Polymeric Nanoparticles for Antimicrobial Therapies: An up-to-date Overview

Despite the many advancements in the pharmaceutical and medical fields and the development of numerous antimicrobial drugs aimed to suppress and destroy pathogenic microorganisms, infectious diseases still represent a major health threat affecting millions of lives daily. In addition to the limitations of antimicrobial drugs associated with low transportation rate, water solubility, oral bioavailability and stability, inefficient drug targeting, considerable toxicity, and limited patient compliance, the major cause for their inefficiency is the antimicrobial resistance of microorganisms. In this context, the risk of a pre-antibiotic era is a real possibility. For this reason, the research focus has shifted toward the discovery and development of novel and alternative antimicrobial agents that could overcome the challenges associated with conventional drugs. Nanotechnology is a possible alternative, as there is significant evidence of the broad-spectrum antimicrobial activity of nanomaterials and nanoparticles in particular. Moreover, owing to their considerable advantages regarding their efficient cargo dissolving, entrapment, encapsulation, or surface attachment, the possibility of forming antimicrobial groups for specific targeting and destruction, biocompatibility and biodegradability, low toxicity, and synergistic therapy, polymeric nanoparticles have received considerable attention as potential antimicrobial drug delivery agents. In this context, the aim of this paper is to provide an up-to-date overview of the most recent studies investigating polymeric nanoparticles designed for antimicrobial therapies, describing both their targeting strategies and their effects.


Introduction
Microorganisms are an essential part of human existence, being responsible for numerous and diverse processes, including nitrogen fixation, vitamin production, photosynthesis, and organic matter decomposition. However, the delicate balance between microorganisms and the immune system may shift in favor of microorganisms, thus causing immune deficiencies [1]. Therefore, diseases caused by pathogenic microorganisms, such as bacteria, viruses, fungi, parasites, protozoa, or algae, can be directly or indirectly (vector-borne) transmitted from one individual to another, which is termed as infectious diseases [2][3][4][5].
In antiquity, around half of the individuals died before reaching sexual maturity, while in late medieval times, one-third of babies died in their infancy, which was mostly due to infectious diseases [6]. Thus, the discovery of antimicrobials, including antibacterial, antiviral, antifungal, and antiparasitic or anthelmintic drugs, has greatly impacted the global health system, as the infection-related mortality was significantly reduced [4,7,8]. Specifically, they have allowed the early treatment of infections without identifying the pathogen, consequently bringing novel possibilities for modern medicine, such as surgery,

Microbial Targeting Strategies
Generally, the design and development of nanosized systems for the delivery of antimicrobial drugs must consider the fulfillment of several characteristics, namely improving the efficiency of the antimicrobial treatment, increasing the local concentration of the antimicrobial drug at the specific infection site, minimizing the accumulation of the antimicrobial drug within healthy tissues, and the exposure of the commensal microflora to sub-lethal doses for avoiding the development of antimicrobial resistance and reducing the associated risks of toxicity [33]. In this context, nanoparticles must be specifically designed and/or improved to achieve an efficient payload binding capacity, targeted delivery, selective recognition, and increased cellular uptake and internalization with no cytotoxicity for the host cells [34][35][36][37][38].
Nanoparticles can target microbial cells through two general possibilities, namely passive or active targeting [39]. On one hand, passive targeting is associated with the accumulation of nanoparticles at the infection site due to a higher vascular permeability and impaired lymphatic system functioning, leading to prolonged drug retention [39][40][41]. This type of delivery is directly influenced by several factors, including hydrophobicity, van der Waals forces, and static electric attraction. Its potency can be increased with the electrostatic interactions between the negative charge of the bacteria surface and the cationic charge of the nanoparticle surface [40,42]. On the other hand, active targeting is a widely employed phenomenon based on the conjugation of active molecules onto the surface of nanoparticles that will specifically bind to the surface molecules overexpressed by microbial cells, such as polysaccharides, proteins, or lipids [39,40,[43][44][45]. In this manner, a wide variety of functional groups and molecules or stimuli-responsive ligands can be attached through chemical, physical, or biological methods [35]. Active targeting is an advantageous method to improve the therapeutic index by increasing the selectivity and recognition properties of nanoparticles and consequently reaching higher concentrations at the specific infection site in shorter periods. Thereby, side effects and their associated socioeconomic costs are considerably reduced [34,35,43,44]. Nonetheless, active targeting nanoparticles' design should ensure an appropriate balance between the interaction strength, the release rate, and the conjugate stability [39]. Common biomolecules used as moieties for active targeting include small molecules, peptides, antibodies, nanobodies, proteins, nucleic acids, carbohydrates, and antimicrobial drugs ( Figure 2) [34][35][36][39][40][41]43,[46][47][48].

