Cytotoxic Evaluation in HaCaT Cells of the Pa.7 Bacteriophage from Cutibacterium (Propionibacterium) acnes, Free and Encapsulated Within Liposomes

Abstract

Introduction:

Acne is a multifactorial disease involving the colonization of skin follicles by Cutibacterium (formerly Propionibacterium) acnes. A combination of different retinoid-derived products, antibiotics, and hormonal antiandrogens are used to treat the disease, but these treatments require extended periods, may have secondary effects, are expensive, and not always effective. Owing to antibiotic resistance, the use of bacteriophages has been proposed as an alternative treatment. However, if they are intended for a cosmetic or pharmaceutical use, it is necessary to evaluate the safety of the phages and the preparations containing them.

Materials and Methods:

In this study, the cytotoxicity of Pa.7 bacteriophage was evaluated in HaCaT cells, along with a liposome suitable for their encapsulation, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays.

Results:

We found that Pa.7 was not cytotoxic for HaCaT cells. Also, 30 mM of liposomes, or below are considered noncytotoxic concentrations.

Conclusion:

Phages encapsulated in the liposomes presented in this study can be used safely for skin treatments.

Introduction

Cutibacterium (formerly Propionibacterium) acnes is an opportunistic, Gram-positive, and microaerophilic bacteria commonly associated with skin infections, such as acne.¹ This multifactorial disease is related to four crucial conditions: an increase in sebum production caused by androgen hormones, the hyperkeratinization and obstruction of sebaceous follicles, the proliferation of C. acnes, and inflammation.²

The disease develops in the hair follicles found in the human skin. Follicles are accompanied by a sebaceous gland that lubricates the follicle and protects the skin from external factors. Acne occurs mainly in adolescence, associated with the increased levels of androgenic hormones, such as testosterone, which augment the sebum secretion causing the plugging of the follicles and generating different types of acne.³ However, this condition can also occur at any time in a person's life due to events that lead to stress, causing an increase in cortisol levels and generating inflammation of the entire body.⁴ In addition to the factors mentioned earlier, other factors influence the development of acne, such as the patient's immune system.⁵

Acne is one of the most frequent skin diseases, occurring in ∼85% of people between 12 and 24 years, at least once in their life.⁶ The clinical manifestations of acne on the patient's mental health have been raised as one important concern.⁷ Acne damages patients' self-esteem, compromising their physical appearance and generating, on some occasions, peers and self-rejection. Appearance may affect one's professional development, as there have been reports in which acne has affected the opportunity to get a job.⁸ Therefore, treatment of acne is of great importance. There are two types of treatments: topical and systemic. Topical treatments are diverse, and their selection depends on the severity of the acne; mild acne is treated with topical retinoids, benzoyl peroxide, and azelaic acid; in moderate acne, topical antibiotics are used combined with anti-inflammatories.

When acne becomes severe and no longer responds to topical treatment, systemic treatment begins with oral antibiotics and contraceptives.⁵ Most frequently prescribed antibiotics for acne treatment are tetracyclines, macrolides, clindamycin, and trimethoprim/sulfamethoxazole due to their antibacterial and anti-inflammatory effects.⁹ It is vital to highlight Isotretinoin as part of the systemic treatments, which is a nonaromatic retinoid¹⁰ used in critical cases of acne. Several adverse effects have been described in patients who use Isotretinoin, such as nasal bleeding, headache, muscle tiredness, and itching.¹¹ There is also an ongoing discussion about the association between this medication and impairments in people's moods.^11,12

These side effects have led researchers to look for alternative methods to treat acne. One of the most promising is phage therapy defined as the use of bacteriophages to control bacterial populations.¹³ Some advantages of phage therapy are that phages are highly specific,¹⁴ and their nature allows them to reproduce when they encounter the susceptible host bacteria. Bacteriophages can be temperate (lysogenic life cycle) or virulent (lytic life cycle). Temperate phages can integrate their genome into the bacterial DNA and remain in this state until they receive a signal to enter the lytic cycle. Then, virulent phages lyse the bacteria, releasing the virions that can now attack the surrounding cells, which causes an exponential propagation of the phages.

