Physicochemical Transformations of ZnO Nanoparticles Dispersed in

Subscriber access provided by Queen Mary, University of London

Article

Physicochemical Transformations of ZnO Nanoparticles Dispersed in Peritoneal Dialysis Fluid: Insights into Nano-Bio Interface Interactions Anupam Guleria, Mukesh Kumar Meher, Narayan Prasad, Krishna Mohan Poluri, and Dinesh Kumar J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b04889 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Physicochemical Transformations of ZnO Nanoparticles Dispersed in Peritoneal Dialysis Fluid: Insights into Nano-Bio Interface Interactions Anupam Guleria1*, Mukesh Kumar Meher2, Narayan Prasad4, Krishna Mohan Poluri2,3* and Dinesh Kumar1* 1

Centre of Biomedical Research, SGPGIMS Campus, Lucknow-226014, India

2

Center for Nanotechnology, 3Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India 4

Department of Nephrology, SGPGIMS Campus, Lucknow-226014, India

*

Authors for Correspondence:

Dr. Anupam Guleria Centre of Biomedical Research (CBMR), SGPGIMS Campus, Raebareli Road, Lucknow-226014 Uttar Pradesh, India Email: [email protected] Mobile: +91-9918004592

Dr. Krishna Mohan Poluri Department of Biotechnology Indian Institute of Technology Roorkee Roorkee – 247667, Uttarakhand, India Email: [email protected], [email protected]

Dr. Dinesh Kumar Centre of Biomedical Research (CBMR), SGPGIMS Campus, Raebareli Road, Lucknow-226014 Uttar Pradesh, India Email: [email protected] 1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Absence of effective antibiotics for the treatment of infectious peritonitis along with the appearance of multidrug-resistance has prompted the use of antimicrobial nanoparticles to impart infection resistant properties to the existing peritoneal dialysis (PD) fluid. To explore this research perspective, we investigated the solubility and physicochemical transformations of Zinc oxide (ZnO) nanoparticles (NPs) dispersed in PD fluid using nuclear magnetic resonance spectroscopy in conjunction with dynamic light scattering, transmission electron microscopy, Fourier transform infrared and UV-Visible spectroscopy. Our results emphasize that ZnO NPs strongly interact with organic acids found abundantly in biological fluids (demonstrated here with lactic and citric acid) and further lead to the formation of bioconjugates. Based on the current detailed investigations, we propose that the surface coating of ZnO NPs would be required to impart agglomeration resistant properties and to inhibit the binding interactions of NPs and thus rendering their safe and efficient intraperitoneal use as antimicrobials.

2 ACS Paragon Plus Environment

Page 2 of 28

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Peritoneal dialysis (PD), a life-sustaining therapy used for treating patients with end-stage renal failure (ESRF), has evolved significantly since its inception.1,2 However, in spite of significant advances in peritoneal dialysis, technique failure remains a common outcome. The most common cause of PD technique failure includes mechanical complications, inadequate solute clearance, ultrafiltration failure, psychosocial issues and infectious complications.3 Among all these, PD-related infections remain a leading issue to successful long-term PD and are responsible for increased morbidity and mortality.4 Bacterial and fungal infections are the common type of infections in PD patients. Bacterial infections generally develop in the peritoneum, subcutaneous tunnel and catheter exit site, and fungal infections may develop subsequent to antibiotic use. 4 If not treated accurately and in a timely fashion, these infections may lead to infectious peritonitis,5,6 an inflammation-related tissue damage of peritoneum which may be associated with severe pain leading to hospitalisation, catheter loss, possible permanent membrane damage and a risk of death. Recent statistics have shown that peritonitis is the main cause of morbidity and mortality in PD patients.4 Clinically, the PD associated infections are mostly treated by the use of antibiotics delivered through the peritoneal route. Clinical improvement of the patient and the pathogenic organisms responsible for the infection determines the duration of antibiotics treatment. However, pathogenic micro-organisms associated with PD infections are becoming increasingly resistant to many commonly used antibiotics which pose a very serious problem to the health of PD patients. Therefore, the development of new approaches other than antibiotics is the focus of extensive research to keep up with the constantly changing and increasing multiple drug resistance (MDR) of bacterial strains. Nanotechnology offers a novel approach for killing or reducing the activity of numerous microbial organisms and has been increasingly utilized in many biomedical applications owing to the exclusive antimicrobial activity against a variety of infections and potential wound healing and anti3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

