Bioinspired Nanotheranostic Agents: Synthesis, Surface

Article pubs.acs.org/journal/abseba

Bioinspired Nanotheranostic Agents: Synthesis, Surface Functionalization, and Antioxidant Potential Jayeeta Bhaumik,* Neeraj S. Thakur,† Pavan K. Aili,† Amit Ghanghoriya, Amit K. Mittal, 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: Bioinspired synthesis of nanomaterials is highly advantageous as a natural and cost-effective resource. Development of noble metal nanotheranostic agents was achieved through bioinspired synthetic routes. These biosynthesized nanoparticles were characterized by various analytical techniques including absorption spectroscopy, FTIR and electron microscopy (SEM and TEM). A large number of medicinal plants were screened, among which Potentilla f ulgens (PF, vajradanti) and Camellia sinensis (CS, green tea) were found to produce nanomaterials with higher yields. Plant (PF and CS) mediated metallic nanoparticles had added advantage of metal reduction and simultaneous phytochemical capping over chemically synthesized procedures, which require multiple reagents. Antioxidant potential of the nanomaterials was determined by in vitro antioxidant assays confirming substantial antioxidant properties, which was due to the presence of phytochemicals on the nanoparticle surface. Flavonoids and catechins on the nanomaterial surface served as the supplier of hydroxyl groups for further derivatization. The surface of the nanoparticles was engineered by conjugating imaging and therapeutic moieties, resulting in the formation of theranostic nanoagents. The multimodal agents were characterized and the extent of drug loading was determined to validate the efficacy of those nanoconjugates. These bioinspired multimodal nanoprobes can serve as essential diagnostic and therapeutic tools in ongoing biomedical research. KEYWORDS: metal nanoparticles, plant extract, drug loading, antioxidant, conjugation surface of the nanoparticles are coated.16 These phytochemicals mainly comprise flavonoids and phenolic compounds, which possess strong reducing properties. Additionally, the presence of functional groups (such as hydroxy, amine) make them useful for conjugation. Bioinspired theranostic agents can be developed through the following strategies: (1) selection and screening of plant extracts for the synthesis nanoparticles; (2) optimization of various physicochemical parameters for biosynthesis; (3) functionalization of the synthesized nanoparticles with the therapeutic and imaging agents; (4) characterization of nanoconjugates using various analytical techniques.

1. INTRODUCTION Nanoparticles within the size range of 1−100 nm can be majorly utilized in biomedical applications.1 Among different synthetic routes to nanoparticle formation, bioinspired methods are considered better compared to chemical routes because these avoid the use of toxic chemicals.2−4 Multimodal nanoparticles with large surface area can simultaneously incorporate therapeutic and imaging agents, for which nanomaterials can be termed as theranostic (therapeutic plus diagnostic) nanoagents.5−8 The biosynthesis of noble metal nanoparticles incorporated with therapeutic and imaging agents possess theranostic activities.9−12 These multifunctional nanotheranostic agents can be used in the treatment of cancer, microbial infection, and many other diseases.13−15 The antioxidant capability of the biosynthesized nanoparticle is dependent on the properties of phytochemicals with which the © XXXX American Chemical Society

Received: December 19, 2014 Accepted: May 5, 2015

A

DOI: 10.1021/ab500171a ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Scheme 1. Diagrammatic Representation of Bioinspired Synthesis and Surface Functionalization of the Theranostic Nanoagents

hydroxyethyl] piperazine-N-[2-ethansulfonic acid]) sodium salt [HEPES] (Sigma-Aldrich), 1-(3-(dimethylamino)propyl)3-ethylcarbodiimidehydrochloride [EDC], N-hydroxysuccinimide [NHS] (Spectrochem). 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) [ABTS] and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Life Sciences (St. Louis, MO, USA). Potassium persulfate was purchased from LOBA Chemie Pvt. Ltd. (Mumbai, India). Reference standard ascorbic acid was procured from HiMedia Laboratories (Mumbai, India). HPLC grade methanol was obtained from Merck (Germany). Fresh stock solutions were prepared before each analysis in deionized water (Veolia Water Technologies, UK). The thermo multiskan spectrum reader (Thermo Fisher Scientific Inc., Waltham, MA USA) was used to measure the absorbance of samples and standards in 96 multiwell plates. Incubator Shaker (Innova 4230), U-3010 Spectrophotometer (HITACHI), and UV−vis spectrophotometer (Lab India) were used for the synthesis and characterization of nanoparticles. The size and potential of nanoparticles were estimated by Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Scanning electron microscopy (HITACHI, Japan) and transmission electron microscopy (FEI, USA) were used for the determination of size and shape of the synthesized nanoparticles. Nanoparticles were freeze-dried using lyophilizer (Allied frost, Delhi, India). The functional groups present on the surface of synthesized nanoparticles were confirmed by Fourier transform infrared spectroscopy (PerkinElmer). 2.2. Plants. Several plants were screened among which Camellia sinensis (Green tea) and Potentilla f ulgens (Vajradanti) were chosen for the synthesis of silver (AgNPs) and gold (AuNPs) nanoparticles. C. sinensis leaves were purchased from Reliance Market, S.A.S. Nagar and P. fulgens roots were provided by North-Eastern Hill University, Shilong and the root extract (dried) was prepared by the Department of Natural Products, NIPER, S.A.S. Nagar. 2.3. Preparation of Various Plant Extracts. A sample of 100 g of C. sinensis (green tea) leaf powder was extracted with methanol (200 mL) for 24 h using a Soxhlet apparatus. The extract was then concentrated using a rotavapor and the resulting dry powder was stored at 4 °C. This extract was further used in preparing aqueous stock solution of green tea and used in the synthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs). P. f ulgens extract was prepared by suspending 25 mg of the dried root extract in 25 mL of deionized water in a 100 mL flask and then incubated in a shaker for 6 h at 35 °C and 200 rpm. The extract was filtered and stored at 4 °C for further synthesis of AuNPs. 2.4. Biosynthesis of Silver and Gold Nanoparticles. The aqueous extract of C. sinensis was used for the synthesis of silver and gold nanoparticles. P. f ulgens was used in the synthesis of AuNPs. In order to synthesize nanoparticles these plant extracts were suspended in 25 mL of deionized water in different conical flasks, silver and gold salts were added to these solutions and the reaction mixtures were incubated at 35 °C (200 rpm) under dark condition. 2.5. Optimization of Various Physicochemical Parameters To Enhance the Yield of Biosynthesized Nanoparticles. 2.5.1. Optimization of the Concentration of the Plant Extract. The rate of synthesis as well as size and shape of the nanoparticles were controlled by the ratio of metal ions to reducing agents present in the plant extract. To synthesize AgNPs and AuNPs by C. sinensis, we prepared 40 mg/mL stock solution. Different amounts of stock extract

