Accepted Manuscript

Liposomes for delivery of antioxidants in cosmeceuticals:

Challenges and development strategies

Vinh Van Tran, Ju-Young Moon, Young-Chul Lee

PII: S0168-3659(19)30141-5
DOI: https://doi.org/10.1016/j.jconrel.2019.03.003
Reference: COREL 9686
To appear in: Journal of Controlled Release
Received date: 16 January 2019
Revised date: 5 March 2019
Accepted date: 5 March 2019

Please cite this article as: V. Van Tran, J.-Y. Moon and Y.-C. Lee, Liposomes for delivery
of antioxidants in cosmeceuticals: Challenges and development strategies, Journal of
Controlled Release, https://doi.org/10.1016/j.jconrel.2019.03.003

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 ACCEPTED MANUSCRIPT

Liposomes for Delivery of Antioxidants in Cosmeceuticals: Challenges and

Development Strategies

Vinh Van Trana, Ju-Young Moonb,*, Young-Chul Leea,*

a
Department of BioNano Technology, Gachon University, 1342 Seongnam-Daero, Sujeong-Gu,

Seongnam-Si, Gyeonggi-do 13120, Republic of Korea.

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b
Myeong-dong Campus, Jeonghwa Arts College, 16-gil 21, Toegye-ro, Jung-gu, Seoul 04631,

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Republic of Korea.
*
Corresponding authors:

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Email: dreamdbs@gachon.ac.kr (Y.-C. Lee) NU
Email: bora7033@naver.com (J.-Y. Moon)
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ABSTRACT

Antioxidants (AOs) play a crucial role in the protection and maintenance of health and are

also integral ingredients in beauty products. Unfortunately, most of them are sensitive due to their

instability and insolubility. The use of liposomes to protect AOs and expand their applicability to

cosmeceuticals, thereby, is one of the most effective solutions. Notwithstanding their offered

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advantages for the delivery of AOs, liposomes, in their production and application, present many

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challenges. Here, we provide a critical review of the major problems complicating the development

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of liposomes for AO delivery. Along with issues related to preparation techniques and encapsulation

efficiency, the loss of protective function and inefficiency of skin permeability are the main
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disadvantages of liposomes. Corresponding development strategies for resolving these problems,

with their respective advantages and drawbacks, are introduced, discussed in some depth, and
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summarized in these pages as well. Advanced liposomes have a vital role to play in the

development and delivery of AOs in practical cosmeceutical product applications.

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KEY WORDS

Antioxidant, Liposome, Lipid Particle, Encapsulation, Skin Penetrability, Ethosome, Transfersome

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Table of Contents

1. Introduction ................................................................................................................................. 4

2. Antioxidants in cosmeceuticals .................................................................................................. 5

2.1. Oxidative stress and antioxidant defense.......................................................................... 6

2.2. Classification of antioxidants ........................................................................................... 7

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2.3. Challenges to cosmeceutical applications of antioxidants ............................................... 8

3. Structural components and incorporation mechanism of antioxidants in liposomes ........... 9

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3.1. Structural components of liposomes................................................................................. 10

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3.2. Incorporation mechanism of antioxidants in liposomes ................................................... 11

4. Cosmeceutical benefits of liposomes .......................................................................................... 13

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4.1. Improved stability of antioxidants .................................................................................... 13
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4.2. Enhancement of antioxidant activity and solubility of antioxidants ................................ 14

4.3. Enhancement of skin penetration of antioxidants............................................................. 15

5. Challenges to applicability of liposomes ................................................................................... 17

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6. Development strategies of liposomes for delivery of active anti-oxidants ............................. 19

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6.1. Enhancement of stability of liposomes ............................................................................. 19

6.2. Development of techniques for preparation of liposomes ............................................... 27

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6.3. Development of new generations of liposomes ................................................................ 31

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6.4. Development of advanced materials for delivery of AOs in cosmeceuticals .................. 39

7. Conclusion and perspectives ...................................................................................................... 46

Acknowledgements

Conflict of Interest

References

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1. Introduction
According to the definition of the U.S. Food and Drug Administration (FDA), cosmetics are

“articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise

applied to the human body...for cleansing, beautifying, promoting attractiveness, or altering the

appearance” [1]. During recent decades, the development of new formulations of cosmetics based

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on the use of sensitive ingredients or antioxidants (AOs), such as vitamins, polyphenols and various

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active agents, has been one of the most promising strategies. Such cosmetic formulations also are

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referred to as “cosmeceuticals,” which is defined by the FDA as products that work with both

cosmetic and medicinal functions [2]. Along with the increased demand for beauty products,
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cosmeceuticals have attracted extensive attention in the personal care industry, especially in the

forms of numerous topical products introduced to the market for treatment of photo-aging, hair
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damage, hyperpigmentation, and wrinkles [3].

Recently, the development of AOs in the area of cosmeceuticals has faced several instability-
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and insolubility-related problems that limit their practical applications. Because many AOs are
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unstable and sensitive to temperature, pH, light and oxidation, it is necessary to protect them against
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degradation caused by such factors [4]. Microencapsulation techniques are regarded as an important
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solution to the problem of developing suitable materials for protection and enhancement of AOs’

bioactivity in the cosmeceutical field [5]. Especially, biodegradable polymers-based delivery

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systems have been considered the most suitable for cosmeceuticals applications, owing to their

biocompatibility [3]. It has been reported that a variety of biodegradable polymers-based delivery

systems such as liposomes, nanostructured lipid carriers, solid lipid nanoparticles, and emulsions

are available for cosmeceutical applications [6-8]. Among them, liposomes, microscopic bilayer

vesicles from dispersion of membrane-like lipids in aqueous solvents, have shown great utility for

protection of reactive or sensitive compounds [9]. In recent decades, liposomes have been paid

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considerable attention and regarded as the most commonly used carrier system for encapsulation of

AOs in cosmeceuticals as well as within the food and nutritional industries [10]. The use of

liposomes as encapsulation materials offers several advantages over other carrier materials, such as

(i) highly increase efficacy and bioavailability of entrapped agents, (ii) easy removal from the body

through simple metabolic pathways, (iii) low toxicity, biocompatibility, biodegradability, and (iv)

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the capacity for modification of surface and size [4].

However, the production and application of liposomes in cosmeceuticals face many

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significant problems as well, which fact has significantly limited the development and expansion of

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their practical applications. Generally, the challenges for the development of liposomes in

cosmeceuticals as well as topical applications are related to three major problems, including: (i)
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decreased stability of liposomes over time; (ii) the difficulty of preparation techniques, mass
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production and low encapsulation efficiency, especially for hydrophilic AOs; (iii) limitation of skin

permeability. To broaden liposomes’ applicability, development strategies to tackle such problems

have been gaining considerable attention in the literature. In the present review, we provide a
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comprehensive overview of the challenges and development strategies of liposomes for

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encapsulation and delivery of AOs in the cosmeceuticals industry. Briefly, we emphasize the serious

problems related to the applicability of AOs as well as the benefits and necessary use of liposomes
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in cosmeceutical products. Also, we thoroughly summarize the incorporating mechanisms as well as

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the applications of liposomes for encapsulation of AOs. The main scope of this study was to

identify the remaining challenges to the use of liposomal vesicles in cosmeceutical applications. An

additional goal was to provide a critical update on the effective strategies that have been employed

to resolve these problems.

2. Antioxidants in cosmeceuticals

AOs have been defined as highly active agents that significantly delay or prevent the reaction

chains of oxidations involving free radicals, even at low concentrations [11]. AOs’ main

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mechanisms of action in inhibiting free radicals, as discussed in detail in other reviews [12-16], are

as follows: (i) neutralizing reactive species, (ii) sequestering transition metal ions (chelation

activity), (iii) inhibition of enzymes overproducing reactive oxygen species (ROS) [13-16]. Briefly,

in cosmeceuticals, AOs play a critical role in inhibiting or slowing the progression of oxidative-

damage reactions.

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2.1. Oxidative stress and antioxidant defense

Normally, skin cells generate ROS as a result of normal cellular metabolism (endogenous ROS

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sources) and environmental factors (exogenous ROS sources such as air pollutants or sunlight

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exposition) [16, 17]. The most common ROS types includes superoxide anion (•O2-), peroxide,

hydroxyl radical (•OH), hydrogen peroxide (H2O2), and singlet oxygen (1O2) [18]. These generated
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ROS tend to take electrons from other, nearby stable-molecules to produce more unstable-
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molecules, which causes damage to skin cells (Figure 1a). In healthy skin, endogenous AOs

instantaneously neutralize these free radicals to generate a homeostasis between those free radicals

and endogenous antioxidants (Figure 1b). This balance prevents dermatological disorders. When
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human skin is exposed to solar ultraviolet radiation (UVR) or other pollutants, a dramatic increase
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in the production ROS shifts the natural balance toward a pro-oxidative state, resulting in a

phenomenon called oxidative stress [19]. Oxidative stress expresses an imbalance between ROS
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and AOs due to the presence of numerous ROS (Figure 1b) [20, 21]. Oxidative stress is the main
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cause of various dermatological disorders such as skin aging, deep wrinkles, coarse texture, solar

elastosis, telangiectasia and pigmentation, psoriasis, atopic dermatitis, allergic contact dermatitis,

vitiligo, acne vulgaris, alopecia areata, lichen planus, and melanomas [16, 18, 22-24]. It has been

reported that almost 80% of ROS are generated by UV radiation, mainly UVA (95–98%) [25].

Therefore, it is very important to protect skin from the harmful effects of sunlight.

It is known that the skin, as the outermost human organ, is always influenced by ROS, and

thus, it has its own defense system deriving from endogenous AOs and exogenous AOs for

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modulation of free-radical reactions [26]. On the one hand, skin cells protect themselves from ROS

by using endogenous AOs originated from melanin [27]. Under physiological conditions, excess

ROS can be eliminated by healthy biological systems based on endogenous AOs including

glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH)

[12, 28]. On the other hand, endogenous AO defense systems are incomplete without supplementary

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exogenous AOs [29]. For protection of skin from damage, it is notable that the level of endogenous

AOs must be kept stable; when it is insufficient to maintain the balance with ROS due to oxidative

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stress, exogenous AOs should be administrated either orally or topically [26, 29]. Therefore, the

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development of cosmeceutical products for topical supply of external AOs is needed.

2.2. Classification of antioxidants

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AOs can be categorized in various ways. Firstly, they can be classified as enzymatic or non-
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enzymatic based on their activity [12, 30]. In the presence of cofactors, which normally are trace

elements such as copper, zinc, manganese, and iron, enzymatic AOs convert dangerous oxidative

products to H2O2 and then to water through a multi-step process. Non-enzymatic AOs scavenge
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ROS by interrupting free-radical chain reactions. The common non-enzymatic AOs are vitamin C,
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vitamin E, plant polyphenol, carotenoids, and glutathione. In addition, concentrations of enzymatic

and nonenzymatic AOs have differences in the types of skin cells. Keratinocytes have a lower
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concentration of enzymatic AOs than fibroblasts, while enzymatic AOs are not present in
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melanocytes [31].

The other way of categorizing antioxidants is based on their solubility and permeability.

According to the FDA, AOs have been classified into 4 types based on the biopharmaceutical

classification system (BCS), including: (I) high solubility – high permeability; (II) low solubility –

high permeability; (III) high solubility – low permeability; (IV) low solubility – low permeability

[32]. The high-solubility AOs (vitamin C) often appear in the cellular fluids (i.e. cytosol, or the

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cytoplasmic matrix), while the low-solubility AOs (vitamin E, carotenoids, and lipoic acid) are

mainly present in cell membranes.

As above mentioned, AOs in the skin’s defense systems can also be divided into 2 types

according to their origin: (i) endogenous AOs, which can be enzymatic or non-enzymatic AOs

including intracellular non-enzymatic AOs (ferritin, myoglobin, metallothioneins, coenzyme Q10,

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glutathione, melatonin, polyamines) and extracellular AOs (transferrin, lactoferrin, albumin,

ceruloplasmin, uric acid, bilirubin) [28, 33]; (ii) exogenous AOs such as vitamin C, vitamin E,

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carotenoids, polyphenols, and trace elements. The exogenous AOs used in cosmeceuticals

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formulations are often derived from plant extracts that contain a mixture of compounds.

Finally, AOs can be divided into natural and synthetic types based on their function [19, 20,
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29]. Natural AOs include mineral AOs (i.e. selenium, copper, iron, zinc, and manganese), vitamins,
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polyphenols, and phyto-antioxidants. Mineral AOs act as cofactors of enzymatic AOs. The synthetic

antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyl anisole (BHA), propyl

gallate and metal chelating agent play an important role in capturing free radicals and terminating
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their chain reaction. Synthetic AOs can be regenerated from natural AOs and are often used in
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combination with natural AOs to yield synergistic stabilization effects [34].

