A Novel, Hand-Held, and Low-Level Light Therapy Device for the Treatment of Acne Vulgaris: A Single-Arm, Prospective Clinical Study

Tere is an increasing demand for low-level light therapy devices for the treatment of dermatologic conditions, such as acne, hair loss, undesirable body hair


Introduction
Acne vulgaris is a chronic infammatory disease of the pilosebaceous units, afecting approximately 85% of adolescents and young adults [1]. Although acne vulgaris is not life-threatening, it can signifcantly afect the patients' self-esteem and quality of life. Previous studies have found the impact of acne on the social, emotional, and vocational aspects of quality of life [2][3][4]. Excess sebum production, abnormal keratinization, colonization by Cutibacterium acnes, and skin infammation are the four major pathogenic factors involved in acne vulgaris. Although topical antibi-otics and retinoids are the standard antiacne medications for mild to moderate acne, a variety of side efects, including irritation, antibiotic resistance, and the teratogenicity of isotretinoin, have been challenging [5,6]. Terefore, there is a substantial unmet need for alternative acne treatment options.
With the growing medical demand for light-based therapies, several diferent types of lasers have been used extensively in the treatment of patients with acne [7][8][9]. However, with these laser treatments, it may take at least a few days or weeks for the skin to fully recover. Low-level light therapy or photobiomodulation refers to the use of photons at nonthermal irradiance to alter biological activity, and has been shown to reduce pain and infammation, promote tissue regeneration, and prevent tissue damage [10]. Recently, the efcacy of low-level light therapy has been widely established in dermatology and has shown good efcacy in the treatment of acne vulgaris [11][12][13]. Tere have been several reports on the clinical improvement of acne lesions with blue light treatment, suggesting that low-fuence blue light irradiation induces photoexcitation of protoporphyrin IX and coproporphyrin III, which leads to the destruction of Cutibacterium acnes [14][15][16]. In contrast, red light penetrates deeper and has better anti-infammatory efects, activating the release of multiple transcription factors and cytokines in various cell types. According to some studies, red light has been reported to increase nitric oxide release and reduce TNF-α and COX-2 expression [17][18][19]. In addition to the efcacy of each individual wavelength, numerous attempts have demonstrated that the combination of blue and red light is synergistic in treating acne lesions by integrating antibacterial and anti-infammatory efects [20][21][22]. Papageorgiou et al. compared the efcacy of blue light against mixed blue and red light in the treatment of acne, and the efect of mixed light was evaluated to be better [20]. Nonetheless, clinical studies evaluating the efectiveness of blue-red combined phototherapy in the treatment of acne are lacking. Tere is currently no consensus on a treatment regimen for acne vulgaris with low-level light therapy.
Te COVID-19 pandemic has increased the demand for noncontact medical procedures among healthcare providers and patients, and advancements in solid-state light sources have enabled the commercialization of athome light therapy devices [23,24]. Employing a practical and user-friendly home-based light device signifcantly enhances adherence and compliance among acne patients, owing to its satisfactory efcacy and favorable safety profle [25]. Terefore, in this study, we aimed to evaluate the clinical efectiveness of a hand-held blue-red combined low-level light therapy device for patients with mild to moderate acne using a self-treatment method. In addition, to provide a better understanding of the efect of blue-red combined phototherapy on antibacterial properties and in vivo skin extracellular matrix remodeling, we assessed its antibacterial potential against Cutibacterium acnes and evaluated the role of low-level light therapy in skin extracellular matrix remodeling in SKH1 mice.

Te Blue-Red Combined Photomodulation Device for
Potential Low-Level Light Terapy. Te light source used in this study was a commercially available device, P1-FOX (PAD-FA120, Ptech Corp., Pyeongtaek, Gyeonggi-do, Korea), which is composed of three blue lights (wavelength � 450 nm) of light-emitting diodes and six red lights (wavelength � 680 nm) of laser diodes. Te light-emitting diode emits a power of 11.7 mW/cm 2 and the laser diode emits a power of 13.3 mW/cm 2 . Tis product functions via 625-Hz pulse operation and performs the cross-output of three light-emitting diodes and six laser diodes every four seconds (Supplementary Figure 1).

