A Novel Handrub Tablet Loaded with Pre- and Post-Biotic Solid Lipid Nanoparticles Combining Virucidal Activity and Maintenance of the Skin Barrier and Microbiome

Objective:
This study aimed to develop a holobiont tablet with rapid dispersibility to provide regulation of the microbiota, virucidal activity, and skin barrier protection.

Methods:
A 2³ factorial experiment was planned to define the best formulation for the development of the base tablet, using average weight, hardness, dimensions, swelling rate, and disintegration time as parameters to be analyzed. To produce holobiont tablets, the chosen base formulation was fabricated by direct compression of prebiotics, postbiotics, and excipients. The tablets also incorporated solid lipid nanoparticles containing postbiotics that were obtained by high-pressure homogenization and freeze-drying. The in vitro virucidal activity against alpha-coronavirus particles (CCoV-VR809) was determined in VERO cell culture. In vitro analysis, using monolayer cells and human equivalent skin, was performed by rRTq-PCR to determine the expression of interleukins 1, 6, 8, and 17, aquaporin-3, involucrin, filaggrin, FoxO3, and SIRT-1. Antioxidant activity and collagen-1 synthesis were also performed in fibroblast cells. Metagenomic analysis of the skin microbiome was determined in vivo before and after application of the holobiont tablet, during one week of continuous use, and compared to the use of alcohol gel. Samples were analyzed by sequencing the V3–V4 region of the 16S rRNA gene.

Results:
A handrub tablet with rapid dispersibility was developed for topical use and rinse off. After being defined as safe, the virucidal activity was found to be equal to or greater than that of 70% alcohol, with a reduction in interleukins and maintenance or improvement of skin barrier gene markers, in addition to the reestablishment of the skin microbiota after use.

Conclusions:
The holobiont tablets were able to improve the genetic markers related to the skin barrier and also its microbiota, thereby being more favorable for use as a hand sanitizer than 70% alcohol.

1. Introduction

Hand disinfection is an effective way to prevent a variety of diseases and has received global attention due to the recent pandemic. The Centers for Disease Control and Prevention (CDC) recommend the use of soap with water or alcohol-based handrubs (ABHRs) containing least 60% alcohol [1]. However, these sanitizing agents can negatively affect skin barrier function, causing an increase in transepidermal water loss (TEWL) and an acute loss of surface lipids and denatured proteins of the epidermis, mainly the stratum corneum. This can propagate an inflammatory response in the skin, resulting in skin diseases [1,2]. Although it is a necessary prevention measure, this recommended hygiene practice has, at the same time, increased the prevalence of inflammatory and allergenic contact dermatitis [3]. In addition, when the skin barrier is disrupted by long-term use of sanitizers, the beneficial bacteria in the skin flora can become virulent, increasing the risk of contamination [4]. The use of hand sanitizers with the addition of barrier protectants and avoiding new allergens are therefore recommended as an alternative to common detergents [5].

A plethora of microorganisms can be found on our hands, with bacteria being the most prevalent (>80% relative abundance), then viruses, and then fungi (<5% relative abundance) [6]. The most prevalent bacteria are from the Staphylococcaceae, Corynebaccteriaceae, Propionibacteriaceae, and Streptococcaceae families [7]. Information regarding viruses is more limited as the identification of viral groups on the skin is difficult, due to the low biomass obtained from skin samples [8]. It was reported that up to 94% of the viral sequences did not match a known viral genome in reference databases [9]. It is also worth noting that these studies focused solely on the DNA virome, and the RNA virome remains uncharacterized [8]. However, it is important to note that not all microorganisms are bad. An appropriate balance between a human as the host and its microbiota, which together are described as the holobiont, contributes to health and wellness [10]. Repeated use of hand sanitizer can upset this balance; therefore, one way of promoting or reestablishing the balance of hand microbiota might be to incorporate active ingredients as prebiotics and postbiotics, carefully selected based on their ability to stimulate the growth of natural skin microbiota on human hands.

We have considered the key elements in the design of a new approach for hand sanitizer that effectively disinfects the skin of harmful microorganisms whilst protecting the skin barrier and promoting a healthy skin microbiota for long term skin health.

First, consideration of an effective anti-infective agent. Quaternary ammonium compounds, such as cetyltrimethylammonium chloride, are the most useful and widely accepted antiseptics [11]. They are effective against viruses by the interaction of their cationic charges with the virus lipid membrane, causing membrane disruption and protein denaturation [12].

Second, an improved delivery system for the skin. Whilst most antiseptics are conventional formulations, antimicrobial nanocarriers have been studied for their potential to provide more effective deliveries. For example, Steelandt et al. [13] evaluated a chlorhexidine-loaded polymeric nanocapsule (CHX-NC) suspension formulation that was prepared by a new solvent-free process, easily freeze-dryable, and stable over one month storage. They showed that the in vitro inhibition of two bacterial strains (Escherichia coli and Staphylococcus aureus) and one fungal strain (Candida albicans) was equivalent between the nanocapsule formulation and chlorhexidine solution, with the former providing advantages for biocompatibility and ease of use. Hirao et al. [12] synthesized mesoporous silica particles filled with cetyltrimethylammonium chloride, a cationic surfactant with known antiseptic activity. They showed high loading and retention of the surfactant, with antiviral activity up to 3 months and the potential to use the particles in a range of applications, including wallpaper and air filters.

