Traditional and unmodified PAMAM Ds and PPI Ds possess a wide number of peripheral protonable primary amine groups and additional protonable tertiary amine groups in the inner matrix, which give them high antibacterial activity due to their high density of positive charge. Surprisingly, even though PAMAM Ds were one of the earliest to be synthesized and extensively studied for various biomedical applications, their activity as cationic antimicrobial agents has only recently been evaluated.
Moreover, also results obtained from comparison studies between antimicrobial Ds and hyperbranched polymers reveal conflicting outcomes. Concerning PPI Ds studied by Cooper and colleagues, when colonies of E. coli were exposed to 12 μg/mL of a PPI D and to the same amount of a hyperbranched polymer, with the same number of functional groups and similar MW, all bacteria were destroyed in the first case while in the second case only a reduction of 30–40% was observed. By contrast, the hyperbranched carbosilane polymers, investigated in another study and reported by Mintzer in 2012 , showed higher activity than the second- and third-generation carbosilane Ds with similar molecular weight and ζ potential, being the MIC values of 8–16 μg/mL vs. 16–64 μg/mL against E. coli and of 4 μg/mL vs. 4–8 μg/mL against S. aureus.
Interestingly, in the earliest studies, PAMAM Ds were not evaluated as active antimicrobial agents per se, but rather as carriers for antimicrobial agents, with the purpose of improving their performance.
In this context, Neelgund and colleagues reported the design of antimicrobial nanohybrid Ds, namely f-MWCNTs-CdS and f-MWCNTs-Ag2
S, synthetized by covalent grafting of cationic hyperbranched dendritic PAMAMs onto multiwalled carbon nanotubes (MWCNTs) followed by deposition of CdS and Ag2
S quantum dots (QDs), respectively
. The antibacterial activity of f-MWCNTs-CdS and f-MWCNTs-Ag2
S hybrid Ds (20 μg/mL) was evaluated on E. coli
, P. aeruginosa
and S. aureus
and it was found that the biocidal action of MWCNTs, already improved by grafting of PAMAM, was further enhanced by loading CdS and Ag2
S QDs. In particular, the Ag2
S Ds were more active than the CdS ones and both materials were more active against Gram-negative bacteria than towards the Gram-positive specie S. aureus
. A little perplexity on the efficacy of f-MWCNTs-CdS and f-MWCNTs-Ag2
S hybrid Ds prepared by Neelgund and colleagues arises from the fact that no reference data such as the growth inhibition activity of known antibiotics have been provided. In addition, to assess the real applicability of these interesting Ds, investigations regarding their toxicity towards eukaryotic cells are still lacking.
Despite their established antimicrobial activity, various characteristics limit their potential use, such as the long synthesis process, necessary for high-generation compounds and their non-specific interaction with the negative charges of bacterial or eukaryotic membranes responsible for cytotoxicity and hemolytic toxicity. In addition, fast clearance from the blood circulation, as well as a high level of uptake in the reticuloendothelial system affect their activity. To address these issues, different types of structural modifications or synthetic combinations of PAMAM Ds with more hydrophobic Ds have been developed. In this regard, PAMAM-based dendrimers compounds, with tunable cationic and hydrophobic characters able to self-assemble, were prepared combining poly(aryl ether)-based dendrons with PAMAM-based dendrimer residues, which are different for generation and terminal groups. The obtained poly(aryl ether)-PAMAM-based amphiphilic Ds were assessed for their intrinsic antibacterial activity by determining MIC and MBC values and by time-killing experiments performed at 4 × MIC concentrations on S. aureus and E. coli strains. The best performing Ds were, as expected, the amine terminated ones but interestingly, the best between the two was that of the lower generation. Time-killing experiments established that AD-1 was the dendrimer able to cause the high reduction in the growth of both bacteria along 24 h of exposure, thus confirming its strongest bactericidal effects. The cytotoxicity essay performed on AD-1 highlighted that NIH/3T3 fibroblast cells showed significant damage only when they were exposed to AD-1 concentration four times higher than MIC or to an AD-1 dosage 2-times the MBC for both E. coli and S. aureus species.
