Zoltán Géczi,Ivett Róth,Zsófia Kőhidai,László Kőhidai,Khaled Mukaddam,Péter Hermann,Dániel Végh,Tivadar Zelles
The use of Trojan-horse drug delivery system in managing periodontitis
Periodontitis,Silver,Folic acid,Drug development,Macrophages,Toxicity
The aim of this review is to evaluate the possibility of delivering a silver-acid complex via a Trojan-horse mechanism for managing periodontits. We theroised that the complex could be an effective treatment option for bacterial inflammatory processes in the oral cavity. Searches were conducted using MEDLINE, Embase, Web of Science Core Collection, and Google Scholar search engines. We also reviewed several reference lists of the included studies or relevant reviews identified by the search. By using Medical Subject Headings (MeSH) terminology, a comprehensive search was performed for the following keywords: silver, folic acid, periodontitis, macrophages, Trojan-horse mechanism, toxicity, and targeting. Using the keywords mentioned earlier, we selected 110 articles and after appropriate elimination the review was written based on 37 papers. Accordingly the we noted that silver isons were an effective approach to kill oral pathogens. Secondly the Trojan-horse mechanism. could be used by macrophages (as the Trojan horse) to deliver silver ions in large quantities to the inflammatory focus to kill the periodontopathogens. The Trojan-horse mechanism has never been described in the field of dentistry before. The proposed novel approach using the principle of Trojan Horse delivery of drugs/chemicals could be used to manage oral inflammatory conditions. This method can be used to supplement regular treatments.
The use of Trojan-horse drug delivery system in managing periodontitis

The aim of this review is to evaluate the possibility of delivering a silver-acid complex via a Trojan-horse mechanism for managing periodontits. We theroised that the complex could be an effective treatment option for bacterial inflammatory processes in the oral cavity. Searches were conducted using MEDLINE, Embase, Web of Science Core Collection, and Google Scholar search engines. We also reviewed several reference lists of the included studies or relevant reviews identified by the search. By using Medical Subject Headings (MeSH) terminology, a comprehensive search was performed for the following keywords: silver, folic acid, periodontitis, macrophages, Trojan-horse mechanism, toxicity, and targeting. Using the keywords mentioned earlier, we selected 110 articles and after appropriate elimination the review was written based on 37 papers. Accordingly the we noted that silver isons were an effective approach to kill oral pathogens. Secondly the Trojan-horse mechanism. could be used by macrophages (as the Trojan horse) to deliver silver ions in large quantities to the inflammatory focus to kill the periodontopathogens. The Trojan-horse mechanism has never been described in the field of dentistry before. The proposed novel approach using the principle of Trojan Horse delivery of drugs/chemicals could be used to manage oral inflammatory conditions. This method can be used to supplement regular treatments.

Periodontal disease is mainly caused by oral pathogens. Dental plaque, a biofilm made up of a diverse microbial population that forms on tooth surfaces, has been linked to an inflammatory reaction in periodontal tissues. Periodontal inflammation may cause bleeding, swelling, and discomfort. Gingivitis is a frequent, reversible inflammation of the gingiva's soft tissues, and it affects up to 95% of the world's population., Periodontitis is caused by the invasion of microorganisms or toxins and calculus, although the pathophysiology of the illness is mostly influenced by the human immune response., The way a given host reacts to these bacteria is determined by genetic, immunologic, and environmental variables. Whilst gingivitis may be treated with good dental hygiene and plaque eradication, periodontitis is an irreversible illness that affects half of the world's population. Furthermore, untreated gingival irritation may lead to alveolar bone resorption and tooth loss in the long run. As a result, prevention, early detection, and prompt treatment are always suggested. Folic acid (FA) is a nonimmunogenic, stable, and cost-effective ligand with a high affinity for the cell surface folate receptors (FRs). The folate enters the cells through receptor-mediated endocytosis and deposited in the cytosol. This technique of active targeting through the FRs may be used effectively for site-specific medication delivery whilst avoiding undesired side effects. One of the most commonly used molecule targeting agents is FA., This is a vitamin that is utilised by all cells to synthesise nucleotides. FRs are present in numerous cells and may be addressed selectively. For example, FR is expressed on active macrophages and is found in more than 40% of human malignancies, including ovarian and central nervous system–related tumours. These targets are appropriate for cancer and autoimmune applications. Due to the fact that FA has a very strong affinity for FRs (Kd = 107 mM), it can be used for a wide range of treatment methods.,, The phrase “Trojan horse” refers to a range of tactics used in drug research to deliver medications to the target in a secret, safe, and efficient manner. The main goals of this strategy are (1) to overcome barriers, such as the blood-brain barrier or simply the cell membrane, that protects the target (Troy) from a drug (the Greeks); (2) specifically target the disease rather than the entire body; and (3) to achieve a slow release of the drug over the time with no rapid concentration peaks. This type of medication has already been studied in other fields of medicine. Both theoretical and clinical studies have supported a new type of health care option based on the Trojan-horse mechanism in the care of other health problems (Figure 1).12, 13, 14, 15, 16, 17, 18 In order to manipulate macrophages in the therapies of rheumatoid arthritis (RA), this research is the first to use bioactive nano systems (without drug loading), offering a fresh perspective on using nanomaterials to treat RA. A target site for the therapies of periodontitis is the FR, a receptor on the cell surface. The 4 most prevalent subtypes of FRs are FR-α, FR-β, FR-γ, and FR- δ. For the targeted treatment of tumours, FR-α is applied. Inflammatory synovial tissues produced by activated macrophages overexpress FR-β, which has been used to specifically diagnose and treat RA. Researchers actively targeted M1 macrophages with FA-modified silver nanoparticles (NPs), which caused Ag+ to release under the influence of intracellular glutathione. This resulted in M1 macrophage apoptosis and reactive oxygen species (ROS) clearance, allowing M1-to-M2 repolarisation and decreasing inflammation to increase efficacy and biosafety. The aim of this paper is to illustrate the potential dental use of the Trojan-horse mechanism based on literature data. The role of silver and FA-silver complex in the treatment of the bacterially induced inflammations is explored. Our research group is currently still conducting in vitro tests on the synthesis and physicochemical characterisation of the FA-silver complex. According to our plans, as a next step we will publish the results of the characterisation and plan the cell physiology studies.

