What Are Bioactive Peptides & How They Penetrate the Skin?

What are Bioactive Peptides?

Bioactive peptides are small proteins composed of short sequences of 2-20 amino acids that can naturally occur in the skin or be produced synthetically. Naturally occurring peptides are produced from enzymatic hydrolysis of extracellular matrix (ECM) proteins like collagen, elastin, and fibronectin present in the skin.^[1] They can also be generated from fermentation of animal and plant proteins.^[2] Synthetic peptides can be isolated and purified in the laboratory using membrane separation and chromatography.^[3] Bioactive peptides have a wide variety of biological effects on the skin, including:

• Improved skin elasticity and integrity^[4-7]
• Improved wound healing^[8]
• Increased fibroblast proliferation^[9]
• Growth factors^[10]
• Tensioning/tightening agents^[9]
• Prevention of aging, hyperpigmentation, and wrinkle development^[11]
• Increased collagen and elastin synthesis^[9]

How do Bioactive Peptides Enter the Skin?

The majority of known bioactive peptides are orally ingested and act like hormones or neurotransmitters, indirectly exerting their effects on the skin.^[12] Transdermal administration of bioactive peptides has garnered great interest because it avoids degradation in the GI tract when ingested orally.

Normal skin is impermeable to most molecules, especially those larger than 500 Daltons.^[13] Additionally, the stratum corneum (figure 1), the outermost layer of epidermis, is highly lipophilic. Due to their large molecular size (typically greater than 500 Daltons) and hydrophilic nature, bioactive peptides do not easily penetrate the skin.

Figure 1. Layers of the skin.
Credit: Hoet PH, et al. 2004: 438-470

Several strategies have been developed to overcome the skin barrier and facilitate entry of peptides into the skin. For example, the specific sequence of amino acids can be modified or substituted to increase receptor binding, specificity, and solubility of peptides.^[14] Modification of N- and C- terminals via acetylation, glycosylation, and amidation can increase plasma half-life and thus resist degradation by peptidases. Moreover, conformational changes like replacing the L-amino acids with D-amino acids can reduce proteolysis and degradation of peptides.^[15] Newer strategies to increase delivery of peptides into the skin include lipid conjugation, electroporation, iontophoresis, sonophoresis, and microneedling.

Lipid conjugation

Bioactive peptides have a net charge at physiologic pH, which poses a challenge to transdermal entry. However, conjugating peptides to a lipid can effectively increase peptide delivery across the skin. Lintner et al. conjugated a pentapeptide to a 16-carbon fatty acid to create palmitoyl pentapeptide (pal-KTTKS), which increased delivery across the skin compared to pentapeptide alone.^[16] In a 12-week double blinded, placebo-controlled study of 93 women, topical application of pal-KTTKS increased collagen production and demonstrated a significant reduction in wrinkles and fine lines.^[17]

Electroporation

Electroporation is a noninvasive technique where skin cells are exposed to an electrical, high voltage current (>50V) in brief pulses in order to transiently permeabilize the skin.^[18] The electric current targets the phospholipid bilayer of the stratum corneum which is disrupted, allowing the entry of molecules into the skin.^[19] Recent studies have shown electroporation of the stratum corneum, the main skin barrier to peptides, to be possible.^[20] Though electroporation has been around for many years, it has only recently been applied to skin. Few clinical studies have been conducted on its use with bioactive peptides.

Iontophoresis

Iontophoresis exposes skin cells to a low voltage (<10V) continuous current in order to allow transdermal delivery of charged peptides. This current moves ions across the skin membrane, increasing the potential difference. This potential difference can be applied to charged peptides placed under cathodes and anodes to help peptides move across the skin via electrorepulsion.^[19] Though the new delivery method has yet to be approved by the FDA, one  study showed a 30 times increase in permeability of peptides with iontophoresis relative to passive permeation.^[21]

Sonophoresis

Sonophoresis is a potentially noninvasive technique that utilizes low frequency ultrasound (<100 kHz) to facilitate skin penetration by peptides and other hydrophilic compounds.^[13] While the mechanism of action is still not fully understood, hypothetically ultrasound waves could help facilitate the entry of peptides by disrupting the stratum corneum.^[13]

Microneedling

Microneedling is a minimally invasive technique involving a patch of micro-sized needles that puncture the skin in a controlled manner to mechanically disrupt the stratum corneum without damaging the epidermis. These microinjuries initiate a wound healing cascade which releases growth factors and leads to fibroblast migration and collagen deposition.^[22] Traditionally, this was used as a collagen induction therapy for skin rejuvenation and facial scars. It is now also used as a delivery system to deliver drugs past the stratum corneum directly to the epidermis, as shown in figure 2.^[19]

Figure 2. Microneedling bypasses the stratum corneum to deliver drug products directly to the epidermis. Credit: Alkilani et al. 2015: 438-470

Which Bioactive Peptides are Able to penetrate the Skin?