A highly cationic dendrimer, containing both a positively charged fourth-generation PAMAM architecture and a quaternary ammonium compound covalently linked, known as 1-hexadecyl-azoniabicylo [2.2.2] octane (C16-DABCO), and possessing antibacterial activity per se, was prepared. Additional mannoside end groups have also been incorporated into the antimicrobial dendrimer, to prevent the bacteria adhesion to the surface of eukaryotic cells by an intercepting mechanism. First, in the study of Vankoten and colleagues, the antibacterial activity of the obtained materials was tested against several Gram-positive and Gram-negative strains of pathogenic bacteria. With the exception of Streptococcus oralis, which showed MIC values > 20 μM, the remaining species were reported to be quite susceptible to the dendrimer, displaying MIC values in the range 0.13–2.0 μM. Unfortunately, the observed hemolytic toxicity (MHC = 0.6 μM) limits the clinical use of C16-DABCO dendrimer for the treatment of infections caused by S. aureus and B. cereus (MICs = 0.13 μM). A further study evaluating the tendency of the prepared dendrimer to select resistant strains within a specie was performed on E. coli and B. cereus. The results established that the multivalent antimicrobial dendrimer, due to its ability to deliver a detrimental dose of positive charge to the bacteria, displayed a significantly reduced tendency to develop bacterial resistance. C16-DABCO dendrimer has also been tested for its ability to disrupt biofilm and/or inhibit biofilm formation. No inhibition or reduction of biofilm biomass was observed in any of the species tested. Interestingly, experiments aimed at evaluating the prevention of biofilm formation, proved that on membranes pretreated with a 1 mg/mL solution of C16-DABCO dendrimer (33.3 μM), and inoculated with S. aureus, no visible biofilms were present after three days of incubation.
With the aim of reducing the incidence of implant-associated infections, severe post-surgery complications leading to patient disability and morbidity, and even death, Wang et al., (2011), for the first time, successfully coated three types of titanium-based substrates with PAMAM Ds modified with various percentages of poly(ethylene glycol) (PEG) to limit cytotoxicity 
. Based on their inner and peripheral high content of amine groups, electrostatic interactions were more favored with bacterial membranes characterized by higher negative charge, causing their destruction. Consequently, the PAMAM coatings displayed greater bactericidal activity against Gram-negative P. aeruginosa
, than against Gram-positive S. aureus
, due to the higher negativity of their OM. The dendrimer adsorbed on titanium surfaces, proved durable bacteriophobic abilities, inhibited bacterial colonization even after 30 days and showed good stability and biocompatibility with human bone mesenchymal stem cells (hMSCs).
In the same year, Navath and colleagues developed in situ forming PAMAM Ds-PEG biodegradable hydrogels, loaded with amoxicillin, and proposed these materials as novel agents for the delivery and sustained release of that specific antibiotic in the cervicovaginal region, in order to treat ascending genital infections. Briefly, the PAMAMs-based amoxicillin formulations were prepared by the cross-linking of [(NH2
)49-G4-(NH-PDP)15] dendrimer and 8-arm-PEG via formation of disulfide bridges and were tested both in vitro and in vivo. Amoxicillin release from these hydrogels (3%, 6% and 10% w
) was sustained for more than 240 h, while in vivo assessment of the hydrogels, carried out using a pregnant guinea pig model, showed excellent tolerability, lack of alterations in vaginal pH and/or erythema up to 72 h and slow degradation. The gels were retained in the maternal tissues without being transferred across the fetal membranes 
Recently, another approach has been reported to obtain a synergistic action between cationic Ds and antibiotics. Instead of binding the antibiotic to the dendrimer backbone, thus making a device that possesses both the intrinsic antimicrobial activity of the carrier and that of the antibiotic, synthetic polycationic and polyanionic Ds were administered in combination with levofloxacin (LVFX), in order to reduce the development of resistance to the drug, decrease its dosage and toxicity, as well as the environmental pollution resulting from the wide spread use of fluoroquinolones. The germicidal activity of the combination made was investigated by treating E. coli
ATCC 25922, Proteus hauseri
