Advanced Nanotechnological Approaches for Biofilm Prevention and Control

Abstract

Biofilm-associated infections present a significant challenge in modern medicine, primarily due to their resilience and resistance to conventional treatments. These infections occur when bacteria form biofilms, protective layers formed by bacterial communities, which are notoriously resistant to traditional antibiotics on surfaces such as medical implants and biological surfaces, making eradication with standard antibiotics difficult. This resilience leads to persistent infections, imposing a substantial economic burden on healthcare systems. The urgency to find alternative treatments is critical as current methods are insufficient and costly. Innovative approaches, such as nanotechnology-based therapies, offer promising alternatives by targeting biofilms more effectively and reducing the need for invasive procedures. Nanocarriers hold significant promise in the fight against biofilm-associated infections. Nanocarriers can penetrate biofilms more effectively than conventional treatments, delivering higher concentrations of antibiotics or other antimicrobial agents precisely where they are needed. This targeted approach not only enhances the efficacy of treatments but also minimizes potential side effects. The development of nanocarrier-based therapies is crucial for overcoming the limitations of current treatments and ultimately improving patient outcomes and reducing the economic burden of biofilm-associated infections on healthcare systems. In this review, nanotechnology-based systems, their characteristics, limitations, and potential benefits are explored to address biofilms-related infections. Additionally, biofilm evaluation models and the tests necessary for the preclinical validation of these nanosystems to facilitate their clinical application are addressed.

1. Introduction

A biofilm is defined as a three-dimensional organized structure constituted by microorganism clusters adhered to either a biotic (e.g., host tissue) or abiotic (e.g., biomaterial/implant) surface or interface and encased in a protective self-produced hydrated ECM composed mainly of proteins, polysaccharides, and extracellular DNA. These structures are heterogenous, both in space and time, and reveal “water channels” that have colossal importance to the growth of cells within the biofilm since they allow the passage of essential nutrients and oxygen [1].

Additionally, the development of biofilms is due to diverse environmental cues (e.g., nutrient limitation is the reason why the growth rate of biofilm-associated organisms is typically slower than planktonic organisms) and exhibits extreme resilience, insensitivity, or tolerance to severe conditions, including antibiotic treatment (biofilms are up to 10–1000 times more resistant to antimicrobials than planktonic organisms), UV radiation, pH stress, chemical exposure, dehydration, osmolarity, host immune response, and mechanical and shear forces [2,3]. These features are the natural result of being in a biofilm form, which allows and eases lateral gene transfer, leading to high mutation rates and, thus, high resilience [4,5,6,7,8].

Nanocarriers show great potential in combating infections associated with biofilms. Biofilms are protective layers created by bacterial communities that are highly resistant to traditional antibiotics. Unlike conventional treatments, nanocarriers can more effectively penetrate biofilms, delivering higher concentrations of antibiotics or other antimicrobial agents directly to the affected area. This targeted method increases the effectiveness of treatments while reducing potential side effects. Developing nanocarrier-based therapies is essential for overcoming the limitations of current treatments and ultimately improving patient outcomes and lowering the economic impact of biofilm-associated infections on healthcare systems [9]. This review explores nanotechnology-based systems for addressing biofilms, discussing their characteristics, limitations, and benefits in treating these infections. Additionally, biofilm evaluation models and the tests required for the preclinical validation of these nanosystems to aid their clinical application are also analyzed.

The purpose of this review is to examine nanotechnology-based systems, detailing their characteristics, limitations, and potential advantages in tackling biofilm-related infections. It also discusses biofilm evaluation models and the testing required for preclinical validation of these nanosystems to support their clinical use. The structure of this review is summarized in Figure 1.

2. Biofilm Concepts and Formation

Biofilms can be monomicrobial or polymicrobial. A monomicrobial biofilm encases a community of a single species of microorganisms, while polymicrobial biofilms contain a diverse community of multiple species of microorganisms, conceding different features and properties [1,10]. Polymicrobial biofilms tend to be more robust than monomicrobial biofilms. Moreover, due to symbiotic interactions between distinct species of microorganisms, polymicrobial biofilms have different architectures, and also altered tolerance to antimicrobial agents and other adverse environmental conditions (e.g., limited nutrition supply, the host immune system) [11]. Polymicrobial biofilms are major causes of oral, wound, respiratory, skin, and bone infections [12].

Regarding microorganism behavior within the biofilm, microorganisms can interact with each other in a synergetic/cooperative or inhibitory/antagonistic way, having a significant impact on the pathogenesis of infection [13]. Microbial synergism produces an effect or effects not achieved by individual species alone. These effects include (i) passive resistance—protecting from antibiotic killing activity; (ii) metabolic cooperation—producing adenosine triphosphate (ATP), carbon, and nitrogen to synthesize metabolites and maintain redox balance; (iii) generation of metabolic by-products—a cooperative effect denoting metabolic cross-feeding (or syntrophy) where one species generates a metabolic by-product capable of increasing the growth of the surrounding species; (iv) quorum sensing (QS) systems—signal exchange, allowing cell-to-cell communication and triggering a wide range of biological processes within the biofilm (e.g., detachment) [14,15,16,17,18,19,20,21]. Conversely, microbial antagonism (or antibiosis) is defined as the suppression and inhibition of one microbial species by another. In those scenarios, antagonistic mechanisms can be developed, such as (i) the production of factors that kill or inhibit the growth of the surrounding species; (ii) the production of chemical signals, negatively affecting the behavior and physiology of the surrounding species; and (iii) limitation of nutrients, leading to starvation [18].

Biofilm formation can be divided into four distinguished stages: (i) attachment—organic/inorganic molecules and microorganisms are adsorbed on a surface substrate (e.g., bone) through electrostatics, electric double layer forces, and hydrophobic and van der Waals interactions, leading to the irreversible attachment of microorganisms to the surface mediated by production of extracellular polymeric substance (EPS) matrix; (ii) formation of microcolonies—microorganisms grow within multicellular surface-attached communities; (iii) the biofilm’s maturation—cell-to-cell interactions allow cells within the biofilm to take on specialized functions, secreting mature biofilm components, namely, proteins, polysaccharides, and extracellular DNA; (iv) detachment—microorganisms/microcolonies from the biofilm are dispersed and released into surrounding media as free-floating microorganisms, which can further spread to and colonize another site, causing other surface contamination, systemic infections, or septicemia. Biofilms disperse because of several factors, such as lack of nutrients, intense competition, and an outgrown population [1,2,3,22].

Recent advances in research have significantly deepened our understanding of the genetic and molecular mechanisms that underpin the resilience of biofilms. Biofilms, complex communities of microorganisms embedded in a self-produced matrix, exhibit remarkable resistance to antibiotics and the host immune system. This resilience is largely driven by a variety of genetic and molecular processes [23].

One key discovery is the role of specific gene regulatory networks that enable bacteria within biofilms to enter a dormant state, making them less susceptible to antimicrobial agents [24]. Quorum sensing, a communication process between bacterial cells, has also been identified as a critical factor in biofilm formation and maintenance. This process regulates gene expression in response to cell density, allowing the biofilm to adapt to environmental changes and enhance its defenses [25].

Moreover, research has highlighted the importance of extracellular polymeric substances (EPS) in biofilm resilience. These substances, produced by the bacteria, create a protective barrier that shields the cells from external threats. Advances in molecular biology have revealed how the production of EPS is tightly controlled by specific genetic pathways, which can be potential targets for new therapeutic strategies [26].

Additionally, the discovery of persister cells—dormant variants of regular bacterial cells within the biofilm—has provided insight into the biofilm’s ability to survive even the most potent antibiotics. These cells can repopulate the biofilm once the antibiotic threat has passed, making treatment of biofilm-associated infections particularly challenging [27].

Biofilm heterogeneity significantly impacts treatment efficacy, making infections harder to eradicate. Within a biofilm, microbial cells can differ in terms of metabolic activity, growth rates, and gene expression. This diversity creates a range of microenvironments, with some cells becoming more resistant to antibiotics and immune responses. As a result, standard treatments often fail to penetrate the entire biofilm or kill all the cells, leading to persistent infections. Understanding and targeting this heterogeneity is crucial for developing more effective therapies against biofilm-associated infections [23].

3. Biofilm Characterization

Understanding biofilms is a crucial step as these structures are highly dynamic, constantly changing over time. Multiple approaches can be performed to characterize biofilm density and structure. Quantitative methods enumerate, directly or indirectly, the cells present in biofilms, while qualitative characterization techniques evaluate the structure, morphology, and biofilm composition, as well as provide information about microorganism interactions within biofilms. These characteristics are of high importance since, from these, it is possible to infer new standards to treat, avoid, and control biofilm formation, and, consequently, to reduce costs regarding equipment damage, product contamination, and infections [22,28].

