Fluorescent lactose-derived catanionic aggregates: synthesis, characterisation and potential use as antibacterial agents

Abstract

The spread of infections from multi-resistant bacteria in hospitals around the world is raising at an alarming rate. With the increasing capacity of bacteria to develop resistance to traditional antibiotics that target a particular metabolic pathway inside the cell, the use of nanoparticles as therapeutic agents is gaining importance because of their ability to attack bacterial membranes without evoking resistance. We have synthesized the catanionic surfactants Coum12–Coum18, based on fluorescent lactose-derivative organic salts using low-cost starting materials. In water, they self-assemble spontaneously to form stable aggregates at a physiological pH. The antibacterial properties of Coum12–Coum18 were investigated towards multi-drug-resistant Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Compound Coum18 was found to have both a bacteriostatic and a bactericidal activity towards Gram-positive bacteria, although the values of both the MIC and MBC (32 μg mL⁻¹) suggest only a topical use of the molecule. The valuable results that were found provide a challenging task for further investigation aimed at the development of this class of antibacterial drugs. In vitro fluorescence microscopy gave insight into the interaction between the aggregates and the cellular membranes on HeLa and CHO cells.

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Introduction

Antibiotic use and misuse have caused diffuse resistance¹ with the result that nearly half the deaths from clinical infections in developed countries are associated with multi-drug-resistant bacteria.^2,3 Because antibiotic resistance occurs as part of a natural process in which bacteria evolve, it can be slowed down but not stopped, and it is now recognized as one of the greatest threats to human health. It should also be noted that, despite the need for new antibiotics, the rate of development of new effective molecules is in decline.⁴ Therefore, the need of novel classes of antibiotics to face more and more resistant bacteria is of great importance. However, the cost associated with the development, and the moderate interest of the pharmaceutical companies in the last 20 years to design profitable drugs for the treatment of chronic diseases has greatly reduced the antibiotic discovery process.^4,5 One of the most promising strategies for the development of new classes of antibiotics is the use of nanosized materials and of nanoparticles in particular.^6–8 Generally speaking, the advantage of this approach arises from the direct interaction with microbial cell membranes/walls and key proteins/enzymes that can both inhibit pathogen growth and/or induce cell death through mechanisms different from classic antibiotics. In addition, the size, shape, and chemical characteristics of nanomaterials may be easily modified to facilitate these molecular interactions, optimizing their ultimate action. In the literature, different mechanisms have been described so far to explain how nanomaterials overcome microbial resistance depending on the type of systems proposed.^9–11 In this wide panorama however, to the best of our knowledge, “catanionic” aggregates have never been investigated. This class of compounds can be easily prepared by mixing a cationic and an anionic surfactant. In water, they self-assemble spontaneously to form various microstructures spanning from vesicles, helicoidal fibers, liquid crystals, or icosaedra, just to cite a few.^12,13 They can also form highly stable ionic liquid (IL)-in-water emulsions¹⁴ and can be used to encapsulate fluorescent probes.¹⁵ In particular, catanionic sugar-based amphiphiles are especially appealing because the presence of a sugar moiety should enhance the biocompatibility of these supramolecular assemblies. Rico-Lattes et al. have reported various examples of sugar-derived catanionic vesicles that can be used for drug delivery¹⁶ and they have also proved that they can spontaneously interact with pure lipid systems, used as a cell membrane model, thus emphasizing their use for enhanced direct cytosolic drug delivery.¹⁷ More recently, Blanzat et al. have described the interaction mechanisms between lactose-derived catanionic vesicles and CHO cells¹⁸ and their potential use for photodynamic therapy.¹⁹ The same group also reported an example of a fluorescent catanionic sugar surfactant able to assemble in aqueous solution to form helices and tubules.²⁰ However, the fluorescence properties of these types of assemblies and their potential use as probes for in vitro cell imaging have never been explored, as far as we know. Starting from these results, we report herein the synthesis of a series of new catanionic lactose surfactants, obtainable both in large quantities and at low cost, and featuring a fluorescent coumarin fragment. We discuss their physical chemical and photophysical characterization and their antibacterial activities towards multi-drug-resistant Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Moreover, in vitro fluorescence microscopy was performed on two different cell lines (HeLa and CHO) to highlight the interactions between the cell membranes and the proposed compounds.

