Development and characterization of lactoferrin-GMP nanohydrogels: Evaluation of pH, ionic strength and temperature effect

Accepted Manuscript Development and characterization of Lactoferrin-GMP nanohydrogels: Evaluation of pH, ionic strength and temperature effect Ana I. Bourbon, Ana C. Pinheiro, Maria G. Carneiro-da-Cunha, Ricardo N. Pereira, Miguel A. Cerqueira, António A. Vicente PII:

S0268-005X(15)00087-9

DOI:

10.1016/j.foodhyd.2015.02.026

Reference:

FOOHYD 2901

To appear in:

Food Hydrocolloids

Received Date: 16 September 2014 Revised Date:

4 February 2015

Accepted Date: 9 February 2015

Please cite this article as: Bourbon, A.I., Pinheiro, A.C., Carneiro-da-Cunha, M.G., Pereira, R.N., Cerqueira, M.A., Vicente, A.A., Development and characterization of Lactoferrin-GMP nanohydrogels: Evaluation of pH, ionic strength and temperature effect, Food Hydrocolloids (2015), doi: 10.1016/ j.foodhyd.2015.02.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

Development and characterization of Lactoferrin-

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GMP nanohydrogels: Evaluation of pH, ionic strength

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and temperature effect

Ana I. Bourbon a, Ana C. Pinheiro a, Maria G. Carneiro-da-Cunha b,

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Ricardo N. Pereira a, Miguel A. Cerqueira a, * and António A. Vicente a *

Corresponding author: [email protected], Phone: (+351) 253 601962/601986

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Fax: (+351) 253 604429

CEB – Centre of Biological Engineering, Universidade do Minho, Campus de

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Gualtar, 4710-057 Braga, Portugal

Departamento de Bioquímica, Laboratório de Imunopatologia Keizo Asami

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(LIKA), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, s/n, Cidade Universitária, CEP 50.670-420 Recife, Pernambuco, Brazil

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Abstract Lactoferrin (Lf) and glycomacropeptide (GMP), two bioactive proteins from milk, were used to produce nanohydrogels by electrostatic interaction and thermal gelation. Parameters such as protein concentration, molar ratio, pH,

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temperature and heating time were evaluated in order to develop the nanohydrogels, which were characterized in terms of morphology (Transmission Electron Microscopy - TEM, Dynamic Light Scattering – DLS, Atomic Force Microscopy – AFM and Confocal Laser Scanning Microscopy – CLSM) and stability. The aggregation of the mixture between Lf and GMP increased with

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the increase of temperature, resulting in particles with lower hydrodynamic diameter and polydispersity index (PdI). Nanohydrogels were obtained from the

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mixture of Lf and GMP solutions (0.02% (w/w) in molar ratio 1:7) at pH 5.0, and subsequently stirred and heated at 80 °C, during 20 min. Results showed that nanohydrogels have a spherical shape with a hydrodynamic diameter around 170 nm, a PdI of 0.1 and a swelling ratio of 30. The minimum size of nanohydrogels depends on protein concentration and molar ratio. Decreasing the protein concentrations and increasing the content of GMP molecules in

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solution, the hydrodynamic diameter and PdI of nanohydrogels decreased. The electrical charge values of nanohydrogels at different pH values suggest that Lf molecules are in the surface and GMP molecules are mostly in the core of the

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structure (confirmed by confocal microscopy). Also, it was observed that nanohydrogels’ hydrodynamic diameter and PdI, after formation, are influenced when submitted at different conditions of pH, ionic strength and temperature. Lf-

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GMP nanohydrogels showed properties that indicate that they are a promising delivery system for food and pharmaceutical applications.

Keywords: milk proteins; biopolymer nanoparticles; electrostatic interaction; thermal gelation; whey proteins

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1 Introduction

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One of the challenges in food engineering is the development of safe bio-

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systems that can protect, carry and deliver functional food components, which

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can be achieved using natural polymers. Protein-based nanohydrogels have

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attracted considerable attention due to their non-toxicity, biodegradability and

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small dimension with a large interior network for multivalent bioconjugation,

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which offers several possibilities for the encapsulation of functional components

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by covalent bonds (Bengoechea, Peinado, & McClements, 2011; Daniel-da-

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Silva, Ferreira, Gil, & Trindade, 2011). Food protein nanohydrogels are a

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network stabilized by hydrophobic interactions, disulfide bonds, and hydrogen

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bonds resulting from gelation process. During the heat induced gelation, the

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native protein conformation becomes unfolded (exposing functional groups such

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as sulfhydryl or hydrophobic groups) and the aggregation process occurs

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through disulfide bonds and hydrophobic interactions, in order to minimize the

16

energy of the system. Then a large increase in elasticity occurs, resulting from

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the formation of multiple hydrogen bonds upon cooling (Batista, Portugal,

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Sousa, Crespo, & Raymundo, 2005). However gelling can also occur in the

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absence of sulphydryl/disulphide interchange reactions. In this case hydrogen

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bonding, electrostatic and hydrophobic interactions can be important in protein

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aggregation.

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Lactoferrin (Lf) is a basic, positively charged iron-binding glycoprotein of the

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transferrin family with a molecular weight of 80 kDa and an isoelectric point

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around 7.8-8.5, present in various external secretions of mammals such as milk

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(Brisson, Britten, & Pouliot, 2007; Tokle, Decker, & McClements, 2012; van der

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Strate, Beljaars, Molema, Harmsen, & Meijer, 2001). One of the main interests

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in Lf resides in its various biological activities such as: antimicrobial, anti-

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inflammatory,

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increasing the interest in its use as a natural bioactive ingredient in food and

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health products (Brisson, et al., 2007; González-Chávez, Arévalo-Gallegos, &

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Rascón-Cruz, 2009). Casein-macropeptide (CMP) is one of the most abundant

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peptides in cheese whey with typical concentrations between 15% and 25% of

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total whey proteins (El-Salam, El-Shibiny, & Buchheim, 1996). Glycosylated

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antitumour,

immuno-modulatory

and

enzymatic

activities,

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carbohydrate content (N-acetylneuraminic acid (sialic acid), galactose, and

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Nacetylgalactosamine) without aromatic amino acids. It is an acidic peptide with

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isoelectric point between 4 and 5, highly soluble in water and heat stable

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(Thomä-Worringer, Sørensen, & López-Fandiño, 2006). GMP has been

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reported to have a wide range of bioactive properties such as: antibacterial

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activity, modulation of immune system responses and control of blood

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circulation (Thomä-Worringer, et al., 2006). Due to its biological activities and

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high potential as an ingredient for functional food and pharmaceuticals, great

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attention has been given to GMP application in recent years (Nakano & Ozimek,

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2000; Thomä-Worringer, et al., 2006; Xu, Sleigh, Hourigan, & Johnson, 2000).

