Keto â Enol Tautomerism of Temperature and pH Sensitive Hydrated

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Keto – Enol Tautomerism of Temperature and pH Sensitive Hydrated Curcumin Nanoparticles, Their Role as Nanoreactors, and Compatibility with Blood Cells Rajpreet Kaur, Poonam Khullar, Aabroo Mahal, Anita Gupta, Narpinder Singh, Gurinder Kaur Ahluwalia, and Mandeep Singh Bakshi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03893 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Journal of Agricultural and Food Chemistry

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Keto – Enol Tautomerism of Temperature and pH Sensitive Hydrated Curcumin Nanoparticles, Their Role as Nanoreactors, and Compatibility with Blood Cells

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Rajpreet Kaur3, Poonam Khullar3*, Aabroo Mahal3, Anita Gupta4, Narpinder Singh5, Gurinder Kaur Ahluwalia2, Mandeep Singh Bakshi1* 1

Department of Natural and Applied Sciences, University of Wisconsin - Green Bay, 2420 Nicolet Drive, Green Bay, WI 54311-7001, USA. 2Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, NL A2V 2K7 Canada. 3Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar 143005, Punjab, India. 4Amity Institute of Applied Sciences, AUUP, Noida 201304, India. 5Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, Punjab, India.

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Abstract

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In order to provide a solution for poor aqueous solubility and poor bioavailability of

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curcumin, we present the synthesis and characteristic features of water soluble curcumin

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hydrated nanoparticles (CNPs). They are stable and nearly monodisperse in aqueous phase where

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keto form of curcumin self-assembles into spherical CNPs, but are highly sensitive to the

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temperature and pH variations. The CNPs are quite stable up to 40 oC and at neutral pH. Higher

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temperature range reduces their hydration and makes them unstable, thereby disintegrating them

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into smaller aggregates. Similarly, higher pH converts the keto form of CNPs into enol form

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promoting their inter-particle fusions driven by the hydrogen bonding with remarkable color

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change from yellow to bright orange-red demonstrating their excellent photophysical behavior.

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The stable keto form CNPs are highly efficient nonreactors for the in situ synthesis of Au, Ag,

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and Pd NPs which are simultaneously entrapped in curcumin aggregates, thus promoting the

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metal NPs carrying ability of curcumin aggregates. The CNPs also demonstrate their excellent

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dose dependent biocompatibility with blood cells. A concentration range up to 5 mM of CNPs is

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quite safe for their applications in biological systems.

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ACS Paragon Plus Environment


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Key words: Curcumin nanoparticles, keto – enol form of curcumin, fluorophore, metallic

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nanoparticles, hemolysis

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Introduction

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Curcumin is an active component of turmeric and possesses remarkable antioxidant, anti-

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inflammatory, antitumor, and antimicrobial properties.1-3 Its aqueous insoluble nature is mainly

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responsible for its poor bioavailability and hence, prevents its physiological absorption.1 A large

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amount of curcumin dose usually ≤ 20 g per day is required to demonstrate its potential. Poor

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aqueous solubility of curcumin is an advantage to produce its self-assembled hydrated

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nanoparticles in aqueous phase that can be used as biomarkers because of its excellent

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photophysical properties in addition to several clinical applications including drug delivery

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vehicles.4-6 In aqueous phase, it exists in keto form while enolic form exists in organic solvents

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(Fig 1a).7,8 The keo – enol tautomerism can be easily controlled by the pH and exhibits dramatic

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influence on the photophysical properties of curcumin in aqueous phase. This mechanism even

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becomes more interesting when curcumin exists in the self-assembled nanoparticles as depicted

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in Fig 1a. The shape, size, and stability of the hydrated curcumin nanoparticles (CNPs) in

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aqueous phase are directly related to the pH of the medium. The keto form provides yellow color

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and is highly fluorescent while the enol form is bright red and becomes slowly non-florescent

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with the increase in the pH. Another important parameter which significantly influences the

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stability of CNPs is the temperature. Since CNPs are the self-assemblies where poor or insoluble

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amphiphilic molecules aggregate in such a way so that they can shield their non-polar functional

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groups from the aqueous phase as shown in Fig 1a. By doing so, they entrap a water pool in the

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anterior of the self-assembled state. Thus, the molecular arrangement of these molecules in the

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hydrated nano-assemblies becomes highly temperature sensitive. Such CNPs assemblies have

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dual advantages in terms of indicators or markers because they are both pH as well as

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temperature sensitive.9-11 We want to highlight these remarkable properties of CNPs along with

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their bioapplicability.

