Contemporary human beings are increasingly becoming conscious of their outward display of aging. The human skin is one of the external indicators of aging because it sags and develops wrinkles as the aging process proceeds. However, the latest developments in anti-aging measures employ substances known to retard the aging process by maintaining the integrity of the skin.

1.1 Skin Degradation

The human skin degrades over time due to age and exposure to the atmospheric elements. The aging process is characterized by the thinning of the epidermis, which is the outer skin layer. Nonetheless, the thinning process does not change the number of cells in the skin layer. In addition, the skill looks paler because of the decreased number of melanocytes, which are the cells containing melanin or the skin pigment.

The aging of the skin is accelerated by extended exposure to the sun, particularly the ultraviolet component. Ultraviolet radiation induces a photooxidation process in which the polymers making up the skin, primarily collagen, are broken down (Masaki, 2010). The sun-induced collagen breakdown is often accelerated by extended exposure to the sun. In addition, the ultraviolet radiation from the sun penetrates the skin surface and reaches the dermis, which is the middle layer of the skin. This causes an abnormal accumulation of elastin. Elastins induce the production of enzymes that break down collagen. They also cause the formation of solar scars, which are discolorations on the skin. It is the degradation of the collagen that makes the skin got wrinkle and sag. Similarly, ultraviolet radiation catalyzes the formation of free radicals, which are unstable oxygen species that are activated to contain one outermost electron instead of two (Mumpuni and Mulatsari, 2018). The unpaired electron makes the free radicles to be highly reactive as they strive to stabilize their electronic structure. In turn, this high reactivity accelerates the amount of enzymes that catalyze skin collagen breakdown.

1.2 Skin Degradation Prevention

Although skin aging is a natural process, it can be slowed down by slowing down the processes that cause it. The three most common strategies for preventing premature aging of the skin include neutralizing free radicals, modulating collagen turnover, and protecting the skin from the sun. The most straightforward approach to retarding skin aging is by protecting it from the ultraviolet radiation from the sun. Physical avoidance or physically blocking the sun rays using barriers like hats is effective and should be used when one ventures outdoors. Similarly, sunscreens can be applied to the skin to prevent the penetration of ultraviolet radiation. The sunscreen is a lotion that can be organic or inorganic. Neutralizing free radicles is another anti-aging approach, which involves the reaction of an antioxidant. The antioxidant scavenges for free radicals in the skin formed by the accumulation of elastins (Masaki, 2010). The body has a natural antioxidation mechanism comprising of enzymatic and non-enzymatic systems. The natural enzymatic defense includes superoxide dismutase and glutathione peroxidase. Contrastingly, the non-enzymatic defense system comprises uric acid, glutathione, and thiols. However, this defense can be promoted nutritionally by carotenoids and vitamins C and E. In this regard, consuming foods rich in carotenoids, and vitamins C and E can help slow down skin degradation due to age. Modulating collagen turnover is the other approach to anti-aging. Collagen is the protein that binds the extracellular matrix that holds the skin and disuse together. One way of slowing down this breakdown is by using treatments that arrest the breakdown process. Extracts such as apigenin and curcuma have been evidenced to slow down collagen turnover.

1.3 Apigenin

Apigenin is a flavone found naturally in plants. Plants with a high apigenin concentration include chamomile tea, celery, and parsley, although it is also found in many fruits and vegetables (Shen et al., 2014). Apigenin has antioxidant properties, which leverage its application as an anti-aging substance (Mumpuni and Mulatsari, 2018). Its mechanism of action is to scavenge for free radicles and neutralize them before they degrade the collagen in the skin. Apigenin also has anti-carcinogenic and anti-inflammatory properties, which help soothe the skin from irritations and prevent the development of skin cancer due to excessive exposure to ultraviolet radiation from the sun. The mechanism of apigenin’s action against the aging process has been explained by Arterbery and Gupta (2018), who demonstrated the activity of matrix metalloproteinase (MMP-1) against collagen could be reduced by apigenin. Besides, it has been reported that apigenin employs an enzymatic approach by inhibiting the expression of cyclo-oxygense-2 (COX-2), which acts as a mediating species in inflammations. Delivery of apigenin to the site with many reactive oxygen species is often through the skin’s surface. Apigenin can be formulated into an ointment that is applied on the skin surface from where the active compound is absorbed through.