Microbial Targeting Strategies
Generally, the design and development of nanosized systems for the delivery of antimicrobial drugs must consider the fulfillment of several characteristics, namely improving the efficiency of the antimicrobial treatment, increasing the local concentration of the antimicrobial drug at the specific infection site, minimizing the accumulation of the antimicrobial drug within healthy tissues, and the exposure of the commensal microflora to sub-lethal doses for avoiding the development of antimicrobial resistance and reducing the associated risks of toxicity [33]. In this context, nanoparticles must be specifically designed and/or improved to achieve an efficient payload binding capacity, targeted delivery, selective recognition, and increased cellular uptake and internalization with no cytotoxicity for the host cells [34][35][36][37][38].
Nanoparticles can target microbial cells through two general possibilities, namely passive or active targeting [39]. On one hand, passive targeting is associated with the accumulation of nanoparticles at the infection site due to a higher vascular permeability and impaired lymphatic system functioning, leading to prolonged drug retention [39][40][41]. This type of delivery is directly influenced by several factors, including hydrophobicity, van der Waals forces, and static electric attraction. Its potency can be increased with the electrostatic interactions between the negative charge of the bacteria surface and the cationic charge of the nanoparticle surface [40,42]. On the other hand, active targeting is a widely employed phenomenon based on the conjugation of active molecules onto the surface of nanoparticles that will specifically bind to the surface molecules overexpressed by microbial cells, such as polysaccharides, proteins, or lipids [39,40,[43][44][45]. In this manner, a wide variety of functional groups and molecules or stimuli-responsive ligands can be attached through chemical, physical, or biological methods [35]. Active targeting is an advantageous method to improve the therapeutic index by increasing the selectivity and recognition properties of nanoparticles and consequently reaching higher concentrations at the specific infection site in shorter periods. Thereby, side effects and their associated socio-economic costs are considerably reduced [34,35,43,44]. Nonetheless, active targeting nanoparticles' design should ensure an appropriate balance between the interaction strength, the release rate, and the conjugate stability [39]. Common biomolecules used as moieties for active targeting include small molecules, peptides, antibodies, nanobodies, proteins, nucleic acids, carbohydrates, and antimicrobial drugs ( Figure 2) [34][35][36][39][40][41]43,[46][47][48].

Small Molecules
The use of small molecules for the surface functionalization of nanomaterials has brought a new perspective for biomedical applications, as they possess the ability to

Small Molecules
The use of small molecules for the surface functionalization of nanomaterials has brought a new perspective for biomedical applications, as they possess the ability to modulate the biological properties of nanomaterials. In this manner, binding to the host cell receptors can be mediated, and antimicrobial resistance can be further avoided [40].
One example of such molecules is antifolates, with folic acid as the central molecule of their metabolism [49]. Folic acid or folate is a group of water-soluble metabolites that belong to the B-vitamin family, namely B9. Folates function as enzymatic co-factors in the C1 transfer reactions, playing fundamental roles in amino acid metabolism and purines, pyrimidines, and methionine synthesis. Alternatively termed tetrahydrofolate molecules, they are characterized by a common chemical structure comprising a pteridine ring, a paminobenzoic acid, and one or more γ-linked L-glutamate residues [49][50][51]. By contrast to bacteria, fungi, and some plants, mammalian cells are unable to synthesize folate de novo, but they possess specific folate receptors that allow for its internalization. As microbial infections are characterized by an accelerated folate metabolism rate in order to ensure the proper functioning of the processes involved in cell replication and protein and nucleic acid synthesis, folic acid antagonists have been widely used as alternative antibiotic agents [49][50][51][52]. Moreover, antifolates such as pyrimethamine, proguanil, and sulfadoxine represent an important class of antimalarial drugs [53,54]. Additionally, the specific cyclosporiasis drug treatment is based on the combination of two antibiotics, namely trimethoprim and sulfamethoxazole or co-trimoxazole. Their action involves blocking two consecutive steps required for the biosynthesis of nucleic acids and proteins essential for the Figure 2. The main types of biomolecules used for the surface modification of nanoparticles for active microbial targeting.
One example of such molecules is antifolates, with folic acid as the central molecule of their metabolism [49]. Folic acid or folate is a group of water-soluble metabolites that belong to the B-vitamin family, namely B 9 . Folates function as enzymatic co-factors in the C1 transfer reactions, playing fundamental roles in amino acid metabolism and purines, pyrimidines, and methionine synthesis. Alternatively termed tetrahydrofolate molecules, they are characterized by a common chemical structure comprising a pteridine ring, a p-aminobenzoic acid, and one or more γ-linked L-glutamate residues [49][50][51]. By contrast to bacteria, fungi, and some plants, mammalian cells are unable to synthesize folate de novo, but they possess specific folate receptors that allow for its internalization. As microbial infections are characterized by an accelerated folate metabolism rate in order to ensure the proper functioning of the processes involved in cell replication and protein and nucleic acid synthesis, folic acid antagonists have been widely used as alternative antibiotic agents [49][50][51][52]. Moreover, antifolates such as pyrimethamine, proguanil, and sulfadoxine represent an important class of antimalarial drugs [53,54]. Additionally, the specific cyclosporiasis drug treatment is based on the combination of two antibiotics, namely trimethoprim and sulfamethoxazole or co-trimoxazole. Their action involves blocking two consecutive steps required for the biosynthesis of nucleic acids and proteins essential for the parasite by inhibiting tetrahydrofolic acid and dihydrofolic acid production, respectively [55].