For phage therapy, it is essential that the phages only perform the lytic cycle to prevent the lysogenic conversion of the bacteria and because the bacterial infection must be controlled in the shortest possible time, with the highest efficiency.¹⁴ However, one of the difficulties in the research for lytic phages to implement in acne treatment is the limited diversity of phages.⁹ Few studies have examined the efficacy of C. acnes phagesin diminishing inflammatory lesionsin murine models,^15,16 suggesting their potential use for C. acnes infection control. An important advantage of phage therapy is that phages can be effective against sensitive bacteria as well as those strains that are resistant to antibiotics.^17,18 Some authors have reported a decrease in the virulence of phage-resistant bacteria, adding another possible advantage to phage therapy.¹⁹

Bacteriophage activity can be affected by physicochemical factors such as pH, temperature, and ions in the environment.²⁰ Acne phage treatment is mainly performed topically as the C. acnes population resides inside the pilosebaceous follicles of the skin²¹ and induces the process of hyperkeratinization due to comedogenesis.²² These physical barriers may prevent phage from effectively reaching the site of the bacterial infection on the skin due to their limitations in movement. In addition, phage composition of only proteins and nucleic acids may favor its degradation in the environment due to the action of metabolites normally found in the skin. Because these are factors that cannot be controlled in vivo, several strategies have been proposed to stabilize bacteriophages, including lyophilization, spray drying, biodegradable matrix processing, and encapsulation.²³

Liposome encapsulation is one of the forms of directed delivery that has been successfully developed since 1970. Liposomes are vesicles formed by biocompatible compounds such as cholesterol (CHO) and phospholipids, which are both hydrophobic and hydrophilic, therefore good candidates for the transport of compounds of any of the two natures.²⁴ Another advantage of these lipid-based delivery systems is that they can be used to release antimicrobial compounds inside the cell due to the resemblance of the two phospholipid bilayers that mediate the fusion of the liposome with the bacteria.²⁵ Previous in vivo assays performed in mice with phages against Klebsiella pneumoniae have shown a better performance of the phages in liposomes when compared with free phages.^26,27

Another important aspect to consider refers to the requirements a new product must comply regarding its toxicity.²⁸ There are different cytotoxicity tests that can be performed in vivo or in vitro. Acne-related in vivo tests are performed on animals, such as the rhino mouse, the Mexican hairless dog, the Golden Hamster, the pig, and the guinea pig.²⁹ However, due to the ethical concerns regarding this type of assays, a preferred way to evaluate cytotoxicity in the case of phages intended for use in the human skin is through in vitro tests. The latter are used to evaluate whether certain compounds affect cell proliferation or show direct cytotoxic effects that ultimately lead to cell death.

In addition, phage preparations should include information on the characteristics of the phages to prove that phage therapy is safe and nontoxic in animal and clinical studies. These toxicity endpoints may consider their morphology; titer; genetics, and protein profile; the composition of the phage preparations, including the levels of bacterial contaminants (viable bacteria, endotoxin, enterotoxin B, or bacterial DNA); and other impurities from the purification method.^30,31 Other characteristics such as sterility, pH stability, suspended buffer, and osmotic pressure might be considered.³⁰

Based on the aforementioned, this study evaluates the in vitro cytotoxicity on epithelial cells of an in-house preparation of bacteriophage Pa.7 against C. acnes and the liposome formulation that contain them, to assess their innocuousness and its potential as a new treatment for acne. This phage was isolated from human skin, purified, tested for host range, transmission electron microscopy observed, and sequenced.³² It belongs to the Siphoviridae family and was unable to infect other skin bacteria assayed.

Materials and Methods

Buffers

SM buffer was prepared by adding 5.0 mL 2% (w/v) of Gelatin (OXOID), 50 mL of Tris-Cl (Chem cruz) (1 M, pH 7.5), 2 g de MgSO₄·7H₂O (Panreac), and 5.8 g NaCl (Sharlau) to 1 L of distilled water. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer was prepared using 2.38 g (20 mM) of HEPES acid (Sigma), 4.23 g (145 mM) NaCl and then the solution was aliquoted with Milli-Q water until it reached 500 mL. Buffers were sterilized by autoclave.

