Nanomaterial-based drug-delivery system as an aid to antimicrobial photodynamic therapy in treating oral biofilm

Abstract

Diverse microorganisms live as biofilm in the mouth accounts for oral diseases and treatment failure. For decades, the prevention and treatment of oral biofilm is a global challenge. Antimicrobial photodynamic therapy (aPDT) holds promise for oral biofilm elimination due to its several traits, including broad-spectrum antimicrobial capacity, lower possibility of resistance and low cytotoxicity. However, the physicochemical properties of photosensitizers and the biological barrier of oral biofilm have limited the efficiency of aPDT. Nanomaterials has been used to fabricate nanocarriers to improve photosensitizer properties and thus enhance antimicrobial effect. In this review, we have discussed the challenges of aPDT used in dentistry, categorized the nanomaterial-delivery system and listed the possible mechanisms involved in nanomaterials when enhancing aPDT effect.

Plain language summary

Executive summary.

Background

• aPDT has been advocated as a promising alternative approach against oral biofilm, with the advantages of multiple targets, low possibility of resistance development, and minimal cytotoxicity to host cells.

• Oral biofilm harbors several important traits that make them difficult to amend, including abundant EPS matrix, quorum-sensing systems, multi-species interaction and resistance to drugs.

• The extraordinary locations of the oral cavity such as the lateral canal, deep periodontal pocket, root furcation and screwed-implant surface are not easily accessible for external interventions.

• aPDT is characterized as a procedure that involves the photochemical reaction of PS, light source and oxygen, which ultimately generates ROS.

• PSs can be categorized into porphyrin dyes, chlorophyll dyes, phthalocyanine dyes, phenothiazinium dyes, xanthene dyes, curcumin and phenalenone dyes. These current PS are hydrophobic and tend to aggregate in an aqueous solution, leading to poor bioavailability and fluorescence quenching.

Recent developments of nanomaterials associated with aPDT in the treatment of oral infection

• The versatile features of nanomaterials, such as ultra-small size, large surface area, and highly reactive surface, could be utilized as drug-delivery systems to modify PS properties and improve aPDT effect.

• The encapsulation or incorporation of PS into nanomaterials has achieved several improvements, such as improved bioavailability and solubility of PS, enhanced increase of ROS yield, targeting of infection sites, improved therapeutic action of PS and so on.

Possible mechanisms involved in nanomaterials enhancing aPDT effect

• There are several possible mechanisms involved in nanomaterials enhancing aPDT effect, including increase of microbial cell membrane permeability, disruption of EPS formation, increase of ROS quantum yield and increase of PS's penetration depth.

Future perspective

• Despite a number of studies reporting positive results of nanomaterials-based PSs, there is a great deal of work needs to be undertaken in order to overcome the limitations and challenges of nanomaterials as a drug-delivery system for PS.

Approximately 700 species of microorganisms comprise the whole oral microbiome, with an estimated number of 50–200 species per person. The oral cavity offers different residing sites for microorganisms, such as tooth surfaces, the mucosal epithelium of the cheeks, the hard and soft palate and the gingival crevice. In addition to distinct ecological niches, the oral cavity is an ideal ‘incubator’ where the microbes could survive in a warm, moist and nutritious microenvironment. Under such circumstances, microorganism starts to increase, aggregate in close proximity with each other, and form a well-organized structure community called biofilm. The dental biofilm refers to multi-species microbial communities on oral surfaces encapsulated in a self-produced extracellular polymeric substances (EPS) matrix [1]. These microbial communities generally establish a harmonious relationship with the host. However, disturbances in the interactions between oral microbes, the microenvironment and the host may transform the equilibrium state into a pathogenic state. The imbalanced state in the oral cavity leads to microbial composition change, toxic metabolites accumulation of pathogens and subsequent pathogenic biofilm formation [2,3]. The oral biofilm is considered as the prominent factor responsible for the development of many oral diseases [4]. Other oral diseases, such as endodontic infection, peri-implantitis and denture stomatitis, mainly originate from the oral biofilm.

Mechanical approaches in combination with antimicrobials are well-accepted therapeutic regimens to disinfect oral biofilms, however, cases of secondary biofilm infection are still reported [5]. The resistant oral infection is possibly due to the repeated systemic administration of antimicrobials and the subsequent selection of resistant strains [6]. In the past decades, innovative strategies were widely explored to reduce bacterial resistance. Antimicrobial photodynamic therapy (aPDT) has been advocated as a compelling alternative against oral biofilm and attracted substantial interest, with the advantages of multiple targets, low possibility of resistance development and minimal cytotoxicity to host cells [7,8]. aPDT is a noninvasive, nontoxic and clinically approved approach that mainly involves the interaction of photosensitizer (PS), light source and oxygen. The principle of aPDT is that PS binds to target cells and undergoes a series of photochemical reactions upon the specific wavelength of a light source, resulting in the generation of reactive oxygen species (ROS) that is deleterious to microbes [9]. Figure 1 depicts, in detail, the principles of aPDT. During aPDT process, the photoactivated PS would finally generate hydroxyl radical (OH), superoxide (O^2-), hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂). The burst of oxidative stress due to ROS could cause photo-oxidation of cellular macromolecules such as lipids, proteins and nucleic acids [10]. Mounting evidence suggests that aPDT has the capacity to eliminate biofilms formed by various microorganisms in vitro and improve the clinical outcome of conventional therapy in treating endodontic infection, periodontitis or peri-implantitis [^11-13]. Though the achievements of aPDT in oral infectious diseases treatment are significant, some obstacles still exist and hinder its wide clinical applications.

Figure 1.

The principles of antimicrobial photodynamic therapy reviewed in previous studies.

Initially, the ground state of the PS transforms into the unstable singlet excited state with the irradiation of the light source. Then the excited singlet state of the PS would convert into a triplet state via intersystem crossing. The long-lived triplet state PS undergoes two chemical reactions defined as type I and II photodynamic mechanisms. In type I, PS could undergo electron transfer with the cellular substrates to form PS radicals, and then the PS radicals would react with molecular oxygen, resulting in the formation of hydroxyl radical (OH), superoxide (O^2-) and hydrogen peroxide (H₂O₂). Alternatively, the triplet PS transfers energy directly to the ground state triplet oxygen, yielding a more chemically reactive substance called singlet oxygen (¹O₂).

First, the oral biofilm is embedded in an impermeable and dense EPS matrix which serves as a nutrient source for biofilm cells and acts as a physical and biochemical barrier against exogenous stimuli [14]. Moreover, the biofilm can regrow and persist after anti-biofilm treatment owing to its cell-to-cell communication pathways, presenting a significant challenge in oral biofilm elimination [15]. Bacteria or other microorganisms could communicate with each other via quorum sensing signaling molecules and this behavior could increase their tolerance toward antimicrobials and survival [16].