2.3. Challenges to cosmeceutical applications of antioxidants

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Due to having activities against free radicals, AO compounds have attracted great attention in
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cosmeceutical industries. They can be delivered and supplied to targeted skin cells through diet and

oral supplementation. However, direct applications of AOs to the skin have more advantages, due to

their transport of sufficient amounts of AOs to the targeted skin area. Therefore, the addition of AOs

to cosmeceutical formulations is a potential strategy. Unfortunately, this approach faces several

obstacles. In this review, we discuss only the two main problems directly affecting the development

of liposomes for delivery of AOs in cosmeceutical applications.

Most AOs are inherently unstable compounds under some environmental stress conditions

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such as air, moisture, light, heat, metal ions, oxygen, and alkalinity [26]. Indeed, it is AO instability

that causes many problems in cosmeceuticals formulations. Due to AOs’ instability, it is difficult to

maintain their activities in formulations during the intended shelf life, which fact renders them

problematic for direct use in practical products. For example, vitamin C, a well-known AO with a

variety of biological, pharmaceutical and dermatological functions, is very unstable: it is easily

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oxidized and loses its activities in the presence of oxygen as well as under light and alkalinity

conditions. Similarly, vitamin E, a strong AO in medical and cosmeceuticals applications, is also

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rapidly degraded due to its light, heat and oxygen sensitivity [34]. Therefore, the applications of

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vitamin C and vitamin E within the cosmeceutical field are significantly limited, despite their

interesting functions [7].

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For use in cosmeceutical formulations, AOs must penetrate inside deeper layers of skin and
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reach their target tissue in the active form [34]. However, most of them are absorbed in the

outermost layer of skin due to their low permeability and low water-solubility. To illustrate,

catechins, major AOs in green tea, have thus far been limited in their application to cosmeceuticals
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products, as they suffer from low skin permeability [35]. Resveratrol, a naturally occurring
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polyphenol, has attracted considerable interest for a number of potential skin applications. However,

because it is soluble only in trace amounts in the aqueous and lipid phases [36], resveratrol has
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shown low bioavailability, and in fact, it cannot reach a target site to exert the desired health effect
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[7].

3. Structural components and incorporation mechanism of antioxidants in liposome

Liposome, first introduced by Alec Bangham in 1961 [37], has been known as one of the most

common carriers for encapsulation of active antioxidants in cosmetic and pharmaceutical

applications. Simply, it is a colloidal system based on the formation of a bilayer membrane of

particles through self-assembly and dispersal of phospholipids in aqueous solution [38]. Liposomes

can encapsulate both hydrophilic and hydrophobic AOs due to their bilayer composition [8] (Figure

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2a). Liposomes are often classified into 5 types based on their size and number of bilayers [39]

(Figure 2b): small unilamellar vesicles – SUV or nanoliposome (20-100nm), large unilamellar

vesicles – LUV (>100nm), giant unilamellar vesicles – GUV (>1000nm), oligolamellar vesicles –

OLV (100-1000nm), and multilamellar vesicles – MLV (>500nm). However, according to Table 1’s

data, only two types, SUV and LUV, usually have been used for cosmeceuticals applications, due to

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their small particle sizes.

3.1. Structural components of liposome

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Generally, the main components of lipid bilayers are phospholipids and cholesterol. Cholesterol

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plays a crucial role in the physical stability of liposome bilayers due to its increase of the

phospholipid phase transition temperature [40]. Phospholipids used for fabrication of liposome can
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be natural or synthetic phospholipids, i.e. phophatidylcholine (PC), phosphatidylethanolamine (PE),
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phosphatidylserine, and phosphatidylglycerol [39]. The phase transition temperatures (TM) of

phospholipids are regarded as the most important factors in the choice of suitable phospholipids,
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due to the temperature dependence of its state, either fluid (T > TM) or a gel (T < TM). Because
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liposome will be applied in cosmeceuticals products, phospholipid should be chosen with gel states
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under physiological conditions to ensure the stability of liposome and the bioactivity of AOs. If the

TM of phospholipids is less than or equal to body temperature (T≈ 37 °C), liposome will exist in a
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fluid state, and the incorporated AOs will be expelled before reaching the site of action.
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Phospholipids with a higher TM (>37oC) will be the major ingredient in the fabrication of liposomes

for cosmeceuticals applications. Hence, in the cosmeceuticals field, PC is one of the most suitable

phospholipids commonly used due to the requirement for the satisfaction of TM [41], moreover, it is

the most abundant lipid class in mammalian membranes and a major membrane component in

eukaryotic organisms.

To enhance the stability of the phospholipid bilayer, the addition of cholesterol (Chol) in the

liposome preparing process is a necessary step. However, to optimize the effect of Chol, its ratio to

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phospholipid is an important factor that affects the encapsulation efficiency of liposome and

delivers an active-antioxidant [42-45]. Thus, for formulation of liposomes with the desired

characteristics, the amount of cholesterol employed needs to be carefully considered. For example,

in an experiment evaluating the effect of the Chol/phospholipid ratio on the loading efficiency and

size of liposomes, nanoliposome prepared with different ratios of PC and Chol (20:80, 40:60, 60:40

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and 80:20) by the ethanol-injection method showed that both the size and loading efficiency have a

significant dependence on the ratio [45]. It was indicated that the optimal condition for both the size

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and loading efficiency in this study is formed nanoliposomes with a PC:Chol ratio of 60:40.

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Moreover, the needed amount of cholesterol for liposome formation may also depend on synthesis

methods and antioxidant classes.

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3.2. Incorporation mechanism of antioxidants in liposome
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Various methods including thin-film, ethanol-injection, reverse-phase-evaporation and pH-driven

approaches (Table 1) can be used to fabricate liposomes with different properties including size,

lamellarity, and encapsulation efficiency for cosmeceuticals applications. While some methods are
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easy to perform on the laboratory scale (thin-film, reverse-phase evaporation), other techniques are
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more useful for scale-up but require special equipment (ethanol injection, pH driven, extrusion,

microfluidic). As already mentioned, liposomes potentially can encapsulate both hydrophilic and
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hydrophobic AOs due to the amphiphilic feature of phospholipid. Hydrophilic antioxidants will be
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entrapped in the interior aqueous compartments, while hydrophobic agents will mainly be

incorporated within the lipid bilayers (Figure 2a).

For hydrophilic AOs, there are two common methods of encapsulation in liposome: passive

loading and active loading [46, 47]. Simply, passive loading is based on the dissolution of dried thin

films of lipid in aqueous solutions containing the target antioxidant [48], but this approach often has

only a low loading efficiency. In the passive loading method, moreover, the encapsulation

efficiency of the antioxidants strongly depends on the volume of the water phase during liposome

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formation and the amount of added Chol [46]. By contrast, active loading has been shown to be

exceedingly efficient, resulting in high encapsulation of hydrophilic antioxidants in liposome [47,

49]. For active loading, a transmembrane pH gradient will drive AO internalization into preformed

liposomes, in which setting, the pH outside the liposome make the AOs exist in a united form in

which they can migrate across the lipid bilayer (Figure 3a) [48]. Then, the AOs become ionized due

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to the pH difference, and become trapped upon entering the liposomes.

For hydrophobic AOs, the lipophilic antioxidants become highly embedded in the lipid

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bilayers when the liposomes self-assemble. Besides, it has been shown that the position and

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orientation of the hydrophobic AOs in the bilayer affect the protective effect of membranes against

oxidation [50]. The incorporation of hydrophobic agents in lipid bilayers may, due to their chemical
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structures, cause a contrasting effect on the membrane properties. For example, polar AOs can
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improve the rigidity of the lipid bilayer, but non-polar AOs have a negligible or opposite effect on

the lipid membranes [51]. It is notable that each hydrophobic AO, because of its particular features,
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will possess a specific incorporating ability with regard to concentration and encapsulation
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efficiency in liposomes as well as differences in the linking position and orientation in lipid bilayers.
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This hypothesis was demonstrated in a study that evaluated the incorporating ability of four

different carotenoids including lycopene, lutein, β-carotene and canthaxanthin [38, 52]. The linking
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positions and orientations of these hydrophobic AOs in lipid bilayers are clarified in Figure 3b. This
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study found that lycopene and canthaxanthin have a poor lipid-bilayer-incorporating ability whereas

lutein and β- carotene do so at a high level. It can be explained that the low ability of lycopene is

because of the lycopene molecules positioned deeply in the lipid bilayers due to the strong

lipophilicity. By contrast, the flexible location and orientation of β-carotene in the liposome

membrane due to the presence of two β-ionone rings makes β-carotene easily incorporated into lipid

layers with high ratios [53]. Due to the hydrogen bonding between the polar end groups of lutein

molecules and bilayer’s polar region, lutein is also able to orientate vertically to the membrane

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plane, which contributes to the strong incorporating ability of lutein due to the fact that more

molecules can be incorporated across the bilayers. Despite the similar polar region and orientation

to lutein due to the presence of keto groups, the incorporating ability of canthaxanthin into the

liposomal membrane was really low compared with the others, since the structure of canthaxanthin

molecules cannot fit into the lipid bilayer architectures and lack a lipid soluble antioxidant such as

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β-carotene or lycopene [54].

4. Cosmeceuticals benefits of liposome

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Due to the similarity of structure between bilayer lipid and natural membranes, liposomes have

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been attracting a great attention for their potential applications to skin treatment and cosmeceuticals

[55]. Because lipid vesicles are able to alter cell membrane fluidity and fuse with cells, they easily
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deliver active AOs to the target site [56]. According to experts, liposomes have a great potential as
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active vectors in AO carriers, due to the possibility to enhance encapsulating performance by

improving AO solubility and stability and, thus too, delivering incorporated AOs to specific target

sites and providing sustained AO release [8]. In addition, the main advantage of liposomes used in
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cosmeceuticals applications is that the lipid membrane reduces the danger of acute and chronic
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toxicity, because it is made from physiological lipids. Moreover, compared with other materials,

liposomes are more biocompatible and biodegradable.

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4.1. Improved stability of antioxidants

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Liposomes can remain stable against various environmental and chemical changes, and thus they

are also able to enhance the performance of cosmeceuticals products by increasing the stability of

the active AOs in these products’ mixes [57]. The use of liposomes as effective delivery vehicles to

protect reactive or sensitive compounds against environmental conditions including light,

temperature, pH, humidity, oxygen and others has been demonstrated in many studies with various

AOs, both hydrophilic and hydrophobic agents, such as tea polyphenol [9, 58], retinol [59], vitamin

C [45], vitamin E [60], curcumin [61-64], coenzyme Q10 [65], and others (Table 1). To illustrate,

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Zou et al. (2014) reported that tea polyphenol, a sensitive AO that is strongly affected by an alkaline

pH, oxygen level and even its concentration, as encapsulated in liposome, increased the stability

against the adverse factors [58]. Although tea polyphenol will be more sensitive to degradation

when reducing concentrations or increasing pH from an acid to an alkaline, tea polyphenol

entrapped in liposome was protected, due probably to the hindering of two important factors (i.e.,

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oxygen levels and concentration of tea polyphenol), which significantly enhanced the stability of

the tea polyphenols. Since liposomes are isolated in extra-membrane environments, the protection

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of tea polyphenols can occur due to the interacting limit between oxygen and tea polyphenol. In

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addition, the degradation of tea polyphenols in liposome is also prevented due to its slow release

ratio compared with tea polyphenol solution. The protection of other AOs in liposome can be
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explained by similar mechanisms.
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4.2. Enhancement of antioxidant activity and solubility of antioxidants

Liposomes, with their biphasic characteristic, construction, composition and diversity of design,

have been attracting great interest as a dynamic and adaptable technology for encapsulating and
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improving the solubility of poorly water-soluble AOs [66]. In addition, numerous studies have
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indicated that besides providing a physiochemical barrier for encapsulated and incorporated active

AOs against pro-oxidant elements and environmental conditions, the lipid bilayers are also likely to
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enhance the water-solubility of these active agents and be homogenously dispersed in

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cosmeceuticals formulations for significantly increased bioavailability of AOs [39]. Tan et al.

(2014) demonstrated these properties by incorporating four carotenoids (lutein, β-carotene,

lycopene, canthaxanthin) into a liposomal system and comparing the AO activities of the

carotenoid-loaded liposomes and carotenoid solutions using 2,2-diphenyl-1-picrylhydrazyl (DPPH)

scavenging and ferric-reducing AO powder (FRAP) assays [52]. It was indicated that compared

with the carotenoid solutions, all of the carotenoids, when entrapped in liposomes, expressed a

significant increase in DPPH scavenging activity and FRAP activity, and this was especially true for

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lutein and β-carotene. This improvement can be considered to have been effected via two main

mechanisms: (i) enhancement of water solubility and dispersion of carotenoids due to liposome

encapsulation; (ii) post-encapsulation reduction of the steric hindrance of the AO molecules. In

summary, the development trends of liposome-based cosmeceuticals formulations will be promising

directions for AOs, particularly hydrophobic agents, because of two interesting properties of

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liposomes, namely enhanced solubility and bioactivity.