Cutibacterium acnes Culture and Investigation of the Antibacterial Properties of the Blue-Red Combined Photomodulation Device.
To investigate the antibacterial properties of blue-red combined photomodulation, we serially diluted Cutibacterium acnes and cultured them on Brucella agar plates after 20 min of exposure to the blue-red combined photomodulation (Figure 1(a)). Cutibacterium acnes was used to assess the antibacterial properties of the blue-red combined photomodulation device. Cutibacterium acnes (KCTC 5933) were purchased from the Korean Collection for Type Cultures (Daejeon, Korea). Cutibacterium acnes was grown for 48 to 72 h at 37°C on Brucella agar plates (BANDIO, Pocheon, Gyeonggi-do, Korea) under anaerobic conditions using Oxoid ™ AnaeroGen ™ 2.5 L Sachet (Termo Scientifc, Waltham, MA, USA) in anaerobic jars (Sigma-Aldrich, St. Louis, MO, USA). Single colonies from the plate were picked and inoculated into 5 mL of brain heart infusion broth (Kisan Bio, Seoul, Korea) in a round-bottom tube. Te bacterial suspension was incubated at 37°C in a shaking incubator at 160 rpm until visible growth was observed. Bacteria were diluted to an OD600 value of 0.1. Diluted bacterial suspensions were divided and tested under two conditions: no low-level light therapy (control group) and low-level light therapy treatment (experimental group). Aliquots of 500 μL of the diluted bacterial suspensions were placed into the wells of a 24-well plate, and blue light was placed directly on top of the 24-well plate, approximately 5 mm from the surface of the liquid. Te plates were then incubated in the dark at 37°C. After low-level light therapy exposure, the bacterial suspensions were serially diluted 10fold in brain heart infusion broth. Next, 3 μL of each well was spotted onto Brucella agar plates (BANDIO, Pocheon, Gyeonggi-do, Korea) to quantify the survival after low-level light therapy exposure [26]. After 24 h of incubation under anaerobic conditions, the plates were examined and scored for growth.

RNA Isolation and Quantitative Polymerase Chain
Reaction. RNA was isolated from the mouse skin using the TRIzol reagent (Invitrogen, Carlsbad, USA). RNA was quantifed using a spectrophotometer (Termo Scientifc ™ , To perform low-level light therapy, four 8-week-old SKH1 male mice were anesthetized with 1% isofurane inhalation prior to the treatment. Te low-level light therapy device was placed directly on the surface of the mouse skin, approximately 5 mm away from the surface of the skin, and the mice were exposed to the blue-red combined photomodulation for 20 min. After 6 h, the mice were sacrifced for analysis.

Histological Evaluation.
Te dorsal skin of each mouse was collected at the end of the experiment. Te tissue was fxed with 4% paraformaldehyde in phosphate-bufered saline for 24 h, dehydrated gradually in ethanol, and embedded in parafn. Te parafn-embedded samples were sectioned into 5 μm-thick sections to be processed for hematoxylin and eosin and Masson's trichrome staining for histological evaluation, as previously described [28]. None of the subjects had received laser-or light-based therapy for at least 6 weeks before enrollment, dermabrasion, trichloroacetic acid peel, botulinum toxin type A, dermal fllers, photodynamic therapy, cosmetic surgery, or oral retinoid treatment within the past 6 months, and had not received oral antibiotics or oral contraceptives within the past 3 months. Patients were also excluded if they used topical alpha hydroxy acids, retinoic acid, retinol, salicylic acid, vitamins C and D, or their derivatives within 6 weeks prior to enrollment. Finally, pregnant and breastfeeding patients were excluded from the study. Te baseline characteristics of the 57 patients with mildto-moderate facial acne enrolled in the study are shown in Table 1.

Devices and Treatment
Protocols. All enrolled patients were given a hand-held blue-red combined low-level light therapy device. All patients had mild-to-moderate facial acne and were instructed to turn on the device after adjusting the panel to align the facial acne lesions as closely as possible. Te treatment was performed twice a day for 10 min for each treatment for 4 weeks, regardless of the severity of the acne. Te device has a self-recording program to assess patient compliance and actual usage time. Posttreatment follow-up assessments were conducted two and four weeks later. All patients were encouraged to use a nonirritating cleanser (Zeroid foam cleanser, Neopharm, Daejeon, Korea) and moisturizer (Zeroid soothing cream, Neopharm, Daejeon, Korea), as well as a noncomedogenic sunscreen on their face.