Second, the structures and characteristics of the oral cavity also present a challenge to aPDT application. For instance, the oral biofilm could colonize some extraordinary locations such as the lateral canal, deep periodontal pocket, root furcation and screwed-implant surface [17,18]. When treating oral diseases, the PS or the light source should enter into sites where oral biofilms originated from. However, the wavelengths of light source matching by the current PS are in the scope of visible light whose penetrating depth is limited at 0.5 cm. Thus, the photodynamic effect is hindered by the weakening light intensity accompanied by the increase of tissue depth. Additionally, the dentinal tubules with a narrow lumen and considerable length are not accessible to the reach of PS or a light source. The oxygen concentrations in the deep periodontal pocket and dentinal tubules are too low to provide sufficient oxygen molecules for photochemical reactions.

Thirdly, the current PSs used in dental filed can be categorized into porphyrin dyes, chlorophyll dyes, phthalocyanine dyes, phenothiazinium dyes, xanthene dyes, curcumin and phenalenone dyes. Different physiochemical properties of most PSs compromise their wide application in biological tissue. PSs are hydrophobic and tend to aggregate in an aqueous solution, leading to poor bioavailability and fluorescence quenching. The generated ROS has a short lifespan and diffusion length in the biological environment. Moreover, high concentrations of PSs with dark toxicity to surrounding host cells defy the safety and selectivity of aPDT. Based on the physiochemical challenges of aPDT and the biological challenges in the oral cavity (Figure 2), it is crucial to improve the photochemical properties of current PSs to achieve better antimicrobial performance in oral biofilm elimination.

Figure 2.

Biological and physiochemical challenges of antimicrobial photodynamic therapy when treating oral biofilm.

Nanomaterials, characterized by their homogeneous particles within the 1–200 nm range, are increasingly employed as drug-delivery systems. These include liposomal nanomaterials, micelle-based nanomaterials, polymer nanomaterials and inorganic nanomaterials, leveraging their unique structural, chemical, magnetic and biological properties. The key advantage of nanomaterials lies in their small and controllable size, allowing for the transport of diverse molecules and facilitating entry into anatomical locations inaccessible to traditional drugs. Furthermore, nanomaterials harbor some specific structures or functional ligands, rendering them ideal for drug delivery purposes (e.g., magnetic nanoparticles responding to external magnetic fields). Given the diminishing efficacy of traditional drugs due to their resistance mechanisms and biofilm formation, the application of nanomaterials as carriers represents a promising strategy. Researchers have demonstrated that nanomaterials can overcome challenges like efflux pump resistance and reduced drug uptake in certain bacteria [19]. The use of nanomaterials in drug delivery allows targeted antibiotic concentration at infection sites, minimizing systemic side effects commonly associated with traditional antibiotic use. Owing to the benefits of using nanomaterials as a delivery system, the introduction of nanomaterials into aPDT field has aroused dramatic interest of researchers.

The objectives of this review were to: elucidate the biological and physicochemical challenges associated with aPDT in the context of controlling oral infections; present recent advancements in nanomaterial-based aPDT against oral infections; explore how nanomaterials enhance the effectiveness of aPDT as drug-delivery systems; and discuss the challenges and future perspectives associated with nanomaterials-based aPDT.

The search for this literature review was conducted on PubMed and included publications from January 2012 to December 2022. Search strategies were mainly constructed on these key words: (‘photodynamic therapy’ OR ‘photodynamic inactivation’ OR ‘photodynamic antimicrobial chemotherapy’) AND (‘nanoparticle’ OR ‘nanomaterial’ OR ‘nanoplatform’) AND (‘oral biofilm’ OR ‘oral infection’ OR ‘caries’ OR ‘endodontic infection’ OR ‘periodontitis’ OR ‘peri-implantitis’). Both in vitro and in vivo experiments (animal models and human studies) were included. Only articles published in English were considered. The exclusion criteria included non-English papers, reviews, commentaries, letters to the editor, interviews and updates.

Recent developments of nanomaterials associated with aPDT in the treatment of oral biofilm & its induced diseases

So far, many scientists have been ironing out these issues by introducing nanomaterials into aPDT field (Figure 3). Thanks to the versatile features of nanomaterials, such as ultra-small size, large surface area and highly reactive surface, they could be utilized as drug-delivery systems to modify PS properties and improve aPDT effect. As a number of studies have confirmed the beneficial role of nanomaterials in aPDT field, we would like to give a summary of recent progress on how nanomaterials enhance the effect of aPDT on oral biofilm and its induced diseases. Supplementary Table 1 has listed some recent developments of nanomaterials associated with aPDT in the treatment of oral biofilm and its induced diseases.

Figure 3.

Organic and inorganic nanoparticles for PS delivery in antimicrobial photodynamic therapy field.

Liposomes

Liposomes have emerged as promising candidates for drug-delivery systems against pathogens and biofilms. Liposomes are spherical vesicles composed of one or more phospholipid layers of cholesterol with an aqueous inner core. Concerning the application in aPDT field, liposome encapsulation could offer many advantages. First, liposomes could deliver hydrophobic and hydrophilic PS and enhance PS uptake [20]. As the phospholipid layer of liposomes is similar to the bacteria cell membrane, liposomes with specific composition (phosphatidyl ethanol amine) could fuse with the phospholipid membranes of bacteria and then deliver the PS into the cell directly [21]. Second, liposomes could interact with biofilm in nonspecific and specific ways to achieve targeted affinity with biofilm [22]. The nonspecific interactions are described as the interaction between cationic liposomes and negatively charged bacteria lipopolysaccharides (LPS) or biofilm EPS. As for specific interaction, liposomes are usually grafted with functional groups that express high affinity to specific locations on the target biofilm.

The failure of root canal treatment often lies in the inadequate disinfection of bacterial biofilm in the deeper sites of dentin tubules. Ossmann and co-workers incorporated the PS 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in liposomes and invasomes, two transdermal penetration nanocarrier [23]. They hypothesized that the tissue penetration ability of these two nanocarriers could promote the delivery of mTHPC into dentin tubules. They demonstrated that both formulations of mTHPC result in a significant antibacterial effect against Enterococcus faecalis (E. faecalis) biofilm especially in aPDT with invasomes. mTHPC loading with invasomes presented a higher antibacterial effect than free mTHPC on disinfecting the endodontic biofilm located at the dentin depth of 50 μm. Using liposomes as a nanocarrier presents a notable advantage in enhancing the solubility of mTHPC and facilitating greater penetration of mTHPC molecules into dentin tubules compared with direct injection of mTHPC into the root canal. The surface charge of the liposome determines its behaviors when applied as a PS nanocarrier. The benefit of using liposomes in the aPDT field was supported in a clinical report [24]. The study first investigated the photocytotoxicity of aluminum chloride phthalocyanine entrapped in cationic liposomes (AlClPc) on cariogenic bacteria and eukaryotic cells. Experimental data showed the AlClPc-mediated aPDT induces significant cytotoxicity against bacteria culture, not pulp cells. Additionally, the study also recruited ten patients presenting class I cavitated carious lesions involving two-thirds of the dentine. The cavity was treated with atraumatic restorative treatment (ART) protocol followed by aPDT application mediated by AlClPc or not. The microbial analysis showed that aPDT protocol produced significant disinfection in all cavities treated. Liposomes as a nanodelivery system can improve some features of PS, enhance the bioavailability of PS, and facilitate enhanced cellular uptake of PS by bacteria. These collective findings underscore the efficacy of liposomes as a nanocarrier for optimizing the delivery and performance of PS in oral biofilm treatment.