1.4 Encapsulation

Encapsulation involves coating one substance with another to preserve the chemical, biological and physical properties of the substance of interest. In this case, the active compound is encapsulated using an inactive one to help stabilize it (Sonawane, Bhanvase, and Sivakumar 2020). Encapsulation is critical in ensuring that bioactive substances are delivered to the targets in the body without losing their efficacy (Mota et al., 2021). The bioactive substance forms the core material, while the encapsulating agent is the shell, which serves and the carrier material.

Encapsulating substances is critical in the health and lifestyle industries because it helps deliver medicine and skincare products without diminishing their efficaciousness. However, several characterization parameters and tools determine the efficiency and effectiveness of encapsulation. Critical features of encapsulated species include particle or vesicle size, surface charges, polydispersity, and crystallinity (Chou et al., 2021).

1.5 Delivery Systems

Drugs and cosmetics are delivered in a manner that preserves the activity of the bioactive compounds. Encapsulation is one approach used to deliver unstable drugs into the body. It is used to create nanoparticles that can be incorporated in a cream, lotion, gel, or tablets (Ahmad et al., 2019; Ismail et al., 2021). However, the delivery of drugs and cosmetics is complex because of the delicate balance that needs to be maintained between reactivity and solubility. It is desired to have a highly soluble but unreactive particle for storage.

Ethosomes and liposomes are some commonly used delivery systems. They comprise two layers, with the outer layers being hydrophobic and the inner one hydrophilic (Pilch and Musiał, 2018). These delivery systems are renowned in the cosmetic industry because of their broad applicability in skincare products (Razazi and Janfaza 2015). These delivery systems have an aqueous component and an organic one. While the organic layer is hydrophobic and located outside the vesicle, the aqueous layer is hydrophilic and acts as the carrier of the active compound or drug.

Figure 1. Diagrammatic representation of liposomes and ethosomes

Source: Paiva-Santos et al. (2021)

1.6 Ethosomes

Ethosomes are typically composed of phospholipids suspended in an ethanol/water mixture containing phosphatidylcholine (usually between 2%-5%). The ethanol content usually ranges between 20% and 40%, while the rest is water (Gunjan and Swarnlata 2014). Ethanol serves to enhance skin permeation by interacting with the hydrophilic stratum corneum (SC) lipid, thus enhancing fluidity. By acting as the mixing agent, ethanol gives the vesicles the softness characteristics typical of skincare products (Costa and Santos, 2017). This is because this feature enables increased distribution in the different layers of the skin (Razavi and Janfaza, 2015). Ethanol can also improve the encapsulation efficiency and improve the loading of drugs, especially for drugs with high solubility. According to Sudhakar, Jain and Charyulu (2016), the ethanol in the ethosome penetrates the skin through the lipid perturbation mechanism, as illustrated in figure 2.

Figure 2. Skin permeation mechanism of ethosomes

Source: Paiva-Santos et al. (2021)

In turn, a change in ethanol concentration leads to a change in the size of the ethosome, thus giving it unique characteristics. Besides, it charges the ethosomes’ surface negatively, hence increasing its steric stability, alongside the electrostatic repulsion needed to prevent aggregations of the vesicles (Paiva-Santos 2021). Usually, in practice, other chemicals are added to the ethosome to improve its properties. For instance, propylene glycol is often added to enhance the skin penetration feature of the ethosome. In addition, the vesicle membrane of the ethosome is often stabilized using cholesterol at a proportion range of between 0.1% and 1%.

Ethosomes differ from liposomes by being in a more fluid state compared to liposomes. They also differ in drug permeability, with ethosomes having an enhanced drug penetration compared to liposomes (Pilch and Musiał, 2018). In this regard, ethosomes are preferred for delivering skincare products. The different characteristics of ethosomes and liposomes are compared in table 1.