inflammatory properties of nanoparticles (NPs). Among the various antimicrobial nanomaterials, ZnO nanoparticles have been shown to exhibit a wide range of antibacterial activities against microorganisms including bacteria (both Gram-positive and Gram-negative bacteria, such as Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, Staphylococcus aureus, and Campylobacter jejuni etc.),7-9 fungi,10,11 and algae,12 etc. Though the precise mechanism of action of ZnO nanoparticles against these pathogenic organisms is not clearly understood, however a few studies have suggested the generation of reactive oxygen species including hydrogen peroxide (H2O2), OH(hydroxyl radicals), and O2-2 (peroxide) which are harmful to bacterial cells, as one of the possible mechanisms.7,13-15 Additionally, the enhanced membrane permeability, disorganization of bacterial membrane and cell wall damage upon contact with ZnO nanoparticles were also indicated to inhibit the bacterial growth.16,17 Recently, the use of antimicrobial metallic nanoparticles such as ZnO NPs has been proposed as a compelling alternative for the management of recurrent and persistent infections during long-term PD.18 However, before going into realizing the use of ZnO NPs in the existing peritoneal dialysis fluid composition, it is essential to investigate the properties of nanoparticles dispersed in PD fluid. To date, no study has been carried out to characterize the physicochemical properties NPs in PD fluid. Further, it has been reported that ZnO nanoparticles forms bioconjugates of glucose when added in glucose solution.19 Since the main constituents of PD fluid are glucose and lactic acid, so it become essential to study the interaction and binding of ZnO nanoparticles with the constituents of PD fluid, if any. In this report, we present our results on the solubility and interaction of ZnO Nanoparticles dispersed in PD fluid. Since lactic acid and glucose are the main constituents of PD fluid, the characterization of ZnO Nanoparticles dispersed separately in lactic acid and glucose has also been carried out. Further as the PD fluid enters the peritoneal cavity, various other metabolites including organic acids get secreted into the PD fluid from the tissues in close proximity and may interact with ZnO NPs. Among such organic acids, 4 ACS Paragon Plus Environment

Page 4 of 28

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


citric acid is an important component of the central metabolic pathway i.e. the Kreb’s cycle, and therefore the interaction of ZnO NPs with citric acid has also been studied. Experimental Section

Materials Zinc Oxide Nanoparticles dispersion was purchased from Sigma Aldrich (catalog No. 721077) having a reported particle size < 100 nm as measured by dynamic light scatting (DLS) and an average particle size < 40 nm as measured using an aerodynamic particle sizer (APS) spectrometer. Peritoneal dialysis solution IP (Dianeal PD-2 with 2.5%, w/v Dextrose) was obtained from Baxter Healthcare. Deuterium oxide (D2O) and sodium salt of trimethylsilylpropionic acid-d4 (TSP) used for NMR spectroscopy were purchased from Sigma-Aldrich (Rhode Island, USA). Transmission electron microscopy (TEM) High resolution images of ZnO nanoparticles were recorded with a Tecnai G2 20 S-TWIN Transmission Electron Microscope (TEM) (FEI Netherlands) operated at 200 kV. Samples were prepared by depositing two to three droplets of dilute solution of the nanoparticles onto a Carbon coated copper grids (Icon Analytical Equipment Pvt. Lt). Selected area electron diffraction pattern (SAED) and energy-dispersive X-ray spectroscopy (EDX) was also carried out in situ using the embedded detectors of the same instrument. X-ray diffraction ZnO NPs were also characterized using powder x-ray diffraction (XRD) to determine the crystal structure and purity. XRD was carried out at Bruker D8 X-ray diffractometer equipped with a Cu Kα1 source at 40 kV and 30 mA. ZnO NPs were placed in a glass holder and scanned from 10° to 90° with a scanning rate of 2.0°/min.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Size and surface charge analysis The hydrodynamic size and the zeta potential were measured with a Zetasizer Nano ZS90 (Malvern Instruments Inc, UK) utilizing dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. UV-Visible spectroscopy The UV-Visible spectroscopy was carried out in an Agilent Cary 300 double beam UV-Vis spectrophotometer with a spectral band width of 2 nm using a quartz cuvette having a path length of 1 cm. Spectra were acquired at different time intervals and the wavelength range was scanned from 200-800 nm. Fourier Transform Infrared (FT-IR) Spectroscopy Adsorption of lactic and citric acid onto ZnO nanoparticles was investigated using an FT-IR absorption spectrometer (PerkinElmer Frontier Instruments). For the measurements, the solution samples were dried out and the pellets of dried powder samples in KBr were prepared, and FT-IR absorption spectra were recorded between 450 and 4000 cm-1. Nuclear Magnetic Resonance (NMR) spectroscopy One‐dimensional 1H‐NMR spectra were recorded on Bruker Avance III 800 MHz NMR spectrometer (equipped with Cryoprobe) with a 5 mm broad-band inverse probe-head and Zshielded gradient at 298 K. Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence20 (cpmgpr1d, standard Bruker pulse program) with pre-saturation of the water peak was used to acquire the NMR spectra on PD fluid, lactic acid, PD fluid-ZnO NPs, lactic acid-ZnO NPs, glucose-ZnO NPs and citric acid-ZnO NPs solutions. The 400 µL of the samples were filled in 5 mm NMR tubes (Wilmad Glass, USA) and a sealed capillary tube containing the known concentration of 0.1 mM TSP (Sodium salt of 3-trimethylsilyl-(2,2,3,3-d4)-propionic acid) dissolved in D2O was inserted separately both for the purpose of locking and chemical shift referencing. The following parameters were used 6 ACS Paragon Plus Environment