SLNs composed of biodegradable and safe lipidic components are also promising nanocarriers for controlled delivery of drugs and can be designed to carry a variety of hydrophilic and lipophilic substances within their internal core structures. SLNs also have the advantage of being solvent-free [13]. SLNs are composed of solid lipids, analogous to skin lipids, which are dispersed in an aqueous solution containing a surfactant as a stabilizer [14]. They are frequently used as a formulation strategy for cosmetic and therapeutic materials, to control the release, increase the bioavailability, and enhance the stability of active compounds [14,15]. Given the similarity of their composition to stratum corneum components, they are particularly useful nanocarriers for skin applications. SLNs can be freeze-dried before being incorporated into tablets for later reconstitution. Freeze-dried SLNs combined with surfactants can improve both the tablet formation and their subsequent dissolution [16,17]

Third, key ingredients that can promote the skin microbiota can be identified. Fatty acids are important for the maintenance of normal skin barrier function, reducing the loss of moisture from the skin, maintaining the metabolism of skin cells, and contributing to the skin microbiota [18,19]. Short-chain fatty acids (SCFAs), enzymes, peptides, and vitamins, described as postbiotics, can be collected as metabolic by-products secreted by live bacteria/yeast or released after bacterial/yeast lysis. They can positively affect microbiota homeostasis, thus affecting specific physiological and immunological reactions [20,21], and may be useful in preventing viral infections caused by the transmission of viruses to the hands after touching contaminated surfaces [20]. Prebiotics are non-digestible dietary fibers, including mainly carbohydrates that provide nutrition to selectively stimulate the activity and growth of normal skin microbiota. Some prebiotics, such as oligosaccharides and xylitol, can also promote skin health [22].

A fourth consideration is that the formulation development should be based on sustainability and promoting good environmental principles. There is a drive to reduce water in pharmaceutical, cosmetic, and personal care formulations through the manufacture of anhydrous or waterless products, such as sticks and tablets. These may be more economical and beneficial for skincare and the environment because they are less prone to microbial contamination, have a longer shelf life, need fewer preservatives, need less packaging, and are more potent due to a higher concentration of active ingredients than water-based cosmetics/pharmaceuticals [23].

Applying the considerations discussed above, the aim of this study was to develop a waterless hand sanitizer with easy topical application and storage for skin barrier maintenance, virus protection, and fast microbiome reestablishment. The postbiotics used were tyndallized Lactobacillus sp. and a combination of long-, medium-, and short-chain fatty acids [24], niacinamide, and alpha-glucan oligosaccharide. Polymnia sonchifolia root juice and maltodextrin were selected as prebiotics. A glycyrrhizin salt (dipotassium glycyrrhizinate) was included, as it has been reported to attenuate skin inflammation (such as pro-inflammatory cytokines Il-1b and Il-6) [25,26], thus contributing to skin barrier repair [27].

For a sustainable product, these bioactive compounds were combined in a handrub tablet that was easily dispersed by contact with water (0.5–1.0 mL) and did not require rinse-off after use. It was applied on hands as a leave-on product to continually sanitize the skin and feed the resident microbiota. The tablet was prepared by associating hydrophilic prebiotics with hydrophobic postbiotics that are pre-entrapped in SLNs. These SLNs can be attached to the skin surface due to the cationic properties of quaternary ammonium compounds, which also exert antiseptic activity against temporary viruses such as SARS-CoV-2, pathogenic bacteria, and fungi, together with the lipophilic postbiotics in the core of the nanoparticle responsible for the skin homeostasis recovery and overall skin health.

The approach taken in the study is shown in the flowchart (Figure 1).

2. Materials and Methods

2.1. Materials

Cetyltrimethylammonium chloride (Sunquart CT-50^®), palmitic acid, and stearic acid were purchased from Aqia (Guarulhos, Brazil); poloxamer 188 (Kolliphor^® P 188) and polyvinylpyrrolidone K30 (Kollidon 30^®) from BASF (São Paulo, Brazil); menthol from Mapric (São Paulo, Brazil); fragrance (Sunflower Fav-310939) from Fav 105 (Diadema, Brazil); silicon dioxide (Aerosil^®) from Evonik (São Paulo, Brazil); sodium croscarmellose (Solutab^®), magnesium stearate, and microcrystalline cellulose PH101 (Microcel^® PH101) from Blanver (Taboão da Serra, Brazil); xylitol, lauric acid, and caprylic acid from Química Anastácio (São Paulo, Brazil); hydroxypropyl cellulose (Klucel TM^®) from Ashland (São Paulo, Brazil); and glyceryl monostearate from Labsynth (Diadema, Brazil). Alpha-glucan oligosaccharide, Polymnia sonchifolia root juice, maltodextrin, Lactobacillus (Ecoskin^®), and alpha-glucan oligosaccharide (Bioecolia^®) were from Solabia (Maringá, Brazil); niacinamide (Niacinamide PC^®) from DSM (São Paulo, Brazil); glycyrrhizinate dipotassium from Cosmotec (Guarulhos, Brazil); and valeric acid, butyric acid, and oleic acid from Sigma-Aldrich (São Paulo, Brazil). All the reagents and solvents were analytical grade and were purchased from commercial suppliers.

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Machado, A.C.H.R.; Marinheiro, L.J.; Benson, H.A.E.; Grice, J.E.; Martins, T.d.S.; Lan, A.; Lopes, P.S.; Andreo-Filho, N.; Leite-Silva, V.R. A Novel Handrub Tablet Loaded with Pre- and Post-Biotic Solid Lipid Nanoparticles Combining Virucidal Activity and Maintenance of the Skin Barrier and Microbiome. Pharmaceutics 2023, 15, 2793.
https://doi.org/10.3390/pharmaceutics15122793

excipients formulation