ATCC 15442, and S. aureus
ATCC 6538 strains for 24 h with fixed concentrations of LVFX and Ds. Satisfactory results, in terms of antimicrobial activity, were obtained only against the Gram-negative E. coli
by the third-generation PPI-based cationic dendrimer, the only one of interest for this review, characterized by a dense maltose shell (PPI-G3-DS-Ma) and known to be endowed with a dose-dependent cytotoxicity. In fact, a combination of the dendrimer PPI-G3-DS-Ma (10 μM) and LVFX (0.01 μg/mL) reduced the growth of E. coli
by 80%, while dendrimer or antibiotic alone reduced the same growth by only 6% or 4%, respectively. This strategy, even if limited to E. coli
, has allowed to significantly reduce the LVFX dosage, using the cationic dendrimer at a dose which, for a similar dendrimer of higher generation previously reported, showed very limited cytotoxicity on several human cell lines (cell viability around 80–100%) 
In this last study, in fact, the preparation and evaluation of the germicidal activity of an unmodified fourth-generation PPI D (PPI-G4) and of PPI Ds modified with 25% and 100% maltose attached to their surface (PPI-25%mG4 and PPI-100%mG4, respectively), was reported. The target bacteria were both Gram-positive (S. aureus ATCC 6538 and S. epidermidis ATCC 12228) and Gram-negative (E. coli ATCC 25922 and P. aeruginosa ATCC 15442) strains. In parallel, the cytotoxic effects of all tested Ds were checked on a Chinese hamster fibroblast cell line (B14), a human liver hepatocellular carcinoma cell line (HepG2), a mouse neuroblastoma cell line (N2a) and a rat liver cell line (BRL-3A). The obtained results established that Gram-negative bacteria were refractory to the Ds, also at very high concentrations (100 μM). The unmodified PPI-G4 was the most active dendrimer, killing 90% of S. epidermidis isolates at 30 μM and 60% of S. aureus strains at 30 μM. Unfortunately, due to its IC50 values in the range of 3.18–6.91 μM against the eukaryotic cells analyzed, its use for clinical applications must be excluded. PPI-100%mG4 was practically inactive against all the tested pathogens, while PPI-25%mG4 proved good activity against S. aureus, at concentrations that allowed high viability maintenance of all eukaryotic cell lines essayed.
Ds, due to a high density of exterior functional groups available for nitric oxide (NO) loading, were particularly attractive for preparing (NO)-releasing Ds characterized by large NO payloads and antibacterial activities against a wide range of pathogenic bacteria, including P. aeruginosa
and S. aureus.
The antibacterial activity of a library of (NO)-releasing PPI Ds was evaluated against Gram-positive and Gram-negative isolates, including methicillin-resistant S. aureus
(MRSA) by Sun et al. (2012)
and it was compared with that of analogous PPI Ds, non-NO-releasing, taken as control. From the results, the NO-releasing Ds displayed both enhanced biocidal action directly proportional to Ds size (in terms of MW) and reduced toxicity against mammalian fibroblast cells. In addition, minimal toxicity against fibroblasts and the strongest biocidal activity (≥99.999% killing) was exerted by NO-releasing PPI Ds modified with styrene oxide (SO). The MBC values for each bacterial strain were determined in a non-conventional way, i.e., as the concentration of Ds that caused either a 3 or 5 log reduction in cell viability (compared to untreated cells for a particular bacterial strain) after 2 h. Concerning the cytotoxic effects against L929 fibroblasts cell lines, a very low cell viability (<20%) was observed when cells were exposed to concentrations of all G5-PPI Ds necessary to kill bacteria. On the other hand, both G2-PPI-PO and G2-PPI-NH2
exhibited minimal toxicity to fibroblast cells when tested at bactericidal concentrations. When administered at concentrations 2× MBCs, G2-PEG- and SO-modified Ds (namely 3 and 5) still inhibited fibroblast proliferation by approximately 76% and 64% respectively, therefore showing considerable cytotoxicity. Significantly, the toxicity of NO-releasing Ds against L929 fibroblasts was minimal (≥60% viability) when the devices were administered at both the minimum and twice the minimum concentration required to elicit 3-log killing against all tested bacterial strains. In particular, both G2 and G5 NO-releasing PPI-SO Ds, and their precursors, proved to be non-toxic to the fibroblast cells (>80% viability) even at concentrations necessary to induce 5-log killing against all species studied (10 and 1 μM).
