Propolis Controlled Delivery Systems for Oral Therapeutics in Dental Medicine: A Systematic Review

This systematic review synthesizes the existing evidence in the literature regarding the association of propolis with controlled delivery systems (DDSs) and its potential therapeutic action in dental medicine. Two independent reviewers performed a literature search up to 1 June 2023 in five databases: PubMed/Medline, Web of Science, Cochrane Library, Scopus, and Embase, to identify the eligible studies. Clinical, in situ, and in vitro studies that investigated the incorporation of propolis as the main agent in DDSs for dental medicine were included in this study. Review articles, clinical cases, theses, dissertations, conference abstracts, and studies that had no application in dentistry were excluded. A total of 2019 records were initially identified. After carefully examining 21 full-text articles, 12 in vitro studies, 4 clinical, 1 animal model, and 3 in vivo and in vitro studies were included (n = 21). Relevant data were extracted from the included studies and analyzed qualitatively. The use of propolis has been reported in cariology, endodontics, periodontics, stomatology, and dental implants. Propolis has shown non-cytotoxic, osteoinductive, antimicrobial, and anti-inflammatory properties. Moreover, propolis can be released from DDS for prolonged periods, presenting biocompatibility, safety, and potential advantage for applications in dental medicine.


Introduction
Odontogenic infections, such as dental caries, periodontal diseases, endodontic infections, and dental abscesses are very common problems in dental medicine, with caries as the most common infectious dental disease in the world. They mainly involve an interaction between the microbial biofilm and tooth structure or oral tissues. When not properly treated, these infections can promote inflammation and consequently irreversible damage to oral tissues [1]. In general, conventional treatments involve removing the affected tissue and replacing it with filling materials and surgical approaches [2]. Despite these treatments being effective, they may not be ideal as they do not biologically replace the lost tissue. So, the development of new alternative methods aiming to provide antimicrobial, antiinflammatory, and biological properties during the treatment of oral lesions are considered promising in dental research [2]. However, these methods also include systemic or local administration of high doses of drugs leading to antibiotic resistance, patient sensitivity, and possible side effects. In this context of aiming to reduce these drawbacks, the field of drug delivery systems (DDSs) has grown considerably in the last decades [3].

Protocol and Registration
This systematic review was carried out following PRISMA statement guidelines [30]. It was registered on the Open Science Framework platform under registration DOI 10.17605/OSF.IO/V9FXT.

Research Question (PICO)
The research question (PICO) was "Is there an influence on the incorporation of propolis in drug delivery systems comparable to drug delivery systems with other substances or without propolis?", where the following items where observed: • P: the drug delivery systems DDSs • I: application and efficacy of propolis in drug delivery systems, in dental medicine • C: comparison between DDSs without propolis extracts and/or DDSs with other substances • O: effectiveness of propolis-based DDSs for dental medicine • S: clinical, in vitro and in vivo studies.

Eligibility Criteria and Selection Process
Investigations using propolis in DDSs or combined with other biomaterials, molecules, or stem cells in the dental medicine field were selected. The inclusion criteria were papers evaluating propolis-based DDSs for biomaterials. The following items were considered as exclusion criteria: literature reviews, clinical cases, case reports, dissertations, thesis, conference abstracts, and studies that evaluated the actions of propolis-based DDSs in areas other than dentistry.

Data Collection Process
Abstracts were carefully appraised; studies that met the inclusion criteria or had insufficient data available in the title or abstract were selected for a full-text analysis. Disagreements reported on the eligibility of the included articles were resolved by consensus and by a third reviewer (M.F.). Reference lists of all the included studies were also hand-searched for additional studies.

Data Items
The study information, such as demographic information, enrollment criteria, study design, aims of the study, application in dentistry, type of biomaterials, type of propolis, characterization and origin, toxicity assessment, drug release, main results, presence of controls, and sample size were extracted by the reviewers (A.B. and M.F.).

Study Risk of Bias Assessment and Synthesis of Results
The risk of bias of the included studies was analyzed according to the RoBDEMAT tool [31] for laboratorial analysis. The clinical studies were analyzed according to Robbins-I [32] to non-randomized trials and Rob 2 to randomized clinical trials [33]. In addition, a qualitative synthesis of results was performed based on individual studies and is presented in the next section.

Study Selection
A flowchart illustrating this review's search and selection is presented in Figure 1. The search resulted in the retrieval of 2019 articles. After the database screening and removal of duplicates, 1264 studies were identified. Then, 26 titles were screened, a careful examination of the full texts was performed and assessed to check if they were eligible for this systematic review. As a result, four studies were excluded because they did not fit the inclusion criteria and 21 studies were selected.
A flowchart illustrating this review's search and selection is presented in Figure 1. The search resulted in the retrieval of 2019 articles. After the database screening and removal of duplicates, 1264 studies were identified. Then, 26 titles were screened, a careful examination of the full texts was performed and assessed to check if they were eligible for this systematic review. As a result, four studies were excluded because they did not fit the inclusion criteria and 21 studies were selected.

