Research Article pubs.acs.org/journal/ascecg
Development of Gold-Based Phototheranostic Nanoagents through a Bioinspired Route and Their Applications in Photodynamic Therapy Neeraj S. Thakur, Jayeeta Bhaumik,*,† Seema Kirar, and Uttam C. Banerjee* Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar - 160062, Punjab, India S Supporting Information *
ABSTRACT: Green synthesis of nanoparticles using phytoconstituents has been widely accepted. However, further applications for delivery, diagnosis, and therapy are not yet fully established. Thus, bioinspired nanomedicinal diagnostic agents have been developed with antioxidant, diagnostic, as well as therapeutic properties. The sustainable biological synthesis of fluorescent gold nanoparticles using Syzygium cumini fruit extract presents a simplified process for the production of surface-functionalized nanoparticles with good antioxidant potential. The gold-based phototheranostic nanoagents (PTNAs) were engineered by conjugating nanoparticles either with a photosensitizer (rose bengal or pyridyl porphyrin) or with an imaging agent (rhodamine B). The assembled PTNAs were revealed to possess good fluorescent properties and to generate singlet oxygen, and showed antimicrobial properties when irradiated with low-cost green LED light. All of the processes and properties (synthesis, antioxidant potential, percent conjugation, fluorescence, singlet oxygen generation, and antimicrobial photodynamic therapy) of PTNAs synthesized using S. cumini were characterized and compared with those synthesized using chitosan. The antimicrobial effect of photodynamic therapy using developed PTNAs was characterized by confocal laser scanning microscopy (CLSM). The development of bioinspired PTNAs described herein will find applications of photodynamic therapy for the treatment of cancer and microbial infection. KEYWORDS: Bioinspired nanomaterials, Photosensitizer, Photoluminescence, Singlet oxygen, Antimicrobial PDT
■
INTRODUCTION A theranostic nanoagent (TNA) combines the properties of therapy and diagnostic imaging.1 Emerging nanotechnology techniques offer prospects for the design and development of such agents for the delivery of molecules and act as a detection modality throughout the entire treatment regimen.2,3 Metal nanoparticles have enticed a great deal of speculation due to their fascinating properties, tunability in electronic and electrooptical properties, and the plausibility of increasing luminescence.4 However, for biomedical applications, gold nanoparticles (AuNPs) require inherent or added photoluminescence (PL) and/or therapeutic properties.5,6 Fluorescence of AuNPs increases because of the exhibition of the localized surface plasmon (LSP).7 The metal-enhanced fluorescence has also endowed many applications in biological sciences. PL from gold is due to radiative recombination and consequently interband electronic transitions between the 5d and 6sp electronic bands.8 However, the origin of fluorescence in biosynthesized nanoparticles has not been found out yet and has needed more elaborative research. Among various reported methods, the synthesis and functionalization of metal nanoparticles using plant extracts remain beneficial in terms of ease of preparation, economy, greenness, and sustainability.9−12 Phytochemicals present in the plant extract are mainly © 2017 American Chemical Society
responsible for the synthesis, functionalization, and added medicinal and antioxidant properties of nanoparticles.13−16 The reported strong reducing property of phytochemical contents present in Syzygium cumini fruit extract17 may have played a significant role in the synthesis of gold nanoparticles. The studies of S. cumini phytoconstituents described its antioxidant, antibacterial, antidiabetic, anti-inflammatory, antineoplastic, radioprotective, and chemopreventive properties.17−20 The functional groups (mainly hydroxyl) of these phytochemicals contribute in the surface functionalization of nanoparticles which makes them advantageous for conjugation.10 With the conjugation of a photosensitizer (PS) or an imaging agent on the surface of these nanoparticles, the additional properties could be introduced with the inherent medicinal or diagnostic properties. Photosensitizers (PSs) consist of various types of lightabsorbing chromophores, and can produce local cell killing when irradiated with a particular wavelength of light.21 The applications of PSs in tumor destruction21−24 (lung cancer, Barrett’s esophagus, bladder cancer, head and neck cancers, and Received: May 14, 2017 Revised: July 4, 2017 Published: July 17, 2017 7950
DOI: 10.1021/acssuschemeng.7b01501 ACS Sustainable Chem. Eng. 2017, 5, 7950−7960
Research Article
ACS Sustainable Chemistry & Engineering skin cancer) and bacterial infection25,26 (antibiofilm and treatment of dentin infection) have already been reported. The compounds which act as PSs are derivatives of tetrapyrrolic macrocycles (porphyrins, chlorins, phthalocyanines, naphthalocyanines, etc.) and nonporphyrinic PSs including rose bengal, methylene blue, acridine dyes, etc.21,23 However, insolubility of tetrapyrrolic macrocyclic PSs in aqueous media27 is the major hurdle for the delivery of such agents into the biological system. Many research projects are currently ongoing for the enhancement of the hydrophilicity of such agents by chemical modifications in their core structure.27−31 Although these modifications are beneficial for the increase of the hydrophilicity of the molecules, nevertheless, the photosensitizing properties cannot be compromised. We have already reported the synthesis of functionalized porphyrins and conjugation on the surface of nanoparticles to make them hydrophilic while keeping their photodynamic properties intact.32 Rose bengal (RB), a fluorescein analogue, at present is being used in various biomedical applications including photodynamic therapy.33,34 One of its parenteral formulations is in a phase III clinical trial for the treatment of locally advanced cutaneous melanoma.35 RB is a good PS which can be activated by green light (∼561 nm).36 The acid group conjugation of RB with a suitable moiety makes it useful in the development of multimodal theranostic agents. Rhodamine B (RhB) is a dye and is extensively used in various spectroscopic techniques.37 The conjugated RhB can be used as an imaging agent for the diagnosis of a particular disease at various delivery sites. In the present study, we developed multimodal gold nanoparticles as nanomedicinal diagnostic agents (theranostic nanoagents) using the fruit extract of Syzygium cumini. The study includes the following: the optimization of various conditions for the synthesis of AuNPs and their characterization; the determination of total flavonoid contents, antioxidant activities, and inherent fluorescence properties; the conjugation of diagnostic as well as therapeutic agents and their characterization. The developed TNAs were then investigated for PL properties (fluorescence quantum yield), singlet oxygen generation (singlet oxygen quantum yield), and antimicrobial photodynamic therapy (aPDT) using a green (500−570 nm) LED light source. Overall, this model system can act as a single source of phototheranostic nanoagents (PTNAs) with an added PL property derived from a sustainable bioinspired route using low-cost materials.
