Topical Application of Peptide Nucleic Acid Antisense Oligonucleotide for MMP-1 and Its Potential Anti-Aging Properties

Matrix metalloproteinase-1 (MMP-1) is a zinc-containing endopeptidase that degrades dermal collagen and other extracellular matrix molecules. It is recognized as one of the most important indicators of cellular senescence and age-related skin changes. Here, we introduced a novel MMP-1 peptide nucleic acid (PNA) derivative—PNA-20 carboxyethyl fluorene (CEF)—which can interact with and consequently silence the MMP-1 gene sequence. The investigation on the efficacy of PNA-20 CEF in MMP-1 silencing in human dermal fibroblasts revealed significantly decreased expression of MMP-1 at both gene and protein levels. Treatment with PNA-20 CEF showed significantly increased expression of collagen I protein, indicating its potential role in preventing the degradation of collagen I and consequently combating the skin aging process. Its topical application on 3D human skin tissue showed successful absorption into the epidermis and the upper dermis. Furthermore, the additional 4-week single-arm prospective study on 21 Asian women revealed improvements in facial wrinkles, skin moisture, elasticity, and density after the use of the topical PNA-20 CEF cosmeceutical formulation. Additional in-vitro and ex-vivo studies are needed for a comprehensive understanding of the skin anti-aging effects of MMP-1 PNA.


Introduction
Aging of the skin is a complicated process that occurs because of both natural intrinsic and extrinsic aging processes. With age, the ability of the human body, including that of the skin, to resolve inflammation is markedly reduced, resulting in an imbalance between pro-and anti-inflammation. This causes a chronic low-grade pro-inflammatory status known as "inflammaging", eventually leading to cellular aging [1]. Among the extrinsic factors aggravating cellular aging, ultraviolet radiation (UVR) exposure produces one of the most powerful effects. Repetitive UV irradiation induces large-scale deletions in mitochondrial DNA, resulting in dysfunction and aging owing to reduced collagen synthesis and increased collagen breakdown [2,3]. Moreover, it alters collagen homeostasis itself by UVR-induced hyperactivity of matrix metalloproteinases (MMPs), including MMP-1 (interstitial collagenase), MMP-3 (stromelysin), and MMP-9 (gelatinase), because of the oxidative stress-mediated activity of activator protein-1 (AP-1) and nuclear factor-kappa beta (NF-κB) transcription factors [4].
As the average human life expectancy increases, there is increasing interest globally in the anti-aging field, resulting in the establishment of the aging-related nutraceutical market, PNA-20 CEF was produced by the OliPass Corporation as a 14-mer peptide nucleic acid complementarily targeting a 14-mer RNA sequence in human MMP-1 pre-mRNA. In brief, PNA-20 CEF was synthesized via solid-phase peptide synthesis on an automatic peptide synthesizer by Fmoc chemistry based on the method disclosed in a patent disclosure with minor modifications [15]. H-Rink Amide ChemMatrix resin was purchased from PCAS BioMatrix Inc. (Quebec, Canada) and was used as a solid support. After synthesis, PNA-20 CEF was purified by C18-reverse phase high-performance liquid chromatography (HPLC; water/acetonitrile or water/methanol 0.1% trifluoroacetic acid) and identified using high-resolution mass spectrometry. Its sequence was confirmed by measuring melting temperature (Tm) against complementary DNAs. Detailed manufacturing processes are available on request.

Quantitative Reverse Transcription-PCR (qRT-PCR)
HDFs (5 × 10 4 cells/well) were seeded in 6-well plates and treated with a 6-point concentration of PNA-20 CEF or 0.05% OliPass RNA RS.301 OLV cream when cells reached over 80% confluence. After a 24 h incubation, total RNA was obtained from the incubated cells using RNAiso Plus (Invitrogen, Waltham, MA, USA) and synthesized into cDNA using an RNA to cDNA EcoDry Premix Kit (Takara Sake, Berkley, CA, USA). The Taqman primer of MMP-1 (Hs00899658_m1; Applied Biosystems, Waltham, MA, USA), and Taqman Gene Expression Master Mix were used to perform qRT-PCR. The primers used here were shown to be stable by the manufacturer. For normalization of MMP-1 expression levels, GAPDH (Hs02786624_g1; Applied Biosystems) was used as the control, and these results were analyzed using the 2 −∆∆Ct method.