Rhodamine B is frequently used in diagnostic imaging due to its distinct fluorescent properties. Additionally, rhodamine B contains a carboxyl functionality which can be helpful in conjugating it to nanoparticles containing counter functionalities (e.g., amine or hydroxyl group). Rose bengal is a commercially available photosensitizer which is commonly used in photodynamic therapy for the treatment of cancer and microbial infection.17,18 Rose bengal (RB) also contains a carboxyl functionality and thus it can be attached on a nanoparticle surface.19 Use of RB in photodynamic therapy (PDT) of cancer cells and use of conjugated RB in antimicrobial photodynamic treatment (APDT) has been reported.20 However, to the best of our knowledge, surface conjugation on biosynthesized noble metal nanoparticles (silver and gold) either with RB or rhodamine B to construct theranostic nanoagents are not yet reported. Surface-functionalized silver nanoparticles can be used in antimicrobial and anticancer assays.21 Surface-functionalized gold nanoagents can be used in further photothermal therapy (PTT) studies or in combination with PDT for the treatment of cancer.22 Here we report, simultaneous bioinspired synthesis of noble metal nanoparticles followed by surface engineering to construct nanotheranostic agents. This procedure avoids the use of toxic chemicals since the plant extracts are used as the sole source of active ingredients in entire nanomaterial formation (reduction of metal, aggregation and stabilization). The main advantage of using plant extracts is that, they can be used as reducing and stabilizing agents simultaneously. The phytochemicals present in the plants used in nanoparticle preparation are mainly consisted of polyphenols and flavonoids which are rich in hydroxyl moieties. Thus, any potential molecule (a drug or an imaging agent) with counter functionality (such as −COOH group) can be reacted to form an ester bond and attach on the phytochemical surface. Due to the presence of carboxylic acid groups in rhodamine B and rose bengal both could be successfully conjugated to the surface of bioinspired nanoparticles by the aid of EDC (or EDC/NHS) mediated coupling (Scheme 1). The nanomaterials were completely characterized by means of various spectroscopic techniques. Bioinspired nanoconstructs were further subjected to antioxidant assays to determine whether the phytochemicals retained their antioxidant properties after participation in the nanoformulation. Drug loading on the surface of the nanoconjugates was also estimated by UV−vis spectroscopy and HPLC analysis. Overall, these bioinspired nanotheranostic agents can find potential applications in nanomedicine.

2. MATERIALS AND METHODS 2.1. Chemicals and Instruments. Silver nitrate and gold chloride were purchased from Sigma-Aldrich (Steinheim, Germany); dimethyl sulfoxide (Fisher, India), rose bengal (Sigma-Aldrich), (N-[2B


Article

ACS Biomaterials Science & Engineering (0.5 μL to 1 mL) were then diluted to 25 mL with deionized water to prepare reducing solutions of different concentrations. Silver salt was then added to each these dilutions and the reaction mixtures were incubated at 35 °C (200 rpm) in dark condition. To synthesize AuNPs from P. f ulgens, different volume of stock extract (10 μL to 1 mL) was diluted to 25 mL with deionized water to prepare reducing solutions of different concentrations. Gold salt was added to these solutions and the reaction mixtures were incubated at 35 °C (200 rpm) in dark condition. 2.5.2. Metal Ion Concentration Optimization. To optimize silver ion concentration, the flasks containing different concentrations of silver nitrate (0.2 to 10 mM) along with optimized concentration of plant extract were incubated at 35 °C (200 rpm) in dark condition. To optimize gold ion concentration the flasks containing different concentrations of gold chloride (0.1 to 1 mM) along with optimized concentration of plant extracts were incubated at 35 °C (200 rpm) in dark condition. 2.5.3. Temperature Optimization. To optimize incubation temperature, we incubated reaction flasks containing optimized concentrations of plant extracts and salt solutions at temperatures ranging from 25 to 65 °C under shaking conditions (200 rpm) in the dark. 2.5.4. pH Optimization. To optimize reaction pH, we incubated reaction flasks containing optimized concentrations of plant extracts and salt solutions having initial pH in the range of 3 to 9 at optimized temperatures under shaking condition (200 rpm) in dark. 2.5.5. Optimization of Total Reaction Time. To optimize the reaction time in terms of yield and properties of the nanoparticles, we collected samples at various time intervals (3, 6, 12, 18, 24, 30, 36, and 48 h) from the reaction mixture and subjected to further characterization. 2.6. Characterization of Nanoparticles. 2.6.1. UV−Visible Spectroscopic Analysis. UV−visible spectroscopy is a commonly used technique for the preliminary identification of nanoparticles formation. Different wavelengths in the range 300−800 nm are generally used for characterizing various metal nanoparticles in the size range of 2−100 nm. Spectrophotometric absorption measurements in the wavelength ranges of 400−450 and 500−550 nm are used in characterizing the AgNPs and AuNPs, respectively. 2.6.2. Zetasizer and Zeta Potential Analysis. Dynamic light scattering (DLS) measurements were performed with a fixed wavelength of 532 nm at 35 °C with 90° detection angle. To estimate the particle size and zeta potential, a dilute suspension of nanoparticle was prepared in deionized water and sonicated in order to remove aggregation at 35 °C for 30 min and subjected to Zetasizer analysis. 2.6.3. Scanning Electron Microscopy (SEM). SEM provides important information about the size and morphology of the nanoparticles. In SEM, an electron beam comes from a filament (tungsten) and is condensed by a condenser lens and then projected onto the sample by objective lens. This beam scans over the surface of the sample and various signals including photons and electrons are emitted from the sample surface. The interaction between the sample and beam produces different types of signals providing about the surface structure and morphology of the nanomaterials. The resolution of SEM is few nanometers to micrometers and it can operate at various magnifications, which can be easily adjusted. 2.6.4. Transmission Electron Microscopy (TEM). This technique involves irradiation of a very thin sample by a high-energy electron beam. The topographic information obtained by TEM in the vicinity of atomic resolution can be utilized for structural characterization and identification of various phases of nanomaterials, i.e. hexagonal, cubic or lamellar.19 Samples for high resolution transmission electron microscopic (HR-TEM) analysis were prepared by drop coating AgNP and AuNP solutions onto carbon coated copper TEM grids. The films on the TEM grids were later allowed standing for 2 min following which the extra solution was removed using a blotting paper and the grid was allowed to dry prior to the measurement. 2.6.5. Fourier Transform Infrared (FTIR) Spectroscopy. FTIR analysis of the synthesized nanoparticles were performed in order to confirm the functional groups present on to the surface of the nanoparticles. First, the nanoparticle reaction mixture was centrifuged