Figure 1. (a) Comparative UV−vis spectra of a HAuCl4 salt solution and gold nanoparticles which were synthesized using chitosan (ChAuNPs) and S. cumini fruit extract (ScAuNPs), respectively. Inset images 1, 2, and 3 represent the visual appearance of corresponding solutions of the HAuCl4, ChAuNPs, and ScAuNPs, respectively. (b) Optimization of various factors affecting the synthesis of AuNPs by S. cumini extract.
conjugation with other moieties due to the availability of a higher number of functional groups with an overall increased surface area. It is also well-known that the spherical shape of nanoparticles has the highest surface area compared to other shapes and hence has a higher conjugation efficiency.39 It is reported that the ratio of metal ion to plant extract in the reaction medium determines the size, shape, and yield of synthesized nanoparticles.12 To synthesize small and spherical nanoparticles with the highest yield using a bioinspired route (S. cumini plant extract), we have optimized the various reaction parameters. It was observed that the various factors promisingly influence the synthesis of the nanoparticles (Figure 1b). The increase of plant extract concentration in the reaction mixture boosted the rate of reaction up to a certain value, and thereafter resulted in a lower yield of nanoparticles (Figure S1a in the SI). The S. cumini mediated biosynthesis of AuNPs (ScAuNPs) suggested that lower pH favors the forward reaction toward nanoparticle synthesis, while an increase of pH shifted the UV−vis spectra toward the right with decreasing intensity. The bathochromic shift of the spectra indicated larger nanoparticles, and the hypochromic effect represented the lower yield of nanoparticles (Figure S1c in the SI). The reaction temperature at 30 °C enhanced the synthesis of smaller ScAuNPs while a further increase of temperature triggered a bathochromic shift and a hypochromic effect in the UV−vis spectra (Figure S1d in the SI). Finally, the optimal conditions for the synthesis of a colloidal solution of gold nanoparticles were established with a 12 h duration reaction carried out in an incubator shaker at 200 rpm in 20 mL of reaction mixture (400 μM HAuCl4, 125 μL of extract, pH 3.5, and 30 °C; Figure 1b, and Figure S1 in the SI). Characterization of Gold Nanoparticles. The characterization of AuNPs is essential for the identification of their physical and morphological properties. The benchmark
■
RESULTS AND DISCUSSION Bioinspired Synthesis of Gold Nanoparticles (AuNPs) Using Plant Extract. Biological methods have the added advantage of the acquisition of functionalized nanoparticles over chemical processes, which require multiple steps of synthesis along with the use of harsh reagents. Phytochemicals present in the reaction medium are responsible for the reduction of gold ions to form AuNPs.10 It is evident from Figure 1a that the color of the reaction mixture changed from pale yellow to dark magenta indicating the formation of AuNPs in the solution (Figure 1a3). The color change was due to the unique feature of the metallic nanoparticles as they emit characteristic electromagnetic radiation in the visible light region because of the phenomenon known as “surface plasmon resonance”.38 The size, shape, and yield of nanoparticles play significant roles in the development of such multimodal therapeutic and diagnostic nanoagents. Notably, the smaller size of the nanoparticles increases the possibility of higher 7951
DOI: 10.1021/acssuschemeng.7b01501 ACS Sustainable Chem. Eng. 2017, 5, 7950−7960
Research Article
ACS Sustainable Chemistry & Engineering
Figure 2. (a) Schematic presentation of the synthesis of gold nanoparticles using S. cumini fruit extract. (b) Characteristic TEM image of ScAuNPs at 100 nm scale and its corresponding enlarged view showing tiny multinuclear gold nanocrystal arrangements. (c) ScAuNPs at 50 nm scale. (d) TEM image of ChAuNPs at 50 nm scale. (e) TEM image of ChAuNPs at 100 nm scale and its enlarged view showing a single crystal spherical nanoparticle.
580 nm are used in the characterization of Pyr-Por@ChAuNPs > RB@ChAuNPs > RhB@ScAuNPs > RhB@ChAuNPs > ScAuNPs. From the results of PL studies of all developed gold nanoprobes, it can be concluded that bioinspired nanotheranostics can be potentially used for image-guided delivery and other biomedical applications. The origin of fluorescence in the gold nanoclusters (