By using Medical Subject Headings (MeSH) terminology, a comprehensive search of online databases and literature was performed for the following key words: silver, folic acid, periodontitis, macrophages, Trojan-horse mechanism, toxicity, and targeting. Searches were conducted using the following search engines: MEDLINE, Embase, Web of Science Core Collection, and Google Scholar. Search dates were not limited. To ensure saturation of the literature, we also reviewed reference lists of included studies or relevant reviews identified by the search.

Two independent reviewers conducted a comprehensive search of the databases (TZ and ZG). Duplicates were deleted from all of the detected studies, which were exported to a citation manager.

Data were retrieved separately by 2 reviewers (TZ and ZG). The 2 coauthors (TZ and ZG) assessed the titles and abstracts of the papers and selected those that were suitable for inclusion in the full text, which the 2 authors assessed separately for details. Following a discussion, both reviewers agreed on all eliminated data, and any dispute was sent to a third reviewer (DV). Using the keywords mentioned earlier, TZ selected 110 articles and ZG 97 from the provided databases. Thirty-four of all the articles collected were selected by the 2 reviewers. The third reviewer (DV) helped in 5 cases. Finally, the review was written based on 37 papers (Table).

The current periodontitis diagnostic classification is organised into 4 phases and 3 grades based on severity and development. Once a patient's situation has been classified, a periodontal therapy technique may be selected. Conventional treatments for periodontal disease now include both nonsurgical and surgical methods, as well as extra medications or nutraceuticals. Achieving sufficient oral care at home is a critical component of periodontal disease prevention, effective periodontal treatment, and long-term dentition retention. Practitioners should educate the patient about the necessity of eliminating dental biofilm properly at home, particularly before beginning more invasive periodontal treatments.

The use of NPs including such as nanotubes, nanowires, fullerene derivatives, and quantum dots to produce new forms of analytical instruments in the domains of life science and biotechnology has recently received a lot of interest.,,, Amongst all nanomaterials, silver nanoparticles (AgNPs) are most utilised and may be regarded as one of the most significant. They are increasingly popular as a material for consumer goods., Currently, AgNPs are widely employed in health care and women's hygiene products, paints, skin care, the food sector, biosensors, medical equipment, sunscreen, clothes, electronics, environmental cleanup, and so on.,,, AgNPs are high-demand products in a variety of industries due to their distinct physical and chemical properties, as well as their antibacterial ability, which distinguishes them from bulk materials. For example, a high surface-area-to-volume proportion improves the surface characteristics of AgNPs, boosting their interaction with blood, saliva, mucus, and liquid components of the pulmonary barrier as compared to bulk particles. However, the significant oxidative activity of AgNPs produces silver ions, which cause cytotoxicity, genotoxicity, immunologic reactions, and even cell death in biological systems.,56, 57, 58, 59 It is described that silver has a higher toxicity towards prokaryotic cells than towards mammalian cells. Ag+ can be taken up by the cells and reach the cytoplasm through copper ion transports,; however, AgNPs are too big to pass via ion channels and are instead absorbed by phagocytic and endocytic routes. After uptake, AgNPs seem to be limited to membrane-bound vesicles such as early and late endosomes, as well as autophagosomes. AgNP-induced damage is shown in practically every part of the cell, including nuclei, the mitochondria, and the Golgi apparatus., Most studies concur that Ag+ cytotoxicity is caused by the production of ROS and the creation of oxidative stress.,,, AgNPs also enhance ROS and induce oxidative stress on proteins., It is unclear whether AgNP-induced damage varies from Ag+-induced damage,,, nor is it understood how AgNP or Ag+ damage is transported from the cellular membrane, including endosomes, to other compartments. Several investigations have shown that Ag+ is the major toxicant present in combinations of intact AgNPs and Ag+. Teeguarden et al discovered that the extracellular release of Ag+ was sufficient to elicit cytotoxic reactions of murine macrophages to AgNP stimulation. Finally, our integrated strategy to evaluate AgNP and Ag+ toxicity reveal substantial discrepancies in mammalian cell responses to AgNPs and Ag+. AgNPs cause cell death by a process that includes lipid peroxidation, proteotoxic damage, and necrotic cell death, whilst Ag+ exposure raises H2O2, which causes oxidative stress and apoptotic cell death. This study finds a distinct method by which AgNPs destroy mammalian cells that is not reliant on the contribution of Ag+ produced in the extracellular medium. Understanding exactly which elements induce AgNP toxicity is critical for biomedical applications, including such cancer treatment, as well as determining the implications of unintentional exposures,, (Figure 2).