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In view of the antioxidant properties of curcumin, hydrated CNPs also act as excellent

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nanoreactors for the synthesis of Au, Ag, and Pd nanoparticles (NPs) because curcumin

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possesses a strong reducing ability to convert Au(III) or Ag(I) or Pd(II) into their respective

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Au(0), Ag(0), and Pd(0) oxidation states.12-15 Efficient nanoreactors are those self-assembled


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morphologies which possess the ability to confine the oxidant in self-assembled state and then

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locally induce the reduction to produce nucleating centres which ultimately convert into NPs.16-18

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CNPs are highly efficient in doing so because they provide perfect environment to solubilize and

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collect the metal ions inside the hydrated environment and then initiate the reduction by

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simultaneously controlling their crystal growth without involving any external reducing or

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stabilizing agent.19-21 Thus, both pH and temperature can stimulate the release of metal NPs from

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CNPs and can be simultaneously monitored by the photophysical properties. We demonstrate the

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mechanism of synthesis, stability, and bioapplicability of CNPs in terms of keto – enol

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tautomerism, and their role as nanoreactors for the synthesis of Au, Ag, and Pd NPs.

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Experimental

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Materials

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Curcumin, (1E, 6E) - 1,7 –Bis (4-hydroxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione

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(analytical standard), chloroauric acid (HAuCl4), silver nitrate (AgNO3), and potassium

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tetrachloropalladate (K2PdCl4), all more than 99 % pure, were purchased from Aldrich. Double

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distilled water was used for all preparations.

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Preparation of curcumin nanoparticles (CNPs)

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CNPs were prepared by dissolving required amount of curcumin (2 - 50 mM) in

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chloroform in a screw capped glass tube. Chloroform was evaporated under the flux of pure N2

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leaving a dried curcumin film at the bottom of the tube. Then, 5 ml of pure water was added

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along with few small glass beads and vortexed it for at least 5 minutes to completely disperse the

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curcumin in aqueous phase. This procedure produced a stable suspension of hydrated CNPs. It is

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to be mentioned that CNPs suspensions were quite stable for a period of one month while the

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CNPs produced from the high concentration of curcumin tend to destabilized after 2 – 3 weeks of

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preparation due to the inter-particle fusions. Inter-particle interactions expected to produce small

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aggregates of the CNPs those tend to settle under gravity.

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CNPs as nanoreactors

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In situ synthesis of Au, Ag, and Pd NPs was carried out by using aqueous suspension of

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known concentration of CNPs along with required amount of [HAuCl4 / AgNO3 / K2PdCl4] =



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0.25 – 1.0 mM without using any external reducing or stabilizing agent. Curcumin acts as a weak

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reducing agent while its CNPs assemblies provide necessary colloidal stability for the growing

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NPs. This reaction under constant stirring for 6 hours at 70 oC produce metallic NPs of Au, Ag,

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and Pd which are simultaneously entrapped in the hydrated CNPs because the reduction reaction

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takes place inside the hydrated pool (Fig 1a). The color of the solution changed from light yellow

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to brown black indicating the formation of Au, Ag, or Pd NPs. Purification of CNPs loaded Au,

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Ag, and Pd NPs was done in two steps process. In the first step, as prepared suspension of the

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NPs was centrifuged at 12,000 rpm for 5 min to obtain the concentrated suspension of NPs. In

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the second step, it was further diluted with distilled water and repeated the first step twice to get

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the only CNPs entrapped metallic NPs. This allowed the removal of excess of CNPs without

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metallic NPs and the purified suspension contained only CNPs entrapped tiny Au, Ag, or Pd

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NPs. This suspension was quite stable over the period of one month. A stable colloidal

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suspension allowed us to perform several spectroscopic analyses with required accuracy.

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Methods

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Spectroscopic analysis: The stability of CNPs was studied with respect to pH and

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temperature by simultaneously measuring the UV-visible (Shimadzu-Model No. 2450, double

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beam) and steady state fluorescence (PTI QuantaMaster) spectra. Both instruments were

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equipped with a TCC 240A thermoelectrically temperature controlled Cell Holder that allowed

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to measure the spectrum at a constant temperature within ± 1 oC.

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Microscopy: All samples were characterized by Transmission Electron Microscopic

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(TEM) analysis on a JEOL 2010F at an operating voltage of 200 kV. The samples were prepared

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by mounting a drop of a solution on a carbon coated Cu grid and allowed to dry in the air. DLS

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and zeta potential measurements were performed using a light scattering apparatus (Zetasizer

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Nano series, Nano-ZS, Malvern Instruments) equipped with a built-in temperature controller

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with an accuracy of ± 0.1 oC. The measurements were made using a quartz cuvette with a path

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length of 1 cm. Average of 5 measurements were analyzed using the standard algorithms with an

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uncertainty of less than 7 %.