Preparation of purified phages

Previous to this study, bacteriophage Pa.7 was isolated and identified as a lytic phage against P. acnes.³³ In brief, C. acnes strain Pa.6 was previously isolated and identified.³⁴ Bacteria were grown in Nuevo broth (tryptone 1.5%, yeast extract 0.5%, brain–heart infusion 0.5%, glycerol 1%, NaCl 0.2%, K₂HPO₄ 0.2% L-Cysteine HCl 0.03%, Tween 80 0.025%, and Bromocresol purple 0.002%). Cultures were incubated 16–18 h at 37°C. The phage titer was achieved by the double agar technique³⁵ using Nuevo medium soft agar (0.25%) plus the bacterial culture, poured on Nuevo medium agar (1.5%). Phages were diluted in SM buffer, and 5 μL of each dilution were spotted on the surface of the agar. Spots were allowed to dry, and plates were incubated for 24 h at 37°C with 5% CO₂.³² This phage was stored at 4°C for later use.

Thin film hydration method for the creation of liposomes

The liposome was composed of 40% of 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-CHO, 10% of CHO, 3% of polyethylene glycol, and 47% of 1,2-dioleoyl-sn-glycero-3-phosphocholine.³² The lipids were dissolved in chloroform. Later, this dissolvent was eliminated with nitrogen to provide a dry lipid film, which was later hydrated with the aqueous phage suspension in HEPES at 37°C. During the hydration, the lipid came off the recipient, and they self-assembled into multilamellar structures encapsulating the phage.³⁶ The concentration of the bacteriophages in the stock used to mix with the lipids was 10⁸ PFU/mL. These structures were maintained in the shaker for 12 h at 37°C. Triton X-100 was used in previous experiments to break down liposomes and calculate the encapsulated viable phages.³² This standardized encapsulation protocol was repeated for the experiments presented in this study, and the encapsulation index varied between 63.75% and 70%.

Liposomes characterization

Phage-containing liposomes were prepared as described earlier. To remove media components, the preparation was ultra-centrifuged at 40.000 rpm, 15°C for 10 min and the resulting pellet was resuspended in 1 mL phosphate-buffered saline pH 6.8. This washing procedure was repeated three times. The final suspension was applied to 300 mesh, Formvar-carbon-coated Cu grids. Liposomes were negatively stained with 2% uranyl acetate, pH 4.8, for 30–60 s, and observed using a Focused Ion Beam/Scanning Electron Microscope LYRA3 (TESCAN). Morphology was assessed; size and elemental composition were determined.

Cell culture

Human keratinocytes (HaCaT) were used at a concentration of 1 × 10⁵ cells/mL in a 96-well microplate. The HaCaT cell line was donated from Universidad de Antioquia and was derived from primary epidermal keratinocytes from normal human adult (HEKa; ATCC, PCS-200-011™). HaCaT cells were grown in Dulbecco Modified Eagle's Minimal Essential Medium (DMEM) supplemented with 1 g/L glucose, 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin and streptomycin. All cells were incubated at 37°C with 5% CO₂ in a humidified atmosphere.

Trypan blue exclusion assay

The treatments were carried out when the cells in the microplate formed a monolayer. Concentrations assessed for the free phages were 1 × 10,⁵ 1 × 10,⁶ 1 × 10⁷ PFU/mL; and for phages in liposomes 0, 1 × 10,⁴ 1 × 10,⁵ 1 × 10⁶ PFU/mL. The two buffers, SM and HEPES, used for the different presentations of phages were also evaluated. The negative control contained 100 μL of DMEM without FBS; the test assays had 100 μL of the different concentrations evaluated, which were composed of 90 μL of DMEM without FBS and 10 μL of each phage or phage in liposomes solution.

Cells were incubated at 37°C with 5% CO₂ in a humidified atmosphere. After 24 and 48 h of exposure, the medium was removed and 50 μL of trypsin was added, then the microplate was taken to the incubator for about 10 min. Subsequently, 100 μL of DMEM supplemented with FBS was added by homogenizing the mixture with a micropipette. Then, 10 μL of trypan blue was placed in each well containing 50 μL of the cell suspension. Finally, 10 μL were transferred to the Neubauer chamber to perform cell counting.

The cell counting was done using Equation (1)³⁷:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay

The cell cultures and treatments were carried out in the same way described for the trypan blue exclusion assay. After 24 and 48 h of exposure to phages and phages in liposomes, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) was added to each well and incubated again for 2 h. Later, the medium was removed and 100 μL of dimethyl sulfoxide was added to dissolve the formazan crystals. The well plate was analyzed with a microplate reader (BioRad) at a wavelength of 595 nm and a reference length of 655 nm. The results were presented as a percentage of living cells calculated from the absorbance of the negative control, taking the last one as a 100%.

