Adsorption of Proteins on Dual Loaded Silica Nanocapsules

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Adsorption of Proteins on Dual Loaded Silica Nanocapsules Sathya Ramalingam, Gwenaelle Le Bourdon, Emilie Pouget, Antoine Scalabre, Jonnalagadda Raghava Rao, and Adeline Perro J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b12028 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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The Journal of Physical Chemistry

Adsorption of Proteins on Dual Loaded Silica Nanocapsules Sathya Ramalingama, Gwenaelle Le Bourdonb, Emilie Pougetc, Antoine Scalabrec, Jonnalagadda Raghava Raoa and Adeline Perrod,* a.

Inorganic and Physical Chemistry Laboratory, Council fradiof Scientific & Industrial Research- Central Leather Research Institute, Adyar, Chennai-6000 20, India.

b.

Institut des Sciences Moléculaires (ISM) – CNRS - Université de Bordeaux - Bordeaux INP, UMR 5255, 351 cours de la libération, 33405 Talence, France.

c.

Chimie et Biologie des Membranes et des Nanoobjets (CBMN), CNRS - Université Bordeaux - Bordeaux INP, UMR5248, Allée St Hilaire, Bat B14, 33607 Pessac, France.

d.

Université de Bordeaux, Bordeaux INP, ISM, UMR 5255, Site ENSCBP, 16 avenue Pey Berland, 33607 Pessac, France.

Abstract The design of nanocarriers containing hydrophobic and hydrophilic compounds represents a powerful tool for cocktail delivery. Water-in-oil-in-water emulsions constitute an attractive approach as they offer dual encapsulation and provide a template for the constitution of a capsule. A limitation in the preparation of nano double emulsions is their instability resulting from high curvature radii. In this work, silica nanocapsules (NCs) stable over several months were synthesized. This was achieved by exploiting a double emulsion in which the oil phase is constituted by a combination of oils presenting several volatilities. The decrease of oil droplets size by evaporation favored the deposition of a silica layer at the nanoscale interface. The release of the payload obtained by drying the capsules was investigated by fluorescence spectroscopy. Understanding the interactions between proteins and nanocapsules is a fundamental point for many biological applications. Nanocapsules were exposed to two model proteins, which were Bovine Serum Albumin (BSA) and Lysozyme (Ly). These proteins, presenting differences in charges and size, showed distinctive arrangements onto the nanocapsules. Moreover, we have studied changes

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in α-helix and β-sheet content, which divulged the interactions between the proteins and the nanocapsules.


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Introduction Nanocarriers containing hydrophobic and hydrophilic compartments have attracted a significant attention giving rise to many applications in various fields such as cosmetics, food sciences or pharmaceutical products. 1- 3 The combination of immiscible components in one single carrier requires a fabrication process offering colloidal stability and high encapsulation efficiency in the nanometer range. Innovations in colloidal design have led to an increase in the fabrication of complex capsules loaded with incompatible molecules. A judicious approach is to exploit double emulsions, which acts first for dual encapsulation and serve also as a template for the deposition of a shell. 4 Recently, microfluidic devices were used to produce carriers containing both hydrophilic and hydrophobic molecules. 5,6 Although microfluidic offers an interesting approach allowing an efficient encapsulation process in one step, the diameter of the carriers is directed by a flow process leading to micron-sized cargos. A most conventional method is to use a multi-steps process by first forming an inverse water-inoil emulsion, followed by the emulsification of this mixture in a water phase. 7 In order to fulfill the specifications of multi-encapsulation, the formulation of such double emulsions requires an appropriate cocktail of surfactants or the use of complex designed polymers, which will direct the size and the stability of the double emulsion. 8 Moreover, nanocarriers have to maintain a difficult balance: to be stable enough in order to protect the complex payload and to be breakable in order to allow its liberation. 9 In general, the release of the encapsulated components can be triggered by several mechanisms including physical, chemical or mechanical. 10,11 The choice of the shell nature is directed by the expected properties for the nanocargos in terms of porosity, dimension and hydrophobic/hydrophilic balance character. Silica coating has attracted interest for its biocompatibility and its stable surface potential ideal for protein adsorption under physiological conditions. 12- 15 In this work, nanocarriers loaded with hydrophobic and hydrophilic compartments were prepared via the mineralization of nano-sized double emulsion. The fabrication of such emulsions is based on the preferential evaporation of a volatile solvent from the oil phase. 16 Removing a part of the oil leads to a decrease of the double emulsion droplets diameter, preserving the sequestered aqueous phase. Moreover, we have focused on surfactants, which facilitate the stability of the double emulsion and the silica deposition. The resulting nanocapsules were characterized by TEM and infrared spectroscopy. Then, the efficiency of the nanocarriers to release their payload was