Peptides
The pathway involved in the targeted drug release using nanoparticles aims to accomplish the complex tasks of specifically targeting the cells of interest, crossing the extracellular membrane and internalization into the cell, and localization to specific subcellular organelles. In this context, peptides have been extensively researched and exploited for their capacity to fulfill these aims after their surface attachment onto the surface of the nanoparticles [56,57]. Therefore, antimicrobial peptides have received a great interest in naturally occurring antimicrobials produced by bacteria, fungi, protozoa, and some plants and animals, with an improved biocompatibility, selectivity, and efficiency, safety, and tolerance by the human organism [40,[58][59][60][61]. These peptides comprise 5-50 amino acid chains and are generally composed of L-amino acids, such as lysine, arginine, and histidine, which are defined in α-helices and/or β-sheets secondary structures [60,62]. Their amphipathic or cationic structure allows for an efficient microbial membrane targeting and a broad action spectrum against Gram-positive and Gram-negative bacteria, fungi, viruses, and protozoa [58,60]. Owing to their biochemical properties, the action mechanisms are generally based on their interaction with the microbial phospholipid membranes [60]. Specifically, subsequent to the antimicrobial peptide binding to the target membrane, permeabilization of the membrane occurs, consequently causing cellular component leakage and cell death [63]. This binding is a result of the electrostatic forces between the positively charged antimicrobial peptides, i.e., an overall charge of +1 to +7, and the negatively charged microbial membrane. Moreover, peptide features such as the amino acid sequence, the amphipathic charge, the structure, and the hydrophobicity are key factors in the overall antimicrobial activity. Therefore, the active targeting can be modulated, as antimicrobial peptides have a tendency of binding to microbial cells, rather than mammalian cells, which are zwitterionic. Furthermore, studies have shown that with sufficient positive charges present, reducing their hydrophobicity favors bacterial cell targeting [62].
The models proposed for membrane permeabilization involve (i) the toroidal-pore model based on the accumulation of peptides on the surface, which leads to a continuous lipid monolayer bending through the pore and their building up within the pore, (ii) the barrel-stave model, through which the hydrophilic part of the peptide moves from the core interior region, while the hydrophobic part is localized toward the lipids, thus inserting into the cell membrane, and (iii) the carpet-like model, where the peptides parallelly aggregate to the microbial membrane and cover it without forming any pores, and membrane permeabilization is triggered by peptides attached to the surface, thus causing its disruption in a manner similar to detergent mechanisms that result in micelle formation [63].

Proteins
Nanoparticle functions and targeting can also be enhanced by protein coating [35,64]. The lysozyme is a potential candidate, as it is a biomolecule with key defensive roles in the innate immune system widely distributed in phages, bacteria, plants, vertebrates, and humans [65]. Owing to its antibacterial, antiviral, and anti-inflammatory properties, it has been extensively used in the medical, environmental protection, and food industries [35,[65][66][67]. The lysozyme's antimicrobial activity mainly relies on the degradation of the microbial cell wall through the peptidoglycan hydrolyzation, specifically the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine monosaccharides [35,[65][66][67][68]. Furthermore, there is evidence of lysozymal antiviral character based on its potential activity against the human immunodeficiency virus [65].
The field of antimicrobial therapy has also witnessed the emergence of monoclonal antibodies that are able to target specific microbial phenotypes [69,70]. Their advantages are numerous, including but not limited to high specificity against one specific microbial type, longevity, and multiple antimicrobial mechanisms that limit toxicity and resistance [71]. An example of such types of antibodies is the human monoclonal antibody 3E9-11, which specifically targets the O25b O-antigen present within the Escherichia coli ST131 O25b:H4 clonal group that is associated with extended-spectrum beta-lactamases acquisition and fluoroquinolone resistance [69]. Furthermore, monoclonal and polyclonal antibodies targeting the poly-N-acetyl-d-glucosamine and the deacetylated poly-N-acetyl-d-glucosamine polysaccharides that are highly conserved and expressed by a multitude of microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and protozoa, have shown promising in vitro and in vivo efficiency [72]. Nanobodies, a novel and unique class of single domain antibodies derived from naturally occurring heavy-chain-only antibodies only present within camelid serum, have been widely investigated in recent years for their superior physicochemical properties. Specifically, nanobodies are nanoscaled compounds with a robust structure, high stability, antigen-binding affinity, one cognate target specificity, water solubility, and reversible refolding that have the potential for the development of next-generation biomolecules [73,74]. These polypeptides with a molecular weight of less than 15 kDa have emerged from the phage display process and are an important tool for developing novel nanobiotechnologies, as some are already under clinical investigation for a variety of human diseases, such as chronic inflammation, brain tumors, breast cancer, lung disorders, and infectious diseases [73][74][75]. Specifically, the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) have approved the use of caplacizumab, which is a bivalent nanobody with efficiency in the treatment of thrombotic thrombocytopenic purpura [74]. Nanobodies have also proven their efficacy in the targeting of specific microbial strains, including bacteria, viruses, and protozoa [76,77].