Many nanomaterial combinations have been tested experimentally, utilising a variety of biological models.,, Limbach et al created the term “Trojan-horse mechanism” to describe an increase or prolongation of nanomaterial's damaging action compared to the corresponding metallic ions, either through catalytic properties of the nanomaterial or due to the prolonged cytoplasmic release of ions from the nanomaterial. The use of this phrase was extended to find the optimal carrier function of nanomaterials for chemicals, resulting in more efficient absorption of chemicals into organisms and enhanced toxicity. Similar to the concept we have outlined, FA-silver–containing complexes have already been addressed in the literature. These complexes have been investigated for their applicability in targeting therapy. They were found to be suitable based on the FA receptor–mediated uptake mechanism. Their use in dentistry has not yet been tested, but the same principle suggests that they may be effective for inflammation of the oral cavity. In our further in vitro studies, we aim at synthesis and physiochemical characterisation. To deliver medications directly to target cells, one strategy is to tie them to cell-penetrating NPs or monoclonal antibodies. With 10 US Food and Drug Administration–approved antibody-drug compounds (ADCs), many of which are in clinical trials, this approach has already been shown to be effective, although it is still being refined. Up until recently, all licensed ADCs delivered cytotoxic chemicals that act on either DNA or tubulin and are too cytotoxic to be administered unconjugated and have significant adverse effects. Cetuximab saratolacan sodium was approved in Japan this year, and a worldwide phase 3 study is currently underway. This dye is only cellular-damaging when exposed to locally supplied near-infrared light and hence safe even whilst circulating unbound throughout the body.,, Another strategy is to inject medications directly into or onto entire cells such as erythrocytes, platelets, leukocytes, macrophages, and progenitors or into vesicles (eg, microvesicles and exosomes). The benefit of these cell-based technologies over synthetic nano- and microcarriers is that they are extremely biocompatible, biodegradable, and nonimmunogenic (if donor–recipient paired) and have the ability to pass through otherwise impenetrable barriers. Macrophages are another Trojan horse that is not yet clinically tested but is gaining traction as a possible delivery mechanism., These immune cells’ intrinsic homing ability to target injured, diseased, or cancerous tissue makes them ideal candidates for the guided administration of medications in low concentrations, thereby lowering the likelihood of drug-associated side effects even more. Many methods of macrophage-mediated therapeutic delivery centre on drug-loaded nanoparticles, although anticancer viruses and antisense oligonucleotides are also being researched. Because of their phagocytic qualities, loading into macrophages is easy (in most situations) and may be adjusted by altering the shape, size, or surface property of the cargo; nevertheless, several additional hurdles must be overcome. The limitations of utilising macrophages as carriers include drug toxicity to the carrier cells, disruption of macrophage activities (eg, response to chemotactic cues), achieving regulated treatment release and, most critically, maintaining cargo integrity.,,