Results and discussion

Synthesis and characterization of catanionic surfactants Coum12–Coum18

Compounds Coum12, Coum14, Coum16 and Coum18 (they will be indicated as Coum12–Coum18 in the text) were synthesized as shown in Scheme 1, using a modified literature synthesis.²¹ D-Lactose was first reacted with alkylamine NH₂–(CH₂)_n–CH₃ (n = 11, 13, 15 or 17) to form alkyllactosylamines 1a–1d. NaBH₄ was used as a reduction agent to obtain the reduced alkyllactosylamines 2a–2d. The products were then reacted with the carboxylic coumarin derivative 11-((2-oxo-2H-chromen-7-yl)oxy)undecanoic acid 3 to form the four catanionic surfactants Coum12, Coum14, Coum16 and Coum18 bearing an alkyl chain containing 11, 13, 15, and 17 carbon atoms, respectively. The ion-pair structures of Coum12, Coum14, Coum16 and Coum18 were confirmed via ¹H NMR, ¹³C NMR and IR spectroscopy (see ESI for synthetic details and for a comparison of IR spectra, Fig. S1†).

Scheme 1 Reaction scheme adopted for the synthesis of the catanionic fluorescent surfactants Coum12, Coum14, Coum16 and Coum18, n = 11, 13, 15, and 17 respectively.

When the organic salts Coum12–Coum18 were suspended in water at 25 °C, bluish, stable suspensions were obtained, indicating the establishment of a colloidal suspension. To investigate their surfactant characteristics, we measured the change of surface tension occurring in water solutions upon increasing the concentration of these newly synthesized molecules. The graphs shown in Fig. 1a–d confirm that Coum12, Coum14, Coum16, and Coum18 exhibit surface-active properties, as shown by the lowering of the water surface tension. In the first part of the curve, the catanionic associations show the classical adsorption behavior at the air–water interface leading to a rapid decrease in the surface tension until a break occurs in the curve at log C = −4.51, −4.60, −4.83, and −5.05 for Coum12, Coum14, Coum16 and Coum18, respectively. After this point, the surface tension remains constant. The change in the slope of the curve is interpreted as the formation of self-assembled aggregates.

Fig. 1 Surface tension (TS) vs. log C (M) of (a) Coum12, (b) Coum14, (c) Coum16 and (d) Coum18 in water at 25 °C.

The CAC (critical aggregation concentration) for the four compounds are reported in Table 1, along with their ζ-potential. As expected, the CAC decreases while the hydrophobic chain length increases (Coum12 > Coum14 > Coum16 > Coum18). Alongside the same series, we also detected a marked increase in the (negative) ζ-potential. The ζ-potential reflects the net charge on the aggregates’ surface and accounts for their electrostatic stability. Indeed, the larger the magnitude of the ζ-potential the greater the repulsive force experienced by the aggregates and the smaller the probability that they flocculate/coalesce.²² Accordingly, Coum18 forms the most stable colloidal suspension within the investigated series.

Table 1 Critical aggregation concentration (CAC, M) and ζ-potential (mV) of Coum12–Coum18 in water at 25 °C

Compounds	Coum12	Coum14	Coum16	Coum18
CAC	3.1 × 10^−5	2.5 × 10^−5	1.4 × 10^−5	1.0 × 10^−5
ζ-Potential	−35	−40	−45	−60

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Antibacterial activity of Coum12–Coum18

The colloidal dispersions in water of the catanionic aggregates of Coum12–Coum18 were tested for their antibacterial activity (see ESI† for experimental details). None of the examined compounds show any activity towards Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). This is probably due to the dimension of the aggregates, which did not allow the passage through the external membrane. Remarkably, some activity was observed on Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis. In particular, as shown in Table 2, the most active compound was Coum18 with a minimal inhibitory concentration (MIC) and a minimal bactericidal concentration (MBC) of 32 μg mL⁻¹.

