Development, Characterization and Use of Liposomes as Amphipathic Transporters of Bioactive Compounds for Melanoma Treatment and Reduction of Skin Inflammation: A Review

Figures & data

Figure 1 Skin microstructure and skin penetration mechanisms of nanoparticles. (A) Penetration trough sebaceous glands and hair follicles. (B) Transcellular penetration. (C) Intracellular penetration.

Figure 2 Melanoma stages.

Notes: Reprinted with permission from patient resource LLC. ©2020 Patient Resource LLC.¹⁴

Figure 3 (A) Schematic representation of a liposome. (B) Classification by vesicle number.

Abbreviations: MLV, multilamellar vesicle; MVV, multi-vesicular vesicle; GUV, giant unilamellar vesicle; LUV, large unilamellar vesicles; SUV, small unilamellar vesicles.

Figure 4 Main components of liposomes. (A) Phospholipids (nomenclature=phosphatidyl + choline, + ethanolamine, + glycerol, + inositol, + serine) the molecular weight of the lipids will change depending on the R groups; (B) Chemical structure of cholesterol.

Abbreviation: CPP, critical packing parameter.

Table 1 Physical Properties of Liposomes with Different Compounds and Lipid Ratio After Being Prepared with Thin-Film Hydration

Bilayer Compounds (Best Conditions)	Ratio	Size Reduction Method	Particle Size (nm)	EE%	Zeta Potential	Encapsulated Compound	Generalities	Characterization Methods	Ref
DSPC:Chol	55:45	Extrusion	117.8±2.7 to 143.4±11.4	0 to 49.9±2.1	−13.6±1.4 to −1.0±0.4	5-Fluorouracil, Cetuximab	Drug loaded: Drug-to-lipid ratio at 0.1 and 0.2	DSL (Particle size, Zeta potential, and PDI) EE	[^Citation13]
DPPC	100	Extrusion	108.0±2.0 to 133.0±28.0	31.1±16.9 to 43.0±5.6	−16.7±5.7 to 4.3±1.7	Curcumin	Curcumin loaded: 0.1–15% w/w	DLS (Particle size, Zeta potential, and PDI) Liposome deformability trough extrusion	[^Citation87]
SL:P80	1:48	Extrusion	86.0±1.0 to 90.2±1.6	88.7±0.2 to 92.72±0.1	−33.9±1.4 to −27.7±11	Naringin	Naringin loaded: 3, 6 y 9 mg/mL	DSL (Particle size, Zeta potential, and PDI) EE TEM (negative staining)	[^Citation97]
DPPC:SC,T80 or S80	max 84.5:15.5	Sonication/extrusion	127.0±7.8136.0±10.0	Max 99		Oestradiol	Oestradiol loaded: 1 mg/mL	DSL (Particle size, Zeta potential, and PDI) EE	[^Citation3]
PC:T80 or SC	70:30 and 80:20	Sonication/extrusion	81.6±6.5 to 102.6±3.5	30.6±1.2 to 37.4±1.1	−22.1±2.3 to −11.7±1.2	Methotrexate	Drug loaded: Drug-to-lipid ratio at 1.5 w/w	DSL (Particle size, Zeta potential, and PDI) EE TEM (negative staining) Liposome deformability trough extrusion	[^Citation25]
SPC:SC	86:14	Sonication/extrusion	167.0±6.0 to 191.0±4.0	0.8±0.8 to 22.7±0.63	−58.0±6.0 to −32.0±6.0	Pirfenidone	Drug loaded: 5 mg/mL	DSL (Particle size, Zeta potential, and PDI) EE Stability for a period of 4 weeks	[^Citation86]
DPPC:MPPC: SC	80:10:10 75:15:15	Sonication/extrusion	141.0±20.0 to 190.0±70.0	91.9±1.9 to 93.2±1.2	−6.1±21.5 to 14.3±.3.8	Tamoxifen	Drug loaded: 2 mg/mL	DSL (Particle size,Zeta potential, and PDI) EE FTIR (Interaction of the drugs with the liposomal system)	[^Citation108]
DPPC:MPPC: SM	70:15:15	Sonication/extrusion	156.0±4.0 to 188.0±6.0	54.6±2.7 to 73.2±1.5	12.3±1.1 to 14.7±0.8	Imatinib mesylate	Drug loaded: 2–3 mg/mL
DPPC:MPPC: SM	70:15:15	Sonication/extrusion	168.0±7.0 to 180.0±4.0	Tamoxifen: 92.8±1.5 Imatinib: 73.8±4.4	14.5±1.2 to 16.7±3.6	Tamoxifen: Imatinib mesylate	Drug loaded: 2–3 mg/mL each

Abbreviations: EE, encapsulation efficiency; Chol, cholesterol; DLS, dynamic light scattering; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; FTIR, Fourier transform infrared; MPPC, monopalmitoyl-2-hydroxy-sn-glycero-3-phosphocholine; P80, Polysorbate 80; PDI, Polydispersity Index; PC, phosphatidylcholine; SL, soybean lecithin; SPC, soy phosphatidylcholine; S80, Span 80; SC, sodium cholate; SM, sorbitan monooleate; T80, Tween 80; TEM, transmission electron microscopy; YL, yolk lecithin; w/w, weight/weight.

