The incidence of microbial infections has expanded dramatically, mainly due to the increasing occurrence of resistance among diverse strains of bacteria. The resistance to drugs is described as the tolerance or insensitivity of a microbe to an antimicrobial drug despite earlier susceptibility to it [1,2]^[1][2]. Concerning drug resistance (DR), different definitions are used in the medical literature to characterize the different patterns of resistance found in healthcare-associated, antimicrobial-resistant bacteria. Particularly, bacteria can be multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) ^[3]. So, MDR bacteria are those that have acquired non-susceptibility to at least one antibiotic in three or more antimicrobial categories, XDR bacteria are defined as non-susceptible to at least one antibiotic in all but two or fewer antimicrobial categories, and PDR bacteria are those non-susceptible to all agents in all antimicrobial categories ^[3].

Resistant bacteria, fungi, viruses, and parasites are able to combat the attack of available drugs, which are no longer effective, thus resulting in the persistence and spreading of chronic infections. The development of MDR is a natural phenomenon; however, excessive use of antibiotics, among humans, animals, and plants, incessantly supports its expansion ^[4]. Additionally, the widespread increase in immunocompromised individuals, such as patients affected by HIV infection, diabetic patients, persons who have experienced organ transplantation, and severe burn patients, makes the human body an easy target for hospital-acquired infectious (HAIs), thus contributing to further spread of MDR pathogens. From studies on WHO reports concerning the rates of resistance in bacteria, such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Nontyphoidal Salmonella, Shigella species, Neisseria gonorrhoeae, and Mycobacterium tuberculosis, several fungi, viruses, and parasites, against several different classes of antibiotics, it has emerged that they are responsible for severe infections such as urinary tract infections (UTIs), pneumonia, and bloodstream infections (BSIs) [5,6]^[5][6]. The most common MDR microbes, tolerated drugs, and related hospital-acquired infections are reported in Table 1.

Antifungal drugs available for the treatment of chronic fungal infections are limited and resistance to drugs such as amphotericin B, ketoconazole, fluconazole, itraconazole, voriconazole, flucytosine, and echinocandins was found in isolates of Candida spp., Aspergillus spp., C. neoformans, Trichosporon beigelii, Scopulariopsis spp., and Pseudallescheria boydii [7,8,9,10]^{[7][8][9][10]}. Additionally, antiviral resistance has become a serious matter of concern in immunocompromised patients, including immunosuppressed transplant receivers and oncology patients infected by CMV, HSV, VZV, HIV, influenza A virus, hepatitis C (HCV), or HBV [11,12,13,14,15]^{[11][12][13][14][15]}. Parasitic MDR has been observed in isolates of Plasmodia, Leishmania, Entamoeba, T. vaginalis, Schistosomes [16^{[16][17][18][19][20][21][22][26][27]},17,18,19,20,21,22,26,27], and T. gondii [23,24,25]^[23][24][25] against drugs such as chloroquine, pyrimethamine, artemisinin, pentavalent antimonials, miltefosine, paromomycin, and amphotericin B, in addition to atovaquone and sulfadiazine. A leading example of a parasite with a high tendency to develop MDR is P. falciparum, which is the cause of malaria. Other protozoan MDR parasites are Entamoeba spp., which causes amoebiasis, a major public health threat in many tropical and subtropical countries; and Schitosomes which is responsible for schistosomiasis, considered a global health concern similar to malaria and other chronic diseases ^[27].

2. Prevention and/or Eradication of BF by Dendrimers: A Possibility Still Little Explored

Although cationic dendrimers have been extensively studied as antibacterial agents with high potentiality, as shown by a search of Scopus using “cationic dendrimers” and “biofilm” as keywords, in the last 15 years (2007–2022), only 29 documents concerning this topic have been reported in the literature (Figure 1). This scenario shows that, although good premises exist, the possibility of using dendrimers to prevent the formation of BF or even to promote the breakdown of mature BF is, to date, still little explored. Unfortunately, the research in this field, instead of increasing, was shut down and after 2016, only five jobs were reported. This phenomenon highlights a diminished interest of a significant number of experts in developing new cationic dendrimers active against BF, further underlining the difficulty in being successful in such a challenge compared to being successful in developing new cationic materials, including dendrimers with bactericidal activity against bacteria with other types of resistances.