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studied by fluorescence spectroscopy. Finally, their ability to interact with the two model proteins BSA and lysozyme was apprehended.


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Materials and Methods Rhodamine 6G (Dye Content 95%), Nile Red (Dye content ≥98%), Castor oil, Hexadecane (≥99%), Ethanol (Absolute), Chloroform (≥99%), Oleic acid(≥99%), Sodium Dodecyl Sulfate (SDS), Polyvinyl alcohol (Mw 89,000-98,000), Tetraethylorthosilicate (TEOS) (99%) and Trimethyoxysilyl propylmethacrylate (TSPM) (98%) were purchased from Sigma aldrich. Ultrapure deionized (D.I.) water was generated using a Millipore Milli-Q system.

Double emulsion generation Using a sonicator probe, an inverse emulsion was carried out. 500 µL of the water phase (W1) (containing 10%vol. of ethanol) was dispersed in the oil phase (O) constituted of 0.5g of castor oil, 50µL of oleic acid, used as surfactant and 20 µL of Trimethyoxysilyl propylmethacrylate dissolved in a mixture of hexadecane (50µL) and chloroform (1950µL). To monitor the encapsulation efficiency, water and oil phase are fluorescently labeled using Rhodamine 6G and Nile red respectively. Fluorescent dyes for water and oil phase were dissolved in ethanol and hexadecane, respectively. The inverse emulsion (W 1 /O) was then introduced in the continuous phase (W2) containing 10 wt% Poly(vinyl alcohol) (PVA). The mixture was emulsified using a sonicator probe. To evaporate the solvent, the resulting double emulsion was kept under constant stirring during one night at room temperature. The solvent evaporation process led to the formation of a silica shell at the oil/water interface. Finally, the prepared nanocapsules were washed with DI water for three times under centrifugation for 2000 rpm during 5 min. After proper washing, the measurement of hydrodynamic diameter and ζ potential of nanocapsules was performed by DLS using Zetasizer Nano ZS via 4 mW He/Ne laser (632.8 nm wavelength) with a scattering angle of 175° and the temperature was set at 25 °C. Samples were prepared by dispersing 200 μL of the double emulsion in 2 mL of water (pH=6). A disposable polystyrene cuvette was used to measure the hydrodynamic diameter of the particles. Further, the morphology of the nanocapsules containing oil and water phase was investigated by high-resolution TEM (HR-TEM; JEOL 3010 electron microscope at the accelerating voltage of 200 kV). 100µL of nanocapsules were dispersed in 1 mL of DI water and well dispersed particles were drop casted on carbon coated grid. During the drying process the grid coated with


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nanocapsules revealed oil release over the surface that led to cloudy appearance. To avoid the blur appearance, the grids were dried for two days before their visualization through TEM.

Encapsulation efficiency and release behavior The hydrophilic and hydrophobic contents were labeled with Rhodamine 6G and Nile red, respectively. The payload release was studied by drying the nanocapsules at 70ºC overnight. The dried capsules were dispersed in ethanol to quantify the Rhodamine 6G and in chloroform for the Nile red quantification. The resulting solutions along with nanocapsules were kept under shaking for 5h before measuring their fluorescence emission. The amount of free dye was calculated by measuring the maximum intensity of the fluorescence emission and the concentration of dye was estimated by a calibration curve of the corresponding fluorescent dye. The encapsulation efficiency was calculated by taking the ratio of (total amount of dye - amount of dye remaining in the supernatant) over total amount of dye.

