Silver salts of carboxylic acid terminated generation 1 poly (propyl ether imine) (PETIM) dendron and dendrimers as antimicrobial agents against S. aureus and MRSA

Abstract

Novel therapeutic strategies are essential to address the current global antimicrobial resistance crisis. Branched molecules with multiple peripheral functionalities, known as dendrimers, have gained interest as antimicrobials and have varying levels of toxicity. Silver displays activity against several micro-organisms only in its positively charged form. In this study, silver salts of generation 1 (G1) poly (propyl ether imine) (PETIM) dendron and dendrimers were synthesised and evaluated for their antimicrobial potential against sensitive and resistant bacteria. The purpose was to exploit the multiple peripheral functionalities of G1 PETIM dendron and dendrimers for the formation of silver salts containing multiple silver ions in a single molecule for enhanced antimicrobial activity at the lowest possible concentration. G1 PETIM dendron, dendrimers and their silver salts were synthesised and characterised by FT-IR, ¹H NMR and ¹³C NMR. PETIM silver salts were evaluated against Hep G2, SKBR-3 and HT-29 cell lines for their cytotoxicity using the MTT assay. The G1 PETIM dendron/dendrimers, silver nitrate and silver salts of the G1 dendron (compound 13), G1 dendrimer with an aromatic core (compound 14) and an oxygen core (compound 15) were evaluated for activity against S. aureus and methicillin-resistant S. aureus (MRSA) by the broth dilution method. PETIM silver salts were found to be non-cytotoxic even up to 100 μg ml⁻¹. Minimum inhibitory concentration values of compounds 13, 14 and 15 against S. aureus were 52.1, 41.7 and 20.8 μg ml⁻¹ while against MRSA they were 125.0, 26.0 and 62.5 μg ml⁻¹, respectively. The calculated fractional inhibitory concentration index further indicated that compound 14 specifically displayed additive effects against S. aureus and synergism against MRSA. The enhanced antimicrobial activities of the PETIM dendron/dendrimer-silver salts against both sensitive and resistant bacterial strains widen the pool of available pharmaceutical materials for optimizing treatment of bacterial infections.

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Introduction

Infectious diseases, a significant portion of which are of bacterial origin, are one of the leading causes of death globally for adults and children and remain a major public health issue for developed and developing countries.¹ While antibiotics revolutionized the treatment of infections, thereby saving millions of lives, eighty years after their discovery, their effectiveness is seriously threatened by antimicrobial resistance (AMR).² This nullifies the use of even the most potent antibiotics, which leads to patient suffering and/or dying due to infection control failure, and results in escalated health care costs.³

Globally, resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA),⁴ vancomycin-resistant Enterococcus (VRE)⁵ and vancomycin-resistant Staphylococcus aureus (VRSA),⁶ have become significant threats in community settings and hospitals for treatment of infections. Furthermore, if current escalating trends in AMR continue, several important procedures, such as cancer chemotherapy, organ transplantation and hip and other joint replacements, could no longer be performed for fear that the related compromised immune system might put the patients at severe risk of acquiring a difficult to treat and ultimately fatal infection.⁷ The global AMR crisis is amplified by the decreasing development of new antibiotics by pharmaceutical companies,⁸ with 20 novel classes of antibiotics being developed in between 1930–1962,^9,10 and only two of them have been marketed.^11–14 This decline in drug development is due to the high costs and lengthy delays associated with developing a new chemical entity, high attrition rates at final testing, and increasing AMR, which makes finding a new drug very expensive and limits the return on investment.^3,15

It is therefore essential that alternative novel antimicrobial therapeutic strategies are explored to address the imminent crisis with conventional antibiotics. Alternative options currently being investigated are novel drug delivery systems for existing antibiotics, such as silver nanoparticles,^16–18 solid lipid nanoparticles,^19–21 liposomes^22–24 and the synthesis of new antimicrobial materials, such as dendrimers^25–27 and antimicrobial peptides.²⁸

Silver is a potent antimicrobial agent, particularly in its positively charged ionic form, as it displays a strong toxicity to a wide range of micro-organisms and concurrently has a particularly low human toxicity.^29–31 Antimicrobial silver is widely used to combat organisms associated with burns and wounds.³⁰ In addition, silver-based medical preparations are available and frequently used in coatings of biomedical materials, such as silver impregnated catheters and dressings for wound healing.²⁹ Silver is also capable of disturbing key functions in a microorganism that causes AMR. It has a high affinity for negatively charged side groups on biological molecules, such as carboxyl, phosphate, sulfhydryl and others dispersed throughout microbial cells. It thereby transforms the macromolecule's molecular structure via this binding reaction, rendering it useless to the cell.³⁰ Concomitantly, silver can attack numerous sites within the cell, incapacitating critical physiological functions, such as cell wall synthesis, protein folding and function, membrane transport, nucleic acid (such as RNA and DNA) synthesis, and translation and electron transport, which are vital for cell energy production. Dispossessed of such key functions, bacterial growth can either be inhibited or, more frequently, the microorganism is killed.³⁰ It is highly improbable that resistance to antimicrobial silver could ever develop, as this would mean that an organism would have to undertake concurrent mutations in every critical function within just a single generation to evade the compounds multiple actions.³⁰ This is a crucial factor to consider when developing new antimicrobial materials to overcome resistance. However, it should be noted that silver is nontoxic to human cells only in minute concentrations.³² This clearly limits the use of metallic silver and silver ions as an antibacterial agent only up to concentrations that are non-toxic to eukaryotic cells.