2.1. Antibacterial Cationic Macromolecules

Currently, there are few molecules under clinical development that are active against MDR pathogens and/or BF producers. Concerning the main agents in clinical development (Phase III) in 2020, only caspofungin, which is an antifungal drug, has been proven to inhibit the synthesis of polysaccharide components of the bacterial BF of S. aureus [144]^[28]. Concerning nanoparticles, some research groups are currently studying polymeric lipid nanoparticles, involving the conjugation of rhamnolipids (biosurfactants secreted by the pathogen P. aeruginosa) and polymer nanoparticles made of clarithromycin encapsulated in a polymeric core of chitosan. By the same principle, rhamnolipid-coated silver and iron oxide NPs have been developed, which have been shown to be effective in eradicating S. aureus and P. aeruginosa BFs [144]^[28]. Therefore, to counteract the phenomenon of drug resistance and meet the urgent need for new antibacterial agents that are also active in infections sustained by BF cells, the search for alternative therapeutic strategies capable of acting under mechanisms different from those of conventional drugs is a daily challenge of researchers in this field. In this regard, various natural cationic molecules called antimicrobial peptides (NAMPs) have shown significant capabilities to limit or inhibit bacterial growth and represent excellent candidates for replacing current antibiotics that are no longer effective. The amphiphilic structure and the net positive charge of NAMPs are the two most important requisites necessary for antibacterial effects and decide their mechanism of action [63,145,146,147]^{[29][30][31][32]}. It has, in fact, been shown that NAMPs’ action causes the death of the bacterial cell in a non-specific way from the outside, without having to enter the bacterial cell and interact with its metabolic processes, which is likely to genetically mute the conferring of resistance [63,145]^[29][30]. The action of NAMPs on bacteria is rapid, causes depolarization and destabilization of the membranes, and generates pores that gradually lead to an increase in their permeability, with consequent leakage of essential cations and other cytoplasmic materials and cell death. Since it is not connected to mutable bacterial processes, this mechanism of action generally allows the cationic peptides to induce a lower development of resistance in the target cell compared to traditional antibiotics [63,145]^[29][30]. Although the main target of NAMPs is bacterial membranes, it has been reported that some of these cationic antimicrobial peptides, following the increased permeability of bacterial envelopes, could also enter the cell and irreversibly damage molecules, such as DNA, RNA, and enzymes, thus leading to an improvement in their original therapeutic efficacy [63,146,147,148,149]^{[29][31][32][33][34]}. Unfortunately, the in vivo application of NAMPs is hampered by their early inactivation by peptidases, their high hemolytic toxicity, and high costs of production. Based on this, in the last decades, inspired by NAMPs, new cationic antibacterial macromolecules, such as polymers, copolymers, and dendrimers, have been developed, with proven interesting antibacterial effects. Such macromolecules, unlike small antibacterial compounds, in addition to possess multivalence, have other several advantages, including a limited residual toxicity, greater long-term activity, greater chemical stability, and lesser tendency to develop resistance [63,147 A research line started in the first decade of the 2000s concerned the synthesis and screening of libraries of dendrimer compounds with C-fucosyl and galactosyl residues with an affinity for fucose- and galactose-specific lectins. The scope was to detect potent fucose and/or galactose inhibitors of lectins LecA and LecB produced by P. aeruginosa, which are implicated in tissue binding and BF formation. In this regard, regarding the results obtained the previous year when a very large library of peptide dendrimers was screened to assess their affinity for LecB, the most potent LecB-ligands identified as dendrimers (FD2 (C-Fuc-LysProLeu)4(LysPheLysIle)2 LysHisIleNH2 (IC50 = 0.14 mM by ELLA) and PA8 (OFuc-LysAlaAsp)4(LysSerGlyAla)2 