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Hemolytic assay: In order to determine the bioapplicability of CNPs, hemolytic assay

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was performed on blood group B of red blood cells from a healthy human donor. Briefly, 5%

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suspension of blood cells was used for this purpose after giving three washings along with three


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concentrations (i.e. 25, 50, and 100 µg/ml) of each sample. 1 ml packed cell volume (i.e.

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hematocrit) was suspended in 20 ml of 0.01 M phosphate buffered saline (PBS). The positive

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control was blood cells in water and it was prepared by spinning 4 ml of 5% blood cells

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suspension in PBS. PBS as supernatant was discarded and pellet was resuspended in 4 ml of

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water. The negative control was PBS. All the readings were taken at 540 nm i.e. absorption

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maxima of hemoglobin.

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Results and discussion

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Characteristic features of CNPs

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Concentration effect

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CNPs have been characterized by different techniques. Some of the representative TEM

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images of CNPs are shown in Fig 1b, whereas Fig 1c show the images of CNPs along with tiny

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Au NPs. DLS (Fig 1d, inset, representative size distribution histogram) provides better

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information about the dynamic nature of CNPs in the fluid state and other size distribution

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histograms for some of the samples are shown in the Supporting Information, Fig S1a-e. A

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variation of the CNPs size and zeta potential with concentration are plotted in Fig 1d. A

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somewhat larger size of CNPs from DLS in comparison to TEM is due to the hydrated nature of

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vesicles in the former case in comparison to the dried state in the latter case. Size appears to

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decrease with the increase in the amount of curcumin after passing through a maximum which

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indicates the formation of large aggregates initially that slowly transforms into monodisperse

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CNPs. Zeta potential on the other hand is always negative and does not change significantly. The

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negative zeta potential of CNPs is expected in view of electron rich hydroxyl, ether oxygens, and

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carbonyl moieties.22,23

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Temperature effect

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The stability of the CNPs can be simultaneously monitored by UV-visible and

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fluorescence measurements with respect to temperature, and the results are presented in Fig 2a

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and Fig S2, respectively. The UV-visible absorption spectrum at 20 oC does not show any clear

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characteristic peaks of curcumin in both UV and visible regions except small broad hump around

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400 nm due to the light yellow color of the CNPs suspension. But as temperature increases, the



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intensity of absorbance falls with the emergence of typical curcumin spectrum around 44 oC with

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prominent peaks around 350 and 420 nm,22,24 whose intensity increase with the further increase

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in the temperature. On the other hand, the intensity of the fluorescence emission spectrum of

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CNPs at 540 nm decreases with the increase in temperature from 20 – 70 oC (Fig S2).25-27 A

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variation in the intensity profiles of both UV-visible and fluorescence spectra is depicted in Fig

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2b which indicates that the intensity of the absorbance passes through a strong minimum around

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44 oC while emission intensity also shows a pronounced fall around this temperature. Thus, 44

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o

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results in the de-stability or deformation of the hydrated CNPs that reduces the self-aggregation

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of curcumin molecules and hence, produces characteristic curcumin absorbance spectrum (Fig

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2a). Dismantling or deformation of the CNPs also induces higher randomness and greater degree

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of inter-molecular collisions with the result radiative decay converts to non-radiative decay and

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causes an instant fall in the emission intensity (Fig 2b).

C represents a maximum temperature of CNPs stability and further increase in the temperature

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This is further evident from the simultaneous measurements of size and zeta potential of

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CNPs with temperature (Fig 2c). CNPs size starts decreasing as soon as the temperature

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increases but tends to constant with possibility of formation of small sized aggregates beyond 35

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o

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indicated by Fig 2b from both UV-visible and fluorescence studies. Zeta potential does not show

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any marked variation over the temperature range because a change in the self-aggregation

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behavior from large to small aggregates of curcumin is not supposed to influence its zeta

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potential within the temperature studied.

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pH effect

C which is essentially the same temperature range where CNPs dismantling or deformation is

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CNPs are also highly pH sensitive due to the well-known keto – enol tautomerism.28 Keto

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form is thermodynamically favoured at room temperature. However, a treatment of CNPs

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solution with base converts the keto into enol form with significant change in the fluorescence

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intensity and dramatic color change from bright yellow to orange-red (Fig 3a). It causes a large

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decrease in the fluorescence intensity with a red shift of ~ 50 nm (inset, Fig 3a). Both properties

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show an abrupt change at pH 11.2 where CNPs merge with one another to produce large

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aggregates which is quite clear from the DLS measurements (Fig 3b). In fact, a significant

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degree of self-aggregation converts the radiative decay into non-radiative decay due to enhanced


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inter-molecular collisions and hence, it causes a large drop in the emission intensity in Fig 3a.