Peptides acting on the skin can be categorized based on their mechanism of action as signal peptides, carrier peptides, enzyme inhibitor peptides, or neurotransmitter inhibitor peptides as shown in Table 1. 

ECM = extracellular matrix; MMP = matrix metalloproteinase; Ach = Acetylcholine

 

Signal peptides

Signal peptides are the most widely known peptides. Essentially, they are messengers that instruct cells. They help control the production or breakdown of larger extracellular matrix proteins by stimulating skin cells called fibroblasts, which leads to an increase in collagen and elastic fiber production. They can also act as growth factors by activating protein kinase C, important in cell growth and migration.^[28] Palmitoyl pentapeptide-4 (palmitoyl-KTTKS) is a well-known synthetic peptide derived from procollagen that stimulates collagen types I and III and fibronectin.^[24] In a randomized, double-blind, placebo-controlled study of 93 subjects, topical application of palmitoyl-KTTKS twice daily for 12 weeks was reported to reduce the total length of fine lines and wrinkles on the face.^[17] 

Carrier peptides

Carrier peptides help transport trace elements into the skin, where they activate wound healing pathways and promote collagen synthesis. Additionally, they can stimulate metalloproteinase enzymes responsible for remodeling the extracellular matrix.^[8] For example, the tripeptide-copper complex, glycyl-l-histidyl-l-lysine-Cu2+ (copper peptide GHK-Cu) is a small naturally occurring peptide widely known for its broad range of regenerative effects on the skin. In a study conducted on 71 volunteers for 12 weeks, facial creams containing GHK-Cu demonstrated improved elasticity and tightness of skin, as well as reduced fine lines and deep wrinkles.^[26]

Enzyme inhibitor peptides

Enzyme inhibitor peptides act by directly or indirectly inhibiting enzymes essential for skin health. For example, soy derived proteins act as an enzyme inhibitor to prevent aging, improve skin moisture, and support hair health.^[9] Naturally extracted from soybean seeds, soy peptides prevent proteinase activity from degrading collagen in the skin.^[9] They can also reduce skin pigmentation by inhibiting melanosome phagocytosis by keratinocytes.^[27] In a randomized, double-blind placebo-controlled study of 65 women with facial photodamage, topical application of a soy extract moisturizer over 12 weeks showed significant improvement in pigmentation, blotchiness, dullness, overall texture and skin tone.^[30]

Neurotransmitter inhibitor peptides

Neurotransmitter inhibitor peptides work by inhibiting muscle contraction, which prevent the formation of wrinkles. Their structure is similar to SNARE complexes that interact with the neurotransmitter acetylcholine, thus competing for binding sites and preventing the release of acetylcholine at nerve endings.^[28] Acetyl hexapeptide-3, a peptide similar to botulinum toxin A, decreases muscle contraction by inhibiting acetylcholine formation.^[31] In a study of 20 human subjects for 30 days, the application of hexapeptide-3 on skin reduced wrinkle depth by 59% and 71% on dry and oily skin, respectively, compared to placebo.^[29]

Practical Tips

• Bioactive peptides can be modified via amino acid substitution and lipid conjugation to increase their permeability into the skin.
• Bioactive peptides can be delivered through the stratum corneum via electrical currents, ultrasound waves, or microneedling to reach the epidermis.
• Palmitoyl pentapeptide-4 and palmitoyl oligopeptide are examples of signal peptides that can be applied topically to stimulate collagen synthesis and reduce fine lines and wrinkles.
• Copper GHK is an example of a carrier peptide that can be used in topical preparations to improve wound healing, elasticity, and tightness of skin.
• Acetyl hexapeptide-3 and pentapeptide-3 are examples of neurotransmitter-inhibiting peptides with botox-like activity that can be applied to the skin to prevent wrinkles.
• Soybean proteins and rice peptides are examples of enzyme inhibitor peptides that can be used in skin creams for anti-aging and moisturizing effects.