Nucleic Acids
Nucleic acid-nanoparticle complexes have also attracted significant interest, as most of the small interfering RNA (siRNA) or microRNA delivery systems have been approved for clinical trials for virus infections, cancer therapy, and many other diseases [78].
Moreover, aptamers are a special kind of targeting biomolecules, possessing excellent physicochemical features for superior infectious disease diagnosis and treatment [75]. Precisely, they are short single-stranded nucleic acids or peptides with defined threedimensional structures and the ability to recognize and bind targets with high affinity and specificity [75,[79][80][81][82]. Aptamers can discriminate between molecules structurally different with only one group or even enantiomers, establishing dissociation constants in the picomolar to nanomolar range for high molecular weight targets nanomolar to the micromolar range for low molecular weight targets [81]. They are generally obtained through a process of in vitro selection, following the methodology described by Tuerk and Gold in 1990 of The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [75,[79][80][81][82]. The SELEX scheme involves three main steps, namely library and target incubation, aptamer-target complexes separation from unbound oligonucleotides, and bound molecules amplification [83]. Their small size, low molecular weight, stability under a variety of conditions, and lack of toxicity have allowed for an efficient nanoparticle functionalization for numerous active targeted delivery applications [79][80][81]. Moreover, they are a potential alternative for the use of antibodies, as they are able to overcome their limitations, such as higher temperature stability [80]. Therefore, aptamer-nanoparticle systems' potential relies on the increase of targeting interactions due to a higher aptamer density onto the surface of the nanoparticles while being protected from the nuclease digestion [81].

Carbohydrates
Carbohydrates are an essential type of macromolecules that are ubiquitously found throughout living organisms, playing vital roles in numerous biological recognition processes associated with cell differentiation, development, adhesion, communication, and signaling [84]. In the context of infectious diseases, many pathogenic microorganisms attach onto the surface of host cells through carbohydrate-protein interactions between cell-surface glycans and adhesins or agglutinins. Bacteria, viruses, and fungi express onto their surface a vast number of glycan-binding proteins, which are also known as lectins [84][85][86].
The sugar-binding activity of lectins has led to various in vivo functions, including host-pathogen interactions, nutrition absorption inhibition, intercellular recognition and signal transduction, and cell migration [87,88]. In this manner, glycans could represent an important candidate for nanoparticle surface functionalization for the active targeting of microorganisms. There are many studies proving the potential of glycoprotein binding of many viruses, including human immunodeficiency virus-1, influenza, coronavirus, Ebola, Zika, herpes simplex, etc. [89], and bacteria, e.g., Escherichia coli, through the interaction between gluconamide-functionalized nanoparticles and the lipopolysaccharide molecules present onto the outer membrane of the microorganisms [90].

Antimicrobial Drugs
Nonetheless, the surface of nanoparticles can also be modified using vancomycin, which acts by targeting peptidoglycans present onto the surface of Gram-positive bacteria, polymyxin, which is responsible for targeting lipopolysaccharides found onto Gramnegative bacteria, or zinc(II)-bis(dipicolylamine), which targets phosphatidylserine and is present onto the surface of both Gram-positive and Gram-negative bacteria. Notably, they are able to specifically bind bacteria, since healthy mammalian cells do not express these types of molecules [45].
Vancomycin is a broad-spectrum glycopeptide antibiotic that specifically binds Grampositive bacteria, including staphylococci, streptococci, and most enterococci, through hydrogen bonds between its carbonyl and amine groups and the peptidoglycans found onto the cell wall [91][92][93]. The mechanisms of action mainly involve the inhibition of the cell wall synthesis by forming non-covalent complexes with the C-terminal L-Lys-D-Ala-D-Ala motif within the bacterial peptidoglycan precursors and the inhibition of RNA synthesis [92][93][94]. While the administration of vancomycin has resulted in the development of vancomycin-resistant Staphylococcus aureus, vancomycin-intermediate Staphylococcus aureus, and vancomycin-resistant enterococci [92], its attachment onto the surface of nanoparticles has allowed for the capture of both Gram-positive and Gramnegative bacteria within complex samples, such as urine or blood [91].
Polymyxins are a class of cationic polypeptide antibiotics comprising five types of compounds, namely polymyxin A-E, which is the standard gold treatment against Gramnegative bacterial infections [95][96][97]. While their use has been avoided in the 1970s and 1980s due to the introduction of presumably safer broad-spectrum antibiotics, the emergence of multiple drug-resistant Gram-negative bacteria, especially Pseudomonas aeruginosa and Acinetobacter baumannii, has led to its clinical reintroduction [96,98]. The precise mechanisms involve binding to the bacterial cell wall and subsequently altering the outer and inner membrane permeability to K + and Na + ions. In this manner, the osmotic barrier of the cell is lost, leading to the death of the bacterium through lysis [8].