using an ultra centrifuge at 22000 × g for 15 min. The resulting pellet was then redispersed in deionized water. The entire process was repeated three times to remove excess salt, plant extract and other components which were not responsible for capping. Nanoparticles were then lyophilized using a freeze drier and the solid nanoparticles were analyzed by FTIR spectroscopy (PerkinElmer) at a resolution of 4 cm−1 by KBr pellet method. Simultaneous analysis of lyophilized plant extract was also done to compare the plant constituents, which were responsible for capping of the nanoparticles. 2.7. Antioxidant Assay. 2.7.1. ABTS Assay. Standard solutions of ascorbic acid (5 to 30 μg/mL) and nanoparticle (50 μg/mL) were separately prepared in deionized water. Nanoparticle solution (10 μL) was then added to a solution (100 μL) containing ABTS radical cations with vigorous shaking. Then resulting mixture was then incubated in dark for 6 min at room temperature and absorbance was recorded at 734 nm. Percent scavenging by nanoparticles was calculated with respect to negative control (100% ABTS cationic solution without test sample) using the following eq 1a. percentage scavenging (%) = ⎡⎣(Abscontrol − Abssample )/Abscontrol ⎤⎦ ·100

(1a)

Where Abscontrol is the absorbance of only ABTS cation radical solution and Abssample is the absorbance of ABTS cation radical solution after reaction with nanoparticles. Ascorbic acid (AA) [final concentration 0.25 to 1.5 μg/mL (10 μL AA + 100 μL ABTS solution +90 μL deionized water)] was used to make standard calibration curve for scavenging capacity and also used as positive control. By the use of the linear equation, EC50 value of standard and samples were determined.

2.7.2. DPPH Assay. Standard solutions of ascorbic acid (5 to 40 μg/ mL) and nanoparticles (10 μg/mL) were separately prepared in deionized water. Nanoparticle solution (100 μL) was then added to 100 μL of 200 mM methanolic DPPH radical ion solution with vigorous shaking. The reaction mixture was then incubated in dark for 30 min at room temperature and absorbance was recorded at 517 nm. Radical scavenging capacity of nanoparticles was expressed as percentage effect (E %) and calculated using the following eq 1b percentage scavenging (%) = ⎡⎣(Abscontrol − Abssample )/Abscontrol ⎤⎦ ·100

(1b)

Where Abscontrol is the absorbance of only methanolic DPPH radical solution and Abssample is the absorbance of DPPH radical solution after reaction with nanoparticle solution. Ascorbic acid [final concentration 1 to 8 μg/mL (40 μL AA + 100 μL DPPH solution +60 μL deionized water)] used to make standard calibration curve for scavenging capacity and used as positive control. To calculate IC50 value, different nanoparticle concentrations were used. By using the linear equation, we determined the IC50 value.

2.8. Surface Functionalization of Nanomaterials with Therapeutic and Imaging Agents. For the conjugation of biosynthesized nanoparticles, solutions of various nanoparticles (50 μg/mL), EDC (1 mg/mL), NHS (1 mg/mL) rhodamine B (5 mg/ mL) and rose bengal (5 mg/mL) were prepared in 50 mM HEPES buffer. Conjugation reactions were set up according to Table S9 (see Supporting Information), at 120 rpm in dark condition for 12 h. The reaction progress was monitored by absorption spectroscopy. After completion of the reaction, the nanoparticle-drug conjugate solutions were centrifuged at 10000 rpm for 30 min. Colored supernatants (containing fluorophores) were discarded from each reaction mixture and this procedure was repeated until clear supernatants were observed from each reaction mixture. The resulting pellets were then redispersed in the buffer and analyzed using UV−vis spectroscopy and HPLC. For further confirmation of the conjugation reaction, the pH of conjugate solutions was increased up to 11, using 1 N NaOH. This technique was adopted in order to hydrolyze the ester bond formed between the nanoparticle and the fluorophore. After 5 h C


Article


Figure 1. Visual inspection of the colloidal solutions of silver and gold nanoparticles synthesized using medicinal plant extracts and their corresponding UV−visible spectra. of shaking at 150 rpm and 25 °C the reaction mixtures were centrifuged at 14 000 rpm for 60 min. The UV−vis spectroscopy and HPLC was performed with supernatant solutions to determine the extent of conjugation. 2.8.1. Drug-Loading Calculation Using Absorption Spectroscopy. The percentage loading of the rhodamine B and rose bengal on the nanoparticles surface was calculated using linear equations obtained from the standard calibration curves. To obtain linear graph and equations, we prepared different concentrations of rhodamine B (0.5− 5 μg/mL) and rose bengal (2.5−25 μg/mL) and took absorbance using a multiskan spectrophotometer at 554 and 544 nm, respectively. All the readings were taken in triplicates. Averages of all readings were taken, calibration curves were plotted, and linear equations with R2 values were calculated. Concentrations of unknown rhodamine B and rose bengal were calculated using the linear equations. Further % loading of the drugs/imaging agents on the nanoparticle surface were determined. 2.8.2. Drug-Loading Calculation Using HPLC Method. Rhodamine B and rose bengal loading efficiency on the surface of bioinspired noble metal nanoparticles were studied. The supernatant solutions of the reaction mixtures containing rhodamine B or rose bengal conjugated nanoparticles obtained after the centrifugation at 12000g for 60 min was subjected to quantitative analysis. Shimadzu (Japan) and Waters-e2695 (USA) HPLC systems controlled by Class-VP and Empower pro softwares, respectively, equipped with an auto sampler and analytical C18 column (Phenomenex Luna 5u C18, 250 × 4.6 mm) were used for the quantification studies. Rhodamine B and Rose bengal detection was performed at 30 °C temperature using a Shimadzu fluorescence detector (excitation at 550 nm and emission at 580 nm) and waters 2998 photodiode array detector (400−600 nm), respectively. Rhodamine B (5 μL) and rose bengal (10 μL) were eluted isocratically at a flow rate of 1 mL/min using ammonium acetate buffer (10 mM) and acetonitrile as the mobile phase at the concentration ratio of 60:40 (v/v), respectively. The percent loading on the surface of the nanoparticles was determined by using eq 1.