When evaluating biofilms, both direct and indirect methods have distinct advantages and limitations. Direct methods, such as microscopy and staining techniques, allow for a detailed visualization and quantification of the biofilm structure, composition, and cell viability. They provide precise, real-time insights into biofilm morphology but often require specialized equipment and can be labor intensive. In contrast, indirect methods, like measuring metabolic activity or quantifying extracellular matrix components, offer a simpler, faster way to assess biofilm presence and behavior. However, these methods provide less specific information and may overlook critical structural details, leading to potential inaccuracies in evaluating biofilm characteristics. Choosing the appropriate method depends on the specific research or clinical objective, balancing the need for detail against practicality and speed [29].

3.1. Quantitative Assessment Methods

The number of cells present in a biofilm can be assessed directly or indirectly. Direct methods allow the quantification of the number and type of microorganisms inside a biofilm, while indirect methods allow the inference of biofilm density and proliferation by quantifying proxy markers [28,30].

3.1.1. Direct Methods

Direct methods include (i) plate counting (colony-forming units CFU); (ii) DAPI (4′,6-Diamidino-2-phenylindole dilactate); (iii) Live/Dead assay; (iv) fluorescent in situ hybridization (FISH) [28].

Briefly, plate counting is a traditional microbiological method used to determine the number of viable bacteria in a biofilm. The biofilm is first disrupted, usually by sonication or vortexing, to release the cells. The resulting suspension is then serially diluted and plated on an agar medium. After incubation, the number of colonies that form (colony-forming units, CFU) is counted. This method provides a direct measure of the number of viable, culturable cells within the biofilm. It is widely used due to its simplicity and effectiveness but only counts cells that can grow under the given conditions, possibly underestimating the total cell population [29]. DAPI is a fluorescent stain that binds strongly to DNA, making it useful for visualizing and quantifying cells in a biofilm. When applied to biofilms, DAPI stains the nuclei of all cells, allowing them to be viewed under a fluorescence microscope. DAPI staining is used to estimate the total number of cells in a biofilm, regardless of their viability. This method is particularly useful for imaging biofilms and providing a total cell count, but it does not distinguish between live and dead cells [29]. The Live/Dead assay utilizes two fluorescent dyes to differentiate between live and dead cells within a biofilm. Typically, a green fluorescent dye (e.g., SYTO 9) stains all cells, while a red fluorescent dye (e.g., propidium iodide) penetrates only the damaged membranes of dead cells. The stained biofilm is then observed under a fluorescence microscope or analyzed using flow cytometry. This assay allows for the simultaneous quantification of live and dead cells within a biofilm, providing insights into cell viability and the effectiveness of antimicrobial treatments. It is widely used in biofilm research for assessing cell viability and treatment outcomes [31]. FISH is a molecular technique that uses fluorescently labeled probes that bind to specific nucleic acid sequences within cells. When applied to biofilms, these probes hybridize to the target sequences, allowing the visualization of specific microorganisms or groups of microorganisms within the biofilm using fluorescence microscopy. FISH is particularly useful for identifying and quantifying specific microbial populations within a biofilm, providing both spatial and quantitative information about the biofilm’s microbial composition. It is highly specific but requires knowledge of the target organisms’ genetic sequences [2].

Table 1 summarizes some of the most common direct methods used to quantify biofilm, showing their advantages and disadvantages.

Table 1. Direct methods to quantify biofilms and their advantages and disadvantages.

Method	Advantages	Disadvantages	References
Colony-forming unit (CFU) counting	Allows viable counting. Special equipment or dyes are not required. May be performed in most laboratory scenarios.	Lengthy and requires repetitive tasks. Involves complex and indirect procedures that increase ambiguity and uncertainty. Detects only viable bacteria. Requires removal of bacteria through sonication or matrix-degrading enzymes.	[2,28,29,32,33,34]
4′,6′-diamino-2-fenil-indol (DAPI) staining	Enables measurement of the total cells on the surface. Particularly effective for handling a large number of samples.	Imaging is restricted to two dimensions. Needs the use of fluorochromes, epifluorescence microscopy, and confocal laser scanning microscopy (CLSM). Cannot distinguish between live and dead bacterial cells.	[28,35]
Live/Dead staining	Enables counting and distinguishing between live and dead bacteria. Particularly suitable for handling a large number of samples.	Imaging is restricted to two dimensions. Fluorochromes are sensitive to light. Requires epifluorescence and confocal laser scanning microscopy (CLSM).	[31,36,37]
Fluorescence in situ hybridization (FISH)	Enables distinguishing and quantifying the sessile population. Analyzes the spatial distribution within biofilms without damaging their structure. Discriminates microorganism species using fluorescent labeling with specific oligonucleotide probes.	Requires specific probes, epifluorescence, and CLSM. Involves costly equipment. Protocol optimization is necessary for various microbiology applications.	[2,38]

Table 1. Direct methods to quantify biofilms and their advantages and disadvantages.

Method	Advantages	Disadvantages	References
Colony-forming unit (CFU) counting	Allows viable counting. Special equipment or dyes are not required. May be performed in most laboratory scenarios.	Lengthy and requires repetitive tasks. Involves complex and indirect procedures that increase ambiguity and uncertainty. Detects only viable bacteria. Requires removal of bacteria through sonication or matrix-degrading enzymes.	[2,28,29,32,33,34]
4′,6′-diamino-2-fenil-indol (DAPI) staining	Enables measurement of the total cells on the surface. Particularly effective for handling a large number of samples.	Imaging is restricted to two dimensions. Needs the use of fluorochromes, epifluorescence microscopy, and confocal laser scanning microscopy (CLSM). Cannot distinguish between live and dead bacterial cells.	[28,35]
Live/Dead staining	Enables counting and distinguishing between live and dead bacteria. Particularly suitable for handling a large number of samples.	Imaging is restricted to two dimensions. Fluorochromes are sensitive to light. Requires epifluorescence and confocal laser scanning microscopy (CLSM).	[31,36,37]
Fluorescence in situ hybridization (FISH)	Enables distinguishing and quantifying the sessile population. Analyzes the spatial distribution within biofilms without damaging their structure. Discriminates microorganism species using fluorescent labeling with specific oligonucleotide probes.	Requires specific probes, epifluorescence, and CLSM. Involves costly equipment. Protocol optimization is necessary for various microbiology applications.	[2,38]

3.1.2. Indirect Methods

Indirect assessment methods include (i) dry mass; (ii) Crystal Violet assay (CV); (iii) Tetrazolium salts (e.g., 2,3,5-triphenyl-tetrazolium chloride (TTC) and 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT)); (iv) total protein determination; (v) Quartz Crystal Microbalance (QCM); (vi) Total Organic Carbon; (vii) Resazurin assay (or Alamar Blue) [28,32].

Briefly, the dry mass method involves measuring the total biomass of the biofilm. The biofilm is first collected, typically by scraping it from a surface, and then dried to a constant weight. The dried sample is weighed to determine the total mass of the biofilm. This method provides a direct measure of the total biofilm mass, including both the cellular and extracellular matrix components. It is simple and straightforward but does not differentiate between living and dead cells [39]. The Crystal Violet assay is a widely used technique to quantify biofilm formation. Biofilms are stained with Crystal Violet dye, which binds to the biofilm matrix. The bound dye is then solubilized, and its absorbance is measured spectrophotometrically. The CV assay is a quick and easy method to assess the total biomass of the biofilm, including cells and extracellular polymeric substances (EPS). It is especially useful for high-throughput screening but does not provide information on cell viability [28]. Tetrazolium salts, such as 2,3,5-triphenyl-tetrazolium chloride (TTC) and MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide), are used in colorimetric assays to measure cell viability. In metabolically active cells, these salts are reduced to colored formazan products, which can be quantified by measuring the absorbance. These assays specifically assess the metabolic activity of cells within the biofilm, providing an indirect measure of viable cell biomass. They are useful for distinguishing between live and dead cells in biofilms [28]. Total protein determination involves quantifying the amount of protein within a biofilm using colorimetric assays like the Bradford or BCA assay. The biofilm is lysed to release proteins, which are then quantified. This method provides an estimate of the total cellular content within the biofilm as protein content is directly proportional to the number of cells. It is useful for comparing the cellular density of different biofilms [28]. The QCM is a highly sensitive technique that measures changes in mass on a quartz crystal surface. As biofilm forms on the crystal, the increase in mass causes a change in the frequency of the crystal’s oscillation, which can be precisely measured. The QCM allows for real-time monitoring of biofilm formation and growth. It is particularly valuable for studying the dynamics of biofilm development and the effects of various treatments on biofilm mass [40,41,42]. Total Organic Carbon (TOC) analysis measures the total amount of carbon in the biofilm, representing the organic matter present. The biofilm is oxidized to convert all carbon into CO₂, which is then quantified. TOC provides a measure of the total organic content of the biofilm, including both cellular material and extracellular polymeric substances (EPS). This method is useful for assessing the overall organic load of biofilms [28]. The Resazurin assay, also known as Alamar Blue, is a redox-based assay that measures cell viability. Resazurin is a non-toxic blue dye that is reduced to the pink fluorescent compound resorufin in metabolically active cells. The fluorescence intensity or absorbance is then measured. This assay is a sensitive method for determining the viability and metabolic activity of cells within a biofilm. It is particularly useful for assessing the effects of antimicrobial agents on biofilm-embedded cells [43].