Evaluation of Blue-Red Combined Low-Level Light
Terapy on Skin Regeneration. Efcacy analysis was performed in the intention-to-treat and per-protocol populations. Te intention-to-treat population included patients who received at least one treatment and had at least one onstudy visit upon inclusion in the study. Te per-protocol population included all patients who received at least 80% treatment. Te subjects were carefully assessed at 2-week intervals at baseline (weeks 0, 2, and 4) and followed-up until 4 weeks after the fnal treatment (weeks 6 and 8). Standardized digital photographs capturing the frontal and bilateral 45°sides for assessment of full facial acne lesions were taken during each visit, using identical positions and camera settings (Janus Premier, PIE Co., Suwon, Korea) to ensure the reliability of the evaluation. Efcacy evaluation was performed using the number of acne lesions on the entire face from the hairline to the jawline. Te acne lesions included in the count were comedones, papules, pustules, and nodules. A global severity assessment, according to the Investigator's Global Assessment, was also conducted (Supplementary Table 2). Te Investigator's Global Assessment grade was recorded in a blinded manner by three independent dermatologists (CEH, LSH, and KJE). In addition, patients recorded their self-assessments of the therapeutic efectiveness during each visit using a visual analog scale; at baseline, a disease-free state was designated as 0, and a state of severe acne was designated as 10. All adverse efects including pruritus, pain, erythema, scaling, and hyperpigmentation were recorded in detail throughout the study.

Statistical Analysis.
Demographic characteristics are expressed as means ± standard deviation for continuous variables and as frequencies and percentages for categorical variables. Treatment efects were compared with the baseline scores at each follow-up visit using paired t-tests. Te statistical analysis was performed using both the intention-totreat (ITT) and the per-protocol (PP) analysis sets. Te ITT analysis set included all randomized participants, while the PP analysis set was restricted to participants who remained adherent to the study protocol. We applied the last-observation-carried-forward (LOCF) rule for missing data. Data were analyzed using SPSS Statistics (version 26.0, IBM Corp., Armonk, NY, USA). In all analyses, * * * p < 0.001 was considered to indicate statistical signifcance. Figures 1(b) and 1(c), we observed a signifcant decrease in the number of Cutibacterium acnes colonies upon blue-red combined photomodulation as a function of the initial bacterial concentration. Tese results demonstrated the antibacterial properties of blue-red combined photomodulation, exhibiting the inhibition of Cutibacterium acnes growth.

Investigation of in Vivo Toxicity of Blue-Red Combined Photomodulation Exposure Using an Animal Model.
To confrm the efect of blue-red combined photomodulation, we assessed the mRNA expression and histological changes in SKH1 mouse skin upon blue-red combined photomodulation to precisely evaluate whether 20 min of blue-red combined photomodulation would induce skin damage in both the epidermal and dermal layers of SKH1 mouse skin tissues. As shown in Figure 2(a), genes associated with skin extracellular matrix remodeling, such as Col1α1, fbronectin, EGF, and FGF2, did not show any signifcant changes. Additionally, although COX2, which is an infammation marker, was slightly decreased in skin tissues with blue-red combined photomodulation, it did not show a signifcant diference (p > 0.05).
To further evaluate whether blue-red combined photomodulation could cause damage to the skin tissues and cytotoxic efects on the cells present within the epidermis and dermis, we performed a histological evaluation. As shown in Figures 2(b)-2(d), hematoxylin and eosin and Masson's trichrome staining demonstrated that there was no damage to either the epidermis or dermis, and collagen deposition in the dermis seemed to be intact in both the  control and experimental groups. Moreover, Ki67 immunohistochemistry revealed no signifcant diferences in the numbers of Ki67 positive nuclei, indicating that blue-red combined photomodulation did not cause apoptosis of cells in both the epidermis and dermis layers. Taken together, these results suggest that blue-red combined photomodulation does not have a detrimental efect on skin homeostasis and extracellular matrix remodeling.