We also used the MTT assay for the evaluation of the effect of different concentrations of liposomes (10, 20, 30, 40, 50, 60, 75, 100, 150, 200, 250, and 300 mM per microplate well) on the cell viability, following the same procedure described earlier, and the effect of phages in liposomes (phages in constant concentration and liposomes in 10, 20, 30, 40, 50, and 60 mM per microplate well).

Statistical analysis

For the MTT and the trypan blue assays, each experiment was performed in triplicate wells and was repeated three times. The mean viability percentage of each treatment was compared with the negative control viability by a one-way or two-way analysis of variance (ANOVA) test. Two-way ANOVA was used to determine how viability was affected by the time of exposure to the free and encapsulated phages and by the viral titer. In addition, the Dunnett's test was carried out to compare each treatment with the control because the latter represents 100% of cell viability. The Mann–Whitney U test was used to evaluate the differences between the 24- and 48-h treatments. The data were analyzed with the GraphPad Prism software version 7.00 for Mac (GraphPad, San Diego, CA). The Ethics Committee for Research Affairs approved the experimental procedures of this project (Document code No. 1657).

Results

Buffers effect on cell viability

No significant differences in cell viability (p > 0.5) were found for SM nor HEPES buffers (used for phage suspension and liposomes preparation) when compared with the controls, for both 24 and 48 h, with the two methods evaluated, trypan blue assay (Fig. 1A) and MTT (Fig. 1B).

FIG. 1.

HaCaT cell viability after exposure to the two buffers HEPES and SM buffer evaluated through the (A) trypan blue exclusion assay and (B) MTT assay. The data shown in each bar represents three biological replicates, repeated three times. The comparisons were made between the control versus other concentrations and were evaluated by a two-way ANOVA and Dunnett's multiple comparisons tests. No significant differences were found. ANOVA, analysis of variance; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Bacteriophages effect on cell viability

In the experiment with free bacteriophages, both at 24 and 48 h, there were no significant differences (p > 0.5) in the viability percentages between treatments and controls for the two methods assayed (Fig. 2). In the MTT assay, it was observed that some readings gave a higher value compared with the controls (Fig. 2B). Despite that, no significant differences were found for any of the concentrations evaluated.

FIG. 2.

HaCaT cell viability after exposure to the Pa.7 phage suspended in SM buffer evaluated through the (A) trypan blue assay and (B) MTT assay. The data shown in each bar represents three biological replicates, repeated three times. The comparisons were made between the control versus other concentrations and were evaluated by a two-way ANOVA and Dunnett's multiple comparisons tests. No significant differences were found.

Liposomes effect on cell viability

We found a negative correlation between liposome concentration and cell viability; where the liposome concentration increases, and the cell viability decreases (Fig. 3). The highest liposome concentration at which the cell viability percentage was maintained >75% was 30 mM. Cell viability was significantly different for all liposome concentrations after 24-h exposure (Fig. 3). Also, viability was significantly different for concentrations >150 mM after 48-h exposure (Fig. 3B). Altogether, no significant differences were observed between 24- and 48-h experiments for all the concentrations used (p > 0.05, Mann–Whitney U test).

FIG. 3.

HaCaT cell viability after exposure to the different concentrations of liposomes per well through the MTT assay being (A) 10–60 mM for 24 h, (B) 75–300 mM for 24 and 48 h. The data in this figure represent the three biological replicates of each treatment, performed three times. The comparisons were made between the control versus other concentrations and were evaluated by a one-way ANOVA (A) and two-way ANOVA (B) and Dunnett's multiple comparisons tests. Significant differences: *p < 0.05, **p < 0.01, and ***p < 0.001.

Effect of phages encapsulated in liposomes

When the phages were encapsulated using different concentrations of the liposomes, it was observed that they followed the same tendency of the liposomes alone in their effect on cell viability (Fig. 4): a negative and significant correlation between liposome concentration and cell viability, with 30 mM as the highest liposome concentration at which the cell viability percentage was maintained >75%.

FIG. 4.