A library of amphiphilic NO-releasing PAMAM Ds were synthesized by Lu et al. (2013), through a ring-opening reaction between terminal primary amines present on the dendrimer and propylene oxide (PO), 1,2-epoxy-9-decene (ED) or different ratios of the two, followed by reaction with NO, in order to achieve the NO-releasing derivatives 
. The hydrophobicity of non-NO-releasing and of NO-releasing Ds was tuned by varying the ratio of the external functionalization PO/ED. The bactericidal efficacy of the NO-releasing vehicles obtained was evaluated against planktonic Gram-negative P. aeruginosa
strains and established P. aeruginosa
biofilm. The results were correlated with the Ds exterior hydrophobicity (i.e., ratio of PO/ED), their size (i.e., generation), NO release and was compared to that one of non-NO-releasing compounds with analogous composition, taken as controls. It was observed that both the size and the exterior functionalization of Ds were pivotal factors influencing several parameters affecting the antibacterial activity such as dendrimer-bacteria electrostatic interactions and the capability to disrupt bacterial membranes, the efficacy to deliver NO, their migration within the biofilm, and their toxicity against mammalian cells. All the NO-releasing Ds analyzed displayed higher antimicrobial activity than the non-NO-releasing Ds. In more details, the first-generation (G1) PO-Ds were ineffective both against planktonic cells and biofilm cells of P. aeruginosa
, while the G1 ED-Ds proved to have a potent antimicrobial activity against the same planktonic cells (MBC = 5 μg/mL), associated with low toxicity against L929 mouse fibroblast cells, and good activity against the biofilm cells (MBC = 15 μg/mL), unfortunately associated with considerable toxicity to L929 mouse fibroblast cells (cell viability < 40%). The optimal PO to ED ratios for biofilm eradication with minimal toxicity against L929 mouse fibroblast cells were 5:5 (MBC = 20 μg/mL, cell viability in the range 90–100%). The study by Lu et al. demonstrated the importance of both the D size and its external functionalization (PO/ED) in determining the efficacy of the devices against established biofilms without compromising their biocompatibility with mammalian cells.
In the field of NO-releasing Ds, Worley et al. (2014) 
described the synthesis of NO-releasing quaternary ammonium (QA)-functionalized generation 1 (G1) and generation 4 (G4) PAMAM Ds, where QA groups were supported by different alkyl chain lengths (i.e., methyl, butyl, octyl, dodecyl) via a ring-opening reaction. The secondary amines resulting from these reactions were further modified with N
-diazeniumdiolate NO donors to achieve the NO-releasing QA-modified PAMAM Ds, capable of spontaneous NO release. Differently from non-NO-releasing QA-modified PAMAM Ds, the NO-releasing ones can exert an antibacterial dual action. In detail, the highly cationic structure of the macromolecules can inactivate the pathogens by disrupting their membranes, furthermore the release of NO can disarm bacteria, by causing oxidative and nitrosative stresses, by promoting the production of reactive NO byproducts [(N2
), peroxynitrite (ONOO−)], by causing membrane destruction via peroxynitrite-induced lipid peroxidation and by triggering protein S
-nitrosation and DNA deamination. In this regard, the antibacterial activity of the NO-releasing QA-modified PAMAM Ds was evaluated against the Gram-positive S. aureus
and the Gram-negative P. aeruginosa
species after four hours of exposure. Overall, the bactericidal activity of the devices was found to be influenced by dendrimer generation, QA alkyl chain length, and bacterial Gram class. The presence of shorter alkyl chains conferred an increased bactericidal activity, particularly against P. aeruginosa
, for both generations, with NO-releasing Ds resulting markedly more potent in killing bacteria. The toxicity of non-NO-releasing Ds and of NO-releasing Ds on L929 fibroblasts was minimal (about 60–100% and 80–110% viability respectively) when administered at the MBC concentrations, but a lower toxicity was observed for NO-releasing Ds, if compared to the non-NO-releasing ones.
