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Reborn from the Ashes: Turning Organic Molecules to Antimicrobial Carbon Quantum Dots Scott G. Harroun,† Jui-Yang Lai,∥,⊥,§ Chih-Ching Huang,‡,Δ,# Shou-Kuan Tsai,‡ and Han-Jia Lin*,‡,Δ Department of Chemistry, Université de Montréal, 2900 Boulevard Édouard-Montpetit, Montréal, Québec H3C 3J7, Canada Institute of Biochemical and Biomedical Engineering, Chang Gung University, 259 Wenhua 1st Road, Taoyuan, 33302, Taiwan ⊥ Department of Ophthalmology, Chang Gung Memorial Hospital, 5 Fuxing Street, Taoyuan, 33305, Taiwan § Department of Materials Engineering, Ming Chi University of Technology, 84 Gungjuan Road, New Taipei City, 24301, Taiwan ‡ Department of Bioscience and Biotechnology, National Taiwan Ocean University, 2 Beining Road, Keelung, 20224, Taiwan Δ Center of Excellence for the Oceans, National Taiwan Ocean University, 2 Beining Road, Keelung, 20224, Taiwan # School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung, 80708, Taiwan †
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ABSTRACT: Using polyamines as the initial organic raw material and by applying simple pyrolysis methods, super cationic carbon quantum dots (CQDs) can easily be made. Since polyamines are natural products and the synthesis procedure is green, these polyamine-derived CQDs display low toxicity and high biocompatibility but possess high antibacterial activity. In addition, polyamine-derived CQDs display other unique properties, such as facilitation of wound healing and passage through the tight junction, which make them a very promising bactericide in future clinical applications.
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search for new materials that exhibit low toxicity toward human cells while possessing high activity as bactericides. Carbon nanomaterials, including carbon nanotubes (CNT), fullerenes, graphene, graphene-derived materials, carbon quantum dots (CQDs, also sometimes called C-Dots), and so on, have low cost, are easy to functionalize, possess high biocompatibility and low cytotoxicity, and have many useful properties, such as photoluminescence, photothermal, and photocatalysis.3 These properties make carbon nanomaterials very attractive as potential materials to develop new bactericides. Single-wall monolayer carbon nanotubes (SWCNT) and fullerenes were found to bind to bacterial cell membrane lipids with their hydrophobic surfaces, destroy the integrity of the membrane, and even block the respiratory chain, thus causing subsequent cell death.4 Surfaces functionalized with hydroxyl and carboxyl groups, as well as longer SWCNTs (>5 μm), show better antimicrobial activity.5 After functionalization with hydrophilic groups, it not only improves the solubility but also allows for their application as photosensitizers in photodynamic therapy (PDT).6 Graphene, graphene oxide, and reduced graphene oxide have been applied as antibacterial materials.7 Their antimicrobial mechanism arises mainly from their ability to cause membrane stress and produce ROS. In addition, graphene and its derivatives have high specific surface areas, high abundant reactive surface functionalities, and outstanding mechanical strength. They can also form nanocomposites with other antimicrobial nanomaterials, such as silver nanoparticles (Ag NPs).8 These materials tend to exhibit synergistic effects and have improved antimicrobial activity. For practical applications,
hroughout history, infectious bacteria have been one of the greatest threats to human health. Prior to the discovery of antibiotics, people had tried to produce bactericides from natural herbal extracts, as well as by further processing of these extracts. For example, ancient Chinese people knew to use ash of Mugwort (Artemisia argyi) as a sterilizer and applied it on wound dressings. The invention of antibiotics led to humans having very specific weapons against infectious bacteria for the first time. Notwithstanding the numerous effective antibiotic treatments developed over the years, this advantage may not last indefinitely. Because of antibiotic overuse, the rapid emergence of drug-resistant bacteria, once again, threatens human health. With fewer effective antibiotics currently in our armory, it is now imperative to find new weapons. The progression of nanotechnology has led to increased hope for human beings in the face of this life-threatening challenge. Metal and metal oxide nanoparticles, such as silver (Ag), iron oxide (Fe3O4), titanium oxide (TiO2), copper oxide (CuO), and zinc oxide (ZnO), were found to have good bactericidal activities.1 Most