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Interactions between natural biopolymers, such as peptides or proteins, under

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specific conditions (e.g. pH, temperature, heating time, ionic strength and

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concentration) can promote the formation of hydrogels with improved functional

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properties in comparison with those of proteins alone. These biopolymer-based

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nanohydrogels must be carefully designed and manufactured so that they

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exhibit the required functional attributes within the final product (e.g. optical

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properties,

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properties and physicochemical stability) (Owen Griffith Jones & McClements,

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2010). In this process, the biopolymer selection to form nanohydrogels will

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depend on a number of factors, such as: biopolymers characteristics (molecular

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weight, types of structure (globular or linear proteins), solubility, charge,

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isoelectric point) and preparation technique (heat induced gelation, use of

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cross-linkers, effect of pH and ionic strength). These factors are extremely

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important to control the ability of biopolymers to assemble into particles,

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influencing their functional attributes (e.g. size, structure, charge, permeability,

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and stability to environmental and solution conditions) (Morimoto, Nomura Shin-

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ichiro, Miyazawa, & Akiyoshi, 2006; Schmitt, et al., 2009; Yu, Yao, Jiang, &

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Zhang, 2006). This research work provides important information on the effect

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of specific conditions (such as temperature, concentration and ionic strength) in

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the development of nanohydrogels from a globular protein (Lf) and a

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glycosylated peptide (GMP) which can be useful for the design of e.g. nano-

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structures for food and pharmaceutical applications.

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properties,

release

characteristics,

encapsulation

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2 Materials and Methods

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2.1 Nanohydrogel preparation

Purified Lf powder was obtained from DMV International (USA). The reported

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composition expressed as a dry weigh percentage was: 96% protein, 0.5% ash,

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3.5% moisture and an iron content around 120 ppm. Commercial GMP was

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obtained from Davisco Food International, INC. (Le Sueur, USA) and its

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reported composition was: 82.5% protein, 1% fat, 7% ash and 7% moisture.

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Lf and GMP aqueous solutions were prepared separately (with concentrations

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in the range of 0.02-2.0% (w/w) using deionized water purified to a resistance of

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15 MΩ (Millipore, France). Protein solutions were prepared by adding deionized

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water to a weighted amount of Lf or GMP powder and then stirring (60 rpm) at

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room temperature (25 ºC) for 1 h. The pH values of biopolymer solutions were

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separately adjusted to 5.0, with 0.1 mol/L hydrochloric acid (Panreac, Spain). Lf

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aqueous solution was added dropwise into GMP aqueous solution with gently

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stirring until final molar ratio (MR) of Lf to GMP ranging between 1:7, 1:4 and

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1:2.

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After gentle stirring for 30 min, the pH of the mixture was adjusted to different

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values of pH (ranging between 2.5 and 10.5) with 0.1 mol/L hydrochloric acid or

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sodium hydroxide (Riedel-de Haen, Germany). The mixture was subsequently

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heated at 60, 70 or 80 ºC for 10, 30 or 60 min in a water bath (closed system) to

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obtain a homogeneously dispersed nanohydrogel solution. The solutions were

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stored at 4 °C until further utilization. All the e xperiments in which were tested

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the stability of environmental conditions on nanohydrogels properties, were

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performed with an incubation time of 48 h, in order to ensure the stability of

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particles (i.e. after this time the nanohydrogels did not show any change in

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hydrodynamic diameter and charge).

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2.2 ζ-potential

ACCEPTED MANUSCRIPT ζ-potential measurement was carried out at room temperature (25 oC) on a

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Zetasizer Nano ZS (Malvern Instruments, UK) in a folded capillary cell using a

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He-Ne laser-wavelength of 633 nm and a detector angle of 173°. The

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measurements were made in triplicate, with three readings for each sample.

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The results are given as the average ± standard deviation of nine

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measurements.

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2.3 Nanohydrogel hydrodynamic diameter and polydispersity index

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The mean hydrodynamic diameter of nanohydrogels was determined by

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dynamic light scattering (DLS) in a Zetasizer Nano ZS (Malvern Instruments

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Limited, UK). Cycles of temperature were also performed using DLS; at each

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temperature the sample was equilibrated at least 2 min before measurement.

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Each sample was analysed in cuvettes with a path length of 12 mm. The values

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reported correspond to polydispersity index (PdI) and Z-average diameter, that

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is, the mean hydrodynamic diameter, and represent the average ± standard

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deviation of nine measurements.

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The swelling ratio of the nanohydrogels was calculated based on the ratio of

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the wet volume to the dry volume. Wet volume was estimated based on the

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hydrodynamic diameter obtained by DLS measurements and dry volume was

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estimated based on diameter obtained by TEM and AFM measurements. The

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diameter of dried nanohydrogels was determined by measuring diameters of

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100-150 particles in a TEM and AFM images, using ImageJ 1.48v program.

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2.4 Quartz Crystal Microbalance (QCM)

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QCM measurements were carried out in a multi-frequency quartz crystal

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microbalance (QCM 200, Stanford Research Systems, SRS, USA), equipped

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with AT-cut quartz crystals (5 MHz) with optically flat polished titanium/gold

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electrodes in contact and liquid sides. Adsorption measurements were carried

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out by alternate immersion of the crystal into 0.2 mg/mL Lf (pH 5.0) and GMP

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(pH 5.0) solutions, for 15 minutes. The variations of resonance frequency (∆F)

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and of the motional resistance (∆R) were simultaneously measured as a

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function of time; analyses were made in triplicate at 25 ºC (Pinheiro, et al.,

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2012).