NO-releasing scaffolds, including Ds, were developed by Backlund et al. (2014) and were evaluated as alternatives to current treatments for periodontitis (e.g., scaling/root planning and chlorhexidine), which are affected by limited efficacy, since they fail to suppress microbial biofilms satisfactorily over time, and since the use of adjunctive antimicrobials, in those conditions, can promote the emergence of antibiotic-resistant organisms 
. The engineered NO-releasing G1-PAMAM-PO-Ds demonstrated a 3-log reduction in the growth of periodontal pathogenic bacteria such as Aggregatibacter actinomycetemcomitans
and Porphyromonas gingivalis
(MBC = 2000 and 1000 μg/mL). Streptococcus mutans
and Streptococcus sanguinis
, typical caries-associated organisms, were, on the contrary, substantially less sensitive to NO treatment (MBC = 48,000 μg/mL). NO-releasing Ds showed less toxicity against human gingival fibroblasts at concentrations needed to eradicate periodontal pathogens than clinical chlorhexidine concentrations. Although the authors considered these results promising and suggested NO-release dendrimer scaffolds as novel platforms for the development of periodontal disease therapeutics, in our opinion, the MBC values observed are too high to make these structures usable in clinical practice.
Later, Backlund and colleagues prepared generation 1 (G1) propyl-, butyl-, hexyl-, octyl-, and dodecyl-functionalized PAMAM-D core
scaffolds, subsequently converted to N-diazeniumdiolate NO donors. The killing effect of hydrophobic G1 NO-releasing Ds on S. mutans
and their capability to disperse its biofilm were examined at pH 7.4 and 6.4, the latter being a value promoting the dental caries process 
. The bactericidal action of the NO-releasing Ds against both planktonic and biofilm-producing strains of S. mutans
was shown to be greater by increasing the alkyl chain length and lowering the pH (pH = 6.4). The authors hypothesized that the improvement in bactericidal efficacy at pH 6.4 was attributed to both increased surface positive charge of the scaffold, responsible for a better dendrimer-bacteria interaction, resulting in membrane damage, and to a faster NO-release kinetics from proton-labile N
-diazeniumdiolate NO donors. Specifically, only octyl- and dodecyl-modified PAMAM Ds were actually effective in eradicating planktonic S. mutans
cells (MBC values in the range of 12–25 μg/mL), but were ineffective in eradicating S. mutans
biofilm cells (MBC values in the range of 1000–2000 μg/mL). In addition, even if NO-release Ds showed mitigated cytotoxicity on HGF-1 human gingival fibroblasts, compared with non-NO-releasing Ds, the viability of the cells at the concentrations required for biofilm eradication was in the range of 15–20%. These findings established that octyl- and dodecyl-modified PAMAM Ds can be taken into consideration for clinical application with the aim of killing planktonic forms of S. mutans
strains, but too toxic to counteract its biofilm structure.
The following year, alkyl-modified NO-releasing PAMAM Ds, from first to fourth generation, comprising butyl and hexyl chains, and non-NO-releasing analogs, similar to the PAMAM Ds developed by Backlund et al. (2014), were prepared by Worley et al. 