Clinical Trials.
Te intention-to-treat population consisted of 57 patients, whereas the per-protocol population consisted of 46 patients. Among the 11 patients who were excluded from the per-protocol population, 6 patients failed to comply with the treatment protocol due to nonadherence according to the self-recording program of the device, and the remaining 5 patients were withdrawn from the study due to their inability to visit the hospital. Efcacy variables were analyzed in the intention-to-treat and per-protocol populations.

Evaluation of Treatment Efcacy in the per-Protocol
Population. After two weeks of low-level light therapy treatment, infammatory acne counts were signifcantly reduced (40.6% reduction, from a score of 9.59 to 5.70, p < 0.001). Te number of infammatory lesions decreased gradually during the four weeks of treatment (45.4% reduction, from a score of 9.59 to 5.24, p < 0.001). Eventually, at the fnal visit (week 8, 4 weeks after the fnal treatment), the fnal counts decreased by 64.7% (from a score of 9.59 to 3.39, p < 0.001). A statistically signifcant reduction was evident in the mean infammatory lesion count as early as week 2, and the improvement was maintained at the end of treatment (p < 0.001; Figure 3(a)). Tere were no diferences in the number of noninfammatory lesions (Figure 3(b)). Clinical pictures of the two patients with marked improvement are shown in Figures 3(c) and 3(d). Aligned with the results of the infammatory acne lesion counts, the proportion of the Investigator's Global Assessment grade 3 indicating moderate acne severity decreased signifcantly from 52.2% at the baseline to 15.2% at week 2 and to 0% at fnal visit (Figure 3(e)). Before treatment, patients' selfassessment of their acne was given a visual analog scale score of 10. Troughout the course of treatment, the visual analog scale score decreased signifcantly from 8.24 at baseline to 3.52 at the fnal visit (Figure 3(f )).

Evaluation of Treatment Efcacy in the Intention-to-Treat Population.
Infammatory acne counts were signifcantly reduced by low-level light therapy (38.8% reduction, from a score of 9.54 to 5.84, p < 0.001) after 2 weeks of treatment. During the four weeks of treatment, the counts decreased gradually (42.2% reduction, from a score of 9.54 to 5.51, p < 0.001). Moreover, at the fnal visit (week 8, 4 weeks after the fnal treatment), the counts decreased by 59.3% (from a score of 9.54 to 3.88, p < 0.001). A statistically signifcant reduction was evident in the mean infammatory lesion count as early as week 2, and the improvement was maintained at the end of treatment (p < 0.001; Figure 4(a)).
Tere were no diferences in the number of noninfammatory lesions (Figure 4(b)).

Safety and Patient Compliance.
To assess patient adherence to treatment, we analyzed the usage data recorded on the devices. Four patients did not complete the daily use program, whereas the other patients regularly used the devices throughout the study. Among these 57 patients, neither severe adverse reactions nor adverse efects (e.g., skin dryness, erythema, or desquamation) were reported.