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2.5 Visible (Vis) Absorbance Changes of transparency of Lf-GMP mixtures with a WR of 1:2 were recorded

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at 600 nm in a spectrophotometer with controlled temperature (Lamda 35,

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Perkin-Elmer, Germany) at 25, 60, 70 and 80 ºC with deionized water as blank.

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2.6 Fluorescence measurements Fluorescence

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spectrofluorimeter (Horiba Scientific) equipped with a standard thermostated

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cell holder. The excitation wavelength was 290 nm. Emission spectra were

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recorded between 300 and 400 nm with 1% attenuation, and fluorescence

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intensities were recorded every 0.5 nm. Excitation and emission slits were

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15 nm. Data analysis of fluorescence peak was performed with Peak Fit 4.12

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(SYSTAT Software Inc., Richmond, CA, USA) program.

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2.7 Atomic Force Microscopy (AFM) AFM samples were prepared by drying the solution naturally on a freshly

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cleaved mica surface at room temperature (25 ºC). Images were acquired in

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tapping mode on a Digital Instruments NanoscopeIIIa (Veeco Instruments,

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USA).

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2.8 Transmission Electron Microscopy (TEM)

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TEM micrographs were conducted on a Zeiss EM 902A (Thornwood, N. Y.)

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microscope at an accelerating voltage of 50 kV and 80 kV. The samples were

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prepared by dropping nanohydrogel solutions onto copper grids coated with

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carbon film and followed by natural drying.

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2.9 Confocal laser scanning microscopy Confocal laser scanning microscopy (Olympus Fluoview, FV 1000, USA) was

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used to visualize the distribution of Lf and GMP in nanohydrogels. Lf and GMP

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were labelled with Fluorescein Isothiocyanate - FITC (Fluka, Germany) and

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Rhodamine B Isothiocyanate - RBITC (Sigma-Aldrich, USA), respectively.

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Briefly, the pH of each protein solution (2 mg/ml) was adjusted to 9.3 via dialysis

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against 2 L of 100 mM sodium carbonate pH 9.3, at 24 ºC for 24 hours. 0.0015

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g of a FITC or Rhodamine solution (3 mg of corant in 1 mL of 100 mM sodium

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carbonate pH 9.3) was added to the protein solution and incubated at 20 ºC for

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1 h. Unattached corant was removed by dialysis (cut off 100-500 Da;

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Spectra/Por CE; Spectrum laboratories; USA). After staining proteins,

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nanohydrogels were prepared as described before (please see section 2.1). In

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order to identify each protein, the laser was adjusted to green (FITC-labeled

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lactoferrin) or red (RBITC-labeled GMP) mode which yielded two excitation

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wavelengths at 488 (green laser) and 561 nm (red laser), respectively. The

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superposition of the images obtained in these two channels allowed visualizing

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the protein distribution in nanohydrogels, in the same image.

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2.10 Native polyacrylamide gel electrophoresis (Native-PAGE) In order to evaluate the heat treatment effects on nanohydrogel kinetic

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formation, patterns of Lf, GMP and Lf-GMP mixtures samples were analysed

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using Native-PAGE or “nondenaturing” gel electrophoresis. Native-PAGE

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analyses were carried out using the Mini-Protean II dual slab cell system

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equipped with a PAC 300 power supply (Bio-Rad Laboratories, Hercules, CA,

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USA). The resolving and stacking gel contained 10 and 4% of polyacrylamide,

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respectively. The gels were stained with silver nitrate methodology (Chevallet,

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Luche, & Rabilloud, 2006). Standard marker proteins PageRuler Unstained

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Broad Range Protein Ladder from Thermo Scientific, was used to identify

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molecular weight of samples.

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2.11 Influence of environmental stresses on nanohydrogels stability

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pH stability: The influence of pH on nanohydrogels stability was determinate

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by preparing a series of samples with aqueous solutions adjusted to values

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ranging from pH 2 to 10 using either 0.1 mol/L HCl or 0.1 mol/L NaOH. Salt stability: The influence of ionic strength on nanohydrogels stability (pH

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5.0, at 25 ºC) was examined by adding 0 to 200 mM NaCl to nanohydrogels

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after preparation.

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Temperature stability: The influence of temperature on nanohydrogels stability (pH 5.0) was examined in DLS as explained in section 2.3.

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Stability of nanohydrogels throughout time: Nanohydrogels solutions were

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stored at 4 °C and their hydrodynamic diameter and PdI was evaluated by DLS

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during five months.

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2.12 Statistical analyses

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Statistical analyses of the data were carried out using Analysis of Variance

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(ANOVA) and Tukey mean comparison test (p<0.05) (Statistica® 7, Statsoft,

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Tulsa, OK, USA).

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3 RESULTS AND DISCUSSION

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3.1 Electrostatic properties of the biopolymers

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In order to evaluate the pH conditions that promote the development of

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electrostatic interactions between Lf and GMP, ζ-potentials of individual

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solutions of each biopolymer and of the product of ζ-potential values from the

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two solutions were determined (Fig 1A).

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Lf solution showed a change in its charge from positive ζ-potential (13.5 ±

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1.4 mV) at pH 1.5 to negative ζ-potential (-19.3 ± 1.5 mV) at pH 10.0, with a

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point of zero charge around the pH 7.7, as reported in previous publications

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2014; Tokle, et al., 2012). On the other hand, GMP charges ranged between

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20.81 ± 1.47 and -41.12 ± 4.98 for pH ranging between 1.5 and 10.0. The

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isoelectric point was found to be at pH 3.7, which is in agreement with other

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reported values (Thomä-Worringer, et al., 2006). The product of ζ-potentials

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reaches a minimum at pH 5.0, indicating that the strongest electrostatic

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attraction between these two biopolymers should happen at this pH value.

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Electrostatic interactions between Lf and GMP were clearly seen by QCM

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measurements (Fig. 1B) in which the response signal (of resonant frequency –

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Delta F) changes are assumed to be proportional to the mass of substance

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deposited to the electrode surface (Pinheiro, et al., 2012). Fig. 1B shows the

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consecutive decrease of frequency after the alternate immersion of the crystal

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in Lf and GMP solutions, which means that mass is being subsequently

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deposited confirming the electrostatic interaction between these biopolymers.