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The formation of large aggregates also prompts the increase in the negative zeta potential which

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remains more or less close to ~30 mV after the onset of aggregation (Fig 3b). Enol form is

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obviously expected to have strong intermolecular hydrogen bonding in comparison to keto and

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hence, produces favorable conditions for enhanced self-aggregation to produce large

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aggregates.29

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CNPs as nanoreactors

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Keto form of CNPs makes them as excellent nanoreactors for the in situ synthesis of

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nanomaterials.12-15 This happens when different metal ions driven by the electrostatic interactions

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prefer to solubilize in the hydrated CNPs rather than in the bulk where they get reduce by

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curcumin from Au(III)/Ag(I)/Pd(II) oxidation state to zero oxidation state (Fig 4a). The

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nucleating centres thus created (as shown in the second step of the flow diagram) simultaneously

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attain the colloidal stability because of their solubilisation in the hydrated pool and hence, they

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undergo control nucleation growth to produce respective NPs of small dimensions.19 The

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reduction reaction works well at 70 oC because it provides appropriate annealing to the growing

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metal NPs and is monitored simultaneously by the fluorescence measurements to understand the

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stability of CNPs. Fig 4b shows fluorescence behavior of curcumin in a typical reduction

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reaction from 20 – 70 oC. Curcumin emission shows a dramatic increase with a strong maximum

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within a temperature range of 40 – 50 oC (inset) with a large blue shift of about 35 nm. In fact,

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this temperature range represents the onset of deformation of CNPs as depicted in Fig 2b and c,

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but shows a marked maximum in the fluorescence intensity (Fig 4b, inset) rather than a regular

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fall with temperature as indicated by Fig 2b. The origin of these contrasting behaviors is due to

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the onset of nucleating among the Au atoms and simultaneous adsorption of curcumin on solid –

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liquid interface to provide the colloidal stabilization. This causes a dismantling of the self-

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assembled arrangement that induces an instant increase in the emission intensity.29 However,

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further increase in the temperature increases the intermolecular collisions leading to the

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conversion of radiative decay into non-radiative decay.

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TEM images (Fig 5) indicate the presence of small aggregates of NPs of ≤ 20 nm

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entrapped by the deformed curcumin aggregates. Fig 5a and (Fig S3) show close up images of

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single deformed curcumin aggreate entrapping Ag NPs prepared by taking 27 mM curcumin (see



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experimental for the preparation of vesicles), and 0.5 and 1 mM of AgNO3, respectively. Each

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NP surface is coated with a thick layer of ~ 4 nm curcumin (Fig 5b). The size distribution

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histogram of this sample with size 169±25 nm is shown in Fig 5c along with the negative zeta

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potential of 14.3±7.0 mV which is lower than that of CNPs (~25 mV, Fig 1d). A lower value of

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zeta potential in the former case is expected on the basis of charge neutralization of curcumin in

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the event of its NPs surface adsorption. Similarly, Fig 5d is representing the TEM image of

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curcumin aggregate entrapping Au NPs prepared by under similar reaction conditions, and DLS

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histogram along with the zeta potential are shown in Fig 5e. There is not much difference in the

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shape and size of Ag (Fig 5a) and Au NPs (Fig 5d) though one can observe several Au nanorods

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along with small mainly spherical Au NPs. Similar reaction for the synthesis of Pd NPs do not

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produce such assemblies entrapping NPs rather long interconnected strands of lamellar curcumin

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entrapping tiny Pd NPs (Fig 5f) and this lamellar structure even becomes more intense with the

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increase in the concentration of K2PdCl4 from 0.25 mM to 1 mM (Fig S4). The origin of this is

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related to the highly reactive nature of Pd nano-surface that encourages the adsorption and

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rearrangement of other molecules30,31 in terms of its catalytic behavior. It promotes the excessive

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adsorption that in turn drives the inter-CNPs fusions and thus, producing lamellar structures.

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Bioapplicability

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Bioapplicability of CNPs in systemic circulation in terms of pharmaceutical formulations

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can be evaluated by performing the hemolytic assay.32-34 Usually, charged lipid vesicles exhibit

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high hemolysis in media containing low concentrations of electrolytes, and it is reduced when

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the surface potential of the cells is made more positive or by increasing concentrations of

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electrolytes.35 Hemolysis also depends on the concentration and size of the vesicles. Coated

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vesicles usually cause less hemolysis.35 Hemolysis results of present samples are presented in Fig

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6. Panel a shows a series of samples with different doses of CNPs in blood suspension along with

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positive and negative controls while panel b shows the same samples after centrifugation. Panel c

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depicts the optical micrograph of blood sample with 5 mM of curcumin vesicles. Most of the