Table 2 Values of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) in μg mL⁻¹ for Coum12–Coum18 and precursors 2d and 3

Compounds	Bacteria
	Staphylococcus aureus		Enterococcus faecalis
	MIC	MBC	MIC	MBC
Coum12	128	256	>512	>512
Coum14	64	>512	64	128
Coum16	32	>512	32	>512
Coum18	32	32	32	32
2d	64	64	32	64
3	64	>512	128	256

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The antibacterial activity of the two separated components, namely the alkyllactosylamine 2d and the carboxylic coumarin derivative 3, was also evaluated. The values for both the MIC and the MBC were higher than those observed for Coum18, suggesting a possible synergic effect when the two sub-units are interacting in the catanionic surfactant.

Coum16 was also active with a MIC value of 32 μg mL⁻¹ towards the two examined bacterial strains, however it did not show any bactericidal activity. Coum14 and Coum12 were even less active. These results suggest that the longer the alkyl chain of the surfactant the higher the activity. However, a MIC of 32 μg mL⁻¹ is considered high and suggests a topic use of the formulation.

As Coum18 was the most active compound in the series, we decided to further investigate the nature of the aggregates formed using this surfactant, its photophysical properties and its interaction with the cell membranes.

Morphological characterization of Coum18

The morphological characterization of the self-assembled aggregates originated when Coum18 was dispersed in water and was performed via cryo-transmission electron microscopy (TEM) experiments on a 0.2% wt formulation. Dispersions of Coum18 in water were found to be composed of a mixture of vesicular and tubular structures (Fig. 2). Vesicles ranged in diameter from 100 nm to above 1 μm. They were either isolated or associated in clusters. The tubes were straight, presenting a uniform diameter of about 65 ± 5 nm (mean ± S.D., n = 100) and variable length, up to several μm.

Fig. 2 Cryo-TEM image of a 0.2% wt dispersion of Coum18 in water. Representative images of the vesicular and tubular structures. For clarity, several vesicles and tubes are indicated with a black arrow and a white arrow, respectively. The white asterisks point to areas of the supporting perforated carbon net. Scale bars: 500 nm.

Photophysical properties of Coum18

The UV-vis spectrum of Coum18 in water (see ESI, Fig. S2†) shows an absorption band at 324 nm (ε = 13 000 M⁻¹ cm⁻¹). The photophysical properties of a dispersion of Coum18 in water (1.0 × 10⁻⁷ M) were tested using fluorescence spectroscopy. As shown in Fig. 3 Coum18 presents an emission band at 394 nm (black curve) and an excitation band at 324 nm (red curve). The quantum yield, calculated using quinine sulphate in H₂SO₄ 0.5 M, was found to be 0.352.

Fig. 3 Excitation (red curve) and emission (black curve) spectra of Coum18 (1.0 × 10⁻⁷ M) in water at pH 7.5.

The effect of the concentration of the surfactant on the formulation fluorescence was evaluated in the concentration range 2.0 × 10⁻⁹ M to 1.4 × 10⁻⁵ M. As shown in Fig. 4a a linear increase in fluorescence emission occurred for concentration values under the CAC (1.0 × 10⁻⁵ M), then a plateau was observed at around 5.0 × 10⁻⁵ M followed by a decrease in luminescence intensity at higher concentrations probably due to a self-quenching effect caused by the aggregation (Fig. 4b).

Fig. 4 Plot of the fluorescence intensity vs. concentration of Coum18 in water: (a) concentration range between 2 × 10⁻⁹ M to 3 × 10⁻⁷ M; and (b) concentration range between 4 × 10⁻⁷ M to 1.5 × 10⁻⁴ M.

Interaction with biologically relevant metal cations and anions

In order to understand whether the fluorescence properties of Coum18 were affected by the presence of biologically relevant metal cations and anions, we tested the ability of Coum18 in water to act as a fluorescence sensor for both metal cations and anions (see ESI, Fig. S3 and S4†). No significant fluorescence quenching or enhancement was observed in the presence of essential metal cations such as Na⁺, K⁺, Mg²⁺, Zn²⁺, or anions as Cl⁻, F⁻, NO₃⁻, hydrogen phosphate (H₂PO₄⁻) or hydrogen pyrophosphate (HPpi³⁻).