. They were evaluated for their bactericidal activity within 24 h against biofilms produced by Gram-positive and Gram-negative bacteria, including antibiotic-resistant strains, assuming that the antibiofilm abilities of the alkyl chain modified NO-releasing and non-NO-releasing Ds could be improved by increasing the size of the Ds and the density of the functional groups [69
]. The results showed that the antibiofilm action of the Ds was dependent on their generation, the bacterial species, and the length of the alkyl chain of the devices. In particular, the more effective eradication of the biofilm was associated with the greater infiltration capacity of the Ds into the biofilm biomass. In this regard, the introduction of the ability to release NO has significantly improved the antibiofilm activity of those Ds incapable of effective biofilm penetration. In particular, G1 butyl Ds were practically ineffective against planktonic cells of P. aeruginosa
, S. aureus
and against MRSA strains, while G1/G4-butyl/hexyl non-NO-releasing Ds and the analogous NO-releasing devices showed, against P. aeruginosa
, MBC values in the range of 10–50 μg/mL and of 25–50 μg/mL respectively. The G3-hexyl non-NO-releasing D was recorded as the most active one (MBC value = 10 μg/mL). The same non-NO-releasing and NO-releasing Ds that showed activity against P. aeruginosa
were practically ineffective against S. aureus
and MRSA strains, with the most active one being the G3-hexyl non-NO-releasing D, which showed an MBC value of 50 μg/mL, against both S. aureus
and MRSA strains. Minimum biofilm eradication concentrations after 24 h of exposure (MBEC24h
) against biofilm produced by P. aeruginosa
, S. aureus
, and MRSA isolates were very high for all the tested Ds and, although in general the NO-releasing Ds were better than the non-NO-releasing ones, the most active Ds were the G3 hexyl (MBEC24h
= 200, 100, 100 μg/mL against the three species respectively) and the G3 hexyl/NO ones (MBEC24h
= 100, 100, 100 μg/mL against the three species respectively). Interestingly, the best performant G3 hexyl Ds, against both planktonic cells and biofilm, showed in in vitro cytotoxicity essay on L929 mouse fibroblasts, an IC50
of 450 μg/mL, either for non-NO-releasing and for NO-releasing compounds, thus establishing a low level of cytotoxicity at both MBC and MBEC24h
In the search for new alternatives to overcome bacterial drug resistance, an appealing option could be the application of bacteriophage enzymes, such as endolysins, which are able to degrade bacterial peptidoglycan (PG), and to lead to bacterial cell lysis. Starting from this approach, and in order to help endolysins to gain access to PG, Ciepluch et al. (2019) recently combined the endolysins produced by phage KP27 with unmodified and peripheral modified 20% maltose containing PPI Ds, known for their capacity to destabilize the bacterial OM 
. The antibacterial activities of mixtures containing the modified PPI Ds and the endolysins (12 μM) in a molar ratio of 1/1 and ¼, were essayed against P. aeruginosa
PAO1 (ATCC 15692) wild-type and mutant strains with reduced antigen O in LPS. The results were compared to those obtained by the administration of endolysins 12 μM or PPI Ds (12 μM and 50 μM), administered separately. The findings showed that the co-administration of endolysins and maltose-modified PPI D in ratio of 1/1 (the combination used contained a concentration of the dendrimer not toxic to eukaryotic cells), significantly enhanced the antibacterial activity of the enzyme (40–50%) against P. aeruginosa
PAO1 and WAAL strains.