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3.2

Effect of heating temperature and time on nanohydrogel formation

In order to evaluate the influence of thermal process on nanohydrogels

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formation, a heat treatment at different temperatures and time were carried out

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to induce intermolecular hydrophobic association and promote the association

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between Lf and GMP. Heating individual solutions of Lf or GMP do not show a

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significant change (p>0.05) in transparency values, indicating that no significant

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aggregation happened when protein solutions were treated alone (Fig. S.1).

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From Table S.1 and Table S.2 is possible observe the effect of temperature on

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hydrodynamic diameter and PdI values of Lf and GMP individual solutions,

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respectively. Increasing temperature did not show a significant change in

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hydrodynamic diameter of protein solutions. Furthermore, it is possible observe

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that Lf and GMP solutions present a high value of PdI suggesting that these

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solutions are not monodisperse. However, when the mixture of the two proteins

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is submitted to the heating treatment, a gradual decrease of transparency is

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observed with the increase of temperature from 60 to 80 ºC (Fig. S.1). During

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the gelation process, it is possible observe that the decrease of transparency is

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proportional to the decrease of hydrodynamic diameter and PdI values obtained

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for Lf-GMP mixtures (Table S.3).

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ACCEPTED MANUSCRIPT It also was observed that the increase of temperature led to a faster and more

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extensive aggregation of the mixture. The transparency decrease is presumably

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an indication of changes in the conformation and aggregation state of proteins

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during heating: when proteins unfold, they expose nonpolar groups normally

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buried in their non-polar interior, which leads to aggregation through

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hydrophobic attraction (Hoffmann, Sala, Olieman, & de Kruif, 1997). We believe

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that this behaviour is due the presence of globular protein (Lf) once GMP

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structure did not change with temperature, as expected for a peptide.

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Bengoechea et al., 2011 showed by DSC that native Lf has two thermal

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transitions: the first transition around 65.1 ºC corresponding to the apo-

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lactoferrin form and the second transition around 89-92 ºC which corresponds

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to the holo-lactoferrin form (Bengoechea, Peinado, et al., 2011). In this work,

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the authors pre-heated Lf at 75 ºC for 20 min (a temperature which is between

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the two thermal denaturation peaks) in order to avoid the completely

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denaturation of protein and when analysed the turbidity measurements they

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observed that the turbidity remained relatively constant and close to zero when

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the samples were heated from 25 to 60 °C. However a pronounced increase in

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turbidity occurred from 60 to 80 °C, suggesting tha t the observed increases in

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turbidity correspond to protein aggregation induced by thermal denaturation of

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the globular proteins. We believe that as reported by these authors, the effect of

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this temperature range is due the conformational changes on Lf structure.

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Also, Brisson et al. (2007) analysed that heating holo-Lf at 80 °C led to

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soluble polymer formation whereas apo and native Lf associated into large

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insoluble aggregates. The thermal aggregation of holo-Lf was mainly driven by

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non-covalent interactions, with intermolecular thiol/disulphide reactions also

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observed above 80 °C (Brisson, et al., 2007).

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The effect of heating time and temperature in the Lf-GMP mixture properties

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was also evaluated by DLS (Fig. 2). Fig. 2A shows that after 10 min of heating,

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nanohydrogels are formed and the increase of heating time for each

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temperature

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nanohydrogels solutions (PdI<0.2). Results also show that for a temperature of

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80 ºC during 20 min and a molar ratio of 1:7 Lf to GMP, the nanohydrogels were

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formed with a minimal hydrodynamic diameter and PdI values (320 ± 10 nm and

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0.12 ± 0.03, respectively), with a single peak in the size distributions (results not

results

in

a

PdI

decrease,

resulting

in

homogeneous

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shown). These experiments are useful for determining optimum processing

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conditions required to form Lf-GMP nanohydrogels by thermal processing. If the

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time is too short, then protein aggregation will not have fully occurred. On the

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other hand, if the time is too long, there will be unnecessary energy and time

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costs (Benichou, Aserin, Lutz, & Garti, 2007). In order to analyze the effect of heating time on the conformational and

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structural changes of Lf and Lf-GMP mixtures, intrinsic fluorescence of

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tryptophans (Trp) residues was performed. Trp fluorescence wavelength is

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known to be sensitive to the polarity of its local being a useful tool to monitor

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changes in proteins and to make inferences regarding local structure network

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and dynamics (Uversky, Narizhneva, Kirschstein, Winter, & Löber, 1997; Vivian

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& Callis, 2001)

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It was observed that the averaged emission fluorescence spectra for Trp,

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presented a typical maximum near to 336 nm representing the emission band

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Trp which is presented in different parts of the Lf, in agreement with data

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reported in the literature (Fang, et al., 2014).

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The changes in the fluorescence intensity and wavelength of the maximum

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intrinsic fluorescence spectrum (λmax) was used to follow the structural changes

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of the protein induced by heating time at 80 ºC. An increase of fluorescence

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intensity at λmax was observed increasing the heating time of protein solutions at

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80 °C during 20 min (Fig. 3A). This difference in f luorescence intensity as a

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function of heating time reflects changes in the compactness of the protein

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molecule due to molecules unfolding, and increasing accessibility of Trp

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residues. As shown in Fig. 3A it was also observed that Lf used as control has

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a higher fluorescence intensity than Lf-GMP mixtures, suggesting that the

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interaction between these two molecules are influencing the accessibility of Trp

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residues presented in Lf. For instance, LF used as control had a λmax at about

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336.2 nm while the λmax of LF–GMP mixtures was significantly blue-shifted to

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331.6 nm (see Fig. 3B). The blue-shift of λmax suggests that Trp residues are

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less exposed to the solvent being placed in the nonpolar core of the system Lf-

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GMP (Uversky, et al., 1997).

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A native electrophoreses was performed in order to evaluate the kinetic

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formation of Lf-GMP nanohydrogels during 20 min at 80 ºC (Fig. 4).