Figure 5 Fluorescent images of Nile red stained giant unilamellar liposomes obtained by confocal laser scanning microscopy (multiphoton confocal microscope LSM 710 NLO, Carl Zeiss.

Table 2 Liposomal Formulations for Skin Delivery of Different Compounds

Type	Encapsulated Compound	Aim	Outcomes	References
UDL	Oestradiol Concentration: 1 mg/mL	Evaluate Span 80 and Tween 20 as edge activators against Sodium Cholate for skin delivery in a human epidermal cadaver membrane.	Span 80 and Tween 80 are as effective as sodium cholate in terms of skin permeability if compared with a saturated aqueous solution.	[^Citation3]
UDL	Methotrexate Ratio: drug: lipid 1.5 w/w	Investigate the skin permeation potential of the chemotherapeutic encapsulated in the liposome. The skin used was abdominal male Sprague Dawley rats.	Skin permeation at 24h for UDL (470.3 µg/cm^2) was significant higher compared to the conventional liposomes (around 150 µg/cm^2), due to its deformability. Among the different vesicles that were prepared, PC: T80 ratio 7:3 w/w was more effective in skin permeation and deformability terms.	[^Citation25]
UDL	Psoralen, 5-methoxypsoralen, 8-methoxypsoralen Concentration: 5–15% respect to lipid weight.	Liposomes preparation Psoralen and derivatives-loaded to evaluate their efficacy in vitiligo treatment. B16F10 Melanocyte cells were used for cell uptake study.	UDL derivatives-loaded at a concentration of 2.5 µM shown stimulation of melanin and tyrosinase activity.	[^Citation38]
UDL	Naringin Concentration: 1–24 mg/mL	Formulate liposomes with different amounts of naringin, characterize them, and test their ability to diminish skin inflammation-induced in female CD-1 mice.	The treatment with UDL reaches an edema reduction of ~20% compared with the Naringin dispersion because the UDL promotes the internalization of the compound into epithelial cells.	[^Citation97]
UDL	Curcumin and STAT3 siRNA Concentration: Lipids: Curcumin ratio 5, 10 and 15:1 w/w. liposomes 10:1: STAT3 siRNA ratio 10:1	The co-delivery of curcumin and STAT3 siRNA by topical application for skin cancer treatment in epidermoid carcinoma cells (A431).	Cells treated with liposomes with both curcumin and STAT3 siRNA shows a bigger growth inhibition than the solutions or liposomes just with one of them. The nanoparticles suppressed STAT3 protein and induced apoptosis.	[^Citation94]
UDL	Pirfenidone Concentration: 5 mg/mL	Investigate the feasibility of load pirfenidone into liposomes for skin delivery, also enhance the permeation of the liposomes with a hydrogel with permeation enhancers. The tissue used for the permeation study was pig ear.	The permeation of pirfenidone loaded in liposomes was 251±4.2 µg/cm^2, while free solution had a permeation 85.3±18.8 µg/cm^2. When the vesicles are incorporated into a hydrogel with permeation enhancers such as isopropyl myristate and oleic acid the liposomes shown even more permeation (344±28.8 µg/cm^2).	[^Citation86]
CL	Curcumin	Measure the physical properties and skin permeability of curcumin-loaded liposomes using pig ear skin.	The melting temperature could be affected by the packing reduction of the bilayer when curcumin loading increases, even with that behavior, liposomes with the higher amount of curcumin have better permeability in the epidermis (retention of ~70%) and dermis (retention of 7%)	[^Citation87]
Ethosomes	Rhodamine B	Evaluate the percutaneous efficiency of a drug-loaded model and determine the mechanisms of skin permeation.	The permeation of loaded molecule in ethosomes is 2.8-fold higher than liposomes, and 2.5-fold higher than a mixture of ethanol and water, the authors suggest that the enhanced permeation is due to a synergistic mechanism among skin, ethanol, and phospholipids.	[^Citation28]

Abbreviations: CL, conventional liposomes; UDL, ultradeformable liposome.

Figure 6 Chemical structures of bioactive compounds that have been incorporated into liposomes.

Figure 7 Chemical structures of most used edge activators in the preparation of liposomes.