Stimuli-Responsive Nanosystems
Another approach for microbial targeting is the development of stimuli-responsive nanosystems, which can either recognize specific microenvironmental changes associated with the pathological state of infection or inflammation, such as pH, enzyme, and chemical compound concentrations, and redox state variations, or respond to external physical stimuli, such as thermal, magnetic, light, or ultrasound effects ( Figure 3). Consequently, the nanosystems react dynamically, leading to a controlled release of the drug at the targeted site [33,44,99,100]. Similarly, the targeting characteristics and the efficiency of the antimicrobial therapy are considerably enhanced, while the side effects are significantly minimized [33,99]. Additionally, this approach allows for the reversibility to the nanosystems' initial state to control the antimicrobial effects [33,44].
An example of such nanosystems involved developing gentamicin sulfate-functionalized nanoparticles covalently grafted onto the surface of titanium implants. As the bioactive molecules were linked to the nanoparticles through pH-sensitive imine bonds, a decrease in the local pH induced by bacterial proliferation and infection would lead to the drug's release due to the hydrolysis of the imine bond [93,101]. the nanosystems react dynamically, leading to a controlled release of the drug at the targeted site [33,44,99,100]. Similarly, the targeting characteristics and the efficiency of the antimicrobial therapy are considerably enhanced, while the side effects are significantly minimized [33,99]. Additionally, this approach allows for the reversibility to the nanosystems' initial state to control the antimicrobial effects [33,44]. An example of such nanosystems involved developing gentamicin sulfate-functionalized nanoparticles covalently grafted onto the surface of titanium implants. As the bioactive molecules were linked to the nanoparticles through pH-sensitive imine bonds, a decrease in the local pH induced by bacterial proliferation and infection would lead to the drug's release due to the hydrolysis of the imine bond [93,101].

Antimicrobial Applications of Polymeric Nanoparticles
Due to their insolubility and administration route, bioactive compounds are generally prone to lose their pharmacological activity. Thus, the process of drug discovery and development must be optimized in order to ensure optimal pharmacokinetics, absorption, distribution, metabolism, excretion, toxicity, and therapeutic effect duration [102]. In the context of infectious diseases, the life and bioavailability of antimicrobial drugs must be enhanced, while the administered dose must be reduced [103,104].
After administration, conventional antimicrobial drugs are distributed throughout the body via bloodstream, where a considerable percentage of the drug undergoes rapid clearance and inactivation. By contrast, drug-carrying nanosystems have the capacity to stay in the circulatory system for longer time periods and specifically target the tissue of interest. In this manner, an appropriate drug dose is administered, thus reducing the plasma fluctuations and the associated adverse effects. Furthermore, the nanoscale of these systems allows for an improved penetration through the tissue barriers while ensuring the protection of the drug until cellular uptake and targeted release [102,103,105,106]. The main mechanisms involved in the controlled release of antimicrobial drugs include diffusion-based, elution-based, and chemically-or stimuli-controlled release (Figure 4) [105].

Antimicrobial Applications of Polymeric Nanoparticles
Due to their insolubility and administration route, bioactive compounds are generally prone to lose their pharmacological activity. Thus, the process of drug discovery and development must be optimized in order to ensure optimal pharmacokinetics, absorption, distribution, metabolism, excretion, toxicity, and therapeutic effect duration [102]. In the context of infectious diseases, the life and bioavailability of antimicrobial drugs must be enhanced, while the administered dose must be reduced [103,104].
After administration, conventional antimicrobial drugs are distributed throughout the body via bloodstream, where a considerable percentage of the drug undergoes rapid clearance and inactivation. By contrast, drug-carrying nanosystems have the capacity to stay in the circulatory system for longer time periods and specifically target the tissue of interest. In this manner, an appropriate drug dose is administered, thus reducing the plasma fluctuations and the associated adverse effects. Furthermore, the nanoscale of these systems allows for an improved penetration through the tissue barriers while ensuring the protection of the drug until cellular uptake and targeted release [102,103,105,106]. The main mechanisms involved in the controlled release of antimicrobial drugs include diffusionbased, elution-based, and chemically-or stimuli-controlled release (Figure 4) [105].
In this context, nanostructured systems appear to be an ideal tool for combating antimicrobial resistance and developing efficient treatment options [102]. As previously mentioned, polymeric nanosystems are superior due to many advantages, such as high drug solubility and storage, biocompatibility, biodegradability, and stability, permitting the deliberate and precise drug release at the targeted sites [103,106,107].
This review focuses on polymeric nanoparticles, which are categorized into polymeric nanospheres and polymeric nanocapsules, depending on their internal structure and morphology ( Figure 6). On one hand, nanospheres comprise a continuous polymeric network with a regular sphere structure in which drug molecules are either retained inside the matrix or attached to its surface. On the other hand, nanocapsules consist of a polymeric shell surrounding the liquid/solid oily core. The drug is dissolved and modulates the release profile of the drug [110,111]. In this context, nanostructured systems appear to be an ideal tool for combating antimicrobial resistance and developing efficient treatment options [102]. As previously mentioned, polymeric nanosystems are superior due to many advantages, such as high drug solubility and storage, biocompatibility, biodegradability, and stability, permitting the deliberate and precise drug release at the targeted sites [103,106,107].
Polymeric nanosystems can be synthesized from a variety of natural or synthetic precursors, such as collagen, chitosan, gelatin, or albumin, and polyethylene glycol, polylactic acid, poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA) or polycaprolactone (PCL), respectively [42,108,109]. Additionally, they can be developed in multiple forms, including nanoparticles, micelles, vesicles, dendrimers, or hybrid inorganic-polymer nanosystems ( Figure 5) [17].   In this context, nanostructured systems appear to be an ideal tool for combating antimicrobial resistance and developing efficient treatment options [102]. As previously mentioned, polymeric nanosystems are superior due to many advantages, such as high drug solubility and storage, biocompatibility, biodegradability, and stability, permitting the deliberate and precise drug release at the targeted sites [103,106,107].
Polymeric nanosystems can be synthesized from a variety of natural or synthetic precursors, such as collagen, chitosan, gelatin, or albumin, and polyethylene glycol, polylactic acid, poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA) or polycaprolactone (PCL), respectively [42,108,109]. Additionally, they can be developed in multiple forms, including nanoparticles, micelles, vesicles, dendrimers, or hybrid inorganic-polymer nanosystems ( Figure 5) [17].  This review focuses on polymeric nanoparticles, which are categorized into polymeric nanospheres and polymeric nanocapsules, depending on their internal structure and morphology ( Figure 6). On one hand, nanospheres comprise a continuous polymeric network with a regular sphere structure in which drug molecules are either retained inside the matrix or attached to its surface. On the other hand, nanocapsules consist of a polymeric shell surrounding the liquid/solid oily core. The drug is dissolved and modulates the release profile of the drug [110,111].