Each of these methods provides valuable information about different aspects of the biofilm’s structure and function, allowing researchers to assess biofilm formation, viability, and response to treatments from multiple perspectives.

In Table 2, the advantages and disadvantages of different indirect methods to assess biofilm quantity are presented.

Table 2. Indirect methods to quantify biofilms and their advantages and disadvantages.

Method	Advantages	Disadvantages	References
Dry mass	Rapid technique.	Heat or precipitation is required to fully eliminate water molecules.	[28]
Crystal Violet staining	Enables the measurement of total biomass.	Cannot assess biofilm viability because deceased bacteria retain staining.	[28,32,44,45,46]
Tetrazolium salt	Enables measurement of cellular metabolic activity. Detection and quantification using colorimetric methods.	Expensive. Exhibits challenges related to variability within and between species.	[28,44]
Adenosine Triphosphate (ATP) bioluminescence	ATP detection enables the identification of metabolically active cells involved in both building up and breaking down processes. Rapid, real-time assessment of viable cell density.	Expensive. High variability. Requirement for regular luminometer calibration to maintain precision.	[28,47,48,49]
Total protein determination	Provides a holistic view of the biofilm composition by quantifying all proteins present.	Protein levels vary depending on the species, age, and culture conditions, resulting in fluctuations in the data obtained.	[28]
Quartz Crystal Microbalance (QCM)	Enables continuous monitoring of biofilm growth. Offers detailed insights into how biofilms interact with surfaces. Allows for easy adjustment of variables like flow rate and temperature. Provides a quick and non-invasive way to study biofilms. Highly sensitive for tracking the accumulation of mass (ng/cm2) over time.	Requires specialized equipment, electronics, software, and consumables. Changes in temperature and pressure can affect the resonant frequency and thus impact data collection.	[28,40,41,42]
Resazurin assay	Enables determination of cells’ metabolic activity by measuring the reduction of resazurin, indicating whether cells are active metabolically or not.	Differences in metabolic rates between planktonic and biofilm forms can introduce considerable inaccuracies.	[33,43]

Table 2. Indirect methods to quantify biofilms and their advantages and disadvantages.

Method	Advantages	Disadvantages	References
Dry mass	Rapid technique.	Heat or precipitation is required to fully eliminate water molecules.	[28]
Crystal Violet staining	Enables the measurement of total biomass.	Cannot assess biofilm viability because deceased bacteria retain staining.	[28,32,44,45,46]
Tetrazolium salt	Enables measurement of cellular metabolic activity. Detection and quantification using colorimetric methods.	Expensive. Exhibits challenges related to variability within and between species.	[28,44]
Adenosine Triphosphate (ATP) bioluminescence	ATP detection enables the identification of metabolically active cells involved in both building up and breaking down processes. Rapid, real-time assessment of viable cell density.	Expensive. High variability. Requirement for regular luminometer calibration to maintain precision.	[28,47,48,49]
Total protein determination	Provides a holistic view of the biofilm composition by quantifying all proteins present.	Protein levels vary depending on the species, age, and culture conditions, resulting in fluctuations in the data obtained.	[28]
Quartz Crystal Microbalance (QCM)	Enables continuous monitoring of biofilm growth. Offers detailed insights into how biofilms interact with surfaces. Allows for easy adjustment of variables like flow rate and temperature. Provides a quick and non-invasive way to study biofilms. Highly sensitive for tracking the accumulation of mass (ng/cm2) over time.	Requires specialized equipment, electronics, software, and consumables. Changes in temperature and pressure can affect the resonant frequency and thus impact data collection.	[28,40,41,42]
Resazurin assay	Enables determination of cells’ metabolic activity by measuring the reduction of resazurin, indicating whether cells are active metabolically or not.	Differences in metabolic rates between planktonic and biofilm forms can introduce considerable inaccuracies.	[33,43]

3.2. Qualitative Assessment Methods

Quantitative methods are typically supplemented by qualitative methods, which allow the observation of the dynamics and complexity of biofilms. Qualitative assessment methods evaluate the morphology and structure of biofilms, their arrangement in space, and interactions between biofilms and their surroundings. There are several approaches for biofilm imaging, such as light microscopy, SEM, transmission electron microscopy (TEM), and CLSM, with SEM being the most used for biofilm analysis and observation [28,30].

SEM is a technique performed to observe biofilms without disrupting them. It provides high-resolution imaging of microscopic structures due to its great magnifying power, which allows magnifications ranging from 10 to 500,000 times. It is a well-established technique that permits the collection of high-resolution three-dimensional images of the morphology of a biofilm formed on a given material surface, the morphology of the material surface, and the interactions established between them [28]. Low-vacuum SEM does not need metal or carbon sputtering and is less prone to disrupting the bacteria adhered to a surface or altering the surface. Additionally, the surface chemical composition can be defined by using energy-dispersive X-ray (EDX) [28,29].

Regarding its drawbacks, SEM is an expensive technique that does not allow the enumeration of bacteria due to the reduced field and laborious work. Also, it requires a conductive specimen (essentially “metal sputtered”) and lengthy sample preparation, which consists of a set of procedures that are tedious and labor intensive. Sample preparation may require drying the sample, which may concede undesirable outcomes (e.g., cell shrinkage, damage, and distortion of the biofilm). Differentiation between living and non-living cells is not possible. Performing this technique requires specialized training and equipment [28,29].

Despite the above-mentioned drawbacks of SEM, this approach is seen as a fundamental tool in the study of biofilms as high-resolution three-dimensional images of biofilm are collected and used to evaluate the biofilm morphology and topography. The details revealed by those images are unmatched by other techniques [28].

4. Biofilm-Associated Infections

A significant proportion of microbial infections (65 to 80%) are caused by biofilms [50]. Those infections include tissue (non-device-associated) and device-associated infections, which represent a hard medical challenge, in terms of infection diagnosis and treatment, since they exhibit greater recalcitrance to antimicrobial therapies relative to planktonic organisms [28,32].

The non-device-associated category encompasses biofilm formation on host tissues like epithelial and mucosal surfaces, as well as teeth. These biofilms can cause infections in individuals with cystic fibrosis, diabetic foot ulcers, chronic otitis media or rhinosinusitis, chronic prostatitis, osteomyelitis, recurrent urinary tract infections, and dental issues such as cavities and gum disease [51,52].

Device-associated infections happen when microorganisms colonize non-living surfaces, including medical devices like central venous or urinary catheters, joint or dental prostheses, heart valves, endotracheal tubes, intrauterine devices, and dental implants [53]. Microorganisms can disaggregate from these biofilms, spreading to nearby tissues or entering the bloodstream, worsening the infection [1]. Frequently, hospital-acquired infections are linked to medical devices [54]. Independently of the biomedical implants’ and tissue engineering constructs’ complexity, all medical devices are vulnerable to microbial colonization, eventually leading to infections [55]. Biofilm formation on these devices is notoriously difficult to eliminate, often leaving few options for treatment, such as removing the infected device or administering high doses of antibiotics [56]. These approaches, however, increase treatment costs and may contribute to the development of antibiotic resistance and cytotoxicity [1]. Furthermore, removing medical devices is often not an option for patients who depend on essential medical devices such as pacemakers. The substantial clinical impact of biofilm-associated infections and their inherent resistance to antimicrobials underscore the urgent need to develop new anti-biofilm strategies.