Dendrimers are repeatedly branched molecules or nano-sized, radially symmetric molecules that have a well-defined, uniform and monodisperse structure that consists of branches surrounding a core.^33,34 The availability of several functional surface groups and their low polydispersity make them a rich source for finding novel and unique properties.^33,35 Due to these very distinctive properties, and the fact that they can be adapted to therapeutic needs, they are regarded as model carriers for small molecule drugs and biomolecules.²⁶ Dendrimers have gained further interest as likely antimicrobial agents due to the availability of numerous end groups and their compressed structure.^36,37 Therefore, if any one of the functional groups is capable of interacting with a target, other groups within close proximity of one another could make synergistic interactions for antimicrobial activity possible.³⁶ Specific interactions (e.g. quaternary ammonium based dendrimers) aim to eliminate bacterial/viral infections by inhibiting the growth of microbes, thereby killing them and nonspecific interactions (e.g. oligosaccharide based dendrimers), and preventing the initial attachment between bacteria/viruses and host cells.³⁶

It has also been highlighted that dendrimers show promising biocompatibility in general,²⁶ which is essential for their application, and can themselves be used as antimicrobial agents.^38–41 Consequently, highly potent dendrimer based antibacterial agents have been synthesised.^38,40 Currently, the most extensively used dendrimers in drug delivery include poly propylene imine (PPI), polylysine, triazine⁴² and polyamidoamine (PAMAM), the latter being the first and most commonly studied.²⁶ Unfortunately, its uses are constrained by limitations such as cytotoxicity resulting from its amine-terminated nature⁴³ and as a result, there are no commercially available dendrimer based formulations for systemic administration.⁴² Researchers have recognized the potential of developing complexes of silver and dendrimers to enhance antimicrobial activity, with Balogh et al. having prepared PAMAM dendrimer based silver complexes,⁴⁴ which showed enhanced antimicrobial effect, creating a new and potent antimicrobial agent for biomedical applications.

A fairly new class of dendrimers, known as the poly (propyl ether imine) (PETIM) dendrimers, has been reported to have good biocompatibility when compared to commercial PAMAM dendrimers, and has been effectively applied for encapsulation of ketoprofen for sustained drug delivery.⁴² Although it has several advantages, such as non-cytotoxicity and easy functional group modification at the periphery, its potential for antimicrobial therapy has not been exploited. This study is therefore the first combination of PETIM dendrimers and silver to identify novel antimicrobial materials effective against both sensitive and resistant bacterial strains, and will widen the pool of available pharmaceutical materials to optimize the treatment of bacterial infections.

In this study, a generation 1 (G1) PETIM dendron and two PETIM dendrimers containing a carboxylic acid function at the periphery were synthesised and reacted with silver nitrate to form dendrimer-silver salts. The PETIM dendrimers were used as templates to contain the silver ions. The rationale for using PETIM dendron and dendrimer as a template to contain silver ions were: (i) more than one silver ion can be accommodated on a single PETIM dendron or dendrimer, as it contains multiple carboxylic acid functions at the periphery; (ii) PETIM silver complexes are non-toxic to mammalian cells due to the biocompatibility of PETIM dendrimers; and (iii) PETIM on its own could display antimicrobial activity, thus the potential antimicrobial activity of PETIM silver complexes may display additive or synergistic effects. Published studies on silver complexes of organic compounds as antimicrobial agents mostly include two organic molecules complexed with one silver ion through a chemical bond formation.^45,46 In the present investigation, our goal was therefore to exploit the multiple peripheral functionalities of biocompatible PETIM dendron and dendrimers to form silver salts containing multiple silver ions in a single molecule for enhanced antibacterial activity at the lowest possible concentration. The intention was to study the effect of the number of silver ions per molecule of these dendrimer silver salts on antimicrobial efficacy against both sensitive and resistant strains. For this reason, a G1 PETIM dendron (two carboxylic acid functions at the periphery), G1 PETIM dendrimer with oxygen core (four carboxylic acid functions at the periphery) and G1 PETIM dendrimer with aromatic core (six carboxylic acid functions at the periphery) were selected. The results of the investigations are reported in this paper.

Results and discussion

Synthesis

In this study three different compounds were employed, viz., a G1 PETIM dendron 4, a PETIM dendrimer with an aromatic core 7, and another PETIM dendrimer with an oxygen core 12. Synthetic steps for these compounds are depicted in Schemes 1–3 and explained hereunder.

Scheme 1 Synthesis of G1 PETIM dendron.

Scheme 2 Synthesis of G1 PETIM dendrimer containing an aromatic core.

Scheme 3 Synthesis of G1 PETIM dendrimer containing an oxygen core.

The dendron was prepared using 3-amino-1 propanol 1 and excess tert-butyl acrylate 2 to afford an ester in good yield. Thereafter the resulting ester was deprotected (AcCl, H₂O) to obtain the free carboxylic acid containing G1 PETIM dendron 4 (Scheme 1).

The two dendrimers synthesised were prepared with slight modifications in a previously reported method.⁴⁷ Upon synthesis of the dendron 3, its attachment to a selected core was carried out. Compound 3 was coupled with 1,3,5-benzenetricarbonyl trichloride 5 in the presence of DMAP to attain 6. Thereafter the resulting ester was deprotected via an acetyl chloride and water system to attain the free carboxylic acid containing G1 PETIM dendrimer with an aromatic core 7 (Scheme 2).

