COMPELLING SCIENCE
Tackling the Delivery of Beneficial microRNAs to the Skin
Brianna Scacchi, Julie Fisher, Joseph Ceccoli, and Paul Lawrence
ABSTRACT
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Gene expression can be modulated without alterations to the genetic code by a group of mechanisms collectively called “epigenetics”. One such mechanism is the post-transcriptional down-regulation of protein synthesis via small endogenous RNA molecules called microRNAs (or miRs). This natural regulatory pathway has been investigated for its capacity to be used to ameliorate diseases and conditions at the pharmaceutical and cosmetic level through miR “mimics” and “inhibitors” that toggle expression of specific genes down and up, respectively. Indeed, this has been demonstrated in cell culture, and also systemically with intravenous injection of mimics and inhibitors. However, with topical application of these epigenetic modulators for skin care purposes, there are certain hurdles that must be surpassed including effective penetration and protection from enzymatic and thermal degradation. Mimics and inhibitors of natural miRs have been delivered into multiple skin-relevant cell lines and primary cultures using commercial and novel transfection reagents. Following known miR regulatory pathways, introduction of miR-211-5p into melanocytes and melanocyte-derived cells stimulated melanogenesis. Contrastingly, introduction of miR-218-5p triggered the inhibition of melanin synthesis. Alternately, miR-29a-3p is responsible for the down-regulation in expression of protein important to skin health such as collagen, elastin, and fibrillin. The application of inhibitors of this microRNA in fibroblast cultures reverses the effect. Conventional reagents are not suitable for topical delivery., thus an alternative vehicle was explored. It protected small RNA effectors from thermal and enzymatic degradation over time and showed transfection capability in cell culture. However, upon topical application in a three-dimensional skin model, its efficacy seems varied, requiring additional innovations to be incorporated in future iterations of this reagent.
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HYPOTHESIS
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miR mimic molecules can treat a variety of skin conditions, including aberrant skin pigmentation and wrinkle formation.
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APPROACH
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1) evaluate capacity of mimics of skin-relevant microRNAs to modulate gene expression.
2) evaluate capacity of a novel delivery vehicle to protect and transfect skin-relevant cell cultures and a three-dimensional skin model
Figure 1. Skin-relevant microRNA pathways.
A. Schematic of roles of TGFβR2, MITF, and TYR in melanin production along with indicated miRs. B. Schematic roles of miR-29a-3p and antagomiR anti-miR-29a-3p in elastin (ELN1) and fibrillin (FBN1) production
Figure 2. S91 cell pellets post-miR transfection.
S91 melanocyte-derived cells were transfected with miR-NC, miR-155, miR-211, and miR-218 and incubated for 3 days. Pigmentation changes were observed relative to negative controls.
Figure 3. Western blot of miR-transfected S91 cells. Cell lysates were examined by Western blot probing with anti-TYR. Densitometry scanning was conducted using protein loading to normalize the resulting arbitrary units (AU). Negative control miR was transfected at 6X the concentration of miR-218.
Figure 4. Effect of miR-29a and anti-miR-29a (A29) on ELN1 and FBN1 production. Normal human fibroblasts were transfected with miR-29a and A29 at 50, 150, 250, 350, and 450 nM. Three days post-transfection, cells were collected and examined by Western blot. Bands were examined by densitometry scan and AUs normalized against loading controls. Shown is 1 of 2 replicates.
A. miR-29a – FBN1. B. A29 – FBN1. C. miR-29a – ELN1. D. A29 – ELN1.
Figure 5. Evaluation of an alternative formulation to transfect skin cells. The experiment in Figure 3 was repeated with miR-NC and miR-218 mimics using a commercial transfection reagent and a new in-house formulation (#2). After 3 days, cells were collected and tested by Western blot with anti-TYR production and compared by densitometry scan (A). One representative Western blot result comparing different transfection reagents (B).
Figure 6. miR thermal stability study.
Nonsense miR mimics were mixed with A. water, B. Formulation #1, C. Formulation #2, and D. Formulation #3 at 100 nM. The mixtures were divided 3 ways and incubated continuously at 4º C, 25º C, and 40º C. Periodically, they were evaluated for relative miR concentration using Qubit™ microRNA assay.
Figure 7. Evaluation of Formulation #2 to facilitate the introduction of miR mimics in three-dimensional skin model.
MatTek Melanoderm (MEL-300-A) were treated with cocktails on miR-211 and miR-218 mimics mixed with Formulation #2. Water (H2O) and Formulation #2 alone (VEH) were used as negative controls. Resveratrol (RESV) was used a lightening positive control. The Melanoderm models were examined microscopically for changes in pigmentation levels at 5, 9, and 14 days post-treatment.
CONCLUSIONS
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• miR-211-5p and miR-218-5p mimics can epigenetically regulate the expression of tyrosinase (TYR) in the skin pigmentation pathway as seen molecularly on Western blots and visually with alterations in cell pellet pigmentation.
• miR-29a-3p mimics and their antagomiR counterparts (anti-miR-29a-3p) decreased and increased fibrillin-1 and elastin-1 levels, respectively, as shown by Western blot assay.
• An experimental formulations (#2) for the delivery of microRNA mimics protected the mimics from degradation over time and exhibited transfection capability in cell monolayer culture.
• Treatment of a 3D skin model with mixtures of Formulation #2 and either miR-211-5p or miR-218-5p did not produce pigmentation changes as consistently as observed in cell monolayer cultures.