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Electrophoresis profile of Lf (lane 1) revealed the presence of other proteins

ACCEPTED MANUSCRIPT bands, reflecting the partial purity of the Lf used. Lf was identified as the band at

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79 KDa. This profile and molecular weight were also observed by other authors

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(Bourbon, et al., 2011; Lindmark-Månsson, Timgren, Aldén, & Paulsson, 2005).

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The analysis of GMP (lane 2) gave a unique band with a mass of 25 KDa,

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corresponding to GMP. Lane 3 corresponds to the electrophoresis profile of Lf-

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GMP mixture without heating treatment and it is possible to observe the

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presence of two bands: the lower band indicate the GMP presence and the

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higher band reflects the LF presence. Lane 4, 5, 6 and 7 is the kinetic formation

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of nanohydrogels during the heating time of 5, 10, 15 and 20 min at 80 ºC,

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respectively. It is possible to observe that after 10 min, occurs the aggregation

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of Lf and GMP protein. This is traduced by the appearance of a new smear

343

band with much higher molecular weight, suggesting formation of a

344

nanohydrogel network.

346 347

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3.3 Effect of biopolymer concentration and molar ratio (MR) on the formation of Lf-GMP nanohydrogels

348

In this section, the influence of Lf and GMP concentrations and MR on the

350

hydrodynamic diameter and PdI of the nanohydrogels was analysed. Initially,

351

was evaluated the effect of increasing protein concentrations for a MR of 1:4 of

352

Lf to GMP. Results showed that higher protein concentration leads to increasing

353

hydrodynamic diameter values, from 320 ± 10 nm to 5.0 ± 0.2 µm for 0.02 to

354

2.0% (w/w) protein concentration, respectively. The effect of protein

355

concentration on hydrodinamic diameter was already reported by other authors,

356

which pointed out an increase in particles diameter with increased biopolymer

357

concentration (Hu, Yu, & Yao, 2007).

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The interactions between Lf and GMP depend not only upon the protein

359

concentration of the solution but also upon their ratio of mixture. Therefore,

360

different WR were evaluated for 0.02% of Lf and 0.02% of GMP (concentration

361

that allow the lower values of hydrodynamic diameter and PdI of

362

nanohydrogels). Hydrodynamic diameters decrease when the ratio of Lf to GMP

363

(MR) is 1:7, together with a PdI around 0.1 and a single peak in the size

364

distribution.

ACCEPTED MANUSCRIPT Anema & de Kruift (2012) evaluated the interaction between Lf and Κ-casein

366

in order to obtain stable complexes coacervates, and observed that 1 molecule

367

of Lf interact for 4 molecules of GMP, and at these conditions the positive

368

charges are equal to negative charges and consequently a larger particles were

369

formed. Based on this, and due the fact that in this work we are using a glycol-

370

peptide obtained from K-casein, we believe that the smaller hydrodynamic

371

diameter and PdI obtained for a MR 1:7 of Lf to GMP can be justified by the fact

372

that at this conditions the electrostatic interactions are higher and due the

373

hydrophilic properties and glycosylated part of the peptide, the hydrophobic

374

interactions between Lf and GMP are more evident, reflecting a smaller and

375

stable particle (Anema & de Kruif, 2012). Therefore, the MR of 1:7 was chosen

376

to proceed with the study of Lf and GMP nanohydrogels (Table 1).

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3.4 Characterization of Lf-GMP nanohydrogels

The morphology of nanohydrogels was observed by TEM (Fig. 5). Individual

381

Lf and GMP solutions with the same thermal treatment used to develop

382

nanohydrogels were analyzed and it was observed that no nano spherical

383

particles were formed (Fig. 5A and Fig. 5B). However, when the mixture of Lf

384

and GMP are treated with thermal treatment, nanohydrogels are observed by

385

TEM (Fig. 5C and Fig. 5D), the presence of particles with a spherical shape and

386

with a mean diameter of 80 nm. These images confirm the formation of the Lf-

387

GMP nanogels, corroborating the previous results, however the hydrodynamic

388

diameter is lower than observed by DLS, this can be due to drying process of

389

particles realized to prepare samples for TEM visualization.

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390

Additionally, AFM images were analyzed to determine the average surface

391

roughness of each system (Fig. 6). Solutions were fixed to mica slides by

392

desiccation of dilute sample solutions based on preliminary experiments that

393

indicated particle structure maintenance during lyophilization. The AFM images

394

of Lf-GMP nanohydrogels showed uniform spherical particles, with the

395

dimensions of the structures observed being less than 70 nm and with a smooth

396

surface.

397

ACCEPTED MANUSCRIPT 398

The swelling ratio of the nanohydrogels is about 30 calculated by the ratio of

399

the wet volume to the dry volume (170 nm)3/(70 nm)3. This calculate was used

400

based on DLS measurements for the diameter of particles in solution and in

401

TEM and AFM micrographs for dry samples (Huang, Remsen, Kowalewski, &

402

Wooley, 1999).

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3.5 Effect of environmental conditions on the stability of Lf-GMP nanohydrogels

406

Nanohydrogels may be exposed to a great variety of conditions when they are

408

incorporated into products, such as foods, cosmetics, personal care products

409

and pharmaceuticals (Bengoechea, Jones, Guerrero, & McClements, 2011). It

410

is therefore important to establish the effect of various environmental conditions

411

on their stability and functional properties. In this section, the nanohydrogels

412

produced at 80 ºC during 20 min, with MR 1:2, at pH 5.0 were selected based in

413

their low hydrodynamic diameter distribution and PDI values, and the influence

414

of environmental conditions (pH, ionic strength and temperature) on their

415

stability were evaluated (Fig. 7).

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The pH and ionic strength of the solutions were adjusted after nanohydrogels

417

formation. Fig. 7A shows ζ-potential of nanohydrogels and of lactoferrin and

418

GMP individually, (already showed in Fig. 1 and reported here again for

419

comparison purposes). The electrical charge values of nanohydrogels changed

420

from positive at low pH to negative at high pH, with a neutral charge around pH

421

6.0 which is close to the zero ζ-potential of Lf (Fig. 7A). This result suggests that

422

the surface of the nanohydrogels is enriched with Lf molecules and GMP

423

molecules are mostly in the core of the structure. These results are in

424

agreement with the results suggested by micrographs obtained by CLSM (Fig.