Page 6 of 28

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for 1D CPMG pulse sequence: spectral sweep width: 12 ppm; data points: 32 K; flip angle of radiofrequnecy pulse: 90°; total relaxation delay (RD): 5 sec; number of scans: 128;

window

function: exponential; and line broadening: 0.3 Hz. All the spectra or FIDs (free induction decays) were processed using Topspin-2.1 (Bruker NMR data Processing Software) using standard Fourier Transformation (FT) procedure following manual phase and baseline-correction. Prior to FT, each FID was zero-filled to 4096 data points and a sine–bell apodisation function was applied. After FT, the chemical shifts were referenced externally to TSP methyl protons at 0.0 ppm. Spectral resonances were identified and assigned, by comparing them with the chemical shifts available in the database library of Chenomx Profiler (NMR Suite, v8.1, Chenomx Inc., Edmonton, Canada) and using the existing literature reports.21,22 Results & Discussion The crystalline nature and phase purity of as purchased ZnO NPs was confirmed by the X-ray diffraction (XRD) pattern shown in Figure 1 (A). All detectable peaks in XRD pattern are well indexed to hexagonal wurtzite structure.23 Also the presence of impurities was found to be negligible as no other characteristic impurities peaks were observed in the XRD pattern indicating a high quality ZnO NPs. X-ray diffraction patterns of the ZnO nanoparticles dispersed in PD fluid, lactic acid, citric acid and glucose were also recorded to investigate any influence on the crystalline structure of ZnO nanoparticles. As shown in Figure 1(B), the XRD pattern for ZnO NPs dispersed in glucose is almost similar to that of pure ZnO NPs implying the non-alteration of the crystallinity of ZnO nanoparticles in glucose. However, for ZnO NPs dispersed in PD fluid, lactic acid and citric acid (Figure 1(C-E)), the intensity of the diffraction peaks for ZnO NPs is significantly decreased along with the appearance of some other peaks inferring the effect of surface modifications on the crystallinity of ZnO NPs.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1: XRD pattern of ZnO nanoparticles in (A) de-ionized water, (B) glucose, (C) PD Fluid, (D) lactic acid and (E) citric acid. The size and morphology of the ZnO NPs was further determined by transmission electron microscopy (TEM). Figure 2(A, B) shows the representative TEM images of ZnO NPs at 100 and 500 nm resolutions, respectively. ZnO NPs showed a size distribution in the range of 20-80 nm with the majority of particles having a spherical morphology and a small proportion of elongated particles. TEM images of ZnO NPs suggested that the particles were well dispersed without forming 8 ACS Paragon Plus Environment

Page 8 of 28

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


any fused particle domains. However, strikingly when the ZnO NPs were added to PD fluid, agglomeration of the NPs occur as depicted by TEM images in Figure 2(C, D), obtained at 100 and 500 nm resolutions, respectively. Figure 2 (E and F) show the selected area electron diffraction pattern (SAED) patterns for ZnO nanoparticles in de-ionized water and PD fluid, respectively, revealing the crystalline nature of ZnO NPs.

Figure 2. TEM images of ZnO nanoparticles (A & B) in de-ionized water and (C & D) in PD fluid, scale bars are 100 and 500 nm for (A & C) and (B &D), respectively. Figures (E) and (F) 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

show the selected area electron diffraction (SAED) pattern for ZnO nanoparticles in de-ionized water and PD fluid, respectively.