Table 1. Characteristics of ethosomes and liposomes

Characteristics	Ethosomes	Liposomes
Nature of vesicles	3^rd generation elastic lipid	Bilayer lipid
Flexibility of vesicles	High deformability and elasticity	Rigid
Composition	Phospholipids and ethanol	Phospholipids and cholesterol
Extent of skin penetration	Highly penetrative through the paracellular space	Penetration rate is very low The stiff shape and size are not suitable for penetrating the stratum corneum in the skin
Permeation mechanism	Lipid perturbation	Lipolysis Fusion Diffusion

Source: Sudhakar, Jain and Charyulu (2016)

1.7 Future Proposals

The articles that were used to motivate this study are Costa and Santos (2017), El-Menshawe et al. (2019), and Pilch and Musial (2018). While the methodologies provided valuable insights to the design of this study, their suggestion for further research was used to refine and study topic. These articles were recent and therefore, found relevant to the proposed study. However, some retrieved sources were not cited because either they were older than 10 years and therefore, outdated or they did not contain relevant information or not published in credible journals. These include Dayan and Touitou (2000) and Elsayet et al. (2006). Nonetheless, the literature in the selected articles revealed several knowledge gaps that needed to be filled to enhance understanding of the delivery systems used in anti-aging cosmetics, which justifies this study, while supplying pertinent methodological approaches. For instance, Costa and Santos (2017) notes that there was need to compare the real benefits delivered by biological raw materials and the synthetic ones as cosmetic ingredients. In addition, they noted that the effects of protracted use of nanoparticles as cosmetic delivery systems and their uniformity and reproducibility were largely unknown. Therefore, they recommended long term studies in the stability and controlled release of the nanoparticles. Similarly, El-Menshawe et al. (2019) after demonstrating that the addition of stearylamine improved the skin retention of ethosomes because of its positive charge inducing effect raised curiosity whether this would work for apigenin-induced ethosomes, considering that their studies used nicotinamide-induced ethosomes. It is hypothesized in the proposed study that the enhanced skin retention of positively-charged ethosomes could have improved efficacy against wrinkles. Besides, after Pilch and Musial (2018) demonstrated the effectiveness of ethanol in enabling active ingredients penetrate deep skin layers, they raised concerns related to the possible denaturing of proteins components in bioactive compounds encased in ethosomes. They recommended the conduct of further stability studies related to ethanol-induced denaturation of protein components, which is a concern that this study could address. Therefore, future proposals should include protracted stability tests on ethosome. The storage period should be extended to between 3 and 6 months. Besides, formulated gels and creams containing the apigenin-loaded ethosomes could be tested for optimization and followed up with clinical studies to determine their safety.

2.0 Aim of the Project

This project aims to determine whether a positive charge inducer can enhance the skin penetrability of encapsulated apigenin, an established skin-toning substance, in ethosomes without degrading the stability of the vesicle or the bioactive ingredient.

3.0 Objectives

The objectives of this project are:

To encapsulate Apigenin in ethosomes
To characterize the morphology, entrapment efficiency, and vesicle size of the apigenin-loaded ethosomes
To determination whether the stability of apigenin-loaded ethosomes is affected by stearylamine
To determine the permeation of apigenin-loaded ethosomes in artificial skin
To determine the stability of apigenin encapsulated in ethosomes is affected by stearylamine.

4.0 Methodology

This section describes the methods used to achieve the set objectives, which include preparing ethosomes, loading them with apigenin and characterizing the vesicles, and finally determining the stability of the apigenin-loaded ethosome on artificial skin. Apigenin will be purchased from credible chemical suppliers. However, it will be stored carefully wrapped in foil and placed in an airtight jar before use. Apigenin becomes degraded in the atmosphere due to exposure to ultraviolet light and other physical elements. Therefore, photodegradation of apigenin as an active compound should be prevented using light-blocking strategies, like using aluminium foil. This precaution is critical for ensuring that the photoisomerization of apigenin is prevented to maintain the latency.

4.1 Constituents of the Ethosome

The structural components of the ethosome are summarized in table 2 by stating their function, quantity, and rationale for selection.

Table 2. Ethosome structural components

Constituent	Function	Quantity (% v/v)	Rationale of Use
Lipoid S100	Phospoholipid	5	Influence vesicle’s size stability, entrapment efficiency and penetration efficiency
Ethanol	Skin penetration enhancer	85	Influence vesicle’s size, stability and entrapment efficacy, and softens vesicle
Propylene glycol	Skin penetration enhance	1	Improve the penetration characteristic of ethanol
Stearylamine	Positive charge inducer	<1	Improves vesicle penetration properties
Water	Aqueous layer	5	Dissolves agent
Apigenin	Active agent	1	Antioxidant, slowing down skin sagging and wrinkle formation