Bis-nitrile 9 was attained from acrylonitrile 8 and aqueous NaOH (40%). Bis-nitrile was subjected to successive reactions; i.e. reduction of the nitrile using LiAlH₄ to a diamine 10; Michael addition of tert-butyl acrylate to afford the tetrakis 11; and deprotection of the ester (AcCl, H₂O) to attain the free carboxylic acid containing G1 PETIM dendrimer with an oxygen core 12 (Scheme 3).

Preparation of PETIM-silver salts (Scheme 4). PETIM silver salts (13, 14 and 15) were all prepared in a similar method where silver was reacted with 4, 7 and 12 to afford these PETIM-silver salts.

Scheme 4 Synthesis of silver salts of G1 PETIM dendron and dendrimers.

Characterisation

The synthesised dendron and dendrimers were characterised by FT-IR, ¹H NMR, ¹³C NMR and HRMS and were compared with the literature values.⁴⁷ Synthesis of the silver salts were accomplished via reaction of silver nitrate with the corresponding dendron/dendrimer acid. Formation of silver salts was supported by observing the shifts in the positions of characteristic IR frequencies of carboxylic groups in the dendron and dendrimers.

The main feature which allows one to differentiate a carboxylic acid from all other carbonyl compounds is a broad absorption band due to the strongly hydrogen bonded O–H stretching vibrations which extends from 3300–2500 cm ⁻¹. The transformation of the ester function to a carboxylic acid was confirmed by the presence of this characteristic peak in FT-IR spectrum. In addition, all carboxylic acid terminated dendron/dendrimers exhibited a peak in the range of 1707–1714 cm⁻¹ indicating the presence of a C=O stretching band of the –COOH group. The aliphatic C–H stretching band appeared as a jagged peak near 3000 cm⁻¹. Coupled vibrations involving C–O stretching were observed in the range of 1459–1399 cm⁻¹. Salts of carboxylic acids do not display any of the carbonyl bands rather bands owing to the asymmetric and symmetric stretching vibrations of the equivalent carbon–oxygen bonds. They are observed at 1610–1550 cm⁻¹ and 1420–1300 cm⁻¹ respectively, which provides evidence for the carboxylate anion.⁴⁸ In our study the peaks in the range of 1459–1332 cm⁻¹ from carboxylic acid terminated dendron and dendrimers disappeared and appearance of symmetric stretching vibrations in the range of 1288–1233 cm⁻¹ was observed (Fig. 1–3) after transforming them into their respective silver salts. Thus, the presence of the bands because of symmetric stretching vibrations of the equivalent carbon–oxygen bonds strongly confirms the formation of silver salts of G1 PETIM dendron and dendrimers. Further attempts to characterise silver salts using elemental analysis were not successful because of their hygroscopic nature.⁴⁷

Fig. 1 (a) FT-IR spectra comparing G1 PETIM dendron 4 and G1 PETIM dendron-silver salt 13; (b) G1 PETIM dendrimer (aromatic core) 7 and G1 PETIM dendrimer (aromatic core)-silver salt 14 and (c) G1 PETIM dendrimer (oxygen core) 12 and G1 PETIM dendrimer (oxygen core)-silver salt 15.

Fig. 2 (a) Cytotoxicity assay against Hep G2 cells, displaying percentage cell viability after exposure to various concentrations of PETIM silver salts [G1 PETIM dendron-silver salt 13; G1 PETIM dendrimer (aromatic core)-silver salt 14; G1 PETIM dendrimer (oxygen core)-silver salt 15] to Hep G2 cells. Results are presented as mean ± SD (n = 6). (b) Cytotoxicity assay against HT-29 cells, displaying percentage cell viability after exposure to various concentrations of PETIM silver salts [G1 PETIM dendron-silver salt 13; G1 PETIM dendrimer (aromatic core)-silver salt 14; G1 PETIM dendrimer (oxygen core)-silver salt 15] to HT-29 cells. Results are presented as mean ± SD (n = 6). (c) Cytotoxicity assay against SK-BR-3 cells, displaying percentage cell viability after exposure to various concentrations of PETIM silver salts [G1 PETIM dendron-silver salt 13; G1 PETIM dendrimer (aromatic core)-silver salt 14; G1 PETIM dendrimer (oxygen core)-silver salt 15] to SK-BR-3 cells. Results are presented as mean ± SD (n = 6).

Fig. 3 (a) Cytotoxicity assay on Hep G2 cell lines, comparing percentage cell viability after exposure to PETIM silver salts against their individual parent dendron/dendrimers as well as silver nitrate concentrations. Results are presented as mean ± SD (n = 6). *denotes significant difference compared to the respective silver nitrate (P < 0.05) [G1 PETIM dendron 4; G1 PETIM dendrimer with aromatic core 7; G1 PETIM dendrimer with oxygen core 12; G1 PETIM dendron-silver salt 13; G1 PETIM dendrimer (aromatic core)-silver salt 14; G1 PETIM dendrimer (oxygen core)-silver salt 15]. (b) Cytotoxicity assay on HT-29 cell lines, comparing percentage cell viability after exposure to PETIM silver salts against their individual parent dendron/dendrimers as well as silver nitrate concentrations. Results are presented as mean ± SD (n = 6). *denotes significant difference compared to the respective silver nitrate (P < 0.05) [G1 PETIM dendron 4; G1 PETIM dendrimer with aromatic core 7; G1 PETIM dendrimer with oxygen core 12; G1 PETIM dendron-silver salt 13; G1 PETIM dendrimer (aromatic core)-silver salt 14; G1 PETIM dendrimer (oxygen core)-silver salt 15]. (c) Cytotoxicity assay on SK-BR-3 cell lines, comparing percentage cell viability after exposure to PETIM silver salts against their individual parent dendron/dendrimers as well as silver nitrate concentrations. Results are presented as mean ± SD (n = 6). *denotes significant difference compared to the respective silver nitrate (P < 0.05) [G1 PETIM dendron 4; G1 PETIM dendrimer with aromatic core 7; G1 PETIM dendrimer with oxygen core 12; G1 PETIM dendron-silver salt 13; G1 PETIM dendrimer (aromatic core)-silver salt 14; G1 PETIM dendrimer (oxygen core)-silver salt 15].