of these nanoparticles are able to produce a large number of reactive oxygen species (ROS), release sterilizing metal ions, or destroy the integrity of the cell membrane. Compared with antibiotics, which specifically interrupt one part of the bacterial physiological mechanisms from the inside, nanomaterials usually interact with bacteria from the outside; moreover, they can have multiple antimicrobial effects. Therefore, nanomaterials are efficient bactericides toward which bacteria are less likely to develop resistance.2 Nevertheless, since metal and metal oxide nanoparticles may release metal ions in vivo, which may be toxic for human cells, the clinical applications of metal and metal oxide nanoparticles are restricted. Accordingly, scientists continue to © XXXX American Chemical Society
Received: September 11, 2017
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DOI: 10.1021/acsinfecdis.7b00150 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
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without a coupling agent. The size of the as-synthesized SpdCQDs is around 4.6 nm, which is a little larger than normal ammonium citrate CQDs. Moreover, the Spd-CQDs have a very high density of positive charges on the surface, while the surface charge of CQDs produced from only ammonium citrate is negative. By using FTIR and MALDI-TOF, we demonstrated that spermidine is still present on the surface of the Spd-CQDs. We have found that Spd-CQDs very efficiently killed nonmultidrug-resistant Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa, as well as multidrug-resistant bacteria, namely, methicillin-resistant S. aureus (MRSA). The minimal inhibitory concentration (MIC) value of Spd-CQDs was 25 000-fold lower than that of spermidine and 10-fold lower than Ag NPs, indicating their promising antibacterial characteristics. By using TEM and SEM, we observed that Spd-CQDs cause significant damage to the bacterial membrane. We also used different polyamines and tried different synthesis conditions. It was found that the CQDs with more positive charges on the surface showed stronger antibacterial activity. In addition, SpdCQDs did not evoke much ROS, and therefore, the antimicrobial activity of Spd-CQDs is attributed to damage to the integrity of the bacterial cell membrane. Despite their lethalness to bacteria, in vitro cytotoxicity and hemolysis analysis indicated that Spd-CQDs have high biocompatibility with mammalian cells. Furthermore, we conducted in vivo MRSA-infected wound healing studies in rats. When Spd-CQD was used as a wound dressing material, it showed faster healing, more keratinocytes migration, better epithelialization, and significant collagen fiber formation. Recently, we developed a new synthesis method, whereby polyamine powder was directly pyrolyzed through a simple, one-step dry heating treatment. This method produced CQDs with a positively charged surface, photoluminescence, and great antimicrobial activity.16 Additionally, the solubility and yield of the CQDSpds variant prepared from spermidine were much higher than the other polyamines we tested. The MIC of CQDSpds is similar to that of Spd-CQDs. Experimental results suggested that the supercationic CQDSpds, with its small size (diameter ca. 6 nm) and highly positive charge (zeta potential ca. +45 mV), cause severe disruption of the bacterial membrane, which may explain the possible mechanism behind the antibacterial activity of CQDSpds. In addition to in vitro cytotoxicity and hemolysis tests, we also applied CQDSpds on the rabbit cornea, which is one of the most sensitive tissues to foreign bodies. In vivo morphologic and physiologic cornea change evaluations demonstrated the good biocompatibility of CQDSpds, while on the other hand, Ag NPs caused corneal inflammation. To demonstrate its practical application, we conducted in vivo S. aureus-induced bacterial keratitis (BK) studies in rabbits. To our surprise, simple topical ocular administration of CQDSpds can efficiently treat deep corneal infection of S. aureus. Additionally, CQDSpds demonstrated comparable treatment efficiency to that of commercial sulfamethoxazole (SMX), despite the eye drop formulation being at a 10-fold higher concentration. Further study indicated that CQDSpds could temporarily induce the opening of the tight junction of corneal epithelial cells, thereby leading to the great antibacterial treatment of BK. Our results suggest that both Spd-CQDs and CQDSpds are promising antibacterial candidates for clinical applications in treating bacterial infections and even drug-resistant bacteria-induced infections.