Nanoemulsions

Emulsions are defined as multiphase dispersion systems where one liquid (dispersion phase) is dispersed in another (continuous phase) in the form of droplets in the presence of an emulsifying agent. Nanoemulsions refer to emulsions with dispersible diameter in the nanometer range that is usually below 500 nm [25]. Owing to the small size of liquid droplets, the nanoemulsion system possesses thermodynamic stability, high solubilization characteristics and a large specific surface area to improve bioactive drug-release kinetics [26].

Biphasic nanoemulsions can be classified into water in oil or oil in water, which is used to disperse the encapsulated hydrophobic PS in aqueous solutions and biological environments. With respect to the studies of nanoemulsion-assisted aPDT in oral biofilm, chloroaluminium phthalocyanine was the most widely used PS. The hydrophobic characteristic and aggregation behavior of chloroaluminium phthalocyanine compromise its photophysical properties and restrict its antimicrobial effect. A number of studies have evaluated the effect of nanoemulsion-delivered chloroaluminium phthalocyanine against Porphyromonas gingivalis (P. gingivalis), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Candida albicans (C. albicans) and multispecies biofilm [27,28]. These results suggested that nanoemulsion-based strategy could exert broad antimicrobial capacity. In another study, Carmello et al. evaluated the photoinactivation of C. albicans in a murine model of oral candidiasis using chloro-aluminum phthalocyanine encapsulated in cationic nanoemulsions [29]. The results showed cationic nanoemulsion encapsulation contributes to the dispersion of phthalocyanine, improves the inactivating ability of C. albicans on the tongue dorsum of mice, as well as increasing the remission of candidiasis lesions. These collective findings suggested that nanoemulsion delivery could improve the aggregation behavior of hydrophobic PS and therefore promote aPDT killing on pathogens. Since aggregation would impact the photochemical properties of PS, deactivating the excited state decreases the effectiveness of aPDT.

Polymer

The most extensively explored biodegradable polymer as a nanocarrier is poly-lactide-co-glycolide (PLGA). The characteristic features of PLGA in drug delivery include excellent biocompatibility, biodegradability and minimal biological toxicity [30]. It has been evidenced that PLGA nanoparticles for drug loading or entrapment could guarantee sustained drug release, prolongation of drug release, pharmacokinetics modification, increased antimicrobial effect and reduced dosage for effective treatment [31]. For PS delivery, it also offers the benefit of quenching the excited state of PS to reduce photocytotoxicity during dark incubation.

The encapsulation of various PSs by PLGA-based nanoparticles has been successfully applied in photodynamic treatment (PDT) field for cancer therapy. For instance, the loading of 5-aminolevulinic acid (ALA) into PLGA nanoparticles resulted in an encapsulation efficiency of 65.8% and better photo cytotoxicity than free PS toward a human skin squamous cell carcinoma cell [32]. Another study compared the delivery system based on PLGA nanoparticles and polyvinylpyrrolidone (PVP) nanoparticles for the delivery of hypericin derivative, the results suggested that PLGA nanoparticles achieved superior biodistribution and higher-cancer killing capacity when compared with PVP nanoparticles [33]. In addition to the effectiveness of PLGA nanoparticles in cancer treatment has been well documented, the applications of PLGA nanoparticles encapsulated with PSs have also garnered attention in the aPDT field. Klepac-Ceraj et al. reported functionalized MB with anionic and cationic PLGA and tested these derivatives for aPDT inactivation against dental plaque [34]. Their results showed that cationic nanoparticles released MB oleate much faster than anionic nanoparticles. Besides, MB encapsulated within cationic PLGA exhibited more efficient photosensitization ability on oral microorganisms than anionic PLGA-loaded and free MB. Another research team group synthesized curcumin nanoparticles encapsulated in anionic and cationic PLGA by the nanoprecipitation method [35]. The encapsulation efficiency was high while curcumin release from nanoparticles was relatively slow. They compared the antimicrobial activities of new compounds and free curcumin on planktonic cultures, monospecies biofilm, mixed-species biofilm and in a murine model of oral candidiasis infection. Planktonic and biofilm samples were prepared from Streptococcus mutans (S. mutans), C. albicans and methicillin-resistant Staphylococcus aureus (MRSA) while oral candidiasis was induced by C. albicans. Though cationic PLGA encapsulation showed a comparable or even higher antimicrobial effect as free curcumin, the overall trend was that PLGA encapsulation did not significantly boost the antibacterial effect of curcumin in various infection models. Moreover, the anionic curcumin nanoparticle displayed the lowest antimicrobial photodynamic effect. This finding was explained by the authors who pointed out the fact that the negatively charged biofilm matrix could generate repulsion against anionic external agents and reduce the antimicrobial effect of anionic nanoparticles. With regard to the antimicrobial photodynamic effect of PS encapsulated by PLGA nanoparticles in vivo, a clinical plot study has treated periodontitis patients with mechanical scaling or mechanical scaling plus aPDT mediated by MB-PLGA nanoparticles [36]. They evaluated the clinical parameters (probe pocket depth, gingival bleeding index at probe time) of both groups after 1 month of treatment. Data showed that all parameters in two groups showed a similar increasing trend. However, mechanical scaling plus MB-PLGA nanoparticles photoactivation has a greater effect on the gingival bleeding index than mechanical scaling alone.

In conclusion, these findings suggested that PLGA nanoparticles have the potential to act as a PS delivery system for aPDT. However, the antimicrobial photodynamic effect of PS encapsulated by PLGA nanoparticles is not always inspiring, even sometimes the encapsulation could not give rise to higher antimicrobial effectiveness.

Chitosan

Another widely employed polymeric nanoparticle in biomedical applications is chitosan. Functional groups of chitosan endow it with diversified biological properties including antimicrobial activity, antifungal activity and biocompatible features. It is well documented that chitosan has great potential to inhibit the growth of bacterial, fungal single-species biofilm and even cross-kingdom biofilm in dentistry [37,38]. The most recognized antimicrobial mechanism of chitosan is that it can alter the cell permeability of bacteria cell membranes due to its intrinsic positive charge that could interact with negatively charged bacteria surfaces [39,40]. Furthermore, these functional groups contribute to the modification of chitosan, enhancing its mechanical and physical properties.