In vitro cytotoxicity study

An in vitro cell culture system was used to determine the biological efficacy of the PETIM silver salts. The MTT assay, which is based on the biochemical reduction of MTT by viable cells, was used to determine the cytotoxicities of the PETIM silver salts against Hep G2, HT-29 and SK-BR-3 cell lines.⁴⁹ Determining cell viability using cytotoxicity assays are basic steps in toxicology that explain the cellular response to a compound by providing information on cell death and their metabolic activities.⁵⁰ Cell viability of between 80% and 95% were observed for all the PETIM silver salts across all the cell lines (Fig. 2). The comparative results between the individual PETIM dendron/dendrimers, their respective concentrations of silver nitrate, and their subsequent combinations (PETIM silver salts) were tested for cytotoxicity and are represented in Fig. 3.

The range of cell viability obtained in this study indicates that the PETIM silver salts displayed a low toxicity level on all cell lines studied.⁵¹ The results also showed that the effects of the compounds on the cell line were not dose dependent, as no dose dependent trends were observed for any of the PETIM silver salts at the various treatment concentrations against any of the cell lines (Fig. 2). These PETIM silver salts displayed a greater percentage cell viability when compared to their respective concentrations of silver nitrate (Fig. 3). Reduced cytotoxicity of the PETIM silver salts may be due to their close-to-neutral net surface charge, which had little effect on membrane integrity.⁵² These results are in line with previous findings, where acetamide-terminated G5 PAMAM dendrimers revealed to have little effect on membrane integrity, whereas positively charged G5 PAMAM dendrimers reduced the integrity of the cell membrane and prompted the release of cytoplasmic membrane proteins, lactate dehydrogenase and luciferase.^52,53 The PETIM silver salts therefore have a statistically greater cell viability than the silver nitrate (P < 0.05) (Fig. 3) and slightly higher cell viability when compared to the PETIM dendron/dendrimers. Therefore, they can be considered non-toxic with potential for use in the biomedical and pharmaceutical fields.

In vitro antimicrobial evaluation

The antimicrobial activities of silver nitrate, the PETIM dendron/dendrimers and the PETIM silver salts were investigated against S. aureus and MRSA. A summary of the results for the MIC values for in vitro antimicrobial activity is presented in Table 1. MIC values for the different concentrations of silver nitrate against S. aureus were 112.5, 87.5 and 77.5 μg ml⁻¹ respectively, and against MRSA they were 93.7, 210 and 77.5 μg ml⁻¹ respectively (Table 1). Ionized silver brings about structural changes in bacterial cell walls and nuclear membranes as it is highly reactive when it binds to tissue proteins. Thus it results in cell distortion and even cell death. Silver can also bind to bacterial DNA and RNA, and can therefore inhibit bacterial replication. These antimicrobial properties of silver are dependent on the quantity and the rate at which silver is released.^54,55 The MIC values for the PETIM dendron/dendrimers, i.e. 4, 7 and 12 against S. aureus and MRSA, were all 500 μg ml⁻¹ (Table 1). The MIC values obtained for the PETIM dendron/dendrimers indicate that the PETIM dendron/dendrimers alone do have some antimicrobial activity, although low, against the selected bacteria. Higher antimicrobial activity has been reported for both unmodified dendrimers and dendrimers, with additional surface modifications such as PAMAM dendrimer ammonium salts,³⁸ and PPI dendrimers modified with maltotriose 25% and 100%.³⁹ However, the unmodified dendrimers displayed higher levels of cytotoxicity when compared to the surface modified dendrimers due to the cationic nature of these dendrimers.⁵⁶ As the PETIM dendrimers in our study displayed good cell viability due to their anionic nature, this nullifies the need for surface modification procedures to minimize the toxicity. Although these MIC values are higher when compared to surface modified and unmodified dendrimers, such as PAMAM and PPI against gram positive bacteria, this does confirm for the first time the antimicrobial activity of G1 PETIM dendron and dendrimers (4, 7 and 12).