these materials can also be integrated into paper and cotton fabrics and still exhibit their ability to kill bacteria. Graphene quantum dots (GQDs) are usually manufactured by graphite electrolysis and hydrazine reduction.9 They have graphene lattices inside and contain from one to ten or more layers of graphene sheets. The size of GQDs is tunable and usually from a few to 100 nm in the lateral dimension. GQDs themselves do not have much antibacterial capacity, but photoexcited GQDs can produce lots of ROS in order to kill bacteria.10 CQDs, which resemble GQDs, are more diverse in both production methods and starting raw materials.11 There are two main routes to produce CQDs: bottom-up and topdown synthesis.11 The bottom-up approach includes pyrolysis, combustion, or hydrothermal methods, which build up nanomaterials from small organic molecular precursors, while the top-down route is based on cutting small sheets via physical, chemical, or electrochemical techniques into nanomaterials with a desired size. Besides the choice of synthetic route, the initial material and the follow-up functionalization procedures also contribute to the unique characteristics of each type of CQDs. Hence, it is also possible to synthesize antimicrobial materials from CQDs. Recently, Liu et al. used the antibiotic metronidazole to produce CQDs by a simple hydrothermal process.12 In this way, they synthesized a type of CQDs that are able to detect obligate anaerobes via photoluminescence and are simultaneously effective against these bacteria. Thakur et al. conjugated ciprofloxacin hydrochloride, an antibiotic, onto the surface of CQDs made from gum arabic to produce Cipro@CQDs by a microwave-assisted synthesis method.13 Thus, CQDs may also serve as a drug carrier. The results of their study indicated that Cipro@CQDs showed enhanced antimicrobial activity against tested Gram-positive and Gram-negative bacteria. Elsewhere, it has also been shown in animal studies that, unlike some metal and metal oxide nanoparticles, CQDs are rapidly cleared from tissues.14 Our groups were the first to use polyamines as the raw material to synthesize CQDs.15,16 Polyamines, including putrescine, spermidine, and spermine, are natural substances that exist in nearly all organisms. At the cellular level, they play many important roles, such as regulation of the cell cycle, gene transcription, protein translation, and ion channel activity. All polyamine molecules contain multiple positive charges, facilitating their interaction with negatively charged phospholipids on cell membranes and nucleic acids inside cells. With polyamine binding, these molecules may be protected from ROS attacks.17 The physiological concentration of polyamines in human tissue is usually at the millimolar level, in which the survival rate of human cells is not affected by polyamines.18 However, polyamines in such conditions may have a detrimental effect on the growth of bacteria, especially spermine. It was found that spermine may interfere with gene regulation, metabolic pathways, and the formation of biofilms in many kinds of bacteria.19 Since the formation of biofilms is a key step for bacteria to infect a host and to prevent attacks from antibiotics, spermine was also found to increase the bactericidal effect of antibiotics.20 Our first green chemical approach involved using a two-step method to synthesize spermidine-capped fluorescent carbon quantum dots (Spd-CQDs).15 They were synthesized by the pyrolysis of ammonium citrate in the solid state and then functionalized with spermidine by a simple heating treatment B
DOI: 10.1021/acsinfecdis.7b00150 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