4.3. Enhancement of skin penetration of antioxidants

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Any active molecules in cosmeceuticals products spread out on the skin will pass through the skin

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layers by two major routes: the transappendageal and transepidermal pathways [67], the latter being

primarily responsible for skin permeation. In the transepidermal pathway, active agents may
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permeate across the stratum corneum (SC), but maintain their intact structures. Moreover, this
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pathway includes two routes: intercellular and transcellular (Figure 4a). For the intercellular route,

this pathway offers a continuous and tortuous path across the intercellular lipid domains, while

according to the transcellular route, active agents have to pass the keratinocytes and through the
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intercellular lipids [68]. It is clear that the amount of compounds that can permeate across the skin
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via the transcellular pathway is very low, because they must partition and diffuse through the

keratin bricks and go across the intercellular lipids. Hence, the main pathway for skin permeation of
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active AOs is the intercellular lipids.

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As already defined, liposomes are small, spherical bilayers of phospholipids; as such, they can

mimic the cell membrane constituents and improve dermal and transdermal active-AO delivery

thereby [69]. Some studies have determined that the penetration ability of active-compound-loaded

liposomes can be influenced by the surface charge and particle size of liposomes [69, 70].

The skin is likely to present as a negatively charged membrane, because the lipid layer in the

SC consists of a high content of negatively charged lipids [71, 72]; thus transcutaneous diffusion of

antioxidants can be affected by the charge of the vesicle surface. Compared with positively charged

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liposomes, the negatively charged ones generally have a higher lux, and as a result, the

accumulation of active compounds in the superficial skin strata can be greatly improved [71].

However, the conclusions of many studies have been contradictory. Some studies stated that

negatively charged liposomes were more effective than positively charged and neutral liposomes in

enhancing percutaneous drug absorption [69, 71, 73, 74]. For example, Zhou et al. (2014) [73]

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demonstrated that the skin permeation of negatively charged liposomes encapsulating vitamin C

might reach a very high efficiency due to the change of the negative surface charge from -2.3 to -

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35.5 mV after coating of pectin. Besides the negative surface charge, this study also indicated that

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the bioadhesivity of pectin contributed to the skin-permeation enhancement of the liposomes by

increasing their contact time with the skin. In contrast, negative vesicles can be absorbed in the
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viable epidermis and dermis; thus, compared with positive vesicles, they may result in higher skin
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accumulation of encapsulated AOs with lower skin permeation [71]. In addition, it has been

suggested that positively charged liposomes relative to negative ones have a tendency to interact

more strongly with the skin surface and that the positive polymers coated on the surface of
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liposomes may go deeper and disrupt the tight junctions of the lower epidermis layer [69, 75, 76].
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Therefore, liposomes with positively charged surfaces due to coating of positive polymers may

exhibit more effective skin permeation than those with negatively charged surfaces. For illustration,
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Patra et al. (2016) [76] prepared a novel positive liposome to encapsulate curcumin with high skin
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permeability by conjugating R9 (polyarginine) carbon dots with nanoliposomes-coating cell-

penetrating peptides (CPPs), namely RCDLs. The results showed that free curcumin cannot go

through the SC and is accumulated on the outer layer of skin, while the conventional liposomes

(CLs) and carbon dot liposomes (CDLs) loading curcumin were across the SC, but absorbed in the

viable epidermis and dermis layers, respectively (Figure 4b). Especially, RCDLs can go across the

SC, viable epidermis and even dermis, and reach deeper skin layers. This may be explained by two

exceptional features of RCDLs: (i) the positive charge due to the coating of peptides on the surface

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of nanoliposomes enhances the penetration and long-circulation across the skin; (ii) due to a very

strong power of penetration provided by conjugating CPPs, liposomes can go through the deeper

layers of the skin. In summary, despite much controversy about whether positive or negative

vesicles have a higher skin permeability, it is clear that the encapsulation of active AOs in

liposomes can significantly improve their skin penetration.

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Also, it has been shown that penetration of liposomes through the skin strongly depends on

their size [70]. Generally, liposomes of particle sizes up to 600 nm are able to easily penetrate the

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skin, but they will remain within the SC if their particle size is 1000 nm or more [70]. However, it

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was reported that small liposomes of ~ 600 nm particle size still exhibit many limitations in their

permeability due to their difficulty in reaching the deeper layers of the skin [77]. Therefore, to
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overcome this problem, nanoliposomes or SUV liposomes, which is a type of liposome containing
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small lipid bilayers with nanoscale particle sizes (20-100nm), are commonly used to encapsulate

active AOs. Patra et al. (2016) [76], in explaining the impact factors leading to the enhancement of

skin permeability of RCDL liposomes, stated that the very small particle size and narrow
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distribution of these nanoliposomes afford their easy penetration across the SC and into the deeper
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skin layers.

5. Challenges to applicability of liposomes

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Prior to any cosmeceuticals application, a good delivery system for active AOs needs to satisfy
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several necessary conditions including: (i) high encapsulation efficiency; (ii) long-term stability;

(iii) small particle size with narrow size distribution, and (iii) penetrability to the deeper skin layers.

Although it has been assumed that liposomes have a great potential for delivery of active AOs in

cosmeceuticals, their applicability has come up against many challenges.

Two of the major problems limiting liposomes’ utilization in practical applications are their

low physical and chemical stabilities [78, 79]. The physical instability of liposomes can be

explained by two parameters: (i) changed particle size and size distribution due to the aggregation

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and fusion of vesicles; (ii) leakage of entrapped AOs [80]. Such destabilizations will happen at a

faster rate when the surface charges of liposomes are decreased by pH or the presence of strong ions

[81]. It has been reported that liposomes with no- or weak surface charges will tend to afford easier

aggregation than strongly charged ones [82]. The leakage of encapsulated AOs strongly depends on

the particle size and structural compositions of the liposomes. To insure the physical stability of

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liposomes, therefore, increasing the surface charge and optimizing the structural compositions and

particle size will be necessary approaches. As for the chemical instability of liposomes, it has been

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suggested that oxidation and hydrolysis of the phospholipids may be one of the main pathways

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causing this problem [80, 83, 84]. Also, it has been found that temperature and pH are the two major

parameters significantly influencing the chemical stability of liposomes, and it was suggested that
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for long-term stability, liposomes should be stored only at low temperatures (4-6oC) and neutral pH
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(6.5) [80]. Hence, for use in cosmeceuticals applications, increasing the stability of liposomes at

higher temperatures and within a wide range of pH will be critical steps.

Another problem limiting the application of liposomes in cosmeceuticals products is the low
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encapsulation efficiency of active AOs, especially hydrophilic AOs such as vitamin C. It has been
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shown that by using conventional methods (thin-film, ethanol-injection methods) to prepare

liposomes, hydrophilic AOs may be easily dissolved and retained in the external aqueous phase of
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liposomes, while they are encapsulated in the aqueous cores of liposomes with only low efficiency
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[46]. This is due to the fact that the aqueous cavity of the formed liposome is very low [46]. In

addition, the conventional methods are based only on the self-assembly of phospholipids in an

aqueous environment [85, 86] and thus, because of the random nature of bilayer folding, these

techniques will produce vesicles of non-uniform in size and shape. Several studies stated that the

encapsulation efficiency of these methods for hydrophilic AOs is relatively low, with a maximum

value of approximately 35% [87-89].

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Large-scale production is also a major challenge to the development of liposomes in the

cosmeceutical industry. It has been reported that liposomes used for cosmeceuticals products can be

fabricated by various techniques including: (i) mechanical methods: thin-film, sonication,

microfluidization, extrusion; (ii) techniques using aqueous solution to replace organic solvents:

ethanol injection and reverse-phase evaporation [90, 91]. For large-scale production, however, it

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seems that most of the methods are unsuitable, due to difficult scale-up or complicated operation or

non-cost-effectiveness [91]. Therefore, novel techniques that offer the possibility of industrial

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production in the cosmeceuticals field are indispensable.

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As early noted, liposomes can enhance the skin permeability of AOs. However, the

penetration of AO-loaded liposomes into the deep skin layer only occurs in case of nanoliposomes
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with very small size and modified surface. For conventional liposomes (CLs), therefore, they cannot
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reach to targeted sites and are reduced therapeutic functions. When liposomes are applied on skin

surface, most of them only remain in the upper layer of the SC and they play a role as a drug

reservoir [92]. Because of the lack of deformability, a large amount of CLs is mostly stopped in the
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epidermis upper layers according to the intercellular pathways where disruption of vesicles can
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happen (Figure 4 and Figure 8). This statement is supported by the results of skin penetration

studies of CLs in many researches which CLs show a relatively low elasticity altogether with a high
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skin retention [93-96]. According Sala and coworker’s review [97], a lot of recent researches has
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admitted that conventional types of liposomes are low efficient transdermal delivery systems and

they exhibit an inefficient permeation into the deep skin layers.

For the purposes of overcoming these challenges, the specific strategy formulation will have a

very profound significance. These strategies will be discussed in the following sections.

6. Development strategies of liposomes for delivery of active anti-oxidants

6.1. Enhancement of stability of liposomes

6.1.1. Modified surfaces of liposomes

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To enhance the physical and chemical stability of liposomes, it is generally suggested that

increasing interparticle repulsion, either electrostatic or steric, is a highly effective method by which

the liposome surface zeta potential (ξ-potential) will be increased due to modification with a

strongly ionic substance [98]. The zeta potential of liposomes indicates the stability state of

colloidal systems, because if the potential increases, it is equivalent to greater repulsion between

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particles, which makes colloidal dispersions more stable. In the case of a large negative or positive

zeta potential in all particles of a given liposome dispersion, these particles will repel each other,

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and will show no tendency to aggregate [99]. Polymer coating has been demonstrated to be a

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promising approach for the increase of liposomes’ surface charge. In addition, it has been stated that

this method will form a polymer layer around the liposome surfaces, which is likely to reduce the
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oxidation of lipids and prevent the leakage of antioxidants [100, 101]. Simply, this can be conducted
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by adding a polymer solution to a liposome suspension; there is no need of any chemical bonds

between these two components [101, 102]. In cosmeceuticals products, liposomes will be directly

used on the skin; thus the employed polymers must have nontoxic, biocompatible, and
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biodegradable features. It has been reported that several polymers, both cationic and anionic, that
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have been coated on liposome surfaces can be applied in the cosmeceuticals field; these include

chitosan, peptide, pectin, and alginate (Table 1).

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CS is one of the most popular biopolymers that has been widely utilized in cosmeceuticals;
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[103] reportedly, it is a cationic polymer that can significantly improve zeta-potential values when

interacting with anionic liposomes [73, 104-106]. Also, it has been proposed that successful coating

of CS on liposomes can be achieved through electrostatic interaction, hydrophobic interactions and

hydrogen bonding [107]. M. Hasan et al. (2016) [64] indicated that CS coating had increased the

density of the positive surface charge of a curcumin-loaded liposome. After adsorption of CS, a thin

layer around the surfaces of the liposome particles (Figure 5a) was formed by the strong

electrostatic interaction between the negatively charged surfaces of the liposomes and the

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oppositely charged polyelectrolyte of CS, which led to a large shift of the zeta potential value from

negative (-45.8 mV) to positive charge (+61.9 mV). This study also demonstrated it was the

increase of the zeta potential that enhanced the stability of the liposomes, even when stored at 37 oC

for 30 days. Another biopolymer that has been commonly used as a stabilizer for liposomes is

pectin [108, 109]. Zhou et al. (2014) [73] used anionic pectin including high-methoxyl pectin

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(HMP) and low-methoxyl pectin (LMP) to coat the surface of an anionic vitamin C liposome, and

they demonstrated that the successful coating was due to hydrogen-bonding interactions between

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the anionic components [110]. After coating with HMP and LMP pectin, there was a significant

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increase of the surface charge of the liposome particles corresponding to the decrease of the zeta

potential from −2.3 mV to −23.9 mV and −35.5 mV, respectively. The results of this study showed
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that the chemical stability of vitamin C-loaded liposomes coated with pectin was highly enhanced
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compared with non-coated liposomes, and that this was clearly expressed through the better

protection of vitamin C loaded in HMP-liposomes and LMP-liposomes after 10 weeks’ storage at

250C.
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Another approach for biopolymer coating on liposomes is the combination of polymers to

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form novel multilayered liposomes. It has been reported that the complexation of biopolymers can

produce a synergistic effect by taking all of the advantages and reducing the drawbacks of single
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ones [111]. Recently, chitosan and alginate have been gaining great attention as stability-enhancers
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for coating onto liposome surfaces, and indeed, the concept of a liposome-surface-coating technique

that proceeds by forming an alginate-CS polyelectrolyte layer-by-layer via the self-assembly

method has attracted great interest [106, 112, 113]. To obtain a polyelectrolyte delivery system,

positively charged CS will be coated onto the anionic liposome surface by electrostatic interaction

to form cationic liposomes, and then the outer layer of these cationic liposomes will continue to be

deposited with negatively charged alginates (Figure 5b). Liu et al. [106, 113] demonstrated the

strong effect of the polyelectrolyte delivery system in its protection of vitamin C as well as its

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improvement of liposome physiochemical stability. Interestingly, multilayered liposomes coated

with CS and alginate show better stability against environmental stresses such as pH, ionic strength

(NaCl), and temperature [113].