3. RESULTS AND DISCUSSION 3.1. Plausable Mechanism of Nanoparticle Synthesis via Bioinspired Methods. Bioinspired synthesis of noble multimodal metal nanoparticles is a unique approach to develop multifunctional nanoentities which minimizes the need of using toxic chemicals and uses cheaper active ingredients (e.g., plant material). Biomaterials present in the plant extracts (such as phenols, flavonoids) can serve as reducing, stabilizing, and capping agents for nanoparticles.16 Various plants were screened for the synthesis of noble metal nanoparticles among which C. sinensis (CS) and P. f ulgens (PF) were chosen since they gave better yield of nanoparticles.23,24 Biosynthesis of silver and gold nanoparticles (AgNPs and AuNPs) were successfully achieved using C. sinensis (CS) and P. f ulgens (PF) extracts and all the nanomaterials were characterized by various analytical techniques such as UV− visible spectroscopy, zeta potential analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. The surface of the noble nanomaterials was further functionalized with therapeutic and imaging agents to develop theranostic nanoagents which could be engaged in potential biomedical applications. Incubation of a metal salt in the presence of a particular plant extract at optimized reaction parameters (e.g., reaction time, temperature, pH, concentration of the salt and the plant extract used) results in the formation of nanoclusters. However, role of various organic molecules (such as bioreducing agents, flavonoids, peptides) present as essential plant ingredients can be held responsible for the reduction, stabilization and capping of resulting nanoparticle formation.16 From different characterization data (UV−vis spectroscopy, TEM, FTIR) it can be hypothesized that the metal ions initially present in metal salts are reduced by bioreducing agents, then they are aggregated, capped, and stabilized by bioingredients to grow nanostructures of defined size and shape (eq 2, M = metal atom). The nature of bioreducing agents and reaction conditions (e.g., temperature, time, pH) play essential roles in controlling the morphology of the nanomaterials.

loading (%) = [(total dye added − dye in supernatant) /total dye added]·100

(1)

The standard calibration curves of rhodamine and rose bengal were drawn and linear equations were obtained using HPLC runs at different concentrations of rhodamine B and rose bengal. The calibration curves of rhodamine B and rose bengal are shown in the Supporting Information. D


Article


Figure 2. Effect of various reaction parameters on nanoparticle production (a) silver nanoparticles from C. sinensis (CS-AgNPs); (b) gold nanoparticles from C. sinensis (CS-AuNPs); (c) gold nanoparticles from P. f ulgens (PF-AuNPs).

resulting reduction of metal cations to neutral metal atoms. Reduced metal atoms are further aggregated and stabilized by bioingredients. Taking the advantage of relatively low reduction potential of silver and gold those metals can easily undergo reduction in the presence of antioxidants (such as polyphenols and flavonoids). Our prior work described the isolation and characterization of major bioingredients of P. fulgens plant extract (mainly by NMR and mass spectrometry). To further strengthen our claim that plant ingredients are solely responsible for nanoparticle formation, FTIR spectra were collected for both the plant extracts and metal nanoparticles. FTIR spectra showed the presence of similar functionalities in

3.1.1. Rationale behind Using Plant Extracts for Metal Nanoparticle Synthesis. For the synthesis of metal (silver and gold) nanoparticles, two plant extracts [C. sinensis (Green tea) and P. f ulgens (Vajradanti)] were chosen. Both of these plants are known to be rich in catechin and other polyphenol derivatives.23,24 The polyphenols can undergo oxidation while E


Article


Figure 3. Particle size disrtibution, zeta potential, and TEM images of silver and gold nanoparticles synthesized using different plant extracts.

parameters (concentrations of metal salts and plant extracts, pH of reaction mixture, temperature and time) for the synthesis of silver and gold nanoparticles were optimized in order to maximize the yield of nanoparticle synthesis. Interestingly, the highest optical yield was observed at a moderate temperature range of 45−60 °C for all the nanoparticles (Figure 2a−c). It can be noted that, at higher temperature, nanoparticle yield was low, possibly due to the degradation of phytochemicals. AgNPs were formed at higher (basic) pH, whereas the formation of AuNPs took place at very low (acidic) pH, probably due to the use of chloroauric acid as a source of gold metal (Figure 2a−c). For the synthesis of all types of nanoparticles, reaction time did not seem to have major effect on their yields. However, from various reaction time period, 18 h was chosen because the nanoparticle yield was relatively high (Figure 2a−c). 3.4. Zetasizer and Zeta Potential Analysis. Zetasizer analysis is an essential technique which helps to determine the average hydrodynamic particle size distribution and zeta potential of nanomaterials.16 The AgNPs synthesized using C. sinensis (CS-AgNPs) exhibited narrow size distribution range with an average zeta size of 97 nm (Figure 3, entry 1a), zeta

both plant extracts and metal nanoparticles, which justifies our claim. 3.2. Visual Inspection and UV−Visible Spectroscopic Analysis. Metal nanoparticles are known to emit characteristic colors in the visible region of electromagnetic spectrum because of a phenomenon known as “surface plasmon resonance”.7 Preliminary identification of nanoparticles was therefore performed by observing the change in color of the reaction mixture in an aqueous solution before and after the reaction. Silver nanoparticles showed characteristic color change from clear to yellowish brown (Figure 1, entry 1a). Whereas gold nanoparticles formed purple-red color in aqueous solution (Figure 1a, entry 2−3). It should be noted that the color of colloidal solution of nanoparticles were mostly dependent on their size and shape. UV−visible spectroscopic analysis of nanocolloid solutions showed sharp absorption band ∼400− 440 nm for silver nanoparticles (AgNPs, Figure 1, entry 1b) and 520−550 nm for gold nanoparticles (AuNPs, Figure 1, entry 2b and 3b). 3.3. Optimization of Reaction Parameters for the Synthesis of Metal Nanoparticles. Various reaction F