The primary challenge in eradicating biofilms stems from their intricate three-dimensional architecture [57]. Firstly, the biofilm matrices serve as mechanical barriers, safeguarding bacterial cells from forces like fluid shear and attacks by neutrophils [58,59]. Secondly, EPS can neutralize antimicrobials through direct interactions [60]. Thirdly, bacterial cells in various regions of a biofilm exhibit different metabolic states, with dormant cells being less susceptible to many antimicrobial agents, leading to tolerance [61]. Additionally, traditional resistance mechanisms like decreased drug uptake, increased activity of efflux pumps, and altered antibiotic target sites also play a role in biofilm persistence [62]. The close proximity of bacteria within the biofilm enhances the likelihood of horizontal gene transfer, facilitating the spread of resistance genes through cell-to-cell DNA transfer [63]. This intrinsic resistance of biofilms makes them exceptionally difficult to eliminate, making the combatting of antibiotic resistance a crucial goal in biofilm therapy.

Several promising alternatives to antibiotics for biofilm eradication are currently being explored. These include quorum sensing inhibitors [64], bacteriophages [65,66], enzymes [67], surfactants [68], and antimicrobial peptides [55]. Recent works have used typical and modified strategies, including biofilm-related strategies such as the use of nanoparticles [69,70,71] and the use of alternative drugs [72,73,74] and scaffolds as delivery systems [75,76]. Despite their potential, these bioactive substances often face issues such as poor targeting and difficulty in penetrating the biofilm matrix, and are easily inactivated by EPS. Therefore, improving the delivery methods of these antimicrobial agents is crucial to overcome these challenges.

5. Nanocarriers as a Strategy to Fight Biofilms

Nanocarriers are nanomaterials designed to transport various substances. In the review article, the term “nanocarriers” encompasses different types of drug delivery vehicles. Nanocarriers can act differently to improve their performance, as described below. Biological interactions responsible for anti-biofilm capacity are therefore described in the next sections.

5.1. Targeting of Antimicrobial Substances

The targeting capabilities of drug-loaded nanoparticles (NPs) enable them to be absorbed by organisms in a unique way, allowing them to bypass various physiological barriers and deliver drugs directly to specific sites. Targeting improves the bioavailability of drugs and minimizes toxic side effects [77]. Active targeting involves nanocarriers binding to specific receptors on biofilms through the use of ligands or through electrostatic interactions with the biofilm itself. This approach enhances the precision of drug delivery to targeted sites within the biofilm structure [78]. Physical targeting involves using an external magnetic field to exert force on nanocarriers, guiding them directly to biofilms. This method enables precise localization of nanocarriers to biofilm sites, enhancing their therapeutic effectiveness [79].

5.2. Biofilm-Penetrating Efficiency

EPS matrix serves as a barrier that restricts antibiotics penetration [1]. EPS matrix contains extremely small pores, preventing larger drug molecules from passing through. Also, antibiotics can be adsorbed onto the matrix itself [80]. Thus, enhancing antimicrobials’ ability to penetrate biofilms using nanocarriers represents an effective strategy against biofilm infections.

Nanocarriers with smaller particle sizes can more effectively penetrate biofilms, a characteristic closely tied to the structure of the biofilm. Studies using scanning electron microscopy and confocal microscopy have revealed water channels on the biofilm surface and observed its internal structure facilitating the transfer of nutrients and metabolites [81]. These biofilm channels, crucial for efficient transport processes, typically have a minimum width of approximately 100 nm [82]. Biofilms are structured with water channels, yet many antimicrobials face challenges in penetrating EPS due to their size or adsorption onto the EPS matrix. Notwithstanding, the existence of these channels suggests that nanoparticles smaller than these channels can effectively penetrate biofilms.

5.3. Antimicrobial Protection

Biofilm resistance partly arises from the problematic interaction between EPS and antimicrobial agents, which can lead to the deactivation of these substances [83]. Nanocarriers offer a solution by shielding antimicrobial agents from enzymatic inactivation and preventing their binding to DNA and polysaccharides produced by biofilms. This protective role enhances the effectiveness of antimicrobial treatments against biofilm infections.

5.4. Destabilization of EPS

In addition to effectively penetrating biofilms, breaking down EPS can naturally facilitate the delivery of antimicrobial substances. Nanocarriers can perform multiple functions: targeting bacteria and simultaneously breaking down EPS [84]. One key characteristic of nanocarriers is their ability to target multiple sites, allowing certain formulations to act on both EPS and bacteria simultaneously [85]. Breaking down EPS helps deliver a higher concentration of antibacterial drugs to the bacteria and leads to a more efficient removal of the biofilm [86].

5.5. Affinity to Bacterial Cells

Nanocarriers can improve the internalization of antimicrobial substances by bacterial cells by fusion with the bacterial membranes. The ability to merge with phospholipid membranes is the most distinctive characteristic of liposomes [87]. Transmission electron microscopy reveals that liposomes closely interact with the Gram-negative bacterial outer membrane, inducing changes in the membrane structure. Liposome fusion with the bacterial membrane has also been verified using flow cytometry and lipid-mixing assays [88]. Lipid nanocarriers’ fusion with bacterial cells improves antimicrobial internalization. Additionally, electrostatic attraction or ligand–receptor binding extends the interaction between nanocarriers and bacteria. This enhances the antibacterial effect and results in a more potent anti-biofilm effect [89].

5.6. Synergy with Antimicrobial Drugs

Several materials possess intrinsic antibacterial properties, enhancing the effectiveness of the drugs they carry or encapsulate. Chitosan is a high-molecular-weight cationic polymer known for its biocompatibility which is extensively used as a possible carrier for drug delivery [90]. Moreover, several studies have reported the antibacterial and anti-biofilm properties of chitosan and some of its derivatives [91,92].

As drug delivery systems, nanocarriers possess features such as nanoscale dimensions, a high surface-to-volume ratio, and excellent biocompatibility [93]. These attributes give nanocarriers certain anti-biofilm capabilities, and numerous studies have documented their use in managing biofilm infections [94,95]. Several notable nanocarriers have been identified, including liposomes [96,97,98,99,100,101], quatsomes [102,103,104,105], solid lipid nanoparticles (SLNs) [106,107,108,109,110,111,112,113], micelles [114,115], polymeric nanoparticles [116,117,118,119,120,121,122,123,124], polymeric micelles [125], dendrimers [126,127,128,129,130,131,132], metallic nanoparticles [133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148], magnetic nanoparticles (MNPs) [134,136,140,141,145,149], silica nanoparticles [150,151,152,153,154,155], and quantum dots [156,157,158]. Each type of nanocarrier has unique features and benefits.

Table 3. Benefits and drawbacks of nanocarriers in combating biofilms.

Nanocarriers	Benefits	Drawbacks	References
Liposomes	Easy to modify. Fusion with bacterial cell. Reduce side effects.	Poor storage stability.	[96,97,98,99,100,101]
Quatsomes	Biocompatibility. Good stability. Ability to efficiently deliver therapeutic agents to target sites.	Unknown in vivo targeting.	[102,103,104,105]
Solid lipid nanoparticles	Sustained release. Improved drug stability.	Poor solubility. Limitation of drug load.	[106,107,108,109,110,111,112,113]
Lipid micelles	Effectively deliver antimicrobial agents to the biofilm matrix.	Poor storage stability.	[114,115]
Polymeric nanoparticles	Biocompatibility. Controlled drug release. High stability in the body.	Poor storage stability.	[116,117,118,119,120,121,122,123,124]
Polymeric micelles	Effectively deliver antimicrobial agents to the biofilm matrix.	Poor storage stability.	[125]
Dendrimers	Controlled release of the antibiotic, targeting the bacteria more effectively.	Unknown in vivo behavior.	[126,127,128,129,130,131,132]
Metallic nanoparticles	Potent antimicrobial effects.	Potentially cytotoxic.	[133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148]
Magnetic nanoparticles	Physical targeting. Enhanced capacity to penetrate biofilm. Biofilm mechanical destruction.	Potentially cytotoxic.	[134,136,140,141,145,149]
Silica nanoparticles	Biocompatibility and stability.	Limited understanding of the long-term safety implications.	[150,151,152,153,154,155]
Quantum dots	Photothermal and photodynamic effects. Controlled release. Good penetration capability.	Potentially cytotoxic.	[156,157,158]

presents the various nanocarriers and their anti-biofilm properties which will be detailed in the following sections. This review evaluates the advantages of nanocarriers in fighting biofilms by examining the transport process of nanoparticles within biofilms. Additionally, it discusses the challenges of translating nanocarrier formulations from the laboratory to clinical applications. Details of the several strategies are discussed in Section 6, Section 7, Section 8, Section 9 and Section 10.