Table 3 Patents for Liposome Formulation in Skincare

Patent	Country	Aim of Patent	Entrapped Compounds	Highlights	Inventor	Assignee
US8,846,611B2, 2014^Citation134	United States	To provide a formulation for topical administration for improving skin conditions with human growth hormone as an active ingredient.	Human growth hormone	The liposomes are prepared with soybean or egg yolk lecithin as the lipidic phase, the vesicles are can be used in cosmetics or pharmaceutical products.	Kyun Oh, Dahl; Jung Baik, Sang; Young Lee, Kyung	Regeron Inc
US2012/0201871A1, 2012^Citation23	United States	To provide a method to produce small liposomes with components that enhance skin permeation.	Water	The composition of the liposome includes phospholipids, oil-rich essential, fatty acid, behenic acid, oleic acid, skin lipids, and butter-rich in linoleic and linolenic acid.	Kyeong-Nam, Yu	Professional Compounding Centers of America, LTD.
US2009/0232881A1, 2009^Citation109	United States	To achieve an enhanced delivery of skin lightening or UV blocking agents to the skin.	A skin-lightening agent such as niacinamide and a UV-blocker, as 2-ethylhexyl-p-methoxycinnamate or butylmethoxydibenzoylmethane	The loaded compound was loaded in the range of 0.1 to 40% by weight of liposomes, which were prepared with soybean lecithin; mono-, di- or tri-ester of glycerol	Bandyopadhyay, Prasun; Bandyopadhyay, Punam
WO2009/094238A1, 2009^Citation135	International	The development of a vesicle with exceptional cosmetic, oral, and dermatological properties.	Propose the inclusion of water-soluble ingredients.	The lipidic phase is conformed by fatty acid esters and olivates.	Rosemarie, Lorenzo; Nadya, Lawrence	IGI Laboratories, Inc
WO2007/041627A1, 2007^Citation136	International	To prepare liposomes capable to entrap hyaluronic acid, collagen, and other skincare agents to improve the condition of the skin.	Hyaluronic acid, collagen, and other skincare agents.	The liposomes are used in a day cream.	Mark A. Pinsky
WO2006/014035A1, 2006^Citation137	International	To prepare a multilayered liposome that entrapped active substances	Physiological active substances such as peptides, proteins, nucleic acids, synthetic compounds, natural extracts, sugars, vitamins, and inorganic compounds.	The liposome is prepared with: squalane, sterols, ceramide, neutral lipids, fatty acids, and lecithin as a lipidic phase.	Deok-Hoon, Park; Jong-Sung, Lee; Kwang-Sun, Jung	Biospectrum Inc.
US2006/0292244A1, 2006^Citation138	United States	The use of liposomes carrying PVP-Iodine for acne treatment.	An antiseptic compound such as PVP-Iodine	The antiseptic agent concentration could be between 0.1 and 10 g in 100 g of preparation, which contains 1–5 g of liposomes. The formulations can be emulsions, dispersions, suspensions, solutions, gels, ointments, waxes, stray, lotions, among others.	Reimer, Karen; Fleischer, Wolfgang; Hopp, Michel
US2004/0180082A1, 2004^Citation139	United States	To provide a method for submicrometric liposome preparation containing high concentrations of triterpenoid	Triterpenoid with one or more acid groups such as ursolic acid, Centella asiatica extract, etcetera.	Liposomes are prepared preferably with soybean or egg lecithin. The content of triterpenoid is 0.5–2.5% by weight of total liposome. The liposomes could be incorporated into skincare products such as cosmetic, pharmaceutical o quasi-pharmaceutical.	Hyung-Seok, Kang; Gae-Won, Nam; Sang-Hoon, Han; Ih-Seop, Chang	Amorepacific Corporation
US2002/0128222A1, 2002^Citation140	United States	To create a wound coverage material with liposomes.	Genes encoding growth factors for wound healing.	Cationic liposomes containing cholesterol. The concentration of gene-encoding growth factor is 2.2 µg/mL	Herndon, David N; Perez-Polo, Jose R; Barrow, Robert E	Research Development Foundation
US6,365,145B1, 2002^Citation141	United States	To provide a method for hair removal using a liposomal composition	Photosensitizers such as phthalocyanines, porphyrins purpurins, psoralens, bergapten, angelicins, chlorins, and flavins.	The liposomes are prepared with egg yolk lecithin and cholesterol.	Ben-Hur, Ehud; Zuk, Maria M; Wai-Shun, Chan	New York Blood Center Inc

Figure 8 Melanomagenesis pathway.

Notes: Reprinted with permission from Springer Nature Customer Service Centre GmbH: Springer Nature, Springer eBook, Zaidi MR, Fisher DE, Rizos H, et al. Biology of melanocytes and primary melanoma. In: Balch CM, Atkins MB, Garbe C, editors. Cutaneous melanoma. Cham: Springer International Publishing; 2020:3–40.¹²⁴

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