LysHisIleNH2 (IC50 = 0.11 mM by ELLA)) were tested on BF of P. aeruginosa by Johansson et al. in 2008 [156]^[51]. Particularly, FD2 led to complete inhibition of P. aeruginosa BF formation (IC₅₀ = 10 mM) and induced complete dispersion of mature BFs in the wild-type strain and in numerous clinical P. aeruginosa isolates, thus indicating that LecB inhibition by high-affinity multivalent ligands such as FD2 could correspond to a curative approach against BF-associated chronic infection sustained by P. aeruginosa [156]^[51]. Subsequently, Kolomiets et al. (2009), building on promising results previously published, prepared 10 tetravalent and 3 octavalent water-soluble C-fucosyl peptide dendrimers by solid-phase peptide synthesis (SPPS). By determining the relative affinities of these ligands to LecB using an enzyme-linked lectin assay (ELLA), strong binding of up to a 440-fold enhancement in potency over fucose was observed for the octavalent cationic dendrimer 2G3, mainly due to the multivalency of the dendrimer. Collectively, due to the potent binding and inhibition action on LecB by 2G3 and the versatility and reliability of SPPS to produce tunable multivalent fucosylated peptide dendrimer ligands, the dendrimers reported by Kolomiets et al. seemed excellent candidates for the development of polyvalent inhibitors of P. aeruginosa adhesion and BF for use in therapy to prevent BF-associated infections [157]^[52]. Antibacterial dendrimer peptides, acting in this case as membrane disruptors due to their positive charge ((RW) _4D), were prepared by Hou et al. (2009), which were effective in preventing the formation of E. coli BFs, hindering their development and destroying mature forms [158]^[53]. In 2011, Johansson et al. carried out research on a potent LecB inhibitor active in the prevention and dispersal of BF by P. aeruginosa, proposing a strategy to limit the early in vivo deactivation of FD2 by proteases. In this regard, he synthetized the C-fucosyl peptide dendrimer D-FD2 (CFuc-lys-pro-leu)4(Lys-phe-lys-leu)2Lys-his-leu-NH2 using D-amino acids and obtained the stereoisomer of FD2 [159]^[54]. Although D-FD2 showed a lower affinity for the fucose-specific lectin LecB, it was active as a BF inhibitor and, in addition, showed high stability towards proteolysis with pure proteases and in human serum, thus being conceivable for future clinical applications. In summary, the peptide dendrimer D-FD2 was prepared by substituting D-amino acids for L-amino acids in the branches of the dendrimer FD2. Collectively, the exchanged geometry altered the binding affinity to LecB while retaining the P. aeruginosa BF inhibitory properties and supplying complete resistance to proteolysis [159]^[54]. Establishing that BF formation by P. aeruginosa is mediated in part by the galactose-specific lectin LecA (PA-IL) and the fucose-specific lectin LecB (PA-IIL), and understanding that the glycoconjugate–lectin interaction is a key feature in developing potent BF inhibitors, Kadam et al., deepening their research in the field, in 2011, reported the first case of P. aeruginosa BF inhibition by the β-phenylgalactosyl peptide dendrimer GalAG2 multivalent ligand, this time targeting the galactose-specific lectin LecA [160]^[55]. Since it was observed that hydrophobic groups in the sugar anomeric point improved the affinity of galactosides to LecA, the acetyl-protected 4-carboxyphenyl β-galactoside (GalA) was connected to the peptide dendrimer [160]^[55]. To investigate the effect of the sugar-dendrimer linker on the binding affinity, carboxypropyl β-thiogalactoside (GalB) was also introduced as the last building block in solid-phase peptide synthesis to give the dendrimers GalAG1-G2 and GalBG1-G2, and the linear peptides GalAG0 and GalBG0 [160]^[55]. Based on the reported results, the strongest binding was detected with the second-generation glycopeptide dendrimer GalAG2. Interestingly, both the GalAG2 and GalBG2 dendrimers exhibited potent BF inhibition while the G1 equivalents were much less active and the G0 analogs were ineffective [160]^[55]. In the same year (2011), Chen et al. assessed the activity of a dendrimer peptide with various numbers of Trp/Arg repeated residues ((RW)_4D), which was previously found to have antibacterial