Article

ACS Biomaterials Science & Engineering potential of −14 mV (Table 2, entry 1b) and polydispersity index (PDI) of 0.22. Lower PDI value implied good monodispersity and negative zeta potential assured that the capping on the nanoparticles are mainly constructed by negatively charged groups (e.g., hydroxyl groups). For AuNPs, synthesized from C. sinensis (CS-AuNPs), the average hydrodynamic particle size distribution (Figure 3, entry 2a) and zeta potential (Table 2, entry 2b) were found to be 74 nm and −18 mV (capping of nanoparticles by negatively charged groups) respectively, whereas polydispersity index (PDI) was found to be 0.30 (good monodispersity). For AuNPs synthesized using P. f ulgens (PF-AuNPs), the average hydrodynamic size (Figure 3, entry 3a) and zeta potential (Figure 3, entry 3a) were found to be 61 nm and −10 mV respectively, whereas polydispersity index (PDI) was 0.28, indicating the monodispersity of synthesized nanoparticles with the capping of negatively charged groups on the surface. 3.5. Transmission Electron Microscopy (TEM). Transmission electron microscopy (TEM) is a powerful technique that is used for high-resolution imaging of thin film of a solid sample for structural and compositional analysis of nanomaterials.16 TEM micrograph of silver nanoparticles synthesized using C. sinensis leaf extract, confirmed that the AgNPs were spherical in shape with average size ∼100 nm and capped with plant constituents which prevented the nanoparticles from aggregation (Figure 3, entry 1c). Inherent capping offers additional advantage of stabilizing bioinspired nanoparticles. These stability characteristics of plant constituents are in agreement with the reports by Ahmad et al.25 TEM images of the AuNPs synthesized using C. sinensis demonstrated that AuNPs have spherical morphology with average size of 60−80 nm (Figure 3, entry 2c). AuNPs synthesized using P. f ulgens revealed that the AuNPs are spherical in shape, with size range of ∼60 nm according to the corresponding TEM images (Figure 3, entry 3c). Both AgNPs and AuNPs prepared using P. f ulgens are capped by plant constituents as observed in the TEM images. 3.6. Fourier Transform Infrared (FTIR) Spectroscopy. FTIR spectroscopic analysis is performed in order to identify the possible bioconstituents responsible for the reduction of metal ions to nanoparticles, its effective stabilization and capping. Thus, FTIR data were collected for all three types of phytochemical capped metal nanoconstructs. The synthesized nanoparticles showed peaks around 3400, 2900, 1600 cm−1 some of which were also found in the FTIR spectra of the corresponding plant extracts. The peak at 3400 cm−1 indicates hydrogen bonding present within hydroxyl groups. Individual and overlay FTIR spectra of plant extracts and their corresponding biosynthesized nanoparticles are shown in Figures S10−S12. Overall spectral pattern indicated the presence of catechin (flavonoid) like molecules (from the phytochemical capping) in the plant extracts that might be responsible for the coating of hydroxyl groups on the nanoparticles.16,24 Therefore, it can be concluded that the nanoparticle surface is capped by polyphenolics and flavonoids. In can also be noted that the hydroxyl groups present on the nanoparticle surface could be directly functionalized by reacting with carboxylic acid groups present in drugs or imaging agents to construct nanotheranostic scaffolds. 3.7. Determination of the Antioxidant Potential. Antioxidant activity is a measure of inhibition of oxidation by which various life-threatening diseases including cancer can be treated. Through the inhibition of the oxidation nonreactive

stable radicals are formed. Different bioingredients present in the plant (e.g., flavonoids and polyphenols) have strong antioxidant properties, which help in reducing oxidative stress in cells. Thus, those plant constituents play a key role in healing cancer, and inflammatory diseases.16 Because noble metal nanoparticles were coated with important plant ingredients, determination of antioxidant properties are essential to estimate whether those properties are still retained by the bioinspired nanoparticles. It is known that the synthesis and antioxidant activity of nanoparticles is mainly due to the flavonoid content, which can be calculated as quercetin dihydrate equivalent (QDE%) (quercetin standard).16,24 The QDE% values for CSAgNPs, CS-AuNPs, and PF-AuNPs were calculated to be 18, 11, and 7.9%, respectively (Table 1). Antioxidant activities were Table 1. Total Flavonoid Content in Noble Metal Nanoparticles sample no.

nanoparticle type

QDE%

1 2 3

CS-AgNP CS-AuNP PF-AuNP

18 11 7.9

further confirmed by various assays (namely, ABTS, DPPH) which showed promising results due to the presence of polyphenol and flavonoid capping agents on NPs.26 Experimental details of antioxidant assays are discussed in the Supporting Information. 3.7.1. ABTS Assay. In this colorimetric assay, the oxidation of ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] with potassium persulfate generates ABTS radical cation, hydrogen-donating antioxidants reduce these ABTS cation radicals and decolorize the solution which is measured at 734 nm.26 This assay was performed according to a previously reported method with some modifications.16 The antioxidant activities of bioinspired nanoparticles (CS-AgNPs, CS-AuNPs and PF-AuNPs) were found to be 39, 30, and 8.0% with IC50 values 1.3, 1.7, and 6.2 μg/mL, respectively (Table 2), whereas a standard, ascorbic acid, revealed 59% (IC50 0.85 μg/mL) scavenging at the same concentration. Experimental details and standard curve data are furnished in the Supporting Information. 3.7.2. DPPH Assay. In this colorimetric assay, an antioxidant donates a hydrogen atom to the stable DPPH [1,1-Diphenyl-2picrylhydrazyl] radical, for which the DPPH solution is decolorized.26 This assay was performed according to a previously reported method with some modifications.16,27,28 The antioxidant activity of bioinspired nanoparticles (CSAgNPs, CS-AuNPs and PF-AuNPs) were calculated to be 49, 45 and 35% with IC50 values 5.0, 5.6, and 7.1 μg/mL, respectively (Table 2), whereas ascorbic acid showed 52% (IC50 4.8 μg/mL) scavenging at the same concentration. Biosynthesized nanoparticles are highly valuable compared to the native metal nanoparticles in therapeutics because of their considerable amount of antioxidant potential. Experimental details and standard curve data are furnished in the Supporting Information. 3.8. Surface Functionalization of Biosynthesized Nanoparticles. Noble metal nanoconsructs synthesized using plant extracts are promising candidates as delivery vehicles for the therapeutic and diagnostic agents.1,10 Upon conjugation of both drugs and imaging agents on nanomaterial surface, theranostic nanoagents can be constructed for possible G