The intrinsic antimicrobial activity of chitosan has inspired scientists to encapsulate PS with chitosan to maximize the antimicrobial capacity, which has been tested in several articles. In an attempt to improve the antimicrobial effectiveness of anionic PS, the team group of Shrestha et al. have conjugated Rose Bengal (RB) with chitosan nanoparticle and done a series of work to investigate the potential of this new compound, CSRBnp, on root canal infection. In the first paper, in order to testify to the anti-biofilm efficacy of CSRBnp on Gram-negative bacteria, they established Pseudomonas aeruginosa (P. aeruginosa) biofilm and treated it with CSRBnp photoactivation [41]. The findings showed that the anti-biofilm activity of CSRBnp increased by nearly 5 logs in comparison with free RB against P. aeruginosa biofilm. The increased absorption of RB by biofilm coated with chitosan nanoparticles was believed to be a possible mechanism for enhanced antimicrobial ability. Then they evaluated the antibacterial activity of CSRBnp against mature E. faecalis biofilm formed on dentin slices and the cytotoxicity on mouse fibroblast cells [42]. The results suggested that CSRBnp showed characteristics of higher biocompatibility and antimicrobial activity. The mechanisms proposed for the potentiated antimicrobial activity of CSRBnp include the increased binding of RB to bacterial cells and the reduced release rate of ¹O₂. Interestingly, this study also found that photoactivation of CSRBnp results in crosslinking of dentin-collagen, incorporation of chitosan nanoparticles within the collagen architecture, and improved mechanical properties. Further research by their team demonstrated the ability of photoactivated CSRBnp on multispecies biofilm elimination, endotoxin inactivation and resistance to biological degradation [43,44]. Based on the aforementioned properties of CSRBnp, the team group first treated maxillary anterior teeth with extensive root resorption with CSRBnp photoactivation [45]. Their follow-up results demonstrated that CSRBnp photoactivation could be beneficial for root canal disinfection, dentin matrix stabilization and the deposition of reparative cementum/bone-like tissue on root defects.

Chitosan nanocarriers can also encapsulate other PSs to treat oral infections. Cavalcante et al. incorporated chloroaluminium phthalocyanine into chitosan nanoparticles to allow more interaction between biofilm and PS [46]. They treated S. mutans biofilm by chloroaluminium phthalocyanine conjugating with chitosan nanoparticles, demonstrating the increased anti-biofilm activity due to chitosan encapsulation. Another study has depicted that the encapsulation of Indocyanine Green (ICG) into chitosan nanoparticles significantly enhances the effect of aPDT on A. actinomycetemcomitans biofilm [47]. Furthermore, the ability of new synthesized PS to monitor the virulence factor of A. actinomycetemcomitans was also reported. Chitosan could be employed as a drug carrier in the form of nanoparticles, hydrogels, films and membranes. Özodogan et al. prepared chitosan gels loaded with atorvastatin for rat periodontitis treatment [48]. They demonstrated that the chitosan delivery system could prolong the drug duration, increase the drug dosage of periodontal pockets and thus enhance the anti-inflammatory effect than using atorvastatin alone. The popularity of using chitosan as a nanocarrier in aPDT field may be attributed to: the ability to contribute to PS adsorption by bacterial cells owing to its surface charge; the synergistic antibacterial effects produced by chitosan and aPDT; being biocompatible and biodegradable to make it safer option versus conventional systemic drug delivery.

Upconversion nanoparticles

Upconversion nanoparticles (UCNPs) are a sum of solid-state materials doped with rare earth elements, presenting optical properties for photon upconversion. The structure of UCNP usually consists of an inorganic host matrix, a sensitizer and an activator. The inorganic host matrix provides essential and unique optical properties and the most common inorganic matrix of UCNPs in biological applications is NaYF₄. In order to increase the photon upconversion efficiency, two different lanthanide elements are commonly co-doped into the inorganic matrix. One of them acts as a sensitizer, absorbs the incident light source and is excited to transfer energy to another activator, thus emitting fluorescence [49]. UCNPs can convert near-infrared (NIR) light into UV or visible light that could activate the existing PSs for cytotoxicity ROS generation. This characteristic corresponds to anti-stokes emission, where the higher energy light can be easily achieved with two or low-energy photons absorption [50].

Despite the broad antimicrobial spectrum of aPDT, one of the major limitations of aPDT in vivo is that the photoexcited ROS could not enter deep tissue due to the poor penetration of the light source. The current available PSs (porphyrins, chlorins, phthalocyanines) are best excited by blue and green light rather than red light with good penetration. The advent of lanthanide-doped UCNPs with lanthanide rare earth elements is just solving the problem. To date, a variety of PSs, have been loaded with UCNPs to circumvent the limited penetration depth of the original excited light. For an efficient therapeutic index, the emission spectrum should match the excitation wavelength of the loaded PS. Meanwhile, proximity between PS and UCNPs should be assured for an efficient energy transfer from the core to the PS. UCNPs offer several advantages in PS delivery. First, the increased penetration depth is achieved under the NIR excitation light used for up-conversion luminescence [51]. The optical penetration depth of light into the skin is lower than 1 mm under the blue or green light irradiation (400–500 nm). When receiving NIR light irradiation, the penetration depth into the skin surface increases to 1 cm. Moreover, the small size of UCNPs allows the entrapped PS to surpass the biological barrier, obtaining better penetration into tissues or organs. Second, different emission wavelengths could be exploited simultaneously for the activation of various PSs. For example, Lee and co-workers developed a core@shell structure UCNPs (NaYF₄: Yb, Er, Nd@NaYF₄:Yb, Nd), which emits multiple luminescence bands at 407, 520, 539 and 653 nm [20]. The loaded RB and Chlorin e6 (Ce 6) could be activated by blue and red emissions to generate ¹O₂, respectively. Third, some other advantages should be mentioned, such as narrow emission bandwidth, resistance to photobleaching, high stability and low toxicity [52].

Several groups have investigated the antimicrobial capacities of PSs incorporated by UCNPs. Titanium oxide (TiO₂) is a large band-gap semiconductor that could generate ROS and exert bactericidal activity on oral pathogens [53]. However, due to the excitation spectrum of TiO₂ being limited to UVA region, TiO₂ photocatalytic activity has a poor tissue penetration depth. As periodontitis often occurs in the deep pockets, it is of utmost importance to deliver light into these deep locations. Qi et al. described the antimicrobial activity of UCNPs-doped with TiO₂ against periodontitis-related biofilm pathogens [54]. It was expected to take advantage of the deep penetration of NIR and excellent photocatalytic activity of TiO₂ by coating TiO₂ on the UCNPs surface. They showed a significant decrease in the microbial number of planktonic and biofilm cells (Streptococcus sanguinis, P. gingivalis, Fusobacterium nucleatum) when exposed to UCNPs@ TiO₂ nanoparticles and NIR light source, whose effect is more efficacious than Toluidine Blue O (TBO)-mediated aPDT. The authors also reported that the surface of UCNPs@ TiO₂ exhibited a positive charge conducive to its adherence to Gram-negative pathogens and hydroxyl radicals produced by TiO₂ photocatalytic activity are more sensitive to Gram-negative bacteria. The aPDT effects of UCNPs@ TiO₂ could be beneficial for the treatment of periodontal diseases treatment caused by Gram-negative bacteria. Ce6 is another PS that has been loaded by UCNPs to treat periodontitis-related pathogens. Zhang et al. combined Ce6 and NaYF₄: Yb, Er UCNPs using the amphiphilic silane modification technique [55]. With this modification strategy, the new composite became hydrophilic and stable, which could avoid Ce 6 leakage and potential cytotoxicity. They tested the antimicrobial photodynamic effect of the new compound on three 4-day single-strain biofilm. The outcomes from the CFU assay showed that the three bacterial CFU counts of 4-day biofilm all have a logarithmic reduction of more than 2 logs. Enhancing light penetration into diseased tissue is vital for the aPDT treatment in periodontitis, and so is endodontic infection. Persistent endodontic infection is located in the deeper tissue such as apical periodontium, and E. faecalis is the etiologic pathogen of endodontic infection. Zong and his colleagues prepared a novel triple-layered core-shell nanostructure UCNP@SiO₂/MB@quaternized chitosan (QCh) and investigated the photodynamic potential of a newly synthesized compound [56]. They proved UCNP@SiO₂/MB@QCh could stick close to the bacteria and generate ROS to destroy E. faecalis biofilm.