Antibacterial Nanoparticles
The pathway involved in bacteria growth is based on the conversion of chemical nutrients that enter into the bacterial cell through its pores into biomass. In this manner, the biomass increase leads to increased cell size and bacterial DNA replication, and finally to the division into two daughter cells [112]. There are two types of bacterial growth, namely

Antibacterial Nanoparticles
The pathway involved in bacteria growth is based on the conversion of chemical nutrients that enter into the bacterial cell through its pores into biomass. In this manner, the biomass increase leads to increased cell size and bacterial DNA replication, and finally to the division into two daughter cells [112]. There are two types of bacterial growth, namely the planktonic growth associated with the free-swimming unicellular phase that is not attached to any surfaces, and the biofilm growth phase, which is related to the multicellular sessile state that results in community formation [47,112]. While both are a serious concern, bacterial colonization and biofilm development allow the bacteria to survive in hostile environments and form new and permanent colonies, thus posing the risk of severe systemic infections [47,113].
In this context, the studies discussed below are targeting the application of polymeric nanoparticles against both planktonic and biofilm growth. The criteria involved in the process of article selection involved papers published after 2018 from the Scopus database using the keywords "polymeric nanoparticles" and "antibacterial" or "biofilm". Thus, 24 relevant studies were identified and categorized according to the type of polymer used, either natural or synthetic. Table 1 summarizes all the identified studies investigating the use of natural polymers to develop nanoparticles applied in antibacterial therapies. Among natural polymers, chitosan is the most widely used as a nanocarrier to deliver both antibiotics and alternative antibacterial drugs. For instance, Qiu et al. developed phosphatidylcholine-chitosan hybrid nanoparticles coated with the gentamycin antibiotic. The reason for the introduction of the lipid component is based on the potential of lipid drug carriers to fuse with the bacterial phospholipid membrane. The results confirmed the synthesized system's capacity to inhibit both Gram-positive and Gram-negative bacteria growth and biofilm formation [114]. Alruwaili et al. also investigated the antibacterial effects of gentamycin-containing chitosan nanoparticles that were further dispersed into pH-sensitive Carbopol polymer solutions to obtain sol-gel systems ocular delivery [115]. Ampicillin-loaded chitosan-polyanion nanoparticles were developed by Ciro et al. through ionic gelation and polyelectrolyte complexation using anionic polyelectrolytes corresponding to the sodium and potassium salts of poly(maleic acid-alt-ethylene) and poly(maleic acid-alt-octadecene) and studied for their antibacterial properties [116]. Furthermore, Evangelista et al. also synthesized supramolecular polyelectrolyte complexes based on the interactions between the positivelycharged -NH 3 + groups of the β-cyclodextrin-grafted chitosan and the negatively-charged -SO 3 groups of the carrageenan for antimicrobial applications. Specifically, β-cyclodextrin was used owing to its possibility to form host-guest inclusion complexes with the silver sulfadiazine molecules that will release silver ions against bacterial cells [117]. Another study by Walvekar et al. investigated the antibacterial effects of hyaluronic acid-oleylamine conjugates with different degrees of conjugation as drug nanocarriers against methicillin-resistant S. aureus. Precisely, the vancomycin antibiotic was encapsulated into polymersomes, which are nanocapsules comprising hydrophilic polymers grafted with long fatty acids that have the capacity to self-assemble into spherical drug carriers [118]. Moreover, Oliveira et al. developed a double-layer biomembrane comprising chitosan, hydroxypropyl methylcellulose, and lidocaine chloride as an anesthetic drug as the first layer and polymyxin B sulfate antibiotic-containing sodium alginate nanoparticles as the second layer for wound treatment [119]. MIC values were considerably lower for the free gentamycin, but it lost its activity after 24 h; polymersomes were not as potent as the free vancomycin but were able to improve the antibacterial effects due to a slow and controlled release over a prolonged period of time.
[118] CS-chitosan; NPs-nanoparticles; MIC-minimum inhibitory concentration; ZOI-zone of inhibition; SPECs-supramolecular polyelectrolyte complexes; MRSA-methicillin-resistant S. aureus; n.r.