effects by Hou et al. [158]^[53], against planktonic persister cells and BF-associated persister cells of E. coli HM22 [161]^[56]. The results were then compared with those obtained using three linear synthetic AMPs. Overall, (RW)_4D exhibited potent activities in eradicating BF cells and associated antibiotic tolerance in a dose-dependent manner [161]^[56]. Particularly, after treatment for 1 h with 20, 40, and 80 μM (RW)_4D, the total number of viable BF cells was reduced by 77.1%, 98.8%, and 99.3%, respectively, compared to the untreated control (p < 0.0001). The tolerance to ampicillin was also reduced by 57.2%, 96.9%, and 99.1%, respectively (p = 0.0012). Furthermore, no viable cell was found after treatment of the BFs with 40 or 80 μM (RW)_4D followed by 5 μg/mL ofloxacin, suggesting that all persister cells were eliminated [161]^[56]. On these early results, (RW)_4D may be a pioneer compound for the development of new therapeutics to treat BF-associated infections that is also able to improve the action of antibiotics, including ampicillin and ofloxacin. Wang et al. (2011) presented a report on the antibacterial activity and cytotoxicity of three types of titanium-based substrates with and without calcium phosphate coatings on which poly(amidoamine) (PAMAM) dendrimers were immobilized (named Ti-S(CaPO₄)060-PEGPAMAMs and Ti-S-060-PEGPAMAMs in the table) [162]^[57]. The utilized amino-terminated PAMAM dendrimers were modified with various percentages (0–60%) of PEG to obtain strong absorption on the titanium-based substrates and the formation of dendrimer films. The obtained films effectively inhibited colonization by P. aeruginosa (strain PAO1) and, although to a lesser extent, S. aureus. The antibacterial activity of the films was maintained even after storage of the samples in PBS for up to 30 days [162]^[57]. In addition, the dendrimer-based films showed low cytotoxicity to human bone mesenchymal stem cells (hMSCs) and did not alter the osteoblast gene expression promoted by the calcium phosphate coating [162]^[57]. The next year (2012), Scorciapino et al., using an alternation of hydrophilic and lipophilic amino acids, synthetized a lipodimeric dendrimer (SB056), which demonstrated an antibacterial effect by acting as a membrane disruptor [163]^[58]. The antibacterial activity of SB056 was similar to that of colistin e polimixin B against isolated MDR Gram-negative species while SB056 showed poor activity against strains of Gram-positive species. Interestingly, SB056 showed good antibiofilm effects against BFs produced by S. epidermidis and P. aeruginosa [163]^[58], thus being a future therapeutic weapon against BF-associated infections. The group of Lu et al., in 2013, recovered PAMAM-based dendrimers capable of releasing NO to develop new antibacterial agents, which are hopefully also effective against BFs [164]^[59]. Particularly, a series of amphiphilic PAMAM dendrimers were synthesized by a ring-opening reaction between the primary amine groups on the dendrimers and propylene oxide (PO), 1,2-epoxy-9-decene (ED), or a ratio of the two [164]^[59]. The obtained PAMAM-based dendrimers with different functionalities were then reacted with NO at 10 atm to produce N-diazeniumdiolate-modified scaffolds with a total loading of NO of ~1 μmol/mg [164]^[59]. In sight of a future clinical application of such materials, structure–activity relationship (SAR) studies were carried out to evaluate the bactericidal efficacy of the prepared NO-releasing delivery systems against established BFs by P. aeruginosa as a function of the dendrimer exterior’s hydrophobicity (i.e., ratio of PO/ED), size (i.e., generation), and NO release [164]^[59]. It was revealed that both the size and exterior functionalization of the dendrimer were pivotal for dendrimer–bacteria interaction, the NO delivery efficiency, bacteria membrane disruption, migration within BF, and toxicity to mammalian cells [164]^[59]. In this regard, the best PO/ED ratios for BF eradication with minimal toxicity against L929 mouse fibroblast cells were 7:3 and 5:5 [164]^[59]. The inhibition of BF formation by blocking the action of LecA and LecB lectins from P. aeruginosa was also investigated by Reymond et al. [165]^[60]. So, the four glycopeptide dendrimers