Table 1 MIC results for in vitro antimicrobial activity of PETIM dendron/dendrimers, PETIM silver salts and their corresponding individual silver nitrate concentrations against S. aureus and MRSA

Sample	MIC (μg ml^−1)
	Organism
	S. aureus	MRSA
a Denotes SD = 0.
G1 PETIM dendron 4	500^a	500^a
Silver nitrate	112.5^a	93.7^a
G1 PETIM dendron-silver salt 13	52.1 ± 18.04	125^a
G1 PETIM dendrimer with an aromatic core 7	500^a	500^a
Silver nitrate	87.5^a	210^a
G1 PETIM dendrimer (aromatic core)-silver salt 14	41.7 ± 18.04	26 ± 9.04
PETIM dendrimer with oxygen core 12	500^a	500^a
Silver nitrate	77.5^a	77.5^a
G1 PETIM dendrimer (oxygen core)-silver salt 15	20.8 ± 11.07	62.5^a

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The MIC values for the PETIM silver salts, i.e. 13, 14 and 15 investigated in this study, were 52.1, 41.7, and 20.8 μg ml⁻¹ against S. aureus respectively, while against MRSA they were 125.0, 26 and 62.5 μg ml⁻¹ respectively (Table 1). An increase in antimicrobial activity was observed for all salts when compared to silver nitrate and PETIM dendron/dendrimers alone. This may be a result of a high local concentration of silver ions available at the periphery of the PETIM silver salts. Antimicrobial activity was reported to be less when internal complexes were applied, showing that accessibility of the silver is a vital factor, and that a high local concentration of silver needs to be accessible to have a significant effect on microorganisms.⁴⁴ The MIC values of the salts of PETIM dendron/dendrimers were markedly reduced for G1 PETIM-dendron silver salt 13 and G1 PETIM dendrimer (oxygen core)-silver salt 15 against S. aureus, and G1 PETIM dendrimer (aromatic core)-silver salt 14 against both organisms. Compound 13 and 15 exhibited 42% and 33% greater activity against S. aureus respectively when compared to MRSA. However, compound 14 displayed 62% greater activity against MRSA than S. aureus. The PETIM silver salts showed different degrees of antibacterial activity in relation to the bacterial species used in this study. Compound 14 displayed greater antibacterial activity against MRSA than S. aureus. Certain dendrimers displayed potent and broad antimicrobial activity against S. aureus,³⁷ as well as a selectivity toward this particular bacterial species.³⁹ Polcyn et al. also recently synthesised a range of modified dendrimers and interestingly, they too identified one particular dendrimer as having strong activity against MRSA,³⁷ similar to the antimicrobial activity of compound 14 used in this study. Wang et al., performed antimicrobial testing on norfloxacin-loaded solid lipid nanoparticles for a 144 h time period, and the results indicated antimicrobial activity for an extended time period.¹⁹ Similarly, the antimicrobial activity of 13, 14 and 15 were tested over a 72 h period, with the results being consistent throughout this time span, indicating that they have the potential for sustained antimicrobial activity.

MIC values alone did not contribute toward a clear indication of the combined effects of the PETIM dendron/dendrimers and silver nitrate. Hence, the effects of the combination of G1 PETIM dendron/dendrimers and silver nitrate were also investigated, and these effects were evaluated using ΣFIC. A summary of the results for the ΣFIC values for in vitro antimicrobial activity experiments is presented in Table 2. All of the combinations displayed different degrees of effectiveness against the bacteria tested, and no antagonistic relations were observed. Compound 13 presented a ΣFIC value of 1.58 against MRSA (Table 2), which represents indifference (Table 3). Compound 13 and 14 presented a ΣFIC value of 0.57 and 0.56 against S. aureus (Table 2), and 15 presented a ΣFIC value of 0.93 against MRSA (Table 2), which are all indicative of additive effects (Table 3). Compound 14 presented a ΣFIC value of 0.18 against MRSA (Table 2) and 15 presented a ΣFIC value of 0.31 against S. aureus (Table 2), which signify synergistic effects (Table 3). Of the three PETIM silver salts tested, 13 was observed to be the least active salt, whereas 14 was most active. This pattern of antibacterial activity of G1 PETIM-silver salts against both S. aureus and MRSA can be correlated to the structures of the compounds. The order of antibacterial potency of dendron/dendrimer-silver salts was G1 PETIM dendrimer (aromatic core)-silver salt 14 (six Ag⁺ ions in the structure) > G1 PETIM dendrimer (oxygen core)-silver salt 15 (four Ag⁺ ions in the structure) > G1 PETIM dendron-silver salt 13 (two Ag⁺ ions in the structure). The G1 PETIM-silver salt with the highest number of carboxylic acid functions, and ultimately the highest number of Ag⁺ ions, had the greatest antibacterial activity. As the G1 PETIM-silver salts contain positively charged Ag⁺ ions and the bacterial cell wall has an overall negative charge, which has more affinity towards positively charged compounds, it may be possible that 14 had the best activity because of the highest number of Ag⁺ ions present. The synergistic effect of 14 could therefore be a result of the combination of different mechanisms of actions of both silver and the dendron/dendrimers. Silver is known for its growth inhibitory capacity against microorganisms,⁵⁷ and by using dendrimers as a template to incorporate silver, dendrimers themselves can become potent antimicrobials.⁵⁸ This activity can then be further enhanced if the functional groups of the dendrimers are within close proximity to one another.³⁶

Table 2 ΣFIC results for in vitro antimicrobial activity of the PETIM silver salts

Sample	ΣFIC		Results
	S. aureus	MRSA	S. aureus	MRSA
G1 PETIM dendron-silver salt 13	0.57	1.58	Additive	Indifference
G1 PETIM dendrimer (aromatic core)-silver salt 14	0.56	0.18	Additive	Synergy
G1 PETIM dendrimer (oxygen core)-silver salt 15	0.31	0.93	Synergy	Additive