In comparison to conventional PS delivery, using UCNPs for PS delivery offers many benefits. For instance, UCNPs could help PS penetrate tissues more deeply than visible light. In addition, the use of NIR light sources for excitation reduces the potential for phototoxicity compared with ultraviolet or visible light. UCNPs exhibit high photostability, which means they could protect PS from quick degradation. UCNPs are generally made from hydrophobic organic ligands with poor solubility. This may result in low upconversion efficiency and low aqueous solubility, which limit the practical application of UCNPs. For this fact, many endeavors have been made to conjugate UCNPs with polymeric substances, silica coating, or other functional groups to increase solubility and improve antimicrobial activity [57]. Moreover, the biosafety of UCNPs lacks unanimous conclusions. The composition of UCNPs is some unusual elements in our bodies, which may release due to matrix dissolution and then react with biomacromolecules to cause toxicity.

Magnetic nanoparticles

Magnetic nanoparticles (MNPs) have been extensively explored in biomedical applications such as magnetic resonance imaging, drug delivery, biosensing and magnetic hyperthermia [58]. Being versatile nanoparticles, MNPs harbor a large surface-to-volume ratio to carry drugs or adsorb small molecules. MNPs have an extraordinary superparamagnetic property that responds to external magnetic fields. Under the guidance of external magnetic fields, the MNPs could be directed to specific areas and separated from the mixture timely. This obvious advantage enables MNPs to achieve targeted therapy and detect their biodistribution. Furthermore, MNPs do not tend to aggregate since they do not exhibit any magnetism without an external magnetic field [59]. In addition to these properties, MNPs display their intrinsic antimicrobial activities by releasing metal ions, leading to irreversible protein damage and subsequent microbial cell damage [60]. Superparamagnetic iron oxide NPs (SPIONs) are regarded as one of the most promising MNPs, which could penetrate S. mutans biofilm and cause bacterial cell death [61].

Based on these properties of MNPs, there is increasing interest in applying MNPs as nanocarriers in the aPDT field. For example, Bilici and his co-workers have loaded ICG with 3-amino-propyltrimethoysilane (APTMS)-coated SPIONs and explored the aPDT and photothermal therapy (PTT) potential of newly synthesized compound (ICG-APTMS@SPION) [62]. The distinctive feature of SPION is associated with their absorption in the near-infrared region and conversion into heat. Their results demonstrated that ICG-APTMS@SPION induces more ROS production and an increase in the local temperature upon laser irradiation compared with free ICG. The ICG-APTMS@SPION exhibited a significant bactericidal effect on Gram-positive and Gram-negative bacteria. Another experiment has conjugated curcumin with SPIONs and explored its antibacterial potential [63]. Under SPIONs conjugation, the curcumin became dispersed quickly. Furthermore, the new conjugate displayed heating power and photodynamic action under light irradiation and a magnetic field light. However, to the extent of our literature search, the combination of MNPs with aPDT technology tacking oral biofilm was poorly investigated. Magnetic-responsive PS-based nanoplatforms were first designed and applied to treat oral biofilm in 2018 [64]. Sun et al. developed a multifunctional nanoparticle containing Ce6, Coumarin 6 and Fe₃O₄ MNPs. They found new nanoparticle has an inhibitory effect on periodontal pathogen and reduce biofilm CFU by about four to five orders of magnitude. Additionally, Fe₃O₄ with magnetic field enabled the targeting of infection sites. Balhaddad et al. constructed a nanoplatform integrating TBO and SPIONs via a microemulsion method, which allows TBO molecules to penetrate deep sites under external magnetic forces [65]. In addition to favoring the penetration depth, the new nanoplatform also offered some advantages like enhancement in water solubility and stability of PS, better compatibility, and improved disinfection ability. By establishing S. mutans and saliva-derived multispecies biofilm on dentin slabs, the authors demonstrated the antibiofilm effectiveness of this new platform on monospecice biofilm and mature biofilm. Overall, MNPs present advantages over traditional drug delivery, like magnetic targeting, site-specific targeting, enhanced penetration and retention of PS, implementation of controlled release and imaging capabilities. However, MNPs for drug delivery is still questionable since the manufacturing cost is too high to implement into clinical practice.

Silver nanoparticles

Silver (Ag) nanoparticles are among the most used inorganic nanoparticles in antimicrobial applications. The killing mechanism responsible for Ag nanoparticles is the continual release of Ag ions [66]. Ag ions can be anchored to cell surfaces tightly through electrostatic interactions. The released Ag ions would alter the cell membrane permeability and disrupt the cell envelope. Once Ag ions enter the cell, the respiratory enzymes in the cell membrane are activated to produce a large amount of ROS. The excessive accumulation of ROS, especially hydroxyl radicals, in cells leads to the oxidation and damage of DNA, lipids and proteins. Based on the antimicrobial properties of Ag nanoparticles, many researchers have applied them widely in dentistry.

It is demonstrated that Ag nanoparticles act synergistically with antibiotics, antifungals, bacteriocins, or natural compounds to exert a more potent effect [67]. In this way, it is expected that Ag nanoparticles combined with PSs would give rise to a more pronounced antimicrobial effect. Several papers have focused on coating PS with Ag nanoparticles in the treatment of oral bacteria and biofilm. For instance, Shitomi et al. designed the nanocomposite of sliver nanoclusters (AgNCs) and RB and investigated its antibacterial activity [68]. The characterization experiments demonstrated that AgNCs/RB could generate ¹O₂ and release Ag ions after illumination. The irradiated AgNCs/RB displayed the inhibition ability on S. mutans, P. gingivalis and A. actinomycetemcomitans and had no adverse effect on NIH3T3 mammalian cells. As for oral biofilm, Misba et al. reported the antibiofilm action of TBO-silver nanoparticle conjugate on S. mutans [69]. The authors formulated two kinds of AgNPs, AgNPs coated with citrate and AgNPs coated with dextran. Increased antibiofilm viability against S. mutans was observed on both TBO-AgNP conjugates when compared with TBO alone. Additionally, the inhibition of S. mutans biofilm formation and the destruction of S. mutans biofilm structure of TBO-AgNP conjugates were also confirmed by crystal violet staining and confocal laser scanning microscope.