4.2 Ethosome Preparation

The passive loading method will be used to prepare the ethosomes as described in Shen et al. (2014). Phosphatidyl choline (PC) will be dissolved in a binary alcohol phase (BAP) comprising ethanol and propylene glycol. Citrate buffer will then be added slowly to the BAP drop by drop while the mixture is stirred continuously in a sealed container at 700 using a magnetic stirrer to avoid evaporation. The mixing will be sustained for 5 minutes at a temperature of 30^OC and gradually cooled to room temperature. After that, apigenin will be loaded into the mixture. In this process, 1% (w/v) of apigenin will be added into the empty binary ethosomes dropwise while stirring the mixture using a magnetic stirrer at 700 rpm. After that, another sample of twosomes will be prepared using variations of the phospholipid and ethanol to optimize the formulation. Specifically, phospholipid will be adjusted to 2% and 3%, while and ethanol will be adjusted to 95% and 85% v/v, respectively. During the making the ethosome stock, it will be impregnated with apigenin. After that, the stock will be divided into two portions. One portion will be impregnated with stearylamine to induce a positive charge on the vesicles.

4.3 Characterization of Ethosomes

4.3.1 Morphology

The morphology of the vesicles will be assessed using microscopy. The characterization will be done using a digital microscope fitted with a high-resolution camera (zoom 40x) to view the vesicles. Drops of the ethosomes will be placed on a copper grid coated with carbon to form a thin lamina. This film will be stained with phospotunstic acid (1% v/v) and air-dried before viewing.

4.3.2 Entrapment Efficiency

The vesicle preparation will be placed in a centrifugation tube that is sealed to avoid evaporation. This will then be centrifuged at 15,000 revs per minute for 30 minutes (Li, et al., 2012). Serial dilution will be used to obtain solutions of varing concentrations (El-Menshawe et al., 2019). To do that, a small about of the supernatant (1 mililiter) will be drawn out using a pipette and diluted with phosphate buffer (pH 6.8) to make a 10 milliliter solution. Another 1 milliliter of the diluted solution will be drawn out with a pipette and diluted with the buffer to make 10 milliliters of solution. The last solution will be used to determine the amount of entrapped apigenin. This will be determined using a UV spectrophotometer (Shimadzu Pharmaspec-1700) set at 425.6 nanometers (Mohanty, et al., 2018). The free apigenin in the supernatant will indicate the total amount of apigenin that has not been encapsulated. Contrastingly, triton fusion will help determine the amount of encapsulated. This procedure will be replicated thrice to enhance repeatability. The means and standard deviations will be calculated, representing the entrapment efficiency. The entrapment percentage will be determined using the formula (Pathan et al., 2018)

4.3.3 Vesicle Size

The size of the vesicles will be measured using Malvern Zetasizer DTS version 5.03. This will also be used to measure the size distribution and zeta potential of the vesicles (Frøkjaer, Hjorth, and Wørts, 2019). The zeta potential will inform about the colloidal properties of the ethosome. This property is critical for the permeation and stability of the vesicles.

4.3.4 Degree of Deformability

The ethosome preparation will be passed through several membranes with different number of pores (between 100-200 nm) and thicknesses. The depth of penetration will be measured using a Zetasizer to ensure that the ethosome can deform and penetrate deeply.

4.4 Stability Study

The physical stability of the vesicles will be determined by storing them in different temperatures for 6 months. Vesicles containing stearylamine and those without will be used in this study to determine whether there are any changes vesicle stability. Three storage temperatures, 4^OC, 25^OC and 37^OC will be used. After that, the size of the vesicles and zeta potential will be measured to determine the level of degradation. In addition, the stability of apigenin encapsulated in ethosomes will be determined at varying time intervals: after 1, 2, 3 and 6 months to determine its degradation levels. The amount of apigenin in the ethosome with stearylamine will be compared to that without stearylamine to determine whether stearylamine influences the stability of the bioactive agent.