425

S.2). Two fluorescence channels, green and red lasers, were used to excite Lf

426

and GMP, respectively (Fig. S.2). Images obtained by CLSM allow identifying

427

the localization/distribution of Lf and GMP in the nanohydrogel structure. It was

428

observed that the intensity of FITC-labeled Lf fluorescence was higher than the

429

fluorescence emitted by RBITC-labeled GMP, suggesting that Lf can be more

430

distributed in the surface of nanohydrogels and GMP molecules are enriched in

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ACCEPTED MANUSCRIPT 431

the core of the nanohydrogels. Similar behavior was reported by other authors

432

that developed nanogels by self-assembly and thermal gelification of proteins

433

(Owen G. Jones, Decker, & McClements, 2009; Li, Yu, Yao, & Jiang, 2008; Yu,

434

Hu, Pan, Yao, & Jiang, 2006). Fig. 7B shows an evident effect of pH on nanohydrogels’ structure. After

436

nanohydrogels preparation, the pH of the solution in which these hydrogel

437

particles were dispersed was adjusted to different values to evaluate its

438

influence on their size distribution. Hydrodynamic diameter and PdI of

439

nanohydrogels increased from pH values below 6.0 and higher than 8.0 and

440

then decreased when pH values were in the range of 6.0-7.0. When pH is

441

between 2.0-3.8, Lf is practically fully protonated, while the net negative

442

charges of GMP decrease. For pH values of 8.0-10.0 there are electrostatic

443

repulsions between Lf and GMP because both have net negative charges. As a

444

consequence, the electrostatic attraction decreases causing an expansion of

445

nanohydrogels. In the pH range of 6.0-7.0, the electrostatic attraction between

446

Lf and GMP is higher, resulting in shrinkage of the nanohydrogels.

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The addition of salt to nanohydrogels lead to the particles aggregation for

448

lower salt concentrations, as can be seen by the increase of hydrodynamic

449

diameter and PdI values (Fig. 7C). Ionic strength may influence Lf–GMP

450

interactions by two mechanisms: (1) NaCl may screen the electrostatic

451

interaction, and (2) a high concentration of NaCl may alter the structural

452

organization of water molecules, which alters the strength of hydrophobic

453

interactions between nonpolar groups.

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For low concentrations of salt (< 100 mM) it was observed that the charge of

455

nanohydrogels is near to zero, resulting in more unstable particles which can

456

promote the formation of aggregates (Fig S.3). In terms of structural

457

conformation, it was observed an increase of fluorescence intensity for

458

nanohydrogels with salt concentrations until 100 mM, expressed by higher

459

areas obtained for the fluorescence peak. This behaviour suggest that the Trp

460

residues in Lf moved from an apolar environment to a more polar region and

461

interacted with water molecules (Fig. S.4). Increasing the salt concentration

462

(> 100 mM) a decrease of nanohydrogels hydrodynamic diameter and PdI

463

values was observed, which can be attributed to a new conformational

464

rearrangement of proteins. Fig. S.4 shows a decrease of area peak for this

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ACCEPTED MANUSCRIPT 465

range of NaCl concentration, reflecting the decrease of exposition of Trp

466

residues to polar environmental. This behaviour can be due to reshuffling of

467

protein molecular structure or to the establishment of new intermolecular

468

associations (e.g. hydrophobic interactions) between molecules. The effect of temperature was also assessed by heating and cooling

470

nanohydrogel solutions at temperatures ranging from 20 to 90 ºC at a rate of

471

10 ºC/min. The particle sizes after heating and cooling cycles were analysed by

472

dynamic light scattering (Fig. 7D). When nanohydrogels solutions were heated

473

from 20 to 90 ºC, the hydrodynamic diameter remained constant until 70 ºC and

474

increased slightly from 70 to 90 ºC. This behaviour may be indicative of

475

changes in the conformation state of nanohydrogels, corresponding to the

476

“aggregation temperatures”, as referred above. Fig. 7D shows that after cooling

477

to ambient temperature the hydrodynamic diameter of nanohydrogels

478

decreased.

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This behaviour can be due to the re-organization of molecular structure, once

480

in the cooling stage we are promoting an increase in elasticity, resulting from

481

the formation of multiple hydrogen bonds and this can be reflected by the

482

decrease of nanohydrogels size. This phenomen is usually reported in the

483

development of proteins gels at macro scale in which during cooling stage,

484

occurs the strengthening of the interparticle attractive non-covalent bonds such

485

as hydrogen bonds, as the temperature decreases. This is also reported as the

486

stabilization of the gel structure due the contribution of this kind of bonds (Ould

487

Eleya, Ko, & Gunasekaran, 2004).

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It is therefore important to establish the influence of heating on the stability

489

and physicochemical properties of Lf-GMP nanohydrogels. The PdI values

490

obtained ranged between 0.05 and 0.10 thus showing that size distribution is

491

quite narrow (results not shown). A single peak was observed in the particle

492

size distribution curves, thus leading to the conclusion that the aggregation of

493

particles in solution was minimal.

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494

Finally, it was evaluated the stability of Lf-GMP nanohydrogels after 5 months

495

of storage at 4 ºC (Fig. S.5B). It was observed that nanohydrogels’ suspension

496

does not change its size distribution significantly during the storage time. Also, it

497

was possible to verify that the size distributions of the rehydrated and original

498

nanohydrogels solutions did not show significant differences (p>0.05),

ACCEPTED MANUSCRIPT suggesting that the dissociation or aggregation during the process of

500

lyophilisation and rehydration did not occur (Fig. S.5C). This result confirms that

501

the cross-linking structure of the nanohydrogels can suppress dissociation upon

502

dilution. In another work in which proteins (ovalbumin and lysozyme) were used

503

to develop nanogels, it was observed that the intensities of the lyophilized and

504

original particle solutions are somewhat different; suggesting, a limited

505

aggregation after the lyophilisation process (Yu, Yao, et al., 2006). All these

506

valuable characteristics can offer significant benefits for the industrial

507

application of the nanogels.