HaCaT cell viability after exposure to Pa.7 bacteriophage-containing liposomes, according to the MTT assay. Concentration of liposomes varied between 0 and 60 mM per microplate well. Data shown in this figure represents the three biological replicate of each treatment, performed three times. The comparisons were made between the control versus other concentrations and were evaluated by a one-way ANOVA and Dunnett's multiple comparisons tests. Significant differences: *p < 0.05, **p < 0.01, and ***p < 0.001.

Liposomes characterization

The negative stain scanning electron microscopy images are presented in Figure 5. The images showed that the liposomes were spherical or ellipsoid, discrete particles with defined boundaries that range in size from ∼20 to 350 nm. Liposome composition was C 88.88%, N 1.39%, O 7.93%, Na 0.68%, P 0.52%, and Cl 0.61.

FIG. 5.

Liposomes morphology and size characterization by focused ion beam/scanning electron microscopy.

Discussion

The preparations of bacteriophages intended for phage therapy must comply with various regulatory standards, being its innocuousness of utmost importance. Therefore, a good approximation to evaluate the safety of any substance is to perform cytotoxicity tests. In the case of bacteriophages, despite all the studies that have been conducted on phage therapy, there is scarce information on the effects of its direct addition on immortalized cell lines.³⁸ Performing cytotoxicity assays in human cell lines can be less time-consuming at a relatively low cost when compared with the use of animal models in research.³⁹

In addition, these initial studies can allow the researchers to understand the fundamental dynamics between bacteriophages and mammalian cells. This knowledge will then facilitate the transition into animal studies and clinical trials for phage therapy. In the search for a suitable comparison of bacteriophages as an alternative treatment to antibiotics, we considered the categories of cytotoxicity according to ISO 10993-5, where the percentages of cell viability >70% are considered noncytotoxic.⁴⁰

Our results showed that the free Pa.7 bacteriophage has no cytotoxic effect for the HaCaT keratinocyte cell line, which was an expected outcome since the phages are highly specific for their bacterial host and belong to the normal microbiota of the human body.³³ Other studies have supported the statement that isolated bacteriophages are safe and noncytotoxic for mammalian cell lines by using a similar methodology as performed in this study.^41,42 For example, in a recent study performed by Torabi et al., two bacteriophages isolated from a sewage system were tested for their ability to inhibit multiple drug-resistant Enterobacter cloacae and Enterobacter hormaechei.

Researchers found that besides having the ability to inhibit these bacterial strains, these two phages had no cytotoxic effect in the two cell lines used: A375 and HSFS PI3. Similar results were also found in a study using BALB/c3T3 fibroblasts cultured with bacteriophages at concentrations of 10⁴ or 10⁹ phages per mL and no significant changes in the viability or morphology were found.⁴² Even though different studies have reported the safety of isolated phages, it is important that every newly isolated phage whose goal is to be used as a therapeutic agent must be evaluated for its safety.⁴³

We found that cells treated with bacteriophages presented higher cell viability (>100%) in the MTT assay. This result is comparable with that obtained by Henein et al.,⁴⁴ who tested an Acinetobacter baumannii bacteriophage in mouse fibroblast cells using various cytotoxic tests, including 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and trypan blue. A possible explanation for the increased cellular viability correlated to the presence of bacteriophages is that phage binding to the extracellular matrix⁴⁵ causes a signaling cascade that leads to either the overactivation of the mitochondria or an increase in cellular division. Both events would lead to an increased signal in the MTT assay, since this test measures mitochondrial activity.⁴⁶

However, this hypothesis needs confirmation. A study by Shan et al. evaluated interactions between bacteriophages, human cells, and bacteria. They tested bacteriophages against C. difficile and found that due to phage attachment to HT-29 cells, phages were more efficient at killing the bacteria. Also, there were no cytotoxic effects caused by phage-mediated bacterial lysis. As the authors described in their article, this information could represent a new alternative prophylactic use of bacteriophages. In the context of this study, as a possible alternative for acne treatment, it is an exciting area to explore in the future.³⁸

Bacteriophage encapsulation ensures that phage concentration will persist in the site of infection at a therapeutically effective level.⁴⁷ Polysaccharide polymers, synthetic polymers, liposomes, and micelles have been used for bacteriophage encapsulation and release. Liposomes have been widely used to encapsulate hydrophilic or hydrophobic therapeutic agents, mainly due to their biocompatibility enhancing cargo bioavailability and stability over time.⁴⁸ Liposomes contain natural biodegradable lipids, are nonimmunogenic and nontoxic, and can penetrate the epidermal barrier to treat skin infections.⁴⁹