4.5 Permeation Study (in-vitro release)

The ethosomes (with and without stearylamine) will be exposed to a synthetic skin material (Strat-M ® membrane) representing the human skin. The ethosome will be smeared on 4 square centimeter area (Touitou et al., 2000). The sample will then be loaded to a Franz diffusion cell after preparing it as outlined by Sachan, Parashar and Singh (2013). Specifically, in setting up the Fraz diffusion cell, Strat-M ® membrane will constitute the cell line for the transdermal diffusion test. A 25-square-millimeter diffusion area will be smeared with the preparation of 50 ml containing PBS (pH=7.4) mixed with a magnetic stirrer at 100 rpm at 37OC and placed into the receptor compartment of the test equipment. The apigenin-loaded ethosome will be placed in the donor compartment. It will be sealed in a paraffin film to avoid evaporation while enabling visibility. Aliquots measuring 1 milliliter will be drawn every hour (total 48 hrs) and analyzed. Before analyzing using a spectrophotometer at 427 nm, the samples will be passed through a microfilter with membrane dimensions of 0.22 micrometers. The accumulative amount of the penetrant is the permeation parameter, which will be determined using a plot of the amount of penetrant containing apigenin against time (Niu, et al., 2019).

4.5.1 Penetration Mechanism

The penetration mechanism of the ethosome will be observed using a confocal scanning laser microscope that uses fluorescent markers, in what is known as fluorescence microscopy (Garg, et al. 2016). The high resolution image and videos will help track the ethosome as it penetrated the membranes. To visualize the ethosomes, Rhodamine-123 dye will be used to mart the vesicles.

5.0 Timescale

The timescale describes the allocation of time for every experimental process in the project. It is a sequential process in which the active compound is prepared and characterized to determine its efficaciousness. Nonetheless, this schedule does not focus solely on testing the efficaciousness of the blended form apigenin but also determining the characteristic of the active compound against a skin-like membrane to determine permeability and effectiveness in delivery. The schedule in table 3 outlines the tasks to be undertaken in the project.

Table 3. Gantt chart detailing a 12-week work schedule divided into weeks

Process/ activity	Duration (weeks)
	1	2	3	4	5	6	7	8	9	10	11	12
Review of the literature
Experimental design
Procurement of reagents
Ethosome preparation
Characterization of apigenin Size and chargeEntrapment efficacyPenetration efficiencyStability testIn-vitro testing
Statistical analysis
Report writing and proofing

6.0 Resources

The resources for this project are the equipment and chemicals required to produce and characterize the anti-aging properties of apigenin. The equipment that are needed to conduct this project are detailed in table 4.

Table 4. Instruments and equipment

Equipment	Source/provider
Centrifuge	Laboratory
Rotary Evaporator	Laboratory
Magnetic stirrer	Laboratory
UV Spectrophotometer	Laboratory
TEM Transmission Electron Microscope	Laboratory
Franz-cell Apparatus	Laboratory
Probe Sonicator	Laboratory
Ultraviolet spectrophotometer	Laboratory
HPLC	Laboratory
Laboratory apparatus, computer software and hardware	Laboratory
Zetasizer	Laboratory

The consumables are listed in table 5

Table 5. Consumables

Consumables	Source/provider	Quantity	Price (GBP)
Phosphotidylcholine	Sigma-Aldrich-Merck	100 ml
Artificial skin membrane (Strat-M®)	Sigma-Aldrich-Merck	60 membrane pack	540
Lipoid S100	Sigma-Aldrich-Merck	5 g	230
Stearylamine	Sigma-Aldrich-Merck	1 mg	75
Apigenin (analytical grade from biological sources)	Sigma-Aldrich-Merck	5g	430
Ethanol	Laboratory		–
Phosphate buffer (PBS pH 7.4)	Laboratory	500 ml	–
Rhodamine-123 dye	Rhodamine-123 dye	10mg	45
5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one	Laboratory		–
Tween 100 (non-animal source)	Sigma-Aldrich-Merck	500	44
Sodium chloride	Laboratory		–
Ultra-pure water	Laboratory		–

7.0 Impact Statement

Premature wrinkle formation affects men and women globally due to the aging process that is accelerated by extended exposure to the elements, including the ultraviolet radiation from the sun. Therefore, consumers are keen to use cosmetics that retard wrinkle formation while being safe and environmentally friendly. Researcher has successfully determined the efficacy of apigenin against the skin-aging process by preventing the degradation of the collagen in the deep-skin layer. This proposal aims to formulate an ethosome encapsulating naturally-sourced apigenin. The formulation made is expected to effectively deliver apigenin to the deep-skin layer thus retarding wrinkle formation.

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