Participants
A total of 22 healthy females aged 40-50 years with distinct wrinkles around their eyes were recruited for this clinical study. The exclusion criteria were participants having a history of the following: infectious skin disease, allergies or hypersensitivity, lesions on the test area, adverse responses to medicines, cosmetics, or routine light exposure, using similar medicines as our product, and any form of dermatologic treatment (e.g., Botox, laser, fillers, scaling, and tattoos) up to 3 months before this study. All participants were instructed to apply OliPass RNA RS.301 OLV cream on their faces after washing twice daily for 4 weeks to assess for clinical efficacy and safety. One participant dropped out of because she could not follow the treatment schedule. This clinical study was approved by the Institutional Review Board (IRB) of the Global Medical Research Center (IRB number: GIRB-21N01-GU), performed in compliance with the ethical principles of the Declaration of Helsinki, and informed consent was obtained from each participant. The present clinical study was approved by the Korea Center for Disease Control and assigned a Clinical Research Information Service number (KCT0007742).

Evaluation of Clinical Efficacy
OliPass RNA RS.301 OLV cream was manufactured as a facial anti-aging cream formulation containing PNA-20 CEF. A 24 h pre-clinical cream patch test was performed on the upper back of healthy adult participants between 19 to 59 years of age. The OliPass RNA RS.301 OLV cream showed a skin irritation index of 0.0 and was therefore considered a non-irritating skin product.
After the pre-clinical study, participants in the prospective single-arm clinical study were instructed to apply OliPass RNA RS.301 OLV cream twice daily for 4 weeks. All study participants were investigated at baseline, 2 weeks, and 4 weeks after treatment initiation using the Ultrascan UC22 (Courage Khazaka Electronic GmbH, Köhn, Germany), a device used to measure epidermal and dermal density changes. Skin moisture changes were determined using Corneometer ® (Courage Khazaka Electronic GmbH, Köhn, Germany). The inner and surface elasticity of the skin were measured using the Dermal torque meter (DTM; Dia-Stron, Hampshire, UK) and Cutometer Dual MPA580 (Courage Khazaka Electronic GmbH, Köhn, Germany). For changes in the depth of periorbital wrinkles, the skin analysis camera system (Antera 3D CS; Antera 3D ® , Miravex, Dublin, Ireland) was used. Each measurement was performed after participants were acclimatized to controlled environmental conditions (room temperature: 20-24°C, relative humidity: 45-55%) for 30 min whenever they were evaluated for clinical efficacy. All participants were asked to report any adverse events while using the OliPass RNA RS.301 OLV cream. The participants were surveyed regarding their satisfaction with the anti-aging efficacy of the cream using the following scale: 1 = unsatisfied, 2 = no change, 3 = slightly satisfied, 4 = satisfied, and 5 = very satisfied. The total cream ingredients are shown in Supplementary Table S2.

Statistical Analysis
All data were statistically analyzed for significance using the SPSS Package Program version 25 (IBM Corp., Armonk, NY, USA). Clinical data at baseline and at weeks 2 and 4 were compared using the repeated measures analysis of variance (ANOVA) or the Friedman test followed by the post-hoc Wilcoxon signed-rank test with Bonferroni correction according to the results from the normality test. Data are expressed as mean values ± standard deviation, and statistical significance was set at p < 0.05, p < 0.01, and p < 0.005. All experiments were independently conducted at least three times (n ≥ 3).