-not reported; EOs-essential oils; IH-inhibitory halo; NCs-nanocapsules; MBC-minimum bactericidal concentration. By contrast, many recent studies are focusing on alternative antimicrobial agents in order to avoid the use of antibiotics that are prone to cause the resistance of the bacteria. The study performed by Ejaz et al. is an example of such applications, as they developed mannose-functionalized chitosan nanoparticles with intrinsic antibacterial properties against Gram-positive and Gram-negative bacteria and antibiofilm character [120]. Moreover, Kritchenkov et al. developed betaine-type chitosan derivative nanoparticles through an ultrasound-assisted catalyst-free thiol-yne click chemistry with antibacterial activity [121]. Alternatively, there is an increasing interest in the use of essential oils as antibacterial agents in polymer-based drug delivery systems. In this context, there are two studies performed by Hadidi et al. and Bagheri et al., respectively, that investigated the effects of clove [122] and nettle [123] essential oils encapsulated into chitosan nanoparticles. Another study by Liakos et al. investigated the antimicrobial properties of peppermint, cinnamon, and lemongrass essential oils containing cellulose acetate nanocapsules [124]. Furthermore, Ivanova et al. developed antibody-functionalized self-assembled nanocapsules comprising zein plant protein and containing oregano essential oils. This approach allows for the specific targeting of S. aureus bacterial strains while reducing the dosage and the system's toxicity [125]. Other bioactive compounds that can be used as antimicrobial agents include antimicrobial glycolipids, such as sophorolipids and rhamnolipids encapsulated into chitosan nanoparticles [126], and antimicrobial peptides, such as the SET-M33 peptide, encapsulated into dextran nanoparticles [127].
Another strategy for antibacterial therapies involves developing polymeric nanoparticles as nanocarriers of both antibiotic drugs and alternative biocompounds. For example, de Oliveira et al. developed polymeric nanoparticles consisting of chitosan and hydroxypropylmethylcellulose to administer the ceftriaxone antibiotic and S. brasiliensis extract in antibacterial therapies [128].
Synthetic polymers are also widely used in nanoparticle development for biomedical applications [129]. In this context, Table 2 summarizes all the identified studies investigating their use in antibacterial therapies. Among them, the most commonly used include PLGA, PEG, PLA, and PCL, which have been investigated for the delivery of both antibiotics and alternative antibacterial drugs.
For instance, Ucak et al. investigated the antibacterial effect of teicoplanin-containing PLGA nanoparticles functionalized with S. aureus-specific aptamers [130]. Moreover, Deepika et al. synthesized PEG-PLGA nanoparticles for the co-delivery of rutin, a natural drug, and benzamide, a synthetic compound [131]. Alternatively, Durak et al. studied the effects of PLGA NPs containing natural biocompounds known for their antibacterial activities, namely caffeic acid and juglone [132]. Another study performed by Parmar et al. synthesized hybrid nanocomposites based on biogenic zinc oxide nanoparticles treated with A. indica leaf extract and PLGA [133].
Moreover, Da Costa et al. investigated the potential of rifampicin-containing PLA nanoparticles functionalized with poly-L-lysine. This cationic peptide could reverse the negative nanoparticle surface charge to positive, against planktonic bacteria and biofilm growth [134]. By contrast, Vrouvaki et al. developed PLA nanoparticles encapsulating the Pistacia lentiscus L. var. chia essential oil against Gram-positive and Gram-negative bacteria [135].
PCL was also studied for its potential in polymeric drug delivery systems applications in antibacterial therapies. Specifically, Srisang et al. prepared chlorhexidine-loaded PCL nanospheres for coating urinary catheters using a semi-automatic spray coater. The antibacterial effects of the coating were tested against common uropathogens causing urinary tract infections [136].    Other polymeric nanoparticles include cationic acrylate copolyvidone-iodine nanoparticles with a dual antibacterial activity center comprising the small molecule iodine and quaternary ammonium salt copolymers [137] and polyelectrolyte complex nanoparticles formed between the polymeric salts derived from Eudragit-E100™ and sodium salt of poly(maleic acid-alt-octadecene) for the delivery of ampicillin [138].
It can be observed that all the polymeric nanoparticles-based drug delivery systems exceed 100 nm, which is the generally accepted size limit for nanomaterials. However, considering the significant difficulty associated with the synthesis of small size polymeric nanoparticles, they are generally expected to be smaller than 200 nm in drug delivery applications in order to avoid any adverse reactions to the organism, such as embolisms. Therefore, most of the presented studies describe polymeric nanoparticles that are safe to use as nanocarriers for antimicrobial agents.
Furthermore, zeta potential was considered as the primary indicator for colloidal stability, with an appropriate stability for values other than −30 to +30 mV. Additionally, as it was previously mentioned, particles with positive surface charges exhibit increased antimicrobial potential due to the electrostatic interactions with the negative charge of the bacteria surface. Therefore, the optimum zeta potential values for polymeric nanoparticles for antimicrobial therapies should be higher than +30 mV, ensuring both the stability and the antimicrobial effects of the nanoparticles.
In this manner, chitosan remains a promising candidate for the development of antimicrobial agents due to its positively charged surface, providing intrinsic antimicrobial properties, and the possibility to synthetize nanoparticles with sizes lower than 200 nm. Additionally, chitosan is a natural polymer, thus eliminating the risk of toxicity when introduced into the organism and ensuring a proper biodegradability for a controlled drug release without toxic by-products.