Protein conjugation studies Chicken hen egg white Lysozyme (Ly, lyophilized powder ≥ 90%, ≥ 40,000units/mg) and Bovine Serum Albumin (BSA, lyophilized powder ≥ 98%, Mw = 66 kDa) were purchased from Sigma and used without further purification. The stock solution of both proteins (500 µM) was prepared in a 50 mM sodium phosphate buffer solution pH 7.4 (PBS) and stored at 4 °C, and was used within one week. All adsorption experiments were conducted with a constant concentration of proteins along with different concentrations of nanocapsules in a PBS buffer. The concentration of the nanocapsules was optimized in order to avoid aggregation phenomenon. These aggregates were formed in presence of lysozyme at a concentration above 5 mg/mL and for the BSA above 2 mg/mL. All the vials were mixed uniformly through vortex and incubated at room temperature for 2h.


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Dynamic light scattering The hydrodynamic diameter of the nanocapsules was measured using a Malvern Zetasizer Nano ZS with a fixed detector angle of 173°. Specifically, at pH 7.4, the protein stock suspension was sequentially added with a solution containing the nanocapsules. The mixture was incubated for 2 hours before DLS measurement and a total of 5 measurements were taken. All the samples were filtered through syringe filter (0.25µm pore diameter).

FTIR-ATR analysis FTIR was carried out to analyze the silica layer of the nanocapsules and the interactions with the proteins. Analyses in ATR mode were performed using a Nicolet iS50 FTIR spectrometer (Thermo-Scientific) equipped with a DLaTGS detector and iS50 ATR module. Spectra were collected in the 4000-400 cm-1 spectral range, at a resolution of 4 cm-1 and by averaging 100 scans.

Fluorescence quenching measurements The changes in optical properties arising from the interaction of nanoparticles with these proteins were monitored by optical emission spectroscopy (Varian carry Eclipse fluorescence spectrophotometer). For the fluorescent quenching measurements, the wavelength was set at 265nm, and the emission scan was monitored in the range of 270-450nm. The concentration of lysozyme and BSA was constant at 10µM and the concentration of nanocapsules ranged from 0 to 2 mg/mL in case of BSA and from 0 to 5 mg/mL in case of lysozyme. Blanks corresponding to the buffer were subtracted from the sample spectra to correct the fluorescence background. Duplicate experiments were conducted for each nanocapsules concentration. A Stern-Volmer plot (F 0 /F) versus the concentration of nanocapsules (NCs) provided the kinetic efficiency of static fluorescence quenching. 17 𝐹𝐹0 𝐹𝐹

= 1 + 𝐾𝐾𝑞𝑞 𝜏𝜏0 [𝑄𝑄] = 1 + 𝐾𝐾𝑎𝑎 [𝑄𝑄]

(1)


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Where F 0 and F represent the fluorescence emission intensities in absence and in presence of nanocapsules respectively, K q is the bimolecular quenching rate constant, τ 0 is the lifetime of the fluorophore in the absence of nanocapsules, K a is the association constant and finally (Q) is the nanocapsules/quencher concentration.

In some case, the dependence of the fluorescence as a function of the quencher followed a nonlinear Stern-Volmer equation: 𝐹𝐹0 𝐹𝐹

= �1 + 𝐾𝐾𝑞𝑞 𝜏𝜏𝑜𝑜 [𝑄𝑄]�. (1 + K a [𝑄𝑄])

(2)

The number of binding sites (n) per proteins (BSA and lysozyme) and the individual binding constants were evaluated from the linear double logarithm plot 18 19 𝐹𝐹0 −𝐹𝐹

log �

𝐹𝐹

� = 𝑙𝑙𝑙𝑙𝑙𝑙𝐾𝐾𝑎𝑎 + 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛[𝑄𝑄]

(3)

Time resolved fluorescence measurements

The lifetime measurement of proteins with and without nanocapsules were studied by exciting the tryptophan residues at 265 nm using a picosecond diode (IBH-NanoLED source N-295). Instrument response function (lamp pulse

Adsorption of Proteins on Dual Loaded Silica Nanocapsules

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