Pharmacokinetics and biodistribution are critical factors in the development and effectiveness of nanocarriers for drug delivery in vivo. Nanocarriers, such as liposomes, polymeric nanoparticles, and dendrimers, are designed to enhance the delivery of therapeutic agents by improving their stability, targeting ability, and controlled release profiles. Understanding the pharmacokinetics of nanocarriers is essential to predict how long they will circulate in the bloodstream, how effectively they will reach their target tissues, and how they will be cleared from the body. Factors such as particle size, surface charge, and the presence of targeting ligands on the nanocarrier surface influence these pharmacokinetic properties. For example, smaller particles may circulate longer and penetrate tissues more effectively, while surface modifications can enhance the targeting of specific cells or organs. Biodistribution studies are equally important as they reveal how nanocarriers are distributed across different tissues and organs. Ideally, nanocarriers should accumulate predominantly in the target tissue, such as a tumor or site of infection, while minimizing exposure to healthy tissues to reduce side effects. However, challenges remain in achieving this ideal distribution as nanocarriers can be taken up by the mononuclear phagocyte system (MPS), leading to accumulation in the liver, spleen, and other organs. Recent advances in nanotechnology are addressing these challenges by optimizing nanocarrier design to improve pharmacokinetic profiles and achieve more favorable biodistribution patterns. This includes the development of “stealth” nanocarriers that evade immune detection, as well as responsive nanocarriers that release their payload only in specific environments, such as acidic tumor microenvironments or inflamed tissues. Overall, understanding and optimizing the pharmacokinetics and biodistribution of nanocarriers is crucial for their successful translation into clinical applications and enabling more precise and effective treatments with reduced side effects [159,160].

6. Lipid-Based Nanocarriers

6.1. Liposomes

Liposomes are vesicles made up of a phospholipid bi- or multilayer with an aqueous core [96]. They are widely used as delivery systems for antimicrobial agents because their lipid bilayer structure mimics cell membranes, allowing them to easily fuse with microorganisms’ membranes [97]. Additionally, liposomes can encapsulate both hydrophobic and hydrophilic drugs within their biocompatible and biodegradable structure [96]. Because of these advantages, liposomes are the most prevalent delivery system on the market [100]. Numerous studies are being conducted using these carriers to eradicate biofilms, as depicted in Table 4. Although using liposomes as delivery systems to eradicate bacterial biofilms shows promising results, their lack of stability is still a concern. To address this issue, researchers are exploring surface modifications of liposomes with stabilizer ligands. For example, coating liposomes with calcium phosphate has been shown to enhance their delivery capacity and mechanical stability [99]. While many studies in the literature have concentrated on encapsulating antibiotics, there is growing interest in natural compounds as potential alternatives to traditional antibacterial drugs, which face increasing issues with bacterial resistance [98].

6.2. Quatsomes

Quatsomes are a type of nanostructured lipid carrier that is formed by the self-assembly of cationic bilayers. They are composed of a mixture of lipids and surfactants, where the surfactant is a quaternary ammonium compound (quat) such as cetyltrimethylammonium bromide (CTAB). Quatsomes have a unique structure where the lipid bilayer surrounds an aqueous core, and they can encapsulate both hydrophilic and hydrophobic molecules. These nanostructures have gained attention as potential drug delivery systems due to their biocompatibility, stability, and ability to efficiently deliver therapeutic agents to target sites. Quatsomes are stable structures that maintain their integrity over extended periods of storage, exhibiting uniformity in size, lamellarity, and membrane organization; therefore, these innovative systems offer a promising solution to address the stability limitations often associated with liposomes and similar structures [102]. Recent studies claim that quatsomes can be an important alternative to fight biofilm-related infections [103,104].

6.3. Solid Lipid Nanoparticles

Lipid nanoparticles have gained attention for their potential to facilitate controlled drug release and their cost effectiveness, as well as their scalability [106]. These nanoparticles typically consist of a matrix of physiological or physiologically related lipids known for their versatility and biocompatibility [108]. Moreover, lipid nanoparticles can encapsulate both hydrophobic and hydrophilic drugs [109]. SLNs are composed of a lipid matrix that remains in a solid state at both room temperature and body temperature, and their use in biofilm management has been published [107,112].

Antibiotics like clarithromycin and cefuroxime are commonly used to combat a wide spectrum of pathogens. However, both of these antimicrobial agents suffer from poor systemic bioavailability. Therefore, to address this issue, solid lipid nanoparticles (SLNs) have been developed [110,111], with these studies being a source of evidence that lipid nanoparticles can serve as effective delivery systems, potentially enhancing the therapeutic effects of drugs used to treat bacterial biofilms [110,111].

6.4. Micelles

Micelles are structures formed by amphiphilic molecules, which, in aqueous solutions, self-assemble when they reach critical micelle concentration. In micelles, the amphiphilic molecules arrange their hydrophobic regions to form an inner core, which can be used to load lipophilic drugs [114]. Micelles can effectively deliver antimicrobial agents inside the biofilm matrix and, consequently, the bacteria within it. The amphiphilic nature of micelles allows them to penetrate the biofilm’s EPS, a significant barrier to drug delivery. Once within the biofilm, micelles can release their encapsulated drugs in a controlled manner, ensuring sustained therapeutic levels at the site of infection. This targeted delivery system enhances the efficacy of the antimicrobial agents and reduces the likelihood of systemic side effects [115].

Table 4 summarizes the information concerning using lipid-based formulations to eradicate biofilms. Lipid-based nanocarriers, such as liposomes, quatsomes, and solid lipid nanoparticles, are widely used in drug delivery due to their biocompatibility and ability to encapsulate both hydrophilic and hydrophobic drugs. These nanocarriers mimic biological membranes, which generally leads to lower toxicity compared to other nanomaterials. However, toxicity can still arise from the degradation products of lipid components, the presence of synthetic surfactants, or the accumulation of nanocarriers in tissues. Ensuring biocompatibility involves careful selection of lipid types, optimizing particle size, and avoiding immunogenic responses. Continuous research is needed to balance efficacy with safety, particularly for long-term or repeated use [161].

Table 4. Examples of lipid-based nanocarriers developed to eradicate biofilms.

Type of Nanocarrier	Composition	Encapsulated Compound	Bacteria	Mechanism of Action	References
Liposomes	DOPA coated with CaP	Acridine orange, TMP	Staphylococcus aureus	Controlled drug release, enhanced liposomal stability	[162]
	DPPC and DPPG	Gentamicin	Pseudomonas aeruginosa and S. aureus	Controlled drug release, enhanced liposomal stability	[163]
	Soy phosphatidylcholine, cholesterol, DC-Chol, DSPE-PEG2000	Clarithromycin	MRSA	Enhanced phagocytosis of microbial cells	[164]
	DOPE and DC-Chol	Tobramycin	P. aeruginosa	Enhanced antimicrobial activity	[165]
	Soy lecithin, cholesterol	Cinnamon oil	MRSA	Enhanced chemical stability and antimicrobial activity	[166]
Quatsomes	Cholesterol	Cetylpyridinium chloride	S. aureus and P. aeruginosa	Enhanced anti-biofilm activity	[103]
	Quaternary bicephalic surfactant (StBAclm)	Vancomycin	MRSA	Enhanced antibacterial kinetics	[104]
Solid lipid nanoparticles	GMS, P, SA	Rifampin	Staphylococcus epidermidis	Enhanced efficiency in biofilm degradation and killing bacteria	[167]
	Stearic acid, Tristearin, Soya lecithin	Cefuroxime axetil	S. aureus	Enhanced bioavailability and antimicrobial activity	[110]
	Compritol 888 ATO	Curcumin	S. aureus and Escherichia coli	Enhanced antimicrobial activity	[168]
	Surfactant from Corynebacterium zerosis NS5	-	E. coli, P. aeruginosa, S. aureus, S. mutans	Enhanced biocompatibility Enhanced antimicrobial activity	[169]

CaP, calcium phosphate; DC-Chol, 3β-[N-(N′, N′-dimethyl aminoethane)-carbamoyl] cholesterol hydrochloride; DOPA, 1,2-dioleoyl-sn-glycero-3-phosphate; DOPE, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3-phosphor-(10-racglycerol); DSPE-PEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000; GMS, Glycerylmonostearate.

Table 4. Examples of lipid-based nanocarriers developed to eradicate biofilms.