Study Characteristics
The characteristics of the included studies are described in Table 2. The studies were published between 2007 and 2021. Brazil was the country with the highest number of studies on propolis-based DDSs in dental medicine. Fourteen studies were conducted in vitro, followed by four randomized clinical trials, clinical and two in vitro studies, and only one evaluated under in vitro and in vivo (animal model) conditions.

Study Characteristics
The characteristics of the included studies are described in Table 2. The studies were published between 2007 and 2021. Brazil was the country with the highest number of studies on propolis-based DDSs in dental medicine. Fourteen studies were conducted in vitro, followed by four randomized clinical trials, clinical and two in vitro studies, and only one evaluated under in vitro and in vivo (animal model) conditions.
Regarding application in dentistry, six studies had application in periodontics (periodontal pockets and guided tissue regeneration), followed by six in oral medicine (oral lesions), four in endodontics (pulp protection), two in cariology (anti-cariogenic agent), one in implantodontics, one in regenerative dentistry (hard tissue), and one in control oral infection. Eleven studies used the ethanolic extract of propolis, two used aqueous extract (2), one used ethyl alcoholic, and one used hydroalcoholic solution (1). All studies reported the origin of the propolis used, except three studies [18,34,35].

Results of Individual Studies and Results of Syntheses
Of the twenty-one selected studies, only three studies evaluated the toxicity of the materials. All studies had a control group during the tests pmed. Chlorhexidine was the most commonly found control substance (three studies). Only one study did not report the sample size [19]. The animal model study did not report the sample size calculation, despite reporting that they followed international protocol for studies in animal models.
Concerning drug release, propolis can be released from systems for long periods up to 7 days [20,29,36]. The main results reported the use of propolis in infected periodontal pockets, as it results in the production of higher quality secondary dentin with a lower inflammatory response [29], revealing a role in tissue regeneration as in the study by Simu et al. (2018) [8] which demonstrated an essential osteoinductive effect for mineralized tissue repair.     The results indicated the potential of electrospun fibers to be used as mouthdissolving fibers for effective antibacterial activity in the oral cavity. The ethanol extract of propolis produced the antimicrobial activity in the film as well as provided a better resistance matrix and increased mucoadhesiveness.    In studies involving periodontal [20][21][22] and endodontic [9,14,29] diseases, formulations containing propolis indicate a potentially beneficial anti-inflammatory and antimicrobial effect. Some studies show the antimicrobial activity of propolis against Grampositive [17,18] and Gram-negative bacteria [21], especially Streptococcus mutans [15,18,23], in addition to its antifungal potential against Candida albicans [19]. Some bacteria, such as Streptococcus pyogenes and S. mutans, showed greater susceptibility to propolis compared to metronidazole [24]. Other studies [15,23] have evaluated the ability to prevent cariogenic biofilm compared to gold standard antibacterial agent chlorhexidine and antifungal (nystatin). Further to this, Borges et al. (2015) [25] reported that its incorporation increased the strength of the film matrix and mucoadhesiveness. In addition, it was reported that propolis reduced the inflammatory response and showed no side effects [11,35] which may have promising results especially in the field of stomatology, such as in the treatment of aphthous ulcers and lichen planus [35][36][37][38][39].

Risk of Bias in Studies
In vitro studies exhibited a high risk of bias concerning sample randomization, evaluation blinding, and sample size calculation (Table 3). However, a low risk of bias was observed in terms of presence of control group, statistical analysis, outcome reporting, and analysis standardization between groups. Non-randomized clinical study demonstrated a low risk of general bias (Table 4). For the randomized clinical trials (RCTs), a low risk of bias related to randomization [34,35,37], selection of reported outcomes, and measurement of outcomes was reported (Table 5).