Antiviral Nanoparticles
In the context of antiviral properties, the studies discussed below are targeting the application of polymeric nanoparticles for antiviral therapies. The criteria involved in the process of article selection involved papers published after 2018 from the Scopus database using the keywords "polymeric nanoparticles" and "antiviral". Thus, two relevant studies were identified and briefly described.
Alamdaran et al. developed chitosan nanoparticles with HIV-1 P24 protein-derived peptides adsorbed onto the surface as an alternative to counteract microbial resistance. The nanoparticles' loading and releasing efficiency were investigated on human peripheral blood lymphocyte cells, and results showed reduced toxicity and side effects and a controlled and sustained peptide drug release [145]. Furthermore, Belgamwar et al. investigated the efficiency of dolutegravir sodium-loaded nanoparticles comprising hydroxypropylβ-cyclodextrin cross-linked with diphenyl carbonate in order to enhance the central nervous system uptake through the intranasal administration route. Results proved an improved permeation of the drug through the nasal mucosa and access in the cerebrospinal fluid without damaging the mucosa [146].

Antifungal Nanoparticles
In the context of antifungal properties, the studies discussed below are targeting the application of polymeric nanoparticles for antifungal therapies. The criteria involved in the process of article selection involved papers published after 2018 from the Scopus database using the keywords "polymeric nanoparticles" and "antifungal". Thus, five relevant studies were identified and briefly described.
Costa et al. developed chitosan nanoparticles for the co-administration of miconazole and farnesol for the treatment of vulvovaginal candidiasis, which is mainly caused by the opportunistic fungal strain C. albicans. Results regarding the microorganism growth inhibition on the C. albicans (ATCC28367) showed a minimum inhibitory concentration (MIC) for the nanosystems similar to the values for the miconazole free drug. Moreover, the nanosystems administered in the murine model of vulvovaginal candidiasis were considered the most effective for infection inhibition [147]. Similarly, Charanteja Reddy et al. developed chitosan nanoparticles incorporating itraconazole that could be potentially used against C. neoformans, C. albicans, and A. fumigatus [148]. Furthermore, nanocapsules based on Sterculia striata polysaccharide modified with propionic anhydride through the acylation reaction were synthesized by Sombra et al. for the delivery of amphotericin B. The nanosystems revealed antifungal activity against four C. albicans strains, with MIC values lower for two strains and higher for the other strains when compared to the free drug [149]. Additionally, the previously described studies by Liakos et al. [124] and Srisang et al. [136] also exhibited antifungal activity against C. albicans strains.

Antiparasitic Nanoparticles
In the context of antiparasitic properties, the studies discussed below are targeting the application of polymeric nanoparticles for treatment against parasitic infections. The criteria involved in the process of article selection involved papers published after 2018 from the Scopus database using the keywords "polymeric nanoparticles" and "antiparasitic". Thus, two relevant studies were identified and briefly described.
Specifically, Real et al. studied the effect of chitosan nanocapsules containing triclabendazole, a poorly water-soluble compound used as the drug of choice in the treatment of fascioliasis. Results showed increased stability of over one month and strong interactions with enterocytes, thus enabling a higher uptake and sustained release of the drug [150]. Furthermore, the previously mentioned study performed by Durak et al. also exhibited antiparasitic effects against the Leishmania promastigotes protozoan parasites, with a dose-dependent antileishmanial [132].

Conclusions and Future Perspectives
The antimicrobial resistance of microorganisms that resulted from the overuse and abuse of antimicrobial drugs has become a critical and serious health problem that has led to many deaths worldwide. Nanotechnology has been applied to design alternative antimicrobial agents that could overcome the limitations of conventional drugs. Particularly, polymeric nanoparticles have been widely investigated for their potential to passively or actively target microbial strains and act both as a drug nanocarrier and as an antimicrobial agent due to intrinsic antimicrobial properties. In this context, numerous studies are investigating their application in antimicrobial therapies, but most are targeting bacterial pathogens. Therefore, there is still room for the improvement of polymeric nanoparticles for antiviral, antifungal, or antiparasitic applications, especially concerning the current COVID-19 pandemic situation that has been affected by the lack of effective antiviral agents. In this context, polymeric nanoparticles could efficiently target the coronavirus through active microbial targeting strategies and release appropriate quantities of the antiviral agent to destroy the pathogen.
While polymer-based nanoparticles offer a series of advantages, such as increased biocompatibility, biodegradability, and clearance from the human organisms, there are still some limitations associated with the currently available systems that must be considered. Specifically, the size and the size distribution of polymeric nanoparticles are variables that generally pose significant challenges in the manufacturing process. In this regard, microfluidic approaches could offer a potential alternative that could lead to the development of more uniform and smaller nanoparticles. Additionally, such technologies could allow for the one-step functionalization and drug encapsulation, thus eliminating some of the reaction steps involved in the conventional synthesis and ensure the optimum stability of the nanoparticles through the possibility of modulating the surface charge.
Author Contributions: V.A.S., C.C., A.M.G. and E.A. designed and wrote the paper. All authors have read and agreed to the published version of the manuscript.