In Vitro and 3D Skin Assessments of the Cellular Anti-Aging Effect of PNA-20 CEF
The relative MMP-1 mRNA expression levels in human dermal fibroblasts (HDFs) were measured through quantitative reverse transcription-PCR (qRT-PCR). MMP-1 mRNA expression levels of the control group and after treatment with 1 µM, 10 nM, 100 pM, 1 pM, 10 fM, and 100 aM of PNA-20 CEF were 1, 0.327 ± 0.021, 0.975 ± 0.004, 0.902 ± 0.054, 0.948 ± 0.051, 0.962 ± 0.065, and 0.873 ± 0.029, respectively ( Figure 1A). MMP-1 gene expression levels after treatment with 1 µM or 10 nM PNA-20 CEF were significantly reduced compared to those of the control group (* p < 0.05 and *** p < 0.005, respectively). We also performed a qRT-PCR to evaluate MMP-1 expression levels when treated with PNA-20 CEF concentrations between 10 nM and 1 µM. The result showed that MMP-1 gene expression levels dose-dependently decreased as PNA-20 CEF concentrations increased; the MMP-1 expression levels reduced significantly when treated with 1 µM or 500 nM of PNA-20 CEF ( Figure S1) (*** p < 0.005). MMP-1 concentration was also measured using an ELISA. MMP-1 concentrations of the control group and after treatment with 1 µM, 10 nM, 100 pM, 1 pM, 10 fM, and 100 aM of PNA-20 CEF were 1167.44 ± 106.31 pg/mL, 585.37 ± 58.82 pg/mL, 868.52 ± 72.29 pg/mL, 926.11 ± 84.01 pg/mL, 1132.41 ± 131.13 pg/mL, 1181.48 ± 116.93 pg/mL, and 1197.94 ± 100.35 pg/mL, respectively ( Figure 1B). After 1 µM PNA-20 CEF treatment, MMP-1 concentration was significantly reduced compared to that of the control group (*** p < 0.005). Western blot analysis of MMP-1 from the HDF culture media after treatment with PNA-20 CEF for 24 h and 48 h is shown in Figure 1C. Treatment with 1 µM PNA-20 CEF for 24 h led to significantly reduced MMP-1 levels compared to that of the control group in the HDF cultured media ( Figure 1D, *** p < 0.005). Treatment of 1 µM or 10 nM of PNA-20 CEF for 48 h also significantly reduced the MMP-1 level in the media ( Figure 1E, * p < 0.05). version 25 (IBM Corp., Armonk, NY, USA). Clinical data at baseline and at weeks 2 and 4 were compared using the repeated measures analysis of variance (ANOVA) or the Friedman test followed by the post-hoc Wilcoxon signed-rank test with Bonferroni correction according to the results from the normality test. Data are expressed as mean values ± standard deviation, and statistical significance was set at p < 0.05, p < 0.01, and p < 0.005. All experiments were independently conducted at least three times (n ≥ 3).
Additionally, we investigated the effect of PNA-20 CEF on the expression of collagen I in fibroblasts via western blot analysis (Figure 2A). The media and cell extract samples used were obtained after treatment with 1 µM, 10 nM, 100 pM, 1 pM, 10 fM, or 100 aM of PNA-20 CEF for 24 h or 48 h. In the media, after 24 h treatment with 10 nM or 1 pM PNA-20 CEF, the collagen I protein expression increased significantly compared to that in the control group. After 48 h treatment with 1 µM, 10 nM, or 10 fM PNA-20 CEF, collagen I expression again increased significantly compared to that in the control group ( Figure 2B, * p < 0.05, ** p < 0.01). These results indicated a reduction in collagen I degradation after PNA-20 CEF treatment. In the cell extract, the treatment with 1 µM or 10 nM PNA-20 CEF also showed significantly increased collagen I protein expression after 24 h or 48 h of the treatment ( Figure 2C, * p < 0.05, ** p < 0.01, *** p < 0.005). Additionally, 100 pM PNA-20 CEF treatment showed increased collagen I expression after 48 h compared to the control (*** p < 0.005). In the reconstructed human 3D skin tissue, 1 µM PNA-20 CEF was absorbed in the epidermis up to the upper dermis over time (Figure 3). Thus, topical application of PNA-20 CEF may be able to assist anti-aging of dermal fibroblasts.
Additionally, we investigated the effect of PNA-20 CEF on the expression of collagen I in fibroblasts via western blot analysis (Figure 2A). The media and cell extract samples used were obtained after treatment with 1 µM, 10 nM, 100 pM, 1 pM, 10 fM, or 100 aM of PNA-20 CEF for 24 h or 48 h. In the media, after 24 h treatment with 10 nM or 1 pM PNA-20 CEF, the collagen I protein expression increased significantly compared to that in the control group. After 48 h treatment with 1 µM, 10 nM, or 10 fM PNA-20 CEF, collagen I expression again increased significantly compared to that in the control group ( Figure 2B, * p < 0.05, ** p < 0.01). These results indicated a reduction in collagen I degradation after PNA-20 CEF treatment. In the cell extract, the treatment with 1 µM or 10 nM PNA-20 CEF also showed significantly increased collagen I protein expression after 24 h or 48 h of the treatment ( Figure 2C, * p < 0.05, ** p < 0.01, *** p < 0.005). Additionally, 100 pM PNA-20 CEF treatment showed increased collagen Ⅰ expression after 48 h compared to the control (***p < 0.005). In the reconstructed human 3D skin tissue, 1 µM PNA-20 CEF was absorbed in the epidermis up to the upper dermis over time (Figure 3). Thus, topical application of PNA-20 CEF may be able to assist anti-aging of dermal fibroblasts.