Discussion
Numerous drug delivery systems (DDSs) have been developed for the local treatment and prevention of several diseases in the oral cavity [3,42]. These systems are a safe option as they drastically reduce the adverse reactions due to low doses administered directly at the site of action [11]. In addition, DDSs increase stability and solubility, which is interesting for the use of natural extracts [43]. Among natural compounds, propolis is an advantageous alternative to be used in DDSs due to its biodegradable nature, high tissue compatibility, and long-term release [29]. Therefore, the present study presents scientific evidence for the incorporation of propolis in controlled delivery systems as a therapeutic agent in dental medicine.
Propolis is widely recognized for its antimicrobial and anti-inflammatory properties. Although few studies were found using propolis in controlled drug systems in dentistry, it is possible to observe the diversity of areas in dentistry in which propolis can be applied for therapeutic purposes. Propolis composition can vary according to geographic and environmental conditions in which it is collected, as well as the solvents and parameters used during its extraction [23,25]. Therefore, spectrophotometric methods are important to characterize and standardize compounds present in propolis [29].
Propolis has three main compounds: flavonoids, cinnamic acid derivatives and terpenoids. The cinnamic acid derivatives, also known as phenolic compounds of propolis, include caffeic acid, rutin, quercetin, apigenin, chrysin, ferulic acid, cinnamic acid and galangin [29]. Flavonoids are a very important class of polyphenols, as they are plant compounds that have antimicrobial, antioxidant and anti-inflammatory properties [24]. Their anti-inflammatory property stimulates phagocytic activity and cellular immunity. Propolis contains zinc and iron metal cations, which are essential during collagen synthesis, flavonoids and phenolic acid esters, that are effective in reducing the inflammatory response by inhibiting the arachidonic acid lipoxygenase pathway. In addition to its significant effect on the immune system, they promote cellular phagocytic activities [29]. The caffeic acid phenethyl ester (CAPE) also has a cytoprotective function and protects against the oxidative effects of inflammatory DNA pathologies [8]. One of the discussed possible mechanisms of the antimicrobial activity of propolis is the cinnamic acid and flavonoid components, that changes the ion permeability of the inner bacterial membrane causing membrane potential dissipation and inhibition of bacterial motility [18].
One of the main characteristics attributed to propolis in the literature is its antimicrobial action [13,15,19,23,24]. The oral environment is populated by a multi-species ecosystem, some of the pathogens in the oral cavity are Streptococcus mutans, Staphylococcus aureus, Streptococcus sobrinus, and Candida albicans, which are involved in most infectious diseases of the mouth. It is well known that prevention plays an important role in caries management, therefore, anticaries activity of propolis is also demonstrated in the literature [15,23]. Asawahame et al. (2014) [23] proposed a DDS prepared using electrospinning. This DDS is biodegradable in wet environments, thus when in contact with the saliva it easily degrades. This system showed better antibacterial activity against Streptococcus mutans when compared to commercial mouthwashes and lower activity when compared to chlorhexidine. Additionally, in another study [15], the incorporation of propolis in sustained-release chitosan varnish enabled an increasing antimicrobial activity against Streptococcus mutans, Streptococcus sanguinis, Streptococcus salivarius, Lactobacillus casei, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum compared to chlorhexidine 0.12%, chitosan-based varnish, and nystatin. In this study, sustained-release of propolis from chitosan-based varnishes showed to be a promising alternative for use in biomaterials formulations for dental caries prevention.
Another important application of propolis-based DDSs is in regenerative endodontics. The material of choice for the treatment of infectious endodontics must have an antimicrobial activity without impairing the regenerative process [8]. Studies report that propolis extract as an intracanal medication was more effective against Enterococcus faecalis compared to a mixture of tri-antibiotics [41,44], and that it showed greater antimicrobial activity associated with calcium hydroxide [34,44]. In a study that evaluated the use of propolis in endodontic therapy, the action was equally effective compared to the triple antibiotic, 2% chlorhexidine gel, and calcium hydroxide with propylene glycol against Candida albicans after 7 days at both depths into the dentinal tubules [14].
The lasting antimicrobial effect of propolis is justified by its low solubility, this is an important aspect if the biomaterial is introduced into an area of low vascularization where systemically administered antibiotics can scarcely work [8]. Studies [20,34] have demonstrated that ethanolic extract of propolis was continuously being released after 7 days. In another similar study, release profile studies demonstrated that propolis can be released from systems for an extended period (over 20 days) [45]. The heterogeneity of the results on drug release becomes a limitation of the study. These results are due not only to the origin of propolis but also to the lack of standardization of methodologies and biomaterials (DDSs) tested, according to the purpose of the application. However, there is a clear consensus that propolis can be used for long periods in DDSs.
In the study by Balata et al. (2018) [29], biodegradable chitosan chips loaded with Saudi propolis extract were developed as a controlled delivery system for pulpotomy. In this study, propolis lead to a total or partial reduction in inflammation, absence of necrosis, and greater formation of hard tissue compared to the use of formocresol. This result is consistent with other studies [46,47] that report propolis induced complete hard tissue barrier formation in pulpotomies. This can be elucidated by the anti-inflammatory activity of propolis, which promotes collagen synthesis by dentine pulp cells and stimulates the production of transforming growth factor (TGF)-1 as well as by the free radical and superoxide neutralizing components released by propolis [29].