that were synthesized showed high affinity for the lectins and were efficient in both blocking P. aeruginosa BF formation and inducing its dispersal in vitro [165]^[60]. As mimics of natural cationic amphiphilic peptides (NAMPs) with antifungal activity, eight peptide dendrimers were designed and evaluated for their anti-Candida spp. effects against both the wild-type strains and mutants by Zielinska et al. in 2015 [166]^[61]. Among the synthetized macromolecules, dendrimer 14, containing four tryptophan (Trp) residues and a dodecyl tail, and dendrimer 9, decorated with four N-methylated Trp, was shown to inhibit 100 and 99.7% of Candida growth at 16 µg/mL, respectively [166]^[61]. On these promising outcomes, 9 and 14 were designated for their evaluation against C. albicans mutants with deactivated biosynthesis of aspartic proteases, which are responsible for host tissue colonization and morphogenesis during BF formation (sessile model). According to the reported results, 14 affected C. albicans BF viability and the hyphal and cell wall morphology by membranolytic mechanisms [166]^[61]. By affecting the cellular apoptotic pathway and damaging the cell wall formation in mature BF, 14 may be a potential multifunctional antifungal template compound for the control of C. albicans chronic infections [166]^[61]. C. albicans was also the topic in the study of Lara et al. (2015), where novel antifungal strategies targeting BFs were explored [59]^[62]. Notably, using microwave-assisted techniques, nanosized spherical silver nanoparticles (AgNPs), aiming to investigate their potential biological applications, were prepared by Lara et al. without the addition of contaminants [59]^[62]. A potent dose-dependent inhibitory effect of AgNPs on BF formation of C. albicans was demonstrated (IC₅₀ of 0.089 ppm). Additionally, AgNPs were shown to be efficient when tested against pre-formed C. albicans BFs, resulting in an IC₅₀ of 0.48 ppm while the cytotoxicity assay resulted in a CC₅₀ of 7.03 ppm. Using SEM and TEM analyses, it was evidenced that treatments with AgNPs caused outer cell wall damage, absence of true hyphae, filamentation inhibition, and membrane permeabilization [59]^[62]. The same year (2015), Bahar et al. developed the arginine-tryptophan-arginine 2D-24 dendrimer peptide, which was found to be effective against P. aeruginosa normal planktonic and persister cells, and against P. aeruginosa BF cells [152]^[47]. Particularly, the second-generation peptide dendrimer (2D-24), with residues of arginine and tryptophan, was active both on planktonic and BF cells, and against the MDR isolates of P. aeruginosa PAO1 (ATCC BAA-47) and PDO300 (mutans mucA22 of P. aeruginosa PAO1) [152]^[47]. Collectively, 2D-24 displayed the same efficiency against both planktonic cells and BFs, thus establishing its capability to penetrate the BF’s mass and the alginate layer of mucoid isolates. Concentrations > 20 μM were sufficient to kill 80% of the planktonic and BF cells of PAO1 e PDO300 strains [152]^[47]. Further studies concerning combination therapies were carried out by Bahar et al., observing synergistic effects of 2D-24 with antibiotics such as ciprofloxacin (Cip), tobramycin (Tob), and carbenicillin (Car), all targeting the synthesis of DNA, proteins, and the cell wall [152]^[47]. The best result was obtained by combining 2D-24 with Tob. Additionally, in vitro studies on sheep erythrocytes and cocultures of PAO1 and human IB3-1 cells showed that 2D-24 exerted antibacterial effects at non-toxic concentrations on mammalian cells (25 µg/mL), thus providing encouraging evidence supporting the use of 2D-24 for chronic P. aeruginosa-borne infections [152]^[47]. Using a strategy similar to that used in 2013 by Lu et al. [164]^[59], Worley et al. described the synthesis of NO-releasing alkyl chain modified PAMAM dendrimers of the G1-G4 generations decorated with butyl or hexyl alkyl chains via a ring-opening reaction [167]^[63]. The resulting secondary amines were further reacted with N-diazenium-diolate NO donors, reaching an NO payload of ∼1.0 μmol/mg [167]^[63]. The bactericidal efficacy of these dendrimers was evaluated against BFs produced by isolates of P. aeruginosa, S. aureus, and MRSA. The anti-BF action of the dendrimers