In Vitro Assessment of the Cellular Anti-Aging Effect of OliPass RNA RS.301 OLV Cream
To evaluate the effect of OliPass RNA RS.301 OLV cream on the expression levels of MMP-1 and collagen in HDF cells, an additional qRT-PCR and ELISA were performed. The results indicate that the gene expression level of MMP-1 after treatment with 0.05% cream (0.156 ± 0.001) was significantly reduced compared to that of the control group (1.000 ± 0.009) ( Figure 4A, *** p < 0.005). Accordingly, the ELISA also revealed that the MMP-1 expression level after treatment with 0.05% cream (77.108 ± 0.001 ng/mg) was significantly reduced compared to that of the control group (134.852 ± 0.001 ng/mg; Figure  4B, *** p < 0.005), confirming its effect on silencing MMP-1 gene expression and reducing MMP-1 expression. Meanwhile, an additional ELISA showed significantly increased expression levels of PIP1 α1 after treatment with 0.05% cream (230.169 ± 2.844 ng/mg) compared to that of the control group (195.60 ± 7.602 ng/mg; Figure 4C, * p < 0.05).

In Vitro Assessment of the Cellular Anti-Aging Effect of OliPass RNA RS.301 OLV Cream
To evaluate the effect of OliPass RNA RS.301 OLV cream on the expression levels of MMP-1 and collagen in HDF cells, an additional qRT-PCR and ELISA were performed. The results indicate that the gene expression level of MMP-1 after treatment with 0.05% cream (0.156 ± 0.001) was significantly reduced compared to that of the control group (1.000 ± 0.009) ( Figure 4A, *** p < 0.005). Accordingly, the ELISA also revealed that the MMP-1 expression level after treatment with 0.05% cream (77.108 ± 0.001 ng/mg) was significantly reduced compared to that of the control group (134.852 ± 0.001 ng/mg; Figure 4B, *** p < 0.005), confirming its effect on silencing MMP-1 gene expression and reducing MMP-1 expression. Meanwhile, an additional ELISA showed significantly increased expression levels of PIP1 α1 after treatment with 0.05% cream (230.169 ± 2.844 ng/mg) compared to that of the control group (195.60 ± 7.602 ng/mg; Figure 4C, * p < 0.05).

Participant Characteristics of the Clinical Trial
The clinical study was performed on 22 Asian female participants with distinct periorbital wrinkles. The mean age of the participants was 50.36 years (range, 41-59). The final analysis data were obtained from 21 participants as one participant was dropped because of noncompliance. The demographic data of participants are shown in Supplementary Table S1.

