest level of PHCl diffusion, likely because of its smaller average globule size in comparison to the other NE preparation (NE2). Based on the obtained findings, NE3 was identified as the most suitable NE for incorporation into an NEG framework. The mean globule size and PDI of the varying NEs preparations (NE1–NE3) as provided by Table
The dimensions and structure of PHCl NE3 were verified using high-resolution imaging with the atomic force microscopy (AFM) technique. The results substantiated that the NE globules exhibited a spherical morphology and had dimensions within the nanoscale range (
The systemic NE3 was included in the prepared hydrogel network of Carbopol 934 (1.0% w/w) resulting in the generation of a PHCl-NEG system that demonstrated a transparent, consistent, viscous gel appropriate for topical application. It had an elegant appearance with a transparent golden hue devoid of any gritty particles or aggregate that could be felt by the thumb [Appendix
When it comes to the topical distribution of medications, the pseudoplastic behavior of gel formulations is both practical and preferable (
When constructing a semi-solid pharmaceutical formulation that is meant for cutaneous application, adequate spreadability helps ensure uniform distribution of topical gels; moreover, this aspect is seen as a critical determinant of patient adherence to treatment (
The pH discrepancy between the pH of the generated PHCl-NEG and the pH of skin may lead to skin irritation. Topical formulations should preferably have a pH within the range of the skin’s pH to avoid disrupting the skin’s acid layer. The pH value of the generated PHCl-NEG was determined to be 5.66 +/- 0.04, showing a close resemblance to the pH level of the skin acid shield. Furthermore, the PHCl-NEG exhibited favorable extrudability characteristics, allowing for convenient dispensing by end-users. The generated NEG underwent uniformity and drug content analyses, which revealed a consistent distribution of PHCl throughout the entire system. The uniformity percentage of PHCl in the NEG was evaluated as 99.54%, which was determined based on the drug content of 98.90 +/- 0.46%.
An ex-vivo drug deposition study was conducted to compare the parameters of PHCl-NEG and PHCl gel. The results in Table
Evaluations of the gel systems from skin deposition and permeation investigations.
Variables | PHCl-NEG | PHCl gel |
---|---|---|
Drug deposited in skin (µg/cm2) | 583.38 +/-15.07 | 198.02 +/- 12.24 |
Cumulative amount of drug permeated (µg) | 298.67 +/- 14.87 | 166.54 +/- 7.69 |
Jss (µg/cm2 min) | 4.64 +/- 0.36 | 1.76 +/- 0.28 |
Permeability coefficient (Kp × 10−2) | 4.12 +/- 0.003 | 1.56 +/- 0.002 |
Enhancement ratio (ER) | 2.65 +/- 0.23 |
By analyzing the PHCl-NEG system and conventional PHCl gel, the enhancement ratio (ER) for the permeation of PHCl diffused from the PHCl-NEG system was 2.65 +/- 0.23. The quantity of PHCl that was deposited in the skin by PHCl-NEG was found to be much larger (583.38 +/- 15.07 µg/cm2) compared to the amount deposited by the PHCl gel (198.02 +/- 12.24 µg/cm2), with a more than triple increment. Likewise, dermal drug flux (Jss) of PHCl from PHCl-NEG system was found to be significantly higher (4.64 +/- 0.36) compared to the Jss obtained from PHCl gel (1.76 +/- 0.28), indicating a more than 200% increase in drug flux as displayed in Fig.
After three months of storage at surrounding temperatures (25 +/- 2 °C and 4 +/- 2 °C), the PHCl-NEG was found to exhibit excellent physical stability. Indicated by Table
Storage period | Rheological behavior | PHCl content (%) | Phase separation | pH | Visual appeal |
---|---|---|---|---|---|
1st month | Pseudoplastic | 98.36 ± 1.19 | Nil | 5.66 ± 0.03 | Transparent/Golden |
2nd month | Pseudoplastic | 98.31 ± 0.87 | Nil | 5.64 ± 0.04 | Transparent/Golden |
3rd month | Pseudoplastic | 98.25 ± 1.26 | Nil | 5.62 ± 0.02 | Transparent/Golden |
PHCl was successfully encapsulated in a colloidal dispersion of clove oil, Tween 20, and PEG 400. The characterization approaches indicated that NE3 had superior performance and durability, making it the optimum choice for forming a PHCl-NEG structure, which possesses exceptional stability, spreadability, and desirable rheological behaviors. The ex-vivo skin deposition and permeation assays demonstrated that the nanoemulgel effectively retained the PHCl within the skin layers, the specific location where the medication exerts its therapeutic effects. In addition, the nanoemulgel facilitated the cutaneous permeation of PHCl, albeit in limited quantities.
In the end, the outcomes of our investigation demonstrate that the utilization of the PHCl-NEG system through topical application presents a potentially enhanced substitute for the oral administration of PHCl in instances of complicated IH characterized by severe systemic adverse events and/or failed treatment. This alternative may be particularly beneficial in cases in which complete resolution is not attained leading to permanent cosmetic morbidity. Nevertheless, the research emphasizes the necessity for further clinical and cytotoxicity trials in order to rigorously delve more into the efficacy and safety of this innovative therapeutic strategy.
The authors would like to express their gratitude to Awamedica Pharmaceuticals, Erbil, Iraq, which generously donated samples of the chemicals utilized in this research.
Supplementary information
Data type: docx
Explanation note: The following supporting information regarding PHCl-NE structures includes: fig. S1. Globule size (91.06 Nm with PDI 0.531) of NE1; fig. S2. Globule size (47.70 Nm with PDI 0.352) of NE2; fig. S3. Globule size (14.87 Nm with PDI 0.282) of NE3.