Live cell imaging

Taking advantage of the presence of the fluorescent coumarin fragment, we used fluorescence microscopy to investigate the internalization of the fluorescent aggregates of Coum18 in the cells using two different cell lines, namely HeLa and CHO (see ESI† for details). The cells were incubated with 1 mM Coum18 in water for 2 h. Before the observations, the cells were washed twice with DMEM to remove the excess Coum18 and loaded with nuclear stain Hoechst 33258. We observed that the strongly bound aggregates of Coum18, in spite of the extensive washing with the culture medium, stayed attached to cell plasma membrane. No fluorescence was detected inside cell cytoplasm implicating that internalization of the nanoparticles did not take place. In the overlay of phase contrast and fluorescence, the aggregates can be clearly identified as green fluorescent spots adhering on the surface of both cell types (Fig. 5a).

Fig. 5 Living HeLa and CHO cells incubated for 2 h with 1 mM Coum18 in water: (a) merged images were obtained using different channels: phase contrast (to observe cell morphology), blue fluorescence (to detect HOE-labeled nuclei) and green fluorescence (reporting Coum18 adsorption at cell surface). Scale bar = 10 μm; and (b) quantification of Hoechst fluorescence. The data are expressed as mean ± SD from three independent experiments. Statistically significant differences are indicated by ***p < 0.001; *p < 0.05 versus untreated control cells.

In order to investigate the effect of nanoparticle adhesion to the plasma membrane, the cells were evaluated by measuring the fluorescence intensity of the Hoechst probe, used to detect changes in permeability of the plasma membrane²³ and estimate the amounts of cells undergoing apoptosis. Indeed, with Hoechst stain, healthy cells show weakly stained nuclei, whereas non-healthy cells are identified through an increased blue fluorescence of the nucleus and progressive appearance of condensed and fragmented chromatin, indicating the apoptosis process. When the evaluation of Hoechst fluorescence was assessed in the Coum18-loaded cells in comparison with the non-treated control cells, a significant increase in nuclear fluorescence was detected after 2 h of incubation time (Fig. 5b), although no evidence of chromatin condensation was observed. These results suggest that the aggregation process might prevent nanoparticle internalization at a short time, inducing changes in the plasma membrane. Plasma membrane and intracellular membrane permeabilization processes are well-known to be initiating events of a cell death pathway.

Conclusions

The fluorescent lactose-derivative organic salts Coum12–Coum18 can be prepared via an easy and efficient reaction pathway using low-cost starting materials. Physicochemical characterization showed that these molecules self-assemble in solution forming stable aggregates, particularly in the case of Coum18. Photophysical measurements demonstrated that aggregation causes a partial quenching of the fluorescence emission, while biologically essential metal cations and anions do not cause any significant changes in the luminescence properties of Coum18. Fluorescence microscopy experiments pointed out that the aggregates were not internalized by the HeLa or CHO cells and had an apoptosis effect. Moreover, the antibacterial properties of Coum12–Coum18 were investigated. Coum18 was found to have both a bacteriostatic and a bactericidal activity towards Gram-positive bacteria, although the values of both the MIC and MBC (32 μg mL⁻¹) suggest only a topic use of the molecule. Chemical modifications of the system could improve its efficacy and could bring the development of a new type of antibacterial drugs for many classes of Gram-positive bacteria resistant to classic antibiotics such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococci (VRE), whose diffusion is arising both in hospitals and in everyday life.

Acknowledgements

We would like to thank Regione Autonoma della Sardegna (CRP-59699) and Fondazione Banco di Sardegna for financial support.