Type of Nanocarrier	Composition	Encapsulated Compound	Bacteria	Mechanism of Action	References
Liposomes	DOPA coated with CaP	Acridine orange, TMP	Staphylococcus aureus	Controlled drug release, enhanced liposomal stability	[162]
	DPPC and DPPG	Gentamicin	Pseudomonas aeruginosa and S. aureus	Controlled drug release, enhanced liposomal stability	[163]
	Soy phosphatidylcholine, cholesterol, DC-Chol, DSPE-PEG2000	Clarithromycin	MRSA	Enhanced phagocytosis of microbial cells	[164]
	DOPE and DC-Chol	Tobramycin	P. aeruginosa	Enhanced antimicrobial activity	[165]
	Soy lecithin, cholesterol	Cinnamon oil	MRSA	Enhanced chemical stability and antimicrobial activity	[166]
Quatsomes	Cholesterol	Cetylpyridinium chloride	S. aureus and P. aeruginosa	Enhanced anti-biofilm activity	[103]
	Quaternary bicephalic surfactant (StBAclm)	Vancomycin	MRSA	Enhanced antibacterial kinetics	[104]
Solid lipid nanoparticles	GMS, P, SA	Rifampin	Staphylococcus epidermidis	Enhanced efficiency in biofilm degradation and killing bacteria	[167]
	Stearic acid, Tristearin, Soya lecithin	Cefuroxime axetil	S. aureus	Enhanced bioavailability and antimicrobial activity	[110]
	Compritol 888 ATO	Curcumin	S. aureus and Escherichia coli	Enhanced antimicrobial activity	[168]
	Surfactant from Corynebacterium zerosis NS5	-	E. coli, P. aeruginosa, S. aureus, S. mutans	Enhanced biocompatibility Enhanced antimicrobial activity	[169]

CaP, calcium phosphate; DC-Chol, 3β-[N-(N′, N′-dimethyl aminoethane)-carbamoyl] cholesterol hydrochloride; DOPA, 1,2-dioleoyl-sn-glycero-3-phosphate; DOPE, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3-phosphor-(10-racglycerol); DSPE-PEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000; GMS, Glycerylmonostearate.

Figure 2 depicts a schematic representation of liposomes, quatsomes, and solid lipid nanoparticles.

7. Polymeric-Based Nanocarriers

7.1. Polymeric Nanoparticles

Polymeric nanoparticles (NPs) are made from biodegradable polymers and have strong structural integrity, which helps stabilize volatile drugs and enables their controlled release [119]. Additionally, they are simple to scale up and economical, and maintain high stability during storage [116,120]. Among various polymers, poly(lactic-co-glycolic acid) (PLGA) is the most frequently used for drug delivery because it is both biocompatible and biodegradable [123]. Numerous studies have documented the successful use of PLGA micro- and nanoparticles for delivering antibiotics to bacterial biofilms [121,124,170].

Other polymers, such as chitosan, are extensively used in pharmaceutical and medical fields. Chitosan, derived from chitin, is both biocompatible and biodegradable and is known to have inherent antibacterial properties [122]. Leveraging these characteristics, Tan et al. [117] developed chitosan nanoparticles for the combined delivery of an antibacterial agent (oxacillin) and a biofilm matrix-disrupting agent (Deoxyribonuclease I).

7.2. Polymeric Micelles

Polymeric micelles have gained attention as a potential strategy for combating biofilms due to their unique properties, namely, the fact that they can increase the solubility of hydrophobic antimicrobial agents, making them more effective in targeting biofilms. The ability to functionalize polymeric micelles allows for targeted delivery to biofilms, increasing the concentration of antimicrobial agents at the site of infection while minimizing systemic side effects. Due to their small size and the ability to modify their surface characteristics, polymeric micelles can penetrate the dense extracellular matrix of biofilms more effectively than conventional treatments. Polymeric micelles can be engineered to carry multiple types of therapeutic agents (e.g., antibiotics, enzymes, anti-inflammatory drugs), providing a multifaceted approach to disrupting biofilms. However, the design and synthesis of polymeric micelles can be complex and require precise control over the physicochemical properties of the polymers. Also, they may have stability issues in physiological conditions, leading to premature release of the encapsulated drug before reaching the biofilm. The biocompatibility and potential toxicity of the polymers used in micelle formation need to be thoroughly evaluated. Large-scale production of polymeric micelles with consistent quality can be challenging, potentially limiting their widespread clinical application [125].

7.3. Dendrimers

Dendrimers are highly branched, tree-like macromolecules with a well-defined, three-dimensional architecture. These synthetic polymers are characterized by their multiple layers, called generations, surrounding a central core. The unique structure of dendrimers provides a high degree of surface functionality and versatility, making them suitable for various biomedical applications, including antibacterial treatments [131]. Dendrimers are constituted by a core (the central part of the dendrimer from which branches emanate), branches (repeatedly branched structures extending from the core), and surface groups (functional groups located on the outermost branches, which can be modified to enhance interactions with bacterial cells) [127].

Dendrimers can destroy bacteria through several mechanisms, which can be categorized into direct antibacterial activity and delivery of antibacterial agents [171]. Concerning the direct antibacterial activity, dendrimers can disrupt cell membranes, leading to cell lysis. For example, cationic dendrimers can interact with negatively charged bacterial cell membranes, causing structural damage [128]. Moreover, certain dendrimers can induce the production of reactive oxygen species (ROS), which can damage bacterial DNA, proteins, and lipids, leading to cell death [126]. With regard to the delivery of antibacterial agents, dendrimers can encapsulate or conjugate with antibiotics, enhancing the solubility, stability, and bioavailability of these drugs. The dendrimer structure allows for the controlled release of the antibiotic, targeting the bacteria more effectively [130]. Also, functional groups on the dendrimer surface can be modified to target specific bacterial strains or biofilms. This targeted approach helps in delivering the antibacterial agents directly to the site of infection, reducing the required dosage and minimizing side effects [132]. Poly(amidoamine) (PAMAM) is among the most studied dendrimers for antibacterial applications. PAMAM dendrimers can be functionalized with various antibacterial agents or modified to have intrinsic antibacterial properties [128]. Cationic dendrimers, due to their positive charge, can effectively interact with the negatively charged bacterial cell walls, leading to membrane disruption and bacterial death [129]. Carbohydrate-functionalized dendrimers can target specific bacterial lectins, enhancing the binding and uptake by bacterial cells and delivering antibacterial agents more effectively [132].

Table 5. Examples of polymeric-based nanocarriers developed to eradicate biofilms.

Type of Nanocarrier	Composition	Encapsulated Compound	Bacteria	Mechanism of Action	References
Polymeric nanoparticles	PLGA PVA	Ciprofloxacin	S. aureus and P. aeruginosa	Enhance drug bioavailability, controlled release	[173]
	PLGA	Carvacrol, rifampicin, moxifloxacin, levofloxacin	S. aureus, P. aeruginosa, and S. epidermidis	Enhanced penetration of antibiotics to the deeper layer of the biofilm	[121]
	PLGA	Clarithromycin, rifampicin, ciprofloxacin	-	Enhance drug bioavailability	[170]
Polymeric micelles	PLGA-b-mPEG and PLGA-b-p(Tpy)5	Terpyridine	P. aeruginosa	Fe(II) chelating by Tpy–micelles	[125]
Dendrimers	Liquid crystalline dendrimers, core–shell tecto dendrimer, glycodendrimers, polyglycerol, PAMAM, PEPE, PPI, PAMAMOS	-	S. aureus, E. coli, P. aeruginosa	Enhanced encapsulation	[132]
	PAMAM	-	B. cereus, E. coli, P. aeruginosa, S. aureus, S. oralis	Enhanced antimicrobial activity	[171]

PAMAM, poly(amidoamine); PAMAMOS, polyamido amine organosilicon dendrimers; PEPE, poly ether-copolyester; PLGA, poly(lactic-co-glycolic acid); PLGA-b-mPEG, poly(lactic-co-glycolic acid)-block-methoxy poly(ethylene glycol); PLGA-b-p(Tpy)₅, poly(lactic-co-glycolic acid)-block-poly(terpyridine)₅; PPI, poly(propylene imine); PVA, polyvinyl alcohol.

summarizes the information concerning polymeric-based formulations used to eradicate biofilms. Polymeric-based nanocarriers, such as polymeric nanoparticles, micelles, and dendrimers, are popular in drug delivery systems due to their tunable properties, stability, and controlled release capabilities. While they generally exhibit good biocompatibility, potential toxicity can arise from the polymers themselves or their degradation products, which may cause inflammatory responses or toxicity if not fully metabolized. Additionally, the size, surface charge, and composition of these nanocarriers can influence their interaction with cells and tissues, impacting their safety profile. To enhance biocompatibility, it is crucial to carefully design these nanocarriers, using biodegradable and non-immunogenic materials, and to thoroughly evaluate their long-term effects in vivo [172].

Figure 3 depicts a schematic representation of polymeric nanoparticles, polymeric micelles, and dendrimers.