Quantum dots

Another type of organic nanoparticle, quantum dots (QDs), has also been extensively studied for applications in aPDT. As QDs are created from semiconductors made of different elements, they possess unique optical and emission properties, which enable them to serve as PS. The small size (2–6 nm) of QDs leads to the free distribution of electrons in valence and conduction bonds. Upon appropriate light irradiation, the electrons in valence bands may move to conduction bands, generate free electrons and form the electron–hole pairs, resulting in the yield of free radicals for bacterial killing [70]. Graphene, graphene oxide and TiO₂ are frequent QDs used in biomedical applications. Moreover, QDs are also utilized as a delivery tool to carry antibiotics, antibodies and drugs beyond their photochemical properties [71]. The high surface area, high photostability, and ease of functionalization of QDs contribute to the loading of PSs into the surface [72,73].

Some studies have tested the aPDT effect of QDs in combination with PS and demonstrated their ability on oral biofilm disinfection. For example, Gholibegloo et al. have loaded ICG with various nanocomposites, including graphene oxide (GO), GO-carnosine, and GO-carnosine/ hydroxyapatite [74]. The addition of GO could protect ICG from aggregation, while the presence of hydroxyapatite was confirmed to play a role in enhancing ICG stability. The antimicrobial photodynamic activity of the loaded ICG was significantly higher than the unformulated ICG. Another report synthesized graphene quantum dots (GQD) and coupled them with curcumin, aiming to overcome the shortcomings of curcumin and obtain higher antimicrobial capacity without high concentration usage of curcumin [75]. They conducted a series of experiments to characterize the properties of GQD-Curcumin, and evaluated the biocompatibility and the antibiofilm effect on perio-pathogens mixed biofilm. The findings suggested that GQD-curcumin photoactivation reduces the viable microorganisms of mixed biofilm and downregulates the virulence factors of biofilm.

The potential advantages of QDs as nanodelivery system can be summarized in the following aspects. QDs are highly photostable, meaning they can withstand prolonged exposure to light. Moreover, QDs generally have high quantum yields to maintain longer time for bacterial inactivation. Additionally, QDs exhibit strong and stable fluorescence, making them excellent imaging agents for tracking and visualizing the delivery process. Though these data presented a relatively inspiring outcome, several drawbacks of QDs should bear in mind. The low absorbance of QDs in higher wavelengths of light source may merely be applicable for skin and mucosal infection rather than deep tissue infection. Another important issue that needs to be solved is the toxicity of QDs at the molecular, cellular and tissue level.

Other nanocarriers

Mesoporous silicas nanoparticles (MSNs) have been adopted as a loading platform for various drug delivery. Several features endow MSNs with potential in drug delivery. An ideal drug nanocarrier should meet requirements such as a controlled drug release rate, massive drug adsorption and loading and specific targeting. Concerning the structures of MSNs, the ordered pore structure and narrow pore size distribution make them regulate drug release. Additionally, large surfaces and areas of MSNs contribute to the adsorption efficiency of the drug. Toluidine blue (TB) is sensitized to red light and has a broad antimicrobial spectrum. However, they have poor bioavailability and tend to aggregate in solution. Parasuraman et al. encapsulated TB into MSNs and investigated its antimicrobial photodynamic potential [76]. The loading capacity of MSN is 15.02% and the entrapment efficiency is 74.14%. The encapsulation of TB by MSNs is demonstrated to increase TB binding efficiency and uptake, improve antibacterial capacity against Gram-positive and negative bacteria. Silanol groups in MSNs enable them to conjugate other functional groups and become multifunctional drug carriers. For instance, Dai and his co-workers successfully prepared a novel conjugate by loading ultrasmall copper sulfide nanoparticles (Cu₂–xSNPs), fluorescein isothiocyanate (FITC) and ε-polylysine (PLL) onto MSNs through a layer-by-layer self-assembly approach [77]. Owing to the grafting of the bioactive moieties into MSNs, the synthesized novel conjugate could adhere to the bacteria surface more tightly and damage microbial cells more severely compared with the free form.

As far as we know, carbon nanotubes (CNTs) were discovered and synthesized by Japanese scientist in 1991. Then CNTs have become a research hotspot in the field of nanomedicine materials owing to their unique structures, such as high surface area, high conductivity, and thermal conductivity [78]. Due to the large specific surface area of CNTs, their conjugated bonds can deliver drugs onto the surface through non-covalent interactions such as π–π bond stacking or physical encapsulation. Besides, CNTs could produce free radicals and result in oxidative death of microbial cells by contacting their cell membrane directly [79]. Conjugating PS with CNTs is demonstrated to facilitate bacterial uptake of PS, limit PS efflux and realize controlled PS release. Photoactivated porphyrin–multi-walled carbon nanotubes (MWNTs) conjugates were used to defeat viral infection [80]. The result suggested that porphyrin–MWNTs dramatically reduce the viability of Influenza A virus under light irradiation without triggering antimicrobial tolerance. Anju et al. functionalized ICG with MWNTs and studied its antibacterial effects on P. aeruginosa and Staphylococcus aureus (S. aureus) biofilm [81]. They found that the newly synthesized PS mainly exerts stronger antibacterial effects by increasing ROS production.

MSNs and CNTs are potential candidates for PS delivery with wide biological application. However, the story of these nanomaterials-based aPDT for oral biofilm elimination is not unfolded and future research can be conducted in this direction.

Possible mechanisms involved in nanomaterials enhancing aPDT effect

Though the outcomes of nanomaterial-based delivery systems as an aid to aPDT are encouraging, the involved enhancing mechanisms are barely reviewed. Figure 4 depicts several possible mechanisms of nanomaterial-based delivery systems in improving aPDT effect.

Figure 4.

List of mechanisms involved in nanomaterials enhancing an antimicrobial photodynamic therapy effect.

Increase the permeability of microbial cell

There are three main types of microorganisms in the oral cavity, Gram-positive bacteria, Gram-negative bacteria and fungi. The cell wall components of them are various. For Gram-positive bacteria, their cell wall mainly consist of porous layers of peptidoglycan, teichoic acid and lipoteichoic acid. Since peptidoglycan represents up to 90% of the cell wall of Gram-positive bacteria and is relatively porous, most PSs could pass through the Gram-positive bacteria cell wall with little difficulty. By contrast, the cell wall of Gram-negative bacteria is mainly composed of a unique outer membrane, a thin peptidoglycan layer, and a cytoplasmic membrane. The outer membrane is a bilayer structure consisting of phospholipids molecules, LPS, and lipoproteins, acting as an effective barrier to limit PS penetration. The fungal cell wall consists of mannoproteins, chitin and β-glucans. The fungal cell wall shows an intermediate permeability between Gram-positive and Gram-negative bacteria. Direct photoactivation of Gram-negative bacteria is much harder than Gram-positive bacteria and fungi. Cell permeability alteration is one of the highly antimicrobial resistance mechanisms in microorganisms. Therefore, increasing the permeability of the cell wall or membrane to enable high uptakes of PS might be a potential approach to enhance aPDT effect.