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Table 3 FIC index⁶⁵

Index	Synergy	Additive	Indifference	Antagonism
FIC	≤0.5	>0.5–1	>1 to <2	≥2

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The interesting differences in activity of the three compounds against S. aureus and MRSA as well as specifically the significant synergistic activity against MRSA as compared to S. aureus in 14 may be due to differences in the structure and composition of their cell walls. For example one of the most widely reported mechanisms of resistance in S. aureus is the development of a modified penicillin binding protein (PBP) known as PBP 2a found in MRSA.^59,60 Biosynthesis of peptidoglycan, which comprises the outermost layer of Gram-positive bacteria, is achieved by the membrane-bound enzymes PBP.⁵⁹ With MRSA the modified PBP known as PBP 2a, is intrinsically resistant to inhibition by β-lactams and stays active even in the presence of antibiotics that typically inhibit most endogenous PBP enzymes, thereby replacing their functions in cell wall synthesis and permitting growth in the presence of β-lactam inhibitors such as Methicillin.⁵⁹

The significant increase in activity of 14 against MRSA may be attributed to its higher valency compared to 15. The higher valency of 14 might have resulted in better binding affinity to PBP 2a of MRSA than PBP of S. aureus. This plausible mechanism of action could be supported by the recent findings where multivalent vancomycin-conjugated G5 PAMAM dendrimers exhibited enhancement in avidity in the cell wall models of S. aureus and VRSA as compared to free vancomycin. In this particular study authors have observed that the vancomycin-conjugated PAMAM dendrimers had binding avidity of 2–3 and 5 orders of magnitude with (D)-Ala–(D)-Ala, a cell wall precursor of S. aureus and (D)-Ala–(D)-Lac, a cell wall precursor of VRSA respectively.⁶¹ The absence of PBP 2a in S. aureus could have been the reason behind low activity of 14 against S. aureus as compared to 15. In the case of S. aureus 15 may have greater binding affinity to PBP resulting in its higher antibacterial activity against S. aureus than 14.

Whilst the paper by Choi et al. attempts to provide a mechanistic understanding of the vancomycin-conjugated G5 PAMAM dendrimers against S. aureus and VRSA, there are no such mechanistic studies available in the literature using novel materials and delivery systems against S. aureus and MRSA. There is also the possibility of multiple simultaneous mechanisms of actions of 14 and 15 against both S. aureus and MRSA, therefore, the mechanism of action postulated to explain the differences in antibacterial activity of 14 and 15 against MRSA and S. aureus respectively is a hypothesis based on previous literature and needs to be confirmed by future in depth experimental mechanistic studies.

Experimental

Materials and methods

Acrylonitrile, tert-butyl acrylate and 3-amino-1-propanol were purchased from Alfa Aesar (Germany). 4-(Dimethylamino) pyridine (DMAP), lithium aluminum hydride (LiAlH₄), acetyl chloride (AcCl), 1,3,5-benzenetricarbonyl trichloride, silver nitrate and silica gel were purchased from Sigma-Aldrich (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Merck Chemicals (Germany). All other chemicals and solvents used were of analytical grade, used without further purification and purchased from Merck Chemicals (Germany). Purified water used during the study was produced in the laboratory with a Milli-Q purification system (Millipore corp., USA). Nutrient Broth, Mueller-Hinton Broth (MHB) and Mueller-Hinton Agar (MHA) were obtained from Biolab (South Africa). The bacterial cultures used were Staphylococcus aureus ATCC 25923 and methicillin-resistant Staphylococcus aureus (MRSA) (Staphylococcus aureus Rosenbach ATCC BAA 1683). Optical density (OD) was measured using a Mindray MR-96A microplate spectrophotometer (China). FT-IR spectra of all the compounds were recorded on a Bruker Alpha-p spectrometer with diamond ATR (Germany) as per standard protocols. ¹H NMR and ¹³C NMR measurements were performed on a Bruker 400/600 Ultrashield™ (United Kingdom) NMR spectrometer. HRMS was performed on a Waters Micromass LCT Premier TOF-MS (United Kingdom).

Synthesis of dendron 4 (Scheme 1)

The G1 PETIM dendron with a carboxylic acid function at the periphery was synthesised by hydrolysis of the dendron as reported in the literature.⁴⁷ In summary, a mixture of 3-amino-1-propanol 1 (5 g; 67 mmol) in methanol (20 ml) was added drop wise to a solution of tert-butyl acrylate 2 (51.2 g; 399 mmol) in methanol (100 ml), and was stirred for 6 h at room temperature. Surplus tert-butyl acrylate and solvent were removed in vacuo, with the crude product obtained being diluted with dichloromethane and washed with brine (3 × 25 ml). The organic layer was dried over anhydrous sodium sulphate and concentrated to yield 3 as a clear colourless liquid (21 g; 96%). Acetyl chloride (0.95 g; 12 mmol) and water (0.22 ml; 12 mmol) were added to a solution of 3 (0.5 g; 1.5 mmol) in dichloromethane (30 ml), and the solution was stirred at room temperature for 8 h. Solvents were removed under vacuum to afford 4 as a viscous material (0.3 g; 91%). FT-IR (neat) ν: 2959, 1712, 1399, 1179, 929 cm⁻¹. ¹H NMR (400 MHz, CD₃SOCD₃) δ: 1.82 (q, 2H), 2.50 (t, 4H), 2.82 (t, 2H), 3.47 (t, 4H), 3.76 (t, 2H). ¹³C NMR (100 MHz, CD₃SOCD₃) δ: 28.3, 37.0, 52.2, 58.9, 59.0, 174.4. HRMS (ES-TOF): [M]⁺ calcd for C₉H₁₇NO₅ 220.1185; found 220.1181.