8. Metallic-Based Nanocarriers

Mettalic Nanoparticles

Metallic nanoparticles (NPs) can be used as drug delivery systems because they protect drugs until they reach their target site and avoid triggering the immune system, all while exhibiting low cytotoxicity [1]. Additionally, many metallic NPs have been researched for their inherent antimicrobial properties and the low likelihood of bacteria developing resistance to them [96]. Table 6 provides an overview of metallic NPs developed to eradicate biofilms.

While the precise antibacterial mechanisms of metallic nanoparticles are still under study, theories include the following: (i) Disruption of the biofilm structure. This can be accomplished by physical interaction (metallic NPs can penetrate and disrupt the extracellular matrix of biofilms due to their small size, effectively breaking down the biofilm structure) or by chemical interaction (NPs can interact with EPS, destabilizing them). (ii) Generation of reactive oxygen species (ROS) by oxidative stress (metallic NPs, particularly silver and zinc oxide NPs, can generate reactive oxygen species, which cause oxidative stress to bacterial cells, leading to cell damage and death) or by localized damage (ROS can damage cellular components such as DNA, proteins, and lipids, leading to the breakdown of the biofilm’s protective environment). (iii) Release of metal ions, not only because of their antibacterial properties (many metallic NPs release metal ions (e.g., Ag⁺, Zn²⁺, Cu²⁺) that have potent antibacterial properties by disrupting bacterial cell membranes, interfering with enzyme function and inhibiting essential cellular processes), but also because of their sustained release ability (the slow release of metal ions ensures prolonged antibacterial activity, preventing biofilm formation and recurrence). (iv) Synergistic effects caused by combination therapy (metallic NPs can be combined with antibiotics or other antimicrobial agents to enhance their effectiveness. The NPs can facilitate better penetration of the antibiotics into the biofilm and reduce the likelihood of bacterial resistance) and functionalization (NPs can be functionalized with ligands, peptides, or other molecules to target specific bacterial strains or biofilm components, improving their efficacy) [148].

Among these metallic nanoparticles, silver and gold have become notable for their potent antimicrobial effects against a broad spectrum of microorganisms, including those resistant to multiple drugs, both in planktonic and biofilm-forming bacteria. Furthermore, these NPs can be easily functionalized with ligands, enhancing their biocompatibility and chemical stability [144].

Silver nanoparticles (AgNPs) have demonstrated effectiveness against a wide range of pathogens, including fungi, viruses, and various bacterial species [138]. Additionally, AgNPs have demonstrated potential as antimicrobial agents against drug-resistant bacteria [143]. Proposed mechanisms include contact killing and ion-mediated killing, membrane damage induction, reactive oxygen species (ROS) production, and cellular activity disruption such as ATP production and DNA replication [135]. AgNPs’ antimicrobial activity is credited both to the released silver ions and to the nanoparticles themselves. AgNPs can attach to bacterial cell membranes and penetrate the cells, causing damage by interacting with phosphorus- and sulfur-containing compounds like DNA. They specifically target the respiratory chain and cell division processes, ultimately leading to cell death [147].

Gold nanoparticles (AuNPs) have recently garnered significant attention due to their biocompatibility. Moreover, their anti-biofilm activity has been documented in recent studies [139]. While the precise mechanism by which they inhibit bacterial growth is still unclear, some reports suggest that damage to the bacterial cell wall is a contributing factor to bacterial cell death [137]. AuNPs are believed to interact with functional groups on the bacterial cell surface, leading to the inactivation and destruction of the bacteria [133].

Metal oxide nanoparticles, such as zinc oxide (ZnO) and titanium dioxide (TiO₂) nanoparticles, are also being extensively studied as potential antimicrobial agents. Zinc oxide nanoparticles exhibit antimicrobial properties by strongly adhering to bacterial cell membranes and disrupting the lipids and proteins, leading to the leakage of intracellular contents and eventual cell death [174]. Additionally, the generation of hydrogen peroxide and Zn²⁺ ions, which damage bacterial cells, is considered a key antibacterial mechanism of ZnO nanoparticles (ZnONPs) [146]. Titanium dioxide nanoparticles are studied for their photocatalytic antimicrobial properties. In a process known as photocatalysis, TiO₂ nanoparticles generate reactive oxygen species (ROS), including hydrogen peroxide and hydroxyl radicals, upon exposure to ultraviolet (UV) light. These ROS damage bacterial cell membranes, disrupt their semipermeability, interfere with oxidative phosphorylation, and can sometimes lead to cell death [142].

The magnetic characteristics of iron oxide nanoparticles (NPs) have been researched for their uses in diagnosis and treatment [134]. Consequently, magnetic nanoparticles (MNPs) present a promising targeted nanosystem as they can be maneuvered by a magnetic field gradient to reach specific biological targets [141]. Moreover, these particles are economical, stable both physically and chemically, biocompatible, and environmentally friendly [134]. The bactericidal action mechanism is the generation of ROS leading to cell death by necrosis or apoptosis [145]. Superparamagnetic iron oxide nanoparticles (SPIONs) are widely utilized in various medical applications, such as imaging, biodetection, and the delivery of drugs and genes [140]. These nanoparticles have also been researched for biofilm eradication [136].

Table 6 summarizes the information concerning polymeric-based formulations to eradicate biofilms. Metallic-based nanocarriers, such as those composed of silver, gold, or zinc oxide, are widely used in drug delivery, imaging, and diagnostic applications due to their unique optical, magnetic, and conductive properties. However, their biocompatibility and potential toxicity are major concerns. These nanocarriers can induce oxidative stress, inflammation, and cytotoxicity, especially if they accumulate in tissues or organs. The size, shape, surface coating, and dose of metallic nanoparticles significantly influence their interaction with biological systems. To minimize toxicity, it is essential to use biocompatible coatings, optimize particle size, and ensure the safe clearance of these nanocarriers from the body, as well as conduct thorough in vivo studies to assess their long-term safety [175].

Table 6. Examples of metallic-based nanocarriers developed to eradicate biofilms.

Type of Nanocarrier	Composition	Bacteria	Mechanism of Action	References
AgNPs	Silver and leaf extract of Convolvulus arvensis	S. aureus and P. aeruginosa	Enhanced chemical stability	[176]
	Silk fibroin/nanohydroxyapatite hydrogels with silver nanoparticles	S. aureus, MRSA, S. epidermidis, E. coli, and P. aeruginosa	Intrinsic antimicrobial action	[137]
AuNPs	Silk fibroin/nanohydroxyapatite hydrogels with gold nanoparticles	S. aureus, MRSA, S. epidermidis, E. coli, and P. aeruginosa	Intrinsic antimicrobial action	[137]
ZnONPs	Hydroxyapatite porous granules and zinc oxide nanoparticles	S. aureus and S. epidermidis	Non-specified	[177]
	Nanohydroxyapatite with zinc oxide nanoparticles	S. aureus and E. coli	Intrinsic antimicrobial action	[2]
	Iron oxide	S. epidermidis	Non-specified	[136]
Magnetic nanoparticles	Iron oxide	E. coli and S. aureus	Enhanced antimicrobial activity	[178]

Table 6. Examples of metallic-based nanocarriers developed to eradicate biofilms.

Type of Nanocarrier	Composition	Bacteria	Mechanism of Action	References
AgNPs	Silver and leaf extract of Convolvulus arvensis	S. aureus and P. aeruginosa	Enhanced chemical stability	[176]
	Silk fibroin/nanohydroxyapatite hydrogels with silver nanoparticles	S. aureus, MRSA, S. epidermidis, E. coli, and P. aeruginosa	Intrinsic antimicrobial action	[137]
AuNPs	Silk fibroin/nanohydroxyapatite hydrogels with gold nanoparticles	S. aureus, MRSA, S. epidermidis, E. coli, and P. aeruginosa	Intrinsic antimicrobial action	[137]
ZnONPs	Hydroxyapatite porous granules and zinc oxide nanoparticles	S. aureus and S. epidermidis	Non-specified	[177]
	Nanohydroxyapatite with zinc oxide nanoparticles	S. aureus and E. coli	Intrinsic antimicrobial action	[2]
	Iron oxide	S. epidermidis	Non-specified	[136]
Magnetic nanoparticles	Iron oxide	E. coli and S. aureus	Enhanced antimicrobial activity	[178]

9. Silica-Based Nanocarriers

Silica nanoparticles (SiNPs) are promising drug delivery systems because of their biocompatibility and stability, which allow for controlled drug release [151]. Additionally, their large surface area, due to uniform pores, enables them to carry a high drug payload and be effectively functionalized with various groups for targeted delivery [151]. Even though these nanoparticles lack inherent antimicrobial properties, they hold significant potential for delivering antibiotics to fight multidrug-resistant infections. This is because they can be designed to carry multiple drugs within their structure [152].