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Photodynamic antibacterial effect of graphene quantum dots. Biomaterials 35 (15), 4428−4435. (11) Zheng, X. T., Ananthanarayanan, A., Luo, K. Q., and Chen, P. (2015) Glowing graphene quantum dots and carbon dots: properties, syntheses, and biological applications. Small 11 (14), 1620−1636. (12) Liu, J., Lu, S., Tang, Q., Zhang, K., Yu, W., Sun, H., and Yang, B. (2017) One-step hydrothermal synthesis of photoluminescent carbon nanodots with selective antibacterial activity against Porphyromonas gingivalis. Nanoscale 9 (21), 7135−7142. (13) Thakur, M., Pandey, S., Mewada, A., Patil, V., Khade, M., Goshi, E., and Sharon, M. (2014) Antibiotic conjugated fluorescent carbon dots as a theranostic agent for controlled drug release, bioimaging, and enhanced antimicrobial activity. J. Drug Delivery 2014, 282193. (14) Huang, X., Zhang, F., Zhu, L., Choi, K. Y., Guo, N., Guo, J., Tackett, K., Anikumar, P., Liu, G., Quan, Q., Choi, H. S., Niu, G., Sun, Y.-P., Lee, S., and Chen, X. (2013) Effect of injection routes on the biodistribution, clearance, and tumor uptake of carbon dots. ACS Nano 7 (7), 5684−5693. (15) Li, Y.-J., Harroun, S. G., Su, Y.-C., Huang, C.-F., Unnikrishnan, B., Lin, H.-J., Lin, C.-H., and Huang, C.-C. (2016) Synthesis of selfassembled spermidine-carbon quantum dots effective against multidrug-resistant bacteria. Adv. Healthcare Mater. 5 (19), 2545−2554. (16) Jian, H.-J., Wu, R.-S., Lin, T.-Y., Li, Y.-J., Lin, H.-J., Harroun, S. G., Lai, J.-Y., and Huang, C.-C. (2017) Super-cationic carbon quantum dots synthesized from spermidine as an eye drop formulation for topical treatment of bacterial keratitis. ACS Nano 11 (7), 6703−6716. (17) Rider, J. E., Hacker, A., Mackintosh, C. A., Pegg, A. E., Woster, P. M., and Casero, R. A., Jr (2007) Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide. Amino Acids 33 (2), 231−240. (18) Igarashi, K., and Kashiwagi, K. (2010) Modulation of cellular function by polyamines. Int. J. Biochem. Cell Biol. 42 (1), 39−51. (19) Karatan, E., and Michael, A. J. (2013) A wider role for polyamines in biofilm formation. Biotechnol. Lett. 35 (11), 1715−1717. (20) Kwon, D.-H., and Lu, C.-D. (2007) Polyamine effects on antibiotic susceptibility in bacteria. Antimicrob. Agents Chemother. 51 (6), 2070−2077.
From ancient wisdom to modern technology, which requires well-defined raw materials, accurate production control, and solid scientific evidence, these findings suggest that CQDs have much potential as antimicrobial materials. Moreover, our polyamine-derived CQDs also possess other unique properties, such as the promotion of wound healing and the ability to penetrate the tight-junction. CQDs may have stronger, or even very different, antibacterial characteristics compared to their raw organic starting material. Plenty of avenues remain for us to further study the applications of polyamine-derived antimicrobial CQDs, including a more detailed study of their sterilization mechanism, whether they can inhibit the formation of bacterial biofilms, whether they can produce synergistic effects with antibiotics, and the long-term effects of these materials on human health, before polyamine CQDs can be applied in clinical settings.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: 011-886-2-2462-2192 ext. 5507. Fax: 011-886-2-24622320. E-mail:
[email protected]. ORCID
Jui-Yang Lai: 0000-0002-9227-8549 Chih-Ching Huang: 0000-0002-0363-1129 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the support from Ministry of Science and Technology of Taiwan under contracts 104-2113-M-002-008MY3, 104-2628-M-019-001-MY3, 105-2627-M-019-001-MY3, and 105-2622-M-019-001-CC2.
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DOI: 10.1021/acsinfecdis.7b00150 ACS Infect. Dis. XXXX, XXX, XXX−XXX