Synthesis of G1 PETIM dendrimer with an aromatic core 7 (Scheme 2)

A mixture of 3 (3 g; 9 mmol) and DMAP (3.3 g; 27 mmol) in PhMe (60 ml) was refluxed for 3 h and cooled to room temperature. 1,3,5-benzenetricarbonyl trichloride 5 (0.6 g; 2.3 mmol) was then added to the mixture and the reaction was refluxed for 6 h. PhMe was removed in vacuo and the crude product was purified via column chromatography (silica, mesh size 60–100) (hexane/EtOAc, 4 : 6) to obtain 6 as a colourless oil (1.5 g; 60%). Acetyl chloride (3.63 g; 46 mmol) and water (0.73 ml; 41 mmol) were added to a solution of 6 (1.06 g; 0.92 mmol) in dichloromethane (40 ml), and the resulting solution was stirred vigorously at room temperature for 8 h. Solvents were then removed in vacuo and the subsequent residue was triturated with dichloromethane and hexane to obtain 7 (0.7 g; 93%) as a white foamy solid. FT-IR (neat) ν: 2601, 1710, 1232, 1402, 944, cm⁻¹. ¹H NMR (400 MHz, CD₃SOCD₃) δ: 2.23 (b, 6H), 2.86 (t, 12H), 3.29 (t, 6H), 3.36 (t, 12H), 4.44 (t, 6H), 8.70 (s, 3H). ¹³C NMR (100 MHz, CD₃SOCD₃) δ: 22.8, 28.3, 50.6, 52.1, 62.9, 131.28, 134.3, 167.5, 174.1.

Synthesis of G1 PETIM dendrimer with an oxygen core 12 (Scheme 3)

Acrylonitrile 8 (11.66 g; 0.22 mmol) was added drop wise to aqueous sodium hydroxide (40%) (2 ml), while maintaining the temperature below 30 °C. The reaction mixture was stirred overnight at room temperature and then neutralized with hydrochloric acid (32%) (w/w). The product was extracted with chloroform (3 × 50 ml) and washed with 5% sodium hydroxide (100 ml) followed by brine (50 ml). The organic layer was dried over anhydrous sodium sulphate and concentrated under vacuum to yield 9 (7.44 g; 55%). To a solution of LiAlH₄ (1.38 g; 48 mmol) in dry THF (40 ml) at 0 °C, 9 (3 g; 24 mmol) was added drop wise, a solution of 9 in THF (10 ml). The reaction was allowed to come to room temperature and was then stirred for 1 h, after which cold water (2.2 ml; 122 mmol) was added drop wise to the reaction mixture. The reaction mixture was stirred overnight at room temperature to afford 10, a diamine (2.63 g; 80%) after filtration of the reaction mixture and evaporating the solvent. A solution of 10 (2.63 g; 20 mmol) in methanol (60 ml) was added drop wise to tert-butyl acrylate (14.02 g; 0.11 mmol) in methanol (50 ml), and the reaction was stirred for 6 h at room temperature. After column chromatographic purification (silica, mesh size 60–100) (hexane/EtOAc, 7 : 3) and removal of the solvents, 11 was obtained as a colourless liquid (3.45 g; 27%). Finally, acetyl chloride (1.33 ml; 15 mmol) and water (0.28 ml; 16 mmol) were added to a solution of 11 (0.5 g; 0.77 mmol) in dichloromethane (10 ml), and the solution was stirred vigorously at room temperature for 8 h to afford 12 (0.3 g; 94%) after removing the solvent in vacuo and trituration of residue with hexane and dichloromethane several times. FT-IR (neat) ν: 2931, 1707, 1240, 1400, 930 cm⁻¹. ¹H NMR (400 MHz, D₂O) δ: 2.0 (b, 4H), 2.88 (t, 8H), 2.93 (b, 4H), 3.31 (b, 4H), 3.47 (t, 8H). ¹³C NMR (100 MHz, D₂O) δ: 22.50, 2.28, 52.08, 58.9, 62.7, 176.5.

Silver salt of G1 PETIM dendron 13

To a solution of 4 (0.1 g; 0.46 mmol) in methanol (10 ml), an aqueous solution of silver nitrate (0.154 g; 0.9 mmol) in H₂O (5 ml) was slowly added and stirred vigorously for 2 h. The solvents were removed in vacuo to obtain 13 (0.19 g; 96%). FT-IR (neat) ν: 3028, 1722, 1633, 1284 cm⁻¹.

Silver salt of G1 PETIM dendrimer with an aromatic core 14

An aqueous solution of silver nitrate (0.25 g; 0.31 mmol) in H₂O (30 ml) was slowly added to a solution of compound 7 (0.2 g; 1.18 mmol) in methanol and stirred vigorously for 2 h. The solvents were removed in vacuo to obtain 14 (0.33 g; 92%). FT-IR (neat) ν: 3019, 1713, 1287, 1233, 1181 cm⁻¹.