Besides polymers, silica-coated liposomes, known as Liposil, proposed by Devoisselle et al.

[114], have been becoming one of the most promising approaches to liposome surface modification

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in recent years [115-117]. Silica-coated liposomes are formed by a sol-gel reaction (Figure 5c)

[117] that includes two steps: LUV liposomes are first formed, and then they are enclosed by silica

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layers via interaction with APTES-(3-aminopropyl)triethoxysilane or tetraethyl orthosilicate (TEOs)

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[114, 117]. It has been clearly shown that sol-gel-derived silica possesses some predominant

properties such as biocompatibility, chemical inertness, and inexpensiveness; thus, the silica-coating
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method may be a suitable approach for the development of cosmeceuticals liposome formulations.
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In addition, silica coverage is likely to prevent the aggregation of liposomes as well as the leakage

of the encapsulated compounds, thereby increasing the stability of liposomes. It has been suggested

that synthesis of liposome–silica derivatives should be widely utilized to stabilize lipid bilayers
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[117].
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Currently, polymer-coated liposomes are seldomly used for dermal AOs delivery in

cosmeceutical products, especially for skin diseases relating to bacterial infections such as acne
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vulgaris and staph infections, even though they have shown great stability. It is this stability that has
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led to negative impacts on their delivery function [118-120], because a polymer-coated liposomal

vesicle will be stabilized against fusion with another vesicle, but will also be inhibited from fusing

with bacterial membranes, which prevents contact between encapsulated AOs and target bacteria.

Therefore, a desirable strategy is to develop novel liposomes that have a potential stability against

fusion during the manufacturing and storage periods; however, once applied to the target skin sites,

their fusion activity will be reduced. A new approach to control the fusion activity of liposomes by

modifying their surface using carboxyl-modified gold nanoparticles (Au-COOH) has been

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introduced as a potential method [120]. Due to the non-cytotoxic, biocompatible, non-

immunogenic, and strong antifungal and antibacterial properties, gold nanoparticles have been

exploited in variety of cosmeceutical products [121, 122]. In principle, the fusion activity of this

liposomal system is mediated based on a change of environment pH (Figure 5d) [120]. At pH 7.0 >

pKa of the carboxylic group (~ 5.25), carboxyl-modified gold nanoparticles (Au-COOH) will

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become negatively charged particles (Au-COO-) because of deprotonation, which easily bind to

positively charged liposomes by electrostatic interaction and consequently stabilize the liposomal

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vesicles, the same as in the case of polymer coating. When pH < 5.0, the protonation of the

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carboxylic group will result in neutral Au-COOH nanoparticles that detach from the surface of

liposomes due to the loss of bond force, at the same time releasing liposomes with low stability.
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With a similar approach, Su et al. designed a potential liposome, abbreviated as pepsome, for
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cosmeceutical applications based on coating a serial of peptides on the liposome surface (Figure 5e)

[123]. The water-soluble peptides are also coated on lipid membranes by the electrostatic

interaction. The phase behavior, i.e., the temperature, will govern the permeability of coated
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liposomes and the coating efficiency of peptides. Especially, the peptide layer coated on the surface
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of liposomes can control the release of encapsulated cargos in terms of changes of both pH and

temperature.
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6.1.2. Encapsulation of liposomes in other compartments

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Another suggested strategy for improving the stability of liposomal vesicles is their encapsulation in

another carrier, which is known as the double-encapsulation method of AOs [124]. It was

demonstrated that the structural integrity and functionalities of entrapped vesicles are preserved in

such hybrid systems; and, simultaneously, a synergic efficiency of two distinct delivery platforms

due to the combination of their advantageous properties offers unique benefits such as minimized

burst release, higher thermal stability, and controlled sequential delivery [125-127]. However, it was

indicated that the encapsulation process of a phospholipid bilayer within materials is very

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complicated, because it is necessary to protect the interior contents during the formation of the

exterior membrane [128]. Therefore, it seems that conventional techniques are impossible to be

used in the preparation process of such materials. To synthesize these materials, novel techniques

are required to have a metastable phase wherein encapsulating materials that do not cause

significant damage to liposomal bilayers can be opened and closed.

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Encapsulation of liposomes in hydrogels

As mentioned above, although biopolymers have been used as coating agents on the surface of

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liposomes to provide prolonged release of AOs and significantly enhance long-term stability of

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liposomes, in this case, the protecting effect can be easily defeated by changes of various external

factors due to the weak bond between the polymer matrix and liposomal particles. Therefore, other
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strategies involving encapsulation of liposomal particles within hydrogels have been exploited to
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potentially achieve delivery systems with high flexibility [129-132]. Biopolymer-based hydrogels,

a.k.a. biohydrogels, with their key properties of biocompatibility and biodegradability, recently

have been used in numerous cosmetics, foods, and pharmaceutical applications [133]. Remarkably,
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the encapsulation of liposomes into hydrogels can provide a doubly protecting effect for
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encapsulated AOs. In addition, the combination of liposomes and biohydrogels may produce ideal

candidates for the synthesis of novel AOs delivery systems, due to a tunable liposome release rate
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and controllable viscoelasticity that can respond to environmental stimuli such as light, temperature,
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and pH. Some types of biopolymer-based hydrogels such as chitosan, gelatin, dextran, pullulan,

alginate, carrageenan, methylcellulose, and xanthan gum have proven potential materials for

synthesis of hydrogels that can encapsulate liposomes in cosmeceutical applications [133-135].

To further enhance the efficiency of pH-responsive gold-nanoparticle-stabilized liposomes

mentioned in the above section for topical antimicrobial delivery, it is necessary to develop new

strategies that encapsulate this liposomal system in hydrogels. Recently, Gao et al. introduced a

novel hydrogel formulation incorporating pH-responsive gold-nanoparticle-stabilized liposomes

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(Figure 6a) [136]. In this study, it was indicated that the hydrogel viscoelasticity is easily tuned by

changing the concentration of cross-linkers, which subsequently leads to a controllable release rate

of entrapped AuC-liposomes. The hydrogel formulation exhibits a better preservation of the

structural integrity of the nanoparticle-stabilized liposomes compared with liposomes coated only

with gold nanoparticles. Also, the fusion activity of the released liposomes with bacterial

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membranes will occur when applied to skin, due to the changing of the pH to an acidic condition.

Notably, the hydrogel formulation has been shown to be a non-toxic and effective approach for

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topical treatment of skin infections.

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Recently, Sekine et al. also developed a biodegradable hydrogel encapsulating both nanogel-

coated liposomes and nanogels by crosslinking an acryloyl-group-modified nanogel (CHPOA) and

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terminal thiol groups (pentaerythritol tetra(mercaptoethyl)polyoxyethylene (PEGSH)) (Figure 6b)
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[137]. It was indicated that the nanogel-coated liposomes were stably retained and well dispersed in

the hydrogel matrix without aggregation. Moreover, the hydrogel formulation also possessed a

unique process of nanogel and liposome release under physiological conditions in a two-step
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controllable manner: (i) the gradual release of nanogels from the hydrogel matrix; (ii) the release of
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nanogel-coated liposomes. Especially, this hybrid hydrogel was judged to be a promising candidate

for development of multidrug-delivery systems.

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Encapsulation of liposomes within double-emulsion

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Normally, the structure of liposomes becomes unstable under unfavorable physicochemical

conditions. To prevent interaction between liposomes and these unexpected factors, therefore, a

novel encapsulation system has been developed by incorporating liposomes into a water-in-oil-in-

water (W1/O/W2) double emulsion (Figure 6c) [138, 139]. A W1/O/W2 double emulsion is

constructed from an external aqueous phase (W2) containing oil globules (O), and within each oil

globule, smaller droplets of an internal aqueous phase (W1) are dispersed. It was indicated that

W1/O/W2 double emulsions show a high encapsulation efficiency for various substances in

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different phases, protection of fragile phospholipid vesicles, and controlled release [140]. For

combination of liposomes and a W1/O/W2 emulsion, small liposomal vesicles containing AOs are

entrapped within the W1 phase of W1/O/W2 double emulsions, which creates a double-

encapsulation system [139]. It is expected that the oil phase of double emulsions will act as a layer

to protect the structural integrity of liposomes. In addition, the W1/O/W2 emulsion system exhibits

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a high stability and good protection for liposomal droplets at temperatures below the phase-

transition temperature of the O-phase, and when applied on the skin, the O-phase will be thawed,

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with the result that encapsulated liposomes will be released.

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Encapsulation of liposomes in microspheres

Recently, there are some studies that have reported a new structure for enhancing the stability of
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liposomes by encapsulating them into microspheres of biodegradable polymers, namely liposomes-
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in-microsphere (LIM) [141-143]. In these studies, the double-emulsion solvent

extraction/evaporation method was used to prepare the LIMs (Figure 6d). It was demonstrated that

coating the liposomes with polymers and encapsulating them in microsphere capsules preserved the
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integrity of the lipid bilayer vesicles [141]. Moreover, the release of encapsulated liposomes and
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AOs potentially can be controlled by changing the fabrication parameters and compositions of both

microspheres and liposomes. It was suggested that such a novel system may have a great potential
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to be utilized for drug delivery in the pharmaceutical field owing to some distinguishing features
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[142], including: (i) in LIMs, liposomes are protected by a polymeric matrix of microspheres, while

the biocompatibility of the microspheres is improved by the presence of lipids on their surface; (ii)

compared with liposomes and microspheres, the drug encapsulation efficiency and the drug loading

are significantly improved because of blocking of drug leakage by the lipid layers and the polymer

matrix; (iii) a more controllable release of encapsulated drug and liposomes can be achieved.

However, there has been no study on or use of this system for cosmetic applications, due to the

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complications entailed in the preparation process and its unknown toxicity. Therefore, the

encapsulation of liposomes in microspheres can be a potential strategy for the near future.

6.1.3. Development of ingredients in cosmetic formulations for stability of liposomes

The development strategies for transdermal application of liposomes in cosmetic formulations have

been limited due to their low stability and low transdermal permeability; thus the addition of

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ethanol and synthetic surfactants often has been required. However, Kapoor et al. developed a novel

strategy to overcome these disadvantages based on a new cosmetic formulation called

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nutricosmetics [144]. In this formulation, AOs-entrapped nanoliposomes constructed from

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phosphatidylcholine and fatty acids stabilize within a cosmetic base without any added synthetic

surfactants or alcohol solvents. It is the three-dimensional (3D) matrix of the cosmetic base that
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provides for highly enhanced stability of liposomes at room temperature over a period of six
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months, while the AOs-loaded liposomes alone are stable only at 4°C. In addition, the transdermal

penetration is also highly enhanced by unique compositions of cosmetic bases such as oleic acid

and soya phosphatidylcholine. It is suggested that by using fluidizing liposomes entrapped in

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standard cosmetic bases, this technology can be explored as a platform for transdermal delivery of
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various AOs through the skin. Notably, different types of cosmetics including body lotions, skin

coloring agents and face packs have been used to evaluate the feasibility of this formulation in
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practical applications. The results showed that its compositions are easily available and that the
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cosmetic formulations are socially acceptable amongst African and Asian women due to both low

cost and high suitability.

6.2. Development of techniques for preparation of liposomes

Many techniques have been developed for the fabrication of liposomes; however, the techniques

that can be used to encapsulate antioxidants in the cosmeceuticals industry must satisfy two

mandatory requirements, including: (i) prepared liposomes must have superiorities such as small

particle size with narrow size distribution and high encapsulation efficiency for antioxidants,

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especially hydrophilic antioxidants; (ii) they must be easy to apply for large-scale production. A

comparison of advantages and disadvantages between conventional techniques and advanced

techniques is summarized in Table 2.

These days, the most popular methods that have been used to synthesize liposomes for

cosmeceuticals applications are the thin-film method (Bangham method) [37] and the ethanol-

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injection technique [145]. Although the Bangham method is widespread and easy to apply, the

formed liposomes are heterogeneous both in size and shape, and so reduction techniques, for

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example sonication or extrusion, need to be used in the next step to produce small and uniform

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vesicles [73, 106, 146], which makes this technique difficult to apply on the large scale [147]. It has

been reported that the ethanol-injection technique (Figure 7a) may offer many advantages,
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including simplicity with minimal technical requirements, possibility of scale up, and formulation
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of small-sized liposomes relative to other, conventional techniques [79, 91, 145, 148]. However,

this method is only suitable for encapsulation of hydrophobic AOs, because the encapsulation

efficiency of hydrophilic antioxidants is often very low [87-89]. Because of these limitations of
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traditional methods, recently, another area of research has been developing new engineering tools to
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enhance the physicochemical features of liposomes. These strategies have concentrated on

developing the advanced techniques that can be used to control the physicochemical characteristics
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of liposomes by automatic and programmable systems. Microfluidic technology has been regarded
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as an effective alternative for the assembly process of liposomes [149, 150] due to achieving better

control over the physical properties of the final vesicles so as to optimize size, encapsulation

efficiency and structural heterogeneity [151-153]. In addition, the microfluidic method can easily

incorporate both hydrophilic and lipophilic AOs into the vesicles to a high ratio, due to its

utilization of water-oil-water double emulsion [154].