By divergent growth method, Gholami et al. (2017) synthesized a high MW (116,493 g/mol) G7 PAMAM-D owing 512 positively charged amine groups, thus making the most potent broad-spectrum CAD synthesized to date, endowed with low levels of cytotoxicity on two types of eukaryotic cell lines. Its antibacterial behavior was evaluated on several species such as P. aeruginosa (n = 15), E. coli (n = 15), A. baumannii (n = 15), S. dysenteriae (n = 15), K. pneumoniae (n = 10), P. mirabilis (n = 15), S. aureus (n = 15) and B. subtilis (n = 10). Additionally, representative standard strains for each species were included. G7-PAMAM-D inhibited the growth of both Gram-positive and Gram-negative pathogens, displaying MIC50 and MIC90 values in the range of 2–4 μg/mL and 4–8 μg/mL, respectively, corresponding to very low micromolar concentrations (0.017–0.034 and 0.034–0.068 μM, respectively). MBC50 and MBC90 values were found to be 64–256 μg/mL and 128–256 μg/mL respectively, corresponding to micromolar concentrations of 0.55–2.2 and 1.1–2.2 μM, respectively. The cytotoxicity of G7-PAMAM was evaluated by MTT assay on human intestinal cancer cell lines HCT116 and NIH 3 T3, observing a reduction in viability of 44.6% and 43% respectively at the highest concentration tested (0.85 μM), after 72 h of exposition. It should be kept in mind that cytotoxicity data do not provide information concerning hemolytic toxicity, deriving from a non-specific disruptive action on RBC membranes, which is one of the biggest concerns associated with the possible clinical use of highly cationic and typically membrane-active antimicrobial agents. Based on such considerations, further investigations on the hemolytic toxicity of G7-PAMAM are mandatory to consider it as a promising antimicrobial alternative to conventional antibiotics.
To achieve antimicrobial coatings able to prevent bacterial adhesion, the Zhan’s group (2015) successfully prepared hyaluronic acid/PAMAM dendrimer (HA/PAMAM-D) multilayers on a poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)] substrate by a layer-by-layer self-assembly method 
. By using QCM-D, the authors showed that both the HA outer layer and the PAMAM-D outer layer revealed anti-adhesion activity against E. coli
. Otherwise, by using a live/dead assay, it was observed that while the PAMAM-D outer layer could also exhibit bactericidal activity against E. coli
, the outer layer of HA had no such activity. HA/PAMAM-D coatings were able to maintain the antibacterial and anti-adhesion activity after storage in phosphate-buffered saline for up to 14 days. Moreover, the in vitro MTT assay showed that the multilayers were not cytotoxic against L929 cells, and that HA molecules in the multilayers could also improve the biocompatibility of the film.
In previous studies, with the aim of obtaining a bacteriophobic coating to be applied on cotton fabric, cationic PAMAM-chitosan (CTS) dendrimers (PAMAM-CTS-Ds) were prepared by Klaykruayat et al. (2010) 
. Briefly, a quaternary ammonium hyperbranched dendritic PAMAM of generation 2.5, customized by post-synthesis methylation of a methyl ester terminated PAMAM-D, was employed to modify flake CTS, and the cationic PAMAM-CTS-D obtained was applied to cotton fabric at 1% w
, using a padding method. The antimicrobial performance of the newly modified fabric was assessed against S. aureus
and compared to a similar fabric obtained by applying unmodified CTS. Furthermore, the antimicrobial activity of not neutralized CTS films and of cationic PAMAM-CTS-D films was also evaluated. The antimicrobial evaluations were preliminarily carried out in terms of visual detection and reduction of the percentage of microbial growth, using a single application dose. The 1% w
CTS-impregnated fabric, treated with NaNO2
, according to a necessary procedure, showed no antimicrobial activity, thus indicating that CTS itself was not sufficient to inhibit the growth of S. aureus
, when applied on fabric. Nonetheless, native CTS film (0.33 g) showed an excellent antimicrobial activity (100% reduction of the growth of S. aureus
), due to the positively charged CTS amine groups. The cationic PAMAM-CTS-D film (0.25 g), also exhibited a strong antimicrobial activity against S. aureus
(99.99% reduction), comparable to that of the native CTS film. Significantly, in contrast to cotton fabric coated with CTS alone, cotton fabric treated with cationic PAMAM-CTS-D (1% w
) displayed an excellent antimicrobial activity (98.75% reduction of the growth of the pathogen). Although cationic PAMAM Ds could potentially enhance the performance of CTS as an antimicrobial coating for cotton fabric, probably due to their high cationic character, the preliminary findings highlighted by Klaykruayat et al. require further characterization and verification on other bacterial species. Similarly, more investigations are needed regarding the dose-dependence of the effect observed within the fabric, including its resistance to multiple wash cycles.