Assessment of Safety and Adverse Events
There were no reports from enrolled participants of serious adverse events, and none of the participants dropped out of the study because of adverse events, suggesting that the use of the cream was safe.

Assessment of Safety and Adverse Events
There were no reports from enrolled participants of serious adverse events, and none of the participants dropped out of the study because of adverse events, suggesting that the use of the cream was safe.

Discussion
Here, we constructed a novel MMP-1 oligonucleotide derivative, PNA-20 CEF, which can interact with the MMP-1 gene sequence, specifically with nucleic acids, such as RNAs. The binding of an antisense RNA with sequences complementary to the target mRNA provides a powerful tool to modulate artificial gene expression, called RNA silencing [16]. Antisense oligonucleotides (ASO) can also bind to a pre-mRNA in the nucleus and affect the splicing of the pre-mRNA, producing an altered mRNA sequence [17,18]. However, one of the main barriers in applying ASO technology is the rapid degradation of DNA-based oligonucleotides in cells by nucleases [19].
Reactive oxygen species (ROS) produced by extrinsic aging factors, such as UVR exposure and tobacco smoking, have been known to overexpress MMP-1 [20,21]. MMP-1 has been identified as a skin aging-associated secreted protein (SAASP) which are proteins closely related to matrix degradation and pro-inflammatory processes [22]. Various antioxidants and food supplements with antioxidizing effects-Vitamin A (retinol), Vitamin C (ascorbic acid), Vitamin E (tocopherol), carotenoid, flavonoids, green tea, and selenium-have therefore been widely consumed for skin rejuvenation [23][24][25][26]. Considering the role of MMP-1 in skin aging, it is important to develop pharmaceuticals and cosmetics based on the mechanism of inhibiting MMP-1 activity.
A modified PNA is recognized as one of the most successful ASO derivatives. In PNA, the sugar-phosphate backbone of DNA or RNA is replaced with a pseudo-peptide backbone, while nearly identical geometry and spacing of the bases are retained. This modification can enhance the stability of oligonucleotides against enzymes that degrade DNAs, RNAs, and peptides [18]. It can also increase the PNA-RNA binding affinity, as RNA is negatively charged and PNA is electrically neutral [16]. Unlike small molecule drugs, however, the bioavailability of PNA is considerably limited because of its poor water solubility and cell permeability [27]. The PNA-20 CEF investigated here is a novel form of PNA established by introducing modified nucleobases with a cationic lipid or its equivalent covalently attached to them to overcome the cell permeability barriers.
Investigation of the efficacy of PNA-20 CEF in MMP-1 silencing revealed significantly decreased mRNA expression levels of MMP-1 after treatment with PNA-20 CEF compared to that in the control in HDFs. Moreover, the results of the western blot analysis and ELISA showed decreased MMP-1 proteins detected from the cell internally and externally after treatment with PNA-20 CEF, confirming its action in inhibiting MMP-1 expression. The results suggest that treatment with PNA-20 CEF decreases the MMP-1 gene and protein expression in HDF cells internally and externally by producing non-functional MMP-1 as expected, which is dependent on dose efficacy and time. To further investigate the cellular anti-aging effect of PNA-20 CEF, collagen I protein expression was analyzed via western blot analysis. The analysis showed a significant increase in collagen I protein expression after PNA-20 CEF treatment compared to that in the control group. MMP-1 degrades and fragments collagen synthesized by the cells [28]. The increased collagen I protein expression after PNA-20 CEF treatment was due to the MMP-1 silencing by PNA-20 CEF.