New techniques developed based on microfluidic approaches for liposome production include

electroformation and hydration [155], extrusion [156], pulsed jetting [157], double emulsion

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templating [158], ice droplet hydration [89], transient membrane ejection [159], droplet emulsion

transfer [160] and hydrodynamic focusing [161]. Among them, microfluidic hydrodynamic focusing

(MHF) and double-emulsion templating are commonly used to prepare liposomes for

cosmeceuticals applications. As for the MHF method, it is based on the use of microfluidic devices

with a cross-flow geometry (2D-MHF) or a three-dimensional (3D-MHF) annular coaxial geometry

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[154, 162], 3D-MHF having been developed to overcome the limitations of 2D-MHF such as

expensiveness and time-consumptiveness [162]. Generally, 3D-MHF consists of a concentric

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capillary array (Figure 7b). In this system, the vesicle formulation will occur when an ethanol-lipid

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solution is continuously injected into a central aqueous line [163]. Notably, the size and size

distribution of formed liposomes can be adjusted by control of the volumetric flow rate ratio
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between the lipid phase and water phase streams, as well as the total flow rate.
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In recent years, the employment of templates is a new approach to the development of

microfluidic devices for fabrication of liposomes of highly uniform size and high encapsulation

efficiency. Also, double-emulsion templates in microfluidic devices recently have been utilized for
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preparation of liposomes in the cosmeceuticals field [159, 164]. For example, Shum et al. (2008)
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[154] introduced a microfluidic method based on monodisperse double emulsions with a core-shell

structure as templates for preparation of liposomes. The lipid phase will cover around an aqueous
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core to form the shell of double emulsions of water-in-oil-in-water (W/O/W) which are used as
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templates to directly produce phospholipid vesicles by evaporating the solvent in the lipid phase

(Figure 7c). In this device, a glass-microcapillary microfluidic device plays an important role in

producing monodisperse double emulsions by combining a coflow with a flow-focusing geometry

(Figure 7d, top). The input lines of this device include three phase: (i) inner phase – water phase

containing hydrophilic AOs; (ii) middle phase- lipid phase; (iii) outer phase-aqueous solution

containing poly(vinyl alcohol) and glycerol. The particle size and the thickness of the shell of the

obtained liposomes can be controlled by tuning the flow rates of each fluid phase as well as the

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diameters of each capillary [165]. The obtained liposome particles have a high uniformity in size

and shape (Figure 7d, bottom). The microfluidic devices based on the double-emulsion template

will be ideal methods for the fabrication of uniform phospholipid vesicles.

To optimize liposome production based on the principles of the ethanol-injection method,

there have been several recent studies that have developed a potential technique known as the

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membrane contactor method or the cross-flow injection method, which utilizes an SPG membrane

or a hollow fiber membrane for large-scale production [166-170]. This technique is one of the

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directly scaled-up approaches for large-scale production of liposomes [166]. In this system, an

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aqueous phase containing hydrophilic AOs is pumped through a membrane contactor module, while

an organic phase containing phospholipid and hydrophobic AOs is permeated through the pores of
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the SPG or hollow fiber membrane into the aqueous phase (Figure 7e). The formation of liposomes
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spontaneously occurs as soon as the aqueous phase is in contact with the organic phase. It has been

indicated that the membrane contactor method with a hollow fiber membrane exhibits better

efficiency than the SPG membrane because of a higher membrane area. The above-noted studies
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confirmed that the membrane contactor is an effective alternative to the ethanol injection technique
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in the preparation of liposomes. This method possesses numerous advantages including

reproducibility, facility of use, industrial scaling-up utilities and good stability of formed liposomal
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structure [167]. Moreover, it is a simple and fast method that allows for the production of
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nanoliposomes on a large scale. Therefore, the membrane contactor, especially the hollow fiber

membrane module, creates more opportunities for design, rationalization and optimization of the

preparation process of liposomes in industrial production.

In most of the conventional preparation techniques, hydrophilic AOs are encapsulated within

liposomes in terms of passive loading, in which case, these AOs are entrapped in the phospholipid

bilayer or inner aqueous core of liposomes during self-assembly [48]. Alternatively, the AOs-

incorporating procedure of liposomes, which is known as an active loading procedure, also is

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effective, specifically by controlling the pH condition using transmembrane gradients of pH or

buffers [171]. In this approach, empty liposomes are initially generated in an acidic pH buffer, and

then the extraliposomal phase is removed and titrated to slightly basic pH conditions. The AOs are

added in the extraliposomal phase in the final step, and the remote-loading process occurs when the

liposome solution is incubated over a period of time. Several studies have researched the crucial

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steps and mechanisms of the remote loading process [172-175], it is summarized and described in

Figure 7f. This process includes two main steps: (i) active-AOs transport from the extraliposomal

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phase to the intraliposomal phase due to the pH gradient; (ii) precipitation of AOs within the

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liposome due to the action of a counterion (sulfate) for the ionized AOs. Based on this approach, a

novel, rapid, real-time and single-step technique, called the liposomal electrospray process, recently
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has been developed for preparation of liposomes in terms of active loading (Figure 7g) [171, 176,
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177]. For the electrospray remote-loading process, a lipid ethanol solution is inserted into an inner

needle, while an aqueous buffer is added to the outer solution, which mimics the ethanol injection

process [171]. Liposomes are formed as soon as the ethanol-lipid solution in the inner needle is
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injected into the aqueous solution in the outer needle. Especially, the rapid evaporation of ethanol
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from the droplets significantly enhances hydration of the lipids in the electrospray process.

Hydrophilic AOs will be dissolved in the buffer collection dish solution and the collected solution
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will be incubated over a specific period of time. It has been demonstrated that the formulation and
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remote loading of AOs in liposomes enables a one-step process with a minimum total production

time and high AOs retention, compared with conventional methods. In addition, the electrospray

technique is available for mass production with more stable liposomes. It is offered as a unique

continuous method for synthesis of liposomes on a large scale [171, 176].

6.3. Development of new generations of liposomes

Although liposomes have been one of the most ideal delivery systems for topical drug delivery, it

has been proposed that their penetration ability might be hindered by their rigid structure [178]. It

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has reported that when applied to the skin surface, liposomes can be accumulated in the SC, which

potentially leads to minimal penetration to deeper skin layers [179-181]. As a result, it seems that

traditional liposomes have not yet achieved the highest effect as transdermal delivery systems.

Therefore, various attempts have been made to innovate the standard liposomes, such as modifying

their surface with surfactants, optimizing the content of cholesterol into the bilayer and others,

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which have created new generations of liposomes including niosomes, transferosomes, ethosomes,

cubosomes, vesosomes, novasomes and others [182]. These new-generation vesicles of liposomes

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have been gaining a great deal of interest for cosmeceutical applications due to possessing many

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advantages over conventional liposomes (Table 3). Especially, most of them show a higher skin

penetrability through various mechanisms (Figure 8), which is particular importance for
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cosmeceutical applications because of optimization of entrapped AOs activity.
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6.3.1. Transfersomes

Transfersomes, a new generation of liposomes with flexible features, were developed by

adding edge activators or surfactants to liposomes (Figure 9d). Compared with the structure of
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standard liposomes (Figure 9a), transfersomes consist of a surfactant component, called the edge
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activator, which destabilizes the lipid bilayers and renders the vesicles highly flexible [183].

Generally, there are various substances that can be used as edge activators (softening agents),
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including sodium cholate [184], dipotassium glycyrrhizinate [185], and Span 80 [186], Tween 80,
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Tween 20, and oleic acid [187]. The most interesting feature of all transfersomal systems is their

higher flexibility in comparison with conventional liposomes. Due to the combination of

appropriate lipids and surfactants, transfersomes can easily penetrate pores in the SC that are five

times smaller than their own diameters [188]. In addition, the maintenance of the diameters of

transfersomes after passing through small pores also helps them to resist fragmentation.

Elasticity of the lipid bilayers plays a crucial role in the permeation-enhancing effect of

deformable lipid vesicles. It has been reported that surfactants in tranfersomal systems are able to

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change the packing characteristics of the lipids in the liposomal bilayer, which creates a liposomal

system with high flexibility and elasticity. As a result, transfersomes display higher efficiency for

the skin delivery of AOs [189]. Moreover, the addition of permeation enhancers such as oleic acid

in liposomes also improves the skin delivery of AOs. These enhancers intercalate between the

phospholipid bilayers, causing a decrease in phase transition temperature and an increase in the

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fluidity of skin lipids at the same time [190]. However, too high or too low levels of surfactants or

enhancers in liposomes may cause inefficient delivery of AOs through the human epidermis. An

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optimum ratio of lipid and surfactant or enhancer is proposed in order to reach the highest bilayer

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elasticity of liposomal membranes, which allows transfersomes to pass easily through the small

pores of the SC [189, 191]. This means that transfersomes possess the distinguishing feature of
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ultra-deformable vesicles, in a word elasticity, that differentiates it from other conventional
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liposomes or non-elastic lipid disperse systems.

The hydration gradient in the skin plays the main role in providing the needed stress for

deformation of transfersomes, and it has been suggested that these vesicles should be used under
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non-occluded conditions in order to enable easy passage through the skin [178, 192]. It has been
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demonstrated that transfersomes possess superior skin penetration compared with liposomes, both

in vitro and in vivo [193], due to the advantage of their high elasticity in contrast to standard
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liposomes.
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However, one of the main disadvantages of transfersomes is that the incorporation of

hydrophobic antioxidants into the vesicles can cause a highly negative effect on deformability and

the elastic properties [187].

6.3.2. Ethosome

Ethosome, another special type of ultradeformable vesicle first introduced by Touitou et al. in

1997 [96], is a novel formulation of liposomes developed to improve the penetration ability of

liposomes through the skin. is a novel formulation of liposomes developed to improve the

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penetration ability of liposomes through the skin. Ethosomes are basically composed of three main

components: phospholipids, water, and relatively high amounts of ethanol (20–45%) [149] (Figure

9b). It has been reported that it is the presence of ethanol in ethosomal dispersion that significantly

improves the permeability of ethosomes, because ethanol has an important function as an efficient

permeation enhancer [194]. Higher concentration of ethanol increases the elasticity, flexibility and

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steric stability of ethosomes, leading to deeper penetration of the loaded drug through skin with

subsequent high transdermal flux [95, 96]. Thus, it is the presence of ethanol that generates

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ethosomes with a soft structure. In addition, ethanol decreases the phase-transmission temperature

SC
of the SC lipid bilayer, which can increase AO solubility and permeability [195]. Finally, the

combination of ethanol and phospholipids in ethosomal vesicles produces a synergistic effect that
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enhances the distribution and penetration of ethosomes into skin [196]. Therefore, compared with
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conventional liposomes or ethanolic AO solution, ethosomes might be regarded as a potential

vesicle for improvement of the penetration ability of both hydrophilic and hydrophobic antioxidants

into deeper skin [92, 197, 198]. In fact, several recent studies have begun to use ethosomes for
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delivery of AOs in cosmeceutical applications [199, 200].

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In an attempt to enhance the vesicular characteristics and skin permeation of ethosomes, new

generations of ethosomes also have been developed and introduced by adding other compounds to
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the classical ethosomes. These include binary ethosomes and transethosomes.

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Binary ethosomes, which is a novel type of deformable liposome and a new generation of

ethosome, were developed by adding another alcohol to the conventional ethosomes and first

introduced by Zhou et al. (Figure 9b) [201]. The common types of employed alcohols in company

with ethanol in binary ethosomes include propylene glycol (PG) and isopropyl alcohol (IPA) [202-

205]. Due to the combination of ethanol and other alcohols, binary ethosomes exhibit some

distinguishing features compared with classical ethosomes such as (i) higher stability and lower

aggregation [206, 207]; (ii) higher skin permeation; (iii) higher entrapment efficiency of AOs [208].

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This is because the presence of other alcohols such as PG or IPA in the structure of binary

ethosomes produces smaller vesicles, which leads to higher amounts of entrapped AOs. PG

possesses higher viscosity and hygroscopicity than ethanol, which can increase affinity to

substances in the dermis layer, prolonging the movement of vesicles through the skin and enhancing

their accumulation in the deeper skin layers [206, 209]. In addition, the synergic effect of

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phospholipid vesicles and PG enhances the skin penetration of AO-loaded binary ethosomes [209].