One of the primary reasons SiNPs are effective against biofilms is their ability to penetrate and disrupt the biofilm structure. Their nanoscale size allows them to infiltrate the biofilm matrix, reaching the embedded microorganisms. Once inside, SiNPs can be engineered to release antimicrobial agents in a controlled manner, ensuring a sustained attack on the biofilm. This targeted approach not only enhances the efficacy of the treatment but also minimizes the potential for developing resistance [154]. The surface of SiNPs can be easily modified to carry a variety of therapeutic agents. This functionalization allows for the attachment of antibiotics, antifungals, or even enzymes that degrade the biofilm matrix. The large surface area provided by the mesoporous structure of SiNPs enables a high payload of these agents, ensuring a potent delivery to the biofilm. Moreover, functional groups can be added to the SiNP surface to target specific components of the biofilm or the microorganisms within it, enhancing specificity and reducing off-target effects [155]. Ongoing research is continually expanding the potential applications of SiNPs in biofilm treatment. Studies have demonstrated the effectiveness of SiNPs in eradicating biofilms formed by various pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, and Candida species [150,153].

Silica-based nanocarriers are valued in drug delivery and biomedical applications for their large surface area, tunable pore size, and ease of functionalization. While generally considered biocompatible, concerns about toxicity arise from factors like particle size, surface properties, and dosage. Smaller particles can penetrate cells more easily but may cause more significant cytotoxic effects, such as oxidative stress and inflammation. Additionally, the degradation of silica into silicic acid in biological environments must be carefully monitored to avoid potential toxicity. To ensure safety, it is crucial to optimize the design and surface modification of silica-based nanocarriers and conduct comprehensive in vivo studies to evaluate their long-term biocompatibility and clearance from the body [179].

10. Quantum Dots

Quantum dots (QDs) are nanoscale semiconductor particles that possess unique optical and electronic properties due to their size, typically ranging from 2 to 10 nanometers. These properties arise from quantum mechanics, specifically the quantum confinement effect, which occurs when the particle size is smaller than the exciton Bohr radius. This confinement leads to discrete energy levels and results in the emission of light at specific wavelengths when excited. The size of the quantum dots determines the color of the light they emit, allowing them to be tuned for various applications by simply changing their size [156].

QDs hold significant promise in biofilm elimination. QDs can be used in photodynamic therapy (PDT), a treatment that involves the use of light-sensitive compounds (photosensitizers) and light to produce reactive oxygen species (ROS) that can kill bacteria and disrupt biofilms. QDs are excited by light, and transfer energy to oxygen molecules, creating ROS that can damage the biofilm structure and kill the embedded microorganisms. Moreover, quantum dots can be functionalized with various molecules, including antibiotics, peptides, or antibodies, to target specific components of the biofilm or the bacteria within it. This targeted approach ensures that the therapeutic agents are delivered precisely where needed, increasing their efficacy and reducing the likelihood of off-target effects. The small size and surface properties of quantum dots enable them to penetrate the dense extracellular matrix of biofilms. This penetration is crucial for delivering antimicrobial agents directly to the bacteria and for the physical disruption of the biofilm structure. Additionally, the interaction of QDs with biofilms can lead to the release of encapsulated drugs in a controlled manner, ensuring a sustained attack on the biofilm [157,158].

QDs are nanoscale semiconductor particles with unique optical properties, making them valuable for imaging, diagnostics, and drug delivery. However, their toxicity and biocompatibility are significant concerns, primarily due to the heavy metals (like cadmium) often used in their core. These metals can leach out, leading to oxidative stress, inflammation, and cytotoxicity. Additionally, the surface coatings used to stabilize QDs can influence their interaction with biological systems, potentially triggering immune responses. To enhance biocompatibility, researchers focus on developing QDs with safer materials, improving surface modifications, and ensuring rapid clearance from the body to minimize potential toxic effects. Comprehensive in vivo studies are essential to assess their long-term safety [180].

11. Challenges and Future Perspectives

Despite the potential, the research and application of nanocarriers face several challenges. Biofilms are highly heterogeneous and dynamic, with variations in microbial composition, extracellular matrix components, and microenvironmental conditions. This complexity poses a significant challenge in designing nanocarriers that can effectively penetrate and disrupt the biofilm matrix. The varying pH levels, oxygen gradients, and nutrient availability within biofilms also affect the efficacy of nanocarriers. Another challenge is nanocarrier delivery and penetration. Effective delivery of nanocarriers to biofilms is a critical challenge. The dense extracellular matrix acts as a barrier, hindering the penetration of nanocarriers. Moreover, the nanocarriers’ size, surface charge, and hydrophobicity influence their interaction with the biofilm [181]. Ensuring that nanocarriers reach the target site in sufficient concentrations while maintaining their functional integrity is a complex task. There is also a potential risk that microorganisms may develop resistance to nanocarriers, which is similar to antibiotic resistance. Continuous exposure to sublethal concentrations of nanocarriers might induce adaptive mechanisms in biofilms, leading to the emergence of resistant strains. Research into the mechanisms of resistance development and strategies to prevent it is essential. The translation of nanocarrier-based technologies from the laboratory to large-scale production faces significant hurdles. The manufacturing processes must be optimized for consistency, scalability, and cost effectiveness. Additionally, regulatory challenges related to the approval of nanocarrier-based therapeutics need also to be addressed [182].

While many nanomaterials have shown potent anti-biofilm activity, their potential toxicity to human cells and tissues raises concerns. Ensuring the biocompatibility of nanocarriers while maintaining their efficacy is crucial. The long-term effects of nanocarrier accumulation in the body and the environment also need thorough investigation [183].

Future research should focus on designing multifunctional nanocarriers that combine targeting, penetration, and antimicrobial activities. Innovations such as stimuli-responsive nanocarriers that release their payload in response to specific biofilm conditions (e.g., pH, enzymes) hold great promise. The use of natural or biomimetic materials to enhance biocompatibility and reduce toxicity is another avenue for exploration [78].

The development of personalized anti-biofilm nanocarriers tailored to the specific biofilm composition and the patient’s microbiome could enhance therapeutic outcomes. This approach requires a deep understanding of the individual’s biofilm characteristics and the ability to rapidly design and produce customized nanocarriers [145].

Combining nanocarriers with traditional antibiotics, enzymes, or other antimicrobial agents could enhance their efficacy and reduce the likelihood of resistance development. Synergistic approaches targeting multiple aspects of biofilm structure and function simultaneously are likely more effective [78].

The integration of nanocarriers with imaging agents or sensors could enable real-time monitoring of biofilm disruption and treatment efficacy. This would provide valuable feedback for clinicians, allowing for the optimization of therapeutic strategies in real time.

The development and application of anti-biofilm nanocarriers hold great promise in addressing the persistent challenge of biofilm-associated infections. However, overcoming the current challenges requires a multidisciplinary approach, integrating advances in nanotechnology, microbiology, materials science, and clinical research. With continued innovation and collaboration, the future of anti-biofilm nanocarriers looks promising, offering new hope for more effective and sustainable treatments.

12. Conclusions

Nanocarriers represent a vital advancement in the battle against biofilm-associated infections, which are a significant and growing challenge in medical settings. Biofilms are structured communities of bacteria that adhere to surfaces, such as medical implants, and produce a protective matrix that shields them from conventional antibiotics. This protection makes biofilms particularly difficult to eradicate, often resulting in chronic infections that necessitate surgical intervention and prolonged antibiotic use.

The importance of nanocarriers lies in their unique ability to effectively penetrate these resilient biofilm structures. These microscopic delivery systems can transport antimicrobial agents directly to the site of infection, ensuring higher local concentrations of the drug and enhancing its therapeutic effectiveness. Nanocarriers can be engineered to have properties such as targeted delivery, controlled release, and the ability to overcome bacterial defenses, which are critical for disrupting biofilms and killing the embedded bacteria.

By using nanocarriers, it is possible to potentially reduce the need for invasive procedures and lower the risk of antibiotic resistance, which is exacerbated by the overuse and misuse of antibiotics. Additionally, the targeted approach of nanocarriers minimizes damage to surrounding healthy tissues and reduces side effects, leading to improved patient outcomes. As biofilm-associated infections continue to pose a significant threat to patient health and place a heavy economic burden on healthcare systems, nanocarriers offer a promising and innovative solution to enhance the efficacy of antimicrobial treatments and improve overall healthcare efficiency.