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4 Conclusions

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This work has shown that at specific conditions Lf and GMP can form stable

512

nanohydrogels. The size of the particles formed during the corresponding

513

thermal treatment depended on protein concentration, molar ratio and

514

temperature/time of the heating process. Results also showed that the

515

produced nanohydrogels are stable at pH values ranging from 5.0-8.0 and

516

under high temperatures and high salt concentrations. The nanohydrogels

517

formed by thermal treatment may be useful as functional ingredients in

518

commercial products, such as foods, cosmetics and pharmaceuticals but may

519

also be used as a smart vehicle for bioactive compounds delivery.

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Acknowledgments

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The authors Ana I. Bourbon, Ana C. Pinheiro, Ricardo N. Pereira and Miguel

524

A. Cerqueira are recipient of fellowships from the Fundação para a Ciência e

525

Tecnologia,

526

SFRH/BD/73178/2010, SFRH/BD/48120/2008, SFRH/BPD/81887/2011 and

527

SFRH/BPD/72753/2010, respectively. Maria G. Carneiro-da-Cunha express is

528

gratitude to the Conselho Nacional de Desenvolvimento Científico e

529

Tecnológico (CNPq) for research grant. The authors would like to acknowledge

530

to Jorge Padrão from Centre of Biological Engineering, University of Minho and

POPH-QREN

and

FSE

(FCT,

Portugal)

through

grants

ACCEPTED MANUSCRIPT André Coelho from Federal University of Ceará for helping in electrophoresis

532

measurements and to Rui Fernandes from IBMC, University of Porto for

533

assistance in taking the TEM pictures. Also, the authors thank the FCT

534

Strategic Projects PEst-OE/EQB/LA0023/2013 and PEst-C/EQB/LA0006/2013

535

(FCT and FEDER funds through COMPETE). The work also received financial

536

support from the European Union (FEDER funds) under the framework of

537

QREN (Programa Operacional Regional do Norte (ON.2 – O Novo Norte;

538

FEDER) through projects NORTE-07-0124-FEDER-000028 and NORTE-07-

539

0124-FEDER-000069.

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Appendix A. Supplementary data

543

Transparency measurements (Fig. S.1), micrographs obtained by confocal laser

544

scanning microscopy of

545

nanohydrogels as a function of NaCl concentration (Fig. S.3), area peak of

546

fluorescence emission as a function of NaCl concentrations (Fig. S.4) and size

547

distributions of Lf-GMP nanohydrogels (Fig. S.4). Effect of temperature on Lf-

548

GMP dispersion (at pH 5.0) (Table S.1), Effect of temperature on Lf solutions

549

(0.02 % (w/w) at pH 5.0) (Table S.2), Effect of temperature on GMP solutions

550

(0.02 % (w/w)) at pH 5.0) (Table S.3).

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(Fig.

S.2),

ζ-Potential of

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the nanohydrogels

References

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ACCEPTED MANUSCRIPT Table captions

Table 1. Effect of different weight ratio of Lf:GMP solutions in nanohydrogels

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formation (0.02 % (w/w) at pH 5.0)

ACCEPTED MANUSCRIPT

Tables  

Table1. Hydrodinamic diameter (nm)

1:7

170 ± 3

1:4

320 ± 10

1:2

487 ± 9

0.08 ± 0.01

0.12 ± 0.03

0.32 ± 0.01

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PdI

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molar ratio (Lf:GMP)

 

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ACCEPTED MANUSCRIPT Figure captions

Figure 1. Electrostatic properties of biopolymers: A) ζ-Potential of Lf (●) and GMP (●) solutions as a function of pH. Inset figure shows the product of these ζ-potentials (molar ratio of 1:4) from pH 1.5 to 10.0. Each data point is the average and the error bars show the standard deviation,

GMP onto a gold electrode surface at pH 5.0.

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B) QCM response signal (resonant frequency, Delta F) for the sequential adsorption of Lf and

SC

Figure 2. Effect of temperature and heating time on the hydrodynamic diameter (size) (A) and polydispersity index (PdI) (B) of Lf-GMP nanogels (0.02 % (w/w), molar ratio of 1:4 and at

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pH 5.0).

Figure 3. Structural changes of Lf and Lf-GMP mixture monitored by: A) peak area of intrinsic fluorescence emission during heating time at 80 ºC for Lf (●) and Lf-GMP mixture (●); B) Intrinsic fluorescence emission spectra of Lf solution (●) and LF-GMP mixture (●) heated at

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Figure 4. Native-PAGE analysis of Lf (1), GMP (2), unheated mixture of Lf and GMP (3) and

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heated mixture of LF-GMP mixture during 5, 10, 15 and 20 min for lane 4, 5, 6 and 7,

Fig. 5 TEM micrographs of Lf (A), GMP (B), Lf-GMP nanohyrogels (C) and detail (higher magnification) of Lf-GMP nanohydrogels (D). The scale bar is (A) 0.5 µm, (B) 0.5 µm, (C) 1 µm and (D) 50 nm.

Fig. 6 AFM image of the Lf-GMP nanohydrogels.

ACCEPTED MANUSCRIPT Fig. 7 Effect of environmental conditions on the stability of nanohydrogels: A) ζ-potential curves of Lf-GMP nanohydrogels (●) (curves of individual Lf (♦) and GMP (♦) are shown for comparative purposes); B) Effect of pH adjustment after heat treatment on mean particle diameter (●) and PdI (■) for Lf-GMP nanohydrogels; C) Effect of NaCl concentration on mean particle diameter (●) and PdI (■) for Lf-GMP nanogels and D) Mean particle diameter of Lf-GMP

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by DLS. Dashed lines are shown for visual guidance only.