Based on the idea of producing a novel elastic liposome for delivery of AOs to the dermis

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layer through the SC barriers, Song et al [210] first introduced another new generation of

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ethosomes, namely transethosomes (Figure 9b). This ethosomal system was produced by combining

the advantages of both tranfersomes and ethosomes. For structure, transethosomes comprise the
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basic components of conventional ethosomes and a penetration enhancer or an edge activator
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(surfactant) (EA) [208]. Surfactants are used in the transethosomal system may be cationic

surfactants, i.e. hexadecyl trimethyl ammonium bromide (CTAB) [211], stearylamine [212]; anionic

surfactants, i.e. cremophor EL-35 [211], sodium stearate [213], deoxycholic acid [212], sodium
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taurocholate and oleic acid [210] or neutral surfactants, i.e. SPACE peptide [214], Tween 80 [210].
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Since transethosomes consist of a high content of ethanol in company with an edge activator or

permeation enhancer, it was desired to provide a significant improvement in the skin permeation of
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AOs. Indeed, it has been reported that transethosomes show an irregular spherical shape and much
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higher values in vesicle elasticity, skin permeability and deposition compared with conventional

liposomes, transfersomes, and classical ethosomes [210]. These can be explained by the

combination of ethanol and EA, which causes a rearrangement in the lipid bilayer of

transethosomes [92]. In addition, due to containing higher amounts of ethanol, these vesicles often

have a smaller size relative to conventional liposomes and transfersomes, and as such, exhibit

higher skin permeability.

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Also, it has been found that transethosomes may improve the stability of encapsulated AOs.

This fact may be due to the presence of edge activators. It is notable that the zeta potential value of

vesicles depends on the nature of surfactants or permeation enhancers [210]. A vesicle with a high

zeta potential value possesses a high electrostatic repulsion to surrounding vesicles, which can

significantly decrease the mutual aggregation and fusion of vesicles and improve the stability of

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transethosomes thereby [211]. Moreover, the presence of surfactants also improves the durability of

vesicles and reduces AO leakage from vesicles [214].

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6.3.3. Niosome

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Niosomes are analogous structures of liposomes that are obtained by self-assembly non-ionic

amphiphiles in aqueous solution, which results in closed bilayer structures [215-217]. The formed
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vesicular system consists of an aqueous interior core containing nonionic surfactants, and it
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constitutes the bilayer of these vesicles (Figure 9c). It was found that, compared with liposomes,

niosomes offer distinguishing features with respect to ease of production, low production cost, high

chemical stability, and storage [215, 218]. Therefore, niosomal vesicles have recently found
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potential applications not only in the pharmaceutical and food industries but also in cosmeceuticals
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[219]. Many studies have demonstrated the effect of niosome vesicles by using niosomes to

encapsulate numerous active agents such as β-carotene [220], gallic acid, quercetin, ascorbic acid,
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α-tocopherol, curcumin, and caffeine [221] for cosmeceuticals products [222, 223], and caffeine
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[221].

In view of the physical properties and preparation methods, the niosomal vesicles have many

parallels with liposomes [224]. In the structure of niosomes, nonionic surfactants known as the most

common type of surface active agent are comprised of both polar and nonpolar segments and

possess high interfacial activity [225]. Compared to anionic, amphoteric or cationic surfactants,

nonionic surfactants are preferred for use in the preparation of vesicles, due to superior benefits

including high stability, compatibility and lesser toxicity [225, 226]. Besides, nonionic surfactants

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also contribute to many interesting functions such as those of solubilizers, emulsifiers, wetting

agents, and permeability enhancers. Therefore, incorporation of non-ionic surfactants into niosomal

vesicles enhances the skin penetration of AOs [227, 228]. This phenomenon is due to the fusion of

the vesicles with the lipid of the SC and the direct transfer of AOs from the vesicle formulations to

the skin [221], which highly enhances the ability of the vesicle to change the structural-barrier

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properties of the skin and allows the AOs encapsulated in vesicles to pass more readily into the

intercellular regions of the SC.

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A variety of surfactants can be used to construct niosomes. They have a hydrophilic head

SC
group and a hydrophobic tail. The hydrophilic head groups include glycerol, ethylene oxide, sugars,

amino acids, polyhydroxy groups, and crown ethers, while the hydrophobic tail often consists of
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one or two alkyl or perfluoroalkyl groups with an alkyl chain length from C12–C18 [229, 230]. The
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most commonly nonionic surfactants used to construct niosomes comprise ester of sorbitan (Spans)

and polyoxyethylene alkyl ether surfactants [231]. The choice of surfactant types has a significant

factor that assigns the nature of the membrane and affects the stability of vesicles. For instance, it
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was found that the leakiness of caffeine-loaded Span surfactant niosomes shows a difference
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between used Span surfactants, following the trend Span 80 < Span 20 < Span 40 < Span 60 [224]

and is determined by the degree of membrane fluidity. To prevent aggregation, cholesterol also is
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incorporated into niosomes as a steric stabilizer [232]. Cholesterol increases the phase transition and
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reduces the leakage of AOs from niosomes.

6.3.4. Cubosomes

Cubosomes, known as the next generation of smart lipid nanoparticles, are formed from the

lipid cubic phase and stabilized by a polymer-based outer corona [233]. Cubosomes comprise

curved bicontinuous lipid bilayers organized in three dimensions (3D) as honeycombed structures

and separated into two congruent networks of water channels for encapsulation of hydrophilic,

amphiphilic, and hydrophobic substances (Figure 9g) [182, 234]. Compared with liposomes,

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cubosomes have a significantly higher membrane surface area for encapsulation of active AOs

[233], hence, their high AO-encapsulation efficiency. Due to their good ability to improve skin

permeability as compared with liposomes, cubosomes have been researched recently as topical

delivery systems [235]. In addition, cubosomes exhibit a better storing stability [236, 237], higher

water-solubility, and lower leakage of encapsulated AOs in comparison with liposomes [238]. These

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days however, the bottleneck in the development of strategies for their applications in

cosmeceuticals remains the lack of fundamental knowledge on their physical properties,

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composition, and interaction with cells [233].

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6.3.5. Bilosomes

Bile salts, physiological surfactants, have attracted huge interest in the drug delivery field for their
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outstanding physicochemical properties and biocompatibility. In fact, they enable improved
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bioavailability of drugs by enhancement of aqueous solubility and skin permeability [239].

Bilosomes, bile-salt-containing vesicles, have been developed based on the basic structure of

niosomes. They are designed by incorporating bile salts as EAs into the basic components of
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niosomes to generate more deformable vesicles than conventional niosomes (Figure 9e) [240, 241],
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which can be squeezed through SC intercellular lipids for better penetration into the deep skin

layers. It has been noted that bilosomes have higher stability than their parent liposomes and
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niosomes as well as more efficiency in transmembrane transport and absorption of drugs [239].
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Similarly to liposomes and niosomes, bilosomes are available for encapsulation and stabilization of

both hydrophilic and lipophilic drug molecules. In recent years, bilosomes have gained a great

attention for their applicability to the development of new carriers for transdermal drug delivery

[241, 242]. Some of the research results confirmed the hypothesized superiority of bilosomes for

enhancing drug flux across the skin as compared with niosomes [242]. This shows the large

potential of bilosome-based delivery of AOs in cosmeceutical applications.

6.3.6. Novasomes

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“Novasomes,” nonphospholipid paucilamellar vesicles, are one of the new innovations of liposomes

that have tackled problems related to conventional liposomes [243, 244]. The Novasome technology

was initially developed and patented by Novavax. IGI laboratories, Inc [245, 246]. These days,

Universal Cosmetics has applied Novasome delivery technology to the production of

cosmeceuticals products for the market, due to striking features such as enhanced product stability,

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controlled release, non-irritation, skin-deep penetration, inexpensiveness, low cost and easy mass-

production [209]. The Noversomes are made by Micro Vesicular Systems, and are assembled using

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a series of membrane "modules," each one imparting desired characteristics to the Novasome

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vesicles [245]. The Noversomes are made by Micro Vesicular Systems which are assembled using a

series of membrane "modules", each module imparting desired characteristics to the Novasome
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vesicles [244]. As for the structure of Novasomes, they are formed by combination of numerous
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amphiphiles such as fatty alcohols and acids with each other, or with phospholipids. Unlike

conventional liposomes, Novasome systems have very high stability and high density per unit

volume at room temperature, which enable them to encapsulate high levels of AOs in the core with
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better effectiveness and efficiency than other vesicles. Moreover, Novasomes can be applied in a
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wide range of pH without cytotoxicity [246]. Especially, Novasomes are easily integrated into the

formulation process of cosmeceuticals products [245]. Novasomes are among the most promising
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technologies for more effective and efficient delivery of cosmeceuticals products’ AOs to the skin.
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6.3.7. Vesosome

Besides multifunctional strategies, an advanced liposomal system containing multiple

compartments, namely vesosomes, also has been developed recently. In vesosomes, liposomes will

encapsulate smaller liposomes of various sizes [247] (Figure 9f). Compared with standard

liposomes, vesosomes provide double protection to encapsulated AOs due to possessing that

exceptional structure whereby larger liposomes enclose smaller ones. In addition,

multicompartmental systems of vesosomes offer a favorable condition for cocktails of AOs, in

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which various AOs can be simultaneously encapsulated in a vesosome molecule. Moreover,

compared with single-compartment liposomes, the release ability of AOs from vesosomes is likely

to more effective as well as more easily tuned. Therefore, vesosomal systems will be a potential

approach to enhance the stability of entrapped AOs for cosmeceutical applications in the present

and near future.

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6.4. Development of advanced materials for delivery of AOs in cosmeceuticals
The development of advanced materials to resolve the current limitations of liposomes for delivery

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of AOs in cosmeceutical applications can be divided into two approaches: refinement of existing

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constructs and envisioning of new designs.

6.4.1. Development of advanced liposome-based materials

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Liposomal structures with their many outstanding features have been individually proven as ideal

AOs-delivery systems, notwithstanding the remaining several challenges relating to the reduction of
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their protective functions over time, their skin penetrability and their preparation techniques.

Currently, liposomes are the basis of the carrier systems most widely employed for cosmeceutical
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formulations [122]. Therefore, the design of advanced materials that are based on the foundation of
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the liposome but that, in combination with another agent, can minimize or eliminate liposomes’
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drawbacks, has been a very high priority. With this motivation, Hu et al. recently introduced a novel
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nanoparticle called a nanocomposite structure, which is based on a combination of three existing

motifs into a single construct (Figure 10a) [251]. In this nanocomposite, a liposome is encapsulated
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and tethered with various polymers as amphiphilic linker molecules to a solid spherical core. As for

the chemical structure of a linker, it comprises three main components: a headgroup covalently

binding with the core, a hydrophilic polymer spacer of low dispersity and adjustable length, and a

lipid-like hydrophobic anchor that enables insertion into the lipid vesicle. It has been demonstrated

that such a nanocomposite structure exhibits several attractive properties that can benefit the

development of carrier system of AOs in cosmeceuticals, including: (i) the structure considerably

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enhances the stability of liposomes against a variety of external stresses (due to tethering of the

lipid vesicle to a solid core) while preserving the integrity of the liposomal structure; (ii) the

interplay of polymer length with grafting density provides strong constraints on particle size and

thus forms nanoparticles of high uniformity; (iii) the numerous functionalities of the formed

nanoparticles due to the combination of the outstanding properties of the individual constituents

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make the construct more suitable for various applications in the field of cosmeceuticals.

An emerging concept in the development of strategies for formulation of advanced liposomes

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is the design of hybrid liposomes that incorporate functional nanoparticles to improve the

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controllable release of encapsulated AOs in liposomes when reaching a target site [252]. Generally,

this release of AOs from carriers at a specific time and location can be achieved by adding a
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stimulus [253]. Recently, the decoration of liposomes with bound nanoparticles has been regarded
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as a potential strategy for controllable release of AOs from liposomal systems by provision of

stimuli such as heating, changed pH, UV-vis light, or triggering agents to the bilayer [253, 254].

Magnetoliposomes (MLs) is one of the multifunctional hybrid types of liposome/nanoparticle

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assembly that have received great attention in a wide variety of fields, including cosmeceuticals.
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Various approaches to develop and design MLs have been reported; however, the greatest focus has

been on the superparamagnetic iron oxide (SPIO) nanoparticle (i) incorporated into the
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phospholipid bilayer structure, or (ii) encapsulated within the aqueous core of liposomes [253-257].
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For example, to improve the responsiveness and release of MLs by heating of embedded

nanoparticles, Chen et al. [254] and Shaghasemi et al. [257] developed a novel ML incorporating

small hydrophobic SPIO nanoparticles, namely maghemite (Fe2O3) capped with oleic acid (OA),

into lipid bilayers (Figure 10b). To release encapsulated AOs, the local temperature around the

SPION is increased by means of an alternating magnetic field, which leads to line defects that cause

a local phase separation and release of AOs (the drug).