Article


therefore be selected, which can further be covalently attached to the nanoparticle surface via the formation of an ester bond (Figure 4a). Carboxyl containing therapeutic and imaging molecules [rose bengal (a commercial photosensitizer) and rhodamine B (an imaging agent) respectively] were, therefore, reacted with the nanomaterials in the presence of a coupling agent (EDC) (Figure 4b, c). The conjugated nanoparticles were further characterized by absorption spectroscopy (Figure 5), HPLC and FTIR analysis (Figures S20−S25) to confirm the conjugation. 3.8.1. Characterization of Surface-Functionalized Nanoparticles. Biosynthesized nanoparticles were successfully conjugated to an imaging agent (rhodamine B) or a photosensitizer drug (rose bengal). The conjugation was further confirmed by performing UV−vis spectroscopic analysis of the conjugated nanoparticle solutions (Figure 5). In the UV−vis spectra of rhodamine B and gold-containing nanoconjugates (both CS-AuNPs and PF-AuNPs), a broad peak was observed at 554 nm. The broad peak indicated the conjugated form of rhodamine B due to the overlap of two spectral signals (one from rhodamine B and the other from AuNP) (Figure 5a, b). The conjugated nanoconstructs were then treated with an aqueous NaOH, in order to hydrolyze the ester bond formed in the nanoconjugate. After NaOH treatment, rhodamine B got detached from the gold nanoconjugate and released in the solution. The UV−vis spectra of centrifuged supernatant showed an intense peak at 554 nm confirming rhodamine B removal from the nanoparticle surface. Similarly, rose bengalAuNP nanoconjugates showed a broad absorption peak at 544 nm and a short but intense peak at 544 nm after NaOH treatment (Figure 5c, d) confirming the conjugation of rose bengal on the nanoparticle surface. UV−vis spectroscopic analysis of rhodamine B-AgNP nanoconjugate showed two absorbance maxima, one at 434 nm (due to AgNPs) and another at 554 nm (due to rhodamine B). After NaOH treatment and centrifugation, only one short and intense

Table 2. Determination of the Antioxidant Properties of Bioinspired Metal Nanoparticles Using ABTS and DPPH Assay ABTS assay y = 58.52x sample no. 1 2 3 4 5 6 7 8

material type ascorbic acid (standard) CS-AgNPd CS-AuNPe PF-AuNPf CS extractg PF extracth bare AuNPsi bare AgNPsj

DPPH assay y = 10.46x

% antioxidant activitya

IC50 (μg/ mL)

% antioxidant activityb

IC50 (μg/ mL)

59c

0.85

52c

4.8

39 30 8.0 52 21 4.6 4.3

1.3 1.7 6.2 0.96 2.4 11 12

49 45 35 71 59 3.4 5.2

5.0 5.6 7.1 3.5 4.3 72 47

Percentage effect (E%) at concentration 1 μg/mL. bPercentage effect (E%) at concentration 5 μg/mL. cpercentage effect (E%) of standard at concentration 5 μg/mL, calculated using equation y = 10.46x. dC. sinensis mediated silver nanoparticles. eC. sinensis mediated gold nanoparticles. fP. f ulgens mediated gold nanoparticles. gC. sinensis extract. hP. f ulgens extract. iBare gold nanoparticles. jBare silver nanoparticles. a

biomedical applications.29,30 Addition of a photosensitizer drug on nanoparticles surface can give added advantage of performing photodynamic therapy.31−35 Bioinspired metallic nanoconstructs are known to possess polyphenols and flavonoids as capping agents of their surface, which have multiple hydroxy functional groups.16 Choudhary et al. demonstrated that P. f ulgens is mainly comprised of polar flavanols, including oligomeric flavan-3-ols as sources of −OH functional group.23,24 C. Sinensis is also known to contain catechin and epicatechin moieties as the sources for hydroxyl functionality.36 A conjugatable moiety (either an imaging or a therapeutic agent) possessing a carboxylic acid functionality can

Figure 4. (a) Diagrammatic representation of drug/imaging agent conjugation on the nanoparticle surface. Chemical structures of a photosensitizer and an imaging agent used in the biofunctionalization of nanoparticles: (b) rose bengal and (c) rhodamine B. H


Article


Figure 5. Conjugation of rhodamine B and rose bengal with metal NPs; (a) rhodamine B with CS-AuNPs, (b) rhodamine B with PF-AuNPs, (c) rose bengal with CS-AuNPs, (d) rose bengal with PF-AuNPs, (e) rhodamine B with CS-AgNPs, and (f) rose bengal with CS-AgNPs. Purple line: UV−vis spectra of rhodamine B or rose bengal before conjugation (control sample). Red line: UV−vis spectra after bioconjugation. Blue line: UV− vis spectra of supernatant after NaOH treatment.

absorbance peak was observed at 554 nm (Figure 3e). Two absorbance maxima in the single spectrum confirmed that the rhodamine B was conjugated to the surface groups present on the AgNPs. Treatment of nanoconjugates with NaOH solution removed the conjugated rhodamine B from the nanoparticle surface for which the supernatant after the centrifugation showed only one absorbance maxima at 554 nm. Similarly, the rose bengal-AgNP nanoconjugate solution showed two peaks in UV−vis spectra at 450 (due to AgNP) and 544 nm (due to rose bengal) (Figure 5f). After NaOH treatment the free rose bengal released in the solution and after centrifugation the supernatant resulted absorbance at 544 nm. FTIR analysis of nano-

conjugates also confirmed that all the conjugations have been performed successfully (Figures S20−S25). 3.8.2. Determination of Drug Loading on SurfaceFunctionalized Nanoparticles Using Absorption Spectroscopy. Percent loading of rhodamine B and rose bengal derivative on the surface of the bioinspired nanoparticles was calculated (see Table S9B). The standard calibration curves of rhodamine B and rose bengal derivative were prepared (Figures S19 and S20), and linear regression equations and R2 values were obtained (Table S9).16 The absorbance of supernatant solution of detached rhodamine B and rose bengal derivative were taken at 554 and 544 nm, respectively. The concentrations of I


Article


Table 3. Determination of Percent Loading of Rhodamine B and Rose Bengal on the Surface of Various Silver and Gold Nanoparticles (Using HPLC Method) sample no..

params

1 2 3 4 5

nanoparticle AUCa C0b (μg/mL) Cic (μg/mL) Ld (%)d

rhodamine B y = 4541383.5x, R2 = 1 CS-AgNP 61166760 13.5 15 10.2

CS-AuNP 62504280 13.8 15 8.2

PF-AuNP 64352220 14.2 15 5.5

rose bengal y = 46096x, R2 = 0.9991 CS-AgNP 590107 12.8 15 14.5

CS-AuNP 591830 12.8 15 14.2

PF-AuNP 629033 13.7 15 8.8

a

Area under the curve in the HPLC chromatogram of supernatant solutions containing rhodamine B and rose bengal after NaOH treatment and centrifugation. bConcentrations of rhodamine B and rose bengal in the supernatant solution, calculated using linear regression equation y = mx + c. c Initial concentrations of rhodamine B or rose bengal used in the conjugation reaction. dPercent loading of rhodamine B and rose bengal on the surface of nanoparticles. This was calculated using the equation; Ld% = {[Ci−C0]/[Ci]}·100.