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blood cells are intact demonstrating little hemolysis which is also evident from the representative

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UV-visible plots of heme absorption within 500 to 600 nm of wavelength range with positive

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and negative controls (inset).36,37 The percentage hemolysis = [(sample absorbance - negative

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control absorbance)/(positive control absorbance-negative control absorbance) x 100] evaluated


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from the UV-visible spectra for different samples is compared in the corresponding bar graphs

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(Fig 6d). Negligible hemolysis is observed up to 5 mM of CNPs, however, increase in the

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concentration significantly increases the hemolysis (inset Fig 6d). Thus, 5 mM concentration of

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CNPs is the maximum safe concentration for their appropriate applications as drug delivery

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vehicles in the systemic circulations. Previous studies also noted that the curcumin based

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liposomal formulations in fact induce dose-dependent changes in the shape of blood cells and

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allows dose dependent administration intravenously.33,38

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From the above results, we conclude that it is possible to produce well defined nearly

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monodisperse CNPs in an aqueous phase by carefully controlling the concentration so as to

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produce stable suspension that can be used for various applications. The hydrated CNPs thus

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produced are highly sensitive to the temperature and pH change which make them excellent drug

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release vehicles under a change in these parameters. Since, CNPs are highly fluorescent and their

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photophysical behavior again depends on both temperature and pH, therefore, they can be best

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used below 40 oC and at neutral pH as fluorophores for the biological systems. pH has also

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dramatic effect on the fluorescence intensity and color change of CNPs, that makes it excellent

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pH responsive indicator. In relevance to nanomaterials synthesis in biocompatible systems,

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CNPs demonstrate their high potential as nanoreactors for the in situ synthesis of Au, Ag, and Pd

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nanoparticles and confine them in aggregated assemblies. Finally, CNPs are biocompatible and

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show only dose dependent toxicity towards blood cells. A concentration of up to 5 mM is

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considered to be quite safe for using them in systemic circulation without inducing any

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hemolysis.

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Acknowledgment: These studies were partially supported by the financial assistance from

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UWGB, NAS, Green Bay, and DST under nanomission research project [ref no: SR/NM/NS-

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1057/2015(G)], New Delhi. Dr Gurinder Kaur thankfully acknowledges the financial support

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provided by the Research and Development Council (RDC) of Newfoundland and Labrador,

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NSERC, and the Office of Applied Research at CNA. P.K. acknowledges the TEM studies done

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by SAIF Lab, Nehu, Shillong.

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Supporting Information: UV-visible, TEM images, and DLS plots. This information is

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available free of charge via the internet at https://pubs.acs.org/.


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References

268

1. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Bioavailability of

269

Curcumin: Problems and Promises. Mol. Pharmaceutics 2007, 4, 807-818.

270

2. Yang, H.; Du, Z.; Wang, W.; Song, M.; Sanidad, K.; Sukamtoh, E.; Zheng, J.; Tian, L.; Xiao,

271

H.; Liu, Z.; Zhang, G. Structure Activity Relationship of Curcumin: Role of the Methoxy

272

Group in Anti-Inflammatory and Anticolitis Effects of Curcumin. J. Agric. Food Chem. 2017,

273

65, 4509-4515.

274 275

3. Nelson, K. M.; Dahlin, J. L.; Bisson, J.; Graham, J.; Pauli, G. F.; Walters, M. A. The Essential Medicinal Chemistry of Curcumin. J. Med. Chem. 2017, 60, 1620-1637.

276

4. Bhawana; Basniwal, R. K.; Buttar, H. S.; Jain, V. K.; Jain, N. Curcumin Nanoparticles:

277

Preparation, Characterization, and Antimicrobial Study. J. Agric. Food Chem. 2011, 59, 2056-

278

2061.

279

5. Loo, C. Y.; Rohanizadeh, R.; Young, P. M.; Traini, D.; Cavaliere, R.; Whitchurch, C. B.; Lee,

280

W. H. Combination of Silver Nanoparticles and Curcumin Nanoparticles for Enhanced Anti-

281

Biofilm Activities. J. Agric. Food Chem. 2016, 64, 2513-2522.

282

6. Gangwar, R. K.; Tomar, G. B.; Dhumale, V. A.; Zinjarde, S.; Sharma, R. B.; Datar, S.

283

Curcumin Conjugated Silica Nanoparticles for Improving Bioavailability and Its Anticancer

284

Applications. J. Agric. Food Chem. 2013, 61, 9632-9637.

285

7. Santin, L. G.; Toledo, E. M.; Carvalho-Silva, V. H.; Camargo, A. J.; Gargano, R.; Oliveira, S.

286

S. Methanol Solvation Effect on the Proton Rearrangement of Curcumin Enol Forms: An Ab

287

Initio Molecular Dynamics and Electronic Structure Viewpoint. J. Phys. Chem. C 2016, 120,

288

19923-19931.