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Liposomes are well-known as a passively targeted drug carrier. To innovate the drug release

of liposomes in terms of an active targeting modality, many recent studies have reported a novel

liposome-based material, namely eLiposome, that takes advantages of the passive release of

liposomes along with the notable strengths of using ultrasound [258, 259]. The eLiposome is

defined as a liposome that encapsulates an emulsion droplet within its aqueous core (Figure 10c)

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[259]. The preparation process of eLiposomes includes two steps: (i) formation and unfolding of

SUV liposomes by addition and removal of ethanol to generate stable sheets at temperatures below

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the Tm of the lipids; (ii) combination of the sheets and a suspension of nanoemulsions followed by

SC
entrapment of the nanoemulsions inside the liposomes. Ultrasound has been widely applied as a

common method to actively release entrapped drugs in liposomes to specific sites of interest, thanks
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to its efficiency due to the presence of gas bubbles in the aqueous compartment [260, 261]. The gas
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bubbles can increase the volume inside the vesicle, disrupt the vesicle structure, and, thereby,

release the drug. In addition, a great advantage of using ultrasound is the easy energy transmittance

across the skin and the controllability of the timing and rate of drug release. Therefore, eLiposome
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is a promising material that can create, for cosmeceutical applications, stable liposomes with easy
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ultrasound-enabled activated release.

In the last several years, mixed nanocarrier systems developed based on a combination of
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liposomes and another material have gained significant attention with a wide range of applications
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in the delivery and controlled release of cosmeceutical ingredients [262]. Carbon nanotubes (CNTs),

a class of rolled graphene including their two main types (single-walled CNTs (SWCNTs) and

multi-walled CNTs (MWCNTs)) offer many unique electrical, thermal, mechanical, and optical

properties that make them a potential material for numerous applications [122, 263-265]. CNTs

have been applied in various cosmeceutical products, including hair coloring and cosmetic

compositions [122]. Recently, the combination of liposomes and CNTs has attracted great attention

for development of drug-delivery platforms in the pharmaceutical filed. For illustration, innovative

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delivery platforms for active ingredients made of liposomes and MWCNTs [266] or SWCNTs [267]

have recently been designed based on strong interactions between polar groups of phospholipids

and the functionalized CNTs. It was demonstrated that the presence of CNTs impacted the surface

charge, size, and size-distribution of the liposomes. Notably, these systems have shown a high

suitability for entrapment of active pharmaceutical ingredients, due specifically to their low toxicity

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even at high concentrations. In addition, these platforms also offer controlled release of the

properties of encapsulated active pharmaceuticals to target sites. Although CNT-liposome materials

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have not been widely applied in the cosmeceutical field, it is predicted that they will be a potential

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material and provide many additional options in the formulation of liposome-development

strategies for cosmeceutical applications, due especially to their low toxicity and controllable
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release.
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6.4.2. Development of novel carriers as alternatives to conventional liposomes

These days, carrier technology has offered an intelligent approach to the development of the

delivery platforms of active ingredients in cosmeceuticals. Following the rapid takeoff of

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nanotechnology, a large range and variety of nanocarriers with potential applications for delivery of
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cosmeceuticals have been successfully developed, such as polymersomes, advanced lipid

nanoparticles (solid lipid nanoparticles, SLNs and nanostructured lipid carriers, NLCs), emulsions,
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polymeric micelles, dendrimers, silica nanoparticles, carbon nanotubes, and so on [122]. According
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to our knowledge, however, there are only several novel carriers that have the possibility to be

regarded as advanced carriers that can actually be utilized as alternatives to conventional liposomes.

They are discussed in the following paragraphs.

Advanced lipid nanoparticles

Advanced lipid nanoparticles generally include two classes, solid-lipid nanoparticles (SLNs) and

nanostructured lipid carriers (NLCs), both of which have been widely utilized in the cosmetic and

pharmaceutical industries. Whereas SLNs, designed and developed by Stefan Lucks and Rainer

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Muller at the beginning of the 1990s, are a first-generation lipid nanoparticle technology, NLCs are

well-known as a second-generation lipid nanoparticle technology [6, 268-270]. Due to taking the

advantages and avoiding the disadvantages of polymeric nanoparticles, liposomes and emulsions,

SLNs and NLCs have been considered as potential alternatives to colloidal drug-carrier systems,

especially liposomes and emulsions [271, 272]. Lipid nanoparticles have afforded many cosmetic

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benefits including improvement of the chemical stability of AOs, film formation, enhanced skin

bioavailability, controlled occlusion, skin hydration, and so on [268]. Generally, SLNs are prepared

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by using a solid lipid or a blend of solid lipids to replace a liquid lipid of an o/w emulsion at room

SC
temperature (Figure 11a). SLNs represent a combination of the advantages of solid particles,

emulsions and liposomes [273, 274]. Compared with liposomes, SLNs possesses several
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advantages, such as greater flexibility in the control of the release of encapsulated AOs, better
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protection of entrapped AOs against stress conditions, easy large-scale production and sterilization,

avoidance of organic solvents, negligible skin irritation, and higher percutaneous absorption and AO

accumulation in the skin. As for the second generation of advanced lipid nanoparticles, NLC
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particles are generated by producing blends of liquid lipids (oils) and solid lipids (Figure 11a).
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These blends are obtained by mixing solid lipids with liquid lipids (oils) in ratios of 70:30 up to

99.9:0.1. Especially, NLCs have been developed to modulate the structure and overcome the several
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disadvantages of SLNs. Compared with SLNs, NLCs can be considered to be an upgrade, due to
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possessing more advantages, including higher loading capacity for numerous active AOs, a lower

amount of water in the particle suspension, and minimized potential expulsion of particles during

storage [271]. It is clear that both SLNs and NLCs are advanced core-shell materials that offer

many distinguishing features to overcome the obstacles and limitations of liposomal systems.

Therefore, it is predicted that advanced lipid particles will be one of the most effective alternative

materials to liposomes in cosmeceutical applications.

Colloidosome

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Colloidosomes, templated from Pickering emulsions, are well-known as hollow microcapsules the

shell materials of which are composed of colloid particles [275, 276]. It has been indicated that

these systems exhibit a huge potential for development of microencapsulation materials for delivery

of various active ingredients in a large range of pharmacy, food, personal care, and cosmetics

applications [277, 278]. Generally, colloidosomes are produced by stabilizing emulsions using

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colloidal particles instead of surfactants [278, 279]. In colloidosomal systems, amphiphilic particles

with both oil and aqueous phases are entrapped at the oil/ water or water/oil interface and are closed

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together to create a solid shell. It is notable that these special particles can be used as solid

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surfactants that originate from both inorganic and organic colloidal particles such as silica,

polymers, metal oxides, polysaccharides, and carbon nanotubes [278, 280, 281]. In addition, it was
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indicated that the most outstanding property of the colloidosomal systems is the adjustability of
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their size, mechanical strength, shell structure, and permeability by changing of the preparation

conditions (i.e. solvent, type of colloidal particles, locking methods of colloidal particles, etc) [282,
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283].
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Similarly to liposomes, colloidosomes also can potentially encapsulate both hydrophilic AOs
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and hydrophobic AOs but with higher flexibility and encapsulation efficicency. For illustration, a

novel type of colloidosome, called the all-silica colloidosome, was recently reported for
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microencapsulation of AO-containing hydrophobic liquids for controllable size, structural

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properties, and permeability [276, 281]. The hydophobic liquids are encapsulated in colloidosomes

with almost 100% efficiency through the formation of O/W Pickering emulsions that are stabilized

by amphiphilic silica nanoparticles, based on a silica precursor polymer-hyperbranched

polyethoxysiloxane (PEOS), at the W/O interface (Figure 11b) [276]. Another type of colloidosome

with an aqueous core has also been developed to encapsulate hydrophilic active agents [284]. This

colloidosome is formed by using CaCO3, formed by precipitation between an outer phase of CaCl2

and an inner phase of Na2CO3, to seal a polymer latex shell (Figure 11c). It was demonstrated that

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colloidosomes, with their high activity, can retain the stability of small active molecules for several

months [284]. In addition, this approach provides a promising strategy for fabricating microcapsules

of both hydrophobic and hydrophilic substances with high mechanical stability, biocompatability,

controllable release, and high encapsulation capacity [276]. Therefore, with their special properties,

colloidalsomes should be considered as a effective alternative to liposomes in cosmeceutical

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applications.

Polymersome

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Polymersomes, well-known as synthetic mimic types of liposomes, are constructed by amphiphilic

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block copolymers comprising and enclosing an aqueous lumen; thus, they can encapsulate both

hydrophobic and hydrophilic cargo by means of a mechanism similar to that of liposomes (Figure
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11d) [285-288]. Generally, polymersomes can be prepared by techniques common to liposomes,
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including film rehydration and solvent displacement techniques [285]. Relative to liposomes,

polymersomes possess enhanced variability and higher physical and chemical stability, thanks to
D

the unique properties of the polymer matrix [286, 289-291]. The membrane thickness of
E

polymersome membranes (>5 nm) is often higher than those of liposomes (4-5 nm), due to the high
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molecular weights of block copolymers [288], which leads to better stability and mechanical

strength in comparison with liposomes. In addition, because they are comprised of block
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copolymers, polymersomes can respond and modulate to external stimuli such as oxidative species,
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pH, and enzymatic degradation [285]. Recently, polymersomes because they have been regarded as

models for biological membranes as well as versatile structures, have been the focus of significant

attention for the purposes of several practical applications. Moreover, it has been indicated that

polymersomes are excellent materials in a large range of environmantal, medical and

pharmaceutical fields, due to their numerous distinguishing features such as high mechanical

stability, encapsulation of both hydrophilic and hydrophobic active ingredients, and inhibition of

various external stimuli [291]. We believe that they deserve to be considered as a component of

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potential alternative development strategies for advanced materials utilized in cosmeceutical

applications.

7. Conclusion and perspectives

Due to possessing high antioxidant activity, AOs are among the crucial candidate ingredients in the

development and production of cosmeceuticals. Unfortunately, several problems relating to

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instability and insolubility have limited their practical application, which dilemma has led to the use

of liposomes as a carrier system to encapsulate and deliver AOs for cosmeceutical applications.

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Liposomal vesicles have played important roles and made huge contributions in the enhancement of

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AOs’ bioactivity as well as the expansion of their applicability within the cosmeceutical industry.

However, it has been found that liposomes face several challenges to their application. In this
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review, we focused on discussing the major problems of liposomes that affect their development for
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AO delivery. Chemical instability and low skin permeability are two important disadvantages of

liposomal vesicles, besides the problems related to fabrication techniques and encapsulation

efficiency.
E D

Also, we underlined recently reported development strategies for resolution of liposomes’

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drawbacks. Each strategy has both advantages and disadvantages, but all of them have been meeting

the development needs of the cosmeceuticals industry in the present, and keep on growing at least
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the near future. All of the strategies relevant to the development of liposomes in cosmeceutical
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applications are summarized (Figure 12). Recently, there are four main approaches to develop and

extend applications of liposomes in the cosmeceutical field. The first approach aims to enhance the

stability of the liposomal structure by strengthening the surface charge using polymer coating,

encapsulating liposome particles within other carriers, or developing new cosmetic formulations.

This type of strategy has been widely applied in both research and practical applications due to its

simplicity and efficiency. The second approach focuses on researching and developing novel

techniques that provide the possibility for scale-up in industrial production and improvement of the

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quality of formed liposomes. By the third approach, new generations of liposomes for AO delivery

and development of cosmeceutical products in practice have been continuously discovered and

introduced with numerous outstanding features: high skin penetrability, flexibility, elasticity, and

deformability. In the fourth and final approach, advanced carriers with enhanced properties relative

to liposomes have been developed as alternatives to liposomes in cosmeceutical applications.

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However, whereas these new carriers can, indeed, be regarded as ideal alternatives to liposomes,

more research on their potential toxicity to humans is necessary.

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We predict that these four groups of development strategies will be continuously developed to

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adapt the higher requirements of the advancement of the cosmeceutical industry. Among them, it is

believed that in the future, many more advanced liposomes such as aquasomes, ufasomes,
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sphingosomes, lipospheres, archaesomes, dendrosomes, cryptosomes and others will be
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continuously introduced and used for delivery of AOs in cosmeceutical products. Besides, the

combination of the technical methods such as ethanol injection, membrane contactors, microfluidic

machines, and electrosprays will be potential approaches for production of both conventional and
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advanced liposomes in the industrial scale.

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Acknowledgements

This work was supported by the Basic Science Research Program through the National
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Research Foundation of Korea funded by the Ministry of Education (NRF-

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2017R1D1A1A09000642).

Conflict of Interest

The authors declare no conflicts of interest.