reacted with carboxyl-containing imaging and therapeutic moieties (rhodamine B and rose bengal, respectively). Surface functionalization was successfully achieved through EDC mediated coupling with rose bengal and rhodamine B to construct the theranostic nanoagents. Investigation of the extent of drug loading was done in order to calculate the extent of surface conjugation on nanomaterials which showed significant binding of those molecules. To the best of our knowledge this is the first example of direct surface functionalization on the biologically synthesized nanomaterials. This method does not require any additional chemical coating agents for surface functionalization after nanoparticle formation. Overall, these biofunctionalized multimodal theranostic nanoagents developed through green techniques can find potential applications in biomedical research.

rhodamine B and rose bengal derivatives in the supernatant solution were calculated using linear regression equation. Finally, from the initial concentrations of rhodamine B and rose bengal, the percentage of the conjugated rhodamine B and rose bengal on the surface of nanoparticles were calculated. Experimental details and results are furnished in the Supporting Information. 3.8.3. Determination of Drug Loading on Surface Functionalized Nanoparticles Using HPLC Method. Because photosensitizers are required in lower concentration to show therapeutic benefits, one of the fundamentals for the successful delivery of photosensitizers using nanoparticles is sufficient loading efficiencies. The surface modified nanoparticles have shown fairly good loading efficiencies (Table 3). Loading efficiencies of rhodamine B were 10.2, 8.2, and 5.5% for CSAgNP, CS-AuNP and PF-AuNP respectively. Similarly the loading efficiencies of rose bengal were 14.5, 14.2, and 8.8% for CS-AgNP, CS-AuNP, and PF-AuNP respectively. The loading efficiency can be attributed to the extent of interaction, size and shape of the biosynthesized nanoparticles. It was observed that the loading of rhodamine B and rose bengal are relatively low on PF-AuNP compared to CS-AuNP and CS-AgNP (Table 3). This indicates that more conjugatable handles are present on the surface of the CS-AuNP and CS-AgNP as compared to PFAuNP. The drug loading capacity on the nanoparticle surface, which is in the range of 5−15%, is quite promising for biomedical applications wherever nanotheranostic agents can be improvised.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ab500171a. Optimization of various reaction conditions of nanoparticle synthesis; harvesting of synthesized nanoparticles; scanning electron microscopy; FTIR analysis data; differential scanning calorimetry (DSC) of nanoparticles; antioxidant assays; total flavonoid content of nanoparticles; surface functionalization of noble metal nanoparticles; characterization of nanoconjugates by FTIR spectroscopy (PDF)

■

4. CONCLUSIONS In summary, protocols for the bioinspired synthesis of noble metal nanoparticles using medicinal plants were developed. Optimization of important reaction parameters (time, temperature, and pH of the reaction medium) afforded bioconjugatable noble metal nanoparticles in high yields with controlled size and shape. These biosynthesized nanoparticles were preliminarily identified by visual inspection and absorption spectroscopy. Average size and potential of the nanoparticles were determined by using a Zetasizer. Electron microscopic techniques (SEM and TEM) confirmed the size (∼60−100 nm) and shape (mostly spherical) of the biosynthesized nanoparticles. FTIR spectroscopy was utilized to determine structure and further confirm the presence of −OH functional groups of biomolecules capped on to the nanoparticles. Determination of antioxidant potential via ABTS and DPPH assay confirmed significant retention of antioxidant activities by the biosynthesized nanoconstructs. Taking advantage of the hydroxyl functionalities present on the nanomaterial surface (which was attributed to the phytochemical capping), they were

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

N.S.T. and P.K.A. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS J.B. gratefully acknowledges financial support from the Department of Science and Technology Women Scientists Scheme A (DST WOS-A), Government of India. N.T. is grateful for the support through DST Inspire Scheme. Authors would like to thank Dr. Anupam Chatterje of North-Eastern Hill University, Shillong, India, for providing P. f ulgens plant. The authors also thank Dr. I. P. Singh of Department of Natural Products, NIPER, S.A.S. Nagar for preparing P. fulgens root extract. J


Article


■

antioxidant triterpenes from Potentilla fulgens. Fitoterapia 2013, 91, 290−297. (24) Choudhary, A.; Manukonda, R.; Chatterjee, A.; Banerjee, U. C.; Singh, I. P. Qualitative and quantitative analysis of Potentilla f ulgens roots by NMR,Matrix-assisted Laser Desorption/Ionisation with Time-of-Flight MS, Electrospray Ionisation MS/MS and HPLC/UV. Phytochem. Anal. 2015, 26, 161−170. (25) Ahmad, N.; Sharma, S.; Alam, M. K.; Singh, V.; Shamsi, S.; Mehta, B.; Fatma, A. Rapid synthesis of silver nanoparticles using dried medicinal plant of basil. Colloids Surf., B 2010, 81, 81−86. (26) Krishnaiah, D.; Sarbatly, R.; Nithyanandam, R. A review of antioxidant potential of medicinal plant species. Food Bioprod. Process. 2011, 89, 217−233. (27) Chen, Z.; Bertin, R.; Froldi, G. EC50 estimation of antioxidant activity in DPPH assay using several statistical programs. Food Chem. 2013, 138, 414−420. (28) Sharma, O. P.; Bhat, T. K. DPPH antioxidant assay revisited. Food Chem. 2009, 113, 1202−1205. (29) Wang, S. Y.; Kim, G.; Lee, Y. E. K.; Hah, H. J.; Ethirajan, M.; Pandey, R. K.; Kopelman, R. Multifunctional biodegradable polyacrylamide nanocarriers for cancer theranosticsA “see and treat” strategy. ACS Nano 2012, 6, 6843−6851. (30) Choi, K. Y.; Liu, G.; Lee, S.; Chen, X. Y. Theranostic nanoplatforms for simultaneous cancer imaging and therapy: Current approaches and future perspectives. Nanoscale 2012, 4, 330−342. (31) Bhaumik, J.; McCarthy, J. R.; Weissleder, R. Synthesis and photophysical properties of sulfonamidophenyl porphyrins as models for activatable photosensitizers. J. Org. Chem. 2009, 74, 5894−5901. (32) McCarthy, J. R.; Bhaumik, J.; Merbouh, N.; Weissleder, R. Highyielding syntheses of hydrophilic, conjugatable chlorins and bacteriochlorins. Org. Biomol. Chem. 2009, 7, 3430−3436. (33) Mroz, P.; Bhaumik, J.; Dogutan, D. K.; Aly, Z.; Kamal, Z.; Khalid, L.; Kee, H. L.; Bocian, D. S.; Holten, D.; Lindsey, J. S.; Hamblin, M. R. Imidazole metalloporphyrins as photosensitizers for photodynamic therapy: role of molecular charge, central metal and hydroxyl radical production. Cancer Lett. 2009, 282, 63−76. (34) Kee, H. L.; Bhaumik, J.; Diers, J. R.; Mroz, P.; Hamblin, M. R.; Bocian, D. F.; Lindsey, J. S.; Holten, D. Photophysical characterization of imidazolium-substituted Pd(II), In(III), and Zn(II) porphyrins as photosensitizers for photodynamic therapy. J. Photochem. Photobiol., A 2008, 200, 346−355. (35) Zhang, Y.; Das, G. K.; Vijayaragavan, V.; Xu, Q. C.; Padmanabhan, P.; Bhakoo, K. K.; Selvan, S. T.; Tan, T. T. ″Smart″ theranostic lanthanide nanoprobes with simultaneous up-conversion fluorescence and tunable T1-T2 magnetic resonance imaging contrast and near-infrared activated photodynamic therapy. Nanoscale 2014, 7, 12609−11. (36) Perva-Uzunalic, A.; Skerget, M.; Knez, Z.; Weinreich, B.; Otto, F.; Grüner, S. Extraction of active ingredients from green tea (C. sinensis). Food Chem. 2006, 96, 597−605.