289 290

8. Payton, F.; Sandusky, P.; Alworth, W. L. NMR Study of the Solution Structure of Curcumin. J. Nat. Prod. 2007, 70, 143-146.

291

9. Luo, Z.; Tikekar, R. V.; Nitin, N. Click Chemistry Approach for Imaging Intracellular and

292

Intratissue Distribution of Curcumin and Its Nanoscale Carrier. Bioconjugate Chem. 2014, 25,

293

32-42.

294 295

10.

Vandita, K.; Shashi, B.; Santosh, K. G.; Pal, K. I. Enhanced Apoptotic Effect of

Curcumin Loaded Solid Lipid Nanoparticles. Mol. Pharmaceutics 2012, 9, 3411-3421.



Page 12 of 23

12

296

11.

Liu, K.; Guo, T. L.; Chojnacki, J.; Lee, H. G.; Wang, X.; Siedlak, S. L.; Rao, W.; Zhu,

297

X.; Zhang, S. Bivalent Ligand Containing Curcumin and Cholesterol As a Fluorescence Probe

298

for Aβ Plaques in Alzheimer’s Disease. ACS Chem. Neurosci. 2012, 3, 141-146.

299

12.

El Kurdi, R.; Patra, D. Tuning the Surface of Au Nanoparticles Using Poly(Ethylene

300

Glycol)-Block-Poly(Propylene Glycol)-Block-Poly(Ethylene Glycol): Enzyme Free and Label

301

Free Sugar Sensing in Serum Samples Using Resonance Rayleigh Scattering Spectroscopy.

302

Phys. Chem. Chem. Phys. 2018, 20, 9616-9629.

303 304 305 306 307 308 309 310 311

13.

Sindhu, K.; Rajaram, A.; Sreeram, K. J.; Rajaram, R. Curcumin Conjugated Gold

Nanoparticle Synthesis and Its Biocompatibility. RSC Adv. 2014, 4, 1808-1818. 14.

Bettini, S.; Pagano, R.; Valli, L.; Giancane, G. Drastic Nickel Ion Removal From

Aqueous Solution by Curcumin-Capped Ag Nanoparticles. Nanoscale 2014, 6, 10113-10117. 15.

Yang, X. X.; Li, C. M.; Huang, C. Z. Curcumin Modified Silver Nanoparticles for Highly

Efficient Inhibition of Respiratory Syncytial Virus Infection. Nanoscale 2016, 8, 3040-3048. 16.

Voronov, A.; Kohut, A.; Peukert, W. Synthesis of Amphiphilic Silver Nanoparticles in

Nanoreactors From Invertible Polyester. Langmuir 2007, 23, 360-363. 17.

Zhang, G.; Tang, S.; Li, A.; Zhu, L. Thermally Stable Metallic Nanoparticles Prepared

312

Via Core-Cross-Linked Block Copolymer Micellar Nanoreactors. Langmuir 2017, 33, 6353-

313

6362.

314

18.

Khullar, P.; Singh, V.; Mahal, A.; Kumar, H.; Kaur, G.; Bakshi, M. S. Block Copolymer

315

Micelles As Nanoreactors for Self-Assembled Morphologies of Gold Nanoparticles. J. Phys.

316

Chem. B 2013, 117, 3028-3039.

317 318 319

19.

Bakshi, M. S. How Surfactants Control Crystal Growth of Nanomaterials. Crystal

Growth & Design 2016, 16, 1104-1133. 20.

Khullar, P.; Mahal, A.; Singh, V.; Banipal, T. S.; Kaur, G.; Bakshi, M. S. How PEO-

320

PPO-PEO Triblock Polymer Micelles Control the Synthesis of Gold Nanoparticles:

321

Temperature and Hydrophobic Effects. Langmuir 2010, 26, 11363-11371.

322

21.

Khullar, P.; Singh, V.; Mahal, A.; Kaur, H.; Singh, V.; Banipal, T. S.; Kaur, G.; Bakshi,

323

M. S. Tuning the Shape and Size of Gold Nanoparticles With Triblock Polymer Micelle

324

Structure Transitions and Environments. J. Phys. Chem. C 2011, 115, 10442-10454.


Page 13 of 23


13

325

22.

Singh, P.; Wani, K.; Kaul-Ghanekar, R.; Prabhune, A.; Ogale, S. From Micron to Nano-

326

Curcumin by Sophorolipid Co-Processing: Highly Enhanced Bioavailability, Fluorescence,

327

and Anti-Cancer Efficacy 2014, 4, 60334-60341.