Discussion
Te versatility of low-level light therapy has been proven by its application in various conditions that require stimulation of healing, pain relief, and infammation [29,30]. Low-level light therapy has positive efects on wrinkles, acne scars, hypertrophic scars, and burn healing. In addition, it might beneft infammatory diseases such as acne. Te noninvasive nature and absence of side efects promotes the use of lowlevel light therapy [10].
Although numerous clinical studies of low-level light therapy in treating acne vulgaris have been extensively conducted during the past decade [13,31,32], only three randomized controlled clinical studies have investigated at-home light-emitting diode devices for the treatment of acne [13,23,32]. Low-level light therapy uses either coherent light sources (lasers) or noncoherent light sources consisting of fltered lamps or light-emitting diode, or a combination of both. Laser diodes are the light source used in home devices for the treatment of alopecia diseases, such as androgenetic alopecia, female pattern alopecia, and alopecia areata; however, there have been no studies using home-based devices for acne treatment [33].
Tis prospective study showed that phototherapy with this novel, hand-held, blue light-emitting diode-red laser diode combined low-level light therapy device is a safe and efective treatment option for mild-to-moderate acne. After the 4-week treatment period, we observed signifcant decreases in infammatory acne lesions, and these improvements were maintained for up to 4 weeks after the fnal treatment. At the fnal 8-week follow-up, substantial reductions of 64.7% and 59.3% in infammatory lesions were observed in the per-protocol and intention-to-treat groups, respectively, without any serious side efects. Although the total dose of light irradiated to acne patients was much lower than that in previous studies on combination phototherapy, the overall improvement in infammatory lesions was not inferior to that in earlier reports [13,[20][21][22]34]. One study reported a signifcant reduction in infammatory and noninfammatory acne lesions by 77% and 54%, respectively, in 35 light-emitting diode-treated patients for 2.5 min twice daily for 4 weeks [13]. Another randomized controlled trial demonstrated a signifcant improvement in acne lesion resolution compared with sham-irradiated treatment [31,32]. In contrast to previous studies, we used laser diodes instead of light-emitting diodes as the red-light source. 6 Dermatologic Terapy Previous studies have reported that laser diodes have better wound healing efects than light-emitting diodes [35]. Additionally, the clinical improvement in the study was comparable to that observed with the use of topical antibiotics and retinoids as the frst-line treatment for mild-tomoderate acne. While topical antibiotics and retinoids cause antibiotic resistance and irritation, respectively, our patients experienced no such side efects during the treatment and follow-up periods [36][37][38]. If further large-scale studies show similar efectiveness and safety, this blue-red combined low-level light therapy device could become a reliable and safe alternative for the treatment of mild-to-moderate acne. Despite its efectiveness in various clinical settings, the mechanism underlying combined blue-and red-  lightirradiation has not been elucidated. Only a handful of authors have proposed possible mechanisms for this phenomenon. Blue light has been shown to activate coproporphyrin III and protoporphyrin IX, leading to the destruction of Cutibacterium acnes [14,[39][40][41]. Similarly, red light has been shown to be efective in accelerating wound healing and reducing the infammatory response, possibly by stimulating mitochondrial activity and modulating the release of cytokines from macrophages [42,43]. Kwon et al. proposed that the anti-infammatory properties of mixed blue and red light may induce the suppression of activated NF-κB and activator protein 1 pathways [13].
We assumed that the reason for the substantial improvement despite treatment completion was mainly related to photobiomodulation, which initiates the excitation of endogenous chromophores to elicit photophysical and photochemical events [44,45]. Although the exact mechanism associated with cellular photobiostimulation by lowlevel light therapy is not yet fully understood, low-level light therapy enhances collagen synthesis and fbroblast proliferation and promotes various growth factors and extracellular matrix production by activating cellular mitochondrial respiratory pathways [19,45,46]. Te improvement of infammatory acne lesions could have been sustained by the remodeling of the key epidermal and dermal components.
Although the bactericidal efect of light-emitting diode phototherapy depends on wavelength, power density, bacterial viable number, and bacteria species, it has been demonstrated to have an antibacterial efect. Te bactericidal efect of blue light-emitting diode irradiation depends on the strains and conditions of bacterial inoculation; however, wavelengths of 425 and 525 nm have a bactericidal efect [47]. Similarly, de Oliveira Assunção et al. investigated the efect of wavelengths and energy densities of light-emitting diode irradiation, ranging from the blue (465 nm) to the red spectrum (630 nm), which could suppress the growth of various bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli [48].
Te limitations of this study are as follows: Since the device was designed for in-home use, safety issues associated with long-term use should be tested more rigorously in subsequent studies. In addition, given the absence of a control group or split-face design, our study is unable to rule out the potential efects of cleansers and moisturizers on acne reduction. Although the results of this study are promising, the optimal irradiation fuence and treatment regimen should be determined in future studies. Further studies are required to investigate the exact mechanism of this lowlevel light therapy device.

Conclusion
In conclusion, this study showed that this hand-held bluered combined low-level light therapy device had benefcial efects on infammatory acne lesions with excellent patient compliance and satisfaction. Tis device appears to be both safe and efective for the treatment of mild-to-moderate acne and could provide alternative strategies for treating acne against conventional acne treatments.

Data Availability
Te data that support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
All procedures performed in this study involving human participants were in accordance with the ethical standards of the Institutional and/or National Research Committee, and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.   Dermatologic Terapy