depended on the dendrimer generation, bacterial species, and alkyl chain length, with the most effective BF eradication occurring when antibacterial agents were capable of efficient BF infiltration. Regardless, the ability of dendrimers to release NO also helped the antibiofilm activity of those dendrimers incapable of effective BF penetration [167]^[63]. To better understand the binding mode of the tetravalent glycopeptide dendrimer (TGPD) GalAG2 to its target lectin LecA, the first crystal structures of LecA complexes with the divalent analog GalAG1 were obtained, revealing strong LecA binding and absence of lectin aggregation [168]^[64]. Secondly, a model of the chelate-bound GalAG27LecA complex was also obtained, rationalizing its unusually tight LecA binding (K_D = 2.5 nM) and aggregation by lectin cross-linking [168]^[64]. By evaluating the BF inhibition with divalent LecA inhibitor (GalAG1), it was indicated that lectin accumulation is necessary for BF inhibition by GalAG2, thus establishing that multivalent glycoclusters represent a unique opportunity to control P. aeruginosa BFs [168]^[64]. The next year (2016), the same group of Worley, this time headed by Becklund, studied in more detail the modification of (G1) (PAMAM) dendrimers with several alkyl epoxides to generate propyl-, butyl-, hexyl-, octyl-, and dodecyl-functionalized dendrimers [169]^[65]. The resultant secondary amines were treated with NO to form N-diazeniumdiolate NO donor-modified dendrimer scaffolds (total NO ∼1 μmol/mg) [169]^[65]. In tThis study, the bactericidal action of the NO-releasing dendrimers was tested against both planktonic and BF cells of S. mutans, with the greatest efficiency observed with the increase in the alkyl chain length and at lower pH. Particularly, the best bactericidal efficacy was obtained at pH 6.4, probably due to the increase in the cationic scaffold surface charge, which promoted dendrimer–bacteria association and the ensuing membrane damage. For shorter propyl and butyl chain modifications, however, the increased antibacterial action at pH 6.4 was due to the faster NO-release kinetics from proton-labile N-diazeniumdiolate NO donors. Octyl- and dodecyl-modified PAMAM dendrimers proved to be the most effective in both eradicating S. mutans BFs with the help of NO release and displaying mitigated cytotoxicity [169]^[65]. Inserting their studies in the research line concerning dendrimers that act as ligand inhibitors of LecA and/or LecB, Bergman et al. (2016) synthetized G3 and G4, analogous to GalAG2 and GalBG2, using a convergent synthetic technique and multivalent chloro-acetylcysteine (ClAc) thioether as the linker [170]^[66]. According to the reported results, G3 dendrimers showed an improved ability to link LecA with respect to the parent dendrimers G2 and were capable of exerting total inhibition of BF produced by P. aeruginosa, and causing its disaggregation [170]^[66]. The same year (2016), Michaud, Visini, Bergaman et al. focused on carrying out several changes in the structures of the earlier reported TGPDs acting as ligands of lectins LecB and/or LecA produced by P. aeruginosa to increase their BF inhibition activity [171]^[67]. As examples, they investigated heteroglycoclusters (Het1G2-Het8G2 and Het1G1Cys-Het8G1Cys), each having one pair of LecB-specific fucosyl groups and LecA-specific galactosyl groups, capable of simultaneously binding both lectins [171]^[67]. Interestingly, one of these ligands gave the first fully resolved crystal structure of the complex peptide dendrimer–LecB, providing a structural model for dendrimer–lectin interactions (PDB 5D2A) [171]^[67]. Furthermore, BF inhibition was increased by introducing additional cationic residues in these dendrimers. In a second approach, dendrimers with four copies of the natural LecB ligand (Lewis^a) were prepared, which were able to strongly bind LecB and allowed higher BF inhibition effects. Finally, in this study, syynergistic application of the previously reported dendrimer FD2 with the antibiotic tobramycin at sub-inhibitory concentrations of both compounds was carried out, demonstrating effective BF inhibition and dispersal [171]^[67]. The group of Scorciapino, this time headed by Batoni, in 2016, studied linear and dendrimer