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nanogels during alternate increasing (♦) and decreasing (■) temperature cycles, as measured

ACCEPTED MANUSCRIPT   A) 

1000 800 600

(mV)2

30 20

400 200 0 ‐200

0

2

4

6

8

10

12

pH

‐400 ‐600

0 0

2

4

6

pH

8

10

12

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ϛ‐Potential (mV)

10

‐10 ‐20 ‐30 ‐40

B) 

Lactoferrin 

 

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GMP

Lactoferrin GMP

 

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A) 

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Fig. 2

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ACCEPTED MANUSCRIPT 150  KDa  100  KDa  70  KDa  70  KDa  50  KDa  40  KDa 

20  KDa  15 KDa  1

2

3

 

       

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6

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Fig.4

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30  KDa 

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B)

A) 

 

D)

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C)  C) 

Fig. 5

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Fig. 6

 

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A) 

 

500 400 300 200 100 0 0

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Size (d. nm)

0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

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600

5

PDI

B) 

10

 

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C) 

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pH

 

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D) 

 

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Fig.7

 

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Highlights Lactoferrin and Glycomacropeptide were used to produce stable nanohydrogels Interactions between proteins proved be extremely relevant in design of nanohydrogels

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Increasing gelation temperature led to a faster and more extensive aggregation of the mixture

Interactions between proteins depend not only upon the protein concentration but also

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upon their ratio of mixture

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Effect of environmental conditions were evaluated on Lf-GMP nanohydrogels stability

ACCEPTED MANUSCRIPT Table captions supplementary data Table S.1 Effect of temperature on size and polydispersity (Pdl) average of Lf solution (0.02 % at pH 5.0) and respective standard deviation (SD)

(0.02 % at pH 5.0) and respective standard deviation (SD)

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Table S.2 Effect of temperature on size and polydispersity (Pdl) average of GMP solution

Table S.3 Effect of temperature on size and polydispersity (Pdl) average of Lf-GMP mixture (at

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pH 5.0) and respective standard deviation (SD)

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Heating measurements

Table S.1 Effect of temperature on size and polydispersity (Pdl) average of Lf solution (0.02 % at pH 5.0)

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and respective standard deviation (SD) Temperature (º C)

Size average (nm)

SD

PdI average

SD

20

153

73

0,6

0,1

30

152

6

0,5

40

174

10

0,5

50

160

20

0,5

60

153

23

0,5

0,1

70

147

13

0,5

0,1

80

148

33

0,5

0,1

90

154

37

0,4

0,1

0,0

0,0

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0,1

3

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Table S.2 Effect of temperature on size and polydispersity (Pdl) average of GMP solution (0.02 % at pH 5.0) and respective standard deviation (SD) Size average (nm)

SD

PdI average

SD

20

423

26

0,4

0,6

30

357

14

0,5

0,1

40

370

19

0,5

50

331

10

0,5

60

314

20

0,5

70

327

11

0,5

80

327

11

0,4

90

330

12

0,4

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Temperature (º C)

0,3 0,1 0,1 0,1 0,1

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0,1

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Table S.3 Effect of temperature on size and polydispersity (Pdl) average of Lf-GMP mixture (at pH 5.0) and respective standard deviation (SD)

40 50 60 70 80

SD

0.7

0.3

800

127

0.4

796

207

0.6

700

142

0.6

397

35

0.6

521

41

320

10

486

39

0.1 0.3 0.2

0.2

0.4

0.1

0.1

0.0

0.3

0.0

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90

PdI average

121

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30

SD

1000

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20

Size average (nm)

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Temperature (º C)

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ACCEPTED MANUSCRIPT Figure captions Supplementary data

Figure S.1 Transparency of lactoferrin (♦), GMP (■) and lactoferrin-GMP mixtures (molar ratio of 1:7 at pH 5.0) (▲), at temperatures of 25 ºC (A); 60 ºC (B), 70 ºC (C) and 80 ºC (D) as a

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function of time. Dashed lines are shown for visual guidance only.

Figure S.2 Micrographs obtained by confocal laser scanning microscopy of the nanohydrogels: A) FITC-labeled lactoferrin and B) RBITC-labeled GMP and C) superposition of the images A

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and B.

Figure S.3 ζ-Potential of nanogels as a function of NaCl concentration in nanohydrogels

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solution.

Figure S.4 Peak Area of emission fluorescence of nanohydrogel solutions at different concentrations of NaCl.

Figure S.5 Size distributions of lactoferrin-GMP nanohydrogel: A) as prepared; B) after 5

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months of storage and C) after lyophilization and then rehydration (the three curves

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corresponds to the three true replicates of nine measurements realized for each one).

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Supplementary data

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Development and characterization of Lactoferrin-GMP nanogels: Evaluation



of pH, ionic strength and temperature effect

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Ana I. Bourbon,a Ana C. Pinheiro, a Maria G. Carneiro-da-Cunha b, Ricardo N. Pereira a, Miguel A. Cerqueira a* and António A. Vicente a

a CEB – Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal; E-mail: [email protected]

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b Departamento de Bioquímica, Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, s/n, Cidade Universitária, CEP 50.670-420 Recife, Pernambuco, Brazil

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*To whom correspondence should be addressed:

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E-mail: [email protected]

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Transparency measurement

A)

D)

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C)

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B)

Fig. S.1 Transparency of lactoferrin (♦), GMP (■) and lactoferrin-GMP mixtures (molar ratio of 1:7 at pH

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5.0) (▲), at temperatures of 25 ºC (A); 60 ºC (B), 70 ºC (C) and 80 ºC (D) as a function of time. Dashed

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lines are shown for visual guidance only.

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Characterization of lactoferrin-GMP nanohydrogels

Fig. S.2 Micrographs obtained by confocal laser scanning microscopy of the nanohydrogels: A) FITC-

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labeled lactoferrin and B) RBITC-labeled GMP and C) superposition of the images A and B.

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Effect of environmental conditions on the stability of lactoferrin-GMP nanohydrogels

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Fig. S.3 ζ-Potential of nanogels as a function of NaCl concentration in nanohydrogels solution.

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Fig. S.4 Peak Area of emission fluorescence of nanohydrogel solutions at different concentrations of NaCl

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Fig. S.5 Size distributions of lactoferrin-GMP nanohydrogel: A) as prepared; B) after 5 months of storage and C) after lyophilization and then rehydration (the three curves corresponds to the three true replicates of nine measurements realized for each one).

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