In addition, the liposome surface could be functionalized to improve its performance by attaching hydrophilic polymers to increase circulation time and slow the release of bacteriophages, by labeling bacteriophages-containing liposomes to follow their fate within the cell, by adding specific ligands to target delivery, by protecting bacteriophage from chemical and physical stress, and by modifying charge to promote adhesiveness and modulating the immune response to the presence of bacteriophage in the body.⁴⁷ However, also some limitations can arise: some liposomes may interact with phages and compromise its biostability during storage; differences in size due to different methods used to produce liposomes can influence the release profile, loading capacity, biodegradation rate, stability over time, and biodistribution.^50,51

Regarding cell exposure to bacteriophages-containing liposomes, the MTT assay at 24 and 48 h showed that they are strongly cytotoxic at concentrations of 100 mM and higher (viability <40%). However, the trypan blue results (not shown) showed the opposite tendency since the viability percentages are high and no significant differences are found between the treatments and controls for any of the two times evaluated. When the trypan blue assay was performed, the total number of cells was very small because the cells were already lysed just after the treatment; therefore, we considered that the relationship between live and dead cells was not very accurate and decided to exclude it from the analyses.⁵²

In addition, it should be noted that the lipids used in this study are used in commercial formulations and do not represent any associated toxicity.⁵³ However, the shown cytotoxicity can be associated with the characteristics of the liposome, such as size and charge. In different studies, it has been found that liposomes with a positive charge and a large size (100 nm) can lead to cytokine activation,^54,55 which can implicate some drawbacks in their use.⁵⁶

Beyond the characteristics mentioned earlier, generally, the liposomes are considered safe, nontoxic, and biocompatible. Different formulations have been assayed together with some compounds to improve aspects such as drug delivery in cosmetic and pharmaceutical companies,²⁴ and they even have been used to trap toxic or unstable compounds. Some limitations in using bacteriophage therapy can be associated with the instability that those viral particles can suffer due to changes in pH, temperature, and ions.⁵⁷ Given this, it is necessary to improve phage availability in the desired site, which can be done by encapsulating them in a delivery vehicle such as a liposome. This approach was tried recently in 2018 by Chhibber et al., in which they demonstrated that encapsulation of the bacteriophages in liposomes could improve wound healing in diabetic mice with methicillin resistant Staphylococcus aureus infection. This indicates that the liposomes can help in avoiding the clearance of the bacteriophages, and thus help in the treatment of bacterial skin infections.⁴⁹

When studied in vitro, it has been observed that specific liposome formulations have different effects on the cell lines evaluated.⁵⁸ Owing to this, it is relevant to assess the different liposome-containing formulations with the target cell line, as performed in this study. We also evaluated the effect of all compounds used, such as the bacteriophages and both buffers, independently. As a result, we were able to determine that the liposomes at higher concentrations have a cytotoxic effect on cell viability and that this effect was not due to the nonliposome components of the treatment. Considering this, we found that 30 mM would be the recommended liposome concentration, since it leads to a cell viability reduction of <30%, which corresponds to a classification of noncytotoxic, according to ISO 10993-5.

In conclusion, our study showed that the bacteriophage Pa.7, in its free form and encapsulated in a controlled-30 mM concentration of liposome, is safe to be used on epithelial cells and opens possibilities for their use as an acne treatment according to the tests performed.

Authors' Contributions

D.T.D.B., M.J.V., and H.G.R. designed the study. D.T.D.B. performed the cell culture and viability tests. D.T.D.B. and D.M.N. analyzed the data and wrote the first draft of the article. D.T.D.B., D.M.N., M.J.V., and H.G.R. revised, corrected, and approved the final version of the article.

Author Disclosure Statement

M.J.V. declares she was in the past a member of the spin-off company SciPhage S.A.S., which works for the development of phage therapy in Colombia. She is also among the inventors of the patent no. NC2018/0008178, entitled “Composicionestópicas que comprendenbacteriófagos que se encapsulanenliposomas” (Topical compositions comprising bacteriophages encapsulated in liposomes). Other authors declare no competing interests.

Funding Information

This project was partially funded by “Fondo deInvestigaciones,” School of Sciences, Universidad de los Andes, project code INV-2020-105-2055.