Anaya et al. demonstrated that AgNPs increase the permeability of bacterial biofilm [82]. In analogous to AgNPs, positively charged glucosamine groups of chitosan cause it to attach to negatively charged cell membranes and then damage cell permeability. In addition to the electrostatic interaction effect, some nanoparticles would alter cell permeability via other approaches. It has been demonstrated that the presence of surfactants and cosurfactants in nanoemulsions could increase the cell membrane permeability of microbes and thus permit an enhanced penetration of PS [83]. Sehmi and co-workers have developed two forms of ZnO nanoparticles, one was incorporated with crystal violet-coated polyurethane and oleic acid, and the other was incorporated with crystal violet-coated polyurethane plus magnesium oxide nanoparticles [84]. They found that, under the white light irradiation, the formulated crystal violet exhibited higher antimicrobial against S. aureus and E. coli when compared with crystal violet. They attributed this result to the ability of ZnO nanoparticles to enhance cell membrane permeability.

Disrupt the formation of EPS

As the oral microorganisms are embedded in a highly organized EPS matrix, which confers a physical shield for microorganisms from external stimuli, host immune attacks, or antibiotic assaults. Overcoming the physical barrier provided by EPS is a possible method to increase PS uptake and thus enhance aPDT effect. The EPS matrix is abundant in negatively charged components and hydrophobic groups, with pores filled with water facilitating transport of nutrients. The process of interaction between nanoparticles and biofilm is summarized as follows: nanoparticles are transported near the biofilm, initially deposited on the surface of the biofilm, and nanoparticles migrate to deeper regions of the biofilm [85].

Regarding the studies where nanomaterials could disrupt the EPS matrix, there are some examples. It is reported that Ag ions could bind to electron donor groups of biological molecules and then destabilize the biofilm matrix [86]. The swarm motion of Fe₃O₄ and Fe₂O₃ in these nanomaterials could destroy the EPS matrix by generating strong convection and mechanical force under the control of the magnetic field [87]. Also, some self-assembled surfactants or polymeric nanoparticles with small sizes are able to respond to external stimuli and alter their hydrophobicity or surface charges to interact with EPS matrix components [88]. The interaction would eventually result in EPS perforation and allow more diffusion or bypass of PS into microbial cells.

Understanding the role of nanoparticles in the elimination of the EPS matrix, some reports have tried to take advantage of nanoparticles and further enhance the antimicrobial capacity of aPDT. Exposure to external magnetic forces has been reported to be beneficial for improved penetration of PS since magnetic forces could disrupt biofilm matrix and weaken the protective role of EPS matrix [89]. The experiment conducted by Balhaddad et al. has just proved this phenomenon [65]. They used SPIONs to overcome high-level protection provided by the EPS matrix and thus achieved a more satisfactory aPDT effect on multispecies biofilm.

Increase the quantum yield of ROS

After the light activation, the excited PS could undergo a series of photochemical reactions with oxygen to produce ROS rapidly. ROS production and its diffusion rate are determinants of the final antimicrobial outcome. It is demonstrated that aPDT effect will vanish quickly after light illumination owing to the short lifetime of ROS (<3 μs) [90]. One of the most important ways to improve aPDT effect is to increase ROS quantum yield and slow the diffusion rate of ROS.

PS conjugated with heavy metal atoms was previously shown to enhance ROS generation. For instance, Sun and his co-workers have used Ce6-modified polyethyleneimine as the ligands of Ag nanoparticles and prepared AgNPs-PEI-Ce6 [91]. They evidenced that the surface plasma resonance of silver enhances the generation of ¹O₂, and the ROS can in turn stimulate the oxidative dissolution of the bactericidal Ag+. Similar results were observed by another group, which coated the Ag nanoparticle onto the phthalocyanine zinc/fabric and investigated the photo-inactivation effect of new compound (AgNPs/PS/Fabric) on different microorganisms [92]. Higher production of ROS of AgNPs/PS/fabric after illumination was found when compared with PS alone. The authors attributed this result to the strong electric field around the Ag nanoparticle islands, which could promote the optical absorption of PS and/or the generation of ¹O₂. In addition to high atoms, QDs could also help the loaded PS produce more ¹O₂, which is likely associated with excited state energy transfers between QDs and PS [93]. Narband et al. reported that the presence of QDs in TBO solution contributes to the further killing in S. aureus and Streptococcus pyogenes [94]. They found the enhanced killing was due to a non-Forster resonance energy transfer mechanism, whereby the QD converts part of the incident light to the absorption maximum for TBO; hence more light energy is harvested, resulting in increased concentrations of bactericidal radicals.

Increase the penetration depth

Due to the poor permeability of PS to highly organized biofilm, the microorganisms in the deep biofilm layer usually remain intact after aPDT treatment, which increases the risk of infection persistence. The shallow penetration depth of conventional aPDT light sources still weakens its therapeutic efficacy for periodontal pocket or dentin tubule disinfection. Though the near-infrared light (NIR) source has a penetration depth of more than 1 cm, its energy is not high enough to activate the current available PSs. Moreover, the hydrophobic features of PSs also limit their penetration into the biofilm.

UCNPs have been developed in the hope of absorbing NIR light to emit visible light to trigger PSs for generating ROS. The study by Liu et al. has prepared a delivery system consisting of UCNPs and polyvinylpyrrolidone (PVP) to load β-carboxyphthalocyanine zinc (CPZ), and then investigated its antimicrobial photodynamic effect on bacteria and fungus [95]. To further demonstrate the photochemical effectiveness of UCNPs-CPZ-PVP in deep infection, the aPDT experiments were carried out upon illumination through a layer of 5-mm thick pork tissue. Their findings suggested that UCNPs-CPZ-PVP still present a significant antibacterial effect equal to 3.3 log inhibition on bacteria in the presence of 5-mm thick pork tissue. Consistent with this finding, another report demonstrated that with the assistance of UCNPs, the curcumin–UCNPs could be activated under a NIR light source to exert a photodynamic effect on rats' knee joint tissue (1 cm) infection [51]. These reports indicated UCNPs could be used as a strategy to improve the penetration of PS through dense biofilm. MNPs are also reported to be able to deepen the drug penetration into multiple layers of biofilm and thus result in efficient biofilm elimination. It is proposed that the magnetically forced movement of nanoparticles creates artificial channels in infectious biofilm to enhance drug or antibiotic penetration and bacterial killing [96].