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Table Captions

Table 1. Summary of applications of liposomes for encapsulation and stabilization of AO

Table 2. Comparison of advantages and disadvantages of liposomal preparation techniques
Table 3. Vesicle architecture of new generations of liposomes and their specific features over
liposomes

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Table 1.
Anti-oxidants Preparation method Types Efficiency Size EE (%) ξ-potential Refs
(nm) (mV)
Conventional liposomes
[58]
T
Tea polyphenol Ethanol injection method SUVs Enhances stability and solubility 66.8 78.5 (-) 6.16
with dynamic high-pressure
microfluidization I P
Thin-film and ultrasonic LUVs
C R160.4 60.09 (-) 67.2 [9]

Vitamin E
dispersion method
Ethanol injection and SUVs
U S
Enhances stability 144 99 (+) 53 [10]

Tea polyphenol
sonication method
Reverse-phase evaporation LUVs
A N
Sustains transdermal penetration 202.8 TP: 50.81 (-) 47.5 [60]
(TP) and method
M
and improves solubility VE: 94.05
Vitamin E (VE)
Retinol Thin-film method SUVs
E D
Improves solubility and stability 98 99 - [59]

Vitamin C Ethanol injection and

EPT SUVs Enhances stability
-
95.8
94.5
>90 -
[292]
[45]
sonication method

C C
Arbutin

Curcumin
Thin-film method
and sonication
pH-driven
A LUVs

LUVs
Enhances skin deposition and
skin-whitening activity
Improves water-solubility, stability
179-212

217.5
17.63

62.8
-

(-) 53.1
[293]

[61-64]
Thin-film method MLVs and bioavailability 453.3 78.6 (-) 54
Ethanol injection method SUVs 115.1 46.6 (-) 52.9
Extrusion technique SUVs 93-112 > 85 (-) 3.5 – 2.1

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Epigallocatechin Ethanol injection and SUVs Enhances stability and 71.7 92.1 (-) 10.81 [294]
gallate dynamic high-pressure bioavailability
microfluidization
Carotenoids Thin-film method SUVs Improves antioxidant activity and 66 – 80 80 - [38, 52]
(lycopene, β- LUVs water-solubility 140-95
carotene, lutein)

P T
Coenzyme Q10 Ethanol injection and
sonication techniques
SUVs Enhances stability, solubility and
skin penetration
R I68 95 - [65]

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Catechins Thin-film and sonication SUVs, LUVs Highly enhances effect on 135 - 268 38 - 75 (-) 66 - (-)7.2 [295]

Tretinoin
method
Thin-film and sonication UVs
N U
intratumor distribution
Enhances skin penetration 135-293 70-97 - [71]
method
Thin-film method
MLVs

M A 536-598 75-93 -

Surface-modified liposomes
Curcumin Thin-film method
E
SUVs coating CS D Enhances skin penetration, 221 - (+) 60.9 [64]

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Thin-film method SUV conjugating stability, solubility 128 >90 (+) 29.86 [76]

C E
with carbon dot,
coating peptides
Thin-film and

A C
homogenization method
SUV coating
silica
157 90.62 - [296]

Vitamin C Thin-film method SUVs coating Enhances stability 129 48.3 (-) 35 [73]
pectin
Ethanol injection and SUVs coating CS 95-97 96.5 - [45]
sonication
Thin-film and SUVs coating CS Enhances stability 297 - (-) 15 [106]

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microfluidization methods and alginate
Vitamin E Ethanol injection and SUVs coating CS Enhances stability, solubility 87.5 99 (-) 29.5 [10]
sonication
New generation of liposome
Curcumin Thin-film method and Transfersome Enhances permeability, stability 200 85 (-) 30 [297]
sonication

P T
Vitamin E Ultrasonication method Transfersome Enhances antioxidant activity, water
solubility, permeability
R I
76 - 87 72 - 90 (-) 78 – (-)86 [298]

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Caffeine Thin-film method Transfersome Enhances permeability, stability - 48.82 - [299]
Curcumin Thin-film method and Ethosome Enhances permeability, stability 228.8 81.2 (-) 12 – (-) 29 [199]
sonication
N U
Green tea Thin-film method and
sonication
Ethosome

M A
Enhances permeability, stability 129 - 232 33 - 54 (-) 60 – (-) 66 [200]

[95]
D
Rutin Thin-film method Ethosome Enhances water solubility and 189-225 65 -

Apigenin Sonication method

T E
Binary ethosome
permeability
Enhances water solubility and 36 61-85 (+) 10 - 27 [206]

Hyaluronic acid Thin-film method

E P
Transethosome
permeability, stability
Enhances skin penetration 110 - (-) 40 [214]
Curcumin Thin-film method

C C Binary ethosome Enhances skin penetration 182 92.74 - [300]

Vitamin D3

Curcumin
method A
Supercritical carbon dioxide

Thin-film method and

Niosome

Niosome
Enhances stability

Enhances skin permeability

1,400

82-92
95.9

80-82
(-) 57

(-) 10 – (-)12
[301]

[302]
sonication
SUVs: small unilamellar vesicles; LUVs: large unilamellar vesicles; MLVs: multilamellar vesicles; CS: Chitosan; EE: encapsulation efficiency

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Table 2.

Preparation Advantages Disadvantages Refs

techniques

Conventional techniques

Thin-film - Simple operation - Difficulty to scale up; [37,

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method - Low encapsulation efficiency; 147]

- Large particle-size

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Ethanol - Possibility for large-scale - Difficulty of completely [87-89]

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injection production; ethanol removal;

- Easy handling with minimal - Low encapsulation efficiency

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technical requirements for hydrophilic AOs
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Advanced techniques

Microfluidic- - High encapsulation efficiency; - Requirement of equipment [154,

159,
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based methods - Large-scale production; 164,

165]
- Controllable sizes and uniform
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phospholipid vesicles

Membrane - Directly scaled-up approaches; - Requirement of equipment [166,

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167]
contactor - A simple and fast method for large-
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scale production;

- High unity and controlled size;

- High encapsulation efficiency

Electrospray - Continuous method for producing - Requirement of equipment [171,

liposomes on a large scale; 176]

- Formed liposomes are stable;

- High encapsulation efficiency

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Table 3.

Types Structure and components Advantages Disadvantages Refs

Niosome - Vesicles composed of non- - Penetrate through the - Toxicity due [182,
ionic surfactants, amphipathic deeper skin layers; to the use of 225,
compounds with an overall - Chemical stability; esters 226,
neutral charge; 248,

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- Easy fabrication with
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- Major components: higher purity over
surfactants, cholesterol, charge liposomes;

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inducers, and water
- Mass production and

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Transfersome - Transfersomes consist of - High flexibility, high - Chemical [183,

double chain lipids and an elasticity, instability; 249,
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edge activator; deformability; 250]

- Incorporation
- Phospholipid, cholesterol, - Biodegradable and of hydrophobic
edge activator , water biocompatibility; antioxidants
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- High encapsulation affects their

elasticity
efficiency
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Ethosome - Structure: a vesicular system - Penetrate through the - Low yield, [95,
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manufactured with deeper skin layers; non-economic 149,

phospholipid and high - High deformability; effect; 199,
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concentrations of alcohol; 249]

- High encapsulation
- Phospholipid, cholesterol,
efficiency
water, ethanol;

Vesosome Liposomes encapsulate - A double protection Vesicles with [247]

smaller liposomes of various to encapsulated AOs; large size
size - High encapsulation

efficiency.

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Cubosome Curved bicontinuous lipid - High AOs - Difficulty in [182,

bilayers organized in three encapsulation large scale 233-
dimensions as honeycombed efficiency; production; 238]
structures - High stability and - High
skin permeability. viscosity

Bilosomes Incorporation of bile salts to - Higher penetrability - [239-

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basic components of niosomes through the deeper skin 242]
layers than niosomes;

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- High stability.

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Novasomes Combination of amphiphiles High stability, - [243-
with each other (variety of controlled release, non- 246]
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fatty alcohols and acids), or irritating, skin deep
with phospholipids penetration,
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inexpensive, low cost

and easy mass-
production
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Figure Captions

Figure 1. (a) ROS scavenging mechanism of antioxidants; (b) Oxidative stress and related skin

problems. Modified and adapted from ref. [21]

Figure 2. Schematic drawing of (a) liposomes structure and lipophilic or hydrophilic antioxidant

entrapment models (b) Different liposome types based on size and lamellarity. Reprinted with

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permission from ref [39]. Copyright 2015, American Chemical Society

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Figure 3. Schematic representation of (a) active loading of a hydrophilic AO in liposomes.

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Modified and adapted from ref [48]; (b) Localization and orientation of carotenoid pigments in egg-

yolk phosphatidylcholine membrane. Reprinted with permission from ref [52]. Copyright 2014,
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American Chemical Society.

Figure 4. Schematic presentation of (a) Possible antioxidant permeation pathways through intact
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stratum corneum; transcellular or tortuous intercellular pathways. Modified and adapted from ref.

[70]; (b) possible mechanism for transdermal delivery of curcumin loaded liposomes and free
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curcumin. Reprinted with permission from ref [76]. Copyright 2016, Royal Society of Chemistry.
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Figure 5. (a) Schematic illustration of curcumin-loaded nanoliposome and chitosan-coated

curcumin-loaded nanoliposome. Reprinted with permission from ref. [64]. Copyright 2016, Royal
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Society of Chemistry; (b) Polyelectrolyte delivery system by layer-by-layer self-assembly of

chitosan and alginate onto nanoliposome. Reprinted with permission from ref [112]. Copyright
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2013, American Chemical Society; (c) Scheme of synthesis of silica-coated liposomes loaded with

APTES – (3 aminopropyl)triethoxysilane. Reprinted with permission from ref [117]. Copyright

2015, Royal Society of Chemistry; (d) Destabilization of carboxyl-modified gold nanoparticle (Au-

COOH)-stabilized liposomes in term of pH. Reprinted with permission from ref [120]. Copyright

2010, American Chemical Society; (e) Peptide coated liposome. Reprinted with permission from ref

[123]. Copyright 2014, American Chemical Society.

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Figure 6. (a) Schematic illustration of hydrogel containing gold nanoparticle-stabilized liposomes.

Reprinted with permission from ref. [136]. Copyright 2014, American Chemical Society; (b)

Hybrid hydrogel. Reprinted with permission from ref. [137]. Copyright 2012, Wiley-VCH; (c)

Scheme illustration of the double-encapsulation system of liposomes in W1/O/W2 double emulsion.

Modified and adapted from Ref. [138]; (d) Detailed preparation procedure for LIM. Modified and

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adapted from ref. [143].

Figure 7. (a) Representation of ethanol injection procedure. Reprinted with permission from ref

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[153]. Copyright 2016, Nature; (b) a three-dimensional-microfluidic hydrodynamic focusing (3D-

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MHF) liposome formation device. Reprinted with permission from ref [162]. Copyright 2014,

Royal Society of Chemistry; (c) preparation of liposomal vesicles using double emulsion templates;
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(d) formation of liposome based W/O/W double emulsion in glass microcapillary device. c),d)
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Reprinted with permission from ref. [154]. Copyright 2008, American Chemical Society; (e)

Experimental set-up for the membrane contactor method. Reprinted with permission from ref.

[170]. Copyright 2019, Elsevier; (f) Schematics for remotely loading a weak-base drug into a
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liposome: D stands for drug. D-N symbolizes drug that is a weak base with an amine group. D-NH+
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is the protonated form of the drug. D-NH2SO4 is the sulfate salt precipitate that may be formed with

the amine portion of the drug; (g) Electrospray remote-loading setup for producing liposomes.
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Reprinted with permission from ref. [171]. Copyright 2016, American Chemical Society.
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Figure 8. Schematic representation of the main permeation mechanisms of lipid-based vesicles

Figure 9. Structural illustration of (a) Conventional liposome (b) ethosomes, binary ethosomes,

transethosomes. Adapted from ref [208]; (c) noisome; (d) transfersome; (e) bilosome. Adapted from

ref [239]; (f) vesosme. Adapted from ref [247]; (g) a 3D network of typical cubosomes. Reprinted

with permission from ref [182]. Copyright 2017, American Chemical Society

Figure 10. (a) Proposed nanocomposite structure: nanoparticle core (blue sphere in center);

polymers (green) are tethered that terminate in lipid molecule (cyan head and green tail), which in

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turn anchors the enclosing bilayer of lipids (blue heads and yellow tails). Reprinted with a

permission Ref. [251]. Copyright 2015, American Chemical Society; (b) Drug-magnetoliposome-

association constructs. Reprinted with a permission Ref. [257]. Copyright 2017, Nature; (c)

Illustration of the ideal eLiposome. Reprinted with a permission Ref. [170], Copyright 2019,

Elsevier.

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Figure 11. (a) Structures of solid lipid nanoparticles (SLNs) and nanostructured lipid carriers; (b)

Schematic illustration of formation process of silica colloidosomes templated by o/w Pickering

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emulsions and chemical structure of hyperbranched polyethoxysiloxane (PEOS). Reprinted with a

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permission Ref. [276]. Copyright 2014, American Chemical Society; (c) Schematic of method used

to encapsulate enzymes in CaCO3 sealed colloidosomes. Reprinted with a permission Ref. [284].
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Copyright 2014, American Chemical Society; (d) Schematic of polymersome. Reprinted with a
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permission Ref. [287]. Copyright 2009, Royal Society of Chemistry.

Figure 12. Schematic summary of liposome development strategies for cosmeceutical applications.
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Figure 1.

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Figure 3.
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Figure 4.

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Figure 7.

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Graphical abstract:

Highlights:

 Classification of antioxidants and their action mechanism in scavenging ROS.

 Benefits of liposome for antioxidant delivery in cosmeceutical applications.
 Challenges to expansion of liposomal applications in cosmeceuticals.
 Development strategies of novel techniques and advanced liposome-based carriers.

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 New generations of liposome for delivery of antioxidants in cosmeceuticals.

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