Aged dermal fibroblasts play a major role in dermal-epidermal interactions and overall skin tissue decline [29]. Previous research indicates that skin aging is primarily initiated at the dermis level, where macromolecular damage via extrinsic aging factors accumulates, causing resident fibroblasts to become senescent [7]. Therefore, a skin antiaging pharmaceutical agent should enter the dermis level adequately. To confirm the enhanced efficacy of PNA-20 CEF in topical skin absorption after modification of its PNA molecular structure, we performed skin absorption tests on reconstructed human 3D skin tissue. The results revealed that PNA-20 CEF was effectively absorbed into the epidermis and the upper part of the dermis after OliPass RNA RS.301 OLV cream treatment. In addition, treatment with the cream in HDF cells reduced the MMP-1 mRNA expression level and protein production and increased the production of the PIP1 α1 protein. Hence, PNA-20 CEF inhibits the production of MMP-1 in epidermal keratinocytes and dermal fibroblasts, thereby increasing collagen production and leading to anti-aging clinical manifestations. Correspondingly, the 4-week single-arm prospective study revealed improvements in facial wrinkles, skin moisture, elasticity, and density without any adverse effects after the use of topical PNA-20 CEF. The satisfaction scores of the participants also reflected the overall improvement in aged skin.
Our study had several limitations. First, there were a limited number of samples and a short-term follow-up period. A further prospective controlled study with a larger sample size and a longer follow-up period is required. A well-controlled study design should be constructed in the future to confirm the effect of PNA-20 CEF on MMP-1 silencing. Second, the in vitro study as well as the clinical study designs did not involve sufficient control data, such as scrambled PNA or a vehicle cream without the MMP-1 silencing effect. To test the penetration ability of PNA-20 CEF into the skin, we utilized a reconstructed skin model (Neoderm-ED). Although the skin equivalent is consistent with the epidermis and the dermis, its use to test penetration is still controversial [30]; therefore, an additional study with human ex vivo skin is needed to provide a better understanding of skin absorption. UV irradiation induces the synthesis and expression of MMP-1 by dermal fibroblasts, which is stimulated by the generation of excess ROS [8]. In vitro experiments with a UV-irradiated group as the positive control would have been instructive to evaluate the effect of PNA-20 CEF on MMP-1 and collagen I expressions. Furthermore, we only confirmed the anti-aging effect of PNA-20 CEF by observing collagen I expression. As MMP-1 degrades both collagen I and III, an additional investigation on the expression level of collagen III would have been informative. Further in vitro and ex vivo studies to observe changes in other molecules that are well known as markers of cellular senescence, such as IL-6, p53, and p16, as well as β-galactosidase staining and tissue collagen density measurements, can be added to provide a comprehensive understanding of the effect of PNA-20 CEF targeting MMP-1 on skin anti-aging. PNA-20 CEF was designed only to target the exon-intron junction of MMP-1 to produce non-functional MMP-1 by inducing exon skipping. Future investigations into the effects of PNA-20 CEF that can also inhibit the activities of other MMPs involved in photoaging, including MMP-2, -3, and -9, would be useful to generate additional products against skin aging.

Conclusions
Our study showed the anti-aging effects of a novel ASO derivative targeting the MMP-1 oligonucleotide derivative via both laboratory and clinical studies. As many pharmaceutical and cosmeceutical agents with antioxidizing effects have been developed to treat skin aging with only limited efficacy, an innovative method targeting the mechanism of skin aging is appealing. Although the MMP-1 small interfering RNA (siRNA) is already reported in the literature to inhibit the expression of MMP-1 mRNA and protein levels in vitro [31], siRNAs are not only very expensive to manufacture but also possess poor cellular permeability. Moreover, the common carrier to transfer siRNAs to cells or the skin, lipofectamine, can induce cellular toxicity. Therefore, developing a novel agent, such as PNA-20 CEF with high biocompatibility to inhibit MMP-1 expression efficiently is critical.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jcm12072472/s1, Table S1: Participant demographic data; Table S2: Total ingredients of OliPass RNA RS.301 OLV cream; Figure S1: Relative mRNA expression levels of MMP1 in the HDF cells. The MMP1 gene expression levels decreased dose-dependently as PNA-20 CEF concentration (*** p < 0.005). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data employed and/or studied in this research can be obtained from the corresponding author upon request.