Silver salt of G1 PETIM dendrimer with an oxygen core 15

Compound 12 (0.25 g; 0.59 mmol) was dissolved in acetone (50 ml), to which an aqueous solution of silver nitrate (0.405 g; 2.38 mmol) in H₂O (30 ml) was slowly added and stirred vigorously for 2 h. The solvents were removed in vacuo to afford 15 (0.5 g; 99%). FT-IR (neat) ν: 2932, 1714, 1265, 1210 cm⁻¹.

In vitro cytotoxicity study

Cell culture against hepatocellular carcinoma (Hep G2), colorectal adenocarcinoma (HT-29) and breast adenocarcinoma (SK-BR-3) cell lines were cultured with complete medium (minimum essential medium, supplemented with 10% bovine calf serum, 100 units per ml of penicillin, and 100 mg ml⁻¹ of streptomycin). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂ in air.

Solutions: The compounds were dissolved in DMSO and distilled water as a stock solution,⁶² and diluted in the culture medium at concentrations of 20, 40, 60, 80 and 100 μg ml⁻¹ as working-solutions.⁶³

MTT assay: The cell lines were harvested from the exponential phase, were seeded equivalently into a 96-well plate (2.2 × 10³) and incubated for 24 h to allow for adherence. Thereafter, the culture medium was removed and replaced with fresh medium (100 μl per well), with the samples being added to the wells to achieve final concentrations. The control wells were prepared by adding the culture medium only. Wells containing the culture medium without cells were used as blanks. All experiments were performed with six replicates. Upon completion of the incubation for 48 h, the culture medium and compounds were removed and replaced with fresh medium (100 μl) and 100 μl of MTT solution (5 mg ml⁻¹ in PBS) in each well. After 4 h incubation, the media and MTT solution was removed and 100 μl of DMSO was added to each well to solubilize the MTT formazan. The OD of each well was measured on a microplate spectrophotometer at a wavelength of 540 nm.⁶⁴ The percentage cell viability was calculated as follows:

%cell survival = [A540 nm treated cells]/[A540 nm untreated cells] × 100 (1)

A540: absorbance at a wavelength of 540 nm.

Antimicrobial evaluation

Determination of minimum inhibitory concentrations (MICs): the MICs of the PETIM dendron/dendrimers, silver nitrate and dendrimer-silver salts were determined in triplicate using the broth dilution method. Stock solutions of 4 (0.4 mg ml⁻¹), 7 (0.44 mg ml⁻¹) and 12 (0.38 mg ml⁻¹), as well as silver nitrate in three different concentrations (0.60 mg ml⁻¹, 0.56 mg ml⁻¹, 0.62 mg ml⁻¹), were prepared in dimethyl sulfoxide (DMSO). The quantities were equivalent to the amount of individual components present in 1 mg ml⁻¹ solutions of the respective dendrimer-silver salt. Stock solutions of the various dendrimer-silver salts (1 mg ml⁻¹) were prepared in distilled water 13 and DMSO 14 and 15. The compounds were tested against S. aureus and MRSA, which were grown overnight in Nutrient Broth at 37 °C and adjusted to 0.5 McFarlands standard with distilled water. Serial dilutions of the dendron/dendrimers, silver nitrate and dendrimer-silver salts were prepared in MHB from the stock solutions. The test bacteria were added to each dilution and incubated overnight at 37 °C. Thereafter, each dilution was spotted on MHA plates and incubated overnight at 37 °C. After incubation, the MHA plates were examined for growth and the MIC's was determined, with DMSO being used as a control.

Determination of fractional inhibitory concentration (FIC): the effects of the combination of G1 PETIM dendron/dendrimers and silver nitrate were investigated by determining the Σ FIC. The European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID)⁶⁵ described the method for quantifying MIC results in terms of the FIC index, defined as the sum of FIC values of two drugs in combination. An example of the method used to calculate the ΣFIC is as follows:

For two antibacterials A and B alone and in combination (4 and silver nitrate).

ΣFIC = FIC_(A) + FIC_(B) = 0.10416 + 0.46293 = 0.56709 (4)

The FIC index is shown in Table 3. Indifference is when the effect of a combination of antimicrobials is equal to the effects of the most active compound. The additive effect refers to the effect of a combination of antimicrobials, where the effect of the combination is equal to that of the sum of the effects of the individual components. Synergistic action of a combination of two antimicrobials is present if the effect of the combination exceeds the additive effects of an individual compound.⁶⁵

Statistical analysis

The results are expressed as mean ± standard deviation (SD) and were analysed using one-way analysis of variance (ANOVA), followed by the Mann–Whitney test using GraphPad Prism® (Graph Pad Software Inc. Version 5, San Diego, CA). A p value of less than 0.05 was considered to be statistically significant.

Conclusion

The results obtained in the present study confirm the enhanced antimicrobial activity of the PETIM-silver salts at low concentrations against both S. aureus and MRSA. These results also demonstrate that the PETIM-silver salt with the highest number of Ag⁺ ions, had the greatest antibacterial activity. At the same time these salts display low cytotoxicity, which paves the way to synthesise silver salts of higher generation PETIM dendrimers, and to evaluate them as effective antimicrobials against a range of sensitive and resistant micro-organisms. A combination of such antimicrobial agents increases the spectrum of organisms that can be targeted and circumvent the emergence of resistance in microorganisms. The synthesised G1 PETIM-silver salts in this study show potential for applicability in pharmaceutical as well as biomedical fields.

Acknowledgements

The authors are thankful to National Research Foundation of South Africa and University of KwaZulu-Natal for financial support. Ms Carrin Martin is acknowledged for proof reading the manuscript.

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