Figure 3. TEM images of ZnO nanoparticles (A & B ) in glucose, (C & D) in lactic acid, and (E & F) in citric acid. Scale bars are 100 nm and 200 nm for (A, C & E) and (B, D & F), respectively.

TEM images were also acquired on ZnO NPs dispersed in lactic acid, citric acid and glucose solution. While no change in the morphology of ZnO NPs was observed in glucose solution


Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Figure 3(A, B)), a complete agglomeration forming a cluster was seen for ZnO NPs dispersed in lactic acid and citric acid as revealed from Figure 3(C, D) and Figure 3(E, F), respectively. A series of 1D 1H nuclear magnetic resonance (NMR) spectra of PD fluid were further recorded in the presence of increasing amount of ZnO NPs (Figure 4(A)). The lowermost spectrum shown in Figure 4(A) corresponds to PD fluid which mainly contains the resonances of lactic acid and glucose. We find that when ZnO NPs were dispersed in PD fluid, the intensity of lactic acid resonances exhibit a significant decrease without any effect in the chemical shifts and this decrease continues with increase in ZnO NPs concentration (see the expanded version in Figure 4(B)). A very small change in the intensity of glucose resonances were also observed as ZnO NPs were dispersed in PD fluid (Figure 4(C)). The inset of figures 4 (B) and (C) shows the structure of lactic acid and glucose molecule, respectively. The significant decrease in the intensity of lactic acid resonances suggests the interaction of lactate with ZnO NPs leading to the formation of bioconjugates which also explains the agglomeration of NPs in PD fluid and lactic acid as depicted by TEM. The 1D 1H NMR spectra of lactic acid, glucose and citric acid recorded in the presence of increasing amount of ZnO NPs are shown in supplementary Figures S1-S3, respectively. While we observed a clear decrease in the lactic acid and citric acid resonances similar to as observed in PD fluid, no significant changes were seen in glucose resonances. Table 1. Size distribution and zeta potential of ZnO nanoparticles in de-ionized water, PD fluid, lactic acid, glucose and citric acid (suspension concentration for ZnO NPs was 2mg/ml in each cases). Sample Average Size Zeta Potential Distribution (nm) (mV) ZnO NPs in water 36.3 69.20.31 ZnO NPs in PD fluid 13.4 2076103 ZnO NPs in Lactic acid (Lactic acid =4.48 mg/ml) 1.27 89845 ZnO NPs in Glucose (Glucose=25 mg/ml) 27.7 73.80.58 ZnO NPs in Citric acid (Citric acid =4.48 mg/ml) -0.454 2855142



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 28

Figure 4: (A) Stacked 1H NMR spectra of PD fluid containing different concentrations of ZnO NPs (0, 2, 4, 7 and 14 mg/ml). Quantitative decrease in the intensity of proton resonances of lactic acid with increase in ZnO NPs concentration is clearly visible as marked by *. (B) Expanded version of chemical shift regions corresponding to lactic acid and (C) glucose. Inset of figures B and C show the structure of lactic acid and glucose, respectively. The formation of ZnO NPs bioconjugates in PD fluid was further confirmed by dynamic light scattering (DLS) using a Zetasizer Nano. Samples were prepared by dissolving 2 mg/ml ZnO NPs in de-ionized water, PD fluid, lactic acid, glucose and citric acid followed by ultra-sonication for 30 minutes and further incubation for 10 minutes and respective particle size distribution (PSD) is shown in supplementary Figures S4 (A-E) and the corresponding average particle size has been enlisted in Table 1. The ZnO NPs dispersed in de-ionized water gave an average particle size of 69 nm which is in close agreement with TEM data. We found that the average ZnO NPs size was significantly increased from 69 nm to 2076 nm when dispersed in PD fluid inferring the formation of bioconjugates in PD fluid. Similar increase in average size was observed for ZnO NPs dispersed 12 ACS Paragon Plus Environment

Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in lactic acid (size ~ 898 nm) and citric acid (size ~ 2855 nm), while there was no significant change in size when dispersed in glucose (size ~ 73.8 nm). Also the zeta potential of ZnO NPs in deionized water was +36.3 mV which decreased to +13.4 mV, +1.27 mV and -0.454 mV for ZnO NPs dispersed in PD fluid, lactic acid and citric acid, respectively, suggesting the interaction of positively charged NPs with negatively charged lactate and citrate ions and hence leading to the formation of ZnO-lactate and ZnO-citrate nanocomposites, respectively. The influence of pH on the zeta potential of ZnO nanoparticles in deionized water and PD fluid has also been studied and is presented in supplementary Figure S5. The value of zeta potential was + 36.3 mV for ZnO NPs having pH 7.65 which was found to decrease with the addition of NaOH. The isoelectric point (IEP) for ZnO NPs was found to be at pH 10.4 which is in agreement with the values reported in various studies.24-27 When dispersed in PD fluid, the zeta potential was shifted towards negative values due to the adsorption of lactate ions on ZnO NPs and the new value of IEP was pH 9.4. The UV-Vis absorption spectra were further recorded on ZnO NPs dispersed in deionized water, glucose, PD fluid, lactic acid and citric acid as shown in Figure 5 (A), (B), (C), (E) and Figure S6, respectively. The UV-Vis spectrum reveals a characteristic absorption peak of ZnO NPs at a wavelength of 358 nm (in water and glucose) due to excitonic transition at room temperature.23,28 We did not find any change in the intensity and/or any shift in absorption maxima of ZnO NPs in deionized water and glucose with increase in time as depicted from Figure 5(A) and (B), respectively. However, when the ZnO NPs were dispersed in the PD fluid, lactic acid and citric acid, we observed a red shift in the absorption peak to 368, 366 and 376 nm, respectively. The band gap energy was calculated using the Tauc relation29 εhυ=C(hυ-𝐸𝑔 )1/2

(1)

where C is a constant, ε is molar extinction coefficient and Eg is the band gap of the material. The band gap energy of ZnO NPs was found to be 3.29 eV which decreased to 3.19, 3.23 and 2.88 eV 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

when dispersed in PD fluid, lactic acid and citric acid, respectively. A bathochromic shift of the absorption peaks (~ 10-18 nm) infer the binding of lactate and citrate ions with ZnO NPs leading to the formation of the bio-conjugates.


Page 14 of 28

Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5: Time course of absorption spectra of ZnO nanoparticles in (A) de-ionized water, (B) glucose, (C) PD fluid and (E) lactic acid. No change in absorption with time was observed in deionized water (inset of figure (A)) and glucose. (D) and (F) shows the change of absorption intensity of ZnO NPs with time in PD fluid and lactic acid, respectively. Red line in inset of figure (D) and (F) shows the fitting to the equation (2).

Figure 6: FT-IR absorption spectra of ZnO nanoparticles dispersed in (A) PD fluid, (B) Glucose, (C) Lactic acid and (D) Citric acid. The FT-IR absorption spectra of free lactic acid, glucose and citric acid has also been shown in the respective plots and have been shifted vertically for the purpose of clarity. Arrows in (A), (C) and (D) indicate the red-shifts of bonded COOH with respect to free COOH. The time course of absorption spectra of ZnO NPs in PD fluid, lactic acid and citric acid revealed the decrease in the intensity of absorption peak with increase in time. The kinetics of ZnO NPs bioconjugates formation in PD fluid, lactic acid and citric acid was studied by fitting the change in



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 28

absorption intensity vs. time curve with a non-linear least square fitting based on LevenbergMarquardt algorithm of Microcal Origin 7.5 with the following exponential association equation (inset of Figures 5(D) and (F)): 𝑡

𝐼𝑡 = 𝐼0 + 𝐴1 [1 − exp (1 − 𝑡 )]

(2)

1

where I0 and It are the absorption intensities at time zero and t, respectively. The constant A 1 is the relative contribution and t1 is the corresponding time constant of mechanism involving in interaction between ZnO and the constituents. The value for t1 comes out to be 29.32.6, 2473.7 and 21.10.9 minutes for PD fluid, lactic acid and citric acid, respectively. The exponential association confirms that the binding starts immediately after incorporation of ZnO NPs into PD fluid and/or lactic acid. Surface adsorption of lactic acid and citric acid on ZnO NPs was further investigated by recording FT-IR absorption spectra (Figure 6). The characteristic stretching frequency at 1600-1620 cm-1, corresponding to the C=O stretch confirm the adsorption of lactic acid and citric acid moleclues on ZnO NPs dispersed in PD fluid, lactic acid and citric acid soltuions, respectively. The red shift in the C=O stretch by ∼120-130 cm-1 from ∼1728 cm-1 of a free lactic and citric acid as indicated by arrows in Figures 6(A), 6(C) and 6(D) has been observed. Similar red shift has been reported in various metal oxide nanoparticles coated by various ligands with carboxylic acid groups. 30-33 The observed red shift in the C=O stretch in FT-IR spectra confirms interaction and chemical binding of the carboxylic acid (COOH) group with ZnO NPs leading to the formation of ZnO-lactate and ZnO-citrate nanocomposites. Figure 7(A) shows the schematic representation of agglomeration of ZnO NPs dispersed in PD fluid due to lactate adsorption on the surface of NPs. Figure 7 (B) and (C) shows the most probable bonding structures of lactic acid and citric acid with ZnO NPs according to the FT-IR results. Further, the estimation of number of lactic acid and citric acid molecules adsorbed on ZnO nanoparticles, when dispersed in PD fluid, lactic acid and citric acid


Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


solutions (2 mg/ml in each case) has been carried out and is given in Table 2. The surface area normalized uptake has also been enlisted in Table 2. The surface area of ZnO nanoparticles was approximately equal to 1541.3 cm2/mg. The initial and final concentrations were first quantified from the NMR spectra of various sample solutions prepared before and after the addition of ZnO NPs, respectively and the difference of the duo gives a rough estimate of the concentration of adsorbed lactic and citric acid molecules on the surface of ZnO NPs. Table 2. Surface coverages for lactic acid and citric acid on ZnO nanoparticles in PD fluid, lactic acid and citric acid solutions (suspension concentration for ZnO NPs was 2mg/ml in each cases). Solution Number of molecules adsorbed Surface coverage (molecules cm-2) PD fluid 1.9×1018 6.3  0.057×1015 19 Lactic Acid 1.0×10 3.2  0.029×1016 Citric Acid 6.9×1018 2.2  0.021×1016 Generally, lactic acid and citric acid can deprotonate in suspensions resulting in carboxylate groups, which can interact and form a strong coordinating complex with the surface of Zinc oxide nanoparticles. The adsorption of lactate and citrate on ZnO NPs has been confirmed from zeta potential measurements as a significant decrease in the surface potential was observed due to partial neutralization of positive charge upon interaction with negatively charged carboxylate groups. Studies on the adsorption of citric acid on TiO2 NPs surface has also been reported in literature.34 The observed shift in the surface charge alongwith the ionic strength of PD fluid (~135.5 mM) seems to be enough to overcome the activation barrier for aggregation which is further supported by Derjaguin, Landau, Verwey, and Overbeek theory (DLVO theory), according to which the nanoparticle-nanoparticle interactions and thus the stability of a colloidal suspensions are determined by the sum of van der Waals attractive forces and electrostatic repulsion as shown in Figure 7(D).34,35 While the van der walls attractive forces are a function of particle size and the interparticle interactions, the electrostatic repulsive forces are due to surface charge and electrostatic interactions as measured by the zeta potential.34 Therefore, the ZnO nanoparticle suspensions in 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 28

distilled water are stable with the large nanoparticle-nanoparticle repulsive barrier (as demonstarted by high zeta potential) and thus we do not see any aggregation of nanoparticles. Due to the adsorption of lactate and citrate on NPs, the magnitude of repulsive forces are not strong enough to prevent particle coming together and the attractive van der Waals forces dominates eventually leading to the agglomeration of ZnO-lactate and ZnO-citrate nano-composites (as seen in the TEM images) reducing their high surface energy. Similar agglomeration of ZnO NPs in biological media has also been reported due to the binding of proteins and/or biomolecules onto the surface of NPs.36 However, contrary to the reported interaction of glucose with ZnO NPs by Samanta et al.19 we have not observed any interaction and/or binding of glucose with ZnO NPs (Figure 7(E)) in PD fluid and glucose solution as clearly revealed by DLS, TEM, UV-Vis., FT-IR and NMR spectroscopy. The surface chemistry of ZnO nanoparticles also plays an important role to control the nano-bio interface interactions as the molecules in media may interact with nanoparticles surface through chemical bonding. It is well established that the hydrolysis of ZnO particle surface takes place in deionized water due to adsorption of water molecules with simultaneous formation of Zn(OH)2(s) surface (s) layer (equation 3).37,38 The formed zinc hydroxide is soluble in water and becomes more soluble with increase and decrease in pH. The surface chemistry involved at low pH for ZnO-NPs in aqueous (aq) solution has already been well described previously via the following equations (36):37,38 ZnO(s)  H 2O  Zn(OH)2(s)

water adsorption on the surface

(3)

2+ 2Zn(OH)2(s)  Zn (aq) +O(aq) +H 2 O

metal oxide dissolution

(4)

At low pH (

Physicochemical Transformations of ZnO Nanoparticles Dispersed in

Recommend Documents