The literature also reported the application of propolis-based DDSs in periodontics. Periodontal disease is a chronic infection resulting from a tissue response to a complex biofilm, and affects the supporting structures of the teeth (periodontium) [18,21]. One of the treatments for this condition is the systemic administration of medications and local mouthwash solutions. However, in this therapy high concentrations are used for prolonged periods, posing the risk of side effects and the emergence of resistant strains [20,22,36].
This condition also arouses interest in dentistry in the development of DDSs. Propolis becomes an excellent option due to its prolonged release in these systems and, especially, its antimicrobial activity. De Souza Ferreira et al. (2013) [24] investigated mixed propolis and metronidazole microparticles, which demonstrated in vitro antimicrobial activity against all tested strains, namely E. faecalis ATCC 51299, E. faecalis ATCC 29212, S. pyogenes ATCC 19615, S. mutans ATCC 25175, S. aureus ATCC 25923, K. pneumoniae ATCC 700608, and E. coli ATCC 25922. These microparticles have the advantages of low cost and a variety of dosage forms that can be incorporated as semi-solid systems, and administered in periodontal pockets more easily and safely. In one study [18], biodegradable polymeric PLLA/PCL films with propolis were developed for the application of guided tissue engineering and showed antibacterial activity against Staphylococcus aureus. The porosity of the substrate is essential to promote an environment of cell proliferation, the formation of new tissue, and improve vascular invasion [48]. The incorporation of propolis modified the surface topography of the films, increasing the porosity, which may be beneficial for adhesion [18]. It was also demonstrated that the addition of propolis increased the surface area associated with a fiber morphology arrangement, allowing the encapsulation and fixation of cells, which also allows prolonged release of propolis in periods longer than 48 h, making it a promising material in the engineering of mineralized dental tissues [8,11].
Due to its anti-inflammatory property, propolis can also be a support therapy in cases of oral lesions which inflicts pain and discomfort, such as recurrent aphtous stomatitis (RAS), denture stomatitis (DS), and other ulcerative conditions [35,39,40,49]. RAS has an unknown aetiology and is symptom-based, it presents as a painful rounded ulcer surrounded by an erythematous halo, while DS is a chronic disorder that affects denture-bearing patients and is associated with fungal infection (Candida albicans) [35,[38][39][40]49]. Besides promoting antimicrobial activity to fight candida infection, the anti-inflammatory activity of propolis has been shown to reduce outbreak frequency, reduce ulcer size, promote a faster healing and pain relief, and therefore improve quality of life in those patients. A muco-adhesive film was prepared with propolis extract and applied to the lesion site and patients reported a longer lasting pain relief and higher overall satisfaction with the treatment, compared to placebo [38]. A 500 mg propolis or placebo capsule was administered to RAS patients for six months. Patients who received propolis daily presented a reduction in outbreak frequency and improvement in quality of life [37].
Among the engineering of mineralized dental tissues, the prolonged release of propolis over a month indicated that it could inhibit these dental pathogens in implants long-term, according to Son et al. (2021) [19]. One of the compounds responsible for the antimicrobial action of propolis, cinnamic acid derivatives, showed good stability in orally disintegrating films over twelve weeks, thus, proving to be an ideal substance for release in the oral mucosa and to control infections [25]. The use of natural actives in nanofibers has been validated for the manufacture of adhesives for oral mucosa abrasions and to fight inflammation. Propolis reduced the size of the fibers and, when released, activated hydroxyl groups present in the oral mucosa that tend to form deprotonated species at alkaline pH, providing negative charges with the ability to increase drug solubility and bioaccessibility [11].
According to the main characteristics needed to succeed in DDSs, nanosized particles are advantageous due to their size and are therefore easier to penetrate and overcome barriers at the cellular level. To provide a more efficient pharmacological therapy of oral pathologies, they can also provide bioadhesive properties that respond to stimuli through intelligent systems, as long as the particles are biocompatible and biodegradable [32]. Incorporation of new drug delivery technologies for natural products actives reduces drug degradation, minimizes side effects from cytotoxic products in non-target regions, and facilitates administration in pediatric and geriatric patients [7].
It is expected that shortly, the use of controlled delivery systems for the treatment of odontogenic and non-odontogenic diseases, in particular the use of nanoparticulate formulations, will become routine in clinical practice. It is irrefutable that there are some complexities involved in translating laboratory-developed biomaterials to industry. For this to occur, more methodologies evaluating these materials are needed as well as more government efforts to make the legislation more efficient in approving biomaterials aiming to amplify the development and commercialization of advanced drug delivery platforms.

Conclusions
It can be concluded that there is a beneficial impact on the incorporation of propolis in drug delivery systems. Although there is evidence of antimicrobial, anti-inflammatory, and regenerative activities in preclinical studies, more in-depth studies including the toxicity of this substance, a detailed physicochemical characterization, and genotoxicity assessment of biomaterials containing propolis as DDSs are necessary to ensure safe use in humans. Moreover, clinical studies must be performed to confirm the effectiveness of propoliscontaining delivery systems. Overall, the authors envisage that this systematic review can aid and orientate further studies concerning the use of propolis in dental applications.