REFERENCES

(1) Mittal, A. K.; Chisti, Y.; Banerjee, U. C. Synthesis of metallic nanoparticles using plant extracts. Biotechnol. Adv. 2013, 31, 346−356. (2) Lin, N.; Huang, J.; Dufresne, A. Preparation, properties and applications of polysaccharide nanocrystals in advanced functional nanomaterials: a review. Nanoscale 2012, 4, 3274. (3) Raveendran, P.; Fu, J.; Wallen, S. L. Completely “green” synthesis and stabilization of metal nanoparticles. J. Am. Chem. Soc. 2003, 125, 13940−13941. (4) Narayanan, K. B.; Sakthivel, N. Biological synthesis of metal nanoparticles by microbes. Adv. Colloid Interface Sci. 2010, 156, 1−13. (5) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F. Theranostic nanomedicine. Acc. Chem. Res. 2011, 44, 1029−1038. (6) Kelkar, S. S.; Reineke, T. M. Theranostics: combining imaging and therapy. Bioconjugate Chem. 2011, 22, 1879−1903. (7) Rao, J. Shedding light on tumors using nanoparticles. ACS Nano 2008, 2 (10), 1984−1986. (8) Mura, S.; Couvreur, P. Nanotheranostics for personalized medicine. Adv. Drug Delivery Rev. 2012, 64, 1394−1416. (9) Mittal, A. K.; Kaler, A.; Banerjee, U. C. Free radical scavenging and antioxidant activity of silver nanoparticles synthesized from flower extract of Rhododendron dauricum. Nano Biomed. Eng. 2012, 4, 118− 124. (10) Kaler, A.; Nankar, R.; Bhattacharyya, M. S.; Banerjee, U. C. Extracellular biosynthesis of silver nanoparticles using aqueous extract of Candida viswanathii. J. Bionanosci. 2011, 5, 53−58. (11) Shankar, S. S.; Rai, A.; Ahmad, A.; Sastry, M. Rapid synthesis of Au, Ag, and bimetallic Au core−Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. J. Colloid Interface Sci. 2004, 275, 496− 502. (12) Jain, N.; Bhargava, A.; Majumdar, S.; Tarafdar, J. C.; Panwar, J. Extracellular biosynthesis and characterization of silver nanoparticles using Aspergillus flavus NJP08: a mechanism perspective. Nanoscale 2011, 3, 635−41. (13) McCarthy, J. R.; Bhaumik, J.; Karver, M. R.; Erdem, S. S.; Weissleder, R. W. Targeted nanoagents for the detection of cancers. Mol. Oncol. 2010, 4, 511−528. (14) Kievit, F. M.; Zhang, M. Cancer nanotheranostics:improving imaging and therapy by targeted delivery across biological barriers. Adv. Mater. 2011, 23, H217−H247. (15) Gong, Y.; Winnik, F. M. Strategies in biomimetic surface engineering of nanoparticles for biomedical applications. Nanoscale 2012, 4, 360−368. (16) Mittal, A. K.; Bhaumik, J.; Kumar, S.; Banerjee, U. C. Biosynthesis of silver nanoparticles: elucidation of prospective mechanism and therapeutic potential. J. Colloid Interface Sci. 2014, 415, 39−47. (17) Bhaumik, J.; Mittal, A. K.; Banerjee, A.; Chisti, Y.; Banerjee, U. C. Applications of phototheranostic nanoagents in photodynamic therapy. Nano Res. 2014, DOI 10.1007/s12274-014-0628-3. (18) Josefsen, L. B.; Boyle, R. W. Unique diagnostic and therapeutic roles of porphyrins and phthalocyanines in photodynamic therapy, imaging and theranostics. Theranostics 2012, 2, 916−966. (19) Guo, Y.; Rogelj, S.; Zhang, P. Rose Bengal-decorated silica nanoparticles as photosensitizers for inactivation of gram-positive bacteria. Nanotechnology 2010, 21, 065102. (20) Mousavi, S. H.; Tavakkol-Afshari, J.; Brook, A.; Jafari-Anarkooli, I. Direct toxicity of rose bengal in MCF-7 cell line: role of apoptosis. Food Chem. Toxicol. 2009, 47, 855−859. (21) Mittal, A. K.; Kumar, S.; Banerjee, U. C. Quercetin and gallic acid mediated synthesis of bimetallic (silver and selenium) nanoparticles and their antitumor and antimicrobial potential. J. Colloid Interface Sci. 2014, 415, 39−47. (22) Rai, P.; Mallidi, S.; Zheng, X.; Rahmanzadeh, R.; Mir, Y.; Elrington, S.; Khurshid, A.; Hasan, T. Development and applications of photo-triggered theranostic agents. Adv. Drug Delivery Rev. 2010, 62, 1094−1124. (23) Chaudhary, A.; Mittal, A. K.; Monukonda, R.; Tripathy, D.; Chatterjee, A.; Banerjee, U. C.; Singh, I. P. Two new stereoisomeric K


Bioinspired Nanotheranostic Agents: Synthesis, Surface

Recommend Documents