compounds derived from the previously reported lin-SB056 and den-SB0569 by changing the first two residues [KWKIRVRLSA-NH ₂] of the original sequence [WKKIRVRLSA-NH ₂] and obtained new compounds, namely lin-SB056-1 and den-SB056-1, which possessed an improved amphiphilic profile, and explored the effects of this modification [172]^[68]. Den-SB056-1 showed antibacterial effects against both Gram-negative and Gram-positive bacteria, based on the disruption of bacterial membranes. The results obtained against E. coli and S. aureus planktonic strains confirmed the added value of the dendrimer structure over the linear one, and the impact of the higher amphipathicity on enhancement of the peptide performances [172]^[68]. Unfortunately, while SB056 peptides exhibited enthralling antibiofilm properties mainly against sessile cells of S. epidermidis, SB056-1 showed reduced antibiofilm effects if compared with that of SB056 [172]^[68]. In a study of the same year by VanKoten et al. (2016), a highly cationic fourth-generation PAMAM dendrimer (G4-PAMAM), functionalized with 1-hexadecyl-azoniabicylo [2.2.2] octane (C16-DABCO), a quaternary ammonium compound known to have antibacterial activity, was synthetized [173]^[69]. According to the authors, the dendrimer activity depended on both the presence of mannose residues and the ammonium quaternary groups. The PAMAM dendrimer was active against both Gram-positive and Gram-negative species, especially Gram-positive isolates such as S. aureus and B. cereus [173]^[69]. Although the C₁₆-DABCO-dendrimer did not show intrinsic antibiofilm effects, it was observed that membranes treated with 1 mg/mL C₁₆-DABCO-dendrimer inhibited the formation of S. aureus BF, thus establishing its future use in pre-treating membranes and preventing BF formation [173]^[69]. Fuentes-Paniagua et al. focused on the antibacterial activity against S. aureus and E. coli of two types of cationic carbosilane (CBS) dendrimers and dendrons and their hemolytic properties [78]^[70]. SAR studies were carried out to evaluate the influence of the generation, type of peripheral groups near the cationic charges, core of dendrimers, and focal point of dendrons (-N₃, -NH₂, -OH) on the antibacterial activity of such compounds. These studies evidenced the importance of an adequate balance between the hydrophilic and lipophilic fragments of these molecules. The lowest hemolytic toxicity was registered for dendrimer systems with a sulfur atom close to the surface and when dendrons had a hydroxyl central point. One dendrimer and one dendron, both bearing a sulfur atom close to the surface, scored best in the activity–toxicity relationship analyses, and were chosen for resistance assays [78]^[70]. No changes in the inhibitory and bactericidal capacity were observed in the case of the dendron while only a slight increase in these values was noted for the dendrimer after 15 subculture cycles. Furthermore, these two compounds remained active towards different strains of resistant bacteria and prevented the formation of BF at concentrations over the minimum inhibitory concentration (MIC) [78]^[70]. In their study in 2019, Barrios-Gumiel and co-workers reported on the preparation of silver nanoparticles (AgNP) covered with cationic CBS and poly (ethylene glycol) (PEG), having antibacterial properties, antifouling effects, and improved biocompatibility due to the presence of PEG [174]^[71]. The new family of hetero-functionalized AgNPs was directly synthesized using silver precursor and cationic CBS and PEG ligands containing a thiol moiety. AgNPs were characterized by TEM, TGA, UV, 1H NMR, DLS, Z potential, and XRD. The antibacterial ability of these systems was evaluated against E. coli and S. aureus, evidencing that the effects obtained for PEGylated systems were slightly lower than those observed for non-PEGylated AgNPs, compensated for by the greater biocompatibility [174]^[71]. Furthermore, the authors tested one non-PEGylated and one PEGylated AgNP dendron for their tendency to induce resistance in a planktonic state. Both AgNP-based dendrons barely affected the minimum inhibitory concentration (MIC), whereas the reference antibiotics generated significant resistance [174]^[71]. Concerning the topic of the present paper, a