328

23.

Xu, Y. Q.; Chen, W. R.; Tsosie, J. K.; Xie, X.; Li, P.; Wan, J.; He, C. W.; Chen, M. W.

329

Niosome Encapsulation of Curcumin: Characterization and Cytotoxic Effect on Ovarian

330

Cancer Cells. Journal of Nanomaterials 2016, 1-9.

331

24.

Dutta, A.; Boruah, B.; Manna, A. K.; Gohain, B.; Saikia, P. M.; Dutta, R. K. Stabilization

332

of Diketo Tautomer of Curcumin by Premicellar Anionic Surfactants: UV−Visible,

333

Fluorescence, Tensiometric and TD-DFT Evidences. Spectrochimica Acta Part A: Molecular

334

and Biomolecular Spectroscopy 2013, 104, 150-157.

335

25.

Singh, D.; Jagannathan, R.; Khandelwal, P.; Abraham, P.; Poddar, P. In Situ Synthesis

336

and Surface Functionalization of Gold Nanoparticles With Curcumin and Their Antioxidant

337

Properties: An Experimental and Density Functional Theory Investigation. Nanoscale, 2013, 5,

338

1882-1893.

339

26.

Prasad, S.; Achazi, K.; Bottcher, C.; Haag, R.; Sharma, S. Fabrication of Nanostructures

340

Through Self-Assembly of Non-Ionic Amphiphiles for Biomedical Applications. RSC Adv.,

341

2017, 7, 22121-22132.

342

27.

Gogoi, B.; Sen Sarma, N. Curcumin−Cysteine and Curcumin−Tryptophan Conjugate As

343

Fluorescence Turn On Sensors for Picric Acid in Aqueous Media. ACS Appl. Mater.

344

Interfaces 2015, 7, 11195-11202.

345

28.

Kharat, M.; Du, Z.; Zhang, G.; McClements, D. J. Physical and Chemical Stability of

346

Curcumin in Aqueous Solutions and Emulsions: Impact of PH, Temperature, and Molecular

347

Environment. J. Agric. Food Chem. 2017, 65, 1525-1532.

348

29.

Jagannathan, R.; Abraham, P. M.; Poddar, P. Temperature-Dependent Spectroscopic

349

Evidences of Curcumin in Aqueous Medium: A Mechanistic Study of Its Solubility and

350

Stability. J. Phys. Chem. B 2012, 116, 14533-14540.

351

30.

Long, R.; Mao, K.; Ye, X.; Yan, W.; Huang, Y.; Wang, J.; Fu, Y.; Wang, X.; Wu, X.;

352

Xie, Y.; Xiong, Y. Surface Facet of Palladium Nanocrystals: A Key Parameter to the

353

Activation of Molecular Oxygen for Organic Catalysis and Cancer Treatment. J. Am. Chem.

354

Soc. 2013, 135, 3200-3207.



Page 14 of 23

14

355

31.

Moreno, M.; Ibanez, F. J.; Jasinski, J. B.; Zamborini, F. P. Hydrogen Reactivity of

356

Palladium Nanoparticles Coated With Mixed Monolayers of Alkyl Thiols and Alkyl Amines

357

for Sensing and Catalysis Applications. J. Am. Chem. Soc. 2011, 133, 4389-4397.

358

32.

Yallapu, M.; Ebeling, C.; Chauhan, N.; Jaggi, M.; Chauhan, C. Interaction of Curcumin

359

Nanoformulations With Human Plasma Protein and Erythrocytes. Int J Nanomedicine 2011,

360

6, 2779-2790.

361

33.

Storka, A.; Vcelar, B.; Klickovic, U.; Gouya, G.; Weisshaar, S.; Aschauer, S.; Helson, L.;

362

Wolzt, M. Effect of Liposomal Curcumin on Red Blood Cells In Vitro. Anticancer Res. 2013,

363

33, 3629-3634.

364

34.

Toda, S.; Ohnishi, M.; Kimura, M.; Nakashima, K. Action of Curcuminoids on the

365

Hemolysis and Lipid Peroxidation of Mouse Erythrocytes Induced by Hydrogen Peroxide.

366

Journal of Ethnopharmacology 1988, 23, 105-108.

367 368 369

35.

Martin, F. J.; MacDonald, R. C. Lipid Vesicle-Cell Interactions. I. Hemagglutination and

Hemolysis. J Cell Biol 1976, 70, 494. 36.

Bakshi, M. S. Nanoshape Control Tendency of Phospholipids and Proteins:

370

Protein−Nanoparticle Composites, Seeding, Self-Aggregation, and Their Applications in

371

Bionanotechnology and Nanotoxicology. J. Phys. Chem. C 2011, 115, 13947-13960.