Notes and references

1. J. M. Rolain, S. M. Diene, M. Kempf, G. Gimenez, C. Robert and D. Raoult, Antimicrob. Agents Chemother., 2013, 57, 592–596.
2. A. R. Coates, G. Halls and Y. Hu, Br. J. Pharmacol., 2011, 163, 184–194.
3. R. Watson, Br. Med. J., 2008, 336, 1266–1267.
4. N. Cassir, J. M. Rolain and P. Brouqui, Front. Microbiol., 2014, 5, 551.
5. B. Spellberg, J. H. Powers, E. P. Brass, L. G. Miller and J. E. Edwards Jr, Clin. Infect. Dis., 2004, 38, 1279–1286.
6. N. Beyth, Y. Houri-Haddad, A. Domb, W. Khan and R. Hazan, J. Evidence-Based Complementary Altern. Med., 2015, 2015, 246012.
7. M. J. Hajipour, K. M. Fromm, A. Akbar Ashkarran, D. Jimenez de Aberasturi, I. R. D. Larramendi, T. Rojo, V. Serpooshan, W. J. Parak and M. Mahmoudi, Trends Biotechnol., 2012, 30, 499–511.
8. R. Y. Pelgrift and A. J. Friedman, Adv. Drug Delivery Rev., 2013, 65, 1803–1815.
9. A. J. Friedman, J. Phan, D. O. Schairer, J. Champer, M. Qin, A. Pirouz, K. Blecher-Paz, A. Oren, P. T. Liu, R. L. Modlin and J. Kim, J. Invest. Dermatol., 2013, 133, 1231–1239.
10. H. H. Lara, N. V. Ayala-Núñez, L. C. I. del Turrent and C. R. Padilla, World J. Microbiol. Biotechnol., 2010, 26, 615–621.
11. D. O. Schairer, J. S. Chouake, J. D. Nosanchuk and A. J. Friedman, Virulence, 2012, 3, 271–279.
12. I. Rico-Lattes, M. Blanzat, S. Franceschi-Messant, É. Perez and A. Lattes, C. R. Chim., 2005, 8, 807–814.
13. C. Tondre and C. Caillet, Adv. Colloid Interface Sci., 2001, 93, 115–134.
14. A. Bettoschi, A. Bencini, D. Berti, C. Caltagirone, L. Conti, D. Demurtas, C. Giorgi, F. Isaia, V. Lippolis, M. Mamusa and S. Murgia, RSC Adv., 2015, 5, 37385–37391.
15. A. Bencini, F. Caddeo, C. Caltagirone, A. Garau, M. B. Hurstouse, F. Isaia, S. Lampis, V. Lippolis, F. Lopez, V. Meli, M. Monduzzi, M. C. Mostallino, S. Murgia, S. Puccioni, J. Schmidt, P. P. Secci and Y. Talmon, Org. Biomol. Chem., 2013, 11, 7751–7759.
16. S. Consola, M. Blanzat, E. Perez, J. C. Garrigues, P. Bordat and I. Rico-Lattes, Chem.–Eur. J., 2007, 13, 3039–3047.
17. C. Mauroy, P. Castagnos, M. C. Blache, J. Teissié, I. Rico-Lattes, M. P. Rols and M. Blanzat, Chem. Commun., 2012, 48, 6648–6650.
18. C. Mauroy, P. Castagnos, J. Orio, M.-C. Blache, I. Rico-Lattes, J. Teissié, M.-P. Rols and M. Blanzat, Mol. Pharm., 2015, 12, 103–110.
19. P. Castagnos, M. P. Siqueira-Moura, P. L. Goto, E. Perez, S. Franceschi, I. Rico-Lattes, A. C. Tedesco and M. Blanzat, RSC Adv., 2014, 4, 39372–39377.
20. M. Blanzat, S. Massip, V. Spéziale, E. Perez and I. Rico-Lattes, Langmuir, 2001, 17, 3512–3514.
21. M. Blanzat, E. Perez, I. Rico-Lattes, D. Prome, J. C. Prome and A. Lattes, Langmuir, 1999, 15, 6163–6169.
22. A. V. Delgado, F. González-Caballero, R. J. Hunter, L. K. Koopal and J. Lyklema, J. Colloid Interface Sci., 2007, 309, 194–224.
23. M. E. Lalande, V. Ling and R. G. Miller, Proc. Natl. Acad. Sci. U. S. A., 1981, 78, 363–367.

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Footnote

† Electronic supplementary information (ESI) available: experimental details on the synthesis of Coum12–Coum18 and their precursor, cryo-TEM, cell culture and live cell imaging, antibacterial activity; IR spectra; UV-vis spectrum of Coum18; and histograms describing the fluorescence response of Coum18 towards metal ions and anions. See DOI: 10.1039/c6ra02511k

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