relevant improvement in BF inhibition was achieved by dendronized AgNPs after PEGylation [174]^[71]. Heredero-Bermejo and Casanova focused on the discovery of dendrimers active against Candida spp., which is one of the most common fungal pathogens, the BFs of which, especially those by C. albicans, offer resistance mechanisms against most antifungal agents [60]^[72]. In their work of 2020, the authors carried out a study concerning the in vitro effects of different cationic CBS dendrimers against BF formation and mature BFs by both Colección Española de Cultivos Tipo (CECT) 1002 and clinical C. albicans strains [60]^[72]. One out of 14 dendritic molecules tested, BDSQ024 showed the highest activity with a minimum BF inhibitory concentration (MBIC) for BF formation and a minimum BF damaging concentration (MBDC) for existing BF of 16–32 and 16 mg/L, respectively [60]^[72]. Additionally, synergistic effects at non-cytotoxic concentrations were detected both with amphotericin (AmB) and caspofungin (CSF) [60]^[72]. Subsequently, the research group of Gómez-Casanova et al., using in vitro tests, evaluated the ability of three different generations of cationic CBS dendrons to inhibit the initial formation of C. albicans BF and disaggregate the mature BF [175]^[73]. Concerning their synthesis, the authors obtained highly water-soluble dendrons (ArCO₂G_n(SNMe₃I)_m) starting from 4-phenylbutyric acid. The interaction with the cell membranes of the generated systems was found to be dependent on the hydrophilic/lipophilic balance (HLB), which increases with the number of the dendritic generation [175]^[73]. Although the compounds showed some toxicity, they showed good antifungal activity against C. albicans by inhibiting both the first formation of BF and causing its dispersal [175]^[73]. Furthermore, among all the tested dendrons, the second-generation sendron with four positive charges, ArCO₂G2 (SNMe₃I)₄, was found to be the most effective in inhibiting the formation of BF, thus suggesting its hypothetical use as a disinfectant solution for nosocomial surfaces or as a lotion to treat skin infections [175]^[73]. Furthermore, ArCO₂G2 (SNMe₃I)₄, when combined with ethylenediaminetetraacetic acid (EDTA) and silver nitrate (AgNO₃), was able to work on a mature BF, deforming the cell wall and morphology of C. albicans [175]^[73]. Combination therapy by the association of conventional antibiotics with new compounds represents one of the main strategies used for countering infections by MDR pathogens that produce BFs. Since CBS dendrimers have been proven to be a promising solution for counteracting the formation of BFs, Fernandez et al. developed a new strategy to prevent the formation and/or disaggregation of S. aureus BF using a combination of a second-generation cationic CBS dendron with a maleimide group in the focal point and four positive charges MalG₂(SNHMe₂Cl)₄ and levofloxacin (LEV) [176]^[74]. With the same purpose, LEV was also combined with a nanoconjugate, formed by a CBS dendron and a cell-penetrating peptide (gH625), called dendron-gH625 nanoconjugate (DPC). The data obtained by Fernandez et al. demonstrated that the combination therapy led to a greater anti-BF effect compared to the concentrations tested individually [176]^[74]. In particular, the highest percentages of inhibition were obtained from the combination of DPC-LEV, indicating it is a possible alternative option for BF-associated infections [176]^[74]. Moreover, Galdiero et al. (2020) [177]^[75] studied the antimicrobial and antibiofilm activities of an analogue of the peptide gH625 (namely gH625-M), a membranotropic peptide described to be a cell-penetrating peptide capable of interacting and disrupting the bilayers of membranes without pore formation. gH625-M was obtained by binding a sequence of lysine residues at the C-terminus, responsible for specific interactions with the negative charges of bacterial membranes [177]^[75]. The results obtained by Galdiero et al. showed that the gH625 peptide possessed low antibacterial activity against planktonic cells while it inhibited the initial BF formation of C. tropicalis, C. serratia, C. marcescens, and S. aureus and was also capable of disintegrating mature BFs formed on silicone surfaces [177]^[75].