372 373 374 375

37.

Bakshi, M. S. Nanotoxicity in Systemic Circulation and Wound Healing. Chem. Res.

Toxicol. 2017, 30, 1253-1274. 38.

Ramvilas, N.; Verma, R. Aflatoxin Induced Hemolysis and Its Amelioration by Turmeric

Extracts and Curcumin in Vitro. Acta Poloniae Pharmaceutica 2007; 165-168.

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Figure caption

385

Fig 1. (a) Keto – enol tautomerism of curcumin driven by the pH. Inset shows the possible

386

arrangement of curcumin molecules in the CNPs. (b) and (c) TEM images of CNPs prepared by

387

using 16.5 mM of curcumin in pure water and in the presence of tiny Au nanoparticles,

388

respectively. (d) shows the variation in the size and zeta potential of CNPs with respect to their

389

concentration. Inset shows a representive size distribution histogram of CNPs of (b). See details

390

in the text.

391

Fig 2. (a) shows the UV-visible absorbance of CNPs of 40.7 mM with respect to temperature

392

variation. (b) depicts the variation in the absorbance and emission intensities with temperature.

393

(c) shows the variation in the size and zeta potential of CNPs with respect to temperature. See

394

details in the text.

395

Fig 3. (a) Fluorescence emission spectra of CNPs of 2.7 mM with respect to pH variation. Note

396

the color change from light yellow to dark orange red. Inset shows the variation of emission

397

intensity and WL shift with pH. (b) shows the variation of size and zeta potential measurements

398

with pH. See details in the text.

399

Fig 4. (a) Fluorescence emission spectra of in situ reaction of CNPs of 40.7 mM and 0.25 mM

400

AgNO3 with temperature. Inset shows the variation of the emission intensity with temperature.

401

(b) Proposed mechanism of the in situ synthesis of Ag NPs in the presence of CNPs. See details

402

in the text.

403

Fig 5. (a) TEM image of curcumin aggregates containing Ag NPs of less than 50 nm in size

404

prepared by taking the concentration of 40.7 mM of CNPs and 0.5 mM AgNO3. (b) High

405

resolution image showing the 4 nm thick curcumin coating on Ag NPs. (c) shows the size

406

distribution histogram and negative zeta potential of this sample. (d) TEM image of curcumin

407

aggregates containing Au NPs of ~20 nm in size prepared by taking the concentration of 40.7

408

mM of CNPs and 0.5 mM HAuCl4. (e) shows the size distribution histogram and negative zeta

409

potential of this sample. (f) TEM image of curcumin strands containing tiny Pd NPs of ~5 nm in

410

size prepared by taking the concentration of 40.7 mM of CNPs and 0.5 mM K2PdCl4. See details

411

in the text.



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412

Fig 6. Panels (a) and (b) show the blood samples + CNPs, positive control (1), negative control

413

(2), 5 mM CNPs (3), 8 mM CNPs (4), and 24 mM CNPs (5), before and after the centrifugation,

414

respectively. (c) shows the optical micrograph of sample (3) showing maximum of the blood

415

cells are intact. Inset, typical UV-visible profiles of heme absorption of blood suspension in the

416

presence of different amounts of CNPs along with positive and negative controls. (d) Plots of

417

hemolysis % versus dose of CNPs in the concentration range of 0.4 – 5 mM at different

418

incubation times. Inset, shows the hemolysis % at higher concentrations with incubation time of

419

1 hour. See details in the text.

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434


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Fig 2 0.7

20 22 24 26 28 30 32 34 36 38 40 44 48 52 56 60 64 68 70

0.65

Absorbance

0.6 0.55 0.5 0.45 0.4 0.35 0.3 200

a 300

400

500

600

700

800

Wavelength / nm 0.6

80

0.55

70 0.5 60 50 40 10

0.45

b 20

Absorbance

Intensity (fluorescence) / a.u.

90

30

40

50

60

70

0.4 80

Temperature / oC 350

0 -5

Size / nm

-10 250

-15

200

-20 -25

150

-30 100 50 10

436

c 20

-35 30

40

50

60

Temperature / oC


70

-40 80

Zeta potential / mV

300

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TOC

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Keto – Enol Tautomerism of Temperature and pH Sensitive Hydrated Curcumin Nanoparticles, Their Role as Nanoreactors, and Compatibility with Blood Cells

448 449 450

Rajpreet Kaur, Poonam Khullar, Aabroo Mahal, Anita Gupta, Narpinder Singh, Gurinder Kaur Ahluwalia, Mandeep Singh Bakshi

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Keto â Enol Tautomerism of Temperature and pH Sensitive Hydrated

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