There is still no gold standard for the treatment of periodontitis that can be applied in all cases and solve all problems. In addition to the surgical line, there is an increasing emphasis in research on the development of nonsurgical therapies. The topic examined by our group is the application of the Trojan-horse mechanism in periodontal lesions. We would now like to formulate a new approach on a theoretical level for the treatment of bacterially induced inflammation. There is a growing focus in research on the development of nonsurgical treatments. Our research group is looking at the use of the Trojan-horse mechanism in periodontal diseases. The essence of the novel strategy is that, on the one hand, we would have a direct toxic impact on pathogenic bacteria by utilising the existing well-proven silver ions and AgNPs reported in the literature., Silver ions have this harmful impact by entering the cell via ion channels and increasing the concentration of H2O2 there, therefore activating apoptotic processes. AgNPs, on the other hand, cannot enter ion channels due to their size. These are more likely to enter the cell through the endosomes. Once within the cell, silver ions can be released, causing the aforementioned apoptotic route to be activated, or AgNPs can activate the necrotic pathways. Cell death is involved in both of these methods. This harmful impact is shared by pathogenic flora as well as the body's own cells. Regarding the use of our material, we would apply it directly in the case of periodontal inflammation. So, it is effective locally at killing pathogenic flora and also against other cells that keep inflammation going. Second, this effect should be mentioned as a side effect. As with all antimicrobial substances, side effects must be considered. This duality defines all similar forms of research, in which the composition must be found when it has the highest antibacterial activity whilst having the least toxic effect. One essence of our concept is to exploit the mechanism of action described below for pathogens. The other novel mechanism needs to be approached from 2 directions. One is the Trojan-horse mechanism. This essentially means creating a complex with one component that is responsible for the antimicrobial effect and the other component helping to get that substance into the cell. In our case, the substance responsible for the effect is silver. The substance responsible for entering the cell, however, is FA. The number of FA receptors on the surface of oral pathogens is not significant, because they are able to produce themselves. This shows the antimicrobial effect of free silver ions and AgNPs against pathogenic flora. However, the number of macrophages in active inflammatory processes increases drastically. On the other hand, these macrophages can also be used as a tool for therapy. Another mechanism of the complex we have outlined is the regulation of these macrophages. The formation of highly changeable phenotypes in answer to microenvironmental cues is required for the macrophage's functional variety. Macrophages are divided into 2 types: classically activated macrophages, known as M1 macrophages, which are associated with proinflammatory activation and mediate host defense against viruses and bacteria, and alternatively activated macrophages, known as M2 macrophages, which have anti-inflammatory and pro-healing functions. The M1 macrophages are activated in the presence of interferon (IFN)-γ and increased concentrations of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-β, IL-1β, and IL-6, whereas M2 macrophages can be primed by IL-4/13 and are related with anti-inflammatory metabolic markers arginase (Arg)-1, CD206, CD163, and IL-10., Macrophages definitely play an important role in generating inflammatory illness and oral cancers, as shown by the wide range of behaviours they exhibit in response to specific variables in their microenvironment. The ratio of macrophages is greater in patients with chronic periodontitis than in those with gingivitis, and it is associated significantly with pro-inflammatory cytokines matrix metalloproteinases -9 and interleukin-1β. Meanwhile, in the chronic periodontitis group, probing depth is associated with an increase in the ratio of M1/M2 phenotypes of macrophages. In periodontitis, the phenotypic flip seems to be towards M1 macrophages, whilst in gingivitis, it appears to be towards M2 macrophages. As a result, analysing the M1/M2 ratio may offer significant information on the health of periodontal tissue. The number of FA receptors (FR-β) on the surface of these macrophages increases significantly when they are activated. The FA-based silver complex theoretically has the ability to bind to these FA receptors.,, After binding, it may cause toxicity after entering the cytoplasm (Figure 3). When combined with other periodontal therapies, the complex has the potential to decrease inflammation and enhance healing. This material does not solve the periodontal disease on its own. Professional and at-home cleaning of tooth surfaces as well as surgery in more severe instances are required. On the other hand, the FA-silver combination employs novel mechanisms of action not previously exploited in periodontology rather than traditional mechanisms of action. The next step is the process of laboratory synthesis and physicochemical characterisation. The synthesis is followed by characterisation of the material (dynamic light scattering, atomic force microscope, infrared spectroscopy, 3D spectroscopy, antimicrobial studies, etc).

This work shows the potential of FA-AgNPs for targeted periodontal treatment via synchronous M1 macrophage apoptosis and M1-to-M2 macrophage repolarisation and declares the first instance of using bioactive NPs for treating periodontitis without loading any drugs. The mechanism of action of the FA-silver complex involves mechanisms that have already been widely described in the literature (silver ion, AgNP), as well as a novel research possibility, the Trojan-horse mechanism. The Trojan-horse mechanism has never been described in the field of periodontology before. This is a novel approach to treating bacterially caused oral inflammatory conditions. The mechanism of action is based on the control of M1 and M2 macrophages, which are engaged in inflammation and hence impact inflammation indirectly. This methodology has already appeared in other areas of medical research but has not yet been associated with oral inflammation. This innovative method might be used to supplement mechanical cleaning and surgical treatment. Our next goal is to clinically evaluate the hypothesis of trojan horse mechanism described above.

None disclosed.