Conclusion

The treatment of oral biofilm has been challenging for decades. The presence of EPS matrix and synergistic relationship between species of oral biofilm trigger tolerance to antibiotics and somewhat lead to the emergence of resistance species. The specific anatomy and physiological characteristics of oral cavity also present challenges to current antimicrobial approaches. APDT is increasingly regarded as promising technique against oral biofilm given its well-described properties. However, intrinsic drawbacks of current PSs restrict the antibacterial efficiency of aPDT in clinics. The advent of nanotechnology has opened up the possibility for better delivery of PSs and the improvements in aPDT. The encapsulation or incorporation of PS into nanomaterials has achieved several improvements, such as improved bioavailability and solubility of PS, enhanced increase of ROS yield, targeting of infection sites, improved therapeutic action of PS and so on. The utilization of nanomaterials into aPDT procedure holds great potential for solving oral infection, however, these studies are in basic-research stages and will probably require more years of development for clinical translation.

Future perspective

However, utilizing nanomaterials as a drug-delivery system still possesses some limitations. The most essential aspect is the toxic effects of nanomaterials, which are related to the poor biocompatibility of raw chemicals. CNTs, heavy metals and silicates are toxic to different organs and capable of inducing systemic toxicity at high doses. Moreover, the unsatisfactory loading capacity of nanomaterials when loading PS would impact therapeutic outcomes. Apart from these mentioned points, the stability issue is also a critical aspect of novel nanomaterial based PS. Novel PS may undergo changes in structure or stability during storage, transportation, or in vivo condition. Despite the recent achievements of nanomaterial-based drug-delivery system for enhancing aPDT that are summarized in the present review, it should be noted that many aspects are little addressed.

Confirmation of the versatile properties of new nanomaterial-based PS

As summarized in many reviews, an ideal antimicrobial PS should fulfil characteristics such as solubility, high photostability, high ROS yield, great selectivity to microbial cells or biofilm, assured stability in body microenvironment, and minimal but acceptable cytotoxicity toward human tissues [^97-99]. However, it seems that not all studies have fully explored and confirmed the essential properties of the newly synthesized PSs, but simply validated that the aPDT effect of them is superior to the free form. It has been demonstrated that different features of nanomaterials, such as size, shape, surface charge, surface-to-volume ration will influence their antimicrobial efficiency [100]. It would be necessary for researchers to characterize the physical and optical characteristics of the newly synthesized PS in order to reasonably analyze and speculate on the later experimental results.

Potential cytotoxicity on cells & tissues

One of the most important concerns in applying nanomaterial-based PSs is their potential cytotoxicity. The major nanomaterial-based PSs tend to have a longer release and incubation time. The topical injection of these PSs into oral biofilm may unavoidably result in systemic circulation and tissue distribution. Therefore, systemic and local cytotoxicity of nanomaterial-based PSs are needed to explore thoroughly and carefully before clinical application. Though the potential cytotoxicity of different formulated nanomaterials has been investigated, data are sparse on the cytotoxicity of nanomaterial-based PSs on various cells related to the oral cavity. The cytotoxicity or mutagenic effect of newly synthesized PSs under irradiation or not should be investigated.

Possible drug resistance & disturbance of oral flora

Previous data suggest nanoparticles have a lower potential for bacterial resistance and represent an effective method to overcome antimicrobial resistance [101]. The resistance of oral microorganisms toward nanoparticles is rarely reported previously, nevertheless, there exist some papers reporting the possible antimicrobial resistance of oral bacteria to metal nanoparticles [102]. The proposed resistance mechanisms include the downregulation of porins, upregulation of resistance genes, adaptive mutations, and plasmids with resistance genes. In addition to the possible resistance of nanoparticles, there is accumulating evidence showing oxidative stress induced by aPDT could result in gene mutation in bacteria [103]. Hence, the clinical use of nanoparticle-based aPDT should test the possibility of resistance development to ensure long-term availability.

Additionally, the oral microbiome is highly controlled and balanced to ensure homeostasis. The equilibrium state of oral species within the host has a beneficial effect on maintaining oral health and oral mucosal barriers. It would be shallow to only consider the antibacterial property of the new agent without thinking about the possible influence on oral microbiome ecology. Due to the broad-spectrum antibacterial properties of aPDT and nanomaterials, it is reasonable to suspect that nanomaterial-based aPDT may impact the oral beneficial microbiota. The research and development of newly synthesized PSs should not only focus on antimicrobial properties but also the effect on oral ecological balance.

Assured stability & effectivity

The most common problem of current antimicrobials in dentistry is their short retention time due to saliva clearance. It can be inferred that topical application of PS may not retain at adequate concentrations unless PS has a great affinity to oral tissues. In addition to avoiding saliva clearance, nanomaterial-based PSs should remain within the biofilm matrix and endure extreme conditions like acidic and low oxygen. It is reasonable to use stimuli-responsive polymers such as pH and enzyme-responsive nanoparticles with targeting potential to ensure the long-term stability of newly synthesized PS in the oral cavity. However, most articles lack the exploration of the long-term stability and effectiveness of newly synthesized PSs in saliva.

Controlled release rate & period of PS

Loading PSs into nanomaterials can alter the morphology and chemical structure of the PS, thereby influencing its responsiveness to light irradiation and the release rate of internal active substances. When incorporated into polymer, the permeability and solubility of the polymer will impact the release speed of PS. If the release rate of the PS slows down or the release period extends, it can affect the therapeutic efficacy and lead to the excessive accumulation of the PS in non-therapeutic areas, potentially inducing systemic toxicity. Thus, in the design of novel nano drug-delivery systems, a delicate balance of various factors is essential to ensure the intended therapeutic effects while minimizing potential adverse consequences.

Limited research on clinically relevant biofilm or clinical studies

Apart from testing the photodynamic antimicrobial efficacy of nanomaterial-based PS on laboratory cultures, it is paramount to obtain detailed information on clinically relevant biofilm. Generally, the microbial biofilm presented in the mouth tends to be more complex and resilient than the artificial cultures produced in the lab. It is suggested that clinically relevant strains would be better options for testing the antimicrobial efficiency of novel products since the phenotype of lab reference strain does not exist the same as clinically relevant strain. Furthermore, most reports about nanomaterial-based aPDT are limited to in vitro assay for observing antimicrobial activity rather than ex vivo and in vivo models. Further work is hence recommended to extend the investigation of nanomaterial-based aPDT on in vivo models, which shall help in introducing nanomaterial-based aPDT into clinical application.

Concerns in pharmacoeconomic aspect of nanomaterial as drug-delivery system

It has been reported that nanomaterials as a drug-delivery system is strategically designed to reduce the expenses associated with drug administration, expedite the recovery period, and enhance patient compliance. The above benefits are reflected within pharmacoeconomics, a discipline capable of providing detailed and reliable information on costs and choosing the best one at the lowest possible costs. Consequently, a comprehensive cost-benefit analysis of nanomaterial-based PS is essential to ascertain whether the cost-benefit ratio can surpass that of traditional PSs or alternative drug-delivery systems.

Author contributions

Y Li and G Sun contributed to the conception and design of the study and wrote the first draft of the manuscript. S Huang and S Wang performed the data searching and wrote sections of the manuscript. Y Li performed the final corrections. All authors contributed to the article and approved the submitted version.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.